CaseCHN1.1: Canada, White

CasePresentingHPOs: HP:0008353 (Neutral hyperaminoaciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Variants segregate with the disease, Has a sibling with the same variant and amino acid profile

CasePreviousTesting: Hartnup aminoaciduria profile was confirmed by ion exchange high performance liquid chromatography;

CaseGenotypingMethod: All SLC6A19 exons and flanking introns were directly amplified and sequenced from genomic DNA.

Variant1: NM_001003841.3:c.196G>A (p.Gly66Arg)

CAID: CA359061114 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA359061114

gnomAD: Not in gnomAD

Variant2: NM_001003841.3:c.982C>T (p.Arg328Cys)

CAID: CA3182972, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA3182972

gnomAD: 0.0002479, https://gnomad.broadinstitute.org/variant/5-1216767-C-T

CaseAHN16.1: Australia, White

CasePresentingHPOs: HP:0008353 (Neutral hyperaminoaciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Variants segregate with the disease

CasePreviousTesting: Hartnup aminoaciduria profile was confirmed by ion exchange high performance liquid chromatography;

CaseGenotypingMethod: All SLC6A19 exons and flanking introns were directly amplified and sequenced from genomic DNA.

Variant1: NM_001003841.3:c.277G>A (p.Gly93Arg)

CAID: CA3182636 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA3182636

gnomAD: 0.0001195, https://gnomad.broadinstitute.org/variant/5-1208935-G-A

Variant2: NM_001003841.3(SLC6A19):c.517G>A (p.Asp173Asn)

ClinVar: 2019 https://www.ncbi.nlm.nih.gov/clinvar/variation/2019/

gnomAD: 0.003016, https://gnomad.broadinstitute.org/variant/5-1212453-G-A

Case# P1, Male, Chinese, 5 months

CasePresentingHPOs HP:0006986, HP:0002373, HP:0008947, HP:0001249

CaseMethod1 PCR

CaseGenotypingMethod PCR of sevon exons of QDPR gene

Variant NM_000320.2:c.661C>T(p.R221X), NM_000320.2:c.515C>T(p.P172L)

CAID CA2868005, CA2868085

gnomAD (Latino) 0.00002978, (Ashkenazi Jewish) 0.0001019

VariantEvidence null

CasePMIDs 19099731

Case# PA2

Variant NM_000709.3:c.409G>A(p.Glu137Lys)

CAID CA406007842

VariantEvidence none

Case# PA1

Variant NM_000709.3(BCKDHA):c.347A>G (p.Asp116Gly)

ClinVar 93354

VariantEvidence none

Case1: Male, 15 months onset, Iran

CasePresentingHPOs: HP:0006579, HP:0001324, HP:0001336, HP:0000639, HP:0002134, HP:0002282, HP:0002453, HP:0002540, HP:0002463, HP:0006801, HP:0001252, HP:0011443, HP:0001251, HP:0001310, HP:0000565, HP:0001510 (Neonatal jaundice, Muscle weakness, Myoclonus, Nystagmus, Abnormality of the basal ganglia, Heterotopia, Abnormality of the globus pallidus, Inability to walk, Language impairment, Hyperactive deep tendon reflexes, Muscular hypotonia, Abnormality of coordination, Ataxia, Dysmetria, Esotropia, Growth delay)

HPOsFreeText:

CaseNotHPOs: HP:0012443, HP:0012447 (Abnormality of brain morphology, Abnormal myelination)

NotHPOsFreeText:

CaseAddInfo: No parental consanguinity,

CasePreviousTesting: Plasma amino acids and plasma acylcarnitine profile were normal. The organic acid in the urine showed an elevated 3‐hydroxyisovaleric acid (110 mmol/mol creatinine, normal <44 mmol/mol creatinine). Serum biotinidase activity was normal. Blood lactate and ammonia were normal in repeated measurements.

CaseGenotypingMethod: Patient had the IVD exons and exon-intron boundaries analyzed using PCR/Sanger sequencing to follow up on suspected IVA, with no variants found. Whole Exome Sequencing was performed to determine the correct genetic cause of the disorder by evaluating all known exons and their flanking regions. This found two heterozygous missense mutations in HIBCH consistent with the diagnosis. Trio PCR/Sanger sequencing confirmed parental heterozygosity and the variants in the patient.

Variant1: NM_014362.3:c.641C>T (p.Thr214Ile)

CAID: CA349890265 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_014362.3%3Ac.641C%3ET

gnomAD: Not in gnomAD

Variant2: NM_014362.3:c.913A>G (p.Thr305Ala)

CAID: CA62635843 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_014362.3%3Ac.913A%3EG

gnomAD: European (Non-Finnish) 1.767e-5, Overall 7.978e-6 https://gnomad.broadinstitute.org/variant/2-191077780-T-C

Case2: Male, Caucasian, 9 months onset

CasePresentingHPOs: HP:0011968, HP:0001270, HP:0001251, HP:0002376, HP:0002121, HP:0000737, HP:0002317, HP:0002599, HP:0002078, HP:0012389, HP:0001310, HP:0001337, HP:0001254, HP:0001942, HP:0002329, HP:0001308, HP:0002599, HP:0001332, HP:0002181, HP:0002134 (Poor feeding, Delayed motor milestones, Ataxia, Developmental regression, Absence seizures, Irritability, Unsteady gait, Head titubation, Truncal ataxia, Appendicular hypotonia, Dysmetria, Tremor, Lethargy, Metabolic acidosis, Drowsiness, Tongue fasciculation, Head titubation, Dystonia, Cerebral edema, Abnormality of the basal ganglia)

HPOsFreeText: Head bobbing at 4 months, Rolled over at 5 mo but lost the ability at 10 mo, Reaching out and grasping objects at 4 mo but lost the ability at 13 mo, Eye rolling episodes at 9 months, Lost ability to finger feed at 10 months, Inability to sit unsupported, At 14 months blood pH was 7.29 with a base deficit of 15.8 mmol/L, Sluggish papillary responses, Pooling of secretions in the mouth, No improvement on treatment with thiamine or a combination of vitamin C, vitamin E, and ubiquinone, Signal abnormalities in the globus pallidus and the midbrain

CaseNotHPOs: HP:0000639, HP:0002151, HP:0001987, HP:0003116, HP:0012443 (Nystagmus, Increased serum lactate, Hyperammonemia, Abnormal echocardiogram, Abnormality of brain morphology)

NotHPOsFreeText: N/A

CaseAddInfo: Parents were nonconsanguinous, Proband is compound heterozygous for these mutations with parental testing

CasePreviousTesting: Pyruvate dehydrogenase and pyruvate carboxylase in cultured human skin fibroblasts, Activities of respiratory chain complexes in a muscle biopsy, Marked reduction of complex I (patient 0.089, controls 0.104–0.268) and a borderline reduction of complex IV (patient 0.013, controls 0.014–0.034), Organic acid analysis during ketotic episodes showed excessive excretion of 3-hydroxybutyrate and acetoacetate and moderate excretion of lactate, 2-hydroxyisovalerate, 2-oxoisocaproate, dicarboxylic acids (C6, C8, C10), and methylmalonate, with normal levels of methylcitrate as opposed to normal levels when well, Urine amino acid analysis, Elevated levels of hydroxy-C4-carnitine (patient 0.45-1.73 μM, controls <0.4 μM) even when not ketotic

CaseGenotypingMethod: After confirming low HICBH activity in fibroblasts of the patients, the gene was analyzed at the genomic level and the mRNA level in both patients using sequence analysis.

Variant1: NM_014362.4:c.365A>G (p.Tyr122Cys)

ClinVar: 1145 https://www.ncbi.nlm.nih.gov/clinvar/variation/1145/

gnomAD: Latino 4.338e-4, Overall 6.373e-5 https://gnomad.broadinstitute.org/variant/2-191155151-T-C

Variant2: NM_014362.3:c.79-3C>G

ClinVar: 1146 https://www.ncbi.nlm.nih.gov/clinvar/variation/1146/

gnomAD: Not in gnomAD

Case8: Female

CasePresentingHPOs: HP:0001270, HP:0001252, HP:0011342, HP:0007479 (Motor delay, Muscular hypotonia, Mild motor developmental delay, Congenital nonbullous ichthyosiform erythroderma)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Consanguineity is suspected, Proband is homozygous

CasePreviousTesting: Patient fibroblasts were analyzed for mutations in SLC25A21 and activities of KATII with no abnormalities; Urine AAA and KAA were elevated. Oxidation of radiolabeled AAA in fibroblasts was deficient.

CaseGenotypingMethod: All 17 exons and flanking intronic sequences of DHTKD1 were Sanger sequenced.

CasePMIDs: 1112064, 1112065

Variant: NM_018706.7(DHTKD1):c.2134C>T (p.Arg712Ter)

ClinVar: 572241 https://www.ncbi.nlm.nih.gov/clinvar/variation/572241/

gnomAD: 0.00003267, https://gnomad.broadinstitute.org/variant/10-12149994-C-T

Case6: Female, Japan, Asian

CasePresentingHPOs: HP:0001290, HP:0003324, HP:0002093, HP:0011968, HP:0001283, HP:0003798, HP:0001561, HP:0001558, HP:0001789, HP:0000252, HP:0001622, HP:0001371 (Generalized hypotonia, Generalized muscle weakness, Respiratory insufficiency, Feeding difficulties, Bulbar weakness, Nemaline bodies, Polyhydramnios, Decreased fetal movement, Hydrops fetalis, Microcephaly, Preterm delivery, Contractures)

HPOsFreeText: Atrophic myofibers

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Severe congenital myopathy, Alive at 10 months

CasePreviousTesting: N/A

CaseGenotypingMethod: After LMOD3 was identified as a potential causal gene, bidirectional Sanger sequencing was performed on DNA in genetically unresolved patients using PCR amplification covering 3 exons.

Variant: NM_198271.4:c.349C>T (p.Gln117*)

CAID: CA353477271 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_198271.4%3Ac.349C%3ET

gnomAD: Not in gnomAD

Case3: Female, Portugal

CasePresentingHPOs: HP:0001290, HP:0003324, HP:0002093, HP:0011968, HP:0001283, HP:0002804, HP:0001558, HP:0001561, HP:0001371 (Generalized hypotonia, Generalized muscle weakness, Respiratory insufficiency, Feeding difficulties, Bulbar weakness, Decreased fetal movement, Polyhydramnios, Contracture)

HPOsFreeText: N/A

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Severe congenital myopathy, Deceased in neonatal period

CasePreviousTesting: N/A

CaseGenotypingMethod: After LMOD3 was identified as a potential causal gene, bidirectional Sanger sequencing was performed on DNA in genetically unresolved patients using PCR amplification covering 3 exons.

Variant: NM_198271.4:c.154del (p.Met52*)

CAID: CA909810717 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_198271.4%3Ac.154delA

gnomAD: Not in gnomAD

Case12a: Male, Afghanistan

CasePresentingHPOs: HP:0001290, HP:0003324, HP:0002093, HP:0011968, HP:0001283, HP:0002804, HP:0001558, HP:0001622, HP:0001623, HP:0003798 (Generalized hypotonia, Generalized muscle weakness, Respiratory insufficiency, Feeding difficulties, Bulbar weakness, Arthrogryposis, Decreased fetal movement, Preterm delivery, Breech presentation, Nemaline bodies)

HPOsFreeText: Atrophic myofibers

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Severe congenital myopathy, Deceased in 2 months, Has 2 siblings with same genotype/phenotype

CasePreviousTesting: N/A

CaseGenotypingMethod: After LMOD3 was identified as a potential causal gene, bidirectional Sanger sequencing was performed on DNA in genetically unresolved patients using PCR amplification covering 3 exons.

Variant: NM_198271.4:c.1099_1100delAA (p.Asn367Glnfs*11)

ClinVar: 162218 https://www.ncbi.nlm.nih.gov/clinvar/variation/162218/

gnomAD: South Asian 4.575e-4, Overall 5.625e-5 https://gnomad.broadinstitute.org/variant/3-69168405-GTT-G

Case14a: Female, Australia

CasePresentingHPOs: HP:0001290, HP:0003324, HP:0002093, HP:0011968, HP:0001283, HP:0001561, HP:0001622, HP:0002804, HP:0003798, HP:0001558, HP:0000602 (Generalized hypotonia, Generalized muscle weakness, Respiratory insufficiency, Feeding difficulties, Bulbar weakness, Polyhydramnios, Nemaline bodies, Decreased fetal movements, Ophthalmoplegia)

HPOsFreeText: Atrophic myofibers, Percutaneous endoscopic gastrostomy performed, Nocturnal noninvasive ventilation

CaseNotHPOs: HP:0001627, HP:0002540 (Cardiac abnormality, Inability to walk)

NotHPOsFreeText: N/A

CaseAddInfo: Typical congenital nemaline myopathy, Alive at 10 yrs, Parents are not consanguineous

CasePreviousTesting: N/A

CaseGenotypingMethod: Linkage analysis combined with homozygosity mapping was used to identify candidate chromosomal loci. Whole exome sequencing in this family revealed likely pathogenic mutations in LMOD3 which agreed with the aforementioned analysis and was consistent with autosomal recessive inheritance.

