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

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

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

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 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

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/

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

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

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

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

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

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

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

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

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.

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)

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

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

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

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

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# 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

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)

CaseGM01454: Male, 7 years report, Asian, China

CasePresentingHPOs: HP:0003153 (Cystathioninuria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is a homozygote for this variant.

CasePreviousTesting: N/A

CaseGenotypingMethod: Primers were developed and the CTH exons 1-12 and exon-intron boundaries were PCR amplified and sequenced. All sequence variations were verified by restriction enzyme analysis and/or repeat sequencing.

Variant: CTH, 2-BP DEL, 940CT AKA: NM_001902.5:c.784_785del (p.Leu262Thrfs*21)

ClinVar: 2937 https://www.ncbi.nlm.nih.gov/clinvar/variation/2937/

gnomAD: East Asian 1.088e-4, Overall 7.960e-6 https://gnomad.broadinstitute.org/variant/1-70897822-ACT-A

Case14 age/gender unknown, Caucasian, Bosnia

CasePresentingHPOs HP:0001250

(Seizures)

HPOsFreeText seizure onset in neonatal period from minutes after birth until 3 weeks of age. Definite PDE: formal pyridoxine withdrawal had resulted in reoccurrence of seizures that again responded to pyridoxine.

CaseNotHPOs n/a

CasePreviousTesting post treatment testing: Pipecolic acid in plasma 2.8(nv 0.7-2.6µmol/liter), AASA in plasma 0.5(nv<0.2µmol/liter), urine AASA 1.6(nv<1mmol/molcreatinine). Normal urine PA after pyridoxine treatment.

Variant 1. NM_001182.4:c.605G>T (p.Gly202Val ), 2. NM_001182.4:c.1184T>G (p.Val395Gly), compound heterozygous

ClinVar n/a

CAID 1. CA360731073, 2. CA360723766

gnomAD neither in gnomAD

CaseAddInfo n/a

CasePMIDs n/a

Case13 age/gender unknown, Caucasian, Germany

CasePresentingHPOs HP:0001250

(Seizures)

HPOsFreeText seizure onset in neonatal period from minutes after birth until 3 weeks of age. Possible PDE: no trial of pyridoxine withdrawal performed and no antecedent affected.

CaseNotHPOs n/a

CasePreviousTesting pre-treatment testing: Pipecolic acid in plasma 53(nv 1-3.2µmol/liter), PA in urine 410µmol/mmol creati-nine(nv 0.55–24.1), AASA in plasma 14(nv<0.2µmol/liter), urine AASA 342(nv<1mmol/molcreatinine).

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

ClinVar 17994

gnomAD not in gnomAD

CaseAddInfo n/a

CasePMIDs n/a

Case1 age/gender unknown, Caucasian, German

CasePresentingHPOs HP:0001250

(Seizures)

HPOsFreeText seizure onset in neonatal period from minutes after birth until 3 weeks of age. Possible PDE: no trial of pyridoxine withdrawal performed and no antecedent affected

CaseNotHPOs n/a

CasePreviousTesting post treatment testing: Pipecolic acid in plasma 16(nv 0.4-4.9µmol/liter), AASA in plasma 3.9(nv<0.2µmol/liter), urine AASA 62(nv<1mmol/molcreatinine). Normal urine PA after pyridoxine treatment.

Variant 1. NM_001182.4(ALDH7A1):c.902A>T (p.Asn301Ile), 2. NM_001182.4(ALDH7A1):c.1279G>C (p.Glu427Gln), compound heterozygous

ClinVar 1. 18001, 2. 17994

gnomAD neither in gnomAD

CaseAddInfo n/a

CasePMIDs n/a

Case12 age/gender unknown, Caucasian, Italy

CasePresentingHPOs HP:0001250

(Seizures)

HPOsFreeText seizure onset in neonatal period from minutes after birth until 3 weeks of age. Definite PDE: formal pyridoxine withdrawal had resulted in reoccurrence of seizures that again responded to pyridoxine.

CaseNotHPOs n/a

CasePreviousTesting post treatment testing: Pipecolic acid in plasma 5.4(nv 0.7-2.6µmol/liter), AASA in plasma 9.6(nv<0.2µmol/liter), urine AASA 24(nv<1mmol/molcreatinine). Normal urine PA after pyridoxine treatment.

