US Patent Application for METHOD OF SCREENING NEWBORNS FOR GENE VARIANTS Patent Application (Application #20160068906 issued March 10, 2016) (2024)

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, are methods for screening a newborn infant for one or more gene variants comprising, obtaining a genomic DNA containing sample from the newborn infant; sequencing at least one target region of each of two or more genes selected from the group consisting of PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8 in the genomic DNA; and screening for a gene variant from the sequenced target regions of each gene to identify gene variants present in the genomic DNA, wherein the sequencing does not include whole genome sequencing or whole exome sequencing. In some embodiments, there are provided methods for screening a newborn infant for one or more gene variants comprising, obtaining a genomic DNA containing sample from the newborn infant; sequencing at least one target region of each of genes PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8 in the genomic DNA; and screening for a gene variant from the sequenced target regions of each gene to identify gene variants present in the genomic DNA, wherein the sequencing does not include whole genome sequencing or whole exome sequencing. In some embodiments, the one or more gene variants are associated with one or more diseases or disorders. In some embodiments, the infant is asymptomatic for a disease or disorder. In some embodiments, the method is completed in less than 96 hours. In some embodiments, the at least one target region of each of genes PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8 is sequenced. In some embodiments, the method further comprises sequencing at least one target region of one or more genes selected from the group consisting of MCCC1, MCCC2, HMGCL, HLCS, GCDH, SLC22A5, HADHB, ASS1, CBS, MTHFR, MTR, MTRR, MMADHC, PAH, FAH, DUOX2, PAX8, SLC5A5, TG, TPO, TSHB, TSHR, HBB, BTD, CFTR, GJB2, GJB3, GJB6, ADA, and IL2RG. In some embodiments, the method further comprises sequencing at least one target region of one or more genes selected from the group consisting of MLYCD, ACADSB, AUH, DNAJC19, OPA3, TAZ, HSD17B10, ACADS, HADH, ETFA, ETFB, ETFDH, DECR1, CPT1A, CPT2, SLC25A20, ARG1, SLC25A13, AHCY, GNMT, MAT1A, PAH, GCH1, PCBD1, PTS, QDPR, TAT, HPD, HBA1, HBA2, HBB, GALE, GALK1, GALC, GBA, NPC1, NPC2, GAA, GLA, IDUA, ABCD1, and NGLY1. In some embodiments, at least one target region of each of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more genes is sequenced. In some embodiments, the target region comprises all or a portion of an exon. In some embodiments, the target region comprises about 50 bases to about 1000 bases. In some embodiments, the target region comprises about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500 or more bases. In some embodiments, two or more target regions for each gene are sequenced. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more target regions for each gene are sequenced. In some embodiments, the gene variants are selected from among gene variants listed in Table 5. In some embodiments, the target region that is selected comprises all or a portion of an exon encoding a portion of a gene selected from among the genes listed in Table 4. In some embodiments, the gene variants are selected from a group consisting of a splice site mutation, an in-frame mutation, a nonsense mutation, a mutation comprising an unknown nucleic acid base, and a frameshift mutation. In some embodiments, the gene variants are located in an exon, an intron, a splice site, a codon, a regulatory element, or a non-coding region. In some embodiments, the sample is a blood sample. In some embodiments, the blood sample is dried blood sample. In some embodiments, the sample is from a newborn infant between 0 and 72 hours after birth. In some embodiments, the sample is from a newborn infant less than 48 hours, less than 24 hours, less than 12, less than 6, less than 4, less than 2 hours, or less than 1 hour after birth. In some embodiments, the variant is identified less than 60 hours following collection of the sample. In some embodiments, the variant is identified less than 50 hours following collection of the sample. In some embodiments, the number of sequence reads per target region is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. In some embodiments, the variant is identified using a computer software module. In some embodiments, the method further comprises repeating the method one or more times at predetermined intervals after birth of the newborn infant. In some embodiments, the method further comprises repeating the method at 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, or one month after birth of the newborn infant. In some embodiments, the method further comprises repeating the method prior to discharge of the newborn infant from a care facility after birth. In some embodiments, the newborn infant does not exhibit symptoms of a metabolic disease or condition. In some embodiments, the sample is from an infant receiving care in a newborn intensive care unit (NICU). In some embodiments, the method further comprises providing a report comprising a list of variants identified in the genomic DNA. In some embodiments, the report includes a list of diseases or disorders associated with each variant. In some embodiments, the method further comprises selecting the infant for diagnostic assay for the disease or disorder if a gene variant associated with the disease or disorder is identified. In some embodiments, the diagnostic assay comprises detecting a biomarker indicative of the disease or disorder associated with the gene variant identified. In some embodiments, the detecting is by mass spectrometry. In some embodiments, the detecting is with an antibody. In some embodiments, the disease or disorder is a metabolic disorder. In some embodiments, the metabolic disorder is an organic acid disorder. In some embodiments, the organic acid disorder is propionic acidemia (PROP), methylmalonic acidemia (MUT), isovaleric acidemia (IVA), 3-methylcrotonyl-CoA carboxylase deficiency (3-MCC), 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (HMG), multiple carboxylase deficiency (MCD), beta-ketothiolase deficiency (βKT), or glutaric acidemia type I (GA1). In some embodiments, the metabolic disorder is a fatty acid oxidation disorder. In some embodiments, the fatty acid oxidation disorder is primary carnitine deficiency (CUD), medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, or trifunctional protein deficiency (TFP). In some embodiments, the metabolic disorder is an amino acid disorder. In some embodiments, the amino acid disorder is argininosuccinic aciduria (ASA), citrullinemia (CIT) type I, maple syrup urine disease (MSUD), hom*ocystinuria (HCY), phenylketonuria (PKU), or tyrosinemia (TYR I, II, III). In some embodiments, the disease or disorder is an endocrine disorder. In some embodiments, the endocrine disorder is congenital hypothyroidism (CH) or 21-hydroxylase deficiency (CAH). In some embodiments, the disease or disorder is a hemoglobin disorder. In some embodiments, the hemoglobin disorder is sickle cell disease, metheglobinemia beta-globin type, or beta thalassemia. In some embodiments, the beta thalassemia is thalassemia major or thalassemia intermedia. In some embodiments, the disease or disorder is biotinidase deficiency (BIOT), cystic fibrosis (CF), galactosemia type I, hearing loss, severe combined immunodeficiency (SCID), or X-linked severe combined immunodeficiency (SCID). In some embodiments, the hearing loss is nonsyndromic deafness, palmoplantar karatoderma, hystrix-like ichthyosis, Bart-Pumphrey syndrome, Vohwinkel syndrome, karatitis-ichthyosis-deafness (KID), erythrokeratodermia variabilis et progressive (EKVP), or Clouston syndrome. In some embodiments, the disease or disorder is malonyl-CoA decarboxylase deficiency (MAL), isobutyryl-CoA dehydrogenase (IBD) deficiency, 2-methylbutyryl-CoA dehydrogenase deficiency, 3-methylglutaconic aciduria (3MGA) type I, 3-methylglutaconic aciduria (3MGA) type V, 3-hydroxy-2-methylbutyryl-CoA dehydrogenase deficiency (2M3HBA), short-chain acyl-CoA dehydrogenase (SCAD) deficiency, 3-hydroxyacyl-CoA dehydrogenase deficiency (M/SCHAD), glutaric acidemia type II (GA2), glutaric acidemia type II (GA2), carnitine palmitoyltransferase I deficiency (CPT IA), carnitine palmitoyltransferase II deficiency (CPT II), carnitine-acylcarnitine translocase (CACT), arginase deficiency (ARG), citrullinemia type II (CIT II), hypermethioninemia (MET), disorders of biopterin regeneration, tyrosinemia (TYR I, II, III), alpha thalassemia (hemoglobin disorder-Var-Hb), galactosemia type II, or galactosemia type III. In some embodiments, the disease or disorder is X-linked adrenoleukodystrophy adrenomyeloneuropathy Addison disease (X-ALD), 2,4 dienoyl-CoA reductase deficiency, Pompe disease (GAA deficiency), Krabbe Disease, Gaucher disease (types I, II, & III), Fabry disease, mucopolysaccharidosis type I (MPS I), congenital disorder of deglycosylation type 1v, Niemann-Pick disease (type C1), or Niemann-Pick disease (type C2). In some embodiments, the disease or disorder is congenital adrenal hyperplasia (CAH), medium chain acyl-CoA dehydrogenase deficiency (MCAD), long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHAD), very long chain acyl-CoA dehydrogenase deficiency (VLCAD), beta-ketothiolase deficiency (BKD), isobutyryl CoA dehydrogenase deficiency (IBD), isovaleric acidemia (IVA), maple syrup urine disease (MSUD), methylmalonic acidemias (MMA/8 types), propionic acidemia (PROP), argininosuccinate lyase deficiency (ASA), or galactosemia. In some embodiments, there are provided methods of screening a newborn for one or more gene variants comprising, identifying a gene variant by sequencing at least one target region from each of genes PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8, in a genomic DNA containing sample from the newborn infant, wherein the sequencing does not include whole genome sequencing or whole exome sequencing.

In some embodiments there are provided methods of screening newborn infants for a newborn infant for one or more gene variants comprising (a) generating a genomic library pool from genomic DNA containing sample from a newborn infant; (b) performing a plurality of DNA sequencing reactions on the genomic library pool to determine the DNA sequence of at least one target region in each of two or more genes selected from the group consisting of PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8, wherein DNA containing the target regions is simultaneously sequenced to produce a plurality of sequencing reads for each target region, wherein the sequencing reactions do not comprise whole genome sequencing or whole exome sequencing; (c) identifying gene variants in the two or more genes by comparing the plurality of sequencing reads for each target region to a reference sequence; and (d) generating a report that all identified gene variants. In some embodiments, the generating a genomic library pool comprises amplifying the genomic DNA. In some embodiments, the generating a genomic library pool comprises: (a) fragmenting the isolated genomic DNA to produce fragmented genomic DNA; (b) ligating adaptors to the fragmented genomic DNA to produce adaptor-modified genomic DNA; and (c) amplifying the adaptor-modified genomic DNA. In some embodiments, the adaptor comprises a barcode. In some embodiments, the genomic DNA is amplified by polymerase chain reaction. In some embodiments, the adaptor-modified genomic DNA is amplified using oligonucleotide primers specific to the target region. In some embodiments, the oligonucleotide primers are labeled. In some embodiments, the one or more gene variants are associated with one or more diseases or disorders. In some embodiments, the infant is asymptomatic for a disease or disorder. In some embodiments, the method is completed in less than 96 hours. In some embodiments, the at least one target region of each of gene PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8 is sequenced. In some embodiments, the method further comprises identifying a gene variant by sequencing at least one target region of one or more genes selected from the group consisting of MCCC1, MCCC2, HMGCL, HLCS, GCDH, SLC22A5, HADHB, ASS1, CBS, MTHFR, MTR, MTRR, MMADHC, PAH, FAH, DUOX2, PAX8, SLC5A5, TG, TPO, TSHB, TSHR, HBB, BTD, CFTR, GJB2, GJB3, GJB6, ADA, and IL2RG. In some embodiments, the method further comprises identifying a gene variant by sequencing at least one target region of one or more genes selected from the group consisting of MLYCD, ACADSB, AUH, DNAJC19, OPA3, TAZ, HSD17B10, ACADS, HADH, ETFA, ETFB, ETFDH, DECR1, CPT1A, CPT2, SLC25A20, ARG1, SLC25A13, AHCY, GNMT, MAT1A, PAH, GCH1, PCBD1, PTS, QDPR, TAT, HPD, HBA1, HBA2, HBB, GALE, GALK1, GALC, GBA, NPC1, NPC2, GAA, GLA, ID UA, ABCD1, and NGLY1. In some embodiments, at least one target region of each of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more genes is sequenced. In some embodiments, the target region comprises all or a portion of an exon. In some embodiments, the target region comprises about 50 bases to about 1000 bases. In some embodiments, the target region comprises about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500 or more bases. In some embodiments, two or more target regions for each gene are sequenced. In some embodiments, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more target regions for each gene are sequenced. In some embodiments, the gene variants are selected from among gene variants listed in Table 5. In some embodiments, the target region that is selected comprises all or a portion of an exon encoding a portion of a gene selected from among the genes listed in Table 4. In some embodiments, the gene variants are selected from a group consisting of a splice site mutation, an in-frame mutation, a nonsense mutation, a mutation comprising an unknown nucleic acid base, and a frameshift mutation. In some embodiments, the gene variants are located in an exon, an intron, a splice site, a codon, a regulatory element, and a non-coding region. In some embodiments, the sample is a blood sample. In some embodiments, the blood sample is dried blood sample. In some embodiments, the sample is from a newborn infant between 0 and 72 hours after birth. In some embodiments, the sample is from a newborn infant less than 48 hours, less than 24 hours, less than 12, less than 6, less than 4, less than 2 hours, or less than 1 hour after birth. In some embodiments, the gene variant is identified less than 60 hours following collection of the sample. In some embodiments, the gene variant is identified less than 50 hours following collection of the sample. In some embodiments, the number of sequence reads per target region is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. In some embodiments, the gene variant is identified using a computer software module. In some embodiments, the method further comprises repeating the method one or more times at predetermined intervals after birth of the newborn infant. In some embodiments, the method further comprises repeating the method at 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, or one month after birth of the newborn infant. In some embodiments, the method further comprises repeating the method prior to discharge of the newborn infant from a care facility after birth. In some embodiments, the newborn infant does not exhibit symptoms of a metabolic disease or condition. In some embodiments, the sample is from an infant receiving care in a newborn intensive care unit (NICU). In some embodiments, the method further comprises providing a report comprising a list of variants identified in the sample. In some embodiments, the report includes a list of diseases or disorders associated with each identified gene variant. In some embodiments, the method further comprises selecting the infant for diagnostic assay for a disease or disorder if a gene variant associated with the disorder is identified. In some embodiments, the diagnostic assay comprises detecting a biomarker indicative of the disease or disorder associated with the gene variant identified. In some embodiments, the detecting is by mass spectrometry. In some embodiments, the detecting is with an antibody. In some embodiments, the disease or disorder is a metabolic disorder. In some embodiments, the metabolic disorder is an organic acid disorder. In some embodiments, the organic acid disorder is propionic acidemia (PROP), methylmalonic acidemia (MUT), isovaleric acidemia (IVA), 3-methylcrotonyl-CoA carboxylase deficiency (3-MCC), 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (HMG), multiple carboxylase deficiency (MCD), beta-ketothiolase deficiency (βKT), or glutaric acidemia type I (GA1). In some embodiments, the metabolic disorder is a fatty acid oxidation disorder. In some embodiments, the fatty acid oxidation disorder is primary carnitine deficiency (CUD), medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, or trifunctional protein deficiency (TFP). In some embodiments, the metabolic disorder is an amino acid disorder. In some embodiments, the amino acid disorder is argininosuccinic aciduria (ASA), citrullinemia (CIT) type I, maple syrup urine disease (MSUD), hom*ocystinuria (HCY), phenylketonuria (PKU), or tyrosinemia (TYR I, II, III). In some embodiments, the disease or disorder is an endocrine disorder. In some embodiments, the endocrine disorder is congenital hypothyroidism (CH) or 21-hydroxylase deficiency (CAH). In some embodiments, the disease or disorder is a hemoglobin disorder. In some embodiments, the hemoglobin disorder is sickle cell disease, metheglobinemia, beta-globin type, or beta thalassemia. In some embodiments, the Beta thalassemia is thalassemia major or thalassemia intermedia. In some embodiments, the disease or disorder is biotinidase deficiency (BIOT), cystic fibrosis (CF), galactosemia type I, hearing loss, severe combined immunodeficiency (SCID), or X-linked severe combined immunodeficiency (SCID). In some embodiments, the hearing loss is nonsyndromic deafness, palmoplantar karatoderma, hystrix-like ichthyosis, Bart-Pumphrey syndrome, Vohwinkel syndrome, karatitis-ichthyosis-deafness (KID), erythrokeratodermia variabilis et progressive (EKVP), or Clouston syndrome. In some embodiments, the disease or disorder is malonyl-CoA decarboxylase deficiency (MAL), isobutyryl-CoA dehydrogenase (IBD) deficiency, 2-methylbutyryl-CoA dehydrogenase deficiency, 3-methylglutaconic aciduria (3MGA) type I, 3-methylglutaconic aciduria (3MGA) type V, 3-hydroxy-2-methylbutyryl-CoA dehydrogenase deficiency (2M3HBA), short-chain acyl-CoA dehydrogenase (SCAD) deficiency, 3-hydroxyacyl-CoA dehydrogenase deficiency (M/SCHAD), glutaric acidemia type II (GA2), glutaric acidemia type II (GA2), carnitine palmitoyltransferase I deficiency (CPT IA), carnitine palmitoyltransferase II deficiency (CPT II), carnitine-acylcarnitine translocase (CACT), arginase deficiency (ARG), citrullinemia type II (CIT II), hypermethioninemia (MET), disorders of biopterin regeneration, tyrosinemia (TYR I, II, III), alpha thalassemia (Hemoglobin Disorder-Var-Hb), galactosemia type II, or galactosemia type III. In some embodiments, the disease or disorder is X-linked adrenoleukodystrophy, adrenomyeloneuropathy, Addison disease (X-ALD), 2,4 dienoyl-CoA reductase deficiency, Pompe disease (GAA deficiency), Krabbe Disease, Gaucher disease (types I, II, & III), Fabry disease, mucopolysaccharidosis type I (MPS I), congenital disorder of deglycosylation type 1v, Niemann-Pick disease (type C1), or Niemann-Pick disease (type C2). In some embodiments, the wherein the disease or disorder is congenital adrenal hyperplasia (CAH), medium chain acyl-COA dehydrogenase deficiency (MCAD), long chain 3 hydroxyacyl-COA dehydrogenase deficiency (LCHAD), very long chain acyl-COA dehydrogenase deficiency (VLCAD), beta-ketothiolase deficiency (BKD), isobutyryl COA dehydrogenase deficiency (IBD), isovaleric acidemia (IVA), maple syrup urine disease (MSUD), methylmalonic acidemias (MMA/8 types), propionic acidemia (PROP), argininosuccinate lyase deficiency (ASA), or galactosemia.

In some embodiments, there are provide computer-implemented systems including (a) a digital processing device comprising an operating system configured to perform executable instructions and a memory and (b) a computer program including instructions executable by the digital processing device to create an application comprising: (i) a software module configured to receive a plurality of sequence reads, the sequence reads obtained from sequencing target regions of each of genes PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8; (ii) a software module configured to perform an alignment of the plurality of sequence reads to a reference sequence; (iii) a software module configured to identify gene variants and, optionally, characterize the gene variants as pathogenic or likely pathogenic or associated with a disease or disorder; and (iv) a software module configured to generate a report providing a list of the gene variants identified. In some embodiments, the system further comprises a sequence analyzer communicatively connected with the software module configured to receive a plurality of sequence reads, wherein the sequence analyzer is configured for sequencing a plurality of target regions to provide a plurality of sequence reads. In some embodiments, the system further comprises a database, in computer memory, of gene variants selected from the gene variants listed in Table 5.

In some embodiments, there are provided genetic screening platforms comprising: (a) a processor configured to provide an application comprising: (i) a software module configured to receive a plurality of sequence reads, the sequence reads obtained from sequencing target regions of each of genes PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8; (b) a server processor configured to provide a server application comprising: (i) a database, in computer memory, of gene variants selected from the gene variants listed in Table 5; (ii) a software module configured to perform an alignment of the plurality of sequence reads to a reference sequence; (iii) a software module configured to identify gene variants and, optionally, characterize the gene variants as pathogenic or likely pathogenic or associated with a disease or disorder; and (iv) a software module configured to generate a report providing a list of the gene variants identified.

In some embodiments, there are provided non-transitory computer-readable storage media encoded with a computer program including instructions executable by a processor to create an application comprising: (a) a database, in computer memory, of gene variants selected from the gene variants listed in Table 5; (b) a software module configured to receive a plurality of sequence reads, the sequence reads obtained from sequencing target regions of each of genes PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8; (c) a software module configured to perform an alignment of the plurality of sequence reads to a reference sequence; (d) a software module configured to identify gene variants and, optionally, characterize the gene variants as pathogenic or likely pathogenic or associated with a disease or disorder; and (e) a software module configured to generate a report providing a list of the gene variants identified.

In some embodiments, there are provided compositions comprising a collection of oligonucleotide primers for selective amplification of plurality of target regions of each of PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8.

In some embodiments, there are provided kits comprising a collection of oligonucleotide primers for sequencing of each of PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8. In some embodiments, the kit further comprises one or more reagents for performing a sequencing reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a process flow diagram of an exemplary method for targeted high-throughput screening of newborn samples.

DETAILED DESCRIPTION Certain Terminology

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. All patents, patent applications, published applications and publications, GENBANK sequences, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers change and particular information on the internet is removed, but equivalent information is known and is readily accessed, such as by searching the internet and/or appropriate databases. Reference thereto evidences the availability and public dissemination of such information. Generally, the procedures for antibody production and molecular biology methods are methods commonly used in the art. Such standard techniques are found, for example, in reference manual, such as, for example, Sambrook et al. (2000) and Ausubel et al. (1994).

As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms (e.g., “include”, “includes”, and “included”) is not limiting.

As used herein, ranges and amounts are expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 bases” means “about 5 bases” and also “5 bases.” In some embodiments, “about” includes an amount that would be expected to be within experimental error. In some embodiments, “about” means plus or minus 10% of the expressed value.

As used herein, “optional” or “optionally” means that the subsequently described event or circ*mstance does or does not occur, and that the description includes instances where said event or circ*mstance occurs and instances where it does not. For example, an optionally substituted group means that the group is unsubstituted or is substituted.

As used herein, the terms “subject”, “individual” and “patient” are used interchangeably. None of the terms are to be interpreted as requiring the supervision of a medical professional (e.g., a doctor, nurse, physician's assistant, orderly, hospice worker).

The terms “target region” and “targeted region” are used interchangeably herein and refer to a region of a gene that contains one or more locations of relevant gene variants. In some embodiments, the target region is an exon of a gene associated with a disease or condition. In some embodiments, the target region is of a gene associated with a disease or disorder listed in Table 1. In some embodiments, the target region is of a gene listed in Table 2. In some embodiments, the target region is an exon listed in Table 4. In some embodiments, the target region contains one or more variant set forth in Table 5.

As used herein, a “reference genome” (also simply called “reference”) is any known sequence to which a sequence read is aligned and contains the wild type sequence. In some embodiments, the reference genome corresponds to all or only part of the genome. In some embodiments, the reference is a gene or a target region of a gene.

The terms “gene variant” and “genetic variant” are used interchangeably and refer to mutation in a gene sequence compared to a wild type sequence. In some embodiments the gene variant is associated with a disease or disorder. In some embodiments, a variant is a change of one base to one or more other bases, an insertion of one or more bases, or a deletion of one or more bases. In some embodiments, a variant occurs in one chromosome. In some embodiments, a variant occurs in both chromosomes.

The term “obtaining” as used herein with reference to a genomic DNA containing sample includes receiving the sample by a testing facility. In some embodiments, the sample is collected from a newborn infant by a third party health care practitioner using known techniques and is shipped to the testing facility.

Overview

Provided herein are methods for the rapid screening of newborn infants for gene variants associated with inherited diseases and conditions using a high-throughput targeted genomics-based sequencing assay. In some embodiments, a report of the results of the screening is provided to the newborn's parents or caregiver within a few days of birth, allowing the parents to seek medical advice regarding diagnostic testing or medical intervention as quickly after birth as possible to avoid the development of potentially debilitating disease. In some embodiments, the methods provided identify pathogenic or likely pathogenic genetic variants in the genome of the infant within a few days of birth, e.g., 48-72 hours after birth.

In some embodiments, the newborn screening assay provides comprehensive coverage of genetic conditions recommended for screening of all newborn infants, regardless of whether the infant is exhibiting symptoms of a disease or disorder. In some embodiments, the newborns are asymptomatic and thus, the method provides a means of identifying those infants carrying potentially pathogenic gene variants for diagnostic testing or monitoring prior to the presentation of any symptoms. The assay is adaptable to the addition of new gene targets for screening. The methods provided herein do not require whole genomic or whole exome sequencing, and therefore provides a low-cost primary screening approach for all newborns of high accuracy and sensitivity. In some embodiments, the methods allow for stratification of at risk infants for diagnostic screening for a diseases or conditions, or further monitoring for development of clinical symptoms. In some embodiments, the methods provided herein are performed prior to or in conjunction with current newborn diagnostic screening methods. The present targeted genomic screening allows for a rapid analysis of sequencing results and return of the results to the patient or care provider. In some embodiments, the patient is selected for diagnostic testing based on the screening results. In some embodiments, the methods provided herein further comprise additional testing for diagnosis of a disease or condition.

Currently, primary screening for genetic conditions in newborn infants is accomplished using a metabolic test for each metabolic condition of interest that each measure the concentration of one or more analytes in blood samples obtained from infants shortly after birth. Because the newborn screening tests are prescribed as part of state-based public health programs, the number and type of genetic conditions varies by state and jurisdiction. In most instances, the test for each condition is a separate assay for a particular metabolite or enzymatic activity for each disease or condition. Thus, coverage of all recommended condition requires multiple assays, which increases the likelihood of testing errors and test sample contamination. Newborn screening of premature, low birth weight, or sick infants is complex and test parameters are not optimized for these patient groups. For example, newborn intensive care unit (NICU) infants are more likely to generate false positive or false negative results and repeated screening is often necessary to obtain acceptable results. In turn, because of inaccurate results, necessary or life-saving treatment may be delayed. Exemplary metabolic tests include tandem mass spectrometry (MS/MS), time resolved fluoro-immunoassay, isoelectric focusing (IEF), fluorometric assay, or real time polymerase chain reaction (rtPCR). Validity of each individual test is subject to confounding factors, such as when and how the sample was collected. Further, sample rejection rate is high due to potential analyte contamination or interference, e.g. due to uneven application or layering in the assay, contamination from other samples, alcohol, glove powder and other collection contamination sources (e.g., improper handling), improper drying of the sample, over-application of the sample, serum or tissue contamination, improper storage of the sample or aging of the sample. In addition, because of the variability in the emergence of phenotypic symptoms of the particular disease or condition, multiple samples often are needed for proper screening. For example, a primary test is performed on a sample collected 24-48 hours after birth and a follow-up test is perform on a sample collected about 10-14 days after birth. On average, results are acquired within 7-14 days of birth.

The current newborn screening approaches also preclude the screening for genetic conditions without a metabolic marker or analyte, or conditions with delayed onset phenotypes. In addition, adding conditions to a recommended testing panel is time consuming and expensive. For example, certain tests, such as the test for severe combined immunodeficiency (SCID), require a specialized assay with specific equipment. As a result, a major cost/benefit decision must be made when adding specialized assays, resulting in the lack of universal adoption. Using a single genomics-based test for primary screening avoids such costly decisions by providing a convenient way to add additional conditions by simple addition of target screening regions to the panel. Thus, in some embodiments, the present methods are tailored to the newborn screening requirements for each state or jurisdiction.

In some embodiments, the methods provided herein screen for gene variants associated with each disease in a panel of diseases as prescribed by a particular state or jurisdiction. In some embodiments, the methods provided herein screen for gene variants associated with each disease in a panel of diseases including congenital adrenal hyperplasia (CAH), medium chain acyl-CoA dehydrogenase deficiency (MCAD), long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHAD), very long chain acyl-CoA dehydrogenase deficiency (VLCAD), beta-ketohiolase deficiency (BKD), isobutyryl-CoA dehydrogenase (IBD) deficiency, isovaleric acidemia (IVA), maple syrup urine disease (MSUD), methylmalonic acidemias (MMA/8 types), propionic acidemia (PROP), argininosuccinate lyase deficiency (ASA), and galactosemia. In some embodiments, the disease or condition is one or more conditions listed in listed in Table 1. In some embodiments, the methods provided herein screen for gene variants associated with each core condition listed in Table 1. In some embodiments, the methods provided herein screen for gene variants associated with each core condition and each secondary condition listed in Table 1. In some embodiments, the methods provided herein screen for gene variants associated with each core condition, each secondary condition, and each added condition listed in Table 1. In some embodiments, the diseases or conditions included in the panel of diseases include the primary and secondary conditions recommended for screening by the American College of Genetics for the screening of all newborn infants. In some embodiments, the diseases or conditions panel includes diseases or conditions that are recommended, but not yet incorporated into state-based screening newborn screening programs. In some embodiments, the methods provided herein screen for gene variants associated with a disease or condition.

Exemplary Methods

Provided herein are exemplary methods for screening genomic DNA containing samples from newborn infants for gene variants associated with or likely to be associated with a disease or condition. In some embodiments, the methods provided herein involve screening for the presence or absence of a gene variant that is pathogenic or likely pathogenic. The methods provided herein involve targeted sequencing of genomic DNA containing samples from newborns, and do not comprise whole genome or whole exome sequencing. In some embodiments, the methods provided herein involve screening for the presence or absence of a gene variant associated with a disease or condition. In some embodiments, the disease or condition is a disease or condition listed in Table 1. In some embodiments, the disease or condition is a metabolic disorder. In some embodiments, the disease or condition is an organic acid disorder, a fatty acid oxidation disorder, an amino acid disorder, an endocrine disorder, a hemoglobin disorder, or a combination of any of these disorders.

In some embodiments, the methods provided herein involve sequencing target regions of selected genes for the presence or absence of a gene variant that is pathogenic or likely pathogenic. In some embodiments, the methods provided herein involve sequencing target regions of selected genes for the presence or absence of a gene variant associated with a disease or disorder. In some embodiments, the methods provided herein involve sequencing target regions of selected genes for the presence or absence of a gene variant associated with a disease or disorder listed in Table 1. In some embodiments, a target region of a gene associated with a gene or disorder listed in Table 1 is screened. In some embodiments, a gene selected from among the genes listed in Table 2 is screened. In some embodiments, two or more genes selected from among the genes listed in Table 2 are screened. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more genes are screened.

In some embodiments, the method comprises sequencing all or a portion of a targeted region of a selected gene or panel of genes. In some embodiments, all or a portion of the targeted region of each selected gene is sequenced. In some embodiments, at least one targeted region of each selected gene is sequenced. In some embodiments, two or more targeted regions of each selected gene are sequenced. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more target regions of each selected gene is sequenced. In some embodiments, the targeted regions are located within an exon. In some embodiments, the exons are selected from exons listed in Table 4.

In some embodiments, the method comprises screening for one or more variants in a selected gene or panel of genes. In some embodiments, the method comprises screening for one or more variants in a selected gene or panel of genes selected from among the genes lists in Table 2.

In some embodiments, the steps of the method involve (a) generating a genomic library pool from a genomic DNA containing sample from a newborn infant; (b) performing a plurality of DNA sequencing reactions on the genomic library pool to determine the DNA sequence of at least one target region in each of two or more genes selected from the genes listed in Table 2, wherein the DNA encoding the target regions is simultaneously sequenced to produce a plurality of sequencing reads for each target region; (c) identifying gene variants in the two or more genes by comparing the plurality of sequencing reads for each target region to a reference sequence; and (d) generating a report that characterizes all or a subset of identified gene variants as pathogenic or likely pathogenic or associated with the disease or disorder. In some embodiments, the genomic DNA containing sample is an isolated genomic DNA isolated from a biological sample from the infant.

In some embodiments, a target region of a gene selected from a gene listed in Table 2 is sequenced. In some embodiments, a target region of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more genes listed in Table 2 is selected. In some embodiments, a target region of a gene selected from PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, MCCC1, MCCC2, HMGCL, HLCS, ACAT1, GCDH, SLC22A5, ACADM, ACADVL, HADHA, HADHB, ASL, ASS1, BCKDHA, BCKDHB, DBT, DLD, CBS, MTHFR, MTR, MTRR, MMADHC, PAH, FAH, DUOX2, PAX8, SLC5A5, TG, TPO, TSHB, TSHR, CYP21A2, HBB, BTD, CFTR, GALT, GJB2, GJB3, GJB6, ADA, IL2RG, MLYCD, ACAD8, ACADSB, AUH, DNAJC19, OPA3, TAZ, HSD17B10, ACADS, HADH, ETFA, ETFB, ETFDH, DECR1, CPT1A, CPT2, SLC25A20, ARG1, SLC25A13, AHCY, GNMT, MAT1A, PAH, GCH1, PCBD1, PTS, QDPR, TAT, HPD, HBA1, HBA2, HBB, GALE, GALK1, GALC, GBA, NPC1, NPC2, GAA, GLA, ID UA, ABCD1, and NGLY1 is sequenced. In some embodiments, a target region of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more genes selected from PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, MCCC1, MCCC2, HMGCL, HLCS, ACAT1, GCDH, SLC22A5, ACADM, ACADVL, HADHA, HADHB, ASL, ASS1, BCKDHA, BCKDHB, DBT, DLD, CBS, MTHFR, MTR, MTRR, MMADHC, PAH, FAH, DUOX2, PAX8, SLC5A5, TG, TPO, TSHB, TSHR, CYP21A2, HBB, BTD, CFTR, GALT, GJB2, GJB3, GJB6, ADA, IL2RG, MLYCD, ACAD8, ACADSB, AUH, DNAJC19, OPA3, TAZ, HSD17B10, ACADS, HADH, ETFA, ETFB, ETFDH, DECR1, CPT1A, CPT2, SLC25A20, ARG1, SLC25A13, AHCY, GNMT, MAT1A, PAH, GCH1, PCBD1, PTS, QDPR, TAT, HPD, HBA1, HBA2, HBB, GALE, GALK1, GALC, GBA, NPC1, NPC2, GAA, GLA, IDUA, ABCD1, and NGLY1 is sequenced.

In some embodiments, a target region of a gene selected from PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, MCCC1, MCCC2, HMGCL, HLCS, ACAT1, GCDH, SLC22A5, ACADM, ACADVL, HADHA, HADHB, ASL, ASS1, BCKDHA, BCKDHB, DBT, DLD, CBS, MTHFR, MTR, MTRR, MMADHC, PAH, FAH, DUOX2, PAX8, SLC5A5, TG, TPO, TSHB, TSHR, CYP21A2, HBB, BTD, CFTR, GALT, GJB2, GJB3, GJB6, ADA, and IL2RG is sequenced. In some embodiments, a target region of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more genes selected from PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, MCCC1, MCCC2, HMGCL, HLCS, ACAT1, GCDH, SLC22A5, ACADM, ACADVL, HADHA, HADHB, ASL, ASS1, BCKDHA, BCKDHB, DBT, DLD, CBS, MTHFR, MTR, MTRR, MMADHC, PAH, FAH, DUOX2, PAX8, SLC5A5, TG, TPO, TSHB, TSHR, CYP21A2, HBB, BTD, CFTR, GALT, GJB2, GJB3, GJB6, ADA, and IL2RG is sequenced.

In some embodiments, a target region of a gene one or more additional genes is sequenced. In some embodiments, the one or more additional genes is selected from among MLYCD, ACAD8, ACADSB, AUH, DNAJC19, OPA3, TAZ, HSD17B10, ACADS, HADH, ETFA, ETFB, ETFDH, DECR1, CPT1A, CPT2, SLC25A20, ARG1, SLC25A13, AHCY, GNMT, MAT1A, PAH, GCH1, PCBD1, PTS, QDPR, TAT, HPD, HBA1, HBA2, HBB, GALE, and GALK1. In some embodiments, the one or more additional genes is selected from among GALC, GBA, NPC1, NPC2, GAA, GLA, IDUA, ABCD1, and NGLY1.

An exemplary illustration of the steps of the method is provided in FIG. 1. In some embodiments, all or some of the steps illustrated are performed. In some embodiments, the step performed include all or some of the following 1) sample input (e.g. receiving and preparation of the biological sample from a newborn infant), 2) genomic DNA extraction, 3) genomic library preparation, 4) genomic library pool preparation and gene sequencing, 5) results analysis, and 6) client report generation. In some embodiments, selected steps (e.g., results analysis, including sequence alignment, identification of variants, classification of variants) are performed with a computer system. In some embodiments, the results of method are used by a doctor in determining a diagnosis of the newborn infant.

In some embodiments, the biological sample includes genomic DNA of a newborn infant. In some embodiments, the genomic DNA is in the form of genomic segments of chromosomes. In some embodiments, genomic DNA is in the form of intact chromosomes. In some embodiments, the biological sample contains cells from the newborn infant. In some embodiments, the biological sample is a fluid or a tissue sample. Biological samples include, but are not limited, to whole blood, dissociated bone marrow, bone marrow aspirate, pleural fluid, peritoneal fluid, central spinal fluid, abdominal fluid, pancreatic fluid, cerebrospinal fluid, brain fluid, ascites, pericardial fluid, urine, saliva, bronchial lavage, sweat, tears, ear flow, sputum, hydrocele fluid, sem*n, vagin*l flow, milk, amniotic fluid, and secretions of respiratory, intestinal or genitourinary tract. In particular embodiments, the sample is from a fluid or tissue that is part of, or associated with, the lymphatic system or circulatory system. In some embodiments, the sample is a blood sample that is a venous, arterial, peripheral, tissue, cord blood sample.

In some embodiments, the samples are obtained from the subject by any suitable means of obtaining the sample using well-known and routine clinical methods. Procedures for obtaining fluid samples from a subject are well-known. For example, procedures for drawing and processing whole blood and lymph are well-known and are employed to obtain a sample for use in the methods provided. In some embodiments, the sample is a blood sample obtained from the heel puncture of the newborn infant.

In some embodiments, for collection of a blood sample, the blood is dried on an absorbable medium such as a filter paper.

In some embodiments, for collection of a blood sample, an anti-coagulation agent (e.g. EDTA, or citrate and heparin or CPD (citrate, phosphate, dextrose) or comparable substances) is added to the sample to prevent coagulation of the blood. In some examples, the blood sample is collected in a collection tube that contains an amount of EDTA to prevent coagulation of the blood sample.

Methods for the isolation of nucleic acids from cells contained in tissue or fluid samples are well-known in the art. In particular embodiments, the genomic DNA is isolated from cells contained in a blood sample collected from the newborn infant. In some embodiments, the genomic DNA extracted from the sample is quantified following extraction.

In some embodiments, the genomic DNA of the sample is fragmented, e.g., by sonication or other suitable methods to obtain smaller genomic segments. In some embodiments, genomic segments of about 200 to about 1000 bases long are generated. In some embodiments, genomic segments of less than about 200 bases long are generated. In some embodiments, genomic segments of greater than 1000 bases long are generated. In some embodiments, the ends of the fragmented genomic DNA are then prepared for adaptor ligation. Methods for end repair of fragments genomic DNA are well-known in the art. In some embodiments, the ends of the fragmented genomic DNA are blunted to prepare for adaptor ligation.

In some embodiments, the genomic segments are tagged with a barcode or multiplex identifier (MID). In some embodiments, a sequence of 10 bases are added (e.g., using a ligase) to the end of a genomic segment. In some embodiments a sequence of 10 bases is added using the primers provided in an ILLUMINA Index 2 Barcode Adaptor reaction packet. In this manner, segments from various samples are sequenced in parallel during a same sequencing run using the ID to multiplex. In some embodiments, the ID is read as part of a sequence read, and reads with the same ID are attributed to a same sample and analyzed as a group.

In some embodiments, the percentage of genomic segments representing a selected target region in the genomic sample is increased. In some embodiments, the percentage of genomic segments representing two or more selected target regions in the genomic sample is increased. In some embodiments, the percentage is increased by amplifying and/or enriching the sample for DNA from one or more targeted regions of the genome. In some embodiments, the resulting amplified sample is referred to as a target-increased sample. In some embodiments, a target region is selected from a gene listed in Table 2. In some embodiments, two or more target regions are selected from a gene listed in Table 4. In some embodiments, the target region is about a few hundred bases, e.g., 150-250 bases, 150-400 bases, or 200-600 bases.