Variant1: NM_198271.4:c.1103_1105del (p.Asn368del)

ClinVar: 162216 https://www.ncbi.nlm.nih.gov/clinvar/variation/162216/

gnomAD: Not in gnomAD

Variant2: NM_198271.4:c.1201C>T (p.Arg401Ter)

ClinVar: 162217 https://www.ncbi.nlm.nih.gov/clinvar/variation/162217/

gnomAD: African 4.137e-5, Overall 7.135e-6 https://gnomad.broadinstitute.org/variant/3-69168305-G-A

Case5: Female, Japan, Asian

CasePresentingHPOs: HP:0001290, HP:0003324, HP:0002093, HP:0011968, HP:0001283, HP:0003798, HP:0001561, HP:0001558, HP:0100309 (Generalized hypotonia, Generalized muscle weakness, Respiratory insufficiency, Feeding difficulties, Bulbar weakness, Nemaline bodies, Polyhydramnios, Decreased fetal movements, Subdural hemorrhage)

HPOsFreeText: Atrophic myofibers

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Severe congenital myopathy, Alive at 2 months

CasePreviousTesting: N/A

CaseGenotypingMethod: After LMOD3 was identified as a potential causal gene, bidirectional Sanger sequencing was performed on DNA in genetically unresolved patients using PCR amplification covering 3 exons.

Variant1: NM_198271.4:c.300_304delGACTC (p.Thr101Argfs*4)

CAID: CA913203453 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA913203453

gnomAD: Not in gnomAD

Variant1: NM_198271.4:c.601_602delGA (p.Asp201Glnfs*9)

CAID: CA913203454 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA913203454

gnomAD: Not in gnomAD

CaseST-8: Female, 1 day onset, Germany

CasePresentingHPOs: HP:0006579, HP:0001643, HP:0001410, HP:0001319, HP:0006236, HP:0001786 (Prolonged neonatal jaundice, Patent ductus arteriosus, Decreased liver function, Neonatal hypotonia, Slender metacarpals, Narrow foot)

HPOsFreeText: N/A

CaseNotHPOs: HP:0001250 (Seizure)

NotHPOsFreeText: N/A

CaseAddInfo: Family is non-consanguineous, Proband is compound heterozygous for these variants

CasePreviousTesting: See Group

CaseGenotypingMethod: See Group

Variant1: NM_001123.3:c.893C>A (p.Ala298Asp)

CAID: CA377399006, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA377399006

gnomAD: Not in gnomAD

Variant2: NM_001123.3:c.925C>T (p.Gln309Ter)

CAID: CA377398330, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA377398330

gnomAD: Not in gnomAD

CaseST-6: Male, 1 day onset, Morocco

CasePresentingHPOs: HP:0006579, HP:0001249, HP:0001270, HP:0001324, HP:0004322, HP:0000256, HP:0000316, HP:0006236, HP:0001786, HP:0008070, HP:0000708, HP:0002355, HP:0003202, HP:0002071 (Prolonged neonatal jaundice, Intellectual disability, Motor delay, Muscle weakness, Short stature, Macrocephaly, Hypertelorism, Slender metacarpals, Narrow foot, Sparse hair, Behavioral abnormality, Difficulty walking, Skeletal muscle atrophy, Abnormality of extrapyramidal motor function)

HPOsFreeText: N/A

CaseNotHPOs: HP:0001250 (Seizure)

NotHPOsFreeText: N/A

CaseAddInfo: Family is consanguineous, Proband is homozygous for the variant

CasePreviousTesting: See Group

CaseGenotypingMethod: See Group

Variant: NM_001123.3:c.685_687delinsCC (p.Thr229ProfsTer16)

CAID: CA1139768931, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA1139768931

gnomAD: Not in gnomAD

Case 15 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: PCR

CaseGenotypingMethod: same as group

__

Variant1: NM_015506.2(MMACHC):c.394C > T (p.Arg132Ter)

ClinVar: 1423

CAID: CA251787

gnomAD: 0.0007190 (South Asian)

Variant2: NM_015506.2(MMACHC):c.615C > A (p.Tyr205Ter)

ClinVar: 558292

CAID: CA340133688

gnomAD: 0.0006427 (East Asian)

CaseAddInfo: proband genotype, unknown cis or trans, no phenotypic info, sibling (fetus) carries both variants

Case 13 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: PCR

CaseGenotypingMethod: same as group __

Variant1: NM_015506.2(MMACHC):c.609G > A (p.Trp203Ter)

ClinVar: 30800

CAID: CA259906

gnomAD: 0.0005562 (East Asian)

CaseAddInfo: proband genotype, homozygous, no phenotypic info, sibling (fetus) carrier for p.W203X variant

Case 16 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: PCR

CaseGenotypingMethod: same as group

Variant1: NM_015506.2(MMACHC):c.217C > T (p.Arg73Ter)

ClinVar: 203825

CAID: CA312728

gnomAD: 0.00005560 (East Asian)

CaseAddInfo: proband genotype, homozygous, no phenotypic info, sibling (fetus) is a carrier of variant

Case 5 (Level 2)

**CasePreviousTesting: ** same as group

CaseMethod1: PCR

CaseGenotypingMethod: same as group

Variant1: NM_015506.2(MMACHC):c.626dupT (p.Thr210Aspfs*35)

ClinVar: not in ClinVar

CAID: Not in ClinGen

gnomAD: not in gnomAD

Variant2: NM_015506.2(MMACHC):c.609G > A (p.Trp203Ter)

ClinVar: 30800

CAID: CA259906

gnomAD: 0.0005562 (East Asian)

CaseAddInfo: proband genotype, unknown cis or trans, no phenotypic info, sibling (fetus) carrier for c.626dupT maternal variant

Case 6 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: PCR

CaseGenotypingMethod: same as group

Variant1: NM_015506.2(MMACHC):c.658_660delAAG (p.Lys220del)

ClinVar: 95707

CAID: CA223197

gnomAD: 0.0003071 (East Asian)

Variant2: NM_015506.2(MMACHC):c.609G > A (p.Trp203Ter)

ClinVar: 30800

CAID: CA259906

gnomAD: 0.0005562 (East Asian)

CaseAddInfo: proband genotype, unknown cis or trans, no phenotypic info, sibling (fetus) carrier for both variants

Case4: Female, 2 wks at initation of follow up, European ancestry

HPOsFreeText: NBS C4-acylcarnitine 2.0 umol/L, Plasma C4-acylcarnitine 1.52 umol/L

CaseAddInfo: no data for urine excretion, unremarkable clinical condition

Case4 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_014384.2:c.443C>T (p.Pro148Leu)

ClinVar: n/a

CAID: CA6372945

gnomAD: not in gnomAD

Variant2: NM_014384.2(ACAD8):c.455T>C (p.Met152Thr)

ClinVar: 5355

CAID: CA117433

gnomAD: 0.0001395 (European Non-Finnish)

CaseAddInfo: compound heterozygote, indicated alleles 1 and 2 but not specified maternal and paternal, potential splice site variant of ACAD8 at exon3 (+3A>G)

Case3a: Male, 5d at initiation of follow up, East Indian Ancestry, sibling of Case 3b (family 1)

HPOsFreeText: NBS C4-acylcarnitine 2.4 umol/L, Plasma C4-acylcarnitine 1.14 umol/L, in vitro probe isoC4-acylcarnitine 0.133 umol/ g protein,

NotHPOsFreeText: normal urine isobutyrylglycine ug/mg creatinine

CaseAddInfo: unremarkable clinical condition

Case3a (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant: NM_014384.2(ACAD8):c.867C>A (p.His289Gln)

ClinVar: 5358

CAID: CA117441

gnomAD: not in gnomAD

CaseAddInfo: homozygous mutations

Case9: Male, 2 mo at initiation of follow up, Hispanic ancestry

HPOsFreeText: NBS C4-acylcarnitine 1.8 umol/L, Plasma C4-acylcarnitine 3.28, umol/L, in vitro probe isoC4-acylcarnitine 0.074 umol/g protein, urine C4-acylcarnitine 7.86 mmol/mol creatinine, Urine C4/C3 ratio 63

NotHPOsFreeText: normal urine isobutyrylglycine

CaseAddInfo: unremarkable clinical condition

Case9 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant: NM_014384.2(ACAD8):c.233T>C (p.Met78Thr)

ClinVar: 430160

CAID: CA6372883

gnomAD: n/a

CaseAddInfo: homozygous variants, no potential splice site alterations, no data for ACADS (SCAD-deficient) gene variants

Case2: Female, 3 m at initiation of follow up, European ancestry

HPOsFreeText: NBS C4-acylcarnitine 2.5 umol/L, plasma C4-acylcarnitine 10.00 umol/L, isoC4-acylcarnitine 0.120 umol/g protein, in vitro prob C4-acylcarnitine 0.220 umol/ g protein

CaseAddInfo: no data for urine excretion, unremarkable clinical condition

Case2 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_014384.2(ACAD8):c.289G>A (p.Gly97Arg)

ClinVar: 281037

CAID: CA6372897

gnomAD: 0.0002175 (East Asian)

Variant2: NM_014384.2(ACAD8):c.455T>C (p.Met152Thr)

ClinVar: 5355

CAID: CA117433

gnomAD: 0.0001395 (European Non-Finnish)

CaseAddInfo: compound heterozygous, potential splice-site alteration on ACAD8 at Exon 3 (+3A>G), homozygous for two common ACADS (SCAD-deficient) gene variants (625G>A/625G>A) which has 22% frequency in general population and correlates with increased accumulation of butyrylcarnitine

Case467 Caucasian

CasePresentingHPOs HP:0003571 (Propionicacidemia)

CaseNotHPOs n/a

CasePreviousTesting none reported

Variant 1. NM_000532.4(PCCB):c.1218_1231delGGGCATCATCCGGCinsTAGAGCACAGGA (p.Gly407Argfs), 2. NM_000532.4:c.1222del (p.Ile408SerfsTer?)

ClinVar 1.38875, 2. n/a

CAID 2. unregistered

gnomAD 1. not in gnomAD

CaseAddInfo parents NOT tested, phase unknown

CasePMIDs n/a

Case# 18804, Spanish, 3yo report

CaseHPOs asymmptomatic

Variant NM_000709.3:c.647C>T, NM_000709.3:c.889C>T

CAID CA9461230(c.647), CA9461294(889)

gnomAD c.647=0.0001332(African), 0.00002463(Overall), c.889=0.00006534(African), 0.00001624(Overall)

VariantEvidence reduced BCKD activity to 30% of normal in patient fibroblasts

CaseAddInfo in trans

Case# 20493, Spanish, 1yo reported

CasePresentingHPOs HP:0008344

CasePreviousTesting categorized into classical and variant phenotypes based on BCKD deficiency assessed by measuring the L-[1-14C] leucine decarboxylation rate by intact cells in absence of BCKD kinase inhibitors (Duran, et al., 1978), plasma amino acid levels, and mode of clinical presentation

CaseMethod1 Direct Sequencing

CaseMethod2 PCR

CaseGenotypingMethod direct sequencing of PCR fragments obtained after amplification of the entire coding sequence of BCKDHA, BCKDHB or DBT genes. sequenced both strands (forward and reverse) of corresponding exons and their intronic flanking sequence for confirmation.

Variant NM_000709.3:c.757G>A, NM_000709.3:c.848G>A, NM_000709.3:c.898G>A

CAID CA9461255(757), [CA406013082(848)&CA406013351(898)]

gnomAD c.757=0.00006681(European(Non-Finnish)), 0.00003236(Overall)

VariantEvidence Patient fibroblasts had <1% of normal BCKD activity

CaseAddInfo parents genotypes confirmed (in trans)

Case# 5888, Spanish, 10mo death

Variant NM_000709.3(BCKDHA):c.940C>T (p.Arg314Ter), NM_000709.3(BCKDHA):c.1037G>A (p.Arg346His)

ClinVar 224072(c.940), 93336(c.1037)

gnomAD c.940=0.000008958(European (Non-Finnish)), 0.000004064(Overall)

CaseAddInfo not confirmed in trans

Case# 16243, Spanish, 5yo reported

CasePresentingHPOs HP:0008344, HP:0001315

CasePreviousTesting categorized into classical and variant phenotypes based on BCKD deficiency assessed by measuring the L-[1-14C] leucine decarboxylation rate by intact cells in absence of BCKD kinase inhibitors (Duran, et al., 1978), plasma amino acid levels, and mode of clinical presentation

CaseMethod1 Direct Sequencing

CaseMethod2 PCR

CaseGenotypingMethod direct sequencing of PCR fragments obtained after amplification of the entire coding sequence of BCKDHA, BCKDHB or DBT genes. sequenced both strands (forward and reverse) of corresponding exons and their intronic flanking sequence for confirmation.