Variant NM_001182.4:c.1003C>T (p.Arg335Ter), homozygous

ClinVar n/a

CAID CA126889823

gnomAD MAF= 0.0001307(South Asian), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

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

Patient4: Male, 2.5yo

CasePresentingHPOs: HP:0002033, HP:0001254, HP:0001987, HP:0001873, HP:0002013 (poor sucking, lethargy, hyperammonaemia, thrombocytopenia, vomiting)

HPOsFreeText: acute neonatal IVA, treated with IVA special formula along with sodium benzoate and glycine (benzoate and glycine discontinued after 1yr), elevated isovalerylglycine, C5-carnitine = 17.4 µmol/l, birth weight = 3kg, able to sit at 6mo, walked at 13mo, began to babble at 17mo, did not have bladder/bowel control until 2.5yo, current weight = 14kg, current height = 86.5cm

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: symptom onset at 5do, diagnosis at 30do via clinical presentation, consanguineous family

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_002225.4(IVD):c.863C>T (p.Ala288Val) listed in paper as c.872C>T (p.Ala291Val)

ClinVar: 281076, https://www.ncbi.nlm.nih.gov/clinvar/variation/281076/

gnomAD: n/a

Variant2: NC_000015.9:g.40699940T>C (NM_002225.4(IVD):c.234+14C=)

CAID: CA147572, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NC_000015.9%3Ag.40699940T%3EC

ClinVar: 94053, https://www.ncbi.nlm.nih.gov/clinvar/variation/94053/

gnomAD: 0.6830, https://gnomad.broadinstitute.org/variant/15-40699940-T-C

Patient1: Male, 4yo, Twin A, Family A (fig. 2)

CasePresentingHPOs:

HPOsFreeText: acute neonatal IVA, treated with IVA special formula, elevated isovalerylglycine in urine organic acids, C5-carnitine = 6.15 µmol/l, at 2yo stopped formula and switched to vegetarian diet with L-carnitine supplements, admitted every 3-4mo due to metabolic crisis, at 4yo began formula again along with vegetarian diet and L-carnitine supplements, sat alone at 6mo, walked alone at 1yo, spoke first word at 1yo, unremarkable systemic examination at 4yo

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: diagnosed at 3 days old via NBS (positive family history and 3 sibling deaths), consanguineous family

CasePMIDs: PMID: 24516753

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_002225.4(IVD):c.1184G>A (p.Arg395Gln) listed in paper as c.1193G>A (p.R398Q)

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

gnomAD: n/a

Variant2: NC_000015.9:g.40699940T>C (NM_002225.4(IVD):c.234+14C=)

CAID: CA147572, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NC_000015.9%3Ag.40699940T%3EC

ClinVar: 94053, https://www.ncbi.nlm.nih.gov/clinvar/variation/94053/

gnomAD: 0.6830, https://gnomad.broadinstitute.org/variant/15-40699940-T-C

CaseTS: Male, Poland

CasePresentingHPOs: HP:0000256, HP:0001263, HP:0000729, HP:0001992 (Macrocephaly, Global developmental delay, Autistic behavior, Organic aciduria)

HPOsFreeText: Apgar score of 9

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are non-consanguineous though grandfathers were from neighboring villages, Older brother is healthy, Proband is confirmed homozygous via parental testing

CasePreviousTesting: Biochemical tests included urinary organic acid analysis, amino acid analysis, transaminases, transferrin isoforms, phosphoribosylaminoimidazole-succinocarboxamide (SAICAr), and D-arabinitol; Urinary organic acids analysis by GC-MS was performed repeatedly and revealed N-acetylated derivatives of methionine, glutamic acid, alanine, and glycine in large amounts, as well as leucine, valine, and isoleucine in smaller amounts with brother and parent analysis being unremarkable

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

CaseOS-120_II-1: Female, Birth onset, German/Italian

CasePresentingHPOs: HP:0001992, HP:0011343 (Organic aciduria, Moderate global developmental delay)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Younger brother is unaffected; Proband is confirmed compound heterozygous as mother is heterozygous for R353C but father is unavailable

CasePreviousTesting: GS-MS analysis identified urinary organic acids dominated by N-aceylated AAs, supported by NMR spectroscopy of urine samples with serum samples being negative; Values of excreted AAs are in Table 1; Neuropediatric evaluation performed

CaseGenotypingMethod: Genomic DNA was extracted from blood samples or after EBV transformation of peripheral blood lymphocytes. Exons and exon-intron boundaries were amplified and bidirectionally sequenced.