In some embodiments, the addition of a sample-specific ID occurs at different steps of the method. For example, in some embodiments, the ID is added after the amplification/enrichment step and then the samples are mixed together. In this way, the different samples are amplified or enriched for different target regions. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more target regions are used.

In some embodiments, forward and reverse primers are used to amplify a target region. In some embodiments, the forward and reverse primers are selected from various lengths, e.g., about 15-30 bases long. In some embodiments, the set of forward and reverse primers only amplify one part of the genome. Methods are available in the art for determining optimal primer length and specificity for amplification of genomic DNA segments.

In some embodiments, probes are used to capture genomic segments that correspond to the target region (i.e., enrichment of the target regions). In some embodiments, probes that are designed to hybridize to the target region are placed on a surface. Then, the genomic segments are placed over the surface and the segments of the target region are preferentially be hybridized. For example, a microarray with the probes are constructed, and the segments washed over the microarray. In some embodiments, the probes are about 25-75 bases long for a target region of about 200-550 bases long. As the probe can capture either end of a genomic segment, the segments, in some embodiments, for example, span a region of 200 bases to about 550 bases for genomic segments of up to 250 bases. Methods are available in the art for determining optimal length and specificity of nucleic acid probes for enrichment of genomic DNA segments

In another embodiment, both amplification and enrichment are performed. In some embodiments, the prepared amplified and/or enriched genomic DNA containing various targeted regions is referred to as the genomic library pool.

In some embodiments, the prepared genomic library pool is then sequenced. Sequence reads are determined from amplified and/or enriched genomic segments in the sample. In some embodiments, in the sequencing process, the clones of a same segment created in an amplification process are sequenced separately and optionally, counted later. In some embodiments, about 3,000 reads per sample are obtained. The number of reads depends on a number of factors, such as the size of the sample, amplification of the target region, and the bandwidth of the sequencing process (i.e., how much sequencing the apparatus is set for, e.g., how many beads are used). In some embodiments, not all of the segments in a sample are sequenced. In some embodiments, the sequence reads are about 150-250 bases long. One skilled in the art will appreciate the varied techniques available for performing the sequencing. In some embodiments, the sequencing is performed using an ILLUMINA MISEQ genome sequencer.

The sequencing process is performed by various techniques in various embodiments. In some embodiments, the fragments are amplified during the sequencing process. Where amplification was used to create a target region-increased sample, this amplification would be a second amplification step. The second amplification provides a stronger signal (e.g., a fluorescent signal corresponding to a particular base: A, C, G, or T) than if the second amplification was not performed and, the different amplicons do not result in separate sequence reads.

In some embodiments, the amplified genomic fragments (e.g., where amplification occurred in a solution) are each be attached to a bead. In some embodiments, the attached fragment is then amplified on the bead, and one sequence read is obtained from each bead. In some embodiments, for those that use a surface, a fragment is attached to a surface and then amplified to create a single cluster on the surface. In some embodiments, a single sequence read is obtained for each cluster. In some embodiments, a sequence read is for an entire length of a genomic segment, part of one end, or part of both ends.

In some embodiments, a sequence read includes the bases correspond to the actual segment and optionally the bases corresponding to a sample-specific ID and/or unique sequence tags (e.g., 25 bases long) that were used as part of the sequencing. In some embodiments, the unique sequence tags include part of an adapter that is ligated to the end of a fragment for receiving a universal primer, and part of the adapter is read during the sequencing.

In some embodiments, a plurality of sequence reads are aligned to a target region of a reference genome. By aligning, the process compares the sequence reads to the target region to determine the number of variations between the sequence read and the target region. A perfect match would show no variations. In some embodiments, a portion or all of the sequence reads obtained are used in the alignment process. For example, if the length of a read is too short or too long, then it is removed before alignment.

In some embodiments, the alignment is made so as to minimize the number of variations between the sequence read and the target region. In some embodiments, the sequence read is smaller than the target region or larger. In some embodiments, where the sequence read is larger, the number of variations is counted only in the target region.

In some embodiments, the reads are aligned to a target region only, thereby saving computational effort. As the alignment is specific to only the one or more target region(s), the alignment is performed in a short amount of time as the entire genome does not have to be searched. Also, as the percentage of segments corresponding to a target region is increased, a substantial number of the reads should match favorably to the target region (e.g., relatively few variations).

In some embodiments, where multiple target regions are used, a sequence read is compared to each target region, and the target region that provides the best alignment is identified. In some embodiments, the different target regions are different genes or different exons with a gene. Thus, in some embodiments, the exon with the best alignment is identified.

In some embodiments, where a barcode or ID is used, it is removed before aligning. In some embodiments, where a barcode or ID is used, it is not removed before aligning. In some embodiments, the barcode or ID is used to organize all of the reads for a particular sample into one group. In this manner, mutations from other samples do not impact the analysis of the present sample. This grouping is referred to as demultiplexing. In some embodiments, each sample is aligned to a different reference genome or different part of the reference genome. As different samples may have different target regions, the ID is used to determine which target region(s) of a reference genome should be compared for the alignment.

In some embodiments, sequence reads that differ from a target region by more than a first threshold number of variations are discarded from analysis for the target region. In some embodiments, where the number of variations is more than the threshold, it is an indication that the genomic segment corresponding to the sequence read did not come from the target region, given that the read was so different. In some embodiments, an allowance is made for some variations, so that a later analysis is used to identify mutations, which otherwise would be missed.

Example values for the threshold are 5-10 bases. In some embodiments, the threshold is dependent on the size of the target region. For example, in some embodiments, where the target region is about 200 bases, then the number of variations is capped at about 20 bases, or about 10%. If the target region was 150 bases, then the threshold could be 15 bases.

In some embodiments, for each target region, the reads that have less than or equal to the threshold are identified, e.g., as a group. In some embodiments, this group of reads is then analyzed further in relation to the target region. In some embodiments, where a read satisfies the threshold for more than one target region, it is then added to both groups. Such a read is tracked such that it is not ultimately counted as a mutation for more than one target region.

In some embodiments, accuracy is evaluated in multiple stages, through the library preparation and sequencing of gDNA of well-established samples (Coriell Institute NA12878) with known genomic variants. The ARCHER pipeline (using publicly available tools and algorithms, e.g., BWA, samtools, and freebayes) is used to identify or “call” all genomic variants in comparison to the human reference genome. All detected variants for each sample are compared to the known variants to determine if the sequenced variant(s) agree with the reported variant(s). These known mutations are available for data analysis on the GeT-RM database. The accuracy data is then used to evaluate the sensitivity. The sensitivity data, of the analyzed sample set, is accepted only if at least 85% sensitivity is achieved with 95% confidence interval. Variants are called for replicates of samples, run either on the same sequence runs or multiple sequence runs, and all detected variants for each replicate sample are compared to determine the repeatability and precision of variant calling. The variant calls are accepted if they agree 90% between replicate samples. These verification samples are run on a regular basis to ensure that the system is performing at an acceptable sensitivity/specificity level. Client samples are run similar to the verification samples in that gDNA extraction, library preparation and sequencing are all the same. The resultant raw sequence reads are aligned to the human genome (GCRh37) using Burrows-Wheeler Aligner (bwa) and variants are called using samtools and freebayes. Variants are then filtered for quality and binned to target regions.

In some embodiments, once the variants have been identified within a particular target region, the variants are further characterized as pathogenic or likely pathogenic based on factors, including, but not limited to, current knowledge of the particular gene function or association with a particular disease or condition, nature of the mutation, or a combination thereof.

In some embodiments, the identified variant or variants are compared to known databases of variants. In some embodiments, the variants are for the same target region. In some embodiments, the variants occur for a certain population or subpopulation of people, which is different than the reference genome used.

In some embodiments, the sequence reads from the target region are used to identify mutations in the target region. In some embodiments, the frequency of each variation is determined. For example, for a particular position in a target region, the number of times a G nucleotide variation appears instead of a normal or wild type A nucleotide is counted. A percentage of times the G mutation is seen is determined from the total reads that aligned to that position. In one embodiment, the percentage for a particular variation is required to be greater than a threshold (abundance filter) to be considered an actual mutation. In some embodiments, variations that occur together are identified. In some embodiments, variations that occur together are categorized as part of a same mutation.

In some embodiments, a report is generated which summarizes the identified variants. In some embodiments, the report lists the genes in which each variant was found. In some embodiments, the report lists the genomic location (e.g., chromosome number and numerical location) in which each variant was found. In some embodiments, the report lists the type variant (e.g., single nucleotide change or deletion). In some embodiments, the report lists the identity of the variant (e.g., an A to G mutation). In some embodiments, the report provides information on which variants are pathogenic or likely to be pathogenic. In some embodiments, the report provides information on which variants are associated with a disease of condition. In some embodiments, the disease or condition is a disease or condition listed in Table 1. In some embodiments, the report provides information on the disease or condition, including, but not limited to symptoms, pathology, diagnostic testing, and treatment. In some embodiments, the report provides recommendations for diagnostic genetic testing or diagnostic metabolic testing.

In some embodiments, the time from receipt of the newborn sample to the generation of a report is less than 96 hours. In some embodiments, the time from receipt of the sample to the generation of a report is less than 72 hours. In some embodiments, the time from receipt of the sample to the generation of a report is less than 48 hours.

Exemplary Computer Systems and Software Modules

In some embodiments, there are provide computer-implemented systems including (a) a digital processing device comprising an operating system configured to perform executable instructions and a memory and (b) a computer program including instructions executable by the digital processing device to create an application comprising: (i) a software module configured to receive a plurality of sequence reads, the sequence reads obtained from sequencing target regions of each of genes PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8; (ii) a software module configured to perform an alignment of the plurality of sequence reads to a reference sequence; (iii) a software module configured to identify gene variants and, optionally, characterize the gene variants as pathogenic or likely pathogenic or associated with a disease or disorder; and (iv) a software module configured to generate a report providing a list of the gene variants identified. In some embodiments, the system further comprises a sequence analyzer communicatively connected with the software module configured to receive a plurality of sequence reads, wherein the sequence analyzer is configured for sequencing a plurality of target regions to provide a plurality of sequence reads. In some embodiments, the system further comprises a database, in computer memory, of gene variants selected from the gene variants listed in Table 5.

Digital Processing Device

In some embodiments, the computer-implemented systems described herein include a digital processing device, or use of the same. In further embodiments, the digital processing device includes one or more hardware central processing units (CPU) that carry out the device's functions. In still further embodiments, the digital processing device further comprises an operating system configured to perform executable instructions. In some embodiments, the digital processing device is optionally connected a computer network. In further embodiments, the digital processing device is optionally connected to the Internet such that it accesses the World Wide Web. In still further embodiments, the digital processing device is optionally connected to a cloud computing infrastructure. In other embodiments, the digital processing device is optionally connected to an intranet. In other embodiments, the digital processing device is optionally connected to a data storage device.

In accordance with the description herein, suitable digital processing devices include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles. Those of skill in the art will recognize that many smartphones are suitable for use in the system described herein. Those of skill in the art will also recognize that select televisions, video players, and digital music players with optional computer network connectivity are suitable for use in the system described herein. Suitable tablet computers include those with booklet, slate, and convertible configurations, known to those of skill in the art.

In some embodiments, the digital processing device includes an operating system configured to perform executable instructions. The operating system is, for example, software, including programs and data, which manages the device's hardware and provides services for execution of applications. Those of skill in the art will recognize that suitable server operating systems include, by way of non-limiting examples, FreeBSD, OpenBSD, NetBSD®, Linux, Apple® Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell® NetWare®. Those of skill in the art will recognize that suitable personal computer operating systems include, by way of non-limiting examples, Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX-like operating systems such as GNU/Linux®. In some embodiments, the operating system is provided by cloud computing. Those of skill in the art will also recognize that suitable mobile smart phone operating systems include, by way of non-limiting examples, Nokia® Symbian® OS, Apple® iOS®, Research In Motion® BlackBerry OS®, Google® Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile® OS, Linux®, and Palm® WebOS®. Those of skill in the art will also recognize that suitable media streaming device operating systems include, by way of non-limiting examples, Apple TV®, Roku®, Boxee®, Google TV®, Google Chromecast®, Amazon Fire®, and Samsung® HomeSync®. Those of skill in the art will also recognize that suitable video game console operating systems include, by way of non-limiting examples, Sony® P53®, Sony® P54®, Microsoft® Xbox 360®, Microsoft Xbox One, Nintendo® Wii®, Nintendo® Wii U®, and Ouya®.

In some embodiments, the device includes a storage and/or memory device. The storage and/or memory device is one or more physical apparatuses used to store data or programs on a temporary or permanent basis. In some embodiments, the device is volatile memory and requires power to maintain stored information. In some embodiments, the device is non-volatile memory and retains stored information when the digital processing device is not powered. In further embodiments, the non-volatile memory comprises flash memory. In some embodiments, the non-volatile memory comprises dynamic random-access memory (DRAM). In some embodiments, the non-volatile memory comprises ferroelectric random access memory (FRAM). In some embodiments, the non-volatile memory comprises phase-change random access memory (PRAM). In other embodiments, the device is a storage device including, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, magnetic disk drives, magnetic tapes drives, optical disk drives, and cloud computing based storage. In further embodiments, the storage and/or memory device is a combination of devices such as those disclosed herein.

In some embodiments, the digital processing device includes a display to send visual information to a user. In some embodiments, the display is a cathode ray tube (CRT). In some embodiments, the display is a liquid crystal display (LCD). In further embodiments, the display is a thin film transistor liquid crystal display (TFT-LCD). In some embodiments, the display is an organic light emitting diode (OLED) display. In various further embodiments, on OLED display is a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display. In some embodiments, the display is a plasma display. In other embodiments, the display is a video projector. In still further embodiments, the display is a combination of devices such as those disclosed herein.

In some embodiments, the digital processing device includes an input device to receive information from a user. In some embodiments, the input device is a keyboard. In some embodiments, the input device is a pointing device including, by way of non-limiting examples, a mouse, trackball, track pad, joystick, game controller, or stylus. In some embodiments, the input device is a touch screen or a multi-touch screen. In other embodiments, the input device is a microphone to capture voice or other sound input. In other embodiments, the input device is a video camera or other sensor to capture motion or visual input. In further embodiments, the input device is a Kinect, Leap Motion, or the like. In still further embodiments, the input device is a combination of devices such as those disclosed herein.

Computer Program

In some embodiments, the computer-implemented systems disclosed herein include at least one computer program, or use of the same. A computer program includes a sequence of instructions, executable in the digital processing device's CPU, written to perform a specified task. Computer readable instructions may be implemented as program modules, such as functions, objects, Application Programming Interfaces (APIs), data structures, and the like, that perform particular tasks or implement particular abstract data types. In light of the disclosure provided herein, those of skill in the art will recognize that a computer program may be written in various versions of various languages.

The functionality of the computer readable instructions may be combined or distributed as desired in various environments. In some embodiments, a computer program comprises one sequence of instructions. In some embodiments, a computer program comprises a plurality of sequences of instructions. In some embodiments, a computer program is provided from one location. In other embodiments, a computer program is provided from a plurality of locations. In various embodiments, a computer program includes one or more software modules. In various embodiments, a computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof.

Web Application

In some embodiments, a computer program includes a web application. In light of the disclosure provided herein, those of skill in the art will recognize that a web application, in various embodiments, utilizes one or more software frameworks and one or more database systems. In some embodiments, a web application is created upon a software framework such as Microsoft®.NET or Ruby on Rails (RoR). In some embodiments, a web application utilizes one or more database systems including, by way of non-limiting examples, relational, non-relational, object oriented, associative, and XML database systems. In further embodiments, suitable relational database systems include, by way of non-limiting examples, Microsoft® SQL Server, mySQL™, and Oracle®. Those of skill in the art will also recognize that a web application, in various embodiments, is written in one or more versions of one or more languages. A web application may be written in one or more markup languages, presentation definition languages, client-side scripting languages, server-side coding languages, database query languages, or combinations thereof. In some embodiments, a web application is written to some extent in a markup language such as Hypertext Markup Language (HTML), Extensible Hypertext Markup Language (XHTML), or eXtensible Markup Language (XML). In some embodiments, a web application is written to some extent in a presentation definition language such as Cascading Style Sheets (CSS). In some embodiments, a web application is written to some extent in a client-side scripting language such as Asynchronous Javascript and XML (AJAX), Flash® Actionscript, Javascript, or Silverlight®. In some embodiments, a web application is written to some extent in a server-side coding language such as Active Server Pages (ASP), ColdFusion®, Perl, Java™, JavaServer Pages (JSP), Hypertext Preprocessor (PHP), Python™, Ruby, Tcl, Smalltalk, WebDNA®, or Groovy. In some embodiments, a web application is written to some extent in a database query language such as Structured Query Language (SQL). In some embodiments, a web application integrates enterprise server products such as IBM® Lotus Domino®. In some embodiments, a web application includes a media player element. In various further embodiments, a media player element utilizes one or more of many suitable multimedia technologies including, by way of non-limiting examples, Adobe® Flash®, HTML 5, Apple® QuickTime®, Microsoft® Silverlight®, Java™, and Unity®.

Standalone Application

In some embodiments, a computer program includes a standalone application, which is a program that is run as an independent computer process, not an add-on to an existing process, e.g., not a plug-in. Those of skill in the art will recognize that standalone applications are often compiled. A compiler is a computer program(s) that transforms source code written in a programming language into binary object code such as assembly language or machine code. Suitable compiled programming languages include, by way of non-limiting examples, C, C++, Objective-C, COBOL, Delphi, Eiffel, Java™, Lisp, Python™, Visual Basic, and VB .NET, or combinations thereof. Compilation is often performed, at least in part, to create an executable program. In some embodiments, a computer program includes one or more executable complied applications.

Software Modules

In some embodiments, the computer-implemented systems disclosed herein include software, server, and/or database modules, or use of the same. In view of the disclosure provided herein, software modules are created by techniques known to those of skill in the art using machines, software, and languages known to the art. The software modules disclosed herein are implemented in a multitude of ways. In various embodiments, a software module comprises a file, a section of code, a programming object, a programming structure, or combinations thereof. In further various embodiments, a software module comprises a plurality of files, a plurality of sections of code, a plurality of programming objects, a plurality of programming structures, or combinations thereof. In various embodiments, the one or more software modules comprise, by way of non-limiting examples, a web application, a mobile application, and a standalone application. In some embodiments, software modules are in one computer program or application. In other embodiments, software modules are in more than one computer program or application. In some embodiments, software modules are hosted on one machine. In other embodiments, software modules are hosted on more than one machine. In further embodiments, software modules are hosted on cloud computing platforms. In some embodiments, software modules are hosted on one or more machines in one location. In other embodiments, software modules are hosted on one or more machines in more than one location.

Databases

In some embodiments, the computer-implemented systems disclosed herein include one or more databases, or use of the same. In view of the disclosure provided herein, those of skill in the art will recognize that many databases are suitable for storage and retrieval of gene variant information. In various embodiments, suitable databases include, by way of non-limiting examples, relational databases, non-relational databases, object oriented databases, object databases, entity-relationship model databases, associative databases, and XML databases. In some embodiments, a database is internet-based. In further embodiments, a database is web-based. In still further embodiments, a database is cloud computing-based. In other embodiments, a database is based on one or more local computer storage devices. In some embodiments, the database includes gene variants of target regions of each of genes PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8 that are associated with a disease or disorder. In some embodiments, the database includes target regions of all or a subset of genes from the genes provided in Table 2. In some embodiments, the database includes the gene variants listed in Table 5.

In some embodiments, there are provided genetic screening platforms comprising: (a) a processor configured to provide an application comprising: (i) a software module configured to receive a plurality of sequence reads, the sequence reads obtained from sequencing target regions of each of genes PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8; (b) a server processor configured to provide a server application comprising: (i) a database, in computer memory, of gene variants selected from the gene variants listed in Table 5; (ii) a software module configured to perform an alignment of the plurality of sequence reads to a reference sequence; (iii) a software module configured to identify gene variants and, optionally, characterize the gene variants as pathogenic or likely pathogenic or associated with a disease or disorder; and (iv) a software module configured to generate a report providing a list of the gene variants identified.

In some embodiments, there are provided non-transitory computer-readable storage media encoded with a computer program including instructions executable by a processor to create an application comprising: (a) a database, in computer memory, of gene variants selected from the gene variants listed in Table 5; (b) a software module configured to receive a plurality of sequence reads, the sequence reads obtained from sequencing target regions of each of genes PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8; (c) a software module configured to perform an alignment of the plurality of sequence reads to a reference sequence; (d) a software module configured to identify gene variants and, optionally, characterize the gene variants as pathogenic or likely pathogenic or associated with a disease or disorder; and (e) a software module configured to generate a report providing a list of the gene variants identified.

Exemplary Diseases and Conditions for Monitoring

Organic Acid Disorders

Organic acid disorders result from enzyme deficiencies involved in the catabolism any of a number of organic compounds and metabolites. Organic acid disorders are those conditions that lead to an abnormal buildup of particular acids known as organic acids. Abnormal levels of organic acids in the blood (organic acidemia), urine (organic aciduria), and tissues can be toxic and can cause serious health problems. Present screening tests for organic acid disorders are MS/MS detection of acylcarnitines. Currently a diagnosis is confirmed with quantitative acylcarnitines, organic acids, enzyme assay and/or mutation analysis.

In some embodiments, the organic acid disorder is propionic acidemia (PROP), methylalonic acidemia (MUT), isovaleric acidemia (IVA), 3-methylcrotonyl-CoA carboxylase deficiency (3-MCC), 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (HMG), multiple carboxylase deficiency (MCD), beta-ketothiolase deficiency (BKT), or glutaric acidemia type I (GA1).

In some embodiments, the organic acid disorder is malonyl-CoA decarboxylase deficiency (MAL), isobutyryl-CoA dehydrogenase (IBD) deficiency, 2-methylbutyryl-CoA dehydrogenase deficiency, 3-methylglutaconic aciduria (3MGA) type I, 3-methylglutaconic aciduria (3MGA) type V, 3-hydroxy-2-methylbutyryl-CoA dehydrogenase deficiency (2M3HBA).

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant associated with an organic acid disorder. In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant in at least one target region of at least one gene selected from the group consisting of PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, MCCC1, MCCC2, HMGCL, HLCS, ACAT1, and GCDH. In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant in at least one target region of at least one gene selected from the group consisting of MLYCD, ACAD8, ACADSB, AUH, DNAJC19, OPA3, TAZ, HSD17B1OH. In some embodiments, the methods provided further include a diagnostic test for an organic acid disorder. In some embodiments, the methods further include a diagnostic test for an organic acid disorder if a gene variant is detected in at least one target region of at least one gene selected from the group consisting of PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, MCCC1, MCCC2, HMGCL, HLCS, ACAT1, and GCDH. In some embodiments, the methods further include a diagnostic test for an organic acid disorder if a gene variant is detected in at least one target region of at least one gene selected from the group consisting of MLYCD, ACAD8, ACADSB, AUH, DNAJC19, OPA3, TAZ, HSD17B1OH. In some embodiments, the diagnostic test includes an acylcarnitine profile analysis of a biological sample from the newborn.

Beta-Ketothiolase Deficiency (BKD)

Beta-ketothiolase deficiency is an inherited disorder in which the body cannot effectively process a protein building block (amino acid) called isoleucine. Signs and symptoms typically appear between the ages of six to 24 months. Episodes called ketoacidotic attacks may occur causing symptoms such as vomiting, dehydration, difficulty breathing, extreme lethargy, and occasionally seizures. Infections, fasting, or increased intake of protein rich foods frequently triggers these ketoacidotic attacks. Attacks can also lead to coma. Present screening tests include assays for elevated levels of tiglylcarnitine (C5:1) and 3-hydroxyisovalerylcarnitine (C5OH).

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for at least one gene variant in at least one target region of the acetyl-CoA acetyltransferase 1 (ACAT1) gene at chromosome 11q22.3. In some embodiments, the methods provided further include a diagnostic test for BKD. In some embodiments, the methods further include a diagnostic test for BKD if at least one gene variant in at least one target region of ACAT1 is detected. In some embodiments, the diagnostic test includes an acylcarnitine profile analysis of a biological sample from the newborn. In some embodiments, the diagnostic test includes analysis of tiglylcarnitine (C5:1) and/or 3-hydroxyisovalerylcarnitine (C5OH) levels in a biological sample from the newborn.

Isobutyryl CoA Dehydrogenase Deficiency (IBD)

Isobutyryl-CoA dehydrogenase (IBD) deficiency is a condition that disrupts the breakdown of certain proteins. In particular, patients with IBD deficiency have inadequate levels of an enzyme that helps break down the amino acid valine. Most effected individuals do not experience symptoms. A few children with IBD deficiency have developed features such as a weakened and enlarged heart, weak muscle tone, developmental delay, and anemia. IBD is currently detected by measuring elevated levels of isovalerylcarnitine (C5).

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for at least one gene variant in at least one target region of the acyl-CoA dehydrogenase family, member 8 (ACAD8) gene at chromosome 11q25. In some embodiments, the methods provided further include a diagnostic test for IBD. In some embodiments, the methods further include a diagnostic test for IBD if at least one gene variant in at least one target region of ACAD8 is detected. In some embodiments, the diagnostic test includes an acylcarnitine profile analysis of a biological sample from the newborn. In some embodiments, the diagnostic test includes analysis of isovalerylcarnitine (C5) levels in a biological sample from the newborn.

Isovaleric Acidemia (IVA)

Isovaleric acidemia is a rare disorder in which the body is unable to process certain proteins properly. Patients with isovaleric acidemia have inadequate levels of an enzyme that helps break down the amino acid called leucine. Cases vary from mild to life threatening and in severe cases the features of the disorder become apparent within days after birth. Symptoms include poor feeding, vomiting, seizures, lethargy, coma and possibly death. An odor of sweaty feet is present with acute illness. IVA is currently detected by measuring elevated levels of C4.

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for at least one gene variant in at least one target region of the isovaleryl-CoA dehydrogenase (IVD) gene at chromosome 15q14-q15. In some embodiments, the methods provided further include a diagnostic test for IVA. In some embodiments, the methods further include a diagnostic test for IVA if at least one gene variant in at least one target region of IVD is detected. In some embodiments, the diagnostic test includes an acylcarnitine profile analysis of a biological sample from the newborn. In some embodiments, the diagnostic test includes analysis of isobutyryl (C4) levels in a biological sample from the newborn.

Fatty Acid Disorders

Fatty acid disorders are those disorders in which an enzyme deficiency prevents the body from converting certain fats to energy. Mitochondrial beta-oxidation of fatty acids is important in the body's ability to produce energy during fasting. In infants, a “fasting” state can be produced in as little as four hours. Fatty acids must be transported into the cytoplasm and then into the mitochondria for oxidation; carnitine is required for these transport steps. Once in the mitochondria, fatty acid chains 4-18 carbons in length must be oxidized, two carbons at a time, each reaction using a chain-specific enzyme, before ketogenesis can occur. There are over 20 individual steps in beta oxidation some with multiple enzyme complexes. An enzyme block or deficiency anywhere in this process or a carnitine deficiency results in hypoketotic hypoglycemia and tissue damage related to the toxic accumulation of unoxidized fatty acids. At least 16 separate enzyme disorders have been identified within this oxidation process, which are currently identified by measuring the accumulation of various acylcarnitines.

In some embodiments, the fatty acid disorder is primary carnitine deficiency (CUD), medium chain acyl-CoA dehydrogenase deficiency (MCAD), long chain 3 hydroxyacyl-CoA dehydrogenase deficiency (LCHAD), very long chain acyl-CoA dehydrogenase deficiency (VLCAD), or trifunctional protein deficiency (TFP).

In some embodiments, the fatty acid disorder is short chain acyl-CoA dehydrogenase (SCAD) deficiency, 3-hydroxyacyl-CoA dehydrogenase deficiency (M/SCHAD), glutaric acidemia type II (GA2), carnitine palmitoyltransferase I deficiency (CPT IA), carnitine palmitoyltransferase II deficiency (CPT II), or carnitine-acylcarnitine translocase (CACT).

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant associated with a fatty acid disorder. In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant in at least one target region of at least one gene selected from the group consisting of SLC22A5, ACADM, ACADVL, HADHA, and HADHB. In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant in at least one target region of at least one gene selected from the group consisting of ACADS, HADH, ETFA, ETFB, ETFDH, DECR1, CPT1A, CPT2, and SLC25A20. In some embodiments, the methods provided further include a diagnostic test for a fatty acid disorder. In some embodiments, the methods further include a diagnostic test for a fatty acid disorder if a gene variant is detected in at least one target region of at least one gene selected from the group consisting of SLC22A5, ACADM, ACADVL, HADHA, and HADHB. In some embodiments, the methods further include a diagnostic test for a fatty acid disorder if a gene variant is detected in at least one target region of at least one gene selected from the group consisting of ACADS, HADH, ETFA, ETFB, ETFDH, DECR1, CPT1A, CPT2, and SLC25A20. In some embodiments, the diagnostic test includes an acylcarnitine profile analysis of a biological sample from the newborn.

Medium Chain Acyl-CoA Dehydrogenase Deficiency (MCAD)

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a condition that prevents the body from converting certain fats to energy. Signs and symptoms typically appear during infancy or early childhood and include vomiting, lack of energy, and low blood sugar, seizures, breathing difficulties, liver problems, brain damage, coma, or sudden death. MCAD is the most common of the fatty acid oxidation conditions. Present screening methods include assays for detecting elevated levels of hexanoylcarnitine (C6), octanoylcarnitine (C8), decanoyl (C10), and/or C8/10.

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for at least one gene variant in at least one target region of the acyl-CoA dehydrogenase, C-4 to C-12 straight chain (ACADM) gene at chromosome 1p31. In some embodiments, the methods provided further include a diagnostic test for MCAD. In some embodiments, the methods further include a diagnostic test for MCAD if at least one gene variant in at least one target region of ACADM is detected. In some embodiments, the diagnostic test includes an acylcarnitine profile analysis of a biological sample from the newborn. In some embodiments, the diagnostic test includes analysis of hexanoylcarnitine (C6), octanoylcarnitine (C8), decanoyl (C10), and/or C8/10 levels in a biological sample from the newborn

Long Chain 3 Hydroxyacyl-CoA Dehydrogenase Deficiency (LCHAD)

Long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency prevents the body from converting certain fats to energy. Symptoms include feeding difficulties, lethargy, low blood sugar, weak muscle tone, retinal abnormalities, muscle pain or breakdown of muscle tissue and loss of sensation in arms and legs. Present screening methods include assays for detecting elevated levels of tetradecenolycarnitine (C14:1), hexadecanoylcarnitine (C16), 3-hydroxyhexadecanoylcarnitine (C16OH), octadecanoylcarnitine (C18), and/or 3-hydroxyoctadecanoylcarnitine (C18OH).

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for at least one gene variant in at least one target region of the hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit (HADHA) gene in chromosome 2p23 and/or hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), beta subunit (HADHB) at chromosome 2p23. In some embodiments, the methods provided further include a diagnostic test for LCHAD. In some embodiments, the methods further include a diagnostic test for LCHAD if at least one gene variant in at least one target region of HADHA and/or HADHB is detected. In some embodiments, the diagnostic test includes an acylcarnitine profile analysis of a biological sample from the newborn. In some embodiments, the diagnostic test includes analysis of tetradecenoylcarnitine (C14:1), hexadecanoylcarnitine (C16), 3-hydroxyhexadecanoylcarnitine (C16OH), octadecanoylcarnitine (C18), and/or 3-hydroxyoctadecanoylcarnitine (C18OH) levels in a biological sample from the newborn.

Very Long Chain Acyl-CoA Dehydrogenase Deficiency (VLCAD)

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a disorder in which the body is unable to convert very long-chain fatty acids into energy. Characteristic signs and symptoms of this disorder include lack of energy, and low blood sugar. Very long-chain fatty acids or partially metabolized fatty acids may also build up in tissues and damage the heart, liver, and muscles. Present screening tests include assays for elevated tetradecanolycarnitine (C14), tetradecenolycarnitine (C14:1), hexadecanoylcarnitine (C16), hexadecenoylcarnitine (C16:1), octadecanoylcarnitine (C18), and/or octadecenoylcarnitine (C18:1) levels.

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for at least one gene variant in at least one target region of the acyl-CoA dehydrogenase, very long chain (ACADVL) gene in chromosome 17p13.1. In some embodiments, the methods provided further include a diagnostic test for VLCAD. In some embodiments, the methods further include a diagnostic test for VLCAD if at least one gene variant in at least one target region of ACADVL is detected. In some embodiments, the diagnostic test includes an acylcarnitine profile analysis of a biological sample from the newborn. In some embodiments, the diagnostic test includes analysis of tetradecanolycarnitine (C14), tetradecenolycarnitine (C14:1), hexadecanoylcarnitine (C16), hexadecenoylcarnitine (C16:1), octadecanoylcarnitine (C18), and/or octadecenoylcarnitine (C18:1) levels in a biological sample from the newborn.

Amino Acid Disorders

Amino acid disorders result from enzyme deficiencies involved in the catabolism any of a number of amino acids. Amino acid disorders are those conditions that lead to an abnormal buildup of particular amino acids. Present screening tests for amino acid disorders are MS/MS detection of particular amino acids. Currently a diagnosis is confirmed with quantitative amino acids, enzyme assay and/or mutation analysis.

In some embodiments, the amino acid disorder is arginosuccinic aciduria (ASA), citrullinemia (CIT) type I, maple syrup urine disease (MSUD), hom*ocystinuria (HCY), phenylketonuria (PKU), or tyrosinemia (TYR I, II, III).

In some embodiments, the amino acid disorder is arginase deficiency (ARG), citrullinemia type II (CIT II), hypermethioninemia (MET), or disorders of biopterin regeneration.

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant associated with an amino acid disorder. In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant in at least one target region of at least one gene selected from the group consisting of ASL, ASS1, BCKDHA, BCKDHB, DBT, DLD, CBS, MTHFR, MTR, MTRR, MMADHC, PAH, and FAH. In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant in at least one target region of at least one gene selected from the group consisting of ARG1, SLC25A13, AHCY, GNMT, MAT1A, PAH, GCH1, PCBD1, PTS, QDPR, TAT, and HPD. In some embodiments, the methods provided further include a diagnostic test for an amino acid disorder. In some embodiments, the methods further include a diagnostic test for an amino acid disorder if a gene variant is detected in at least one target region of at least one gene selected from the group consisting of ASL, ASS1, BCKDHA, BCKDHB, DBT, DLD, CBS, MTHFR, MTR, MTRR, MMADHC, PAH, and FAH. In some embodiments, the methods further include a diagnostic test for an amino acid disorder if a gene variant is detected in at least one target region of at least one gene selected from the group consisting of ARGL SLC25A13, AHCY, GNMT, MAT1A, PAH, GCH1, PCBD1, PTS, QDPR, TAT, and HPD. In some embodiments, the diagnostic test includes an amino acid profile analysis of a biological sample from the newborn. In some embodiments, the diagnostic test includes an assay of the affected amino acid in a biological sample from the newborn. In some embodiments, the diagnostic test includes an analysis of leucine, methionine, and/or tyrosine.

Maple Syrup Urine Disease (MSUD)

Maple syrup urine disease is an inherited disorder in which the body is unable to process certain protein building blocks (amino acids) properly. Symptoms commonly begin in early infancy and include urine of a distinctive sweet odor, poor feeding, vomiting, lack of energy and developmental delay. If untreated, maple syrup urine disease can lead to seizures, coma, and death. This disorder may also be caused by mutations in the BCKDHB, DBT, and DLD genes. Maple syrup urine disease is currently detected by an elevation of the amino acid leucine and an abnormal leucine/alanine ratio.

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for at least one gene variant in at least one target region of the branched chain keto acid dehydrogenase E1, alpha polypeptide (BCKDHA) gene in chromosome 19q13.1-q13.2; the branched chain keto acid dehydrogenase E1, beta polypeptide (BCKDHB) gene in chromosome 6q14.1; the dihydrolipoamide branched chain transacylase E2 (DBT) gene in chromosome 1p31; and/or the dihydrolipoamide dehydrogenase (DLD) gene at chromosome 7q31-q32. In some embodiments, the methods provided further include a diagnostic test for MSUD. In some embodiments, the methods further include a diagnostic test for MSUD if at least one gene variant in at least one target region of BCKDHA, BCKDHB, DBT, and/or DLD is detected. In some embodiments, the diagnostic test includes analysis of leucine levels in a biological sample from the newborn. In some embodiments, the diagnostic test includes analysis of leucine/alanine ratio in a biological sample from the newborn.

Propionic Acidemia (PA)

Propionic acidemia is an inherited disorder in which the body is unable to process certain parts of proteins and lipids (fats) properly. Mutations in the PCCA or PCCB genes prevent the production of functional propionyl-CoA carboxylase or reduce the enzyme's activity. The altered or missing enzyme prevents certain parts of proteins and lipids from being broken down properly. As a result, propionyl-CoA and other potentially toxic compounds can build up to toxic levels in the body. Within the first few days of life initial symptoms may arise including poor feeding, vomiting, loss of appetite, weak muscle tone (hypotonia), and lack of energy (lethargy). These symptoms sometimes progress to more serious medical problems, including heart abnormalities, seizures, coma, and possibly death. This condition can also be caused by mutation in the PCCA gene or the PCCB gene. PA is currently detected by measuring elevated levels of propionylcamitine (C3) and/or propionylcarnitine/acetylcarnitine (C3/C2).

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for at least one gene variant in at least one target region of the propionyl CoA carboxylase, alpha polypeptide (PCCA) gene at chromosome 13q32 or a mutation in the propionyl CoA carboxylase, beta polypeptide (PCCB) gene at chromosome 3q21-q22. In some embodiments, the methods provided further include a diagnostic test for PA. In some embodiments, the methods further include a diagnostic test for PA if at least one gene variant in at least one target region of PCCA and/or PCCB is detected. In some embodiments, the diagnostic test includes analysis of propionylcamitine (C3) and/or propionylcarnitine/acetylcarnitine (C3/C2) levels in a biological sample from the newborn.

Argininosuccinate Lyase Deficiency (ASA)

Argininosuccinic aciduria is an inherited disorder that causes ammonia to accumulate in the blood. Ammonia, which is formed when proteins are broken down in the body, is toxic if the levels become too high. Argininosuccinic aciduria belongs to a class of genetic diseases called urea cycle disorders. The urea cycle is a sequence of reactions that occur in liver cells. It processes excess nitrogen, generated when protein is used by the body, to make a compound called urea that is excreted by the kidneys. In argininosuccinic aciduria, the enzyme that starts a specific reaction within the urea cycle is damaged or missing. The urea cycle cannot proceed normally, and nitrogen accumulates in the bloodstream in the form of ammonia. Mutations in the ASL gene cause argininosuccinic aciduria. Argininosuccinic aciduria usually becomes evident in the first few days of life. Symptoms include lack of energy, unwilling to eat, poorly controlled respiratory rate or body temperature, seizures, and coma. Currently, ASA is detected by measuring elevated levels of argininosuccinic acid/citrulline.

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for at least one gene variant in at least one target region of the argininosuccinate lyase (ASL) gene at chromosome 7q11.21. In some embodiments, the methods provided further include a diagnostic test for ASA. In some embodiments, the methods further include a diagnostic test for ASA if at least one gene variant in at least one target region of ASL is detected. In some embodiments, the diagnostic test includes analysis of argininosuccinic acid/citrulline levels in a biological sample from the newborn.

Endocrine Disorders

Endocrine disorders are diseases related to the endocrine glands of the body. The endocrine system produces hormones, which are secreted into the bloodstream and affect other organs within the body to regulate processes, such as appetite, breathing, growth, fluid balance, feminization and virilization, and weight control. In some embodiments, the endocrine disorders are congenital adrenal hyperplasia (CAH) or congenital hypothyroidism (CH).