Variant NM_000709.3(BCKDHA):c.117delC (p.Arg40Glyfs), NM_000709.3(BCKDHA):c.117dupC (p.Arg40Glnfs)

ClinVar 93342(del), 166741(dup)

gnomAD del=0.0007494(African), 0.0001696(Overall)

CaseAddInfo in trans

CasePMIDs duplication variant in 8037208

Case23 5yo diagnosis, Anglo-Celtic

CasePresentingHPOs HP:0001083, HP:0001256, HP:0002751, HP:0000766

(Lens dislocation, Mild intellectual disability, Kyphoscoliosis, Abnormality of the sternum)

HPOsFreeText Patient was diagnosed on the basis of dislocated lenses. Treatment began with 100-200mg/day for two weeks followed by an addition of 5mg/day folic acid. Patient was not responsive to the treatment, plasma homocysteine levels went unchanged. Patient was given additional oral betaine(3g twice per day) with a low protein diet

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting Free homocystine, methionine, cystine, and cysteine-homo-cysteine mixed disulfide levels were measured by amino acid analyzers and total (free plus bound)homocysteine (tHcys) was measured. Exact values were not provided.

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified then direct sequenced the entire coding region of CBS.

Variant 1. NM_000071.2(CBS):c.430G>A (p.Glu144Lys), 2. IVS1+1G>A, compound heterozygous

ClinVar 1. 122, 2. n/a

CAID 2. need more info

gnomAD 1. MAF=0.00009799(South Asian), no homozygotes, 2. n/a

CaseAddInfo n/a

CasePMIDs n/a

FamilyLabel Family 1

Family1PresentingHPOs n/a

HPOsFreeText plasma free homocystine was reduced from >80μmol to <10μmol (nv <60μmol) in response to treatment with folate and pyridoxine. Treatment began with 100-200mg/day for two weeks followed by an addition of 5mg/day folic acid.

FamilyNotHPOs HP:0001249

(Intellectual disability)

NotHPOsFreeText n/a

CountryOrigin n/a

Ethnicity Anglo-Celtic

Race n/a

Family1PreviousTesting Free homocystine, methionine, cystine, and cysteine-homo-cysteine mixed disulfide levels were measured by amino acid analyzers and total (free plus bound)homocysteine (tHcys) was measured. Exact values were not provided.

FamilyMethod1 PCR

FamilyMethod2 Sanger

FamilyGenotypingMethod PCR amplified then direct sequenced the entire coding region of CBS.

ProbandLabel Case 1

Variant NM_000071.2(CBS):c.833T>C (p.Ile278Thr), homozygous

ClinVar 120

gnomAD MAF=0.001432(European(non-Finnish)), no homozygotes

FamilyAddInfo n/a

FamilyPMIDs n/a

Case13 8yo diagnosis, Anglo-Celtic

CasePresentingHPOs HP:0001083, HP:0000767

(Lens dislocation, Pectus excavatum)

HPOsFreeText borderline intellectual disability. Diagnosis was based on dislocated lenses. Treatment began with 100-200mg/day for two weeks followed by an addition of 5mg/day folic acid. This patient had a partial response to the treatment. Homocystine levels were sharply reduced on pyridoxine and folate treatment, but did not levels did not fall below 20μmol (nv <60μmol) on this treatment alone.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting Free homocystine, methionine, cystine, and cysteine-homo-cysteine mixed disulfide levels were measured by amino acid analyzers and total (free plus bound)homocysteine (tHcys) was measured. Exact values were not provided.

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified then direct sequenced the entire coding region of CBS.

Variant 1. NM_000071.2(CBS):c.430G>A (p.Glu144Lys), 2. NM_000071.2(CBS):c.1111G>A (p.Val371Met), compound heterozygous

ClinVar 1. 122, 2. 212862

gnomAD 1. MAF=0.00009799(South Asian), no homozygotes, 2. MAF= 0.00006219(African), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

FamilyLabel Family 2

Family2PresentingHPOs n/a

HPOsFreeText plasma free homocystine was reduced from >80μmol to <10μmol (nv <60μmol) in response to treatment with folate and pyridoxine. Treatment began with 100-200mg/day for two weeks followed by an addition of 5mg/day folic acid.

FamilyNotHPOs HP:0001249

(Intellectual disability)

NotHPOsFreeText no other sequelae

CountryOrigin n/a

Ethnicity Anglo-Celtic

Race n/a

Family2PreviousTesting Free homocystine, methionine, cystine, and cysteine-homo-cysteine mixed disulfide levels were measured by amino acid analyzers and total (free plus bound)homocysteine (tHcys) was measured. Exact values were not provided.

FamilyMethod1 PCR

FamilyMethod2 Sanger

FamilyGenotypingMethod PCR amplified then direct sequenced the entire coding region of CBS.

ProbandLabel Case 3

Variant 1. NM_000071.2(CBS):c.833T>C (p.Ile278Thr), 2. NM_000071.2(CBS):c.302T>C (p.Leu101Pro), compound heterozygous

ClinVar 1. 120, 2. 189185

gnomAD 1. MAF=0.001432(European(non-Finnish)), no homozygotes, 2. not in gnomAD

FamilyAddInfo n/a

FamilyPMIDs n/a

Case27 6yo diagnosis, Anglo-Celtic

CasePresentingHPOs HP:0001083, HP:0001256, HP:0002616, HP:0006280, HP:0002583

(Lens dislocation, Mild intellectual disability, Aortic root dilation, Chronic pancreatitis, Colitis)

HPOsFreeText Patient diagnosed on the basis of presenting marfanoid features. Patient had chronic pancreatitis requiring TPN and colitis requiring colostomy. Final CT scan of patient looking like multi-infarct dementia. Treatment began with 100-200mg/day for two weeks followed by an addition of 5mg/day folic acid. Patient was not responsive to the treatment, plasma homocysteine levels went unchanged. Patient was given additional oral betaine(3g twice per day) with a low protein diet

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting Free homocystine, methionine, cystine, and cysteine-homo-cysteine mixed disulfide levels were measured by amino acid analyzers and total (free plus bound)homocysteine (tHcys) was measured. Exact values were not provided.

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified then direct sequenced the entire coding region of CBS.

Variant 1. NM_000071.2:c.684C>A (p.Asn228Lys), 2. IVS11+1G>A, comp het

ClinVar 1. n/a, 2 n/a

CAID 1. CA410600617, 2. not enough info

gnomAD 1. MAF=0.00001759(European(non-Finnish)), no homozygotes, 2. n/a

CaseAddInfo n/a

CasePMIDs n/a

CaseLE12: Female, 23 yrs report

CasePresentingHPOs: HP:0002725 (Systemic lupus erythematosus)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Variant 1 homozygous, with variant 2 in-cis with one copy confirmed via parental testing

CasePreviousTesting: Specific plasma MMA levels, urine MMA levels, urine MA levels, and MMA/MA ratio available in Table 2

CaseGenotypingMethod: See Group

Variant1: NM_001243279.3:c.1553C>A (p.Ala518Asp)

CAID: CA8238286, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA8238286

gnomAD: 0.00006153, https://gnomad.broadinstitute.org/variant/16-89212397-C-A

Variant2: NM_001243279.3:c.473C>T (p.Pro158Leu)

CAID: CA8237662, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA8237662

gnomAD: 0.0002823, https://gnomad.broadinstitute.org/variant/16-89167562-C-T

FamilyBJ1: Sweden

FamilyPresentingHPOs: HP:0003235, HP:0011344, HP:0001410, HP:0001508, HP:0001252, HP:0000256, HP:0002007, HP:0000316, HP:0001533, HP:0001250, HP:0002463, HP:0003323, HP:0003202, HP:0002910 (Hypermethioninemia, Severe global developmental delay, Decreased liver function, Failure to thrive, Muscular hypotonia, Macrocephaly, Frontal bossing, Hypertelorism, Slender build, Seizure, Language impairment, Progressive muscle weakness, Skeletal muscle atrophy, Elevated hepatic transaminase)

HPOsFreeText: N/A

FamilyNotHPOs: N/A

NotHPOsFreeText: N/A

FamilyGenotypingMethod: The siblings were subject to whole exome sequencing. Variants were extracted, aligned, and filtered to build a list of candidate genes. These were filtered through a series of restrictions until only five candidate genes were left, with ADK being the only one related to methionine metabolism. To confirm the results, Exon 10 of ADK was Sanger sequenced in the family members and the variant was confirmed in a heterozygous state in both parents.

FamilyPreviousTesting: Parents are nonconsanguineous and healthy; Maximum plasma Met values were 455 and 886 μmol/liter, respectively (reference range: 15–35 μmol/liter); Elevated AdoMet and AdoHcy; Sequency analysis of AHCY and enzyme activity was negative for SAHH deficiency; More detailed test values in Table 1

FamilyAddInfo: N/A

Variant: NM_006721.4(ADK):c.953C>A (p.Ala318Glu)

ClinVar: 29603, https://www.ncbi.nlm.nih.gov/clinvar/variation/29603/

gnomAD: 0.00001761, https://gnomad.broadinstitute.org/variant/10-76430016-C-A

Affected: 2

Unaffected: 0

Segregations: 1

AddSegregationInfo: Not enough info

Case# GM-649, M

CaseMethod1 Sanger

CaseMethod2 PCR

Variant NM_000709.3:c.284C>A(p.Pro95His)

CAID CA406005099(p.Pro95His)

gnomAD p.Pro95His=0.0003330(African), 0.00002887(Overall)

VariantEvidence total BCKDH activity (4) in fibroblast extracts from the patient (GM649) is < 10% of normal (Table 1) in 2703538

CaseAddInfo examined patient fibroblasts here and in the previous article

CasePMIDs 2703538

RescuePatientCell CL:0000057

GeneAlteration Patient fibroblasts transfected with EBO vector containing either p.Tyr438Asn or WT cDNA.

RescuePhenotype HP:0008344

RescueMethod Measured decarboxylation rate of patient cells with various EBO vectors(empty, WT, or with variant) in comparison to an untransfected normal cell.

RescueEvidence Patient fibroblasts with the variant, empty vector, and vector containing the variant all showed virtually no decarboxylation activity. Vector containing WT restores decarboxylation rate to that of the normal cell (Figure 6). Figure 7 western blot shows that E1b subunit levels are also rescued.

RescueVariants NM_000709.3(BCKDHA):c.1312T>A (p.Tyr438Asn)

Case7: Male, 6m, Chinese

CasePresentingHPOs: HP:0002248, HP:0001290, HP:0008872, HP:0002572 (haematemesis, hypotonia, feeding difficulties in infancy, recurrent vomiting)

HPOsFreeText: (at 19 days) 1.01 C4 umol/L, 0.38 C4/C2, 1.61 C4/C3

NotHPOsFreeText: normal urine isobutyrylglycine

Case7 (Level 2):

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_014384.2:c.444G>T (p.Pro148=)

ClinVar: n/a

CAID: CA6372946

gnomAD: 0.0002772 (Other)

Variant2: NM_014384.2:c.1176G>T (p.Arg392Ser)

ClinVar: n/a

CAID: CA6373249

gnomAD: 0.0001088 (East Asian)

CaseAddInfo: trans heterozygous, inherited one allele from each parent

Case# Case 5, M, Chinese, 4day onset

CasePresentingHPOs HP:0008344, HP:0040168, HP:0008947, HP:0011968

CasePreviousTesting Tested blood aa levels with liquid chromatography-mass spectrum spectrometry, several branched chain amino acids were overly abundant. Whole exome targeted capture sequencing was applied for the mutation screening of the suspicious patients. A total of 712 genes associated with inherited metabolic diseases were selected and their coding regions were captured.

CaseMethod1 Whole Exome

CaseMethod2 Sanger

CaseGenotypingMethod validated whole exome findings with sanger and qPCR.

Variant NM_000056.3:c.863C>A(p.Ala288Asp), ?

CAID CA364660141 (miss), wait for follow up (exon del)

Case# Case 2, M, Chinese, 1day old Age of Onset

CasePresentingHPOs HP:0001276, HP:0000737, HP:0008344

CasePreviousTesting Tested blood aa levels with liquid chromatography-mass spectrum spectrometry, several branched chain amino acids were overly abundant. Whole exome targeted capture sequencing was applied for the mutation screening of the suspicious patients. A total of 712 genes associated with inherited metabolic diseases were selected and their coding regions were captured.

CaseMethod1 Whole Exome

CaseMethod2 Sanger

CaseGenotypingMethod validated whole exome findings with sanger and qPCR.

Variant NM_000056.3:c.523T>C

CAID CA364657780, exon1-7 del waiting on follow up

VariantEvidence in trans with massive deletion

PMID: 14742428

Gene: BCKDHB

Disease: MSUD, type 1a

MonDO: MONDO:0023691

InheritancePattern: AR

CaseOS-138_II-1: Female, 4 yrs report, Asian

CasePresentingHPOs: HP:0001992, HP:0002373, HP:0001263 (Organic aciduria, Febrile seizure (within the age range of 3 months to 6 years), Global developmental delay)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are consanguineous, Proband is homozygous for this variant

CasePreviousTesting: Routine urine screening for organic acidurias by gas chromatography–mass spectrometry showed markedly increased urinary excretion of several N-acetylated amino acids

CaseGenotypingMethod: PCR-amplification and bidirectional sequencing of all exons and exon-intron boundaries was performed on genomic DNA.