Variant1: NM_000666.3(ACY1):c.699A>C (p.Glu233Asp)

ClinVar: 18111, https://www.ncbi.nlm.nih.gov/clinvar/variation/18111/

gnomAD: 0.0001846, https://gnomad.broadinstitute.org/variant/3-52021204-A-C

Variant2: 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

Case# F1-2:1, Female, European, 3wk

CasePresentingHPOs HP:0001254, HP:0002789, HP:0001943, HP:0001987, HP:0001950

CasePreviousTesting sequencing and deletion/duplication analysis of known urea cycle defects and primary causes of hyperlactatemia. metabolic analysis of blood (Table 1).

CaseMethod1 Sanger

CaseMethod2 WES

CaseGenotypingMethod Sanger of CA5A

Variant NM_001739.1(CA5A):c.697T>C (p.Ser233Pro)

ClinVar 127087

VariantEvidence COS-7 cells with the p.Ser233Pro substitution showed 20% of the activity of the WT and quickly denatured at body temperature (Figure 1)

CaseAddInfo nonconsanguineous family, WES performed on heterzygous parents and affected siblings.

CaseF-2 male, 14mo onset, Ethnicity: North Indian, Hindu

CasePresentingHPOs HP:0001263, HP:0003571

(Developmental delay, Propionicacidemia)

HPOsFreeText normal plasma amino acids, acylcarnitines 13.9(normal <3.6nmol/mL), normal development by 6.5 years

CaseNotHPOs HP:0001942, HP:0001987

(Metabolic acidosis, Hyperammonemia)

CasePreviousTesting Also screened for variants in PCCA

Variant NM_000532.4(PCCB):c.1210G>A (p.Glu404Lys)

ClinVar 217891

gnomAD MAF= 0.00003266(South Asian), no homozygotes

CaseAddInfo nonconsanguineous, no abnormality in family history noted

CasePMIDs n/a

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

PMID: 17883863

Gene: ACADSB

Disease: 2-methylbutyrylglycinuria

MonDO: MONDO:0012392

InheritancePattern: AR

Case1: Newborn

CasePresentingHPOs:

HPOFreeText: Identified by NBS and is healthy with normal development.

CaseNotHPOs:

CasePreviousTesting: DBS C5 carnitine 1.2, 2.5, 1.1 umol/L (ref <0.46); plasma alloisoleucine 2.6 umol/L (ref <2.1); urine C5 carnitine 84.4, 58.1, 64.8 umol/mmol creatine (ref <3.0); urine 2-EHA 36, 31, 22 U (ref <5); urine 2-MBG 36.3, 29.3, 35.2 (ref <2.3); urine 2-methylbutyric acid conjugates as R-isomer 45%, 40% (ref 43-56%).

CaseGenotypingMethod: PCR amplification and sequencing of genomic DNA (entire coding region) from patient and available family members.

Variant: 1. NM_001609.3:c.1165A>G (p.Met389Val), 2. NM_001609.3:c.848A>G (p.Tyr283Cys)

CAID: 1. CA5730948; 2. CA5730830

gnomAD: 1. East Asian 0.0005513; 2. East Asian 0.0007068

VariantEvidence:

CaseAddInfo: In trans

CasePMIDs:

Case1: Female, Mixed, Birth onset

CasePresentingHPOs: HP:0001508, HP:0000252, HP:0002076, HP:0002018, HP:0002013, HP:0011342, HP:0000750, HP:0001270, HP:0007018 (Failure to thrive, Microcephaly, Migraine, Nausea, Emesis, Mild global developmental delay, Delayed speech and language development, Motor delay, Attention deficit hyperactivity disorder)

HPOsFreeText: Seizure-like episodes, Persistent head tilting, Symptoms impreoved over time

CaseNotHPOs: HP:0001252 (Muscular hypotonia)

NotHPOsFreeText: No brain MRI abnormalities

CaseAddInfo: Parents are non-consanguineous

CasePreviousTesting: Born at full term after a pregnancy complicated by maternal hypertension with no postnatal complications; Elevated levels of plasma 2-aminoadipate (37 μmol/L, nl < 4) with elevated 2-ketoadipate (434 mmol/mol creatinine, nl < 2) and 2-hydroxyadipate (28 mmol/mol creatinine, nl < 2) in urine; Electroencephalogram, magnetic resonance imaging, and magnetic resonance angiogram of the brain were unremarkable

CaseGenotypingMethod: Whole exome sequencing was performed as a trio with both parents. Identified DHTKD1 mutations were evaluated for possible effects and filtered for frequencies and predicted pathogenicity. No other candidate genes were found during the WES analysis for either patient sequenced.