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant associated with an endocrine disorder. In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant in at least one target region of at least one gene selected from the group consisting of DUOX2, PAX8, SLC5A5, TG, TPO, TSHB, TSHR, and CYP21A2. In some embodiments, the methods provided further include a diagnostic test for an endocrine disorder.

In some embodiments, the methods further include a diagnostic test for an endocrine disorder if a gene variant is detected in at least one target region of at least one gene selected from the group consisting of DUOX2, PAX8, SLC5A5, TG, TPO, TSHB, TSHR, and CYP21A2. In some embodiments, the diagnostic test includes a test for congenital hypothyroidism (CH). In some embodiments, the diagnostic test includes a test for congenital adrenal hyperplasia (CAH). In some embodiments, the diagnostic test includes an assay of thyroid hormones, T4 and TSH, in a biological sample from the newborn. In some embodiments, the diagnostic test includes an analysis 17-OH-progesterone in a biological sample from the newborn.

Congenital Adrenal Hyperplasia (CAH)

CAH is an inherited defect of cortisol synthesis, in which the adrenal gland cannot make cortisol and overproduces male hormones. Without cortisol, infants are at risk of death due to adrenal crisis and inability to regulate salt and fluids. The most common disorder is 21-hydroxylase deficiency. is an inherited disorder that affects the adrenal glands. Three types of 21-hydroxylase deficiency include the salt-wasting, simple virilizing, and non-classic types.

Infants may be symptomatic at birth, due to diminished cortisol production during gestation, which stimulates the fetal pituitary gland to produce ACTH resulting in excessive adrenal androgens. The androgens virilize female external genitalia, but ovaries and uterus are unaffected. Male infants may have increased scrotal pigmentation or may be asymptomatic. In 75 percent of cases, the 21-hydroxylase deficiency causes reduced production of mineralocorticoids. This reduction leads to a hypotensive, hyperkalemic, salt-losing crisis with rapid onset of adrenocortical failure within 7-28 days of birth, which can be fatal. In 25 percent of cases, the infant has a “non-salt losing” or “simple virilizing form.”

Currently screening is based on an immunoassay for a precursor steroid, 17-hydroxyprogesterone (17-OHP). Affected infants have high levels of 17-OHP, Infants with milder disorders have intermediate levels. Chromosome analysis is used to confirm gender if genitalia are ambiguous.

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for at least one gene variant in at least one target region of the cytochrome P450, family 21, subfamily A, polypeptide 2 (CYP21A2) gene at chromosome 6p21.3. In some embodiments, the methods provided further include a diagnostic test for CAH. In some embodiments, the methods further include a diagnostic test for CAH if at least one gene variant in at least one target region of CYP2.1A2 is detected. In some embodiments, the diagnostic test includes analysis of 17-OH-progesterone levels in a biological sample from the newborn.

Hemoglobin Disorders

Hemoglobin disorders are those disorders that affect the production of function of hemoglobin. In some embodiments the hemoglobin disorder is sickle cell disease, metheglobinemia (beta-globin type), or beta thalassemia (thalassemia major and thalassemia intermedia).

In some embodiments the hemoglobin disorder is alpha thalassemia (hemoglobin disorder-Var-Hb).

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant associated with a hemoglobin disorder. In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant in at least one target region of HBB. In some embodiments, the methods provided further include a diagnostic test for a hemoglobin disorder. In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant in at least one target region of at least one gene selected from the group consisting of HBA1 and HBA2. In some embodiments, the methods further include a diagnostic test for a hemoglobin disorder if a gene variant is detected in at least one target region of HBB. In some embodiments, the methods further include a diagnostic test for a hemoglobin disorder if a gene variant is detected in at least one target region of at least one gene selected from the group consisting of HBA1 and HBA2. In some embodiments, the diagnostic test includes a test for sickle cell disease, metheglobinemia (beta-globin type), or beta thalassemia (thalassemia major and thalassemia intermedia), In some embodiments, the diagnostic test includes an assay for hemoglobinopathies using isoelectric focusing (IEF) of a biological sample from the newborn. In some embodiments, the diagnostic test includes an assay for hemoglobinopathies using high performance liquid chromatography (HPLC) of a biological sample from the newborn. In some embodiments, the diagnostic test includes an assay for hemoglobinopathies using both IEF and HPLC of a biological sample from the newborn.

Other Disorders

Other disorders include those conditions which are genetic disorders that fall outside of the categories of disorders listed above. In some embodiments, the other condition is biotinidase deficiency (BIOT), cystic fibrosis (CF), galactosemia type I, hearing loss ((1) nonsyndromic deafness, (2) palmoplantar karatoderma, (3) hystrix-like ichthyosis, (4) Bart-Pumphrey syndrome, (5) Vohwinkel syndrome, (6) karatitis-ichthyosis-deafness (KID), (7) erythrokeratodermia variabilis et progressive (EKVP), (8) Clouston syndrome), severe combined immunodeficiency (SCID), or X-linked severe combined immunodeficiency (SCID).

In some embodiments, the other disorder is galactosemia type III or galactosemia type II.

In other embodiments, the other disorder is X-linked adrenoleukodystrophy, adrenomyeloneuropathy Addison disease (X-ALD), 2,4 dienoyl-CoA reductase deficiency, Pompe disease (GAA deficiency), Krabbe disease, Gaucher disease (types I, II, & III), Fabry disease, mucopolysaccharidosis type I (MPS I), congenital disorder of deglycosylation type 1v, Niemann-Pick disease (type C1), or Niemann-Pick disease (type C2).

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for a gene variant in at least one target region of at least one gene selected from the group consisting of BTD, CFTR, GALT, GJB2, GJB3, GJB6, ADA, and IL2RG. In some embodiments, the methods further include a diagnostic test for the associated disorder if a gene variant is detected in at least one target region of at least one gene selected from the group consisting of BTD, CFTR, GALT, GJB2, GJB3, GJB6, ADA, and IL2RG. In some embodiments, the diagnostic test includes a test for biotinidase deficiency (BIOT), cystic fibrosis (CF), galactosemia type I, hearing loss, severe combined immunodeficiency (SCID), or X-linked severe combined immunodeficiency (SCID). In some embodiments, the diagnostic test includes an assay for immunotrypsinogen, biotinidase, and/or GALT enzyme activity.

Galactosemia Type I

Galactosemia is a disorder that prevents the body from processing a simple sugar called galactose into energy. Galactosemia type I is the most common and most severe form of the disorder. If infants are not promptly treated complications can arise within the first few days of life. Complications include feeding difficulties, lack of energy, failure to thrive, jaundice, liver damage and bleeding. Mutations in the GALT gene are responsible for classic galactosemia (type I). Most of these genetic changes almost completely eliminate the activity of the enzyme produced from the GALT gene, preventing the normal processing of galactose and resulting in the life-threatening signs and symptoms of this disorder. Another GALT gene mutation, known as the Duarte variant, reduces but does not eliminate the activity of the enzyme. People with the Duarte variant tend to have much milder features of galactosemia. Currently, two screening tests are used to detect galactosemia in a two-tiered sequence, the GALT activity test and the galactose (Hill) test. The GALT enzyme activity test depends upon fluorescence produced by the normal galactose enzyme cascade in red blood cells. It does not differentiate milder variants from severe defects. All infants are screened with the GALT test. The Hill test is a fluorometric chemical spot test that measures galactose and galactose-1-phosphate. Galactose metabolites are greatly elevated in infants with galactosemia if they are receiving a lactose-containing formula or breast milk. All infants with an abnormal GALT or who have been transfused are screened with the Hill test.

In some embodiments, a genomic sample from a newborn is screened according to the methods provided herein for at least one gene variant in at least one target region of the galactose-1-phosphate uridylyltransferase (GALT) gene at chromosome 9p13. In some embodiments, the methods provided further include a diagnostic test for Galactosemia. In some embodiments, the methods further include a diagnostic test for galactosemia if at least one gene variant in at least one target region of GALT is detected. In some embodiments, the diagnostic test includes analysis of GALT enzyme activity level and/or galactose and galactose-1-phosphate levels in a biological sample from the newborn. In some embodiments, a biological sample from the newborn is analyzed using a CALF enzyme activity test and/or a Hill test. In some embodiments, the method further includes treatment of the disorder using the standard treatment for galactosemia, if a gene variant in GALT is identified.

Kits and Articles of Manufacture

In some embodiments, there are provided compositions comprising a collection of oligonucleotide primers for selective amplification of plurality of target regions of each of PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8. In some embodiments, the composition further includes oligonucleotide primers for target regions of additional genes listed in Table 2.

In some embodiments, there are provided kits comprising a collection of oligonucleotide primers for sequencing of each of PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8. In some embodiments, the kit further includes oligonucleotide primers for target regions of additional genes listed in Table 2. In some embodiments, the kit further comprises one or more reagents for performing a sequencing reaction.

TABLE 1 Gene and Diseases Reference: Genes, Cytogenetic Location, Disease, Brief Description, Signs and Symptoms Cytogenic Gene Location Disease Brief Description Signs & Symptoms Core Conditions Organic PCCA 13q32 Propionic acidemia Propionic acidemia is an Within the first few days of life initial symptoms Acid (PROP) inherited disorder in which may arise including poor feeding, vomiting, loss of Disorders the body is unable to appetite, weak muscle tone (hypotonia), and lack process certain parts of of energy (lethargy). These symptoms sometimes proteins and lipids (fats) progress to more serious medical problems, properly. including heart abnormalities, seizures, coma, and possibly death. This condition can also be caused by mutation in the PCCB gene. PCCB 3q21-q22 Propionic acidemia Propionic acidemia is an Within the first few days of life initial symptoms (PROP) inherited disorder in which may arise including poor feeding, vomiting, loss of the body is unable to appetite, weak muscle tone (hypotonia), and lack process certain parts of of energy (lethargy). These symptoms sometimes proteins and lipids (fats) progress to more serious medical problems, properly. including heart abnormalities, seizures, coma, and possibly death. This condition can also be caused by mutation in the PCCA gene. MCEE 2p13.3 Methylmalonic acidemia Methylmalonic acidemia is Effects of the disorder, which normally arise during (MUT) an inherited disorder in infancy, range from mild to life threatening. which the body is unable to Symptoms include vomiting, dehydration, weak process certain proteins muscle tone, developmental delay, lethargy, and fats (lipids) properly. enlarged liver, failure to thrive. Long-term complications can lead to intellectual disability, kidney disease, and pancreatitis and can lead to coma or death. This disorder may also arise due to mutations in the MUT, MMAA, MMAB, and MMADHC genes. MMAA 4q31.21 Methylmalonic acidemia Methylmalonic acidemia is Effects of the disorder, which normally arise during (MUT) an inherited disorder in infancy, range from mild to life threatening. which the body is unable to Symptoms include vomiting, dehydration, weak process certain proteins muscle tone, developmental delay, lethargy, and fats (lipids) properly. enlarged liver, failure to thrive. Long-term complications can lead to intellectual disability, kidney disease, and pancreatitis and can lead to coma or death. This disorder may also arise due to mutations ain the MCEE, MUT, MMAB, and MMADHC genes. MMAB 12q24 Methylmalonic acidemia Methylmalonic acidemia is Effects of the disorder, which normally arise during (MUT) an inherited disorder in infancy, range from mild to life threatening. which the body is unable to Symptoms include vomiting, dehydration, weak process certain proteins muscle tone, developmental delay, lethargy, and fats (lipids) properly. enlarged liver, failure to thrive. Long-term complications can lead to intellectual disability, kidney disease, and pancreatitis and can lead to coma or death. This disorder may also arise due to mutations in the MCEE, MUT, MMAA, and MMADHC genes. MMADHC 2q23.2 (I)Methylmalonic (I) Methylmalonic acidemia (I) Effects of the disorder, which normally arise acidemia (MUT) is an inherited disorder in during infancy, range from mild to life threatening. (II)hom*ocystinuria (HCY) which the body is unable to Symptoms include vomiting, dehydration, weak process certain proteins muscle tone, developmental delay, lethargy, and fats (lipids) properly. enlarged liver, failure to thrive. Long-term (II) hom*ocystinuria is an complications can lead to intellectual disability, inherited disorder in which kidney disease, and pancreatitis and can lead to the body is unable to coma or death. This disorder may also arise due to process certain building mutations in the MCEE, MUT, MMAA, and blocks of proteins (amino MMAB genes. acids) properly. (II) Multiple forms of this disorder exist but most common symptoms include nearsightedness, dislocation of the lens at the front of the eye, increased risk of abnormal blood clotting, and brittle bones. Although most often mutations in the CBS gene cause this disorder, it may also arise from mutations in the CBS, MTHFR, MTR, and MTRR. MUT 6p12.3 Methylmalonic acidemia Methylmalonic acidemia is Effects of the disorder, which normally arise during (MUT) an inherited disorder in infancy, range from mild to life threatening. which the body is unable to Symptoms include vomiting, dehydration, weak process certain proteins muscle tone, developmental delay, lethargy, and fats (lipids) properly. enlarged liver, failure to thrive. Long-term complications can lead to intellectual disability, kidney disease, and pancreatitis and can lead to coma or death. This disorder may also arise due to mutations in the MCEE, MMAA, MMAB, and MMADHC genes. IVD 15q14-q15 Isovaleric acidemia Isovaleric acidemia is a Cases vary from mild to life threatening and in (IVA) rare disorder in which the severe cases the features of the disorder become body is unable to process apparent within days after birth. Symptoms include certain proteins properly poor feeding, vomiting, seizures, lethargy, coma and possibly death. An odor of sweaty feet is present with acute illness. MCCC1 3q27 3-methylcrotonyl-CoA 3-methylcrotonyl-CoA Affected infants often appear normal at birth but carboxylase deficiency carboxylase deficiency is develop signs and symptoms in infancy or early (3-MCC) an inherited disorder in childhood. Symptoms include vomiting and which the body is unable to diarrhea, lethargy, weak muscle tone, delayed process certain proteins development, seizures and coma. Many problems properly. can be prevented with early detection. This disorder may also be caused my mutations in the MCCC2 gene. MCCC2 5q12-q13 3-methylcrotonyl-CoA 3-methylcrotonyl-CoA Affected infants often appear normal at birth but carboxylase deficiency carboxylase deficiency is develop signs and symptoms in infancy or early (3-MCC) an inherited disorder in childhood. Symptoms include vomiting and which the body is unable to diarrhea, lethargy, weak muscle tone, delayed process certain proteins development, seizures and coma. Many problems properly. can be prevented with early detection. This disorder may also be caused my mutations in the MCCC1 gene. HMGCL 1p36.1- 3-hydroxy-3- 3-hydroxy-3- Characteristics of this disorder generally arise p35 methylglutaryl-CoA lyase methylglutaryl-CoA lyase within the first year of life. Signs and symptoms deficiency deficiency (also known as include vomiting, diarrhea, dehydration, lethargy, (HMG) HMG-CoA lyase weak muscle tone, hypoglycemia. Can lead to deficiency) is an breathing problems, convulsions, coma and death. uncommon inherited Commonly mistaken for Reye syndrome. disorder in which the body cannot process a particular protein building block (amino acid) called leucine. HLCS 21q22.13 Multiple Carboxylase Another name for MCD is The signs and symptoms of this disorder generally Deficiency (MCD) holocarboxylase appear within the first few months of life. They synthetase deficiency, a include breathing problems, skin rash, hair loss, disorder where the body is lethargy. Biotin supplements may prevent unable to use the vitamin complications. biotin effectively. ACAT1 11q22.3 Beta-ketothiolase Beta-ketothiolase Signs and symptoms typically appear between the deficiency (βKT) deficiency is an inherited ages of six to 24 months. Episodes called disorder in which the body ketoacidotic attacks may occur causing symptoms cannot effectively process such as vomiting, dehydration, difficulty breathing, a protein building block extreme lethargy, and occasionally seizures. (amino acid) called Infections, fasting, or increased intake of protein isoleucine. rich foods frequently triggers these ketoacidotic attacks. Attacks can also lead to coma. GCDH 19p13.2 Glutaric acidemia type I Glutaric acidemia type I is Most often signs and symptoms first occur in (GA1) an inherited disorder in infancy and early childhood. Symptoms vary from which the body is unable to mild to severe and may result in poor motor control process certain proteins and intellectual disability. Some individuals develop properly. bleeding in the brain or eyes. Fatty Acid SLC22A5 5q23.3 Primary carnitine Primary carnitine Signs and symptoms typically appear during Disorders deficiency (CUD) deficiency is a condition infancy or early childhood and can include severe that prevents the body brain dysfunction, a weakened and enlarged heart, from using certain fats. confusion, vomiting, muscle weakness, and low blood sugar, heart failure, liver problems, and sudden death. ACADM 1p31 Medium-chain acyl-CoA Medium-chain acyl-CoA Signs and symptoms typically appear during dehydrogenase (MCAD) dehydrogenase (MCAD) infancy or early childhood and can include deficiency deficiency is a condition vomiting, lack of energy, and low blood sugar, that prevents the body seizures, breathing difficulties, liver problems, brain from converting certain fats damage, coma, and sudden death. to energy. ACADVL 17p13.1 Very long-chain acyl-CoA Very long-chain acyl-CoA Characteristic signs and symptoms of this disorder dehydrogenase (VLCAD) dehydrogenase (VLCAD) include lack of energy, and low blood sugar. Very deficiency deficiency is a disorder in long-chain fatty acids or partially metabolized fatty which the body is unable to acids may also build up in tissues and damage the convert very long-chain heart, liver, and muscles. fatty acids into energy. HADHA 2p23 Trifunctional protein (I) Long chain 3- (I) Symptoms include feeding difficulties, lethargy, deficiency (TFP) hydroxyacyl-CoA low blood sugar, weak muscle tone, retinal dehydrogenase abnormalities. May experience muscle pain or (LCHAD) deficiency breakdown of muscle tissue and loss of sensation prevents the body in arms and legs. from converting (II) Symptoms include feeding difficulties, lethargy, certain fats to energy. low blood sugar, and liver problems. High risk for (II) Mitochondrial heart problems, breathing difficulty, coma and trifunctional protein sudden death. This disorder can also arise from deficiency prevents the mutations in the HADHB gene. body from converting certain fats into energy. HADHB 2p23 Trifunctional protein Mitochondrial trifunctional Symptoms include feeding difficulties, lethargy, low deficiency (TFP) protein deficiency prevents blood sugar, and liver problems. High risk for heart the body from converting problems, breathing difficulty, coma and sudden certain fats into energy. death. This disorder can also arise from mutations in the HADHA gene. Amino ASL 7q11.21 Argininosuccinic aciduria Argininosuccinic aciduria is Argininosuccinic aciduria usually becomes evident Acid (ASA) an inherited disorder that in the first few days of life. Symptoms include lack Disorders causes ammonia to of energy, unwilling to eat, poorly controlled accumulate in the blood. respiratory rate or body temperature, seizures, and coma. ASS1 9q34.1 Citrullinemia (CIT) Citrullinemia is an inherited Type I (classic citrullinemia): usually becomes type I disorder that causes evident in the first few days of life. Symptoms ammonia and other toxic include lack of energy, poor feeding, vomiting, substances to accumulate seizures, and loss of consciousness later in in the blood. childhood or adulthood. This later-onset form is associated with intense headaches, partial loss of vision, problems with balance and muscle coordination (ataxia), and lethargy. Type II is caused by mutations in the SLC25A13 gene. BCKDHA 19q13.1- Maple Syrup Urine Maple syrup urine disease Symptoms commonly begin in early infancy and q13.2 Disease (MSUD) is an inherited disorder in include urine of a distinctive sweet odor, poor which the body is unable to feeding, vomiting, lack of energy and process certain protein developmental delay. If untreated, maple syrup building blocks (amino urine disease can lead to seizures, coma, and acids) properly. death. This disorder may also be caused by mutations in the BCKDHB, DBT, and DLD genes. BCKDHB 6q14.1 Maple Syrup Urine Maple syrup urine disease Symptoms commonly begin in early infancy and Disease (MSUD) is an inherited disorder in include urine of a distinctive sweet odor, poor which the body is unable to feeding, vomiting, lack of energy and process certain protein developmental delay. If untreated, maple syrup building blocks (amino urine disease can lead to seizures, coma, and acids) properly. death. This disorder may also be caused by mutations in the BCKDHA, DBT, and DLD genes. DBT 1p31 Maple Syrup Urine Maple syrup urine disease Symptoms commonly begin in early infancy and Disease (MSUD) is an inherited disorder in include urine of a distinctive sweet odor, poor which the body is unable to feeding, vomiting, lack of energy and process certain protein developmental delay. If untreated, maple syrup building blocks (amino urine disease can lead to seizures, coma, and acids) properly. death. This disorder may also be caused by mutations in the BCKDHA, BCKDHB, and DLD genes. DLD 7q31-q32 Maple Syrup Urine Maple syrup urine disease Symptoms commonly begin in early infancy and Disease (MSUD) is an inherited disorder in include urine of a distinctive sweet odor, poor which the body is unable to feeding, vomiting, lack of energy and process certain protein developmental delay. If untreated, maple syrup building blocks (amino urine disease can lead to seizures, coma, and acids) properly. death. This disorder may also be caused by mutations in the BCKDHA, BCKDHB, and DBT genes. CBS 21q22.3 hom*ocystinuria (HCY) hom*ocystinuria is an Multiple forms of this disorder exist but most inherited disorder in which common symptoms include nearsightedness, the body is unable to dislocation of the lens at the front of the eye, process certain building increased risk of abnormal blood clotting, and blocks of proteins (amino brittle bones. acids) properly. Although most often mutations in the CBS gene cause this disorder, it may also arise from mutations in the MTHFR, MTR, MTRR and MMADHC genes. MTHFR 1p36.3 hom*ocystinuria (HCY) hom*ocystinuria is an Multiple forms of this disorder exist but most inherited disorder in which common symptoms include nearsightedness, the body is unable to dislocation of the lens at the front of the eye, process certain building increased risk of abnormal blood clotting, and blocks of proteins (amino brittle bones. acids) properly. Although most often mutations in the CBS gene cause this disorder, it may also arise from mutations in the CBS, MTR, MTRR and MMADHC genes. MTR 1q43 hom*ocystinuria (HCY) hom*ocystinuria is an Multiple forms of this disorder exist but most inherited disorder in which common symptoms include nearsightedness, the body is unable to dislocation of the lens at the front of the eye, process certain building increased risk of abnormal blood clotting, and blocks of proteins (amino brittle bones. acids) properly. Although most often mutations in the CBS gene cause this disorder, it may also arise from mutations in the CBS, MTHFR, MTRR and MMADHC genes. MTRR 5p15.31 hom*ocystinuria (HCY) hom*ocystinuria is an Multiple forms of this disorder exist but most inherited disorder in which common symptoms include nearsightedness, the body is unable to dislocation of the lens at the front of the eye, process certain building increased risk of abnormal blood clotting, and blocks of proteins (amino brittle bones. acids) properly. Although most often mutations in the CBS gene cause this disorder, it may also arise from mutations in the CBS, MTHFR, MTR, and MMADHC genes. PAH 12q22- Phenylketonuria Phenylketonuria is an Classic PKU presents normally until the infant is a q24.2 (PKU) inherited disorder that few months old. Without treatment, these children increases the levels of a develop permanent intellectual disability. Seizures, substance called delayed development, behavioral problems, and phenylalanine in the blood. psychiatric disorders are also common. Other, less severe forms have a smaller risk of brain damage. Untreated individuals may have a musty or mouse- like odor as a side effect of excess phenylalanine in the body. FAH 15q25.1 Tyrosinemia (TYR I, II, III) Tyrosinemia is a genetic Type 1: Symptoms include failure to thrive, disorder characterized by diarrhea, vomiting, jaundice, cabbage odor, and elevated blood levels of nosebleeds. Can lead to liver and kidney failure the amino acid tyrosine. and impact the nervous system. Type II: Symptoms include excessive tearing, sensitivity to light, eye pain, redness, and intellectual disability. Type III: Symptoms include Intellectual disability, seizures, and loss of balance. Mutations in the HPD, and TAT genes may also cause tyrosinemia. Endocrine DUOX2 15q15.3 Congenital hypothyroidism Congenital hypothyroidism If untreated, congenital hypothyroidism can lead to Disorders (CH) is a condition that affects intellectual disability and abnormal growth. infants from birth Other genes that may cause congenital (congenital) and results hypothyroidism include the PAX8, SLC5A5, TG, from a partial or complete TPO, TSHB, and TSHR genes. loss of thyroid function (hypothyroidism). PAX8 2q13 Congenital hypothyroidism Congenital hypothyroidism If untreated, congenital hypothyroidism can lead to (CH) is a condition that affects intellectual disability and abnormal growth. infants from birth Other genes that may cause congenital (congenital) and results hypothyroidism include the DUOX2, SLC5A5, TG, from a partial or complete TPO, TSHB, and TSHR genes. loss of thyroid function (hypothyroidism). SLC5A5 19p13.11 Congenital hypothyroidism Congenital hypothyroidism If untreated, congenital hypothyroidism can lead to (CH) is a condition that affects intellectual disability and abnormal growth. infants from birth Other genes that may cause congenital (congenital) and results hypothyroidism include the DUOX2, PAX8, TG, from a partial or complete TPO, TSHB, and TSHR genes. loss of thyroid function (hypothyroidism). TG 8q24 Congenital hypothyroidism Congenital hypothyroidism If untreated, congenital hypothyroidism can lead to (CH) is a condition that affects intellectual disability and abnormal growth. infants from birth Other genes that may cause congenital (congenital) and results hypothyroidism include the DUOX2, PAX8, from a partial or complete SLC5A5, TPO, TSHB, and TSHR genes. loss of thyroid function (hypothyroidism). TPO 2p25 Congenital hypothyroidism Congenital hypothyroidism If untreated, congenital hypothyroidism can lead to (CH) is a condition that affects intellectual disability and abnormal growth. infants from birth Other genes that may cause congenital (congenital) and results hypothyroidism include the DUOX2, PAX8, from a partial or complete SLC5A5, TG, TSHB, and TSHR genes. loss of thyroid function (hypothyroidism). TSHB 1p13 Congenital hypothyroidism Congenital hypothyroidism If untreated, congenital hypothyroidism can lead to (CH) is a condition that affects intellectual disability and abnormal growth. infants from birth Other genes that may cause congenital (congenital) and results hypothyroidism include the DUOX2, PAX8, from a partial or complete SLC5A5, TG, TPO, and TSHR genes. loss of thyroid function (hypothyroidism). TSHR 14q31 Congenital hypothyroidism Congenital hypothyroidism If untreated, congenital hypothyroidism can lead to (CH) is a condition that affects intellectual disability and abnormal growth. infants from birth Other genes that may cause congenital (congenital) and results hypothyroidism include the DUOX2, PAX8, from a partial or complete SLC5A5, TG, TPO, and TSHB genes. loss of thyroid function (hypothyroidism). CYP21A2 6p21.3 21-hydroxylase deficiency 21-hydroxylase deficiency Signs and symptoms for the salt-wasting type (CAH) is an inherited disorder that include low hormone production, loss of sodium in affects the adrenal glands. urine, poor feeding, weight loss, dehydration, and Three types of this vomiting. Females with the salt-wasting and simple disorder include the salt- virilizing forms typically have external genitalia that wasting, simple virilizing, do not look clearly female or male. Individuals with and non-classic types. these forms may also experience decreased fertility and other hormone related symptoms. Hemoglobin HBB 11p15.5 (I ) Sickle cell disease All three disorders affect Symptoms of the different disorders are as follows: Disorders (II)Metheglobinemia, beta- the production or function (1) Anemia, repeated infections, periodic episodes globin type of hemoglobin. of pain. (III) Beta thalassemia: (2) Bluish appearance of the skin, mucous thalassemia major and membranes, or area under the fingernails. thalassemia intermedia (3) Anemia, pale skin, weakness, fatigue, risk of abnormal blood clots, enlarged spleen, liver, and heart. Other BTD 3p25 Biotinidase deficiency Biotinidase deficiency is an The signs and symptoms of biotinidase deficiency Disorders (BIOT) inherited disorder in which typically appear within the first few months of life, the body is unable to reuse but the age of onset varies. Symptoms often and recycle the vitamin include seizures, muscle weakness, breathing biotin. problems, and delayed development. If left untreated, the disorder can lead to hearing loss, eye abnormalities and loss of vision, problems with movement and balance, skin rashes, hair loss, and a fungal infection called candidiasis. Immediate treatment and lifelong management with biotin supplements can prevent many of these complications. CFTR 7q31.2 Cystic fibrosis (CF) Cystic fibrosis is an The disorder's most common signs and symptoms inherited disease include progressive damage to the respiratory characterized by the system and chronic digestive system problems. buildup of thick, sticky Problems with digestion can lead to diarrhea, mucus that can damage malnutrition, poor growth, and weight loss. In many of the body's organs. adolescence or adulthood, a shortage of insulin can cause a form of diabetes known as cystic fibrosis-related diabetes mellitus (CFRDM). GALT 9p13 Galactosemia type I Galactosemia is a disorder Type I: This is the most common and most severe that prevents the body form of the disorder. If infants are not promptly from processing a simple treated complications can arise within the first few sugar called galactose into days of life. Complications include feeding energy. difficulties, lack of energy, failure to thrive, jaundice, liver damage and bleeding. Type II and III are caused by mutations in the GALE and GALT genes. GJB2 13q11-q12 Hearing Loss Several conditions can (1) Damage to inner ear structure resulting in (1) Nonsyndromic cause hearing damage or permanent hearing loss. (2) Mild to profound deafness loss in infants. Listed are hearing loss, skin abnormalities. Increased risk of (2) Palmoplantar the disorders caused by skin cancer. karatoderma mutations in the GJB2 (3) (3) Hystrix-like ichthyosis gene. Dry, thick scaly skin and profound hearing loss, (4) Bart-Pumphrey which may worsen over time. Increased risk of syndrome skin cancer. (5) Vohwinkel syndrome (4) (6) Karatitis-ichthyosis- Discoloration of nails and wart-like growths on deafness (KID) fingers and toes. Hearing loss is profound. (5) Symptoms include hearing loss, starfish- shaped patches of skin on fingers, toes and knees, and tight fibrous tissue around fingers and toes resulting in amputation. (6) Profound hearing loss, vision loss GJB3 1p34 Hearing Loss Several conditions can (1) Damage to inner ear structure resulting in (1)Nonsyndromic deafness cause hearing damage or permanent hearing loss. (2)Erythrokeratodermia loss in infants. Listed are (2) Symptoms include rough, thickened reddish- variabilis et progressive the disorders caused by brown patches and patches of reddened skin. (EKVP) mutations in the GJB3 gene GJB6 13q12 Hearing Loss Several conditions can (1) Damage to inner ear structure (1) Nonsyndromic cause hearing damage or resulting in permanent hearing loss. deafness loss in infants. Listed are (2) Symptoms include sparse, patchy, fragile scalp (2) Clouston syndrome the disorders caused by hair and abnormal fingernails and toenails. mutations in the GJB3 gene ADA 20q13.12 Severe combined Adenosine deaminase The main symptoms arise due to a very weakened immunodeficiency (SCID) (ADA) deficiency is an immune system and commonly include inherited disorder that pneumonia, chronic diarrhea, and widespread skin damages the immune rashes. Affected children tend to grow slower than system and causes severe healthy children and some experience combined developmental delay. immunodeficiency (SCID). IL2RG Xq13.1 X-linked severe combined X-linked severe combined Individuals with this disorder are prone to infection, immunodeficiency (SCID) immunodeficiency (SCID) which may cause life-threatening illnesses. May is an inherited disorder of experience chronic diarrhea or skin rashes. the immune system that occurs almost exclusively in males. Secondary Conditions Organic MLYCD 16q24 Malonyl CoA Malonyl CoA Symptoms appear in early childhood and result in Acid decarboxylase deficiency decarboxylase deficiency delayed development, weak muscle tone, seizures, Disorders (MAL) is a condition that prevents diarrhea, vomiting, low blood sugar, and the body from converting cardiomyopathy. certain fats to energy. ACAD8 11q25 Isobutyryl-CoA Isobutyryl-CoA Most effected individuals do not experience dehydrogenase (IBD) dehydrogenase (IBD) symptoms. A few children with IBD deficiency have deficiency deficiency is a condition developed features such as a weakened and that disrupts the enlarged heart, weak muscle tone, developmental breakdown of certain delay, and anemia. proteins. ACADSB 10q26.13 2-methylbutyryl-CoA 2-methylbutyryl-CoA The initial symptoms often include poor feeding, dehydrogenase deficiency dehydrogenase deficiency lack of energy, vomiting, and an irritable mood. is a type of organic acid These symptoms sometimes progress to serious disorder in which the body medical problems such as difficulty breathing, is unable to process seizures, and coma. Additional problems can proteins properly include poor growth, vision problems, learning disabilities, muscle weakness, and delays in motor skills such as standing and walking. AUH 9q22.31 3-methylglutaconic The name 3- There are five types of 3-methylglutaconic aciduria aciduria (3MGA) type I methylglutaconic aciduria numbered I, II, III, IV and V. is used to describe five Type I: Mutations in the AUH gene cause Type I 3- different disorders that methylglutaconic aciduria. Symptoms include impair the functioning of speech delay, delay in the development of mental energy-producing centers and motor skills, elevated levels of acid in the within cells (mitochondria). blood, abnormal muscle tone, and spasms and weakness of the arms and legs Types II, III, IV and IV are caused by mutations in the DNAJC19, OPA3, and TAZ genes. DNAJC19 3q26.33 3-methylglutaconic The name 3- There are five types of 3-methylglutaconic aciduria aciduria (3MGA) type V methylglutaconic aciduria numbered I, II, III, IV and V. is used to describe five Type V: Mutations in the DNAJC19 gene cause different disorders that Type V 3-methylglutaconic aciduria. Symptoms impair the functioning of include an enlarged and weakened heart, inability energy-producing centers to control voluntary muscle movements, growth within cells (mitochondria). failure, mild intellectual disability and optic atrophy Types I, II, III, and V are caused by mutations in the AUH, OPA3, and TAZ genes. OPA3 19q13.32 3-methylglutaconic The name 3- There are five types of 3-methylglutaconic aciduria aciduria (3MGA) type I methylglutaconic aciduria numbered I, II, III, IV and V. is used to describe five Type III (Costeff Optic Syndrome): Mutations in different disorders that the OPA3 gene cause the Type III form of this impair the functioning of disorder which is characterized by degeneration of energy-producing centers the optic nerves, inability to maintain posture, poor within cells (mitochondria). muscle tone, gradual increase in involuntary jerking movements, and general decrease in cognitive function. Types I, II, IV and IV are caused by mutations in the AUH, DNAJC19, and TAZ genes. TAZ Xq28 3-methylglutaconic The name 3- There are five types of 3-methylglutaconic aciduria aciduria (3MGA) type I methylglutaconic aciduria numbered I, II, III, IV and V. is used to describe five Type II (Barth Syndrome): Mutations in the TAZ different disorders that gene cause the Type II form of this disorder, which impair the functioning of is characterized by, and enlarge and weakened energy-producing centers heart, reoccurring infections, muscle weakness, within cells (mitochondria). and muscle spasms. Types I, III, IV and IV are caused by mutations in the AUH, DNAJC19, and OPA3 genes. HSD17B10 Xp11.2 3-hydroxy-2-methylbutyryl- Another name for This disorder is more severe in males. Normal CoA dehydrogenase 2M3HBA is early development, but experience developmental deficiency (2M3HBA) 17β-hydroxysteroid regression around age 5 resulting in intellectual dehydrogenase type 10 disability and loss of motor skills. Hearing and (HSD10) deficiency. vision loss may also occur. Fatty Acid ACADS 12q24.31 Short-chain acyl-CoA Short-chain acyl-CoA Signs and symptoms can include vomiting, low Disorders dehydrogenase (SCAD) dehydrogenase (SCAD) blood sugar (hypoglycemia), a lack of energy, poor deficiency deficiency is a condition feeding, failure to gain weight and grow at the that prevents the body expected rate, poor muscle tone, seizures, from converting certain fats developmental delay, and a small head size. into energy HADH 4q22-q26 3-hydroxyacyl-CoA 3-hydroxyacyl-CoA Characteristics of this disorder typically arise dehydrogenase deficiency dehydrogenase deficiency during infancy or early childhood. Signs and (M/SCHAD) is an inherited condition symptoms include muscle weakness, poor that prevents the body appetite, vomiting, diarrhea, lethargy, liver from converting certain fats problems, low blood sugar, seizures, life- to energy, particularly threatening heart and breaking problems, coma, during prolonged periods and sudden death (SIDS). without food (fasting). ETFA 15q23-q25 Glutaric acidemia type II Glutaric acidemia type II is Due to buildup of incompletely processed proteins (GA2) an inherited disorder that and fats symptoms can arise including weakness, interferes with the body's poor feeding, decreased activity, and vomiting. ability to break down Other abnormalities include brain malformations, proteins and fats to an enlarged liver, a weakened and enlarged heart, produce energy. fluid-filled cysts and other malformations of the kidneys, unusual facial features, and genital abnormalities. Mutations in the ETFB and ETFDH genes can also cause this disorder. ETFB 19q13.3 Glutaric acidemia type II Glutaric acidemia type II is Due to buildup of incompletely processed proteins (GA2) an inherited disorder that and fats symptoms can arise including weakness, interferes with the body's poor feeding, decreased activity, and vomiting. ability to break down Other abnormalities include brain malformations, proteins and fats to an enlarged liver, a weakened and enlarged heart, produce energy. fluid-filled cysts and other malformations of the kidneys, unusual facial features, and genital abnormalities. Mutations in the ETFA and ETFDH genes can also cause this disorder. ETFDH 4q32-q35 Glutaric acidemia type II Glutaric acidemia type II is Due to buildup of incompletely processed proteins (GA2) an inherited disorder that and fats symptoms can arise including weakness, interferes with the body's poor feeding, decreased activity, and vomiting. ability to break down Other abnormalities include brain malformations, proteins and fats to an enlarged liver, a weakened and enlarged heart, produce energy. fluid-filled cysts and other malformations of the kidneys, unusual facial features, and genital abnormalities. Mutations in the ETFA and ETFB genes can also cause this disorder. CPT1A 11q13.2 Carnitine Carnitine Signs and symptoms of CPT I deficiency often palmitoyltransferase I palmitoyltransferase I appear during early childhood. Signs and deficiency (CPT IA) (CPT I) deficiency is a symptoms include low blood sugar and low ketone condition that prevents the levels, enlarged liver, liver malfunction, and body from using certain elevated levels of carnitine in the blood. Individuals fats for energy, particularly with CPT I deficiency are at risk for nervous during periods without food system damage, liver failure, seizures, coma, and (fasting). sudden death. CPT2 1p32 Carnitine Carnitine Type I (Lethal Neonatal): Appears soon after birth palmitoyltransferase II palmitoyltransferase II with symptoms including respiratory failure, deficiency (CPT II) (CPT II) deficiency is a seizures, liver failure, various abnormal heart condition that prevents the conditions, and hypoketotic hypoglycemia. body from using certain Type II (Severe Infantile Hepatocardiomuscular): fats for energy, particularly Appears within the first year of life with symptoms during periods without food including hypoketotic hypoglycemia, seizures, (fasting). enlarged liver, and various abnormal heart conditions. Type II (Myopathic): Characterized by reoccurring muscle pain and weakness and discolored urine. SLC25A20 3p21.31 Carnitine-acylcarnitine Carnitine-acylcarnitine Many infants with CACT deficiency do not survive translocase (CACT) translocase (CACT) the newborn period without treatment. Signs and deficiency is a condition symptoms include low blood sugar, low levels of that prevents the body ketones, excess ammonia in the blood, enlarged from using certain fats for liver, and weakened heart muscles. energy, particularly during periods without food (fasting). Amino ARG1 6q23 Arginase deficiency (ARG) Arginase deficiency (ARG) Arginase deficiency usually becomes evident by Acid is an inherited disorder that about the age of 3. It most often appears as Disorders causes the amino acid stiffness, caused by abnormal tensing of the arginine (a building block muscles, slow growth patterns, developmental of proteins) and ammonia delays, intellectual disabilities, seizures, tremors, to accumulate gradually in and difficulty with coordination. the blood. SLC25A13 7q21.3 Citrullinemia type II Citrullinemia is an inherited Type I: Caused by mutations in the ASS1 gene. (CIT II) disorder that causes Type II: Caused by mutations in the SLC25A13 ammonia and other toxic gene and chiefly affects the nervous system, substances to accumulate causing confusion, restlessness, memory loss, in the blood. abnormal behaviors (such as aggression, irritability, and hyperactivity), seizures, and coma. AHCY 20q11.22 Hypermethioninemia Hypermethioninemia is an People with hypermethioninemia often do not show (MET) excess of a particular any symptoms. Some individuals with protein building block hypermethioninemia exhibit intellectual disability (amino acid), called and other neurological problems; delays in motor methionine, in the blood. skills such as standing or walking; sluggishness; muscle weakness; liver problems; unusual facial features; and their breath, sweat, or urine may have a smell resembling boiled cabbage. This disorder may also be caused by mutations in the GNMT and MAT1A genes. GNMT 6p12 Hypermethioninemia Hypermethioninemia is an People with hypermethioninemia often do not show (MET) excess of a particular any symptoms. Some individuals with protein building block hypermethioninemia exhibit intellectual disability (amino acid), called and other neurological problems; delays in motor methionine, in the blood. skills such as standing or walking; sluggishness; muscle weakness; liver problems; unusual facial features; and their breath, sweat, or urine may have a smell resembling boiled cabbage. This disorder may also be caused by mutations in the AHCY and MAT1A genes. MAT1A 10q22 Hypermethioninemia Hypermethioninemia is an People with hypermethioninemia often do not show (MET) excess of a particular any symptoms. Some individuals with protein building block hypermethioninemia exhibit intellectual disability (amino acid), called and other neurological problems; delays in motor methionine, in the blood. skills such as standing or walking; sluggishness; muscle weakness; liver problems; unusual facial features; and their breath, sweat, or urine may have a smell resembling boiled cabbage. This disorder may also be caused by mutations in the AHCY and GNMT genes. GCH1 14q22.1- Disorders of biopterin Tetrahydrobiopterin Infants with this deficiency often appear normal at q22.2 regeneration deficiency is a rare birth but over time begin to exhibit mild to severe disorder characterized by a symptoms and signs such as intellectual disability, shortage of a molecule developmental problems, movement disorders, called tetrahydrobiopterin, difficulty swallowing, seizures, behavioral which leads to an problems, and inability to control body increased level of temperature. phenylalanine in the body. PCBD1 10q22 Disorders of biopterin Tetrahydrobiopterin Infants with this deficiency often appear normal at regeneration deficiency is a rare birth but over time begin to exhibit mild to severe disorder characterized by a symptoms and signs such as intellectual disability, shortage of a molecule developmental problems, movement disorders, called tetrahydrobiopterin, difficulty swallowing, seizures, behavioral which leads to an problems, and inability to control body increased level of temperature. phenylalanine in the body. PTS 11q22.3 Disorders of biopterin Tetrahydrobiopterin Infants with this deficiency often appear normal at regeneration deficiency is a rare birth but over time begin to exhibit mild to severe disorder characterized by a symptoms and signs such as intellectual disability, shortage of a molecule developmental problems, movement disorders, called tetrahydrobiopterin, difficulty swallowing, seizures, behavioral which leads to an problems, and inability to control body increased level of temperature. phenylalanine in the body. QDPR 4p15.31 Disorders of biopterin Tetrahydrobiopterin Infants with this deficiency often appear normal at regeneration deficiency is a rare birth but over time begin to exhibit mild to severe disorder characterized by a symptoms and signs such as intellectual disability, shortage of a molecule developmental problems, movement disorders, called tetrahydrobiopterin, difficulty swallowing, seizures, behavioral which leads to an problems, and inability to control body increased level of temperature. phenylalanine in the body. TAT 16q22.1 Tyrosinemia (TYR I, II, III) Tyrosinemia is a genetic Type 1: Symptoms include failure to thrive, disorder characterized by diarrhea, vomiting, jaundice, cabbage odor, and elevated blood levels of nosebleeds. Can lead to liver and kidney failure the amino acid tyrosine. and impact the nervous system. Type II: Symptoms include excessive tearing, sensitivity to light, eye pain, redness, and intellectual disability. Type III: Symptoms include Intellectual disability, seizures, and loss of balance. Mutations in the FAH and HPD genes may also cause tyrosinemia. HPD 12q24.31 Tyrosinemia (TYR I, II, III) Tyrosinemia is a genetic Type 1: Symptoms include failure to thrive, disorder characterized by diarrhea, vomiting, jaundice, cabbage odor, and elevated blood levels of nosebleeds. Can lead to liver and kidney failure the amino acid tyrosine. and impact the nervous system. Type II: Symptoms include excessive tearing, sensitivity to light, eye pain, redness, and intellectual disability. Type III: Symptoms include Intellectual disability, seizures, and loss of balance. Mutations in the FAH and TAT genes may also cause tyrosinemia. Hemoglobin HBA1 16p13.3 Alpha thalassemia Alpha thalassemia is a This disorder causes a shortage of red blood cells Disorders (Hemoglobin Disorder-Var- blood disorder that (anemia), which can cause pale skin, weakness, Hb) reduces the production of fatigue, along with other complications. hemoglobin. Two types have been observed: (I) Hb Bart: characterized by severe anemia, enlarged liver and spleen, heart defects, abnormalities of urinary system or genitalia. (II) HbH: Characterized by mild to moderate anemia, jaundice, overgrowth of jaw or prominent forehead. This disorder can also be caused by mutations in the HBA2 gene. HBA2 16p13.3 Alpha thalassemia Alpha thalassemia is a This disorder causes a shortage of red blood cells (Hemoglobin Disorder-Var- blood disorder that (anemia), which can cause pale skin, weakness, Hb) reduces the production of fatigue, along with other complications. hemoglobin. Two types have been observed: (I) Hb Bart: characterized by severe anemia, enlarged liver and spleen, heart defects, abnormalities of urinary system or genitalia. (II) HbH: Characterized by mild to moderate anemia, jaundice, overgrowth of jaw or prominent forehead. This disorder can also be caused by mutations in the HBA1 gene. Other GALE 1p36-p35 Galactosemia type III Galactosemia is a disorder Type III: Symptoms include cataracts, delayed Disorders that prevents the body growth and development, intellectual disability, from processing a simple liver disease, and kidney problems. sugar called galactose into Type I and II are caused by mutations in the energy. GALK1 and GALT genes. GALK1 17q24 Galactosemia type II Galactosemia is a disorder Type II: Affected infants may develop cataracts, that prevents the body but otherwise experience few additional from processing a simple complications. sugar called galactose into Type I and III are caused by mutations in the GALE energy. and GALT genes. Additional Conditions ABCD1 Xq28 X-linked X-linked Cerebral form (childhood): learning and behavioral adrenoleukodystrophy adrenoleukodystrophy problems, aggressive behavior, vision problems, Adrenomyeloneuropathy Adrenomyeloneuropathy is difficulty swallowing, poor coordination, and Addison disease a genetic disorder, impaired adrenal function. (X-ALD) effecting primarily males Adrenomyeloneuropathy form (early adulthood to that negatively impacts the middle age): stiffness and weakness in the legs, nervous system and the urinary and genital tract disorders and changes in adrenal glands. behavior and thinking ability. Addison disease: adrenocortical insufficiency only. DECR1 8q21.3 2,4 Dienoyl-CoA reductase 2,4 Dienoyl-CoA reductase Poor appetite, vomiting, irritability, weak muscle deficiency deficiency is a condition tone, short torso, small body, and small head size. associated with respiratory acidosis in infancy. GAA 17q25.2- Pompe disease Pompe disease is an Type I (Classic): Onset begins within a few months 25.3 (GAA deficiency) inherited disorder caused and triggers symptoms such as muscle weakness, by the buildup of a enlarged liver, and heart defects. If untreated, this complex sugar called form leads to death within the first year of life. glycogen in the body's Type II (Non-classic): Typically appears by the age cells causing an inability to of one with characteristics including delayed motor function properly. skills and progressive muscle weakness. This form can cause death within early childhood. Type III (Late-onset): May not become apparent until adulthood and may cause muscle weakness and progressively worsening breathing problems. GALC 14q31 Krabbe Disease Krabbe disease (also Symptoms usually arise before the age of one and called globoid cell include irritability, muscle weakness, feeding leukodystrophy) is a problems, fever, and slowed mental and physical degenerative disorder that development, vision loss and seizures. affects the nervous system. GBA 1q21 Gaucher disease Gaucher disease is an Type I (Non-neuronopathic): Characteristics (types I, II, & III) inherited disorder that include enlarged liver and spleen, anemia, easy affects many of the body's bruising caused by decreased platelets, lung organs and tissues. disease and bone abnormalities. Types II & III (Neuronopathic): Both forms have similar symptoms to Type I but additionally affect the central nervous system. Additional conditions may include abnormal eye movements, seizures and brain damage. Perinatal Lethal: The most severe form that causes life-threatening complications including extensive swelling, skin abnormalities, and neurological complications. Cardiovascular: Causes hardening of heart valves, eye abnormalities, and bone diseases. GLA Xq22 Fabry disease Fabry disease is an With disorder characters beginning in childhood, inherited disorder that complications may include life-threatening kidney results from the buildup of damage, heart attack and stroke. Symptoms a particular type of fat, include pain in hands and feet, dark red spots on called skin, decreased ability to sweat, corneal opacity, globotriaosylceramide, in ear ringing, gastrointestinal problems. the body's cells. IDUA 4p16.3 Mucopolysaccharidosis Mucopolysaccharidosis Typically no signs and symptoms are present at type I type I (MPS I) is a birth. Other characteristics of the disorder that (MPS I) condition that affects many slowly progress include large head, fluid in the parts of the body. bread, heart abnormalities, large tongue, upper respiratory infection, clouding of cornea resulting in vision loss, and hearing loss. Severely affected individuals eventually lose basic motor skills. NGLY1 3p24.2 Congenital Disorder of Congenital disorder of Features include seizures, abnormal eye Deglycosylation type 1v deglysosylation is an movements, liver dysfunction, and microcephaly. autosomal recessive multisystem disorder caused by a defect in glycoprotein synthesis. NPC1 18q11.2 Niemann-Pick disease Niemann-Pick disease is Niemann-Pick disease is divided into four main (type C1) an inherited condition types: A, B, C1 and C2. Mutations in the NPC1 involving lipid metabolism, gene are responsible for type C1. Symptoms for which is the breakdown, this form usually appear in childhood and often transport, and use of fats include severe liver disease, breathing difficulties, and cholesterol in the developmental delay, seizures, poor muscle tone, body. lack of coordination, problems feeding, and inability to move the eyes vertically. Type A and B are caused by mutations in the SMPD1 gene and type C2 is caused by mutations in the NPC2 gene. NPC2 14q24.3 Niemann-Pick disease Niemann-Pick disease is Niemann-Pick disease is divided into four main (type C2) an inherited condition types: A, B, C1 and C2. Mutations in the NPC2 involving lipid metabolism, gene are responsible for type C2. Symptoms for which is the breakdown, this form usually appear in childhood and often transport, and use of fats include severe liver disease, breathing difficulties, and cholesterol in the developmental delay, seizures, poor muscle tone, body. lack of coordination, problems feeding, and inability to move the eyes vertically. Type A and B are caused by mutations in the SMPD1 gene and type C1 is caused by mutations in the NPC1 gene.