Variant: NM_000666.3(ACY1):c.589C>T (p.Arg197Trp)

ClinVar: 18113, https://www.ncbi.nlm.nih.gov/clinvar/variation/18113/

gnomAD: 0.00001764, https://gnomad.broadinstitute.org/variant/3-52021009-C-T

CaseOS-146_II-1: Male, 5 yrs report, Romani

CasePresentingHPOs: HP:0001992, HP:0000954, HP:0000431, HP:0000316, HP:0040194, HP:0004322, HP:0001250, HP:0010819, HP:0032792, HP:0002121, HP:0000752, HP:0002342 (Organic aciduria, Single transverse palmar crease, Wide nasal bridge, Hypertelorism, Increased head circumference, Short stature, Seizure, Atonic seizure, Tonic seizure, Generalized non-motor (absence) seizure, Hyperactivity, Moderate Intellectual disability)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are healthy consanguineous cousins, Two cousins and a niece of the mother have epilepsy, Proband is homozygous for this variant

CasePreviousTesting: Routine urine screening for organic acidurias by gas chromatography–mass spectrometry showed markedly increased urinary excretion of several N-acetylated amino acids; Cerebral MRI was normal at the ages of 17 months and 2.5 years

CaseGenotypingMethod: PCR-amplification and bidirectional sequencing of all exons and exon-intron boundaries was performed on genomic DNA.

Variant: NM_000666.3(ACY1):c.1057C>T (p.Arg353Cys)

ClinVar: 18110 https://www.ncbi.nlm.nih.gov/clinvar/variation/18110/

gnomAD: 0.004135 (4 homozygotes), https://gnomad.broadinstitute.org/variant/3-52022837-C-T

CaseSE5: Male, Australia

CasePresentingHPOs: HP:0008353 (Neutral hyperaminoaciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseGenotypingMethod: Five loci were investigated for evidence of linkage to Hartnup due to their established role in amino acid transport including SLC3A2, SLC7A8, SLC1A5, SLC6A18, and SLC6A19. These 5 regions were studied with microsattelite markers and produced linkage only between D5S392 and D5S417, the region with SLC6A18 and 19. SLC6A18 did not increase neutral amino acid uptake in X. laevis oocytes so SLC6A19 was investigated and directly sequenced in the families each of which displayed a causal variant.

CasePreviousTesting: SLC1A5 had no causative variants detected.

CaseAddInfo: Asymptomatic except for hyperaminoaciduria

Variant1: NM_001003841.3(SLC6A19):c.517G>A (p.Asp173Asn)

ClinVar: 2019 https://www.ncbi.nlm.nih.gov/clinvar/variation/2019/

gnomAD: 0.003016, https://gnomad.broadinstitute.org/variant/5-1212453-G-A?dataset=gnomad_r2_1

Variant2: NM_001003841.3(SLC6A19):c.718C>T (p.Arg240Ter)

ClinVar: 2020 https://www.ncbi.nlm.nih.gov/clinvar/variation/2020/

gnomAD: 0.0001960, https://gnomad.broadinstitute.org/variant/5-1213632-C-T

CaseF5P: 18 yrs report, India

CasePresentingHPOs: N/A

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband for Family 5, Variant has been shown to abolish canonical splice donor site and lead to a null protein

CasePreviousTesting: Blood phenylalanine before treatment was 1260 μmol/l, Blood tyrosine before treatment was 36 μmol/l

CaseGenotypingMethod: N/A

Variant: NM_000277.3(PAH):c.842+1G>A

ClinVar: 599 https://www.ncbi.nlm.nih.gov/clinvar/variation/599/

gnomAD: 0.00007752, http://gnomad.broadinstitute.org/variant/12-103246592-C-T

CaseF1S: 5 yrs report, India

CasePresentingHPOs: N/A

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Variant is demonstrated to activate a cryptic splice site and abolish the canon splice site leading to a premature termination and nonsense-mediated decay

CasePreviousTesting: Blood phenylalanine before treatment was 1431 μmol/l, Blood tyrosine before treatment was 35 μmol/l

CaseGenotypingMethod: N/A

Variant: NM_000277.3(PAH):c.169-2A>G

ClinVar: 555212 https://www.ncbi.nlm.nih.gov/clinvar/variation/555212/

gnomAD: 0.00003267, http://gnomad.broadinstitute.org/variant/12-103288698-T-C

CaseF2P: 2 yrs report, India

CasePresentingHPOs: HP:0001010, HP:0000750, HP:0001249, HP:0006801, HP:0004923 (Hypopigmentation of the skin, Delayed speech and language development, Intellectual disability, Brisk deep tendon reflexes, Hyperphenylalaninemia)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Family is consanguinous, Parents were confirmed heterozygous, Variant may not undergo NMD however would be missing essential domains and is predicted to be null

CasePreviousTesting: Blood phenylalanine before treatment was 1619 μmol/l, Blood tyrosine before treatment was 60 μmol/l, PKU diagnosis was based on ferric chloride test on patient urine samples and detection of elevated Phenylalanine levels in blood samples by using HPLC, in addition to analysis of clinical features.

CaseGenotypingMethod: Genomic DNA was isolated from blood samples of proband and family members. PCR amplification and sequencing was used to screen for mutations. Each mutation was confirmed by bi-directional DNA sequencing.

Variant: NM_000277.3:c.1177_1178insT (p.Asn393Ilefs*2)

CAID: CA16020958 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA16020958

gnomAD: Not in gnomAD

Patient18: Group 3

CasePresentingHPOs: same as group 3

CasePreviousTesting: same as group 3

CaseGenotypingMethod: same as group 3

Variant1: NM_002225.3:c.1136T>C (p.p.Ile349Thr) **listed in paper as (p.Phe350Ser)

CAID: CA391723904, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391723904

gnomAD: n/a

Variant2: IVS3+5G>T no ClinVar or CAID

Patient8: Group 2

CasePresentingHPOs: same as group 2

CasePreviousTesting: same as group 2

CaseGenotypingMethod: same as group 2

Variant1: NM_002225.3(IVD):c.941C>T (p.Ala314Val) listed in paper as c.932C>T (p.Ala282Val)

ClinVar: 100060, https://www.ncbi.nlm.nih.gov/clinvar/variation/100060/

gnomAD: 0.0006507, http://gnomad.broadinstitute.org/variant/15-40707653-C-T

Variant2: NM_002225.3:c.262G>T

CAID: CA391745812, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391745812

gnomAD: n/a

Case: Patient C, 11 years, female CaseHPOs: HP:0003235, HP:0011098, Hypermethioninemia, speech dyspraxia

CaseHPOsFreeText: “pronounced hypermethioninaemia on neonatal screening”, cleft lip and palate; Hypermethioninaemia has persisted; unpleasant odor with high blood concentrations of mercaptans; fine nystagmus, dysdiadochokinesis, and increased tendon reflexes led to brain MRI and CT at age 11 years which showed demyelination, calcification of basal ganglia, IQ 87; normal myelination and all symptoms resolved after 12 months treatment with S-adenosylmethionine toluene sulphonate.

CaseNotHPOs: Homocystine was not detected in plasma or urine,normal liver function tests.

CasePreviousTesting: Plasma methionine 600–1,400 umol/L; otherwise normal amino acids. 5-methyl tetrahydrofolate 329, 232 nmol/L (normal 23-170, mean 85+/-41), normalized after 12 months on S-adenosylmethionine toluene sulphonate; S-adenosylmethionine 70, 87 nmol/L (normal 172-450, mean 255 +/-69), improved but still low after 12 months on S-adenosylmethionine toluene sulphonate.

Variant: c.826dupG (homozygous)

ClinVar: N/A

CAID: CA5576692

gnomAD: The highest population minor allele frequency in gnomAD is 0.00003298 (S. Asian); no homozygotes in any population.

VariantEvidence: When the variant was expressed in bacteria, the amount and size of the protein was reduced on Western blot, and there was no detectable MAT1A activity.

CaseAddInfo: The mother is heterozygous for the variant; DNA testing results for the father are not available.

CasePMIDs: 1683972

Points: 2 points. This patient is homozygous for a frameshift variant in MAT1A. When the variant was expressed in bacteria, there was no detectable MAT activity. The highest population minor allele frequency in gnomAD is 0.00003298 (S. Asian); no homozygotes in any population.

CaseAI-1: N/A

CasePresentingHPOs: N/A

HPOsFreeText: Pretreatment phenylalanine level at 330 umol/L

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: N/A

Variant1: NM_000277.3(PAH):c.143T>C (p.Leu48Ser)

ClinVar: 608 https://www.ncbi.nlm.nih.gov/clinvar/variation/608/

gnomAD: 0.001025, https://gnomad.broadinstitute.org/variant/12-102912816-A-G?dataset=gnomad_r3

Variant2: NM_000277.3(PAH):c.898G>T (p.Ala300Ser)

ClinVar: 92751 https://www.ncbi.nlm.nih.gov/clinvar/variation/92751/

gnomAD: 0.006318, https://gnomad.broadinstitute.org/variant/12-102851701-C-A?dataset=gnomad_r3

CaseDI-1: N/A

CasePresentingHPOs: N/A

HPOsFreeText: Pretreatment phenylalanine level at 365 umol/L

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: N/A

Variant1: NM_000277.3(PAH):c.561G>A (p.Trp187Ter)

ClinVar: 102736 https://www.ncbi.nlm.nih.gov/clinvar/variation/102736/

gnomAD: 0.00001760, https://gnomad.broadinstitute.org/variant/12-103249059-C-T

Variant2: NM_000277.3(PAH):c.898G>T (p.Ala300Ser)

ClinVar: 92751 https://www.ncbi.nlm.nih.gov/clinvar/variation/92751/

gnomAD: 0.006318, https://gnomad.broadinstitute.org/variant/12-102851701-C-A?dataset=gnomad_r3

CaseAII-5: N/A

CasePresentingHPOs: N/A

HPOsFreeText: Pretreatment phenylalanine level at 440 umol/L

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: N/A

Variant1: NM_000277.3(PAH):c.165del (p.Phe55fs)

ClinVar: 611 https://www.ncbi.nlm.nih.gov/clinvar/variation/611/

gnomAD: 0.00002639, https://gnomad.broadinstitute.org/variant/12-103306571-CA-C?dataset=gnomad_r3

Variant2: NM_000277.3(PAH):c.898G>T (p.Ala300Ser)

ClinVar: 92751 https://www.ncbi.nlm.nih.gov/clinvar/variation/92751/

gnomAD: 0.006318, https://gnomad.broadinstitute.org/variant/12-102851701-C-A?dataset=gnomad_r3

CaseAII-3: N/A

CasePresentingHPOs: N/A

HPOsFreeText: Pretreatment phenylalanine level at 370 umol/L

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: N/A

Variant1: NM_000277.3(PAH):c.165del (p.Phe55fs)

ClinVar: 611 https://www.ncbi.nlm.nih.gov/clinvar/variation/611/

gnomAD: 0.00002639, https://gnomad.broadinstitute.org/variant/12-103306571-CA-C?dataset=gnomad_r3

Variant2: NM_000277.3(PAH):c.898G>T (p.Ala300Ser)

ClinVar: 92751 https://www.ncbi.nlm.nih.gov/clinvar/variation/92751/

gnomAD: 0.006318, https://gnomad.broadinstitute.org/variant/12-102851701-C-A?dataset=gnomad_r3

CaseC1: Female, Arab, Israel

CasePresentingHPOs: HP:0010896, HP:0010897, HP:0001638 (Hypersarcosinemia, Hypersarcosinuria, Cardiomyopathy)

HPOsFreeText: Elevated blood Sarcosine (345 μmol/l; reference 0-9), Elevated urine Sarcosine (481 μmol/mmol; reference 0-36)

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are consanguineous and first cousins, sister has the same genotype

CasePreviousTesting: N/A

CaseGenotypingMethod: DNA was extracted from the probands and immediate family. The whole coding region and the exon-intron boundaries of the SARDH gene was then amplified and sequenced.

Variant: NM_007101.3:c.211G>T (p.Val71Phe)

ClinVar: 39448 https://www.ncbi.nlm.nih.gov/clinvar/variation/39448/

gnomAD: Not in gnomAD

Case#: GA3, Turkey, 5 years (onset), gender unknown

CaseHPOs: HP:0012026 (hyperornithinemia)

CaseHPOsFreeText: Presented with visual disturbances at 5 years of age. No further details.

CaseNotHPOs: Neonatal encephalopathy

CasePreviousTesting: No previous DNA testing noted. Plasma ornithine at diagnosis 650umol/L (normal <100)

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: Sequence analysis of the OAT gene from fibroblast or lymphocyte cDNA and genomic DNA. Variant: NM_000274.3(OAT):c.991C>T (p.Arg331Ter) (homozygous)

ClinVar: 56141

gnomAD: The maximum population minor allele frequency in gnomAD is 0.0001446 (Latino); no homozygotes in any population.