Variant1: NM_018706.6:c.2143C>T (p.Arg715Cys)

ClinVar: 242503 https://www.ncbi.nlm.nih.gov/clinvar/variation/242503/

gnomAD: 0.0002007, https://gnomad.broadinstitute.org/variant/10-12150003-C-T

Variant2: NM_018706.6(DHTKD1):c.2185G>A (p.Gly729Arg)

ClinVar: 39564 https://www.ncbi.nlm.nih.gov/clinvar/variation/39564/

gnomAD: 0.002748, https://gnomad.broadinstitute.org/variant/10-12154929-G-A

Case4 Female, 7yo diagnosis, Korean

CasePresentingHPOs HP:0001083, HP:0001249, HP:0003235

(Ectopia lentis, Intellectual disability, Increased blood methionine)

HPOsFreeText Marfan syndrome, vascular occlusion, increased pre-treatment blood homocysteine and post-treatment total homocysteine, patient treated with low-methionine diet, vitamin B6, folate, vitamin B12, and betaine after diagnosis

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting pretreatment/posttreatment methionine: 24/575 μM(nv 7-47), pretreatment/posttreatment: 34/0 μM(nv 0-5), posttreatment total homocysteine 194 μM(nv 5-15)

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified all 16 coding exons of CBS gene, then direct sequenced

Variant 1. NM_000071.2(CBS):c.1006C>T (p.Arg336Cys), 2. NM_000071.2(CBS):c.1058C>T (p.Thr353Met), compound heterozygous

ClinVar 1. 92423, 2. 131

gnomAD 1. MAF=0.00004417(European(non-Finnish)), no homozygotes, 2. MAF=0.0002916(African), no homozygotes

CaseAddInfo R336C variant was paternally inherited, T353M variant was maternally inherited

CasePMIDs n/a

ExperimentName: PAH Variant Rescued by FokI-Cas9

RescueCellLine: Cytotoxic T cell (CL:0000910) AKA COS-7 Cell Line

GeneAlteration: A wild-type PAH cDNA was introduced into a vector via XbaI and BamHI cleavage after amplification from the plasmid. The mutation was introduced via site-directed mutagenesis and confirmed via Sanger sequencing. COS-7 cells were seeded and cultured before being transfected with either the mutant vector or the wild-type control. The resulting clone was characterized through RT-PCR and expanded.

RescueCellPhenotype: HP:0005982 (Reduced phenylalanine hydroxylase activity)

RescueMethod: The FokI-dCas9-IRES-ZsGreen1 plasmid was created by designing gRNAs to target the 1222C>T variant and synthesizing a final sgRNA expression fragment which was cloned into the base vector. Electroporation was then performed on the COS-7 cells with the expression vectors incorporated. Flow cytometry analysis confirmed that 18.4% of cells co-expressed both the FokI-dCas9 and the sgRNA.The cells were treated with the ssODN that represents the replacement sequence and confirmed via Sanger sequencing. Any off-target sites were anlyzed via Sanger sequencing and displayed no evidence of off-target cleavage.

RescueEvidence: Western blot analysis performed on cell expressing the wild-type PAH and those treated with differing levels of ODN and RS-1 showed that those treated with the replacement sequence had significantly higher PAH expression than the mutant cells that were not transfected with the vectors. Further, liquid chromatography-electrospray ionization tandem mass spectrometry was used to evaluate PAH activity after repair showing an activity of 22.08 +/- 1.67 % with the RS-1 expressed as opposed to 16.86+/- 1.31 % without the replacement sequence.