TABLE 2 Exemplary Genes List showing the affected genes and core, secondary and additional conditions. Metabolic Disorder Fatty Acid Endocrine Hemoglobin ACMG Code Organic Acid oxidation Amino Acid Disorder Disorder Other Disorder Genes Affected Core (or Primary) Conditions PROP X PCCA PCCB MUT X MUT MMAA MMAB MMADHC MCEE Cbl A,B X MUT MMAA MMAB MMADHC MCEE IVA X IVD 3-MCC X MCCC1 MCCC2 HMG X HMGCL MCD X HLCS βKT X ACAT1 GA1 X GCDH CUD X SLC22A5 MCAD X ACADM VLCAD X ACADVL LCHAD X HADHA TFP X HADHA HADHB ASA X ASL CIT X ASS1 MSUD X BCKDHA BCKDHB DBT DLD HCY X CBS MTHFR MTR MTRR MMADHC PKU X PAH TYR I X FAH CH X DUOX2 PAX8 SLC5A5 TG TPO TSHB TSHR CAH X CYP21A2 Hb SS X HBB Hb S/β TH X HBB Hb S/C X HBB BIOT X BTD CF X CFTR GALT X GALT HEAR X GJB2 GJB3 GJB6 SCID X ADA IL2RG Secondary Conditions CBL C, D X MMADHC MAL X MLYCD IBD X ACAD8 2MBG X ACADSB 3MGA X AUH DNAJC19 OPA3 TAZ 2M3HBA X HSD17810 SCAD X ACADS M/SCHAD X HADH GA2 X ETFA ETFB ETFDH MCAT X SCL25A20 DE RED X DECR1 CPT IA X CPT1A CPT II X CPT2 CACT X SLC25A20 ARG X ARG1 CIT II X SLC25A13 MET X AHCY GNMT MAT1A H-PHE X PAH BIOPT (BS) X GCH1 PCBD1 PTS QDPR BIOPT (Reg) X GCH1 PCBD1 PTS QDPR TYR II X TAT TYR III X HPD Var Hb X HBA1 HBA2 HBB GALE X GALE GALK X GALK1 ADDED Conditions Krabbe GALC Gaucher GBA Niemann-Pick A/B NPC1 NPC2 Pompe GAA Fabry GLA MPS I IDUA X-ALD ABCD1 NGLY1

TABLE 3 Cytogenic Locations for genes included in Exemplary Genes List Gene Cytogenic location ACAD8 11q25 ACADM 1p31 ACADS 12q24.31 ACADSB 10q26.13 ACADVL 17p13.1 ACAT1 11q22.3 ADA 20q13.12 AHCY 20q11.22 ARG1 6q23 ASL 7q11.21 ASS1 9q34.1 AUH 9q22.31 BCKDHA 19q13.1-q13.2 BCKDHB 6q14.1 BTD 3p25 CBS 21q22.3 CFTR 7q31.2 CPT1A 11q13.2 CPT2 1p32 CYP21A2 6p21.3 DBT 1p31 DECR1 8q21.3 DLD 7q31-q32 DNAJC19 3q26.33 DUOX2 15q15.3 ETFA 15q23-q25 ETFB 19q13.3 ETFDH 4q32-q35 FAH 15q25.1 GALE 1p36-p35 GALK1 17q24 GALT 9p13 GCDH 19p13.2 GCH1 14q22.1-q22.2 GNMT 6p12 HADH 4q22-q26 HADHA 2p23 HADHB 2p23 HBA1 16p13.3 HBA2 16p13.3 HBB 11p15.5 HLCS 21q22.13 HMGCL 1p36.1-p35 HPD 12q24.31 HSD17B10 Xp11.2 IL2RG Xq13.1 IVD 15q14-q15 MAT1A 10q22 MCCC1 3q27 MCCC2 5q12-q13 MCEE 2p13.3 MLYCD 16q24 MMAA 4q31.21 MMAB 12q24 MMADHC 2q23.2 MTHFR 1p36.3 MTR 1q43 MTRR 5p15.31 MUT 6p12.3 OPA3 19q13.32 PAH 12q22-q24.2 PAX8 2q13 PCBD1 10q22 PCCA 13q32 PCCB 3q21-q22 PTS 11q22.3 QDPR 4p15.31 SLC22A5 5q23.3 SLC25A13 7q21.3 SLC25A20 3p21.31 SLC5A5 19p13.11 TAT 16q22.1 TAZ Xq28 TG 8q24 TPO 2p25 TSHB 1p13 TSHR 14q31

TABLE 4 Exemplary Target Regions: Coding Exons NCBI reference Target name sequence Target exons Direction ABCD1 NM_000033 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 3 ACAD8 NM_014384 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 3 ACADM NM_000016 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 3 ACADM NM_001127328 2 3 ACADS NM_000017 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 3 ACADSB NM_001609 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 3 ACADVL NM_001270447 1, 2 3 ACADVL NM_000018 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 3 ACAT1 NM_000019 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 3 ADA NM_000022 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 AHCY NM_000687 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 ARG1 NM_000045 1, 2, 4, 5, 6, 7, 8 3 ARG1 NM_001244438 3 3 ASL NM_000048 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 3 ASS1 NM_000050 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 3 AUH NM_001698 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 BCKDHA NM_000709 1, 2, 3, 4, 5, 6, 7, 8, 9 3 BCKDHB NM_000056 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 3 BTD NM_000060 2, 3, 4 3 CBS NM_000071 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 3 CFTR NM_000492 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 3 23, 24, 25, 26, 27 CPT1A NM_001031847 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 3 CPT1A NM_001876 19 3 CPT2 NM_000098 1, 2, 3, 4, 5 3 CYP21A2 NM_000500 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 3 DBT NM_001918 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 DECR1 NM_001359 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 3 DLD NM_000108 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 3 DNAJC19 NM_145261 1 3 DNAJC19 NM_001190233 6, 5, 4, 3, 2, 1 3 DUOX2 NM_014080 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 3 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 ETFA NM_000126 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 ETFB NM_001985 2, 1 3 ETFB NM_001014763 5, 4, 3, 2 3 ETFDH NM_004453 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 3 FAH NM_000137 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 3 GAA NM_001079803 1 3 GAA NM_000152 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 3 GALC NM_001201402 1 3 GALC NM_000153 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 GALE NM_001008216 1 3 GALE NM_000403 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 GALK1 NM_000154 8, 7, 6, 5, 4, 3, 2, 1 3 GALT NM_000155 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 3 GBA NM_000157 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 3 GBA NM_001005742 2 3 GBA NM_001005741 1 3 GCDH NM_000159 1, 2, 5, 6, 7, 8, 9, 10, 11, 12 3 GCH1 NM_001024024 7 3 GCH1 NM_000161 5, 4, 3, 2, 1 3 GJB2 NM_004004 2, 1 3 GJB3 NM_024009 1, 2 3 GJB3 NM_001005752 1 3 GJB6 NM_001110219 5, 4, 3, 2, 1 3 GJB6 NM_006783 1 3 GLA NM_000169 7, 6, 5, 4, 3, 2, 1 3 GNMT NM_018960 1, 2, 3, 4, 5, 6 3 HADH NM_001184705 1, 2, 3, 4, 5, 6, 7, 8, 9 3 HADHA NM_000182 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 HADHB NM_000183 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 3 HBA1 NM_000558 1, 2, 3 3 HBA2 NM_000517 1, 2, 3 3 HBB NM_000518 3, 2, 1 3 HLCS NM_000411 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 HLCS NM_001242784 3, 1 3 HLCS NM_001242785 1 3 HMGCL NM_000191 9, 8, 7, 6, 5, 4, 3, 2, 1 3 HPD NM_001171993 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 HPD NM_002150 1 3 HSD17B10 NM_001037811 6, 4, 3, 2, 1 3 HSD17B10 NM_004493 5 3 IDUA NM_000203 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 3 IL2RG NM_000206 8, 7, 6, 5, 4, 3, 2, 1 3 IVD NM_002225 2 3 IVD NM_001159508 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 3 MAT1A NM_000429 9, 8, 7, 6, 5, 4, 3, 2, 1 3 MCCC1 NM_020166 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 MCCC2 NM_022132 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 3 MCEE NM_032601 3, 2, 1 3 MLYCD NM_012213 1, 2, 3, 4, 5 3 MMAA NM_172250 1, 2, 3, 4, 5, 6, 7 3 MMAB NM_052845 9, 8, 7, 6, 5, 4, 3, 2, 1 3 MMADHC NM_015702 8, 7, 6, 5, 4, 3, 2, 1 3 MTHFR NM_005957 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 MTR NM_000254 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 3 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 MTRR NM_002454 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 3 MTRR NM_024010 1 3 MUT NM_000255 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 NGLY1 NM_001145294 7, 1 3 NGLY1 NM_001145293 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 NPC1 NM_000271 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 3 5, 4, 3, 2, 1 NPC2 NM_006432 5, 4, 3, 2, 1 3 OPA3 NM_001017989 2, 1 3 OPA3 NM_025136 2 3 PAH NM_000277 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 PAX8 NM_003466 12, 11, 10, 8, 7, 6, 5, 4, 3, 2, 1 3 PAX8 NM_013952 9 3 PCBD1 NM_000281 4, 3, 2, 1 3 PCCA NM_000282 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 3 23, 24 PCCB NM_001178014 4 3 PCCB NM_000532 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 3 PTS NM_000317 1, 2, 3, 4, 5, 6 3 QDPR NM_000320 7, 6, 5, 4, 3, 2, 1 3 SLC22A5 NM_003060 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 3 SLC25A13 NM_014251 10 3 SLC25A13 NM_001160210 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 SLC25A20 NM_000387 9, 8, 7, 6, 5, 4, 3, 2, 1 3 SLC5A5 NM_000453 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 3 TAT NM_000353 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 3 TAZ NM_000116 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 3 TG NM_003235 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 3 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 TPO NM_001206744 1 3 TPO NM_000547 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 3 TSHB NM_001277991 1 3 TSHB NM_000549 1, 2 3 TSHR NM_001142626 9 3 TSHR NM_000369 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 3