Case27 sex unknown, Spain

CasePresentingHPOs HP:0006846, HP:0002540, HP:0001344, HP:0001332

(Acute encephalopathy, Non-ambulatory, Nonverbal, Dystonia)

HPOsFreeText Phenotype was classified as "severe," with passive or static dystonia and an inability to stand up, walk, or speak. Patient may have gone undetected by routine screening as glutarate excretion was normal and 3-hydroxyglutarate was only slightly increased.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 2(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 18(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 10.

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The V400M variant creates an NlaIII site. This would lead to cleaved patient DNA fragments and uncleaved control fragments.

Variant NM_000159.3(GCDH):c.1198G>A (p.Val400Met), homozygous

ClinVar 2088

gnomAD MAF= 0.0002769(Other), no homozygotes

CaseAddInfo n/a

CasePMIDs see note on previous studies

"proband disclosed a heterozygous 18 bp deletion (c.553_570del18) in GCDH (NM_000159) predicted to result in a six amino acids deletion (p.Gly185_- Ser190del)"

I don't see any patient carrying this variant in Table 1

Case38 sex unknown, Spain

CasePresentingHPOs HP:0006846, HP:0002540, HP:0001344, HP:0001332

(Acute encephalopathy, Non-ambulatory, Nonverbal, Dystonia)

HPOsFreeText Phenotype was classified as "severe," with passive or static dystonia and an inability to stand up, walk, or speak. Patient may have gone undetected by routine screens as glutarate excretion was normal and 3-hydroxyglutarate was only slightly increased.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 4(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 43(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 9. Patient tested negative in glutarylcarnitine blood spot test, even under carnitine supplementation.

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The Y155H variant creates a NcoI restriction site. Thus, while the patient's DNA fragments would cleave, control fragments would not be cleaved. The R227P variant creates a NlaIV site. This would lead to cleaved patient DNA fragments and uncleaved control fragments.

Variant 1. NM_000159.3:c.463T>C(p.Tyr155His) , 2. NM_000159.3(GCDH):c.680G>C (p.Arg227Pro), compound heterozygous

ClinVar 1. n/a, 2. 2089

CAID 1. CA404317601

gnomAD 1. MAF=0.000008791(European(non-Finnish)), no homozygotes, 2. MAF= 0.0003566(European(non-Finnish)), no homozygotes

CaseAddInfo n/a

CasePMIDs see notes on previous studies

Case10 sex unknown, Spain

CasePresentingHPOs HP:0006846, HP:0002540, HP:0001344, HP:0001332

(Acute encephalopathy, Non-ambulatory, Nonverbal, Dystonia)

HPOsFreeText Phenotype was classified as "severe," with passive or static dystonia and an inability to stand up, walk, or speak. Diagnosed by routine GC-MS organic acid analysis in urine. There was high excretion of glutarate and 3-hydroxyglutarate with glutarate excretion being higher than that of 3-hydroxyglutarate.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 778(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 264(nv 2–14 mm/mol creatinine).

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The A293T variant, with the addition of a mismatch primer, destroys an HgaI restriction site. Thus, while the patient's DNA fragments would not cleave, control fragments would be cleaved. Likewise, the R402W variant destroys a MspI site. This would lead to uncleaved patient fragments and cleaved control fragments.

Variant 1. NM_000159.3(GCDH):c.877G>A (p.Ala293Thr), 2. NM_000159.3(GCDH):c.1204C>T (p.Arg402Trp), compound heterozygous

ClinVar 1. 2083, 2. 2085

gnomAD 1. MAF=0.0004013(African), no homozygotes, 2. MAF= 0.0007566(European(Finnish)), no homozygotes

CaseAddInfo Sibling (Case 11) was asymptomatic. GC-MS measured glutarate of 2083(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 262(nv 2–14 mm/mol creatinine)

CasePMIDs n/a

Case3 sex unknown, Spain

CasePresentingHPOs HP:0006846, HP:0002540, HP:0001344, HP:0001332

(Acute encephalopathy, Non-ambulatory, Nonverbal, Dystonia)

HPOsFreeText Phenotype was classified as "severe," with passive or static dystonia and an inability to stand up, walk, or speak. Diagnosed by routine GC-MS organic acid analysis in urine. There was high excretion of glutarate and 3-hydroxyglutarate with glutarate excretion being higher than that of 3-hydroxyglutarate.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 1773(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 340(nv 2–14 mm/mol creatinine).In patient fibroblasts, %GCD activity was <0.5.

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The A293T variant, with the addition of a mismatch primer, destroys an HgaI restriction site. Thus, while the patient's DNA fragments would not cleave, control fragments would be cleaved.

Variant NM_000159.3(GCDH):c.877G>A (p.Ala293Thr), homozygous

ClinVar 2083

gnomAD MAF= 0.0004013(African), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Case21 sex unknown, Spain

CasePresentingHPOs HP:0006846, HP:0002540, HP:0001344, HP:0001332

(Acute encephalopathy, Non-ambulatory, Nonverbal, Dystonia)

HPOsFreeText Phenotype was classified as "severe," with passive or static dystonia and an inability to stand up, walk, or speak. Diagnosed by routine GC-MS organic acid analysis in urine. There was high excretion of glutarate and 3-hydroxyglutarate with glutarate excretion being higher than that of 3-hydroxyglutarate.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 204(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 143(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 2.

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The A195T variant destroys a BanI restriction site. Thus, while the patient's DNA fragments would not cleave, control fragments would be cleaved.

Variant 1. NM_000159.3:c.583G>A (p.Ala195Thr), 2. NM_000159.3(GCDH):c.356C>T (p.Ser119Leu), compound heterozygous

ClinVar 1. n/a, 2. 288248

CAID 1. CA404318183

gnomAD 1. not in gnomAD, 2. MAF=0.0001984(Ashkenazi Jewish), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Case29 sex unknown, Spain

CasePresentingHPOs HP:0006846, HP:0002540, HP:0001344, HP:0001332

(Acute encephalopathy, Non-ambulatory, Nonverbal, Dystonia)

HPOsFreeText Phenotype was classified as "severe," with passive or static dystonia and an inability to stand up, walk, or speak. Patient may have gone undetected by routine screens as glutarate excretion was normal and 3-hydroxyglutarate was only slightly increased.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 5(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 114(nv 2–14 mm/mol creatinine).

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The V400M variant creates an NlaIII site. This would lead to cleaved patient DNA fragments and uncleaved control fragments.

Variant NM_000159.3(GCDH):c.1198G>A (p.Val400Met), homozygous

ClinVar 2088

gnomAD MAF= 0.0002769(Other), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Case18 sex unknown, Spain

CasePresentingHPOs HP:0002540, HP:0001344, HP:0001332

(Non-ambulatory, Nonverbal, Dystonia)

HPOsFreeText Clinical onset was "insidious," involving slowly progressive psychomotor delay, dystonic postures, lethargy, or macrocephaly. Phenotype was classified as "severe," with passive or static dystonia and an inability to stand up, walk, or speak. Diagnosed by routine GC-MS organic acid analysis in urine. There was high excretion of glutarate and 3-hydroxyglutarate with glutarate excretion being higher than that of 3-hydroxyglutarate.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 4097(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 212(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was <0.5.

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The R402W variant destroys an MspI restriction site. Thus, while the patient's DNA fragments would not cleave, control fragments would be cleaved.

Variant 1. NM_000159.3(GCDH):c.1204C>T (p.Arg402Trp), 2. NM_000159.3(GCDH):c.1173delG (p.Asn392Metfs), compound heterozygous

ClinVar 1. 2085, 2. 370106

gnomAD 1. MAF=0.0007566(European(Finnish)), no homozygotes, 2. MAF= 0.00005646(Latino), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

CaseF2: Female, 6 yrs report, Italy

CasePresentingHPOs: HP:0004923 (Hyperphenylalaninemia)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents do not appear to be tested, DNA sequence analysis "shows the sense sequence expected of compound heterozygotes with two comigrating bands seen"

CasePreviousTesting: Pre-treatment phenylalanine levels determined soon after birth were above 20 mg / 100 ml, Proband has been under treatment since birth and currently tolerates less than 20 mg Phe/kg bodyweight per day

CaseGenotypingMethod: DNA as isolated from leukocytes and haplotype analysis performed. Enzymatic digestion of PCR-amplified DNA fragments was performed and DGGE analysis of all 13 exons and flanking introns were carried out. The resulting samples containing abberant bands were sequenced and cleaved by MaeI and MaeIII for analysis.

Variant1: NM_000277.3(PAH):c.620A>G (p.Asn207Ser)

ClinVar: 102765 https://www.ncbi.nlm.nih.gov/clinvar/variation/102765/

gnomAD: Not in gnomAD

Variant2: NM_000277.3(PAH):c.842C>T (p.Pro281Leu)

ClinVar: 589 https://www.ncbi.nlm.nih.gov/clinvar/variation/589/

gnomAD: 0.0002016, https://gnomad.broadinstitute.org/variant/12-103246593-G-A

CaseF1: Male, 6 yrs report, Italy

CasePresentingHPOs: HP:0004923 (Hyperphenylalaninemia)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents were confirmed heterozygous, Splicing variant is predicted to lead to a null protein

CasePreviousTesting: Pre-treatment phenylalanine levels determined soon after birth were above 20 mg / 100 ml, Proband has been under treatment since birth and currently tolerates less than 20 mg Phe/kg bodyweight per day

CaseGenotypingMethod: DNA as isolated from leukocytes and haplotype analysis performed. Enzymatic digestion of PCR-amplified DNA fragments was performed and DGGE analysis of all 13 exons and flanking introns were carried out. The resulting samples containing abberant bands were sequenced and cleaved by MaeI and MaeIII for analysis.

Variant1: NM_000277.2(PAH):c.611A>G (p.Tyr204Cys)

ClinVar: 590 https://www.ncbi.nlm.nih.gov/clinvar/variation/590

gnomAD: 0.0001632, https://gnomad.broadinstitute.org/variant/12-103249009-T-C

Variant2: NM_000277.2(PAH):c.442-2A>C

ClinVar: 120276 https://www.ncbi.nlm.nih.gov/clinvar/variation/120276

gnomAD: Not in gnomAD

RescueModel: Mouse (Mus musculus)

GeneAlteration: PAH_enu2 mice were purchased and bred to obtain the homozygotic mice for this study. Original Generation Method: Mutations were induced in mice germline cells at a rate near 10^-3 per locus via an alkylating agent called ethylnitrosourea. Inbred BTBR males were treated with this agent and bred to normal BTBR females. Three hundred and fifty progeny were screened for mutations that did not complement the previous model PAH_enu1 and if present bred to a previous homozygote and tested for hyperphenylalaninemia. A PHE clearance test was performed in questionable cases. For those that tested positive the carrier parents were mated to a normal BTBR partner to establish a cogenic mutant line. Western blot confirmed reduced to absent CRM in liver.

RescuePhenotype: HP:0007513, HP:0004923, HP:0005982, HP:0100610 (Generalized hypopigmentation, Hyperphenylalaninemia, Reduced phenylalanine hydroxylase activity, Maternal hyperphenylalaninemia)

RescueMethod: The human PAH expression vector plasmid pAAV2-EF-hPAH-WPRE was created by subcloning a full-length human PAH cDNA into a preexisitng parent vector by substitution with eGFP cDNA. Alos included is an EF promoter, woodchuck hepatitis virus post-transcriptional element, and bovine growth hormone poly (A) signal. The rep2/cap2 plasmid or rep2/cap8 plasmid was used to package the vector and produce the final rAAV2/2-hPAH and rAAV2/8-hPAH vectors by triple plasmid transfection. Five-to-six week old PAH_enu2 mice were anesthetized with ketamine-xylazine and infused with viral particles of rAAV2/2-hPAH or rAAV2/8-hPAH through the hepatic portal vein or tail vein.

RescueEvidence: PAH activity in the livers of treated mice six weeks after treatment recovered to 58.43±5.73% and 89.25±8.79% of the wild-type activity in female and male PAH_enu2 mice respectively compared to <1% WT in untreated mice. Plasma phenylalanine concentration in female PAH_enu2 mice infused with rAAV2/8-hPAH vectors decreased markedly to 110±34.5 µM at two weeks from 2,891±336.2 µM and showed continued significant phenylalanine clearance for six month after rAAV2/8-hPAH infusion. Male PAH_enu2 showed a similar reduction, though they started at a lower base concentration of 1,800.6±233.7 µM. The fur color of treated mice also changed from greyish to black from one week after delivery with sustained fur color changes over the entire six month period. Investiation of effects on pregnant mice revealed a complete recovery of fetus length and body weight compared to the untreated mice (1.746±0.203 cm and 774±144 mg for untreated VS 2.081±0.269 cm and 916±67 mg for treated). Spontaneous abortion rate also normalized to 4.55±5.89% from 30.95±14.27%. The rAAV-hPAH genome was not found in the placentae or fetuses nor did the genome of fetuses affect the development parameters.