RescueCellVariant: NM_000277.3(PAH):c.1222C>T (p.Arg408Trp)

ClinVarID: 577 https://www.ncbi.nlm.nih.gov/clinvar/variation/577/

CasePA008 10mo report, Taiwan

CasePresentingHPOs HP:0001987, HP:0001943, HP:0011342, HP:0003571

(Hyperammonemia, Hypoglycemia, mild global developmental delay, propionicacidemia)

HPOsFreeText hyperammonemia and hypoglycemia for a few days after birth. Showed mild developmental delay at 10 months old

CaseNotHPOs n/a

CasePreviousTesting coding regions and flanking areas of PCCA also analyzed with PCR and Sanger

Variant 1. NM_000532.4(PCCB):c.1301C>T (p.Ala434Val), 2. NM_000532.4:c.-4156_183+3713del

ClinVar 1. 554490, 2. n/a

CAID 2. n/a

gnomAD 1. MAF=0.0003510(East Asian), no homozygotes, 2. n/a

CaseAddInfo n/a

CasePMIDs n/a

Case2: European

CasePresentingHPOs:

HPOFreeText: Identified by NBS with elevated C5-carnitine levels. Asymptomatic up to 3 year follow up. Levels of C5-carnitine decreased with time.

CaseNotHPOs:

CasePreviousTesting: Followup C5 carnitine 1.89 nM/ml (ref <0.63). Additional testing was performed on patients identified by NBS: all either had elevation of 2-methylbutyrlglycine in urine (with no intermediates consistent with isovaleric acidemia), or increase in 2-methylbutyrylcarnitine in culture from fibroblasts.

CaseGenotypingMethod: Bidirectional sequence analysis of genomic DNA.

Variant: 1. NM_001609.3(ACADSB):c.443C>T (p.Thr148Ile) 2. NM_001609.3(ACADSB):c.1159G>A (p.Glu387Lys)

ClinVar: 1. 9202 2. 9203

gnomAD: 1. South Asian 0.002678 2. Latino 0.0004797

VariantEvidence: 1. Decreases ACADSB enzymatic activity to 2.8% of wild type in vitro 2. In vitro expression of c.1159G>A in E. coli shows no protein on Western blot, reduced enzymatic activity to 2% and defects in protein folding and stabilit

CaseAddInfo: No info on cis or trans. Also heterozygous for p.R13K polymorphism.

CasePMIDs:

Case4:

CasePresentingHPOs:

HPOFreeText: Identified by NBS with elevated C5-carnitine levels. Asymptomatic up to 3 year follow up. Levels of C5-carnitine decreased with time.

CaseNotHPOs:

CasePreviousTesting: Followup C5 carnitine 2.58 nM/ml (ref <0.63). Additional testing was performed on patients identified by NBS: all either had elevation of 2-methylbutyrlglycine in urine (with no intermediates consistent with isovaleric acidemia), or increase in 2-methylbutyrylcarnitine in culture from fibroblasts.

CaseGenotypingMethod: Bidirectional sequence analysis of genomic DNA.

Variant: NM_001609.3(ACADSB):c.303+1G>A (homozygous)

ClinVar: 203367

gnomAD: Other 0.0005549

VariantEvidence: Canonical splice site variant. Discrupts canonical splicing signal of intron 3.

CaseAddInfo:

CasePMIDs:

Family#: JU#0213, Japanese

FamilyHPOs: HP:0012026

FamilyGenotypingMethod: PCR, sequencing of the coding exons (3-11) of the OAT gene from genomic DNA

Variant: 1) c.504_505delAA (p.K169DfsX10) (mother) 2) c.1276C>T (p.R426X) (last exon) (father)

ClinVar: 1) NA; 2) 177

CAID: 1) CA913187453

gnomAD: The maximum population minor allele frequency for c.1276C>T (p.R426X) is 0.00002893 (Latino); no homozygotes in any population. c.504_505delAA is not in gnomAD.

Proband: JU#0213:II-1

CaseII-2: Female, Japanese, 4 years death

CasePresentingHPOs: HP:0001290, HP:0001263, HP:0002453, HP:0004372, HP:0001942, HP:0045011, HP:0002339, HP:0002138, HP:0002795, HP:0007695, HP:0008000, HP:0001635, HP:0001942 (Generalized hypotonia, Developmental delay, Abnormality of the globus pallidus, Lowered consciousness, Metabolic acidosis, Decreased urine bicarbonate concentration, Abnormality of the caudate nucleus, Subarachnoid hemorrhage, Abnormal respiration, Abnormal pupillary light reflex, Decreased corneal reflex, Heart failure, Metabolic acidosis)

HPOsFreeText: Her birth weight was 2714 g, She measured 44 cm (− 2.6 SD) in length, Had an occipitofrontal circumference of 32.5 cm (− 0.5 SD), Could hold her head up at 5 months and roll over at 10 months, Febrile illness at 15 months, Required ventilator support, Absence of brain activity, Auditory brainstem response audiometry showed no response