TABLE 5 Exemplary Gene Variant Locations for Screening Chrom. Location Location Gene #: Start End ID Type ABCD1 X 152990721 152990729 RCV000012069 deletion ABCD1 X 152990752 152990767 RCV000077962 deletion ABCD1 X 152991067 152991067 RCV000077963 SNV ABCD1 X 152991127 152991127 RCV000077964 SNV ABCD1 X 152991142 152991142 RCV000029289; RCV000077965 SNV ABCD1 X 152991164 152991164 RCV000012049 SNV ABCD1 X 152991219 152991241 RCV000077966 deletion ABCD1 X 152991241 152991241 RCV000012050 SNV ABCD1 X 152991335 152991335 RCV000077967 SNV ABCD1 X 152991517 152991517 RCV000012051 SNV ABCD1 X 152991559 152991559 RCV000029290 SNV ABCD1 X 152991592 152991592 RCV000012044 SNV ABCD1 X 152991592 152991594 RCV000012066 deletion ABCD1 X 153001649 153001649 RCV000012048 SNV ABCD1 X 153001686 153001686 RCV000012052 SNV ABCD1 X 153001826 153001826 RCV000012053 SNV ABCD1 X 153001940 153001941 RCV000029284 duplication ABCD1 X 153001964 153001964 RCV000012054 SNV ABCD1 X 153002613 153002613 RCV000077950 SNV ABCD1 X 153002632 153002633 RCV000012055; RCV000077951 deletion ABCD1 X 153002646 153002646 RCV000012056 SNV ABCD1 X 153002668 153002668 RCV000012045 SNV ABCD1 X 153005601 153005601 RCV000012057 SNV ABCD1 X 153005609 153005609 RCV000012058 deletion ABCD1 X 153005609 153005609 RCV000012059; RCV000077954 SNV ABCD1 X 153005610 153005610 RCV000077955 SNV ABCD1 X 153005643 153005643 RCV000077956 SNV ABCD1 X 153005649 153005649 RCV000029285 SNV ABCD1 X 153006053 153006053 RCV000077957 SNV ABCD1 X 153006072 153006072 RCV000077958 SNV ABCD1 X 153006164 153006164 RCV000077959 SNV ABCD1 X 153008462 153008462 RCV000077960 SNV ABCD1 X 153008477 153008477 RCV000012062 SNV ABCD1 X 153008509 153008509 RCV000012065 SNV ABCD1 X 153008510 153008510 RCV000012064 SNV ACAD8 11 134128412 134128412 RCV000081617 SNV ACAD8 11 134128475 134128476 RCV000081619 duplication ACAD8 11 134128483 134128483 RCV000005686 SNV ACAD8 11 134131194 134131194 RCV000005689 SNV ACAD8 11 134131232 134131232 RCV000005690 SNV ACAD8 11 134131680 134131680 RCV000005688 SNV ACAD8 11 134132450 134132450 RCV000005687; RCV000081615 SNV ACADM 1 76198337 76198337 RCV000077881 SNV ACADM 1 76198367 76198367 RCV000077882 SNV ACADM 1 76198409 76198409 RCV000003780 SNV ACADM 1 76198554 76198554 RCV000077885 SNV ACADM 1 76199288 76199288 RCV000003782 SNV ACADM 1 76200535 76200535 RCV000003774 SNV ACADM 1 76205773 76205773 RCV000003778 SNV ACADM 1 76205779 76205779 RCV000003777; RCV000077891 SNV ACADM 1 76211507 76211507 RCV000077892 SNV ACADM 1 76211508 76211508 RCV000077893 SNV ACADM 1 76215125 76215125 RCV000003773 SNV ACADM 1 76215129 76215129 RCV000003781; RCV000077894 SNV ACADM 1 76215194 76215194 RCV000003771 SNV ACADM 1 76215237 76215237 RCV000003779 SNV ACADM 1 76226846 76226846 RCV000003769; RCV000077895 SNV ACADM 1 76226963 76226966 RCV000003775; RCV000077877 deletion ACADM 1 76226985 76226985 RCV000003772 SNV ACADS 12 121164918 121164918 RCV000004029 SNV ACADS 12 121164946 121164946 RCV000023585 SNV ACADS 12 121174846 121174846 RCV000004036 SNV ACADS 12 121174852 121174852 RCV000004031 SNV ACADS 12 121174888 121174890 RCV000004037 deletion ACADS 12 121174897 121174897 RCV000004030 SNV ACADS 12 121174901 121174901 RCV000023587 SNV ACADS 12 121175678 121175678 RCV000004034 SNV ACADS 12 121175696 121175696 RCV000004032 SNV ACADS 12 121175742 121175742 RCV000004038 SNV ACADS 12 121176083 121176083 RCV000004035; RCV000077896 SNV ACADS 12 121176662 121176662 RCV000004039 SNV ACADS 12 121176944 121176944 RCV000023586 SNV ACADS 12 121176971 121176971 RCV000004040 SNV ACADS 12 121177150 121177150 RCV000004041 SNV ACADS 12 121177159 121177159 RCV000004033 SNV ACADSB 10 124800121 124800121 RCV000009780 SNV ACADSB 10 124802643 124802643 RCV000009778 SNV ACADSB 10 124812607 124812607 RCV000009781 SNV ACADSB 10 124812676 124812676 RCV000009777 SNV ACADVL 17 7123838 7123838 RCV000001698; RCV000020076; SNV RCV000077913 ACADVL 17 7123838 7123838 RCV000001698; RCV000020076; SNV RCV000077913 ACADVL 17 7124242 7124242 RCV000001692 deletion ACADVL 17 7124243 7124243 RCV000001691; RCV000077915 deletion ACADVL 17 7124288 7124290 RCV000001693 deletion ACADVL 17 7124899 7124899 RCV000077919 SNV ACADVL 17 7125387 7125387 RCV000001698 SNV ACADVL 17 7125522 7125522 RCV000020080 SNV ACADVL 17 7125591 7125591 RCV000020081; RCV000077925 SNV ACADVL 17 7126002 7126004 RCV000001694 deletion ACADVL 17 7126003 7126005 RCV000077926 deletion ACADVL 17 7126480 7126480 RCV000077900 SNV ACADVL 17 7126518 7126518 RCV000001695 SNV ACADVL 17 7127006 7127006 RCV000020069 SNV ACADVL 17 7127026 7127026 RCV000001700 SNV ACADVL 17 7127184 7127184 RCV000020072; RCV000077903 SNV ACADVL 17 7127303 7127303 RCV000001701 SNV ACADVL 17 7127326 7127326 RCV000001699 SNV ACADVL 17 7127343 7127343 RCV000077905 duplication ACADVL 17 7127359 7127359 RCV000020073 SNV ACADVL 17 7127360 7127360 RCV000077906 SNV ACADVL 17 7128285 7128285 RCV000001690 SNV ACAT1 11 107992335 107992335 RCV000002972 SNV ACAT1 11 108004987 108004987 RCV000002977 SNV ACAT1 11 108005967 108005967 RCV000002980 SNV ACAT1 11 108009661 108009661 RCV000077931 SNV ACAT1 11 108009736 108009736 RCV000002967 SNV ACAT1 11 108012415 108012415 RCV000002974; RCV000077933 SNV ACAT1 11 108013242 108013242 RCV000077934 deletion ACAT1 11 108013272 108013272 RCV000002978 SNV ACAT1 11 108014766 108014766 RCV000002979 SNV ACAT1 11 108016956 108016958 RCV000002975 deletion ACAT1 11 108017006 108017007 RCV000002976 duplication ACAT1 11 108017059 108017059 RCV000002973 SNV ACAT1 11 108017061 108017061 RCV000002966 SNV ADA 20 43249032 43249032 RCV000002036 SNV ADA 20 43249723 43249723 RCV000002035 SNV ADA 20 43249744 43249744 RCV000059115; RCV000002038 SNV ADA 20 43249762 43249762 RCV000002048 SNV ADA 20 43249762 43249762 RCV000029302 SNV ADA 20 43251253 43251253 RCV000059114; RCV000002041 SNV ADA 20 43251552 43251552 RCV000059113; RCV000002057 SNV ADA 20 43251680 43251680 RCV000002045 SNV ADA 20 43251683 43251683 RCV000059112; RCV000002044 SNV ADA 20 43251694 43251694 RCV000002034 SNV ADA 20 43251695 43251695 RCV000059111; RCV000002043 SNV ADA 20 43254221 43254221 RCV000059106; RCV000002061 SNV ADA 20 43254222 43254222 RCV000002047 SNV ADA 20 43254234 43254234 RCV000059105; RCV000002056 SNV ADA 20 43254242 43254242 RCV000059104; RCV000002040 SNV ADA 20 43255139 43255139 RCV000002042 SNV ADA 20 43255143 43255143 RCV000002058 SNV ADA 20 43255157 43255157 RCV000002033 SNV ADA 20 43255158 43255158 RCV000002032 SNV ADA 20 43255169 43255169 RCV000002058 SNV ADA 20 43255233 43255233 RCV000059096; RCV000002039 SNV ADA 20 43255238 43255238 RCV000002054 SNV ADA 20 43280227 43280227 RCV000002050 SNV AHCY 20 32880181 32880181 RCV000013819 SNV AHCY 20 32880273 32880273 RCV000013818 SNV ARG1 6 131894454 131894454 RCV000002494 SNV ASL 7 65546812 65546812 RCV000078010 SNV ASL 7 65547430 65547430 RCV000002499 SNV ASL 7 65547921 65547921 RCV000020416 SNV ASL 7 65551738 65551738 RCV000002503 SNV ASL 7 65551750 65551750 RCV000078012 SNV ASL 7 65554101 65554101 RCV000002500; RCV000078017 SNV ASL 7 65554680 65554680 RCV000020415; RCV000078007 SNV ASL 7 65557065 65557065 RCV000002504 SNV ASL 7 65557553 65557553 RCV000002502 SNV ASS1 9 133327655 133327655 RCV000006696 SNV ASS1 9 133327668 133327668 RCV000006703 SNV ASS1 9 133333869 133333869 RCV000006704 SNV ASS1 9 133333936 133333936 RCV000006706 SNV ASS1 9 133342161 133342161 RCV000006697 SNV ASS1 9 133346260 133346260 RCV000006707 SNV ASS1 9 133346264 133346264 RCV000006698 SNV ASS1 9 133355785 133355785 RCV000078024 SNV ASS1 9 133355791 133355791 RCV000078025 SNV ASS1 9 133355792 133355792 RCV000078026 SNV ASS1 9 133355833 133355833 RCV000006705 SNV ASS1 9 133355834 133355834 RCV000078027 SNV ASS1 9 133364791 133364791 RCV000006702 SNV ASS1 9 133364809 133364809 RCV000006711 SNV ASS1 9 133364851 133364851 RCV000006699 SNV ASS1 9 133370368 133370368 RCV000006708 SNV ASS1 9 133370370 133370370 RCV000006700 SNV ASS1 9 133374932 133374932 RCV000006701; RCV000078019 SNV AUH 9 93976659 93976659 RCV000022984 SNV AUH 9 94058308 94058308 RCV000022983 SNV AUH 9 94060275 94060275 RCV000009623 SNV AUH 9 94060305 94060305 RCV000022982 SNV BCKDHA 19 41903746 41903746 RCV000079231 deletion BCKDHA 19 41916550 41916550 RCV000079223 deletion BCKDHA 19 41916909 41916909 RCV000079238 SNV BCKDHA 19 41925187 41925187 RCV000079247 SNV BCKDHA 19 41928081 41928081 RCV000079249 SNV BCKDHA 19 41928162 41928163 RCV000079252 duplication BCKDHA 19 41928167 41928167 RCV000002479 SNV BCKDHA 19 41928183 41928183 RCV000079253 SNV BCKDHA 19 41928214 41928214 RCV000002481 SNV BCKDHA 19 41928215 41928215 RCV000002478 SNV BCKDHA 19 41928275 41928275 RCV000079257 SNV BCKDHA 19 41928548 41928548 RCV000002475; RCV000079258 SNV BCKDHA 19 41928585 41928585 RCV000079260 SNV BCKDHA 19 41928589 41928590 RCV000079261 deletion BCKDHA 19 41928597 41928597 RCV000079263 deletion BCKDHA 19 41928609 41928609 RCV000002480; RCV000079264 SNV BCKDHA 19 41928644 41928644 RCV000079265 SNV BCKDHA 19 41928659 41928659 RCV000079269 SNV BCKDHA 19 41928943 41928943 RCV000079216 SNV BCKDHA 19 41930401 41930401 RCV000002476; RCV000002477 SNV BCKDHA 19 41930409 41930409 RCV000079225 SNV BCKDHA 19 41930477 41930477 RCV000079227 SNV BCKDHA 19 41930485 41930486 RCV000079228 deletion BCKDHA 19 41930487 41930487 RCV000055825; RCV000002473; SNV RCV000079229 BCKDHA 19 41930489 41930489 RCV000079230 SNV BCKDHB 6 80816443 80816444 RCV000082736 deletion BCKDHB 6 80816503 80816513 RCV000082778 deletion BCKDHB 6 80838905 80838905 RCV000082734 SNV BCKDHB 6 80838945 80838945 RCV000082737 SNV BCKDHB 6 80877394 80877394 RCV000082738 SNV BCKDHB 6 80877407 80877407 RCV000012717 SNV BCKDHB 6 80878593 80878593 RCV000082741 SNV BCKDHB 6 80878602 80878602 RCV000082742 SNV BCKDHB 6 80878622 80878622 RCV000082746 SNV BCKDHB 6 80878622 80878622 RCV000082747 SNV BCKDHB 6 80878622 80878622 RCV000082748 SNV BCKDHB 6 80878623 80878623 RCV000082749 SNV BCKDHB 6 80878640 80878640 RCV000082751 SNV BCKDHB 6 80878661 80878661 RCV000082753 SNV BCKDHB 6 80878662 80878662 RCV000012715; RCV000056008; SNV RCV000082754 BCKDHB 6 80878706 80878707 RCV000082757 deletion BCKDHB 6 80878709 80878710 RCV000082758 deletion BCKDHB 6 80878730 80878730 RCV000012716 SNV BCKDHB 6 80910656 80910656 RCV000082766 SNV BCKDHB 6 80910660 80910660 RCV000082767 SNV BCKDHB 6 80910707 80910707 RCV000082768 SNV BCKDHB 6 80910740 80910740 RCV000056009; RCV000082770 SNV BCKDHB 6 80912831 80912831 RCV000082775 SNV BCKDHB 6 80912862 80912862 RCV000082776 deletion BCKDHB 6 80912880 80912880 RCV000082777 SNV BCKDHB 6 80982851 80982851 RCV000082780 SNV BCKDHB 6 80982870 80982870 RCV000082781 SNV BCKDHB 6 80982916 80982916 RCV000082720 SNV BCKDHB 6 81053388 81053388 RCV000082721 SNV BCKDHB 6 81053456 81053456 RCV000056007; RCV000082726 SNV BTD 3 15676984 15676990 RCV000001972; RCV000078084; indel RCV000021886 BTD 3 15676984 15676990 RCV000001972; RCV000078084; indel RCV000021886 BTD 3 15676986 15676986 RCV000001976 SNV BTD 3 15677014 15677014 RCV000022030 SNV BTD 3 15677019 15677019 RCV000021888; RCV000021890; SNV RCV000021889 BTD 3 15677019 15677019 RCV000021888; RCV000021890; SNV RCV000021889 BTD 3 15677019 15677019 RCV000021888; RCV000021890; SNV RCV000021889 BTD 3 15677022 15677022 RCV000021891 SNV BTD 3 15677045 15677045 RCV000021892 SNV BTD 3 15677046 15677046 RCV000021892 SNV BTD 3 15677057 15677057 RCV000021893 SNV BTD 3 15677070 15677070 RCV000021894 SNV BTD 3 15677070 15677070 RCV000021895 SNV BTD 3 15677076 15677076 RCV000021896 SNV BTD 3 15677078 15677078 RCV000032017 SNV BTD 3 15677080 15677080 RCV000021898 SNV BTD 3 15677098 15677098 RCV000021900; RCV000021886; SNV RCV000021901 BTD 3 15677098 15677098 RCV000021900; RCV000021886; SNV RCV000021901 BTD 3 15677098 15677098 RCV000021900; RCV000021886; SNV RCV000021901 BTD 3 15677121 15677121 RCV000001982; RCV000021903; SNV RCV000032009 BTD 3 15677121 15677121 RCV000001982; RCV000021903; SNV RCV000032009 BTD 3 15677121 15677121 RCV000001982; RCV000021903; SNV RCV000032009 BTD 3 15677122 15677122 RCV000021901; RCV000021905 SNV BTD 3 15677122 15677122 RCV000021901; RCV000021905 SNV BTD 3 15677131 15677131 RCV000021906 SNV BTD 3 15677131 15677131 RCV000021907 SNV BTD 3 15677132 15677140 RCV000021908 deletion BTD 3 15677134 15677134 RCV000021909 SNV BTD 3 15677148 15677148 RCV000021910 SNV BTD 3 15677164 15677164 RCV000021911 SNV BTD 3 15677169 15677169 RCV000021912 SNV BTD 3 15677184 15677184 RCV000021913 SNV BTD 3 15683415 15683415 RCV000021914 SNV BTD 3 15683431 15683431 RCV000021915 SNV BTD 3 15683439 15683439 RCV000021916 SNV BTD 3 15683439 15683439 RCV000021917 SNV BTD 3 15683446 15683446 RCV000021918 SNV BTD 3 15683461 15683461 RCV000021919 SNV BTD 3 15683469 15683469 RCV000021920 SNV BTD 3 15683487 15683487 RCV000021921 SNV BTD 3 15683498 15683498 RCV000021922 deletion BTD 3 15683512 15683513 RCV000032021 duplication BTD 3 15683529 15683529 RCV000021923 SNV BTD 3 15683548 15683548 RCV000021924 SNV BTD 3 15683550 15683550 RCV000021926 SNV BTD 3 15683559 15683559 RCV000021927 SNV BTD 3 15683564 15683564 RCV000021928 SNV BTD 3 15685829 15685829 RCV000021930 SNV BTD 3 15685832 15685832 RCV000021931 SNV BTD 3 15685833 15685833 RCV000021904; RCV000078072; SNV RCV000032009; RCV000021933 BTD 3 15685833 15685833 RCV000021904; RCV000078072; SNV RCV000032009; RCV000021933 BTD 3 15685833 15685833 RCV000021904; RCV000078072; SNV RCV000032009; RCV000021933 BTD 3 15685848 15685848 RCV000021934 SNV BTD 3 15685853 15685854 RCV000021935 deletion BTD 3 15685874 15685874 RCV000021936; RCV000031859; SNV RCV000078073 BTD 3 15685874 15685874 RCV000021936; RCV000031859; SNV RCV000078073 BTD 3 15685878 15685878 RCV000021937 SNV BTD 3 15685881 15685881 RCV000032018 SNV BTD 3 15685891 15685891 RCV000021938; RCV000078074 SNV BTD 3 15685907 15685907 RCV000021939 deletion BTD 3 15685920 15685920 RCV000021940; RCV000078075 SNV BTD 3 15685922 15685922 RCV000021941 SNV BTD 3 15685946 15685946 RCV000021942 SNV BTD 3 15685947 15685947 RCV000021943 SNV BTD 3 15685950 15685950 RCV000021944 SNV BTD 3 15685957 15685957 RCV000021946 deletion BTD 3 15685957 15685959 RCV000021945 deletion BTD 3 15685958 15685958 RCV000021947 SNV BTD 3 15685968 15685968 RCV000021948 SNV BTD 3 15685992 15685992 RCV000021949 SNV BTD 3 15685994 15685994 RCV000021950 SNV BTD 3 15685995 15685995 RCV000021951; RCV000078077 SNV BTD 3 15686004 15686004 RCV000021952; RCV000032022 SNV BTD 3 15686004 15686004 RCV000021952; RCV000032022 SNV BTD 3 15686006 15686006 RCV000021953 SNV BTD 3 15686015 15686015 RCV000021955 SNV BTD 3 15686017 15686017 RCV000021956 SNV BTD 3 15686027 15686027 RCV000022031 SNV BTD 3 15686045 15686045 RCV000021957 SNV BTD 3 15686072 15686072 RCV000021958 SNV BTD 3 15686097 15686097 RCV000021959 SNV BTD 3 15686106 15686106 RCV000021960 SNV BTD 3 15686118 15686118 RCV000001978 SNV BTD 3 15686120 15686120 RCV000021961 SNV BTD 3 15686121 15686121 RCV000032019 SNV BTD 3 15686127 15686127 RCV000021962 SNV BTD 3 15686157 15686157 RCV000021963; RCV000021964 SNV BTD 3 15686157 15686157 RCV000021963; RCV000021964 SNV BTD 3 15686177 15686177 RCV000021965 SNV BTD 3 15686195 15686195 RCV000021966 SNV BTD 3 15686196 15686196 RCV000021967 SNV BTD 3 15686199 15686199 RCV000021968 SNV BTD 3 15686228 15686228 RCV000021889 SNV BTD 3 15686243 15686243 RCV000021969 SNV BTD 3 15686250 15686250 RCV000021970 SNV BTD 3 15686259 15686259 RCV000021971 SNV BTD 3 15686292 15686292 RCV000021972 SNV BTD 3 15686295 15686295 RCV000021973 SNV BTD 3 15686296 15686296 RCV000021964; RCV000021975 SNV BTD 3 15686296 15686296 RCV000021964; RCV000021975 SNV BTD 3 15686296 15686296 RCV000021974; RCV000078082 deletion BTD 3 15686297 15686297 RCV000021976 SNV BTD 3 15686298 15686298 RCV000021977 SNV BTD 3 15686331 15686331 RCV000021978; RCV000078083 SNV BTD 3 15686364 15686364 RCV000022032 SNV BTD 3 15686409 15686409 RCV000022033 SNV BTD 3 15686412 15686412 RCV000021979 deletion BTD 3 15686415 15686415 RCV000021980 deletion BTD 3 15686459 15686459 RCV000021981 SNV BTD 3 15686469 15686469 RCV000021982 SNV BTD 3 15686520 15686520 RCV000021983 SNV BTD 3 15686521 15686521 RCV000032015 SNV BTD 3 15686534 15686534 RCV000021984; RCV000021985 SNV BTD 3 15686534 15686534 RCV000021984; RCV000021985 SNV BTD 3 15686554 15686555 RCV000021986 deletion BTD 3 15686568 15686568 RCV000021987 SNV BTD 3 15686570 15686570 RCV000001981 SNV BTD 3 15686574 15686574 RCV000021990 SNV BTD 3 15686577 15686577 RCV000021991 SNV BTD 3 15686590 15686604 RCV000021992 indel BTD 3 15686600 15686600 RCV000032020 SNV BTD 3 15686602 15686602 RCV000021993 deletion BTD 3 15686602 15686613 RCV000021988 deletion BTD 3 15686603 15686614 RCV000032016 deletion BTD 3 15686612 15686612 RCV000021995 SNV BTD 3 15686615 15686615 RCV000021994 SNV BTD 3 15686616 15686616 RCV000021996 SNV BTD 3 15686627 15686627 RCV000021997 duplication BTD 3 15686630 15686630 RCV000021998 SNV BTD 3 15686631 15686631 RCV000021999 SNV BTD 3 15686634 15686634 RCV000021890; RCV000022001 SNV BTD 3 15686634 15686634 RCV000021890; RCV000022001 SNV BTD 3 15686634 15686634 RCV000022000 SNV BTD 3 15686638 15686638 RCV000022002 SNV BTD 3 15686647 15686647 RCV000022003 SNV BTD 3 15686676 15686676 RCV000022004 SNV BTD 3 15686677 15686677 RCV000022005 SNV BTD 3 15686693 15686693 RCV000001977; RCV000078064; SNV RCV000001981; RCV000021912; RCV000021936; RCV000021952; RCV000021933 BTD 3 15686693 15686693 RCV000001977; RCV000078064; SNV RCV000001981; RCV000021912; RCV000021936; RCV000021952; RCV000021933 BTD 3 15686693 15686693 RCV000001977; RCV000078064; SNV RCV000001981; RCV000021912; RCV000021936; RCV000021952; RCV000021933 BTD 3 15686693 15686693 RCV000001977; RCV000078064; SNV RCV000001981; RCV000021912; RCV000021936; RCV000021952; RCV000021933 BTD 3 15686693 15686693 RCV000001977; RCV000078064; SNV RCV000001981; RCV000021912; RCV000021936; RCV000021952; RCV000021933 BTD 3 15686693 15686693 RCV000001977; RCV000078064; SNV RCV000001981; RCV000021912; RCV000021936; RCV000021952; RCV000021933 BTD 3 15686696 15686696 RCV000022006 SNV BTD 3 15686697 15686697 RCV000022007; RCV000021985 SNV BTD 3 15686697 15686697 RCV000022007; RCV000021985 SNV BTD 3 15686702 15686702 RCV000022008 SNV BTD 3 15686715 15686715 RCV000022009 SNV BTD 3 15686724 15686724 RCV000021903 SNV BTD 3 15686731 15686731 RCV000001979; RCV000078065 SNV BTD 3 15686732 15686732 RCV000022010 SNV BTD 3 15686747 15686747 RCV000022011 deletion BTD 3 15686751 15686751 RCV000022012 SNV BTD 3 15686757 15686757 RCV000032023 duplication BTD 3 15686795 15686795 RCV000022014 SNV BTD 3 15686795 15686795 RCV000022034 SNV BTD 3 15686818 15686818 RCV000022015 SNV BTD 3 15686822 15686822 RCV000022016 SNV BTD 3 15686822 15686822 RCV000022017 deletion BTD 3 15686826 15686826 RCV000022018 SNV BTD 3 15686829 15686829 RCV000001980 SNV BTD 3 15686852 15686852 RCV000022019; RCV000078068 SNV BTD 3 15686856 15686856 RCV000022020 duplication BTD 3 15686871 15686875 RCV000078069 deletion BTD 3 15686874 15686874 RCV000022021 SNV BTD 3 15686894 15686894 RCV000022022 SNV BTD 3 15686958 15686958 RCV000001974 SNV BTD 3 15686973 15686973 RCV000022023 SNV BTD 3 15686975 15686975 RCV000001975; RCV000078070 SNV BTD 3 15686976 15686976 RCV000022024 SNV BTD 3 15686979 15686979 RCV000022025 duplication BTD 3 15686982 15686982 RCV000022026 SNV BTD 3 15686990 15686990 RCV000022027 SNV BTD 3 15686992 15686992 RCV000022028; RCV000078071 SNV CBS 21 44474030 44474030 RCV000000146 SNV CBS 21 44478325 44478325 RCV000000153 SNV CBS 21 44478972 44478972 RCV000000149; RCV000000148; SNV RCV000078108 CBS 21 44479037 44479037 RCV000000152 SNV CBS 21 44479409 44479409 RCV000000145 SNV CBS 21 44480638 44480638 RCV000000154; RCV000078106 SNV CBS 21 44482454 44482454 RCV000078105 SNV CBS 21 44483098 44483098 RCV000000137; RCV000000138; SNV RCV000078112 CBS 21 44483184 44483184 RCV000000142; RCV000000141; SNV RCV000078111 CBS 21 44484041 44484041 RCV000000147 SNV CBS 21 44485591 44485591 RCV000000155 SNV CBS 21 44485755 44485755 RCV000000150 SNV CBS 21 44486370 44486370 RCV000000139 SNV CBS 21 44486374 44486374 RCV000000144 SNV CBS 21 44486389 44486389 RCV000000143 SNV CBS 21 44486463 44486463 RCV000000140 SNV CFTR 7 117120149 117120149 RCV000056356 SNV CFTR 7 117120162 117120162 RCV000029477 SNV CFTR 7 117120167 117120167 RCV000007657 SNV CFTR 7 117144306 117144417 RCV000007648 deletion CFTR 7 117144367 117144367 RCV000029470 SNV CFTR 7 117144368 117144368 RCV000056342 SNV CFTR 7 117144378 117144378 RCV000029472 SNV CFTR 7 117149087 117149196 RCV000007648 deletion CFTR 7 117149094 117149094 RCV000007599 SNV CFTR 7 117149101 117149101 RCV000056355 SNV CFTR 7 117149123 117149123 RCV000056357 SNV CFTR 7 117149146 117149146 RCV000056362 SNV CFTR 7 117149147 117149147 RCV000029494; RCV000119039; SNV RCV000078985; RCV000116686 CFTR 7 117149177 117149177 RCV000007563 SNV CFTR 7 117149185 117149186 RCV000007654 deletion CFTR 7 117149194 117149194 RCV000007603 SNV CFTR 7 117170952 117170952 RCV000056370 SNV CFTR 7 117170953 117170953 RCV000007601 SNV CFTR 7 117170953 117170953 RCV000007606 SNV CFTR 7 117170971 117170971 RCV000056375 SNV CFTR 7 117170992 117170992 RCV000046792 deletion CFTR 7 117171004 117171006 RCV000056378 indel CFTR 7 117171005 117171005 RCV000007618 SNV CFTR 7 117171007 117171007 RCV000007527; RCV000058930 SNV CFTR 7 117171028 117171028 RCV000056382 SNV CFTR 7 117171029 117171029 RCV000007528; RCV000007529; SNV RCV000078997 CFTR 7 117171034 117171034 RCV000029525 SNV CFTR 7 117171045 117171045 RCV000056385 SNV CFTR 7 117171049 117171049 RCV000029528 SNV CFTR 7 117171061 117171062 RCV000029532 duplication CFTR 7 117171103 117171103 RCV000007566 deletion CFTR 7 117171108 117171108 RCV000007567 deletion CFTR 7 117171121 117171121 RCV000056392 deletion CFTR 7 117171138 117171155 RCV000007608 deletion CFTR 7 117174371 117174371 RCV000056393 deletion CFTR 7 117174372 117174372 RCV000056394 SNV CFTR 7 117174417 117174417 RCV000047199; RCV000079010 SNV CFTR 7 117175301 117175301 RCV000056398 SNV CFTR 7 117175314 117175314 RCV000029542 SNV CFTR 7 117175317 117175317 RCV000056399 SNV CFTR 7 117175335 117175335 RCV000056400 SNV CFTR 7 117175339 117175339 RCV000007611; RCV000079011 SNV CFTR 7 117175372 117175372 RCV000007660 SNV CFTR 7 117175380 117175380 RCV000056401 SNV CFTR 7 117175442 117175463 RCV000007565 deletion CFTR 7 117176661 117176661 RCV000047258; RCV000079013 deletion CFTR 7 117176663 117176664 RCV000007627 deletion CFTR 7 117176683 117176683 RCV000029545 SNV CFTR 7 117176704 117176704 RCV000029546 SNV CFTR 7 117176711 117176711 RCV000029547 SNV CFTR 7 117176718 117176719 RCV000007658 duplication CFTR 7 117180217 117180217 RCV000007573 SNV CFTR 7 117180219 117180221 RCV000047290; RCV000079015 deletion CFTR 7 117180232 117180232 RCV000007583; RCV000079016 deletion CFTR 7 117180242 117180242 RCV000029549 SNV CFTR 7 117180272 117180272 RCV000056402 SNV CFTR 7 117180281 117180281 RCV000029550 SNV CFTR 7 117180284 117180284 RCV000007559 SNV CFTR 7 117180291 117180291 RCV000056337 SNV CFTR 7 117180297 117180297 RCV000007614 SNV CFTR 7 117180305 117180305 RCV000056338 SNV CFTR 7 117180306 117180307 RCV000007554 duplication CFTR 7 117180306 117180307 RCV000056339 insertion CFTR 7 117180324 117180324 RCV000007530 SNV CFTR 7 117180324 117180324 RCV000007591 SNV CFTR 7 117180324 117180324 RCV000007602; RCV000029468 SNV CFTR 7 117180330 117180330 RCV000007592 SNV CFTR 7 117180338 117180338 RCV000029469 SNV CFTR 7 117180339 117180339 RCV000007619 SNV CFTR 7 117180359 117180359 RCV000007589 SNV CFTR 7 117180363 117180363 RCV000007589 SNV CFTR 7 117180365 117180365 RCV000007555 deletion CFTR 7 117180367 117180367 RCV000007634 deletion CFTR 7 117180374 117180374 RCV000046229 SNV CFTR 7 117180377 117180378 RCV000007574 deletion CFTR 7 117182080 117182081 RCV000056341 duplication CFTR 7 117182155 117182155 RCV000056343 SNV CFTR 7 117182156 117182156 RCV000056344 SNV CFTR 7 117188812 117188812 RCV000046274; RCV000078976 SNV CFTR 7 117188814 117188815 RCV000056346 insertion CFTR 7 117188849 117188849 RCV000007531 SNV CFTR 7 117188852 117188852 RCV000029474 SNV CFTR 7 117188858 117188858 RCV000007552 SNV CFTR 7 117199517 117199517 RCV000056347 SNV CFTR 7 117199522 117199522 RCV000029475 SNV CFTR 7 117199522 117199522 RCV000056348 SNV CFTR 7 117199525 117199525 RCV000029476 SNV CFTR 7 117199563 117199563 RCV000007607; RCV000078977 SNV CFTR 7 117199591 117199591 RCV000056349 SNV CFTR 7 117199600 117199600 RCV000007575 SNV CFTR 7 117199602 117199602 RCV000007526 SNV CFTR 7 117199602 117199603 RCV000007640 deletion CFTR 7 117199644 117199646 RCV000007525 deletion CFTR 7 117199646 117199648 RCV000007523; RCV000007524; deletion RCV000119038; RCV000058929 CFTR 7 117199670 117199671 RCV000007560 deletion CFTR 7 117199683 117199683 RCV000007570 SNV CFTR 7 117199683 117199683 RCV000046339; RCV000029478; SNV RCV000078979 CFTR 7 117199697 117199697 RCV000007571 SNV CFTR 7 117227792 117227792 RCV000007532 SNV CFTR 7 117227809 117227809 RCV000007593 SNV CFTR 7 117227832 117227832 RCV000007535; RCV000119041; SNV RCV000058931 CFTR 7 117227853 117227853 RCV000043664; RCV000007538 SNV CFTR 7 117227853 117227853 RCV000043664; RCV000007538 SNV CFTR 7 117227854 117227854 RCV000007536 SNV CFTR 7 117227854 117227854 RCV000007537 SNV CFTR 7 117227855 117227855 RCV000056350; RCV000007538 SNV CFTR 7 117227855 117227855 RCV000056350; RCV000007538 SNV CFTR 7 117227859 117227859 RCV000007562 SNV CFTR 7 117227860 117227860 RCV000007540; RCV000119250; SNV RCV000119040 CFTR 7 117227862 117227862 RCV000007622 SNV CFTR 7 117227865 117227865 RCV000007542 SNV CFTR 7 117227866 117227866 RCV000007646 SNV CFTR 7 117227874 117227874 RCV000007617; RCV000029482 SNV CFTR 7 117227881 117227881 RCV000029483 SNV CFTR 7 117227883 117227883 RCV000007543 SNV CFTR 7 117227887 117227887 RCV000007533 SNV CFTR 7 117227887 117227887 RCV000007576 SNV CFTR 7 117230406 117230406 RCV000056352 SNV CFTR 7 117230409 117230409 RCV000007662 SNV CFTR 7 117230414 117230414 RCV000007534 SNV CFTR 7 117230419 117230419 RCV000029485 deletion CFTR 7 117230448 117230448 RCV000007539 SNV CFTR 7 117230454 117230454 RCV000029486; RCV000007585; SNV RCV000078981 CFTR 7 117230458 117230458 RCV000029487 SNV CFTR 7 117230480 117230480 RCV000056353 SNV CFTR 7 117232038 117232121 RCV000007568 deletion CFTR 7 117232074 117232074 RCV000046494; RCV000078982 SNV CFTR 7 117232086 117232086 RCV000046498; RCV000029488 SNV CFTR 7 117232132 117232132 RCV000029489 deletion CFTR 7 117232144 117232152 RCV000046508 indel CFTR 7 117232164 117232164 RCV000007623 SNV CFTR 7 117232233 117232233 RCV000029491 deletion CFTR 7 117232272 117232273 RCV000029492 indel CFTR 7 117232273 117232273 RCV000043563 deletion CFTR 7 117232273 117232274 RCV000029493 duplication CFTR 7 117232346 117232346 RCV000056359 SNV CFTR 7 117232349 117232349 RCV000007624 SNV CFTR 7 117232367 117232367 RCV000007594 SNV CFTR 7 117232396 117232397 RCV000007605 duplication CFTR 7 117232416 117232416 RCV000056360 SNV CFTR 7 117232436 117232436 RCV000056361 deletion CFTR 7 117232511 117232511 RCV000056363 SNV CFTR 7 117232512 117232512 RCV000007641 deletion CFTR 7 117232595 117232595 RCV000046585; RCV000029495 SNV CFTR 7 117232643 117232644 RCV000007541 duplication CFTR 7 117232674 117232674 RCV000056364 deletion CFTR 7 117232685 117232685 RCV000056365 SNV CFTR 7 117232700 117232700 RCV000007577 SNV CFTR 7 117234984 117234984 RCV000056366 SNV CFTR 7 117235030 117235030 RCV000056367 SNV CFTR 7 117235031 117235031 RCV000007547 SNV CFTR 7 117235044 117235044 RCV000007561 SNV CFTR 7 117235076 117235076 RCV000056368 deletion CFTR 7 117235090 117235090 RCV000029498 SNV CFTR 7 117243596 117243596 RCV000007625 SNV CFTR 7 117243663 117243663 RCV000007626; RCV000007661 SNV CFTR 7 117243663 117243663 RCV000007626; RCV000007661 SNV CFTR 7 117243665 117243666 RCV000007569 insertion CFTR 7 117243666 117243666 RCV000007548 SNV CFTR 7 117243696 117243696 RCV000029503 SNV CFTR 7 117243708 117243708 RCV000056371 SNV CFTR 7 117243738 117243739 RCV000029504 duplication CFTR 7 117243741 117243741 RCV000046693; RCV000029505 SNV CFTR 7 117243762 117243762 RCV000056372 SNV CFTR 7 117243773 117243773 RCV000007628 SNV CFTR 7 117243803 117243803 RCV000056373 deletion CFTR 7 117243836 117243836 RCV000056374 SNV CFTR 7 117246728 117246728 RCV000046718; RCV000029509 SNV CFTR 7 117246751 117246751 RCV000029510 SNV CFTR 7 117246807 117246807 RCV000029512; RCV000078990 SNV CFTR 7 117250572 117250572 RCV000056376 SNV CFTR 7 117250575 117250575 RCV000046745; RCV000007650; SNV RCV000007651; RCV000078991 CFTR 7 117250622 117250622 RCV000029514 SNV CFTR 7 117250651 117250656 RCV000046775; RCV000078992 deletion CFTR 7 117250679 117250679 RCV000029516 SNV CFTR 7 117251649 117251649 RCV000046799; RCV000029520 SNV CFTR 7 117251656 117251656 RCV000007642 deletion CFTR 7 117251683 117251684 RCV000007581 duplication CFTR 7 117251689 117251689 RCV000007629 SNV CFTR 7 117251691 117251691 RCV000007582 SNV CFTR 7 117251692 117251692 RCV000007578 SNV CFTR 7 117251694 117251694 RCV000007579; RCV000078995 SNV CFTR 7 117251704 117251704 RCV000046825; RCV000029521 SNV CFTR 7 117251707 117251707 RCV000007630 SNV CFTR 7 117251725 117251725 RCV000056377 SNV CFTR 7 117251749 117251749 RCV000007631 SNV CFTR 7 117251761 117251761 RCV000007615 SNV CFTR 7 117251771 117251771 RCV000056379; RCV000119251 SNV CFTR 7 117251771 117251771 RCV000056379; RCV000119251 SNV CFTR 7 117251771 117251771 RCV000056380; RCV000119251 SNV CFTR 7 117251771 117251771 RCV000056380; RCV000119251 SNV CFTR 7 117251797 117251797 RCV000032712; RCV000078996 SNV CFTR 7 117251805 117251805 RCV000056381 SNV CFTR 7 117254753 117254753 RCV000046895; RCV000029522 SNV CFTR 7 117267579 117267579 RCV000007564 SNV CFTR 7 117267591 117267591 RCV000007557 SNV CFTR 7 117267599 117267600 RCV000007652 duplication CFTR 7 117267635 117267635 RCV000029523 deletion CFTR 7 117267642 117267643 RCV000007643 insertion CFTR 7 117267694 117267694 RCV000056383 SNV CFTR 7 117267718 117267718 RCV000007633 SNV CFTR 7 117267719 117267719 RCV000056384; RCV000078999 SNV CFTR 7 117267766 117267766 RCV000007544 deletion CFTR 7 117267766 117267766 RCV000007635 SNV CFTR 7 117267798 117267798 RCV000029526 deletion CFTR 7 117267807 117267807 RCV000007636 SNV CFTR 7 117267819 117267819 RCV000007596 SNV CFTR 7 117282491 117282491 RCV000007597 SNV CFTR 7 117282505 117282505 RCV000007661; RCV000046963 SNV CFTR 7 117282505 117282505 RCV000007661; RCV000046963 SNV CFTR 7 117282505 117282505 RCV000056386 SNV CFTR 7 117282518 117282518 RCV000007653 deletion CFTR 7 117282520 117282520 RCV000007637 SNV CFTR 7 117282526 117282526 RCV000007638 SNV CFTR 7 117282537 117282537 RCV000007613 SNV CFTR 7 117282538 117282538 RCV000007545 SNV CFTR 7 117282542 117282542 RCV000007598 duplication CFTR 7 117282547 117282547 RCV000046980 duplication CFTR 7 117282582 117282582 RCV000046985; RCV000007584 SNV CFTR 7 117282620 117282620 RCV000007549 SNV CFTR 7 117282622 117282622 RCV000007587 SNV CFTR 7 117282631 117282631 RCV000007604 SNV CFTR 7 117282647 117282647 RCV000007572 SNV CFTR 7 117292900 117292903 RCV000007616 deletion CFTR 7 117292906 117292907 RCV000056388 duplication CFTR 7 117292915 117292915 RCV000029533 SNV CFTR 7 117292929 117292929 RCV000007639 SNV CFTR 7 117292931 117292931 RCV000007556 SNV CFTR 7 117292959 117292959 RCV000007600 SNV CFTR 7 117292969 117292969 RCV000007553 SNV CFTR 7 117292979 117292980 RCV000029534 insertion CFTR 7 117304775 117304775 RCV000029535 SNV CFTR 7 117304824 117304824 RCV000029536 SNV CFTR 7 117304834 117304834 RCV000007659 SNV CFTR 7 117304834 117304834 RCV000029537 SNV CFTR 7 117304855 117304858 RCV000056390 indel CFTR 7 117306970 117306970 RCV000056391 deletion CFTR 7 117306999 117306999 RCV000029541 SNV CFTR 7 117307083 117307083 RCV000047130; RCV000007644 SNV CFTR 7 117307145 117307145 RCV000047135; RCV000079008 SNV CPT1A 11 68527116 68527116 RCV000055865 SNV CPT1A 11 68527706 68527706 RCV000009638 SNV CPT1A 11 68527709 68527709 RCV000009636 SNV CPT1A 11 68527728 68527806 RCV000009635 deletion CPT1A 11 68529002 68529155 RCV000009635 deletion CPT1A 11 68529002 68529004 RCV000055864 deletion CPT1A 11 68530094 68530226 RCV000009635 deletion CPT1A 11 68540736 68540736 RCV000055862 SNV CPT1A 11 68540873 68540873 RCV000055861 deletion CPT1A 11 68542865 68542865 RCV000055859 SNV CPT1A 11 68542865 68542865 RCV000055860 SNV CPT1A 11 68542866 68542866 RCV000009633 SNV CPT1A 11 68548115 68548115 RCV000055858 SNV CPT1A 11 68548130 68548130 RCV000055857; RCV000079911 SNV CPT1A 11 68548141 68548141 RCV000055856 SNV CPT1A 11 68548173 68548173 RCV000055855 SNV CPT1A 11 68548205 68548205 RCV000009628 SNV CPT1A 11 68549252 68549252 RCV000055854 SNV CPT1A 11 68549350 68549350 RCV000009632 SNV CPT1A 11 68552367 68552367 RCV000009629 SNV CPT1A 11 68552377 68552377 RCV000055853 SNV CPT1A 11 68552419 68552419 RCV000055852 SNV CPT1A 11 68560802 68560802 RCV000009637 deletion CPT1A 11 68560804 68560804 RCV000055872 SNV CPT1A 11 68560809 68560809 RCV000055870 SNV CPT1A 11 68560838 68560838 RCV000055869 SNV CPT1A 11 68562328 68562328 RCV000055868; RCV000124597 SNV CPT1A 11 68571545 68571545 RCV000055867 SNV CPT1A 11 68575021 68575021 RCV000055866; RCV000079915 SNV CPT1A 11 68575090 68575090 RCV000009630; RCV000079914 SNV CPT1A 11 68579904 68579904 RCV000079913 SNV CPT1A 11 68579964 68579964 RCV000079912 SNV CPT1A 11 68582847 68582847 RCV000055874 SNV CPT2 1 53662764 53662764 RCV000009512; RCV000009511 SNV CPT2 1 53668099 53668099 RCV000009510; RCV000078121 SNV CPT2 1 53675705 53675705 RCV000009530 SNV CPT2 1 53675866 53675866 RCV000009516 SNV CPT2 1 53675984 53675984 RCV000009529 SNV CPT2 1 53676026 53676026 RCV000009526 SNV CPT2 1 53676494 53676494 RCV000009517; RCV000009518 SNV CPT2 1 53676584 53676585 RCV000009520 deletion CPT2 1 53676585 53676586 RCV000078117 deletion CPT2 1 53676688 53676688 RCV000009520 SNV CPT2 1 53676706 53676706 RCV000009528 SNV CPT2 1 53676853 53676853 RCV000009519 SNV CPT2 1 53678947 53678947 RCV000009513 SNV CPT2 1 53679173 53679173 RCV000009514; RCV000009515 SNV CPT2 1 53679181 53679181 RCV000009508; RCV000009509 SNV CYP21A2 6 32006291 32006291 RCV000012938 SNV CYP21A2 6 32006910 32006917 RCV000012946 deletion CYP21A2 6 32006939 32006939 RCV000012966 SNV CYP21A2 6 32007203 32007203 RCV000012933 SNV CYP21A2 6 32007584 32007584 RCV000012947 SNV CYP21A2 6 32007587 32007587 RCV000012947; RCV000055823 SNV CYP21A2 6 32007587 32007587 RCV000012947; RCV000055823 SNV CYP21A2 6 32007593 32007593 RCV000012947; RCV000055823 SNV CYP21A2 6 32007593 32007593 RCV000012947; RCV000055823 SNV CYP21A2 6 32007887 32007887 RCV000012935; RCV000012936; SNV RCV000012934 CYP21A2 6 32007887 32007887 RCV000055820 SNV CYP21A2 6 32007917 32007917 RCV000012940 SNV CYP21A2 6 32007963 32007964 RCV000012964 duplication CYP21A2 6 32007966 32007966 RCV000055821 duplication CYP21A2 6 32008198 32008198 RCV000012951 SNV CYP21A2 6 32008312 32008312 RCV000012937 SNV CYP21A2 6 32008543 32008543 RCV000012953 SNV CYP21A2 6 32008783 32008783 RCV000012943 SNV CYP21A2 6 32008874 32008875 RCV000012941 indel DBT 1 100661812 100661812 RCV000012725 SNV DBT 1 100661813 100661813 RCV000079937 SNV DBT 1 100661905 100661905 RCV000012730 SNV DBT 1 100661969 100661969 RCV000079933 SNV DBT 1 100672060 100672060 RCV000012727 SNV DBT 1 100680373 100680373 RCV000079960 SNV DBT 1 100680411 100680411 RCV000079959 SNV DBT 1 100680441 100680441 RCV000079958 SNV DBT 1 100680485 100680485 RCV000012721; RCV000079957 SNV DBT 1 100681641 100681641 RCV000079951 SNV DBT 1 100681730 100681730 RCV000012731 SNV DBT 1 100696362 100696362 RCV000079944 deletion DBT 1 100696377 100696383 RCV000079943 deletion DBT 1 100696428 100696428 RCV000012726 SNV DBT 1 100696447 100696450 RCV000079942 deletion DBT 1 100700992 100700992 RCV000079941 SNV DBT 1 100715325 100715325 RCV000079945 SNV DLD 7 107542204 107542204 RCV000033218 SNV DLD 7 107542785 107542785 RCV000012742 SNV DLD 7 107555951 107555951 RCV000012744 SNV DLD 7 107557752 107557752 RCV000012752 SNV DLD 7 107557794 107557794 RCV000012751 SNV DLD 7 107557849 107557849 RCV000012747 SNV DLD 7 107559516 107559516 RCV000033216 SNV DLD 7 107559524 107559524 RCV000033217 SNV DLD 7 107559543 107559543 RCV000012743 SNV DLD 7 107559657 107559657 RCV000012746 SNV DNAJC19 3 180702479 180702479 RCV000106304 deletion DUOX2 15 45396374 45396374 RCV000004279 SNV DUOX2 15 45398415 45398415 RCV000004278 SNV DUOX2 15 45401085 45401085 RCV000004277 SNV DUOX2 15 45402093 45402093 RCV000004280 SNV ETFA 15 76566772 76566772 RCV000002712 SNV ETFA 15 76578804 76578804 RCV000002711 SNV ETFA 15 76584777 76584777 RCV000002713 SNV ETFB 19 51850260 51850260 RCV000018200 SNV ETFB 19 51853639 51853639 RCV000018202 SNV ETFDH 4 159593610 159593610 RCV000012806 SNV ETFDH 4 159603421 159603421 RCV000012808 SNV ETFDH 4 159603551 159603551 RCV000012810 SNV ETFDH 4 159606289 159606289 RCV000012809 SNV ETFDH 4 159606289 159606289 RCV000024282 SNV ETFDH 4 159624588 159624588 RCV000024305 SNV ETFDH 4 159624692 159624692 RCV000081076 SNV ETFDH 4 159627422 159627422 RCV000081077 SNV ETFDH 4 159627503 159627503 RCV000024306 SNV ETFDH 4 159629648 159629648 RCV000081078 deletion FAH 15 80445443 80445443 RCV000012640 SNV FAH 15 80450512 80450512 RCV000020126 SNV FAH 15 80454624 80454624 RCV000012641 SNV FAH 15 80460394 80460394 RCV000078137 SNV FAH 15 80460605 80460605 RCV000012649; RCV000078138 SNV FAH 15 80464582 80464582 RCV000020128 SNV FAH 15 80465431 80465431 RCV000020129 SNV FAH 15 80465435 80465435 RCV000012648 SNV FAH 15 80465485 80465485 RCV000012650 SNV FAH 15 80472514 80472514 RCV000012644 SNV FAH 15 80472526 80472526 RCV000012643; RCV000020125 SNV FAH 15 80473390 80473390 RCV000012646 SNV FAH 15 80473411 80473411 RCV000012642 SNV FAH 15 80473462 80473462 RCV000012647 SNV GAA 17 78078692 78078692 RCV000078179 SNV GAA 17 78078910 78078910 RCV000004248; RCV000078181 deletion GAA 17 78081373 78081373 RCV000004250 SNV GAA 17 78081617 78081617 RCV000004251 SNV GAA 17 78081636 78081636 RCV000004240 SNV GAA 17 78081693 78081693 RCV000004236 SNV GAA 17 78084553 78084553 RCV000078159 SNV GAA 17 78084749 78084749 RCV000004237 SNV GAA 17 78084773 78084774 RCV000004241 indel GAA 17 78084822 78084822 RCV000004247 SNV GAA 17 78086713 78086713 RCV000004238 SNV GAA 17 78086721 78086721 RCV000004244; RCV000055768 SNV GAA 17 78086798 78086798 RCV000078163 SNV GAA 17 78087042 78087046 RCV000078166 duplication GAA 17 78087081 78087081 RCV000078167 SNV GAA 17 78087149 78087149 RCV000004239 SNV GAA 17 78091992 78092156 RCV000004246 deletion GAA 17 78092022 78092022 RCV000078174 SNV GAA 17 78092054 78092054 RCV000078175 deletion GAA 17 78092070 78092070 RCV000004249 SNV GAA 17 78092512 78092514 RCV000004243 deletion GALC 14 88407777 88407777 RCV000023589 SNV GALC 14 88411937 88411937 RCV000023588 SNV GALC 14 88429736 88429736 RCV000004022 SNV GALC 14 88431929 88431929 RCV000023594 SNV GALC 14 88434725 88434725 RCV000078208 SNV GALC 14 88434730 88434730 RCV000023593 SNV GALC 14 88452941 88452941 RCV000078200 SNV GALC 14 88459388 88459388 RCV000023595 SNV GALE 1 24122673 24122673 RCV000003866; RCV000078697 SNV GALE 1 24122692 24122692 RCV000003865 SNV GALE 1 24122724 24122724 RCV000020295 SNV GALE 1 24123212 24123212 RCV000003864; RCV000078693 SNV GALE 1 24123267 24123267 RCV000020294 SNV GALE 1 24123618 24123618 RCV000003860 SNV GALE 1 24124208 24124208 RCV000020293 SNV GALE 1 24124650 24124650 RCV000003863 SNV GALE 1 24124678 24124678 RCV000020292; RCV000003867 SNV GALE 1 24124689 24124689 RCV000003862 SNV GALE 1 24125397 24125397 RCV000003861 SNV GALK1 17 73754172 73754172 RCV000005985 SNV GALK1 17 73759113 73759113 RCV000005987 SNV GALK1 17 73760095 73760095 RCV000005983 SNV GALK1 17 73761124 73761124 RCV000005982 SNV GALK1 17 73761136 73761136 RCV000005984 SNV GALT 9 34646701 34646783 RCV000114378 deletion GALT 9 34646702 34646702 RCV000022038 SNV GALT 9 34646719 34646719 RCV000022039 deletion GALT 9 34646726 34646726 RCV000022040 SNV GALT 9 34646728 34646728 RCV000022041 SNV GALT 9 34646742 34646742 RCV000022043; RCV000078221 indel GALT 9 34646768 34646768 RCV000022044 SNV GALT 9 34646783 34646783 RCV000022046 SNV GALT 9 34646783 34646783 RCV000022047 SNV GALT 9 34646783 34646783 RCV000031852 SNV GALT 9 34647085 34647255 RCV000114378 deletion GALT 9 34647093 34647093 RCV000022048 SNV GALT 9 34647094 34647094 RCV000022049 SNV GALT 9 34647098 34647098 RCV000022050 SNV GALT 9 34647101 34647101 RCV000022051 SNV GALT 9 34647103 34647103 RCV000022052 SNV GALT 9 34647110 34647110 RCV000022053 SNV GALT 9 34647116 34647116 RCV000022054 SNV GALT 9 34647133 34647133 RCV000003795 SNV GALT 9 34647133 34647133 RCV000022056 SNV GALT 9 34647137 34647137 RCV000022059; RCV000078214 SNV GALT 9 34647137 34647141 RCV000022058 deletion GALT 9 34647155 34647155 RCV000022060 SNV GALT 9 34647155 34647155 RCV000022061 SNV GALT 9 34647163 34647163 RCV000022062 SNV GALT 9 34647166 34647166 RCV000022063 SNV GALT 9 34647200 34647200 RCV000022065 SNV GALT 9 34647200 34647200 RCV000022066 SNV GALT 9 34647202 34647202 RCV000022067; RCV000078215 SNV GALT 9 34647206 34647206 RCV000029806 SNV GALT 9 34647210 34647217 RCV000022068 deletion GALT 9 34647223 34647224 RCV000022069 insertion GALT 9 34647223 34647224 RCV000032010 deletion GALT 9 34647224 34647224 RCV000003799 SNV GALT 9 34647241 34647241 RCV000022070 SNV GALT 9 34647244 34647244 RCV000022071 SNV GALT 9 34647250 34647250 RCV000022072 SNV GALT 9 34647488 34647564 RCV000114378 deletion GALT 9 34647501 34647501 RCV000022074 SNV GALT 9 34647501 34647501 RCV000022075 SNV GALT 9 34647521 34647521 RCV000022076 SNV GALT 9 34647525 34647527 RCV000078216 deletion GALT 9 34647526 34647526 RCV000022077; RCV000078217 SNV GALT 9 34647528 34647528 RCV000022079 SNV GALT 9 34647528 34647528 RCV000022080; RCV000078219 SNV GALT 9 34647544 34647544 RCV000022083 SNV GALT 9 34647653 34647702 RCV000114378 deletion GALT 9 34647658 34647659 RCV000022087 duplication GALT 9 34647661 34647661 RCV000022088 SNV GALT 9 34647662 34647662 RCV000022089 SNV GALT 9 34647666 34647666 RCV000022090 SNV GALT 9 34647675 34647675 RCV000022091 SNV GALT 9 34647679 34647679 RCV000022092 SNV GALT 9 34647692 34647692 RCV000022078 SNV GALT 9 34647693 34647693 RCV000022093 SNV GALT 9 34647699 34647699 RCV000022094 SNV GALT 9 34647828 34647958 RCV000114378 deletion GALT 9 34647828 34647958 RCV000022096 deletion GALT 9 34647830 34647830 RCV000022098 SNV GALT 9 34647836 34647836 RCV000029808 SNV GALT 9 34647837 34647837 RCV000022099 SNV GALT 9 34647840 34647840 RCV000022100 SNV GALT 9 34647843 34647843 RCV000022101 SNV GALT 9 34647845 34647845 RCV000022102 SNV GALT 9 34647847 34647847 RCV000022103 SNV GALT 9 34647850 34647851 RCV000032011 duplication GALT 9 34647851 34647851 RCV000022104 deletion GALT 9 34647855 34647855 RCV000003802; RCV000078220 SNV GALT 9 34647855 34647855 RCV000022105 SNV GALT 9 34647861 34647861 RCV000022106 duplication GALT 9 34647864 34647864 RCV000022107 SNV GALT 9 34647867 34647867 RCV000022108 SNV GALT 9 34647875 34647875 RCV000022109 SNV GALT 9 34647876 34647876 RCV000003793; RCV000078222 SNV GALT 9 34647876 34647876 RCV000022110 SNV GALT 9 34647879 34647879 RCV000022111 SNV GALT 9 34647893 34647893 RCV000022112 SNV GALT 9 34647893 34647893 RCV000022113; RCV000078223 SNV GALT 9 34647894 34647894 RCV000022114 SNV GALT 9 34647899 34647899 RCV000022115 SNV GALT 9 34647903 34647903 RCV000022116 SNV GALT 9 34647911 34647911 RCV000022117 SNV GALT 9 34647911 34647911 RCV000022118 SNV GALT 9 34647913 34647913 RCV000022119 SNV GALT 9 34647933 34647933 RCV000022120 SNV GALT 9 34647941 34647941 RCV000022121 SNV GALT 9 34647947 34647947 RCV000022123 SNV GALT 9 34647950 34647950 RCV000022124 SNV GALT 9 34647953 34647953 RCV000022125 SNV GALT 9 34647956 34647956 RCV000022126 SNV GALT 9 34648111 34648168 RCV000114378 deletion GALT 9 34648111 34648168 RCV000022096 deletion GALT 9 34648111 34648111 RCV000078224 SNV GALT 9 34648113 34648113 RCV000022135 SNV GALT 9 34648113 34648113 RCV000022136 SNV GALT 9 34648116 34648116 RCV000003800; RCV000078225 SNV GALT 9 34648128 34648128 RCV000022139 SNV GALT 9 34648132 34648133 RCV000022140 insertion GALT 9 34648140 34648140 RCV000022141 SNV GALT 9 34648143 34648143 RCV000022142 SNV GALT 9 34648145 34648145 RCV000022143 SNV GALT 9 34648146 34648146 RCV000022144 SNV GALT 9 34648151 34648151 RCV000003803 SNV GALT 9 34648154 34648154 RCV000022145 SNV GALT 9 34648156 34648156 RCV000022146 SNV GALT 9 34648157 34648157 RCV000022147 SNV GALT 9 34648158 34648158 RCV000022148 SNV GALT 9 34648158 34648158 RCV000022149 SNV GALT 9 34648160 34648160 RCV000022150 SNV GALT 9 34648167 34648167 RCV000003798; RCV000078227 SNV GALT 9 34648330 34648453 RCV000114378 deletion GALT 9 34648330 34648453 RCV000022096 deletion GALT 9 34648331 34648344 RCV000078228 deletion GALT 9 34648334 34648334 RCV000078229 SNV GALT 9 34648340 34648340 RCV000022155 SNV GALT 9 34648341 34648341 RCV000022156 SNV GALT 9 34648346 34648346 RCV000003808 SNV GALT 9 34648350 34648350 RCV000022158; RCV000078230 SNV GALT 9 34648360 34648360 RCV000022159 SNV GALT 9 34648361 34648361 RCV000022160 SNV GALT 9 34648364 34648364 RCV000022161 deletion GALT 9 34648367 34648367 RCV000022162 SNV GALT 9 34648368 34648368 RCV000022163 SNV GALT 9 34648373 34648373 RCV000003806 SNV GALT 9 34648376 34648376 RCV000022164 SNV GALT 9 34648377 