ModelVariant: NM_000277.3(PAH):c.788T>C (p.Phe263Ser)

ClinVar: 619150 https://www.ncbi.nlm.nih.gov/clinvar/variation/619150/

CaseSA5P: Female, 3 months onset, European

CasePresentingHPOs: HP:0001988, HP:0000825, HP:0001249, HP:0001250 (Recurrent hypoglycemia, Hyperinsulinemic hypoglycemia, Intellectual disability, Seizure)

HPOsFreeText: N/A

CaseNotHPOs: HP:0001987 (Hyperammonemia)

NotHPOsFreeText: N/A

CaseAddInfo: Has father with confirmed variant and similar phenotypes

CasePreviousTesting: Leucine load test showed leucine sensitivity; Protein/leucine restriction and dioxazide was effective at reducing symptoms

CaseGenotypingMethod: Genomic DNA was prepared from all patients via peripheral blood leukocytes. The GLUD1 gene exons 1-13 and the flanking intronic regions were PCR-amplified and directly sequenced. All variants were confirmed via restriction enzyme analysis. Additionally, paternity testing was performed by HLA typing and by southern blot analysis of DNA fragments as well as PCR analysis of STRs.

Variant: NM_005271.5(GLUD1):c.965G>A (p.Arg322His)

ClinVar: 16129, https://www.ncbi.nlm.nih.gov/clinvar/variation/16129/

gnomAD: Not in gnomAD

CaseSA2P: Female, 3 months onset, European

CasePresentingHPOs: HP:0001988, HP:0000825, HP:0001987 (Recurrent hypoglycemia, Hyperinsulinemic hypoglycemia, Hyperammonemia)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is confirmed de novo via parental teting and confirmed paternity

CasePreviousTesting: Leucine load test showed leucine sensitivity in some patients; Protein/leucine restriction was effective at reducing symptoms

CaseGenotypingMethod: Genomic DNA was prepared from all patients via peripheral blood leukocytes. The GLUD1 gene exons 1-13 and the flanking intronic regions were PCR-amplified and directly sequenced. All variants were confirmed via restriction enzyme analysis. Additionally, paternity testing was performed by HLA typing and by southern blot analysis of DNA fragments as well as PCR analysis of STRs.

Variant: NM_005271.5(GLUD1):c.965G>A (p.Arg322His)

ClinVar: 16129, https://www.ncbi.nlm.nih.gov/clinvar/variation/16129/

gnomAD: Not in gnomAD

CaseKLE: Male, Japan, Asian

CasePresentingHPOs: HP:0008353 (Neutral hyperaminoaciduria)

HPOsFreeText:

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo:

CasePreviousTesting: Identified via neonatal mass screening

CaseGenotypingMethod: Both SLC6A18 and SLC6A19 were investigated for possible pathogenicity, with SLC6A19's pattern of expression supporting it as a primary candidate. Intronic primers were used to bidirectionally sequence each exon and flanking intronic regions, locating two compound heterozygous mutations that perfectly segregate with the phenotype.

Variant1: NM_001003841.3:c.340del (L114fs*114)

CAID: CA916079859 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA916079859

gnomAD: Not in gnomAD

Variant2: NM_001003841.3:c.682_683delinsTA (p.Thr228Ter)

CAID: CA916079860 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA916079860

gnomAD: Not in gnomAD

Case#: Patient 3, Lebanon, Arab

CasePresentingHPOs: Intermediate form of MSUD

CasePreviousTesting: Previously diagnosed with MSUD by GC/MS/MS.

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: Genomic DNA extracted, sequencing of BCKDHA, BCKDHB and DBT.

Variant: NM_001918.3:c.1430T>G (homozygous)

ClinVar: 93994

gnomAD: European (Non-Finnish) 8.971e-6, Overall 4.065e-6.

VariantEvidence: Mutation is present in the catalytic domain and would expect a defect in catalytic function (Alamut software analysis).

Patient7: Male, 2yo

CasePresentingHPOs:

HPOsFreeText: Asymptomatic, C5 (DBS) = 1.6 μmol l−1, IVG in urine mmol permol creatinine = 272, IVA incorp (%) = 72, Ammonium (μmol l−1) = 51, pH = 7.39, Protein intake (g kg−1 per day) = 1.5, PDI/IQ in follow-up = 93

CaseNotHPOs: HP:0011968, HP:0002013, HP:0001944, HP:0001254, HP:0001259

(poor feeding, vomiting, dehydration, lethargy, coma)

NotHPOsFreeText: sweaty feet odor

CaseAddInfo: Diagnosed with NBS at 28 days, homozygous

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant: NM_002225.3:c.706G>A

CAID: CA268780419, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA268780419

gnomAD: 0.00003187, http://gnomad.broadinstitute.org/variant/15-40705199-G-A

Patient14: Male, 24yo

CasePresentingHPOs: HP:0011968, HP:0002013, HP:0001944, HP:0001254, HP:0001259, HP:0001987, HP:0001941

(poor feeding, vomiting, dehydration, lethargy, coma, hyperammonemia, acidosis)

HPOsFreeText: intermittent isovaleric acidemia, C5 (DBS) = 4.3 μmol l−1, IVA incorp (%) = 12, IVG in urine mmol permol creatinine = 16594, Ammonium (μmol l−1) = 126, pH = 7.17, Protein intake (g kg−1 per day) = 0.7, treated with carnitine and glycine supplements, PDI/IQ in follow-up = 88

CaseNotHPOs:

NotHPOsFreeText: sweaty feet odor

CaseAddInfo: diagnosed via clinical findings at 1yo, hospitalized outside of detection period

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: c.1-?_c.295+? (Delexons 1–3) no ClinVar or CAID

Variant2: NM_002225.4(IVD):c.1184G>A (p.Arg395Gln) listed in paper as (p.Arg395His)

CAID: CA268781747, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA268781747

ClinVar: 529434, https://www.ncbi.nlm.nih.gov/clinvar/variation/529434/

gnomAD: 0.00001415, http://gnomad.broadinstitute.org/variant/15-40710365-G-A

Patient11: Female, 19yo, sibling to patient 10

CasePresentingHPOs: HP:0011968, HP:0002013, HP:0001944, HP:0001254, HP:0001259

(poor feeding, vomiting, dehydration, lethargy, coma)

HPOsFreeText: intermittent isovaleric acidemia, IVA incorp (%) = 13, IVG in urine mmol permol creatinine = 3488, Ammonium (μmol l−1) = 47 pH = 7.33, Protein intake (g kg−1 per day) = 1.0, treated with carnitine and glycine supplements, PDI/IQ in follow-up = 85, sweaty feet odor

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: Diagnosed via clinical findings at 2yo

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_002225.3(IVD):c.884T>C (p.Leu295Pro)

ClinVar: 555464, https://www.ncbi.nlm.nih.gov/clinvar/variation/555464/

gnomAD: n/a

Variant2: NM_002225.3:c.1211A>G

CAID: CA391724518, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391724518

gnomAD: n/a

Patient13 Male, 9yo

CasePresentingHPOs: HP:0011968, HP:0001254, HP:0001259, HP:0001987

(poor feeding, lethargy, coma, hyperammonemia)

HPOsFreeText: acute isovaleric acidemia, C5 (DBS) = 16.3 μmol l−1, IVA incorp (%) = 5.5, IVG in urine mmol permol creatinine = 3352, Ammonium (μmol l−1) = 606, pH = 7.26, Protein intake (g kg−1 per day) = 0.8, treated with carnitine and glycine supplements, PDI/IQ in follow-up = 87, sweaty feet odor

CaseNotHPOs: HP:0002013, HP:0001944 (vomiting, dehydration)

NotHPOsFreeText:

CaseAddInfo: diagnosed via clinical findings at 20 days old, homozygous

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant: NM_002225.3:c.158G>C listed in clinvar as NM_002225.4(IVD):c.149G>C (p.Arg50Pro)

CAID: CA274129, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA274129

ClinVar: 188922, https://www.ncbi.nlm.nih.gov/clinvar/variation/188922/

gnomAD: 0.00001591, http://gnomad.broadinstitute.org/variant/15-40699841-G-C

FamilyLabel 38/39 Fam

Family38/39PresentingHPOs n/a

FamilyNotHPOs n/a

CountryOrigin n/a

Ethnicity Turkish

Race n/a

Family38/39PreviousTesting Diagnosed by selective metabolic screen

ProbandLabel 38

Variant NM_000532.4(PCCB):c.1534C>T (p.Arg512Cys), homozygous

ClinVar 38879

gnomAD MAF= 0.00005440(East Asian), no homozygotes

FamilyAddInfo parental consanguinity

FamilyPMIDs n/a

Case8 Male, 9.8yo report, Ethnicity:Turkish

CasePresentingHPOs HP:0012523, HP:0001508, HP:0002013, HP:0008947, HP:0002789, HP:0002045, HP:0002014, HP:0003571

(propionicacidemia, oral aversion, failure to thrive, vomiting, infantile hypotonia, tachypnea, hypothermia, diarrhea)

HPOsFreeText feeding refusal, failure to thrive, vomiting, hypotonia, tachypnea, hypothermia, diarrhea (4do). 73 IQ on Culture Free Test.

CaseNotHPOs n/a

CasePreviousTesting Diagnosed by selective metabolic screening

Variant NM_000532.4:c.429+3_6delAAGT (p.Asp125_Lys143del), homozygous

ClinVar n/a

CAID to register

gnomAD not in gnomAD

CaseAddInfo parental consanguinity

CasePMIDs n/a

CaseES: Female, 1 yr onset

CasePresentingHPOs: HP:0002312, HP:0002131, HP:0002719, HP:0002078, HP:0002066, HP:0100560, HP:0002345, HP:0008376, HP:0000666, HP:0001348, HP:0002342, HP:0410217, HP:0012237 (Clumsiness, Episodic ataxia, Recurrent infections, Truncal ataxia, Gait ataxia, Upper limb asymmetry, Action tremor, Dysarthic speech, Horizontal nystagmus, Brisk reflexes, Moderate intellectual disability, Reduced blood 5-methyltetrahydrofolate level, Urocanic aciduria)

HPOsFreeText: Urocanis acid levels 158-202 mmol/mol creatinine (reference <10)

CaseNotHPOs: HP:0000496, HP:0001324, HP:0002160, HP:0001903 (Abnormal ocular movements, Muscle weakness, Hyperhomocystinemia, Anemia)

NotHPOsFreeText: N/A

CaseAddInfo: Parents are healthy and nonconsanguineous, Steady improvement after mid-life, Proband is confirmed compound heterozygous by parental confirmation (father heterozygous for Variant 2)

CasePreviousTesting: Cranial MRI and neurophysiological studies, including electroencephalography, electroretinography, visual and brainstem evoked potentials, electromyography, nerve conduction velocities, fundus oculi and cardiological examinations, gave normal results; Cognitive evaluation rated her IQ at 54; Routine laboratory analyses including serum amino transferases, creatinine, urea and other parameters were normal; Screening for inborn errors of metabolism, including plasma and urine amino acids (no histidine accumulation), blood lactate, sialotransferrin isoelectricfocusing, creatine deficiency syndromes, purine and pyrimidine defects, and organic acidurias, gave normal results; Hypothyroidism and coeliac disease were also excluded; Biochemical and/or genetic screening for early onset forms of inherited ataxia gave normal results; Deficiencies in 5-methyltetrahydrofolate, neopterin and biopterin deficiencies were seen, together with an evident reduction in homovanillic acid (HVA) and the HVA:5-hydroxyindolacetic acid ratio; Other parameters determined in cerebrospinal fluid (CSF), such as glucose, proteins, cells and amino acids were normal.

CaseGenotypingMethod: Genomic DNA was obtained from peripheral white blood cells from the proband and her father. Mutation analysis of UROC1 was performed via direct sequencing and PCR amplification of the 20 exons and flanking exon-intronic sequences. In-silico analysis and modeling was performed on the variants.

Variant1: NM_144639.3(UROC1):c.209T>C (p.Leu70Pro)

ClinVar: 408 https://www.ncbi.nlm.nih.gov/clinvar/variation/408/

gnomAD: 0.00005647, https://gnomad.broadinstitute.org/variant/3-126229555-A-G

Variant2: NM_144639.3(UROC1):c.1348C>T (p.Arg450Cys)

ClinVar: 407, https://www.ncbi.nlm.nih.gov/clinvar/variation/407/

gnomAD: 0.00008012, https://gnomad.broadinstitute.org/variant/3-126216984-G-A

CaseFI2: Male, Turkey, Birth onset

CasePresentingHPOs: HP:0009800, HP:0001195, HP:0000773, HP:0003026, HP:0001650, HP:0003561, HP:0001518, HP:0000252, HP:0004887, HP:0001274, HP:0001250, HP:0000411, HP:0007957, HP:0007495, HP:0000963, HP:0000973, HP:0100678, HP:0010648, HP:0001382, HP:0009815, HP:0001181, HP:0010557, HP:0001374, HP:0012330, HP:0030750, HP:0040187 (Maternal diabetes, Single umbilical artery, Short ribs, Short long bone, Aortic valve stenosis, Birth length less than 3rd percentile, Small for gestational age, Microcephaly, Respiratory failure requiring assisted ventilation, Agenesis of corpus callosum, Seizures, Protruding ear, Corneal opacity, Prematurely aged appearance, Thin skin, Cutis laxa, Premature skin wrinkling, Dermal translucency, Joint hypermobility, Aplasia/hypoplasia of the extremities, Adducted thumb, Overlapping fingers, Congenital hip dislocation, Pyelonephritis, Grade III preterm intraventricular hemorrhage, Neonatal sepsis)

HPOsFreeText: Intrauterine growth restriction birth length: 38 cm (< 3rd percentile), birth weight 1370 g (< 3rd percentile) and microcephaly (OFC at birth was 29.5 cm (3rd–10th percentile)). APGAR scores were 5, 9, and 9 after 1, 5, and 10 min, respectively; Typical facial gestalt

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are first-degree cousins that had three healthy daughters and three early abortions

CasePreviousTesting: N/A

CaseGenotypingMethod: Familial DNA was extracted from peripheral blood samples. Direct sequencing of PYCR1 and exons 2-18 of ALDH18A1 was performed in the affected individual with a homozygous single-basepair deletion found predicted to lead to a frameshift and premature stop codon.