CaseNotHPOs: HP:0009782, HP:0001250, HP:0001332 (Aplasia/Hypoplasia of the biceps, Seizures, Dystonia)

NotHPOsFreeText:

CaseAddInfo: Parents are nonconsanguineous, Older sister of patient II-2 who is also homozygous for the mutation and has the disorder, Parents are confirmed heterozygous for the mutation

CasePreviousTesting: Lactate and pyruvate levels in arterial blood (16.5 mg/dL and 1.48 mg/dL, respectively; normal range: 5–20 mg/dL and 0.3–0.9 mg/dL, respectively) and cerebrospinal liquor (13.5 mg/dL and 1.12 mg/dL, respectively; normal range: 9.1–18.8 mg/dL and 0.26–1.32 mg/dL, respectively) were almost normal, Arterial blood ketone bodies, pH, pCO2, and base excess were (8700 μmol/L; normal range: < 130 μmol/L), 7.051, 6.7 mm Hg, and − 26.9 mEq/L, respectively, CSF lactate at 1.5 (< 2.0 mmol/L reference)

CaseGenotypingMethod: Whole exome analysis was performed on the parents and the proband. Afterwards, genomic DNA was extracted from peripheral blood leukocytes and targeted sequences were amplified and sequenced. All SNVs and Indels were evaluated and filtered to confirm the mutations. Afterwards, after identifying the exon 4 mutation, a DNA segment was amplified via PCR and sequenced to confirm these findings.

Variant: NM_014362.3:c.287C>A (p.Ala96Asp)

CAID: CA349896230 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_014362.3%3Ac.287C%3EA

gnomAD: Not in gnomAD

Case1: Male, 3 months onset, Pakistan,

CasePresentingHPOs: HP:0002376, HP:0008947, HP:0001336, HP:0002197, HP:0002490, HP:0012240, HP:0002572, HP:0000737, HP:0002360, HP:0002453, HP:0002352, HP:0002283, HP:0001332, HP:0002871, HP:0000648, HP:0000365, HP:0000252 (Developmental regression, Infantile hypotonia, Myoclonic jerks, Generalized seizures, Increased CSF lactate, Increased muscle lipid droplets, Episodic vomiting, Irritability, Sleep disturbance, Abnormality of the globus pallidus, Leukoencephalopathy, Global brain atrophy, Dystonia, Central apnea, Optic atrophy, Hearing loss, Microcephaly)

HPOsFreeText: Born at term weighing 3.2 kg, Loss of smile

CaseNotHPOs: HP:0002151, HP:0003200, HP:0003688, HP:0010663, HP:0006846 (Increased serum lactate, Ragged-red fibres, Cytochrome C oxidase-negative muscle fibers, Abnormality of thalamus morphology, Acute encephalopathy)

NotHPOsFreeText: N/A

CaseAddInfo: Parents were distantly related, Died at 3 years, Has a younger brother with the same variant, Has a family history of neurological/neuromuscular disorders, Variant was confirmed by parental testing

CasePreviousTesting: At 8 months Nissen fundoplication was performed because of persistent vomiting, Mildly elevated CSF lactate at 2.2 mmol/l (reference range <2 mmol/l) at 8 months, with a normal venous blood lactate of 1.7 mmol/l (reference range < 1.8 mmol/l), Complex I at 0.068 (Reference; 0.104-0.268 ratio to citrate synthase activity), Complexes II + III at 0.010 (0.040-0.204 ratio to citrate synthase activity), Complex IV (COX) at 0.010 (0.014-0.034 ratio to citrate synthase activity)

CaseGenotypingMethod: Genomic DNA was extracted from skin fibroblasts. Exons and flanking intronic sequences of HIBCH was sequenced after PCR amplification. Sequence data was compared to the reference sequence and filtered for variants.

Variant: NM_014362.3:c.950G>A (p.Gly317Glu)

ClinVar: 187864 https://www.ncbi.nlm.nih.gov/clinvar/variation/187864/

gnomAD: Not in gnomAD

Case#: KM24, M, 7 months (Dx), Japanese

CasePresentingHPOs: HP:0001945, HP:0001250, HP:0002013, HP:0002014, HP:0001254 (Fever, Seizures,Vomiting, Diarrhea, Lethargy)

HPOsFreeText: Newborn screening for MSUD was negative. Diagnosis of intermittent MSUD was established by blood and urine analysis after an illness.