34648377 RCV000022165 SNV GALT 9 34648385 34648385 RCV000022166 SNV GALT 9 34648392 34648392 RCV000022167; RCV000078231 SNV GALT 9 34648392 34648392 RCV000022168 SNV GALT 9 34648400 34648400 RCV000022170; RCV000078232 SNV GALT 9 34648401 34648401 RCV000022171 SNV GALT 9 34648416 34648416 RCV000022172 SNV GALT 9 34648418 34648418 RCV000022173 deletion GALT 9 34648418 34648418 RCV000022175 SNV GALT 9 34648424 34648424 RCV000022176 SNV GALT 9 34648424 34648425 RCV000022177 duplication GALT 9 34648433 34648433 RCV000022178 SNV GALT 9 34648442 34648442 RCV000022179 SNV GALT 9 34648443 34648443 RCV000022180 SNV GALT 9 34648446 34648446 RCV000022181 SNV GALT 9 34648453 34648453 RCV000022182 SNV GALT 9 34648758 34648824 RCV000114378 deletion GALT 9 34648758 34648891 RCV000022096 deletion GALT 9 34648762 34648762 RCV000022184 SNV GALT 9 34648763 34648763 RCV000022185 SNV GALT 9 34648768 34648768 RCV000022186 SNV GALT 9 34648790 34648799 RCV000022188 deletion GALT 9 34648816 34648816 RCV000022189 SNV GALT 9 34648818 34648818 RCV000022190 SNV GALT 9 34648819 34648819 RCV000022191 SNV GALT 9 34648823 34648823 RCV000022192 SNV GALT 9 34648823 34648823 RCV000022193 SNV GALT 9 34648827 34648827 RCV000022195 SNV GALT 9 34648839 34648841 RCV000022198 deletion GALT 9 34648841 34648841 RCV000022199 SNV GALT 9 34648843 34648843 RCV000078235 SNV GALT 9 34648848 34648848 RCV000022202 SNV GALT 9 34648849 34648860 RCV000022203 deletion GALT 9 34648856 34648856 RCV000022204 SNV GALT 9 34648861 34648863 RCV000022205; RCV000078236 indel GALT 9 34648864 34648864 RCV000022206 SNV GALT 9 34648883 34648883 RCV000022208 SNV GALT 9 34648885 34648885 RCV000022209 SNV GALT 9 34648886 34648886 RCV000022210 SNV GALT 9 34648994 34649078 RCV000022096 deletion GALT 9 34648994 34649078 RCV000114378 indel GALT 9 34648998 34648998 RCV000022215 deletion GALT 9 34649007 34649007 RCV000022216 SNV GALT 9 34649010 34649010 RCV000022217 SNV GALT 9 34649018 34649018 RCV000022218; RCV000078238 SNV GALT 9 34649028 34649028 RCV000022220 SNV GALT 9 34649029 34649029 RCV000003805; RCV000078239 SNV GALT 9 34649031 34649031 RCV000022219 SNV GALT 9 34649039 34649039 RCV000022221 SNV GALT 9 34649040 34649040 RCV000022222 SNV GALT 9 34649045 34649045 RCV000022223 SNV GALT 9 34649046 34649046 RCV000022224 SNV GALT 9 34649055 34649055 RCV000022226 SNV GALT 9 34649056 34649056 RCV000022227 deletion GALT 9 34649057 34649057 RCV000022228 SNV GALT 9 34649406 34649561 RCV000022096 deletion GALT 9 34649406 34649561 RCV000114378 indel GALT 9 34649422 34649422 RCV000022231 SNV GALT 9 34649424 34649424 RCV000022232 SNV GALT 9 34649442 34649442 RCV000022233; RCV000003797; SNV RCV000078243; RCV000003804; RCV000128642 GALT 9 34649442 34649442 RCV000022233; RCV000003797; SNV RCV000078243; RCV000003804; RCV000128642 GALT 9 34649442 34649442 RCV000022233; RCV000003797; SNV RCV000078243; RCV000003804; RCV000128642 GALT 9 34649449 34649449 RCV000022235 SNV GALT 9 34649450 34649450 RCV000022236 SNV GALT 9 34649451 34649451 RCV000022237 deletion GALT 9 34649452 34649452 RCV000022238 SNV GALT 9 34649453 34649453 RCV000022239 SNV GALT 9 34649454 34649454 RCV000022240 deletion GALT 9 34649459 34649459 RCV000003801 SNV GALT 9 34649460 34649460 RCV000022243 SNV GALT 9 34649461 34649461 RCV000032024 SNV GALT 9 34649463 34649463 RCV000022244 SNV GALT 9 34649469 34649469 RCV000022245 SNV GALT 9 34649469 34649469 RCV000022246 SNV GALT 9 34649470 34649470 RCV000022247 SNV GALT 9 34649472 34649472 RCV000022248 SNV GALT 9 34649476 34649476 RCV000022250 SNV GALT 9 34649478 34649478 RCV000022251 deletion GALT 9 34649481 34649481 RCV000022252 deletion GALT 9 34649482 34649482 RCV000022253 SNV GALT 9 34649485 34649485 RCV000022254 SNV GALT 9 34649487 34649487 RCV000078245 SNV GALT 9 34649488 34649488 RCV000022255 SNV GALT 9 34649491 34649491 RCV000022256 SNV GALT 9 34649499 34649499 RCV000003794; RCV000078246 SNV GALT 9 34649499 34649499 RCV000003807 SNV GALT 9 34649500 34649500 RCV000022258 SNV GALT 9 34649500 34649500 RCV000022259 SNV GALT 9 34649503 34649503 RCV000022260 SNV GALT 9 34649508 34649508 RCV000022261 SNV GALT 9 34649520 34649520 RCV000022263 SNV GALT 9 34649520 34649520 RCV000022264 SNV GALT 9 34649526 34649526 RCV000022265 SNV GALT 9 34649532 34649532 RCV000022266; RCV000078213 SNV GALT 9 34649536 34649536 RCV000022267 SNV GALT 9 34649549 34649549 RCV000022268 deletion GALT 9 34649550 34649550 RCV000022269 SNV GALT 9 34649553 34649553 RCV000022270 deletion GALT 9 34649559 34649559 RCV000022271 SNV GALT 9 34650365 34650446 RCV000114378 indel GALT 9 34650365 34650365 RCV000022275 SNV GALT 9 34650378 34650378 RCV000022276 deletion GALT 9 34650384 34650389 RCV000022277 indel GALT 9 34650404 34650404 RCV000022278 SNV GALT 9 34650414 34650414 RCV000022279 SNV GALT 9 34650438 34650438 RCV000022280 SNV GALT 9 34650444 34650444 RCV000022281 SNV GALT 9 34650446 34650446 RCV000022282 SNV GBA 1 155204793 155204793 RCV000004553; RCV000020153 SNV GBA 1 155204848 155204848 RCV000004552 SNV GBA 1 155204986 155204986 RCV000020152 SNV GBA 1 155204987 155204987 RCV000004531; RCV000004529; SNV RCV000004530; RCV000004528; RCV000020151; RCV000079343 GBA 1 155204994 155204994 RCV000004534; RCV000004535; SNV RCV000004533; RCV000004536; RCV000079342 GBA 1 155204994 155204994 RCV000004534; RCV000004535; SNV RCV000004533; RCV000004536; RCV000079342 GBA 1 155205008 155205008 RCV000004534; RCV000004535; SNV RCV000004533; RCV000004536; RCV000079341 GBA 1 155205008 155205008 RCV000004534; RCV000004535; SNV RCV000004533; RCV000004536; RCV000079341 GBA 1 155205043 155205043 RCV000004512; RCV000004509; SNV RCV000004510; RCV000004511; RCV000004513; RCV000020150; RCV000079339; RCV000004534; RCV000004535; RCV000004533; RCV000004536 GBA 1 155205043 155205043 RCV000004512; RCV000004509; SNV RCV000004510; RCV000004511; RCV000004513; RCV000020150; RCV000079339; RCV000004534; RCV000004535; RCV000004533; RCV000004536 GBA 1 155205043 155205043 RCV000079340 SNV GBA 1 155205499 155205499 RCV000004514 SNV GBA 1 155205517 155205517 RCV000004527; RCV000020149 SNV GBA 1 155205518 155205518 RCV000004524; RCV000004525; SNV RCV000004523; RCV000004526; RCV000004522; RCV000055773; RCV000079338; RCV000004580; RCV000004581 GBA 1 155205518 155205518 RCV000004524; RCV000004525; SNV RCV000004523; RCV000004526; RCV000004522; RCV000055773; RCV000079338; RCV000004580; RCV000004581 GBA 1 155205541 155205541 RCV000004568 SNV GBA 1 155205543 155205597 RCV000004554; RCV000004555; deletion RCV000020147 GBA 1 155205551 155205551 RCV000004544 SNV GBA 1 155205563 155205563 RCV000004520; RCV000004521; SNV RCV000020148 GBA 1 155205614 155205614 RCV000004572; RCV000004571; SNV RCV000055772 GBA 1 155205620 155205620 RCV000079337 SNV GBA 1 155205632 155205632 RCV000004578 SNV GBA 1 155205634 155205634 RCV000004516; RCV000004515; SNV RCV000004517; RCV000079336 GBA 1 155205634 155205634 RCV000020146 SNV GBA 1 155206052 155206052 RCV000004564 SNV GBA 1 155206068 155206068 RCV000004570; RCV000079334 SNV GBA 1 155206086 155206086 RCV000004567 SNV GBA 1 155206089 155206089 RCV000079333 SNV GBA 1 155206119 155206119 RCV000004563 SNV GBA 1 155206167 155206167 RCV000004538 SNV GBA 1 155206170 155206170 RCV000004562; RCV000079331 SNV GBA 1 155206175 155206175 RCV000041967; RCV000004548 SNV GBA 1 155206200 155206200 RCV000079330 SNV GBA 1 155206207 155206207 RCV000004561 SNV GBA 1 155206211 155206211 RCV000004569 SNV GBA 1 155206217 155206217 RCV000004560 SNV GBA 1 155207148 155207148 RCV000004547 SNV GBA 1 155207244 155207244 RCV000004573; RCV000020159; SNV RCV000079357 GBA 1 155207249 155207249 RCV000004580; RCV000004581 SNV GBA 1 155207261 155207261 RCV000004577 SNV GBA 1 155207367 155207367 RCV000004537 SNV GBA 1 155207368 155207368 RCV000004559 SNV GBA 1 155207932 155207932 RCV000004541; RCV000004540; SNV RCV000004542; RCV000020158; RCV000079355 GBA 1 155207935 155207935 RCV000004551 SNV GBA 1 155207965 155207965 RCV000079354 SNV GBA 1 155207983 155207983 RCV000020157 SNV GBA 1 155208006 155208006 RCV000004558; RCV000004557; SNV RCV000020156; RCV000079352 GBA 1 155208019 155208019 RCV000079351 SNV GBA 1 155208061 155208061 RCV000079349 SNV GBA 1 155208310 155208310 RCV000004539 SNV GBA 1 155208361 155208361 RCV000004538 SNV GBA 1 155208364 155208364 RCV000004566 deletion GBA 1 155208387 155208387 RCV000004574; RCV000020155 SNV GBA 1 155208388 155208388 RCV000079347 SNV GBA 1 155208409 155208409 RCV000079346 deletion GBA 1 155208415 155208415 RCV000004550 SNV GBA 1 155208420 155208420 RCV000004518; RCV000004519; SNV RCV000020154 GBA 1 155208421 155208421 RCV000055774; RCV000079345 SNV GBA 1 155209507 155209507 RCV000004576; RCV000004575; SNV RCV000079344 GBA 1 155209725 155209725 RCV000004565 SNV GBA 1 155209730 155209730 RCV000004532 SNV GBA 1 155209824 155209824 RCV000004556 SNV GBA 1 155210420 155210420 RCV000079332 SNV GBA 1 155210442 155210443 RCV000020160 insertion GBA 1 155210451 155210452 RCV000004543; RCV000079356 duplication GBA 1 155210464 155210464 RCV000004549 deletion GBA 1 155210876 155210876 RCV000004546; RCV000004545; SNV RCV000032094 GCDH 19 13006842 13006842 RCV000078254 SNV GCDH 19 13007018 13007018 RCV000078255 SNV GCDH 19 13007063 13007063 RCV000002170; RCV000078256 SNV GCDH 19 13007748 13007748 RCV000002164 SNV GCDH 19 13007754 13007754 RCV000002162 SNV GCDH 19 13008527 13008527 RCV000002167 SNV GCDH 19 13008632 13008632 RCV000002169 SNV GCDH 19 13008638 13008638 RCV000002166 SNV GCH1 14 55310741 55310741 RCV000009869 SNV GCH1 14 55310817 55310817 RCV000009865; RCV000009866 SNV GCH1 14 55310826 55310826 RCV000009863 SNV GCH1 14 55310855 55310855 RCV000009870 SNV GCH1 14 55312510 55312510 RCV000009856 SNV GCH1 14 55312517 55312517 RCV000009875 SNV GCH1 14 55312526 55312526 RCV000009867 SNV GCH1 14 55312561 55312561 RCV000009873 SNV GCH1 14 55332067 55332067 RCV000009860 SNV GCH1 14 55332094 55332094 RCV000009868 SNV GCH1 14 55332097 55332097 RCV000009854 SNV GCH1 14 55369059 55369059 RCV000009864 SNV GCH1 14 55369120 55369120 RCV000009853 SNV GCH1 14 55369240 55369240 RCV000009871 SNV GCH1 14 55369379 55369379 RCV000009859 SNV GJB2 13 20763104 20763104 RCV000037868 SNV GJB2 13 20763116 20763116 RCV000018544 SNV GJB2 13 20763121 20763129 RCV000037866 indel GJB2 13 20763170 20763170 RCV000018531; RCV000037863 SNV GJB2 13 20763170 20763170 RCV000022511 SNV GJB2 13 20763186 20763186 RCV000018553 SNV GJB2 13 20763234 20763234 RCV000020573; RCV000037860 SNV GJB2 13 20763245 20763245 RCV000018551 SNV GJB2 13 20763265 20763265 RCV000037856 SNV GJB2 13 20763293 20763293 RCV000018543; RCV000018542 SNV GJB2 13 20763294 20763294 RCV000018533; RCV000037853 SNV GJB2 13 20763295 20763295 RCV000037852 SNV GJB2 13 20763305 20763305 RCV000037851 SNV GJB2 13 20763351 20763351 RCV000037846 SNV GJB2 13 20763356 20763356 RCV000037844; RCV000080375 SNV GJB2 13 20763361 20763363 RCV000018530; RCV000037841 deletion GJB2 13 20763382 20763382 RCV000020568 SNV GJB2 13 20763395 20763408 RCV000037836; RCV000080370 deletion GJB2 13 20763421 20763422 RCV000037835 deletion GJB2 13 20763438 20763438 RCV000037834 SNV GJB2 13 20763452 20763452 RCV000018541; RCV000037831; SNV RCV000080369 GJB2 13 20763471 20763471 RCV000018560; RCV000037830 SNV GJB2 13 20763471 20763471 RCV000018564; RCV000037829 SNV GJB2 13 20763486 20763486 RCV000018539; RCV000018538; deletion RCV000037827 GJB2 13 20763490 20763490 RCV000018524; RCV000037826 SNV GJB2 13 20763492 20763492 RCV000018526; RCV000037825; SNV RCV000080368 GJB2 13 20763497 20763497 RCV000018554; RCV000018555 SNV GJB2 13 20763498 20763498 RCV000018535; RCV000037823 SNV GJB2 13 20763503 20763503 RCV000018565 SNV GJB2 13 20763525 20763525 RCV000018536 SNV GJB2 13 20763527 20763527 RCV000037819 SNV GJB2 13 20763545 20763545 RCV000018540 SNV GJB2 13 20763546 20763546 RCV000018562 SNV GJB2 13 20763552 20763552 RCV000037815 SNV GJB2 13 20763554 20763554 RCV000018534; RCV000037817 deletion GJB2 13 20763559 20763559 RCV000018558 SNV GJB2 13 20763573 20763573 RCV000018546; RCV000018547 SNV GJB2 13 20763573 20763573 RCV000018556 SNV GJB2 13 20763582 20763582 RCV000018529; RCV000037816; SNV RCV000080366 GJB2 13 20763587 20763587 RCV000022510; RCV000018561 SNV GJB2 13 20763589 20763589 RCV000018545 SNV GJB2 13 20763590 20763590 RCV000018559 SNV GJB2 13 20763602 20763602 RCV000037813 SNV GJB2 13 20763612 20763612 RCV000018550; RCV000037812; SNV RCV000080365 GJB2 13 20763620 20763620 RCV000018523; RCV000037810; SNV RCV000080364 GJB2 13 20763620 20763620 RCV000037811 SNV GJB2 13 20763626 20763626 RCV000037873 SNV GJB2 13 20763650 20763650 RCV000018525; RCV000037871 SNV GJB2 13 20763653 20763690 RCV000037837 deletion GJB2 13 20763665 20763665 RCV000020575 SNV GJB2 13 20763671 20763671 RCV000018549 SNV GJB2 13 20763677 20763677 RCV000037855 SNV GJB2 13 20763686 20763686 RCV000018528; RCV000018527; deletion RCV000037843; RCV000080373 GJB2 13 20763686 20763686 RCV000020570; RCV000037842; SNV RCV000080372 GJB2 13 20763686 20763686 RCV000080374 duplication GJB2 13 20763687 20763687 RCV000018548 SNV GJB2 13 20763687 20763687 RCV000037839; RCV000080371 SNV GJB2 13 20763702 20763702 RCV000037820 SNV GJB2 13 20763712 20763712 RCV000037874 SNV GJB2 13 20763720 20763720 RCV000037821 SNV GJB3 1 35250397 35250397 RCV000006855 SNV GJB3 1 35250398 35250398 RCV000006856 SNV GJB3 1 35250464 35250464 RCV000006864 SNV GJB3 1 35250488 35250488 RCV000006862 SNV GJB3 1 35250619 35250619 RCV000006857 SNV GJB3 1 35250784 35250784 RCV000006861 SNV GJB3 1 35250860 35250860 RCV000006865 SNV GJB3 1 35250901 35250901 RCV000006859 SNV GJB3 1 35250910 35250910 RCV000006858 SNV GJB3 1 35250943 35250943 RCV000006866 SNV GJB6 13 20796931 20796931 RCV000081460 duplication GJB6 13 20797357 20797357 RCV000005883 SNV GJB6 13 20797359 20797359 RCV000081458 duplication GJB6 13 20797510 20797510 RCV000005886 SNV GJB6 13 20797559 20797560 RCV000038711 indel GJB6 13 20797589 20797589 RCV000005882 SNV GJB6 13 20797606 20797606 RCV000088666 SNV GNMT 6 42928654 42928654 RCV000004386 SNV GNMT 6 42930887 42930887 RCV000004387 SNV HADH 4 108911206 108911206 RCV000008482 SNV HADH 4 108930953 108930953 RCV000008483 SNV HADH 4 108948913 108948913 RCV000032678 SNV HADH 4 108954395 108954395 RCV000008484 SNV HADHA 2 26417450 26417450 RCV000009271 SNV HADHA 2 26418053 26418053 RCV000009266; RCV000009267 SNV HADHA 2 26427019 26427019 RCV000009268 SNV HADHA 2 26432709 26432709 RCV000009273 SNV HADHA 2 26437316 26437316 RCV000009275 SNV HADHA 2 26437359 26437359 RCV000009276 SNV HADHA 2 26437385 26437385 RCV000009274 SNV HADHA 2 26461825 26461825 RCV000078334 SNV HADHB 2 26486320 26486320 RCV000015970 SNV HADHB 2 26502112 26502112 RCV000015971 SNV HADHB 2 26502160 26502160 RCV000015969 SNV HADHB 2 26508381 26508381 RCV000015972 SNV HADHB 2 26508414 26508414 RCV000015974 SNV HBA2 16 222912 222912 RCV000016985 deletion HBA2 16 222913 222913 RCV000016929 SNV HBA2 16 222980 222980 RCV000016977 SNV HBA2 16 222981 222981 RCV000016976 SNV HBA2 16 223206 223206 RCV000016979; RCV000016978 SNV HBA2 16 223214 223214 RCV000016900; RCV000016901 SNV HBA2 16 223519 223519 RCV000016930 SNV HBB 11 5246884 5246887 RCV000016876 indel HBB 11 5246887 5246889 RCV000016665 deletion HBB 11 5246889 5246889 RCV000016664; RCV000022611 SNV HBB 11 5246908 5246908 RCV000016658 SNV HBB 11 5246928 5246928 RCV000016775; RCV000016776; SNV RCV000016777 HBB 11 5246928 5246929 RCV000016681; RCV000016682 indel HBB 11 5246935 5246935 RCV000016393; RCV000016392 SNV HBB 11 5246940 5246940 RCV000016599; RCV000016598; SNV RCV000016600; RCV000029991 HBB 11 5246944 5246944 RCV000016687 deletion HBB 11 5246950 5246951 RCV000016668 duplication HBB 11 5246952 5246952 RCV000016747; RCV000016746 SNV HBB 11 5247806 5247806 RCV000016696; RCV000020332 SNV HBB 11 5247807 5247807 RCV000016789 SNV HBB 11 5247827 5247827 RCV000016446; RCV000016443; SNV RCV000016444; RCV000016445; RCV000016807 HBB 11 5247827 5247827 RCV000016446; RCV000016443; SNV RCV000016444; RCV000016445; RCV000016807 HBB 11 5247835 5247835 RCV000029975 duplication HBB 11 5247838 5247839 RCV000016685 insertion HBB 11 5247851 5247851 RCV000016766 SNV HBB 11 5247871 5247871 RCV000029971 deletion HBB 11 5247875 5247875 RCV000016827 SNV HBB 11 5247892 5247892 RCV000016676 deletion HBB 11 5247901 5247905 RCV000016826 indel HBB 11 5247905 5247906 RCV000016675 duplication HBB 11 5247905 5247906 RCV000016690 duplication HBB 11 5247914 5247914 RCV000016301; RCV000029969 SNV HBB 11 5247918 5247919 RCV000029968 deletion HBB 11 5247921 5247921 RCV000029967 deletion HBB 11 5247929 5247929 RCV000016686 deletion HBB 11 5247938 5247938 RCV000016661 SNV HBB 11 5247940 5247940 RCV000016666; RCV000016667 SNV HBB 11 5247978 5247979 RCV000016744 duplication HBB 11 5247987 5247987 RCV000016671; RCV000020329 deletion HBB 11 5247992 5247992 RCV000016660 SNV HBB 11 5247993 5247995 RCV000016277; RCV000016278 deletion HBB 11 5247993 5247996 RCV000016673; RCV000020328 deletion HBB 11 5247994 5247994 RCV000016373; RCV000016371; SNV RCV000016372 HBB 11 5248002 5248008 RCV000016684 deletion HBB 11 5248004 5248004 RCV000016656; RCV000020327 SNV HBB 11 5248005 5248006 RCV000016817 deletion HBB 11 5248008 5248008 RCV000016659 SNV HBB 11 5248010 5248010 RCV000016688 deletion HBB 11 5248014 5248014 RCV000016662 SNV HBB 11 5248014 5248014 RCV000016680 deletion HBB 11 5248027 5248029 RCV000016700; RCV000030009 deletion HBB 11 5248028 5248029 RCV000016821 insertion HBB 11 5248029 5248029 RCV000016620; RCV000016619 SNV HBB 11 5248159 5248176 RCV000016703 deletion HBB 11 5248159 5248159 RCV000016694; RCV000020340 SNV HBB 11 5248159 5248159 RCV000016695 SNV HBB 11 5248160 5248160 RCV000016432; RCV000030007 SNV HBB 11 5248160 5248160 RCV000030006 SNV HBB 11 5248166 5248166 RCV000016612; RCV000016611 SNV HBB 11 5248167 5248168 RCV000016689 duplication HBB 11 5248170 5248170 RCV000016440; RCV000016439; SNV RCV000016441 HBB 11 5248172 5248172 RCV000016820 SNV HBB 11 5248172 5248173 RCV000016829 insertion HBB 11 5248173 5248173 RCV000016332; RCV000016330; SNV RCV000016329; RCV000016331; RCV000016617 HBB 11 5248173 5248173 RCV000016332; RCV000016330; SNV RCV000016329; RCV000016331; RCV000016617 HBB 11 5248177 5248177 RCV000016717; RCV000030002; SNV RCV000020339 HBB 11 5248193 5248193 RCV000016480; RCV000016479; SNV RCV000016481; RCV000020338 HBB 11 5248200 5248200 RCV000016655; RCV000020337 SNV HBB 11 5248201 5248201 RCV000016670 deletion HBB 11 5248205 5248205 RCV000016657 SNV HBB 11 5248206 5248207 RCV000016683 duplication HBB 11 5248216 5248216 RCV000016679 deletion HBB 11 5248224 5248225 RCV000016672; RCV000029974; duplication RCV000020331 HBB 11 5248226 5248227 RCV000016669; RCV000029972; deletion RCV000020330 HBB 11 5248232 5248232 RCV000016286; RCV000030905; SNV RCV000016575; RCV000016573; RCV000016574; RCV000016576; RCV000016577; RCV000016579; RCV000016580; RCV000016877; RCV000016879 HBB 11 5248232 5248232 RCV000016286; RCV000030905; SNV RCV000016575; RCV000016573; RCV000016574; RCV000016576; RCV000016577; RCV000016579; RCV000016580; RCV000016877; RCV000016879 HBB 11 5248232 5248232 RCV000016286; RCV000030905; SNV RCV000016575; RCV000016573; RCV000016574; RCV000016576; RCV000016577; RCV000016579; RCV000016580; RCV000016877; RCV000016879 HBB 11 5248232 5248232 RCV000016286; RCV000030905; SNV RCV000016575; RCV000016573; RCV000016574; RCV000016576; RCV000016577; RCV000016579; RCV000016580; RCV000016877; RCV000016879 HBB 11 5248232 5248232 RCV000016286; RCV000030905; SNV RCV000016575; RCV000016573; RCV000016574; RCV000016576; RCV000016577; RCV000016579; RCV000016580; RCV000016877; RCV000016879 HBB 11 5248232 5248232 RCV000016286; RCV000030905; SNV RCV000016575; RCV000016573; RCV000016574; RCV000016576; RCV000016577; RCV000016579; RCV000016580; RCV000016877; RCV000016879 HBB 11 5248232 5248232 RCV000016286; RCV000030905; SNV RCV000016575; RCV000016573; RCV000016574; RCV000016576; RCV000016577; RCV000016579; RCV000016580; RCV000016877; RCV000016879 HBB 11 5248232 5248232 RCV000016286; RCV000030905; SNV RCV000016575; RCV000016573; RCV000016574; RCV000016576; RCV000016577; RCV000016579; RCV000016580; RCV000016877; RCV000016879 HBB 11 5248232 5248232 RCV000016286; RCV000030905; SNV RCV000016575; RCV000016573; RCV000016574; RCV000016576; RCV000016577; RCV000016579; RCV000016580; RCV000016877; RCV000016879 HBB 11 5248232 5248232 RCV000016674 deletion HBB 11 5248232 5248233 RCV000029966 indel HBB 11 5248233 5248233 RCV000016251; RCV000016285; SNV RCV000016284 HBB 11 5248233 5248233 RCV000016251; RCV000016285; SNV RCV000016284 HBB 11 5248234 5248235 RCV000016678 deletion HBB 11 5248248 5248248 RCV000016770 deletion HBB 11 5248248 5248248 RCV000016824 SNV HBB 11 5248249 5248249 RCV000016783 SNV HBB 11 5248250 5248250 RCV000016691 SNV HBB 11 5248250 5248250 RCV000016692; RCV000016693; SNV RCV000029976 HLCS 21 38132082 38132082 RCV000001987 SNV HLCS 21 38132112 38132112 RCV000001985 SNV HLCS 21 38137345 38137345 RCV000001988 SNV HLCS 21 38137471 38137471 RCV000001986 SNV HLCS 21 38309035 38309035 RCV000001984 SNV HLCS 21 38309098 38309098 RCV000001991 SNV HMGCL 1 24130931 24130931 RCV000012736 SNV HMGCL 1 24144010 24144010 RCV000012733 SNV HMGCL 1 24147022 24147022 RCV000012735; RCV000078342 SNV HPD 12 122277904 122277904 RCV000001642 SNV HPD 12 122284825 122284825 RCV000001640 SNV HPD 12 122285117 122285117 RCV000001641 SNV HPD 12 122287632 122287632 RCV000001639 SNV HPD 12 122295335 122295335 RCV000001643 SNV HSD17B10 X 53458393 53458393 RCV000012199 SNV HSD17B10 X 53458398 53458398 RCV000012197 SNV HSD17B10 X 53458767 53458767 RCV000012198 SNV HSD17B10 X 53459034 53459034 RCV000012195 SNV HSD17B10 X 53459058 53459058 RCV000012196 SNV IDUA 4 981630 981630 RCV000012689 SNV IDUA 4 981646 981646 RCV000012684; RCV000078386 SNV IDUA 4 981704 981704 RCV000012697 SNV IDUA 4 995263 995263 RCV000078394 SNV IDUA 4 995875 995875 RCV000012698 SNV IDUA 4 995905 995905 RCV000012690 SNV IDUA 4 996121 996121 RCV000012703; RCV000012702 SNV IDUA 4 996129 996129 RCV000078370 SNV IDUA 4 996175 996175 RCV000012700 SNV IDUA 4 996180 996180 RCV000012691 SNV IDUA 4 996535 996535 RCV000012683; RCV000078374 SNV IDUA 4 996555 996555 RCV000012686; RCV000078375 SNV IDUA 4 996555 996555 RCV000012686; RCV000078375 SNV IDUA 4 996890 996890 RCV000012694; RCV000078381 SNV IDUA 4 996896 996896 RCV000012693 SNV IDUA 4 997206 997206 RCV000012685 SNV IDUA 4 997871 997871 RCV000078385 deletion IDUA 4 998074 998074 RCV000012699 SNV IDUA 4 998080 998080 RCV000012692 SNV IDUA 4 998093 998093 RCV000087088 SNV IDUA 4 998179 998179 RCV000012695 SNV IDUA 4 998181 998181 RCV000012686 SNV IL2RG X 70328128 70328128 RCV000010701 SNV IL2RG X 70328173 70328173 RCV000010706 SNV IL2RG X 70328186 70328186 RCV000030058 SNV IL2RG X 70328449 70328449 RCV000010709 SNV IL2RG X 70329125 70329125 RCV000030057 SNV IL2RG X 70329171 70329171 RCV000010710 SNV IL2RG X 70329173 70329173 RCV000030056 SNV IL2RG X 70330142 70330142 RCV000010705 SNV IL2RG X 70330145 70330145 RCV000030055 SNV IL2RG X 70330356 70330356 RCV000010711 SNV IL2RG X 70330453 70330453 RCV000010699 SNV IL2RG X 70330465 70330465 RCV000010708 SNV IL2RG X 70330467 70330467 RCV000010703 SNV IL2RG X 70330494 70330494 RCV000030054 SNV IL2RG X 70330830 70330830 RCV000010702 SNV IVD 15 40698153 40698153 RCV000003743 SNV IVD 15 40699840 40699840 RCV000003748 SNV IVD 15 40702937 40702938 RCV000079998 deletion IVD 15 40703500 40703500 RCV000080000 deletion IVD 15 40703799 40703799 RCV000003744 SNV IVD 15 40707653 40707653 RCV000003749; RCV000080003 SNV IVD 15 40708555 40708555 RCV000079995 SNV MAT1A 10 82034291 82034291 RCV000001263 SNV MAT1A 10 82034355 82034355 RCV000001270 SNV MAT1A 10 82034395 82034395 RCV000001261 SNV MAT1A 10 82034810 82034810 RCV000001264 SNV MAT1A 10 82034933 82034933 RCV000001267 SNV MAT1A 10 82034934 82034934 RCV000001269 SNV MAT1A 10 82045273 82045273 RCV000001262 SNV MCCC1 3 182733325 182733325 RCV000002011 deletion MCCC1 3 182751856 182751856 RCV000002010 SNV MCCC1 3 182755006 182755006 RCV000002008 SNV MCCC1 3 182755220 182755220 RCV000002012 SNV MCCC1 3 182756881 182756881 RCV000002009 SNV MCCC1 3 182756914 182756914 RCV000081990 SNV MCCC1 3 182759467 182759467 RCV000002007 SNV MCCC1 3 182763210 182763210 RCV000081989 deletion MCCC1 3 182763310 182763310 RCV000002006; RCV000081995 SNV MCCC2 5 70895499 70895499 RCV000001997; RCV000082095 SNV MCCC2 5 70895584 70895584 RCV000082096 SNV MCCC2 5 70898413 70898413 RCV000001998 SNV MCCC2 5 70898448 70898448 RCV000002000 SNV MCCC2 5 70900188 70900188 RCV000001996 duplication MCCC2 5 70900240 70900240 RCV000002003 SNV MCCC2 5 70928012 70928012 RCV000002001 SNV MCCC2 5 70930804 70930804 RCV000002004 SNV MCCC2 5 70931003 70931003 RCV000001999 SNV MCCC2 5 70945016 70945016 RCV000002002 SNV MCEE 2 71351575 71351575 RCV000002434 SNV MLYCD 16 83932757 83932757 RCV000004274 SNV MLYCD 16 83932868 83932868 RCV000004275 SNV MLYCD 16 83940623 83940623 RCV000004270 SNV MLYCD 16 83941768 83941768 RCV000081531 indel MMAA 4 146560574 146560574 RCV000003308 SNV MMAA 4 146560724 146560724 RCV000003310 SNV MMAA 4 146567195 146567195 RCV000003309 SNV MMAA 4 146576363 146576363 RCV000082687 deletion MMAB 12 109998861 109998861 RCV000082327 SNV MMAB 12 109998873 109998873 RCV000003241 SNV MMADHC 2 150426603 150426603 RCV000000797 SNV MMADHC 2 150426631 150426631 RCV000000803 SNV MMADHC 2 150426633 150426633 RCV000000799 SNV MMADHC 2 150432289 150432289 RCV000000798 SNV MMADHC 2 150433009 150433010 RCV000000804 duplication MMADHC 2 150435992 150435993 RCV000000802 duplication MMADHC 2 150436157 150436157 RCV000000801 SNV MMADHC 2 150438731 150438738 RCV000000800 deletion MTHFR 1 11851273 11851273 RCV000003705 SNV MTHFR 1 11854823 11854823 RCV000003706 SNV MTHFR 1 11855171 11855171 RCV000003701 SNV MTHFR 1 11855215 11855215 RCV000003700 SNV MTHFR 1 11855218 11855218 RCV000003707 SNV MTHFR 1 11856378 11856378 RCV000003697 SNV MTHFR 1 11860308 11860308 RCV000003695 SNV MTHFR 1 11861223 11861223 RCV000003696 SNV MTR 1 236998886 236998886 RCV000015358 SNV MTR 1 237015878 237015878 RCV000015356 SNV MTR 1 237048502 237048502 RCV000015350 SNV MTR 1 237058770 237058770 RCV000015348 SNV MTR 1 237060320 237060320 RCV000015357 SNV MTRR 5 7891518 7891518 RCV000007449 SNV MTRR 5 7892928 7892928 RCV000007446 SNV MUT 6 49399544 49399544 RCV000001958; RCV000078445 SNV MUT 6 49403186 49403186 RCV000001962 SNV MUT 6 49408008 49408008 RCV000001961 SNV MUT 6 49419381 49419381 RCV000001956 SNV MUT 6 49425502 49425502 RCV000001963 SNV MUT 6 49425514 49425514 RCV000001965 SNV MUT 6 49426831 49426831 RCV000001959 SNV MUT 6 49426858 49426858 RCV000001964 SNV MUT 6 49426867 49426867 RCV000001955 SNV MUT 6 49426902 49426902 RCV000001957 SNV MUT 6 49427089 49427089 RCV000078448 SNV MUT 6 49427128 49427128 RCV000001954 SNV NGLY1 3 25761025 25761025 RCV000043662 deletion NGLY1 3 25761670 25761670 RCV000114364 SNV NGLY1 3 25773865 25773865 RCV000114362 duplication NGLY1 3 25775416 25775418 RCV000114363 deletion NGLY1 3 25775422 25775422 RCV000043663 SNV NPC1 18 21113434 21113434 RCV000003099 SNV NPC1 18 21115443 21115443 RCV000003093 SNV NPC1 18 21115647 21115647 RCV000003098 SNV NPC1 18 21116700 21116700 RCV000003101 SNV NPC1 18 21116722 21116722 RCV000020231 SNV NPC1 18 21116775 21116775 RCV000003092 SNV NPC1 18 21116778 21116778 RCV000003106; RCV000078477 SNV NPC1 18 21118528 21118528 RCV000003100 SNV NPC1 18 21118573 21118573 RCV000003103 SNV NPC1 18 21118573 21118573 RCV000020229 SNV NPC1 18 21118573 21118573 RCV000020230; RCV000003094 SNV NPC1 18 21118615 21118615 RCV000003110 SNV NPC1 18 21119357 21119357 RCV000003102 SNV NPC1 18 21119382 21119382 RCV000003105 SNV NPC1 18 21119775 21119775 RCV000119336 duplication NPC1 18 21119787 21119787 RCV000003091 SNV NPC1 18 21119905 21119905 RCV000003096 SNV NPC1 18 21121277 21121277 RCV000119335 SNV NPC1 18 21121319 21121319 RCV000020226 SNV NPC1 18 21121341 21121341 RCV000119334 duplication NPC1 18 21123433 21123434 RCV000119333 deletion NPC1 18 21123468 21123468 RCV000078472 duplication NPC1 18 21123487 21123487 RCV000119332 SNV NPC1 18 21124310 21124310 RCV000119331 SNV NPC1 18 21124384 21124384 RCV000119330 SNV NPC1 18 21125039 21125039 RCV000119329 SNV NPC1 18 21125071 21125071 RCV000119328 deletion NPC1 18 21134722 21134722 RCV000119327 SNV NPC1 18 21134773 21134773 RCV000119326 SNV NPC1 18 21136400 21136400 RCV000003104 SNV NPC1 18 21136503 21136503 RCV000119325 deletion NPC1 18 21141425 21141425 RCV000003108 SNV NPC1 18 21148834 21148834 RCV000119338 duplication NPC1 18 21148913 21148913 RCV000003112 SNV NPC2 14 74947404 74947404 RCV000087100 SNV NPC2 14 74947410 74947410 RCV000009006 SNV NPC2 14 74951123 74951123 RCV000009007 SNV NPC2 14 74951129 74951129 RCV000009001 SNV NPC2 14 74951149 74951149 RCV000020647 deletion NPC2 14 74951186 74951186 RCV000020646 SNV NPC2 14 74951282 74951282 RCV000009003 SNV NPC2 14 74953081 74953081 RCV000020644 SNV NPC2 14 74953089 74953089 RCV000020643 SNV NPC2 14 74953107 74953107 RCV000009004 SNV NPC2 14 74959920 74959920 RCV000008998 SNV NPC2 14 74959951 74959951 RCV000020645 deletion NPC2 14 74959975 74959975 RCV000119339 SNV OPA3 19 46056897 46056897 RCV000020909 SNV OPA3 19 46056975 46056992 RCV000004464 deletion OPA3 19 46056999 46056999 RCV000004463 SNV OPA3 19 46057035 46057035 RCV000004462 SNV PAH 12 103234177 103234177 RCV000078510 SNV PAH 12 103234250 103234250 RCV000000648; RCV000088815 SNV PAH 12 103234252 103234252 RCV000000624; RCV000078508 SNV PAH 12 103234253 103234253 RCV000106346 SNV PAH 12 103234255 103234255 RCV000000623; RCV000088813 SNV PAH 12 103234270 103234270 RCV000000643; RCV000088806 SNV PAH 12 103234271 103234271 RCV000000607; RCV000078507 SNV PAH 12 103234273 103234273 RCV000000667; RCV000088804 SNV PAH 12 103234285 103234285 RCV000078506 SNV PAH 12 103237427 103237427 RCV000106344 SNV PAH 12 103237443 103237443 RCV000106343 SNV PAH 12 103237452 103237452 RCV000106342 SNV PAH 12 103237454 103237454 RCV000000657; RCV000000656; SNV RCV000078503 PAH 12 103237460 103237460 RCV000106341 SNV PAH 12 103237461 103237461 RCV000000650; RCV000088774 SNV PAH 12 103237484 103237484 RCV000000660; RCV000078502 SNV PAH 12 103237547 103237547 RCV000000658; RCV000088747 SNV PAH 12 103237555 103237555 RCV000000626; RCV000088745 SNV PAH 12 103237555 103237555 RCV000078501 SNV PAH 12 103238134 103238134 RCV000000646; RCV000078499 SNV PAH 12 103238137 103238137 RCV000078498 SNV PAH 12 103238175 103238175 RCV000106338 SNV PAH 12 103238182 103238182 RCV000000655; RCV000089195 SNV PAH 12 103238202 103238202 RCV000000609; RCV000089188 SNV PAH 12 103238205 103238205 RCV000078541 SNV PAH 12 103240677 103240677 RCV000000647; RCV000089177 SNV PAH 12 103240687 103240687 RCV000078539 SNV PAH 12 103240710 103240710 RCV000000608; RCV000089164 SNV PAH 12 103240710 103240711 RCV000106378 deletion PAH 12 103240716 103240716 RCV000078538 SNV PAH 12 103240726 103240726 RCV000000649; RCV000089157 SNV PAH 12 103240726 103240726 RCV000106377 deletion PAH 12 103245464 103245464 RCV000078537 SNV PAH 12 103245479 103245479 RCV000078536 SNV PAH 12 103245481 103245481 RCV000000644; RCV000089148 SNV PAH 12 103245487 103245487 RCV000078535 SNV PAH 12 103245490 103245490 RCV000106372 SNV PAH 12 103245508 103245508 RCV000106371 SNV PAH 12 103246593 103246593 RCV000000620; RCV000078534 SNV PAH 12 103246594 103246594 RCV000106370 SNV PAH 12 103246597 103246597 RCV000000610; RCV000078532 SNV PAH 12 103246598 103246598 RCV000106369 deletion PAH 12 103246606 103246606 RCV000000634; RCV000078531 SNV PAH 12 103246617 103246617 RCV000000629; RCV000089111 SNV PAH 12 103246621 103246621 RCV000000627; RCV000089110 SNV PAH 12 103246623 103246623 RCV000106368 SNV PAH 12 103246639 103246639 RCV000106367 SNV PAH 12 103246650 103246650 RCV000106366 SNV PAH 12 103246653 103246653 RCV000000612; RCV000078530 SNV PAH 12 103246654 103246654 RCV000000641; RCV000089090 SNV PAH 12 103246654 103246654 RCV000119826 SNV PAH 12 103246659 103246659 RCV000000633; RCV000089089 SNV PAH 12 103246671 103246671 RCV000000631; RCV000089083 SNV PAH 12 103246681 103246681 RCV000000614; RCV000089079 SNV PAH 12 103246701 103246701 RCV000000664; RCV000089065 SNV PAH 12 103246704 103246704 RCV000000652; RCV000089061 SNV PAH 12 103246707 103246707 RCV000000622; RCV000089059 SNV PAH 12 103246708 103246708 RCV000000619; RCV000078528 SNV PAH 12 103248938 103248938 RCV000106365 SNV PAH 12 103248958 103248958 RCV000000640; RCV000089024 SNV PAH 12 103248982 103248982 RCV000078527 SNV PAH 12 103248988 103248988 RCV000106364 SNV PAH 12 103249009 103249009 RCV000000621; RCV000089007 SNV PAH 12 103249029 103249029 RCV000106361 SNV PAH 12 103249052 103249052 RCV000106360 SNV PAH 12 103249071 103249073 RCV000106359 indel PAH 12 103249087 103249087 RCV000078526 SNV PAH 12 103249093 103249093 RCV000000663; RCV000088974 SNV PAH 12 103260375 103260375 RCV000078524 SNV PAH 12 103260378 103260378 RCV000111461 SNV PAH 12 103260379 103260379 RCV000106358 SNV PAH 12 103260410 103260410 RCV000000618; RCV000078522 SNV PAH 12 103288515 103288515 RCV000106354 SNV PAH 12 103288515 103288515 RCV000106355 deletion PAH 12 103288534 103288534 RCV000000611; RCV000088898 SNV PAH 12 103288572 103288572 RCV000000659; RCV000088892 SNV PAH 12 103288579 103288581 RCV000078518 deletion PAH 12 103288579 103288581 RCV000106353 indel PAH 12 103288604 103288604 RCV000000613; RCV000088884 SNV PAH 12 103288638 103288638 RCV000000671; RCV000088878 SNV PAH 12 103288655 103288660 RCV000106352 duplication PAH 12 103288661 103288661 RCV000078517 SNV PAH 12 103288669 103288669 RCV000106351 SNV PAH 12 103288671 103288671 RCV000000668; RCV000078516 SNV PAH 12 103288682 103288682 RCV000106350 SNV PAH 12 103288696 103288696 RCV000106349 SNV PAH 12 103306572 103306572 RCV000078512 SNV PAH 12 103306572 103306572 RCV000078513 deletion PAH 12 103306573 103306573 RCV000106347 SNV PAH 12 103306594 103306594 RCV000000639; RCV000078511 SNV PAH 12 103306597 103306597 RCV000000662; RCV000088839 SNV PAH 12 103306601 103306601 RCV000000661; RCV000088836 SNV PAH 12 103306620 103306620 RCV000000636; RCV000078504 SNV PAH 12 103310906 103310906 RCV000000653; RCV000088911 SNV PAH 12 103310908 103310908 RCV000000616; RCV000000617; SNV RCV000088868 PCBD1 10 72643730 72643730 RCV000018290 SNV PCBD1 10 72643763 72643763 RCV000018286 SNV PCBD1 10 72643778 72643778 RCV000018287 SNV PCBD1 10 72643786 72643786 RCV000018288 SNV PCCA 13 100807344 100807344 RCV000032110 SNV PCCA 13 100861608 100861608 RCV000032111 SNV PCCA 13 100920985 100920985 RCV000012803 SNV PCCA 13 100920985 100920985 RCV000032112 SNV PCCA 13 100925472 100925472 RCV000032113; RCV000078552 SNV PCCA 13 100925558 100925558 RCV000078546 duplication PCCA 13 100953766 100953766 RCV000012804 SNV PCCA 13 101020767 101020767 RCV000032109 SNV PCCB 3 135969219 135969219 RCV000079093 SNV PCCB 3 135969220 135969220 RCV000079095 SNV PCCB 3 135974794 135974794 RCV000032131 SNV PCCB 3 135975428 135975428 RCV000032132 SNV PCCB 3 135979334 135979335 RCV000079094 indel PCCB 3 135980821 135980821 RCV000032133 SNV PCCB 3 135980866 135980866 RCV000012795 SNV PCCB 3 136045727 136045727 RCV000012794 duplication PCCB 3 136046016 136046029 RCV000032124 indel PCCB 3 136046026 136046026 RCV000032125 SNV PCCB 3 136046081 136046081 RCV000012796 SNV PCCB 3 136046480 136046480 RCV000012798 SNV PCCB 3 136047696 136047696 RCV000032127 SNV PCCB 3 136048782 136048782 RCV000032128 SNV PCCB 3 136048787 136048788 RCV000012797 insertion PCCB 3 136048804 136048804 RCV000032129 SNV PCCB 3 136048854 136048854 RCV000032130; RCV000079090 SNV PTS 11 112097212 112097212 RCV000000506 SNV PTS 11 112097240 112097240 RCV000000505 SNV PTS 11 112099372 112099372 RCV000000511 SNV PTS 11 112099388 112099388 RCV000000508 SNV PTS 11 112100933 112100933 RCV000000510 SNV PTS 11 112103901 112103901 RCV000000509 SNV PTS 11 112103928 112103928 RCV000000513 SNV PTS 11 112104187 112104187 RCV000000512 SNV QDPR 4 17493951 17493951 RCV000000523 SNV QDPR 4 17503456 17503456 RCV000000520 SNV QDPR 4 17506027 17506027 RCV000000524 SNV QDPR 4 17510986 17510986 RCV000000521 SNV QDPR 4 17513610 17513610 RCV000000519 SNV SLC22A5 5 131705667 131705667 RCV000006794 SNV SLC22A5 5 131705707 131705707 RCV000006798 SNV SLC22A5 5 131705715 131705715 RCV000022297; RCV000080056 SNV SLC22A5 5 131705912 131705912 RCV000022308; RCV000080052 SNV SLC22A5 5 131714072 131714072 RCV000006780 SNV SLC22A5 5 131714100 131714100 RCV000022320; RCV000080055 SNV SLC22A5 5 131719846 131719846 RCV000006791 SNV SLC22A5 5 131719847 131719847 RCV000006790 SNV SLC22A5 5 131719973 131719973 RCV000006789 SNV SLC22A5 5 131721062 131721062 RCV000022339; RCV000080058 SNV SLC22A5 5 131721127 131721127 RCV000006795; RCV000080059 SNV SLC22A5 5 131722736 131722736 RCV000006785 SNV SLC22A5 5 131722737 131722737 RCV000022354; RCV000080062 SNV SLC22A5 5 131724712 131724712 RCV000006792 SNV SLC22A5 5 131726522 131726522 RCV000022365; RCV000080047 SNV SLC22A5 5 131726524 131726524 RCV000006796 SNV SLC22A5 5 131726525 131726525 RCV000006793 SNV SLC22A5 5 131726531 131726532 RCV000006786; RCV000080048 duplication SLC22A5 5 131728181 131728182 RCV000006797 indel SLC22A5 5 131728257 131728257 RCV000022379; RCV000080050 SNV SLC22A5 5 131728290 131728290 RCV000006788 SNV SLC22A5 5 131729380 131729380 RCV000022386; RCV000080051 SNV SLC25A13 7 95750995 95750995 RCV000020704 SNV SLC25A13 7 95751007 95751007 RCV000020702 SNV SLC25A13 7 95751007 95751007 RCV000020703 SNV SLC25A13 7 95751008 95751009 RCV000006374; RCV000006375 duplication SLC25A13 7 95751045 95751045 RCV000006376 SNV SLC25A13 7 95751240 95751241 RCV000006371 duplication SLC25A13 7 95751309 95751309 RCV000020699 SNV SLC25A13 7 95799356 95799356 RCV000006373 SNV SLC25A13 7 95813588 95813588 RCV000006369; RCV000006370 SNV SLC25A13 7 95813688 95813688 RCV000020698 SNV SLC25A13 7 95818685 95818688 RCV000006367; RCV000006368 deletion SLC25A13 7 95820501 95820501 RCV000006372 SNV SLC25A13 7 95822348 95822348 RCV000020706 SNV SLC25A13 7 95822414 95822414 RCV000020705 SNV SLC25A13 7 95951254 95951254 RCV000020700 SNV SLC25A20 3 48895152 48895152 RCV000012915 duplication SLC25A20 3 48896530 48896530 RCV000012921 SNV SLC25A20 3 48897020 48897020 RCV000114402 SNV SLC25A20 3 48900014 48900014 RCV000012918 SNV SLC25A20 3 48921430 48921557 RCV000012916 deletion SLC25A20 3 48936144 48936144 RCV000012919 deletion SLC5A5 19 17983405 17983405 RCV000008107 SNV SLC5A5 19 17988632 17988632 RCV000008105 SNV SLC5A5 19 17988649 17988649 RCV000008104 SNV SLC5A5 19 17992770 17992770 RCV000008103 SNV SLC5A5 19 17992969 17992969 RCV000008109 SNV SLC5A5 19 17999206 17999206 RCV000008106 SNV SLC5A5 19 17999241 17999241 RCV000008108 SNV TAT 16 71610150 71610150 RCV000000429 SNV TAZ X 153640466 153640466 RCV000011850 SNV TAZ X 153640521 153640521 RCV000035087 SNV TAZ X 153641585 153641585 RCV000011859 SNV TAZ X 153641841 153641841 RCV000035089 SNV TAZ X 153641844 153641844 RCV000035090 SNV TAZ X 153641862 153641862 RCV000035088 SNV TAZ X 153641886 153641886 RCV000011857 SNV TAZ X 153648376 153648376 RCV000011854 SNV TAZ X 153648377 153648377 RCV000035097 SNV TAZ X 153648550 153648550 RCV000011861 SNV TAZ X 153648996 153648996 RCV000035098 SNV TAZ X 153649015 153649015 RCV000029171 SNV TAZ X 153649015 153649015 RCV000035099 SNV TAZ X 153649287 153649287 RCV000035102 SNV TG 8 133894854 133894854 RCV000013532 SNV TG 8 133911054 133911054 RCV000013539 SNV TG 8 133919031 133919031 RCV000013530 SNV TG 8 133935642 133935642 RCV000013528 SNV TG 8 133980042 133980042 RCV000013541 SNV TG 8 133984049 133984049 RCV000013531 SNV TG 8 134030185 134030185 RCV000013537 SNV TG 8 134034366 134034366 RCV000013542 SNV TG 8 134042152 134042152 RCV000013540 SNV TPO 2 1488368 1488368 RCV000004258 SNV TPO 2 1488386 1488386 RCV000004259 SNV TPO 2 1491613 1491613 RCV000004257 SNV TPO 2 1491763 1491763 RCV000004260 SNV TPO 2 1497748 1497748 RCV000004263 SNV TPO 2 1497783 1497783 RCV000004268 SNV TPO 2 1499831 1499831 RCV000004266 SNV TPO 2 1507728 1507728 RCV000004261 SNV TSHB 1 115576077 115576077 RCV000013522 SNV TSHB 1 115576128 115576128 RCV000013521 SNV TSHB 1 115576636 115576636 RCV000013524 SNV TSHR 14 81422146 81422146 RCV000006812 SNV TSHR 14 81554306 81554306 RCV000006808 SNV TSHR 14 81558891 81558891 RCV000006805 SNV TSHR 14 81558907 81558907 RCV000006804 SNV TSHR 14 81562985 81562985 RCV000006824 SNV TSHR 14 81606172 81606172 RCV000006823 SNV TSHR 14 81609330 81609330 RCV000006827; RCV000122250 SNV TSHR 14 81609372 81609372 RCV000006810 SNV TSHR 14 81609572 81609572 RCV000006814 SNV TSHR 14 81609630 81609630 RCV000006811 SNV TSHR 14 81609693 81609693 RCV000006828 SNV TSHR 14 81609760 81609760 RCV000006806 SNV TSHR 14 81609802 81609802 RCV000006830 SNV TSHR 14 81609832 81609832 RCV000006826 SNV TSHR 14 81609916 81609916 RCV000006820 SNV TSHR 14 81609928 81609928 RCV000006818 SNV TSHR 14 81609977 81609977 RCV000006813 SNV TSHR 14 81610039 81610039 RCV000006809 SNV TSHR 14 81610059 81610059 RCV000006815 SNV TSHR 14 81610200 81610200 RCV000006829 SNV TSHR 14 81610289 81610289 RCV000006822; RCV000006821 SNV TSHR 14 81610293 81610293 RCV000006803; RCV000006802 SNV TSHR 14 81610299 81610299 RCV000006807 SNV TSHR 14 81610317 81610317 RCV000006825 SNV TSHR 14 81610417 81610417 RCV000006819 SNV PAX8 2 114002071 114002071 RCV000014793 SNV PAX8 2 114004337 114004337 RCV000014795 SNV PAX8 2 114004352 114004352 RCV000014796 SNV PAX8 2 114004362 114004362 RCV000014797 SNV PAX8 2 114004379 114004379 RCV000014799 SNV PAX8 2 114004403 114004403 RCV000014798 SNV PAX8 2 114004430 114004430 RCV000014794 SNV SLC26A1 4 980918 980929 RCV000078393 deletion ARG1 6 131897806 131897806 RCV000002498 SNV ARG1 6 131902418 131902418 RCV000002491 SNV ARG1 6 131902466 131902466 RCV000002495 SNV ARG1 6 131904532 131904532 RCV000002492 SNV ARG1 6 131904948 131904948 RCV000002490 SNV ARG1 6 131904950 131904950 RCV000002489 SNV TAT 16 71602163 71602163 RCV000000431 SNV TAT 16 71603797 71603797 RCV000000432 SNV TAT 16 71606127 71606127 RCV000000430 SNV GCDH 19 13010285 13010285 RCV000002165 SNV GCDH 19 13010300 13010300 RCV000002163 SNV GLA X 100652859 100652859 RCV000011518 SNV GLA X 100652895 100652895 RCV000011493 SNV GLA X 100652992 100652992 RCV000011514 SNV GLA X 100653006 100653006 RCV000011492 SNV GLA X 100653021 100653021 RCV000011459; RCV000035299 SNV GLA X 100653053 100653054 RCV000078263 deletion GLA X 100653062 100653062 RCV000011490; RCV000078262 SNV GLA X 100653063 100653063 RCV000011491; RCV000078261 SNV GLA X 100653067 100653067 RCV000011489 SNV GLA X 100653067 100653068 RCV000078259 insertion GLA X 100653358 100653361 RCV000078308 deletion GLA X 100653374 100653374 RCV000011488; RCV000078307 SNV GLA X 100653375 100653375 RCV000011469 SNV GLA X 100653378 100653378 RCV000011487 SNV GLA X 100653383 100653383 RCV000078306 SNV GLA X 100653384 100653384 RCV000078305 SNV GLA X 100653391 100653391 RCV000078304 SNV GLA X 100653395 100653398 RCV000078303 deletion GLA X 100653420 100653420 RCV000029944; RCV000011486; SNV RCV000035314 GLA X 100653455 100653455 RCV000011462; RCV000011461; SNV RCV000078302 GLA X 100653456 100653456 RCV000078301 SNV GLA X 100653458 100653458 RCV000078300 SNV GLA X 100653467 100653467 RCV000011484 SNV GLA X 100653469 100653469 RCV000011510 SNV GLA X 100653471 100653471 RCV000011464 SNV GLA X 100653492 100653492 RCV000078299 SNV GLA X 100653496 100653496 RCV000011483 SNV GLA X 100653522 100653522 RCV000011468 SNV GLA X 100653534 100653534 RCV000078298 SNV GLA X 100653542 100653542 RCV000011521 SNV GLA X 100653551 100653551 RCV000011482 SNV GLA X 100653777 100653777 RCV000011481 SNV GLA X 100653783 100653783 RCV000011480 SNV GLA X 100653826 100653826 RCV000078297 SNV GLA X 100653840 100653840 RCV000078295 SNV GLA X 100653850 100653850 RCV000035310 SNV GLA X 100653894 100653894 RCV000011478; RCV000078294 SNV GLA X 100653895 100653895 RCV000011479; RCV000078293 SNV GLA X 100653897 100653897 RCV000078292 SNV GLA X 100653908 100653908 RCV000011517 SNV GLA X 100653911 100653912 RCV000078291 deletion GLA X 100653927 100653927 RCV000078290 SNV GLA X 100653930 100653930 RCV000011477; RCV000078289 SNV GLA X 100655663 100655663 RCV000078285 deletion GLA X 100655687 100655687 RCV000011476 SNV GLA X 100655745 100655745 RCV000078283 SNV GLA X 100656658 100656658 RCV000078281 SNV GLA X 100656683 100656683 RCV000011475 SNV GLA X 100656701 100656701 RCV000011474 SNV GLA X 100656731 100656731 RCV000011473 SNV GLA X 100656740 100656740 RCV000011495; RCV000078279 SNV GLA X 100656740 100656740 RCV000011516; RCV000078280 SNV GLA X 100658816 100658816 RCV000078277; RCV000035303 SNV GLA X 100658833 100658833 RCV000078276 SNV GLA X 100658834 100658834 RCV000011470; RCV000078275 SNV GLA X 100658834 100658834 RCV000011470; RCV000078275 SNV GLA X 100658887 100658887 RCV000078274 SNV GLA X 100658926 100658926 RCV000078273 SNV GLA X 100658972 100658972 RCV000011470 SNV GLA X 100662698 100662698 RCV000011513 SNV GLA X 100662726 100662726 RCV000011472 SNV GLA X 100662746 100662746 RCV000078270 SNV GLA X 100662755 100662755 RCV000078268 SNV GLA X 100662761 100662761 RCV000011463 SNV GLA X 100662767 100662767 RCV000078266 SNV GLA X 100662773 100662773 RCV000078264 SNV GLA X 100662774 100662774 RCV000011466 SNV GLA X 100662791 100662791 RCV000011471; RCV000078260 SNV GLA X 100662834 100662834 RCV000011511 SNV GLA X 100662873 100662873 RCV000078272 SNV