Variant: NM_002860.4(ALDH18A1):c.2131del (p.Leu711fs)

ClinVar: 156546 https://www.ncbi.nlm.nih.gov/clinvar/variation/156546/

gnomAD: Not in gnomAD

Case: Patient 4/ BIODEF 263, female, 1 week

CasePresentingHPOs: HP:0008297, HP:0040210, HP:0040206

At 1 week old plasma showed Phe levels at 258 umol/L. Repeat screening at 9 days old showed Phe levels at 900 umol/L. Amino acid quantitation of plasma showd a Phe level of 1200 umol/L. Urine analysis showed low BH4, high neopterin, low biopterin, and the presence of primapterin. A diet of low protein and phenylalanine were begun, and continue with 100 mg/kg of phenylalanine per day. Patient is 6 years old and developing normally.

CaseNOTHPOs:

CasePreviousTesting: n/a 

CaseGenotypingMethod: PCR of exons 2-4 of PCBD1 followed by direct sequencing.

Variant: NM_000281.3(PCBD1):c.292C>T (p.Gln98Ter)

ClinVar ID: 16799

Homozygous

gnomAD: n/a

AddCaseInfo: No information about the family.

VariantEvidence: Expression of recombinant protein in bacteria revealed that both the Q98X variants are present in the insoluble fraction compared to wildtype which is found in the soluble. the little amount retained of each in the soluble fraction showed that they retained activity.

Case: Patient 3/ BIODEF 216, male, Morrocan, 1 month

CasePresentingHPOs: HP:0008297, HP:0040210, HP:0040206

HyperPhe presented a 1 month old with plasma levels of 1850 umol/L. A low Phe diet was initiated for 8 months. Urinary pterin profile showed high neopterin, low biopterin, and elevated primapterin. The diet was ceased at 2 years old, and the child has normal Phe levels (110 umol/L).

CaseNOTHPOs:

CasePreviousTesting: n/a

CaseGenotypingMethod: PCR of exons 2-4 of PCBD1 followed by direct sequencing.

Variant: NM_000281.3(PCBD1):c.289G>A (p.Glu97Lys)

ClinVar ID: 91907

Homozygous

gnomAD: MAF (Euro-NF)= 2.320E-5 in hets only, no homozygous. http://gnomad.broadinstitute.org/variant/10-72643733-C-T

AddCaseInfo: Parents are consanguineous. Older brother (by 17 years) presented with HyperPhe in the neonatal period, but no information on whether he was a carrier of the mutation.

VariantEvidence: Expression of recombinant protein in bacteria revealed that the E97K are present in the insoluble fraction compared to wildtype which is found in the soluble. the little amount retained of each in the soluble fraction showed that they retained activity. Per Ayling et al., 2000 (PMID:10924272), another patient (BIODEF 306) that was homozygous for the same E97K mutation, showed reduced activity of PCBD1 from duodenal mucosa cells compared to wildtype (17% activity), indicating that this mutation severely reduces the activity of PCBD1.

NonHumanModel: Mouse (Mus musculus)

GeneAlteration: Two constructs were created, containing with the WT human GDH sequence or the H507Y variant along with the rat insulin promotoe and human growth hormone genomic DNA sequence to stabilize the construct and drive β-cell-specific transcription of the cDNA sequence. These were microinjected into fertilized eggs of B6SJLF1 mice, transferred into pseudopregnant females, and PCR-amplified/sequenced to confirm the succesful insertion of the transgene. Offspring were crossed with C57BL/6 mice and generated two lines of mutant along with one line of WT transgenic mice: TG-1, TG-2, and huGDH.

ModelPhenotype: MP:0000189, MP:0008770, MP:0002079, MP:0005309 (hypoglycemia, decreased survivor rate, increased circulating insulin level, hyperammonemia)

ModelFreeText: Higher sensitivity to leucine and glutamine ramp stimulation, Increased ATP/ADP ratios

HumanPhenotype: HP:0001987, HP:0001988, HP:0000825 (Hyperammonemia, Recurrent hypoglycemia, Hyperinsulinemic hypoglycemia)

HumanFreeText: N/A

ModelEvidence: The two lines of mice transgnically modified with the H507Y variant were observed compared to the WT mice. Both lines of mice had notable and spontaneous hypoglyemia, specifically affecting both the mouse survivial rate and fertility compared to the control line. Cultured insulin islets were compared and demonstrated significantly increased insulin secretion when exposed to glutamine as well as showing an increased sensitivity to leucine. This also results in elevated ammonia as a byproduct, since the elevated reaction product also yields NH4+. This directly results to the pathogenic mechanism of H507Y, and other pathogenic GLUD1 variants, in humans. These variants reduce the protein's sensitivity to regulation by GTP, causing increased glutamate oxidation and insulin production by raising the ATP/ADP ratio artifically.

ModelVariant: NM_005271.5(GLUD1):c.1519C>T (p.His507Tyr)

ClinVar: 16121, https://www.ncbi.nlm.nih.gov/clinvar/variation/16121/

Case764: Female, 4 years report, Spain

CasePresentingHPOs: HP:0011344, HP:0003153, HP:0001257, HP:0003698, HP:0002474 (Severe psychomotor retardation, Cystathioninuria, Spasticity, Difficulty standing, Communication delay)

HPOsFreeText: Elevated urine cystathionine (1611 umol/g creatine; reference <46) and Elevated tHcys in urine (54.3 umol/g creatine; reference <20), Elevated plasma tHcys (20.5 umol/l; reference <7.5), Post treatment normal urine cystathionine (42 umol/g creatine)

CaseNotHPOs: HP:0003345 (Glutathione synthetase deficiency)

NotHPOsFreeText: N/A

CaseAddInfo: Patient is homozygous for this variant

CasePreviousTesting: Karyotype, magnetic resonance imaging, specific genetic tests for neurogenetic diseases and screening for inborn errors of metabolism (lactate, pyruvate, ammonium, uric acid, amino acids, organic acids, glycosaminoglycanes, creatine and guanidinoacetate, purines and pyrimidines, and isoelectric focusing pattern of transferrin), as well as folate, pyridoxine and cobalamine deficiencies, neural tumors and genetically determined homocystinurias, were all ruled out.

CaseGenotypingMethod: All twelve exons of the CTH gene and the intronic flanking sequences of the subjects and their parents was PCR amplified and directly sequenced.

Variant: NM_001902.5:c.200C>T (p.Thr67Ile)

ClinVar: 2939 https://www.ncbi.nlm.nih.gov/clinvar/variation/2939/

gnomAD: Ashkenazi Jewish 9.934e-3, Overall 6.308e-3 https://gnomad.broadinstitute.org/variant/1-70881670-C-T

CaseVC: Male, Birth onset

CasePresentingHPOs: HP:0030082, HP:0002013, HP:0200048, HP:0001250, HP:0005949, HP:0001290, HP:0001662, HP:0008610, HP:0002120 (Abnormal drinking behavior, Vomiting, Cyanotic episode, Seizure, Apneic episodes in infancy, Generalized hypotonia, Bradycardia, Infantile sensorineural hearing impairment, Cerebral cortical atrophy)

HPOsFreeText: Birth weight of 2565 g, Length of 48 cm, Head circumference of 33 cm, Elevated excretion of TMS-derivatives of N-acetylated amino acids

CaseNotHPOs: HP:0001263 (Global developmental delay)

NotHPOsFreeText: N/A

CaseAddInfo: Parents are non-consanguineous, Proband confirmed homozygous via parental testing

CasePreviousTesting: Cerebral MRI showed abnormal signals in the cortico-subcortical zones of the fronto-parietal and temporo-occipital areas consistent with cortical laminar necrosis; Most presenting phenotypes were ameliorated after 6 weeks of age; N-acetylated amino acid excretion shown in Table 1

CaseGenotypingMethod: RT-PCR confirmed expression of aminoacylase I mRNA was significantly lowered in the proband than in controls. Southern blot and nested PCR confirmed the weak band observed was aminoacylase I mRNA and the relative expression levels. Following this, PCR amplification of all 14 coding exons and flanking introns of ACY1 was performed and sequenced.

Variant: NM_000666.3(ACY1):c.1057C>T (p.Arg353Cys)

ClinVar: 18110 https://www.ncbi.nlm.nih.gov/clinvar/variation/18110/

gnomAD: 0.004135 (4 homozygotes), https://gnomad.broadinstitute.org/variant/3-52022837-C-T

CaseBE2P3: Male, 9 days onset

CasePresentingHPOs: HP:0001399, HP:0006579, HP:0001396, HP:0004914, HP:0002240, HP:0001290, HP:0007772, HP:0000347, HP:0000218, HP:0000954, HP:0012448, HP:0001684, HP:0003265, HP:0002490, HP:0002151, HP:0003235, HP:0001897, HP:0001272, HP:0002119, HP:0001263, HP:0000733, HP:0001414, HP:0002612 (Hepatic failure, Prolonged neonatal jaundice, Cholestasis, Recurrent infantile hypoglycemia, Hepatomegaly, Generalized hypotonia, Impaired smooth pursuit, Micrognathia, High palate, Single transverse palmar crease, Delayed myelination, Secundum atrial septal defect, Neonatal hyperbilirubinemia, Increased CSF lactate, Increased serum lactate, Hypermethioninemia, Normocytic anemia, Cerebellar atrophy, Ventriculomegaly, Global developmental delay, Stereotypy, Microvesicular hepatic steatosis, Congenital hepatic fibrosis)

HPOsFreeText: Increased bile salts, Biliary stones in gallbladder, Severe intrahepatic extramedullary haematopoiesis

CaseNotHPOs: HP:0001744, HP:0000842, HP:0008163 (Splenomegaly, Hyperinsulinemia, Decreased circulating cortisol level)

NotHPOsFreeText: Abnormally coloured stools

CaseAddInfo: Parents are first cousins

CasePreviousTesting: All patients received ursodeoxycholic acid (600 mg.m−2.d−1) and fat-soluble vitamin supplementation; All patients received an enriched diet with medium chain triglycerides and dextrin maltose; Sanger sequencing of POLG, DGUOK, and TRMU in P3 revealed no pathogenic variants; Mitochondrial RC defect was considered but no anomaly was found in isolated lymphocytes and in primary culture of fibroblasts

CaseGenotypingMethod: Sanger sequencing of the ADK gene revealed a homozygous termination variant. Both parents carried this in a heterozygous state and the younger healthy brother did not carry the mutation

Variant: NM_006721.3:c.519G>A (p.Trp173Ter)

CAID: CA377397711, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_006721.3%3Ac.519G%3EA

gnomAD: Not in gnomAD

Case2930: Female, 3 weeks diagnosis, United States

CasePresentingHPOs: HP:0003153 (Cystathioninuria)

HPOsFreeText: Elevated total homocysteine (18.2 μM/l; reference 5.1–13.9), Elevated cystathionine (11.8 μM/l; reference 0.04–0.34), Normal total cysteine (246 μmol/l; reference 203–369)

CaseNotHPOs: HP:0001270, HP:0001263, HP:0100543, HP:0030972 (Motor delay, Global developmental delay, Cognitive impairment, Abnormal systemic blood pressure)

NotHPOsFreeText: Unresponsive to B6 treatment

CaseAddInfo: Patient is compound heterozygous for these mutations, Parental testing has confirmed these variants are in trans

CasePreviousTesting: Cystathionine (Cysta), total cysteine (tCys), and total homocysteine (tHcy) panels

CaseGenotypingMethod: PCR amplification of CTH coding regions and exon-intron boundaries was performed. These fragments were sequenced and aligned with a reference to find mutations. Those found were confirmed by restriction digest of 100 controls.

Variant1: NM_001902.5:c.932C>T (p.Thr311Ile)

CAID: CA340839077 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_001902.5%3Ac.932C%3ET

gnomAD: Not in gnomAD

Variant Too Long

Variant2: NP_001893.2:p.Gly57_Gln196del

ClinVar:

gnomAD:

Variant1: NM_001902.5:c.200C>T (p.Thr67Ile)

Variant2: NM_001902.5:c.589C>T (p.Arg197Cys)

Variant3: NM_001902.5:c.932C>T (p.Thr311Ile)

VariantEvidence: Wildtype for CTH, 3 separate mutations, and an empty vector were all expressed in E. coli vectors using recombinant proteins. This showed a marked decrease in activity in Variants 1 and 2, but no significant change in Variant 3.