CaseNotHPOs: HP:0001263 (Global development delay)

CasePreviousTesting: Lymphoblastoid cells from the patient showed reduced decarboxylation activity (~20% of control).

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: cDNAs from the patient were amplified, subcloned and sequenced. Confirmed by restriction enzyme digestion.

Variant: NM_001918.3(DBT):c.1448G>T (p.Ter483Leu) (homozygous)

ClinVar: 11947

gnomAD: Not in gnomAD

VariantEvidence: The variant adds 7 amino acids at the carboxyl terminus of the protein. No functional evidence.

CaseAddInfo: Consanguineous, parents and 2 siblings were heterozygous.

Case: Cecconi_Patient#905, male, 30 yrs old at onset of HCM

CasePresentingHPOs:

CasePresentingHPOsFreeText: In adults, a maximal LV wall thickness (LVWT) ≥15 mm (not provided for this proband); the maximal LVWT between 10 and 14 mm, in conjunction with other features (i.e., family history, electrocardiogram abnormalities) prompted the diagnosis of borderline HCM. LVEF is not provided. Family history of HCM.

CaseNOTHPOs:

CaseNOTHPOSFreeText: no family history of SCD

CasePreviousTesting: The clinical diagnosis of primary HCM was based on medical history, a physical examination and on the echocardiographic demonstration of a hypertrophied left ventricle (LV) that could not be explained by another cardiac or systemic disease.

CaseGenotypingMethod: NGS, Sanger Targeted NGS panel to simultaneously analyze a total of 19 genes encoding not only TTm proteins, but also other sarcomeric proteins and some non-sarcomeric proteins that cause HCM phenocopies. Sanger sequencing to ensure a depth of coverage >20X for each target nucleotide.

Variant: c.1655G>A p.(Gly552Asp)

ClinVarID: or CAID: CA401714383

gnomAD: This proband has a missense variant, Gly552Asp. Not present in gnomAD. Proband has a variant in another HCM gene-MYH7.

AddCaseInfo: history of heart transplant, automatic implantable cardioverter defibrillator

PreviouslyPublished: no

VariantEvidence: none

Case: Cecconi_Patient#907, male, 37 yrs old at onset of HCM

CasePresentingHPOs:

CasePresentingHPOsFreeText: In adults, a maximal LV wall thickness (LVWT) ≥15 mm (<15mm for this proband-diagnosis of borderline HCM); the maximal LVWT between 10 and 14 mm, in conjunction with other features (i.e., family history, electrocardiogram abnormalities) prompted the diagnosis of borderline HCM. LVEF of 60%

CaseNOTHPOs:

CaseNOTHPOSFreeText: family history of HCM

CasePreviousTesting: The clinical diagnosis of primary HCM was based on medical history, a physical examination and on the echocardiographic demonstration of a hypertrophied left ventricle (LV) that could not be explained by another cardiac or systemic disease.

CaseGenotypingMethod: NGS, Sanger Targeted NGS panel to simultaneously analyze a total of 19 genes encoding not only TTm proteins, but also other sarcomeric proteins and some non-sarcomeric proteins that cause HCM phenocopies. Sanger sequencing to ensure a depth of coverage >20X for each target nucleotide.

Variant: c.432-1G>T

ClinVarID: or CAID: CA401717111

gnomAD: This proband has a splice site variant, c.432-1G>T. MAF=0.00001597 in European (non-Finnish) (2/125212 alleles), total allele frequency=0.000007246. No homozygotes. gnomAD v2.1.1.

AddCaseInfo: history of ECG abnormalities

PreviouslyPublished: no

VariantEvidence: none

GroupPresentingHPOs: HP:0000768, HP:0002857, HP:0008454, HP:0001382, HP:0007957, HP:0000687, HP:0004630, HP:0002869, HP:0008807, HP:0002673, HP:0003311, HP:0003414, HP:0002656,

GroupNotHPOs:

GroupPreviousTesting:

GroupMethod1: single gene seq of GALNS exons and flanking intronic seqs

GroupMethod2: GALNS enzyme assa from leukocytes as prev described - range was 0 - 8 nmol/17hr/mg protein with ref range being 40 - 170s

GroupGenotypingMethod: single gene seq

NumberMales: 34

NumberFemales: 34

CountryOrigin: India

Ethnicity:

Race:

AgeRange: 1 - 26

TotalNumber: 68

FamilyInfo: Family blood samples were obtained and the authors state a 3 generation pedigree was constructed for each family but that data was not provided in the study.