EXAMPLES

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Various modifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1 Procedure for the Receipt of Testing Request, Dried Blood Spot Shipment, and Receipt of Samples

Provided herein is an example protocol for the receipt of sample testing request, shipment of dried blood spot (DBS) collection kits, and receipt of DBS specimens at the laboratory. The collected DBS specimens provide an easy and inexpensive way to collect and store peripheral blood specimens from infants for newborn screening (NBS) test. DBS are prepared, for example, by applying a small amount of peripheral blood collected from infant heel punctures to filter paper cards, or by applying a small amount of blood collected from adult finger pricks. DBS can also be prepared, for example, from coagulated whole blood. DBS specimens are shipped at ambient temperatures as non-hazardous goods. Newborn screening (NBS) test requests are received from individual customers and healthcare facilities (hospitals, clinics, etc.).

Preparing the Test Request Receipt:

Upon receipt of a NBS test request, the record customer and/or facility contact information is recorded in a client access database. The information that is added to the client account access database includes, for example, date of the test request, name of the customer and/or facility requesting the test, name, address, phone, fax, email and preferred contact method of the customer and/or facility requesting the test. The sections with no provided information are labeled not applicable (N/A). If the sections with no provided information include sections which require pertinent information, contact is made with the customer and/or facility requesting the test, and information is obtained prior to sample processing.

Shipping the DBS Collection Kit:

The DBS collection kit is prepared for shipment to a customer and/or hospital facility requesting the test by including, for example, a standard mailing envelope or box, pre-paid, addresses outbound return envelope, blood lancet, Whatman 903 specimen collection paper, resealable impermeable plastic bag, humidity indicator cards, glassine envelope, dessicant packs, BG DBS collection instructions (exemplified in Table 15). The filter paper are identified by unique ID numbers, and the ID number for the filter paper included in the shipped DBS collection kit is recorded in the client account access database. The date of shipment of the DBS kit and the tracking number for the DBS kit are also recorded in the client access database.

Collection of DBS Specimen:

The customer is responsible for collecting the DBS specimen. Recommendations for DBS collection are provided in the DBS collection kit. The customer will complete all applicable information on the DBS submission form (attached to the Specimen Collection Paper). The DBS collection is performed, whenever possible, by a healthcare provider of a facility, and performed per the facilities standard protocols.

Shipping of the Collected DBS Specimen:

The DBS card, with attached submission form, is properly dried, and placed in the provided glassine envelope. The glassine envelope, along with desiccant packets and humidity card, are placed into the provided sealable plastic bag and the plastic bag is sealed tightly. The tightly sealed plastic bag is then to be inserted into the provided outbound envelope and immediately shipped back to the testing facility.

Receipt of Collected DBS Sample Shipment:

Upon receipt of the DBS sample at a testing facility, the date and time of sample receipt are recorded in the client account access database. The access database automatically assigns each client sample with a unique sample identification number, Sample ID, based on the date of sample receipt and the number of samples received that day (BG-MMDDYYYY-#), for example a third sample received on Mar. 28, 2014 will be assigned an ID as BG-03282014-3. The sample ID is then recorded in the data tracking system. The information that are required to be entered into the client account access database include, for example, client name and/or ID, date of sample collection, client date of birth (DOB), client age (hours), client gender, date, and time of sample receipt. Additional information provided on the DBS card is useful, and may optionally be entered into the client account access database. Sections, on the submission form, for which no data are provided are entered as not applicable (N/A) into the client account access database.

Storage of DBS Sample:

After entering the sample data into the client account access database, blood samples are placed in a 4° C. refrigerator in a laboratory, until additional sample processing, as per Example 2, commences.

Example 2 Procedure for gDNA Extraction, Purification, Quantification, and Normalization from Dried Blood Spots (DBS)

Provided herein is an example protocol for the extraction, purification, quantification, and normalization of gDNA from dried blood spot (DBS) samples. DBS samples are collected at an external location and shipped to the testing facility for genetic screening, according to the protocol described in Example 1. Following sample receipt, gDNA is extracted and purified from the DBS. The CHARGESWITCH nucleic acid purification kit is a bead-based technology that alters the reaction pH to facilitate nucleic acid purification. Information is collected during sample processing and stored in the laboratory information management system (LIMS).

Decontamination and Preparation for gDNA Extraction:

Before beginning the gDNA extraction process, the bench top and any necessary equipment is decontaminated with decontamination reagent to void the area of RNAase and DNA. Gloves are lightly sprayed with decontamination reagent. Three nuclease free centrifuge tubes are removed for every sample that is processed. The tubes are labeled with the sample ID, the date, and 1, 2, or 3. An additional set of tubes are labeled as “Blank 1, 2, or 3 to serve as a negative control. There are, for example, five circles for blood deposit on each DBS collection card. At least 15, 4 mm fully saturated punches are used for each extraction reaction. At least 15, 4 mm fully saturated pouches are used as negative control. An unused filter paper is processed alongside the client samples, as a “blank control” sample.

DNA Extraction Process:

DNA is extracted using a nucleic acid purification kit (e.g., CHARGESWITCH nucleic acid purification kit (Life Technologies)). A water bath is heated to 95±5° C. The water bath temperature is also recorded in the LIMS. A 1 mL volume of CHARGESWITCH lysis buffer and 10 μL of proteinase K (stored in a refrigerator) are added to Tube 1. A total of 15 fully saturated circles, are punched from the DBS sample filter paper, with a 4 mm punching device, directly into the tube labeled as Tube 1. If multiple samples are prepared for DNA extraction, the punching device is cleaned between each sample by punching a clean filter paper at least three times. A total of 15 fully saturated circles, are also punched from the Blank filter paper, with a 4 mm punching device, directly into the tube labeled as Blank #1. The tube lids are tightly secured and vortexed briefly for a short period of time, for example, 2-3 seconds. The tubes are then placed in a float tray and the float tray is put into the 95° C. water bath. The lid of the water bath is kept off during incubation to avoid contamination due to condensation. The water bath temperature fluctuates (±10° C.) during sample incubation when the lid is removed from the water bath. The tubes are incubated in the water bath for 30 minutes, with mixing by brief 2-3 seconds vortexing, every 10 minutes. During incubation, the QUBIT dsDNA High Sensitivity Standards #1 and #2 are removed from the refrigerator to equilibrate to room temperature. These are used in gDNA quantification via QUBIT as described in Example 3.

DNA Purification:

The sample(s) are removed from the water bath, following lysis, and centrifuged briefly. The serial numbers of pipettes which are used for the DNA purification procedure are recorded in the LIMS. A 200 μL volume of CHARGESWITCH purification buffer, is added to the tube labeled as Tube 2. The supernatant from the lysis reaction, as described in the DNA Extraction process section, is transferred to Tube 2. The filter paper is not transferred to Tube 2. The CHARGESWITCH magnetic beads, are vortexed for a brief time, for example 15 seconds, to thoroughly resuspend. A 20 μL volume of the magnetic beads is added to each tube labeled as Tube 2 and vortexed for a brief time, for example 2-3 seconds. The solution is incubated at room temperature for five minutes to allow DNA to bind to the magnetic beads. The tubes are placed on the magnetic rack for five minutes.

While the tube(s) remain on the magnet, the supernatant is removed and discarded. The pipette tip is angled to avoid disturbing the pellet containing the magnetic beads bound to the DNA.

DNA Wash:

The tube(s) are removed from the magnetic rack. A 500 μL volume of CHARGESWITCH wash buffer, is added to each tube labeled as Tube 2 and gently pipetted up and down to mix. The tube(s) are placed on the magnetic rack for one minute. The supernatant is removed and discarded while the tube(s) remain on the magnet. The pipette tip is angled to avoid disturbing the pellet containing the magnetic beads bound to the DNA. The wash steps, as described above, are repeated once more for a total of two washes.

DNA Elution:

The tube(s) are removed from the magnetic rack. A 150 μL volume of CHARGESWITCH elution buffer is added to each tube labeled as Tube 2 and gently pipetted up and down, around 10 times, to re-suspend the beads. The tube(s) are incubated at room temperature for five minutes. The tube(s) are placed on the magnetic rack for one minute. The supernatant, which contains the purified gDNA from the DBS sample, is removed and carefully transferred to the tubes labeled as Tube 3 while the tube(s) labeled as Tube 2 remain on the magnet.

Quantification of gDNA:

Each sample of gDNA, extracted following the procedures described above, is quantified before normalization and entrance into the library preparation process.

The process for quantification, using a QUBIT 2.0 fluorometer is described in Example 3. The “blank control” sample is also quantified using the QUBIT 2.0 fluorometer, following the process described in Example 3. The results from quantification of the “blank control” are recorded in the LIMS. The blank result is considered acceptable if it is below the detection limit for the QUBIT 2.0 fluorometer.

Example 3 Procedure for gDNA Quantification Using the LIFE TECHNOLOGIES QUBIT 2.0 Fluorometer

Provided herein is an example protocol for the quantification of double stranded (ds) DNA using the QUBIT 2.0 Fluorometer and dsDNA High Sensitivity (HS) kit (Life Technologies). The dsDNA High Sensitivity Kit contains QUBIT dsDNA HS Reagent, QUBIT dsDNA HS Buffer, QUBIT dsDNA HS Standard 1, and QUBIT dsDNA HS Standard 2. The HS Reagent and buffer are stored at room temperature. The Standards 1 and 2 are stored at 4° C. HS standards are aliquoted into small aliquots to avoid cross-contamination during pipetting.

gDNA is extracted from the DBS Samples, following the protocol described in Example 2. After the extraction process, the DNA samples are quantified prior to entry into library preparation, using the QUBIT 2.0 Fluorometer. The QUBIT 2.0 Fluorometer is also used to quantify completed libraries and pools prior to sequencing, which is described in Example 5. The QUBIT fluorometer is qualified to evaluate 1-500 ng/mL gDNA concentrations. All client unknown samples are diluted within the quantifiable range.

For quantification, a working solution is prepared for dilution of all samples and standards. Each time a new working solution is created, standards are diluted and re-quantified for that specific batch of working solution. A quality control sample is also analyzed on the QUBIT prior to sample analysis. The quality control sample is prepared by diluting the HS Standard 2 to evaluate the normal reading range of the QUBIT (1-500 ng/mL). The acceptable range for the quality control sample is defined as the ±10% of the expected target concentration.

Preparation of the Working Solution:

The QUBIT DNA HS Reagent is diluted 1:200 in the QUBIT DNA HS Buffer. 200 μL working solutions are prepared for each standard and unknown sample. It is recommended to create enough working solution for N+2 to accommodate for possible assay repeats. For example, for the analysis of 10 unknown samples, a total of 2. 8 mL of working solution is prepared (2.0 mL for samples, 400 μl for standards, 200 μl for quality control sample, 200 μl for possible rerun).

Once prepared, the working solution is stable at room temperature for three hours. Due to the nature of the light-sensitive dye, the solution is stored in minimal light exposure.

Preparation of Assay Tubes:

The assay tubes are set up for each unknown sample, two standards (1 and 2), and one quality control sample.

The samples, quality control and standards tubes are prepared according to Tables 6 and 7. The unknown samples are diluted between 1:10 and 1:200 to fall within the quantifiable range of the instrument. The sample dilution varies depending on the concentration of gDNA in the unknown sample.

TABLE 6 QUBIT Assay Preparation Final Working Sample/ Total Dilution Solution Standard Volume (μL) (μL) (μL) (μL) Standards 1:20 190 10 200 Unknown  1:200 199 1 200 Samples  1:100 198 2 200 1:50 196 4 200 1:10 180 20 200

TABLE 7 Quality Control Sample Preparation Quality Volume Volume Final Target Control Standard Working Concentration Acceptable QC Sample 2 (μL) Solution (μL) (ng/mL) Range (ng/mL) Quality 5 195 250 225-275 Control

After preparing the tubes, according to dilutions exemplified in Tables 6 and 7, all the tubes are vortexed for a brief time, for example 2-3 seconds. The tubes are flicked or tapped to ensure all solution is at the bottom of the assay tube and no bubbles are present. The tubes are then incubated at room temperature for two minutes.

QUBIT Reading:

The QUBIT 2.0 fluorometer is powered on and, “DNA” and “High Sensitivity” options are selected. The outside of the tubes are cleaned with a delicate task wipe to remove any marks or debris that interferes with the reading. The standards 1 and 2 are first read followed by the quality control samples. The QUBIT reading for the quality control samples are recorded in the LIMS. It is indicated that the reading is within the defined acceptable range for the instrument. If the quality control sample is not within the defined acceptable range, a new working solution and dilutions must be prepared. The unknown samples are read after reading the quality control samples. If the sample quantification value is outside of the QUBIT HS range (high or low), the sample is re-diluted and the reading is repeated. The sample quantification value (in ng/mL) is recorded in the LIMS.

Concentration Adjustment and Calculation:

The final stock concentration of each unknown sample is calculated using the following formulae:

QUBIT reading value (QF value) is multiplied by the working solution dilution factor, as follows:

1 : 100 Dilution: Sample Concentration ( ng mL ) = QF Value × 100

The value is converted to ng/mL, as follows:

Sample Concentration ( ng µL ) = Sample Conc ( ng mL ) 1000

The total gDNA concentration is calculated by multiplying the ng/μL value by the volume used to elute the DNA during the DNA extraction process.

150 µL Elution Volume : Total gDNA ( ng ) = Sample concentration ( ng µL ) × 150

Maintenance and Storage:

After the reading, the QUBIT is stored in an area with no direct sunlight. The machine is unplugged and cleaned gently with an alcohol swab or delicate task dampened with 20% ethanol solution. It is indicated on Appendix 1, that the cleaning is performed after use.

Example 4 Normalizing gDNA for Library Preparation

Provided herein is an example protocol for normalizing the gDNA concentrations prior to library preparation. All samples entering library preparation should be normalized to a concentration of 2 ng/μL.

Normalizing gDNA for Library Preparation:

An appropriate amount of 10 mM tris-HCL is added to each gDNA sample to obtain the desired concentration using the following formula:

gDN A Concentration ( ng µL ) = ( Qubit Value ( ng mL ) × Qubit Dilution Factor ) 1000 Final Sample Volume ( µL ) = ( gDNA Concentration ( ng µL ) × Sample Volume ( µL ) 2 Tris Volume ( µL ) = Final Sample Volume ( µL ) - Sample Volume ( µL )

Samples, at normalized concentrations, are used for the library preparation process, performed in Laboratory 2, as described in Example 5. The samples are stored at −20° C. in the freezer.

Example 5 Procedure for Library Preparation

Provided herein is an example protocol for the preparation of sequencing library from genomic DNA using an anchored multiplex PCR (AMP) technology (e.g., ENZYMATICS).

Client Samples:

The extracted, quantified, and normalized gDNA samples, prepared following methods described in Examples 2-4 enter the library preparation process which includes the following steps: DNA fragmentation, A-tailing and end repair, adapter ligation, and amplification via PCR steps. The technology used for library preparation is the lyophilized version of ARCHER DNA assay kit.

Quality Control (QC) Samples:

A library preparation QC sample is analyzed no less often than quarterly and whenever a reagent lot number changes, to verify the accuracy of the genomic analysis. A QC sample contains, for example, at least one known variant (within the target gene panel) to be considered a positive control. A QC sample does not display, for example, a known variant (within the target gene panel) to be considered a negative control. A single sample serves as both the positive and negative control if donor sample exhibits a known variant in a target gene and does not display a variant in another target region.

QC samples are obtained from donors with known genomic variants within the target gene panel. In some embodiments, QC samples are derived from the Coriell Institute. In some embodiments, QC samples are derived from the triplicate analysis of unknown samples for the development of a known set of variants within the target gene panel.

QC samples are diluted with 10 mM tris HCl to a final concentration of 2 ng/μL.

Final Sample Volume ( µL ) = ( gDNA Concentration ( ng µL ) × Sample Volume ( µL ) 2 Tris Volume ( µL ) = Final Sample Volume ( µL ) - Sample Volume ( µL )

Diluted samples are aliquoted into cryogenic vials and stored at −20° C. The QC samples are processed under normal conditions with the unknown client samples.

DNA Fragmentation, performed in Laboratory 2:

The DNA Fragmentation reaction packet is removed and allowed to reach room temperature. The following thermal cycling program is started (see Table 8) as described in Example 7 and paused once the block reaches 4° C.