Case# Patient 1, Female, Japanese, 2 yo diagnosis

CasePresentingHPOs HP:0004923

CasePreviousTesting measuring urinarypteridines and assaying for DHPR activity. The DHPR activity inred blood cells was 6–8% of normal control

CaseMethod1 RG-PCR

Variant NM_000320.2(QDPR):c.106T>C (p.Trp36Arg)

ClinVar 492

VariantEvidence sevenfold higher DHPR activity in WT tranfected fibroblasts than W36T transfected cells(Fig. 1).

Case# Patient 2, male, Japanese, 11 yo diagnosis

CasePresentingHPOs HP:0002521, HP:0004923

CaseMethod1 RG-PCR

Variant NM_000320.2:c.461A>G

CAID CA356444851

VariantEvidence intronic splice variant (Fig. 4)

CaseAddInfo consanguineous parents, mother was heterozygous, patient and 2 brothers are homozygous

CasePMIDs 7444937

Biallelic

Case#:15, 5 months (report), Korean, female

CaseHPOs: HP:0003235, (hypermethioninemia)

CaseNotHPOsFreeText: No neurologic problems or developmental delay; no treatment or special diet.

CaseNotHPOs:

CasePreviousTesting: At 5 months and 25 months, respectively, plasma methionine 300.2, 87.7 umol (normal <50), plasma homocysteine 14.4, 5.4 umol (<15 umol)

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: All nine MAT1A exons (exon I to IX) and exon–intron boundaries were amplified by polymerase chain reaction (PCR) and directly sequenced.

Variant: c.487C>T (p.Arg163Trp) (homozygous)

ClinVar: N/A

CAID: CA5576782

gnomAD: The highest population minor allele frequency in gnomAD is 0.00006152 (African); no homozygoutes in any population.

VariantEvidence: None.

CaseAddInfo: The mother is heterozygous, the father had only the wild type allele in leukocyte DNA. Biparental inheritance was proven by microsatellite analysis suggesting that either the father is mosaic or this is a de novo variant.

CasePMIDs:

Points: 0.25 points. This individual is homozygous for a missense variant in MAT1A. The highest population minor allele frequency in gnomAD is 0.00006152 (African); no homozygotes in any population. The score is reduced because no functional evidence is available to support the deleterious impact of this variant.

Biallelic

Case#: 14, Korean, female, 58 months (report)

CaseHPOs: HP:0003235, (hypermethioninemia)

CaseNotHPOsFreeText: No neurologic problems or developmental delay; no treatment or special diet.

CasePreviousTesting: At 20 days and 58 months, respectively, Plasma methionine 1352.6, 815.2 umol (normal <50), plasma homocysteine 21.1, 19.06 umol (<15 umol)

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: All nine MAT1A exons (exon I to IX) and exon–intron boundaries were amplified by polymerase chain reaction (PCR) and directly sequenced.

Variant: 1) c.895C>T (p.Arg299Cys); 2) c.1005C>G (p.Tyr335X)

ClinVar: 1) 418870 2) N/A

CAID: 2) CA377360418

gnomAD: The highest population minor allele frequency in gnomAD for c.895C>T (p.Arg299Cys) is 0.0001431 (East Asian). c.1005C>G (p.Tyr335X) is not in gnomAD

VariantEvidence: When expressed in E. Coli, the variant resulted in reduction of methionine adenosyltransferase (MAT) and tripolyphosphatase (PPPase) activities to ~15% and ~20% of wild type respectively (Fernandez-Irigoyn et al, 2010, PMID 20675163; Fig 2).

CaseAddInfo: The mother is heterozygous for the nonsense variant, methionine levels are not available.

Points: 1 point. This individual and her sister, who also has hypermethioninemia, are compound heterozygous for a missense variant, p.Arg299Cys, and a nonsense variant in MAT1A (p.Tyr335X). While the nonsense variant occurs in the second to last exon of it is further than 50 bp from the 3’ end of the exon (~80 bp), and therefore should be identified by nonsense-mediated decay. The highest population minor allele frequency in gnomAD for c.895C>T (p.Arg299Cys) is 0.0001431 (East Asian). c.1005C>G (p.Tyr335X) is not in gnomAD. The score is reduced because other causes of hypermethioninemia have not been ruled out.

CaseKDC23: Male, 38 yrs onset, Korea, Asian

CasePresentingHPOs: HP:0002131, HP:0002321, HP:0001260, HP:0000640, HP:0011477, HP:0100543 (Episodic ataxia, Vertigo, Dysarthria, Gaze-evoked nystagmus, Upbeat nystagmus, Cognitive impairment)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband has no family history of disease

CasePreviousTesting: Negtive response to acetazolamide

CaseGenotypingMethod: Next-generation sequencing was perfomed on all probands with 40 candidate genes and five known EA genes.

Variant: NM_004172.5:c.952A>G (p.Thr318Ala)

CAID: CA3235581, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA3235581

gnomAD: 0.002656, https://gnomad.broadinstitute.org/variant/5-36679820-A-G NOTE EXTREMELY HIGH gnomAD FREQUENCY

CaseP1 Male, 4.5yr report, Austrian

CasePresentingHPOs HP:0010818, HP:0010851, HP:0001290, HP:0011343, HP:0007359, HP:0001942, HP:0002376, HP:0001290

(Generalized tonic seizures, EEG with burst suppression, Hypotonia, moderate GDD, focal seizures, Metabolic acidosis, Developmental regression, Hypotonia)

HPOsFreeText born 40wk gestation. (neonatal, pre-treatment) apgar scores at 1min:8, 5min:9, 10min: 10, generalized tonic fits at <1d old and burst suppression EEG. (3d:) introduced pyridoxine therapy, 100mg I/v trial dose/route, complete cessation of seizures, apnoea(3d), hypotonia, sleepiness, initial maintenance daily dose of 10 mg/kg. (infancy/childhood:) hypotonia, moderate IQ delay. (4d:) withdrew from pyridoxine trial then focal seizures recurrenced 3 weeks after(definite PDE). (PMID: 0894227) gestational toxicosis, birth weight 3,260g, myoclonic jerks of both arms with hours of birth.Paroxysms of polyspike-wave-activity on EEG, normal cranial imaging,CSF analysis

CaseNotHPOs n/a

CasePreviousTesting Administration of pyridoxine relieved patient's seizures. CSF pipecolic acid: 3.4(nv<0.15μM), urine AASA: 168 before treatment(nv<1 mmol/mol creatinine). Blood lactate: 4.0mM and CSF lactate: 4.2mM (nv <2.5mM). (PMID:17068770:)post treatment testing: Pipecolic acid in plasma 4.7(nv 0.7-2.6mmol/liter), AASA in plasma 2.6(nv<0.2mmol/liter), urine AASA 17(nv<1mmol/molcreatinine). (PMID: 0894227) CSF analysis, organic acids in urine, amino acids in serum, and VLCFAs were normal. Plasma PA at 1st week 49µmol/L(before treatment)(nv 1-3.2), at 1-6mo 14.3µmol/L, 6-12mo 10.1µmol/L, CSF PA at 0-1yr 3.38µmol/L.

Variant NM_001182.4(ALDH7A1):c.1279G>C (p.Glu427Gln), homozygous

ClinVar 17994

gnomAD MAF= 0.0006123(European(Non-Finnish)), no homozygotes

CaseAddInfo n/a

CasePMIDs 10894227 (Patient 2), 17068770 (Patient 11)

FamilyLabel Family C

FamilyCPresentingHPOs HP:0001941, HP:0002098, HP:0000737, HP:0011169, HP:0010818, HP:0001336, HP:0000750

(Acidosis, Respiratory distress, Irritability, Clonic seizures, Generalized tonic seizures, Myoclonic jerks, Speech delay)

HPOsFreeText (neonatal, pre-treatment:) acidosis, respiratory distress, irritability, clonic seizures, generalized tonic seizures, myoclonic jerks. no apnoea, hypotonia, sleepness, or hypotension during pyridoxine therapy. (infancy/childhood:) speech delay.

FamilyNotHPOs HP:0003270, HP:0002013, HP:0001290, HP:0010851, HP:0002521

(Abdominal distension, Vomiting, Hypotonia, EEG with burst suppression, Hypsarrhythmia)

NotHPOsFreeText (neonatal, pre-treatment:) abdominal distension, vomiting, hypotonia, EEG with burst suppression, hypsarrhythmia

CountryOrigin n/a

Ethnicity Turkish

Race n/a

FamilyCPreviousTesting n/a

ProbandLabel C1

Variant NM_001182.4(ALDH7A1):c.1597delG (p.Ala533Profs), homozygous

ClinVar 17999

gnomAD not in gnomAD

FamilyAddInfo 1st cousin parents

FamilyPMIDs n/a

Patient1: Male, 6yo

CasePresentingHPOs:

HPOsFreeText: Blood C5-carnitine (μM) = 18.96, under a protein restricted diet with carnitine and glycine supplementation, normal development

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: 10days old and asymptomatic at diagnosis

CasePMIDs:

CasePreviousTesting: Blood amino acids and acylcarnitine analyzed with ESI-MS/MS, urine organic acid analysis with GC/MS

GroupMethod1: PCR (primers in table 2)

GroupMethod2: Direct sequencing

CaseGenotypingMethod: genomic DNA extracted from peripheral blood leukocytes, IVD exons amplified via PCR, then products were purified and underwent direct sequencing for variant analysis

Variant1: NM_002225.3:c.386A>G (p.Tyr99Cys) listed in paper as (p.His100Arg)

CAID: CA391716511, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391716511

gnomAD: n/a

Variant2: NM_002225.4(IVD):c.1199A>G (p.Tyr400Cys) listed in paper as (p.Tyr371Cys)

ClinVar: 371390, https://www.ncbi.nlm.nih.gov/clinvar/variation/371390/

gnomAD: n/a

Patient5-1: Female, 2.5yo, sibling to patient 5-2

CasePresentingHPOs:

HPOsFreeText: under a protein restricted diet with carnitine and glycine supplementation, normal development

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: 2mo diagnosis

CasePMIDs:

CasePreviousTesting: Blood amino acids and acylcarnitine analyzed with ESI-MS/MS, urine organic acid analysis with GC/MS

GroupMethod1: PCR (primers in table 2)

GroupMethod2: Direct sequencing

CaseGenotypingMethod: genomic DNA extracted from peripheral blood leukocytes, IVD exons amplified via PCR, then products were purified and underwent direct sequencing for variant analysis

Variant1: NM_002225.4:c.347C>T (p.Ser89Phe) listed in paper as (p.Ser87Phe)

CAID: CA391716394, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA391716394

gnomAD: n/a

Variant2: NM_002225.4(IVD):c.1199A>G (p.Tyr400Cys) listed in paper as (p.Tyr371Cys)

ClinVar: 371390, https://www.ncbi.nlm.nih.gov/clinvar/variation/371390/

gnomAD: n/a

FamilySA2: Serbia

FamilyPresentingHPOs: HP:0002197, HP:0001988, HP:0001987, HP:0000825, HP:0001263 (Generalized-onset seizure, Recurrent hypoglycemia, Hyperammonemia, Hyperinsulinemic hypoglycemia, Global developmental delay)

HPOsFreeText: N/A

FamilyNotHPOs: N/A

NotHPOsFreeText: N/A

FamilyGenotypingMethod: Genetic analysis was perfomed on the GLUD1 gene.

FamilyPreviousTesting: Diazoxide treatment proved somewhat effective for each affected individual;

FamilyAddInfo:

Variant: NM_005271.5(GLUD1):c.820C>T (p.Arg274Cys)

ClinVar: 16128, https://www.ncbi.nlm.nih.gov/clinvar/variation/16128/

gnomAD: Not in gnomAD

Affected: 2

Segregations: 1

AddSegregationInfo: Not enough segregations

Case# P7, 5day onset, Bangladesh

CasePresentingHPOs HP:0002151, HP:0001946, HP:0001942, HP:0001987

CasePreviousTesting Of the negative samples, we include here only those negative for NAGS and CPS1 genes. NAGS testing was done by single-exon amplification and DNA sequencing.24 CPS1 testing was done by RNA sequencing, in most cases using stimulated lymphocytes

CaseGenotypingMethod sequence analysis of coding exons, including flanking intronic regions of CA5A and CA5B, was performed.

Variant NM_001739.1(CA5A):c.721G>A (p.Glu241Lys)

ClinVar 388645

gnomAD 0.002694 (South Asian), 0.0003177 (Overall)

VariantEvidence CA5A variant in Sf9 insect cells exhibited 26.1% of WT enzyme activity (Fig 2d).

CaseAddInfo consanguineous

Case# Patient 6, Female, 8mo diagnosis

Variant NM_000320.2:c.148G>A (p.G23D)

CAID CA2868208

gnomAD 0.001332 (African)