TABLE 8 DNA Fragmentation Incubation Step Incubation Temperature Incubation Time 1  4° C.  1 min 2 37° C. 12 min 3 72° C. 20 min 4  4° C. Hold Heated Lid on at 105° C.

The green 8-tube strips are removed. Each tube in the strip provides a single reaction. If necessary, the tubes are centrifuged briefly to collect all lyophilized materials at the bottom of the tube. The tubes are labeled with sample ID and placed in a bench top chiller.

The following steps are performed with a single reaction tube open at a time: The lid of the first reaction tube is opened. A 50 μL aliquot of the 2 ng/μL purified gDNA sample (prepared as described in Examples 2 and 3) into the reaction tube. Contact is avoided between the pellet and pipette tip, while dispensing the solution. The lid of the first reaction tube is closed. The process is repeated for each reaction tube. Once DNA is added to each reaction tube, the tubes are gently tapped, for example, 2-3 times to mix solutions. The tubes are briefly centrifuged to collect contents at the bottom of the tubes. The tubes are placed into the block of the paused thermal cycler and program is resumed.

Index 2 Barcode Adapter Addition:

The Index 2 adapter reaction packet (ILLUMINA) is removed along with the Adapter Ligation reaction packet, and both are allowed to reach room temperature. The INDEX 2 barcode adapter reaction packet is opened and the 8 tube-strip is removed. Each reaction contains a unique Index 2 barcode (1 through 48). It is ensured that each sample is placed into the appropriate reaction tube. If necessary, the tube strip is briefly centrifuged to collect all lyophilized materials at the bottom of the tube. The tubes are labeled with sample ID and placed in a bench top PCR tube chiller. A 50 μL aliquot of fragmented gDNA (from Laboratory 2) is transferred into the Barcode Adapter tubes. Care is taken to avoid touching the lyophilized pellet with pipette tip while dispensing solution. The tube lids are closed securely and the tubes are gently tapped 2-3 times to mix. The tubes are centrifuged briefly and returned to a bench top chiller. If all eight tubes are not utilized, the unused tube(s) are labeled with appropriate adapter number (1-8) and returned to the refrigerator.

Adapter Ligation:

The red 8-tube strip is removed from the Adapter ligation reaction packet. If necessary, the tubes are centrifuged briefly to collect all lyophilized materials at the bottom of the tube. The tubes are labeled with sample ID and placed in bench top PCR tube chiller. A 50 μL aliquot of fragmented DNA with Index 2 Barcode Adapters is transferred into the tubes containing Adapter Ligation mix. Care is taken to avoid touching the lyophilized pellet with pipette tip while dispensing solution. The lids are closed securely and the tubes are gently tapped tubes 2-3 times to mix. The tubes are centrifuged briefly and returned to a bench top chiller. The tube strip is placed in the thermal cycler and the reaction is incubated, as described in Example 7, and exemplified in Table 9.

TABLE 9 Adapter Ligation Incubation Step Incubation Temperature Incubation Time 1 16° C. 30 min 2 22° C. 30 min 3  4° C. Hold Heated Lid Off or Not Closed

Post-Adapter Ligation Purification:

It is ensured that AMPURE XP beads are at room temperature. The tubes are removed from the thermal cycler and briefly centrifuged. The samples are not placed in a bench top chiller, as this purification step occurs at room temperature. The AMPURE XP beads are vortexed for a brief time, for examples, for 15 seconds for thorough re-suspension. A 40 μL volume of AMPURE XP beads is added to each 50 μL reaction for a ratio of 0.8×. All caps are secured and the tubes are vortexed for 2-3 seconds. The tubes are incubated room temperature for 5 minutes. The tubes are placed on magnet for 4 minutes or until solution is clear. The supernatant is carefully pipetted and discarded without disturbing the beads. The beads are washed twice with 200 μL of 70% ethanol while on the magnet (the strip is moved on magnet to thoroughly wash beads). The 70% ethanol, used for washing, is freshly prepared weekly. After the second wash, it is ensured that all solution is removed from tubes and the beads are allowed to dry for 6 minutes at room temperature. The tubes are removed from the magnet and thoroughly re-suspended in 24 μL of 10 mM Tris-HCl. The 10 mM Tris-HCl is freshly prepared weekly. The tubes are placed tubes back on the magnet for 2 minutes.

If it is determined that the second wash is a stopping point, then 24 μL of purified solution is carefully transferred into new 200 μL PCR tubes and stored at −20° C. The tubes are labeled with sample ID.

If it is determined that the second wash is not a stopping point, then the lids of the tubes are securely closed and the tubes are transferred into the PCR workstation, after proper UV irradiation, to prepare for a first PCR.

First PCR:

UV light is activated for decontamination of the PCR workstation, for 15 minutes prior to PCR setup. It is ensured that all necessary supplies and equipment are present before activating UV light. DNA samples and PCR reagents are not placed into the workstation until after UV irradiation. The UV decontamination step is recorded in the LIMS.

The First PCR Reagents are removed from freezer and allowed to thaw in a bench top chiller. The enzyme (PHOENIX HS TAQ POLYMERASE) is kept in the freezer until utilized and always kept in a −20° C. bench top chiller while outside the freezer. Following UV decontamination, the laminar flow is activated. The purified DNA and the First PCR Reagents are moved into the PCR workstation. The operator changes gloves to prevent contamination in the PCR hood.

The tubes are labeled with sample ID and placed in a bench top chiller. The following reagents are added into each tube while in a bench top chiller. A PCR1 master mix is created when preparing multiple samples, as exemplified in Table 10.

TABLE 10 Master Mix preparation for First PCR First PCR Reagent Volume (μL) 5X Phoenix Buffer 4 2 mM dNTP 2 10 μM P5_1 2 Primers GC-content Low GSP-1 (100 μM) 2 OR Primers GC-content High GSP-1 (100 μM) Phoenix HS Taq Polymerase 0.4 Nuclease Free Water 0.6 TOTAL VOLUME 11

The tubes containing purified DNA from previous step are placed onto a magnet. A 9 μL aliquot of purified DNA is transferred from Adapter Ligation into two sets of First PCR reaction tubes (GC-content High and GC-content Low). Each set of tubes is labeled with the sample ID and GC-content. The reactions are mixed by gently pipetting up and down 10 times. The lids of the tubes are closed securely and the tubes are centrifuged briefly.

The PCR tubes are placed in the thermal cycler and the PCR reaction is incubated, as described in Example 7, and exemplified in Table 11.

TABLE 11 First PCR Incubation Incubation Incubation Temperature Time # of cycles 94° C. 30 sec 1 94° C. 30 sec 20 60° C. for GC-content Low samples 90 sec OR 64° C. for GC-content High samples (ramp rate of 2.3° C./sec) 72° C. 3 min 1  4° C. HOLD 1 Heated Lid on at 105° C.

After the First PCR is set up, all equipment remains in the PCR workstation for decontamination. All surfaces and equipment are wiped with RNASE Away. Any waste that leaves the workstation is put in a waste bag and the waste bag is tightly closed with a rubber band.

Post-First PCR Purification:

It is ensured that AMPURE XP beads are at room temperature. The tubes are removed from the thermal cycler and briefly centrifuged. The samples are not placed in a bench top chiller, as this purification step occurs at room temperature. The AMPURE XP beads are vortexed for a brief time, for example, for 15 seconds for thorough re-suspension. A 16 μL volume of AMPURE XP beads is added to each 20 μL First PCR reaction for a ratio of 0.8×, and the reaction mixture is pipetted 10 times to mix. The lids are secured and the tubes are incubated room temperature for 5 minutes. The tubes are placed on magnet for 4 minutes or until solution is clear. The supernatant is carefully pipetted and discarded without disturbing the beads The beads are washed twice with 200 μL of 70% ethanol while on the magnet (the strip is moved on magnet to thoroughly wash beads). The 70% ethanol, used for washing, is freshly prepared weekly. After the second wash, it is ensured that all solution is removed from tubes and the beads are allowed to dry for 5 minutes at room temperature. The tubes are removed from the magnet and thoroughly re-suspended in 9 μL of 10 mM Tris-HCl. The 10 mM Tris-HCl is freshly prepared weekly. The tubes are placed tubes back on the magnet for 2 minutes.

If it is determined that the second wash is a stopping point, then 9 μL of purified DNA solution is carefully transferred into 200 μL PCR tubes and stored at −20° C. The tubes are labeled with sample ID and GC-content Low or High.

If it is determined that the second wash is not a stopping point, then the lids of the tubes are securely closed and the tubes are transferred into the PCR workstation, after proper UV irradiation, to prepare for a second PCR.

Second PCR:

UV light is activated for decontamination of the PCR workstation, for 15 minutes prior to PCR setup. It is ensured that all necessary supplies and equipment are present before activating UV light. DNA samples and PCR reagents are not placed into the workstation until after UV irradiation.

The Second PCR Reagents are removed from freezer and allowed to thaw in a bench top chiller. The enzyme (PHOENIX HS taq polymerase) is kept in the freezer until utilized and always kept in a bench top chiller while outside the freezer. Following UV decontamination, the laminar flow is activated. The purified DNA and the Second PCR Reagents are moved into the PCR workstation. The operator changes gloves to prevent contamination in the PCR hood.

The tubes are labeled with sample ID and placed in a bench top chiller. The following reagents are added into each tube while in a bench top chiller. A master mix is created when preparing multiple samples, as exemplified in Table 12.

TABLE 12 Master Mix preparation for Second PCR Second PCR Reagent Volume (μL) 5X Phoenix Buffer 4 2 mM dNTP 2 10 μM P5_2 2 Primers GC-content Low GSP-2 (100 μM) 2 OR Primers GC-content Low GSP-2 (100 μM) Phoenix HS Taq Polymerase 0.4 Nuclease Free Water 0.6 10 μM P7 Index (#1-12) 2 TOTAL VOLUME 11

The tubes containing purified DNA from previous step are placed onto a magnet. A 7 μL aliquot of purified DNA is transferred from Adapter Ligation into each GC-content Low or High Second PCR reaction tube. The reaction is mixed by gently pipetting up and down 10 times. The lids of the tubes are closed securely and the tubes are centrifuged briefly.

The GC-content Low and High PCR tubes are placed in the thermal cycler and the PCR reactions are incubated, following the procedure described in Example 7, and exemplified in Table 13.

TABLE 13 PCR 2 Incubation Incubation Incubation Temperature Time # of cycles 94° C. 30 sec 1 94° C. 30 sec 20 60° C. for GC-content Low samples 90 sec OR 64° C. for GC-content High samples (ramp rate of 2. 3° C./sec) 72° C. 3 min 1  4° C. HOLD 1 Heated Lid on at 105° C.

Library Normalization: Following the second PCR, the individual samples are set to the same concentration via bead normalization.

Samples are treated with Exonuclease I to remove unwanted single-stranded DNA (ssDNA) and primer extension is performed on the completed AMP libraries.

A master mix containing the following reagents is prepared:

VoL per Concentration Component Reaction (μL) Protected P5/P7 capture 0.192 8 nM oligonucleotides (1 μM stock, diluted in 1X Phoenix Taq Buffer) 10 mM dNTPs 0.4 0.167 mM 5x Phoenix Buffer 0.8 1x H2O 0.66 20 units/μL Exonuclease I 2 1.67 units/μL

4 uL of the master mix is added to each 20 uL post-second PCR reaction and incubated according to the condition listed below:

Incubation Conditions 37° C. 30 min 94° C.  5 min 55° C. 30 min 72° C.  1 min  4° C. hold

A buffer solution is prepared by combining the following reagents:

Volume to add (for 10 mL Component total Vol.) Working Concentration 5M NaCl 2 mL 1M 1M Tris pH 100 μl 10 mM 8.0 0.5M EDTA 20 μL  1 mM Tween 10 μL 0.1%

Following the exonuclease treatment and primer extension, the samples are removed from the thermal cycler and placed in a benchtop chiller.

To perform streptavidin bead equilibration, the magnetic beads are resuspended by thorough vortexing. 10 uL of beads are removed from resuspended bead stock and placed into an empty PCR tube. The tube containing the beads is placed on a magnet for one minute and then the supernatant is discarded. The beads are washed by suspending with 20 uL of the previously prepared buffer solution. The samples are placed on the magnet and supernantant is removed and discarded. The wash is repeated two more times for a total of three washes. Resuspend in 10 uL of the Buffer solution and allow to sit off of the magnet.

0.2M NaOH is freshly prepared and the HT1 Buffer is thawed.

Equal volumes of each library are combined into a new PCR tube. 48 μL of the combined pool is transferred into a new PCR tube. The following amounts of each sample are combined depending on the primer set utilized:

Vol. per High # of Libraries to pool GC sample (μL) Vol. per Low GC sample (μL) 10 × 2 5.9 4.1

10 uL of the beads are added to the 48 uL of combined-sample pool and mixed well with pipetting. The mixture is incubated for 15 minutes at room temperature with intermittent mixing to resuspend the beads. Following the incubation, the tubes are briefly spun down and then placed on the magnet. After the beads migrate to the magnet, the supernatant is removed and discarded. The beads are washed on the magnet with 50 uL of the buffer solution by moving the tubes from one side of the magnet to the other. Once the beads have migrated to the magnet, the supernant is removed and discarded. The wash is repeated twice for a total of three washes.

The samples are briefly spun down and any residual supernantant is removed. The beads are resuspended in 15 uL of the freshly prepared 0.2 M NaOH off the magnet and incubated at room temperature for 10 minutes. The tubes are gently flicked one to two times during incubation to mix the beads. While sample is incubating, 185 uL of HT1 Buffer is added to a new tube.

After the 10 minute incubation, the tube containing the beads is placed on the magnet until the supernatant is clear. All 15 ul of the supernatant is removed and placed into the tube containing the HT1 Buffer. Now the normalized, denatured, and diluted pool is stored at −20±5° C. if needed.

Example 6 Procedure for Use of the ILLUMINA MISEQ Sequencer in an Exemplary Method

Provided herein is an example protocol for the use of the ILLUMINA MISEQ sequencer. This protocol is used to perform targeted gene sequencing on patient samples following gDNA extraction, as described in Example 2, and sample library preparation, as described in Example 5. The reagents and kits used in the protocol are, for example, the PhiX control (10 NM), the MISEQ V2 reagent kit which include a reagent cartridge (stored at −20° C.), hybridization buffer (HT1) (Stored at −20° C.), PR2 bottle (stored at 4° C.), and flow cell (stored at 4° C.).

Sample Sheet Preparation (Data Tracking System):

A fresh solution of 0.2 N NaOH is prepared to properly denature samples. Once prepared, the Nao dilution is stable for 12 hours.

PhiX Control Preparation:

The 10 NM Phi Control is thawed in a bench top chiller. The hybridization buffer (HT1) is thawed at room temperature. The HT1 is stored at 4° C. until ready to use.

PhiX Denaturation:

A 4 nM dilution of PhiX control is prepared by combining the following, 10 nM PhiX Control (2 μL), 10 mM Tris-HCl, pH 8.0 with 0.1% tween 20 (3 μL). The solution is pipette up and down to mix. The following are combined to denature the 4 nM PhiX Control, 4 nM PhiX Control (5 μL), 0.2 N NaOH (5 μL), Vortex to mix and centrifuge briefly. The mixture is incubated at room temperature for 5 minutes to denature. A 20 pM dilution of denatured PhiX is prepared by combining the following, HT1 (990 μL), 4 nM denatured PhiX Control (10 uL). This 20 pM PhiX stock is stored at −20° C. for up to 21 days for multiple uses.

If performing a MISEQ sequencer Run: A 11-14 pM dilution of denatured PhiX is prepared by combining the following, HT1 (45 μL) and 20 pM denatured PhiX Control (45 μL). The final volume of PhiX dilution should be 90 μL.

Reagent Cartridge Thaw:

After the library pool is equilibrated to a 2-6 nM solution as described in Example 5, the reagent cartridge (MISEQ sequencer V2 Reagent Kit Box 1 of 2) is thawed in a room temperature water bath for one hour. Care is taken to ensure that he water bath level does not exceed the “max fill” mark indicated on the side of the cartridge. If the reagent cartridge is thawed prior to library preparation completion, the reagent cartridge can be placed at 4° C. for up to 24 hours.

Final Library Pool Preparation:

The tubes are vortexed to mix and centrifuged briefly.

Preparation of 11-14 pM Library: A 600 μL volume of diluted library, with a concentration of 11-14 pM, is prepared by adding an appropriate volume of hybridization buffer (HT1) to dilute, according to the following equation:

Volume of 13 pM Library ( µL ) = ( 600 µL ) ( 13 pM ) Denatured Pool Conc . ( pM )

Preparation of 11-14 pM Library with 15% PhiX: A diluted library, with a concentration of 11-14 pM, is prepared by combining the following, 11-14 pM Library (510 μL, 10 pM denatured PhiX control (90 μL. The PhiX control is freshly diluted to 11-14 pM. The final dilution of the PhiX control must equal the final library concentration.

The tubes are vortexed to mix and microcentrifuged briefly.

Loading Reagent Cartridge:

After cartridge reagents have thawed, they are slowly inverted 10 times to mix. The foil of well 17 is punctured with a new pipette tip, in preparation for library loading. 600 μL of the 11-14 pM Library with 15% PhiX is loaded into the well. The cartridge is firmly tapped on the table to ensure all air pockets are removed from the bottom of the cartridge wells.

Starting a MISEQ Sequencer Run:

The MISEQ control software is initiated and the Welcome screen appears. The “Sequence” option is selected to proceed with. Appropriate account information is used for logging in for Base space monitoring.

Preparation of the Flowcell:

The flowcell is removed (Box 2 of 2) and rinsed thoroughly with nuclease-free water. All moisture on the plastic of the flowcell is removed delicate task wipe. The glass of the flow cell is cleaned using an alcohol wipe or 70% ethanol solution. Care is taken to avoid leaving residue or streaks. It is recommended to use only lens paper on the glass of the flow cell to avoid any damage to the flow cell surface. Before loading the flow cell, the flow cell stage is briefly wiped with an alcohol wipe or 70% ethanol. Ensure no excess moisture, streaks or debris is present between the lanes of the flow cells before loading it onto the stage of the MISEQ sequencer. The flow cell is loaded carefully the cover is gently latched before closing the flow cell compartment door.

Loading PR2 Wash Solution:

The wash solution bottle in the left position of the reagent chiller is removed and replaced it with the new PR2 bottle (Box 2 of 2). The waste container is emptied prior to starting each individual run. Liquid waste is collected in the waste container is disposed of in a liquid hazardous waste container due to the presence of formamide in Well 8 of the MISEQ V2 cartridge. It is ensured that the sippers are lowered into the appropriate container and have no obstructions.

Loading the Reagent Cartridge:

The wash cartridge from the MISEQ sequencer is removed and replaced with the recently loaded reagent cartridge.

Sample Sheet Recognition:

If the sample sheet is saved correctly in the designated folder, the software recognizes and uploads the information. If the sample sheet is not recognized it is due to improper sample sheet naming, wrong location of the saved sample sheet, or formatting error of the sample sheet.

Pre-Run Check:

Once the sample sheet is loaded, the MISEQ sequencer automatically begins a pre-run check. Once the pre-run check is successfully completed, the next step is to proceed with “Sequence” to begin the run. The date of the sequencing run is recorded on Appendix 1.

Post-Run Wash:

The post-run wash is the standard instrument wash performed between sequencing runs and consists of a single wash cycle. The instrument automatically prompts the user to perform a post-run wash using the following steps: a 10% tween solution is prepared by combining, 5 mL 100% tween 20 (5 mL, lab grade water (45 mL, a 0.5% tween solution by combining, 10% tween solution (25 mL, lab grade water (475 mL). The 0.5% tween solution is added to each reservoir of the wash tray. A 50 mL volume of 0.5% tween solution is added to the modified wash solution bottle. When the sequencing run is complete, “Start Wash” is selected to initiate the post-wash run. The wash tray and modified wash solution bottle are inserted. It is ensured that the waste bottle is empty. (see hazardous waste warning, above). The subsequent step is to select “Next” to begin the post-wash run. The date of the post-wash run is recorded on Appendix 1.

Example 7 Procedure for the APPLIED BIOSYSTEMS VERITI Thermal Cycler

Provided herein is an example protocol for the general operation, maintenance, and calibration of the APPLIED BIOSYSTEMS VERITI thermal cycler. The VERITI thermal cycler is used during the sample library preparation process, as described in Example 5. The VERITI thermal cycler is used for DNA fragmentation, adapter ligation, as well as the first and second PCR processes. The library preparation process, including use of the Veriti system, is internally validated during the MISEQ sequencer instrument validation.

Performing a Run:

Samples are prepared per the sample preparation protocol, described in Example 5, and placed in a bench top cooler. The cover is closed and Browse/New Methods are touched. The desired run method is located and selected. The reaction volume is edited and/or cover temperature, if necessary. It is ensured that the cover temperature is properly heated to the desired temperature prior to loading samples into the thermal cycler. The unique Run ID is entered. A run is initiated by pressing by pressing Start Run Now. The sample temperature is monitored until it reaches the desired Stage 1 temperature. For example: If Stage 1 requires a temperature of 95° C., monitor the sample temperature until the desired temperature is reached. When desired temperature is reached, the prepared sample tubes from the bench top chiller are immediately loaded into sample block. If a single sample tube strip is processed, an empty tube strip is inserted next to the sample tube strip to ensure that the thermal cycle cover does not damage the sample tube strip.

Monitoring a Run:

The run screen displays run details including the temperature, time, and current run stage. A status report which is displayed at the bottom of the screen displays any errors that occurred during the run. If necessary, the run is paused or stopped.

Run Report:

The run report is displayed at the end of the run. The run report is saved until the next run is finished and must be saved or printed if documentation is required.

Example 8 Validation Plan for the ILLUMINA MISEQ Sequencer Instrument

Provided herein is an example validation plan to validate the performance of the ILLUMINA MISEQ sequencer instrument to identify genomic variants in human DNA samples in comparison to a reference genome.

For Purposes of the Validation Plan:

False Negative (FN) is defined as Negative result (no variant found) when test is positive (variant present).

False Positive (FP) is defined as Positive result (variant found) when test is negative (no variant is present).

Precision is defined as the closeness of agreement between values obtained by replicate measurements on the same or similar objects under specified conditions.

Within-Run Variability (Repeatability) is defined as the degree to which the same sequence is derived when sequencing the same reference sample many times under the same conditions.

Between-Run (Reproducibility) is defined as the degree to which the same sequence is derived when sequencing the same reference sample many times under variable conditions (multiple days and/or multiple operators).

Sensitivity is defined as the ability to detect all confirmed variants in a sample (true test result) if the variant is present.

Specificity is defined as the proportion of samples that have a negative test result when no variant is present.

True Negative (TN) is defined as Negative result (no variant found) when test is negative (variant not present).

True Positive (TP) is defined as Positive result (variant found) when test is positive (variant is present).

TABLE 14 Known Sample Panel (Coriell Institute) Coriell Targeted Institute Gene Condition/Disease Gene Mutation ID 1 ACADM Medium-Chain Deficiency of Acyl- 985A > G/Lys304Glu NA07523 CoA Dehydrogenase 2 ACADM Medium-Chain Deficiency of Acyl- c. 166G > C/Ala56Pro NA11319 CoA Dehydrogenase 3 ACADS Short-Chain Deficiency of Acyl- 319C > T/Arg107Cys NA17470 CoA Dehydrogenase 4 BCKDHA Maple Syrup Urine Disease Type 8-bp deletion in exon 7 NA00651 (BCKAD) 1A 5 CFTR Cystic Fibrosis c. 1811G > C/Arg560Thr NA11284 Phe508Del 6 CFTR Cystic Fibrosis c. 1496C > A/Ala455Glu NA08211 Phe508Del 7 CFTR Cystic Fibrosis Phe508DEL NA00897 c. 1172C > G/Arg347Pro 8 CYP21A2 Adrenal Hyperplasia 30kb deletion of CYP21A2 NA14734 c. 292G > C 9 GALT Galactosemia - not affected Asp98His NA01212 10 GALT Galactosemia c. 2744A > G Asn314Asp NA01996 11 GALT Galactosemia c.563A > G/Gln188Arg NA00422 c. 1030C > A/Gln344Lys 12 GALT Galactosemia c. 855G > T/Lys285Asn NA00149 13 GBA Gaucher 1448T> C/Leu444Pro NA10915 14 GBA Gaucher c. 1226A > G/Asn370Ser NA00372 84GG 15 GLA Fabry c. 485G > A/Trp162Ter NA00107 16 GLA Fabry c. 658C > T NA00881 17 NPC1 Niemann-Pick C1 c. 709C > T/Pro237Ser NA03124 c. 3182T > C/Ile1061Thr 18 PAH Phenylketonuria 896T > G/Phe299Cys NA02406 1222C > T/Arg408Trp 19 PAH Phenylketonuria c. 755G > A/Arg252Gln NA01565 20 SLC22A5 Primary Carnitine Deficiency No mutations in SLC22A5 NA10665 21 CYP2C19 Unknown c.681G > A NA12878 40bpDEL

Validation Overview:

The validation and verification are performed in multiple stages to evaluate accuracy, sensitivity, and specificity, as defined above.

Validation Plan:

Triplicate Sample Analysis: Coriell institute samples (NA12878) are prepared and analyzed in triplicate via the standard library preparation, as described in Example 5, and sequencing procedures, as described in Examples 5 and 6. The variant results acquired are compared to the human reference genome (GRCh37) and variant results are comprised using the ARCHER Pipeline. Variant results acquired are filtered and analyzed according to the data processing procedure. Final variant results are also analyzed using the GetRM database, as well.

Validation Criteria:

The following validation plan is performed per the established library preparation and MISEQ sequencer operation standard operating procedures, as described in Examples 5 and 6 respectively. Deviations to the established protocols are reported in the final validation report.

Overall MISEQ Sequencer Performance Requirements:

To be considered acceptable and included in the validation, all sample data collected are going to meet the following criteria: 80% bases are going to demonstrate a Q-score of at least 30 or above.

Sensitivity:

Sensitivity is evaluated through the analysis of samples with known genomic variants (true positives). Coriell sample NA12878 will be used to perform the initial sensitivity analysis. Additional Coriell samples will be analyzed, as needed, to reach the minimum variant requirement. Identified variants will be compared to high quality variant data available through the Get-RM database (including Sanger sequenced data) to accurately identify true positive and false negative results. Sensitivity will be calculated using the following formula: (TP/TP+FN)×100=% Sensitivity

Acceptance criteria: To be considered acceptable, at least 85% or higher sensitivity must be achieved with 95% confidence in the analyzed sample set.

Specificity:

Specificity will be evaluated through the analysis of samples with positions of no known variation within the targeted genomic region (true negative). Coriell sample NA12878 will be used to perform the initial specificity analysis. Additional samples will be analyzed, as needed, to reach the minimum variants requirement. As specified in the verification overview, additional samples with known variant(s) will be analyzed blindly to ascertain the sample based on the identified variant(s). Specificity will be calculated using the following formula:


(TN/TN+FP)×100=% Specificity.

Acceptance criteria: To be considered acceptable, at least 85% or higher specificity must be achieved with 95% confidence in the analyzed sample set.

Repeatability:

Repeatability is assessed through the library preparation and sequencing of three (N=3) replicate samples (uniquely indexed) in a single sequencing run. The Archer pipeline analysis is used to call all genomic variants in comparison to the human reference genome. All detected variants for each replicate sample are then compared to determine the closeness of results between replicate samples in the same sequencing run.

Acceptance criteria: All variant calls must agree 90% between replicate samples.

Reproducibility:

Reproducibility is assessed through the library preparation and sequencing of three (N=3) replicate samples (uniquely indexed) by multiple (>2) operator(s) on at least three processing days. The Archer pipeline analysis is used to call all genomic variants in comparison to the human reference genome. All detected variants for each replicate sample are then compared to determine the closeness of results between replicate samples across the multiple sequencing runs.

Acceptance criteria: All variant calls must agree 90% between replicate samples.

Example 9 Gene Sequencing Newborn Screening Report

Provided herein is an example of a gene sequencing newborn screening report.

TABLE 15 Client information for gene sequencing newborn screening Client Name* MH Specimen Type* BLOOD, DBS Parent Name Not Provided Date Specimen 5JUN2014 Collected* BG Sample ID* BGTEST_19 Date Specimen 10JUN2014 Received* Date of Birth* 28DEC1984 Report Date* 7JUL2014 Gender* FEMALE Referring Physician Not Provided Race/Ethnicity* CAUCASIAN Physician Phone Not Provided Number Birth Weight Not Provided Referring Facility Not Provided *Required fields

The test performed is NGS newborn screening panel. The indications for test include, for example, identification of pathogenic or likely pathogenic genetic variants correlated with correlated with clinical metabolic conditions Newborn Screening Panel.

TABLE 16 Test results for a normal screen Variant Variant Protein Chromosomal Gene Classification Identified Change Zygosity Location ACAD8 VOUS* NM_014384.2: p. Ser171Cys Hetero 134128923 c. 512C > G ACADS Pathogenic NM_000017.2: p. Gly209Ser Hetero 121176083 c. 625G > A CPT1A VOUS* NM_001031847.2 p. Ala275Thr Hetero 68562328 c. 823G > A *Variant of unknown significance

Interpretation Summary:

The test results for targeted gene sequencing and variant analysis of genes associated with newborn screening metabolic conditions, as exemplified in Table 16, did not identify pathogenic or likely pathogenic variants. On average, the targeted amplicons in this sample are covered at 87.0%. A report containing pathogenic or likely pathogenic variants indicates that a variant has been identified in the client sample that has been published as being associated with a specific condition. A report containing variant(s) identified as known pathogenic or likely pathogenic is not a diagnosis of a specific metabolic condition. It is recommended that any variants reported as VOUS be periodically reviewed to ensure the clinical significance has not changed. The laboratory does not provide medical advice and recommends that the client contact their physician or medical provider to discuss the results of this test.

In some embodiments, a physician, medical provider, or genetic counselor review the gene variant results reported in the laboratory report. Because the test is not a diagnostic test; a pathogenic or likely pathogenic gene variant is not a diagnosis of a medical condition.

A medical provider may request reanalysis of the genomic variant data for the presence of any pathogenic or likely pathogenic variants that is linked to disorders identified since the date of the report, as exemplified in Table 16 is linked to the client's phenotype based on currently available scientific information.

Test Method:

Genomic DNA is extracted from the submitted specimen and the NBS Panel reagent kit is used to target specific exonic regions of the specimen's genome. These targeted regions are sequenced using lumina sequencing technology with 2×150 bp paired-end reads. The DNA sequence is mapped to, and analyzed in comparison with, the published human genome build UCSC hg19 reference sequence. The targeted coding exons are assessed for average depth of coverage and data quality scores. All sequence variants are compared to the Gene Variant list to identify pathogenic and likely pathogenic variants based on current, published genomic data.

Table 17 exemplifies representative metrics from the client's targeted gene sequencing. Mean depth of coverage refers to the sequence mean read depth across the targeted region, defined as coding exons of the NBS panel reagent kit coding NBS Gene Panel. The quality score is logarithmically related to the base calling error probabilities. A quality score of 30 (Q30) is equivalent to the probability of an incorrect base in 1 in 1000 times, or a base call accuracy of 99.9%. Quality scores must meet the following minimum criteria (>80%>Q30).

TABLE 17 *The values below represent metrics from the client's targeted gene sequencing: Mean Depth of Coverage1 TBR Quality Score (% >Q30) TBR %

NBS Gene Panel: Targeted genes are correlated with metabolic conditions according to the American College of Medical Genetics (ACMG) recommendations. Exonic coding regions of the following genes are targeted in the NBS Gene Panel: ABCD1, ACAD8, ACADM, ACADS, ACADSB, ACADVL, ACAT1, ADA, AHCY, ARG1, ASL, ASS1, AUH, BCKDHA, BCKDHB, BTD, CBS, CFTR, CPT1A, CPT2, CYP21A2, DBT, DECR1, DLD, DNAJC19, DUOX2, EFTA, EFTB, EFTDH, FAH, GAA, GALC, GALE, GALK1, GALT, GBA, GCDH, GCH1, GJB2, GJB3, GJB6, GLA, GNMT, HADH, HADHA, HADHB, HBA1, HBA2, HBB, HLCS, HMGCKM HPD, HSD17B10, IDUA, IL2RG, IVD, MAT1A, MCCC1, MCCC2, MCEE, MLYCD, MMAA, MMAB, MMADHC, MTHFR, MTR, MTRR, MUT, NPC1, NPC2, OPA3, PAH, PAX8, PCBD1, PCCA, PCCB, PTS, QDPR, SLC22A5, SLC25A13, SLC25A20, ALC5A5, TAT, TAZ, TG, TPO, TSHB, TSHR. Limitations: Exonic coding regions of the targeted gene panel are sequenced and analyzed in the NBS Gene Panel; intronic regions are not targeted. The NGS Newborn Screen ensures, for example, 95% coverage of the targeted gene panel; due to inherent limitations in the biological testing system, a TBD % risk of false-negative or false-positive results exists within the test system. Some types of the genetic abnormalities, such as copy number changes, are detectable with the technologies performed by this analysis test. It is possible that the genomic coding region where a mutation exists in the proband is not captured using the current technologies and therefore is not detected. In this example, variants categorized as pathogenic or likely pathogenic are indicated on the client report, variants of unknown significance, likely benign, and benign are not reported. The variant(s) indicated on this example are identified based on current scientific information and are updated as new information becomes available.

Example 10 Sample Processing

Provided herein is an example of the sample handling and screening process the newborn screening. A blood sample is collected from a newborn within 24-48 hours of birth by a standard method such as heel puncture and collected on filter paper resulting in a dried blood spot (DBS) sample. The sample is shipped to a testing laboratory where the newborn screening method is conducted. Upon receipt of the sample by the testing facility, genomic DNA is extracted and purified from the DBS sample using a nucleic acid purification kit (e.g., CHARGESWITCH nucleic acid purification kit (Life Technologies)). Each sample of extracted and purified gDNA is quantified (e.g., using the QUBIT 2.0 fluorometer (Life Technologies) and a dsDNA High Sensitivity (HS) (Life Technologies) kit). gDNA concentrations are normalized prior to library preparation (e.g., to a concentration of 2 ng/μL). The library preparation process is then conducted, which includes the following steps: DNA fragmentation, A-tailing and end repair, adapter ligation, and amplification via PCR steps (e.g., using the lyophilized version of ARCHER DNA assay kit). Targeted sequencing is then conducted on the prepared library using a DNA sequencer to screen for one or more gene variants comprising, identifying a gene variant by sequencing at least one target region of each of gene PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8 in genomic DNA from the newborn infant, wherein the sequencing does not include whole genome sequencing or whole exome sequencing. A report is provided to the newborn's parent or caregiver, which provides a list of gene variants identified in the sample, for those variants categorized as pathogenic or likely pathogenic are indicated on the client report.

Example 11 Newborn Screening Process—Uniform Screening Panel

Provided herein is an example of the process flow for the newborn screening. A blood sample is collected from a newborn within 24-48 hours of birth. The sample is shipped to a testing laboratory where the newborn screening method is conducted. The sample from the newborn is screened for one or more gene variants comprising, identifying a gene variant by sequencing at least one target region of each of gene PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, and ACAD8 in genomic DNA from the newborn infant, wherein the sequencing does not include whole genome sequencing or whole exome sequencing. A report is provided to the newborn's parent or caregiver, which provides a list of gene variants identified in the sample, for those variants categorized as pathogenic or likely pathogenic are indicated on the client report.

Example 12 Newborn Screening Process—Core Conditions Panel

Provided herein is an example of the process flow for the newborn screening. A blood sample is collected from a newborn within 24-48 hours of birth. The sample is shipped to a testing laboratory where the newborn screening method is conducted. The sample from the newborn is screened for one or more gene variants comprising, identifying a gene variant by sequencing at least one target region of each of gene PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, ACAD8, MCCC1, MCCC2, HMGCL, HLCS, GCDH, SLC22A5, HADHB, ASS1, CBS, MTHFR, MTR, MTRR, MMADHC, PAH, FAH, DUOX2, PAX8, SLC5A5, TG, TPO, TSHB, TSHR, HBB, BTD, CFTR, GJB2, GJB3, GJB6, ADA, and IL2RG in genomic DNA from the newborn infant, wherein the sequencing does not include whole genome sequencing or whole exome sequencing. A report is provided to the newborn's parent or caregiver, which provides a list of gene variants identified in the sample, for those variants categorized as pathogenic or likely pathogenic are indicated on the client report.

Example 13 Newborn Screening Process—Core and Secondary Conditions Panel

Provided herein is an example of the process flow for the newborn screening. A blood sample is collected from a newborn within 24-48 hours of birth. The sample is shipped to a testing laboratory where the newborn screening method is conducted. The sample from the newborn is screened for one or more gene variants comprising, identifying a gene variant by sequencing at least one target region of each of gene PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, ACAD8, MCCC1, MCCC2, HMGCL, HLCS, GCDH, SLC22A5, HADHB, ASS1, CBS, MTHFR, MTR, MTRR, MMADHC, PAH, FAH, DUOX2, PAX8, SLC5A5, TG, TPO, TSHB, TSHR, HBB, BTD, CFTR, GJB2, GJB3, GJB6, ADA, IL2RG, MLYCD, ACADSB, AUH, DNAJC19, OPA3, TAZ, HSD17B10, ACADS, HADH, ETFA, ETFB, ETFDH, DECR1, CPT1A, CPT2, SLC25A20, ARG1, SLC25A13, AHCY, GNMT, MAT1A, PAH, GCH1, PCBD1, PTS, QDPR, TAT, HPD, HBA1, HBA2, HBB, GALE, and GALK1 in genomic DNA from the newborn infant, wherein the sequencing does not include whole genome sequencing or whole exome sequencing. A report is provided to the newborn's parent or caregiver, which provides a list of gene variants identified in the sample, for those variants categorized as pathogenic or likely pathogenic are indicated on the client report.

Example 12 Newborn Screening Process—Core, Secondary, and Added Conditions Panel

Provided herein is an example of the process flow for the newborn screening. A blood sample is collected from a newborn within 24-48 hours of birth. The sample is shipped to a testing laboratory where the newborn screening method is conducted. The sample from the newborn is screened for one or more gene variants comprising, identifying a gene variant by sequencing at least one target region of each of gene PCCA, PCCB, MUT, MMAA, MMAB, MMADHC, MCEE, IVD, ACAT1, ACADM, ACADVL, HADHA, ASL, BCKDHA, BCKDHB, DBT, DLD, CYP21A2, GALT, ACAD8, MCCC1, MCCC2, HMGCL, HLCS, GCDH, SLC22A5, HADHB, ASS1, CBS, MTHFR, MTR, MTRR, MMADHC, PAH, FAH, DUOX2, PAX8, SLC5A5, TG, TPO, TSHB, TSHR, HBB, BTD, CFTR, GJB2, GJB3, GJB6, ADA, IL2RG, MLYCD, ACADSB, AUH, DNAJC19, OPA3, TAZ, HSD17B10, ACADS, HADH, ETFA, ETFB, ETFDH, DECR1, CPT1A, CPT2, SLC25A20, ARG1, SLC25A13, AHCY, GNMT, MAT1A, PAH, GCH1, PCBD1, PTS, QDPR, TAT, HPD, HBA1, HBA2, HBB, GALE, GALK1, GALC, GBA, NPC1, NPC2, GAA, GLA, IDUA, ABCD1, and NGLY1 in genomic DNA from the newborn infant, wherein the sequencing does not include whole genome sequencing or whole exome sequencing. A report is provided to the newborn's parent or caregiver, which provides a list of gene variants identified in the sample, for those variants categorized as pathogenic or likely pathogenic are indicated on the client report.

The examples and embodiments described herein are for illustrative purposes only and various modifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.

US Patent Application for METHOD OF SCREENING NEWBORNS FOR GENE VARIANTS Patent Application (Application #20160068906 issued March 10, 2016) (2024)
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