NoFamily:

VariantCount: 40

NonVariant:

OtherVariant:

NonPatientCell: EFO:0005214

GOterm: GO:0016192

GeneAlteration: Undifferentiated N2A cells (murine neuroblastoma cell line) were infected with shRNAs to downregulate endogenous Rab39b expression by 45%.

FunctionalEvidence: N2A cells 4d after treatment with shRNA showed a change in morphology. Clumps of actin and reduced and/or altered membrane protrusions were observed. Rescue with WT RAB39B was able to restore the normal actin and cytoskeletal morphology, indicating that Rab39b may alter intracellular vesicular trafficking steps essential for membrane remodeling.

FAVariants:

NonPatientCell: CL:0002608

GOterm: GO:0016192

GeneAlteration: Rab39b expression was knocked down in mouse primary hippocampal neurons using shRNA

FunctionalEvidence: Expression of Rab39b was reduced by 40% as determined by RT-PCR. Morphological analysis showed significant alteration and the number of growth cones was decreased at each neurite terminal. Authors also show that RAB39B was localized to growth cones and could be involved in intracellular trafficking related to membrane recycling of Golgi-derived vesicles. Therefore RAB39B gene alterations can lead to impaired trafficking and lead to accumulation in membrane content and disorganized growth of growth cones.

FAVariants:

PMID: 34504088

Gene: GALNS

Disease: Mucopolysaccharidosis IVA

MonDO: MONDO:0009659

InheritancePattern: AR

Notes:

genotype phenotype information for probands

Case#: Consequence 13 Table 1 in Data Supplement (2 cases)

DiseaseAssertion: HCM

FamilyInfo: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Cardiac magnetic resonance imaging, BSA, NYHA functional class, obstructive HCM, atrial fibrillation, coronary artery disease, NSVT, Syncope, SCD family hitory, wall thickness

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: Additional oligonucleotide hybridization–based DNA sequencing and dideoxy-based DNA sequencing for MYBPC3, TMP1, TNNC1, TNNI2, TNNI3, TNNT2, ACTC1, MYL3

CaseGenotypingMethod: oligonucleotide hybridization–based DNA sequencing and dideoxy-based DNA sequencing

Variant: NM_000257.4(MYH7):c.1358G>A (p.Arg453His)

ClinVar: 42838 https://www.ncbi.nlm.nih.gov/clinvar/variation/42838/

CAID: n/a

gnomAD: 0.0000 African https://gnomad.broadinstitute.org/variant/3-49167319-C-T

Case#: Consequence 22 Table 1 in Data Supplement (6 cases)

DiseaseAssertion: HCM

FamilyInfo: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Cardiac magnetic resonance imaging, BSA, NYHA functional class, obstructive HCM, atrial fibrillation, coronary artery disease, NSVT, Syncope, SCD family hitory, wall thickness

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: Additional oligonucleotide hybridization–based DNA sequencing and dideoxy-based DNA sequencing for MYBPC3, TMP1, TNNC1, TNNI2, TNNI3, TNNT2, ACTC1, MYL3

CaseGenotypingMethod: oligonucleotide hybridization–based DNA sequencing and dideoxy-based DNA sequencing

Variant: NM_000257.4(MYH7):c.2722C>G (p.Leu908Val)

ClinVar: 14097 https://www.ncbi.nlm.nih.gov/clinvar/variation/14097/

CAID: n/a

gnomAD: 0.00006482 European (non-Finnish) https://gnomad.broadinstitute.org/variant/14-23893316-G-C

Case#: Consequence 32 Table 1 in Data Supplement (1 cases)

DiseaseAssertion: HCM

FamilyInfo: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Cardiac magnetic resonance imaging, BSA, NYHA functional class, obstructive HCM, atrial fibrillation, coronary artery disease, NSVT, Syncope, SCD family hitory, wall thickness

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: Additional oligonucleotide hybridization–based DNA sequencing and dideoxy-based DNA sequencing for MYBPC3, TMP1, TNNC1, TNNI2, TNNI3, TNNT2, ACTC1, MYL3

CaseGenotypingMethod: oligonucleotide hybridization–based DNA sequencing and dideoxy-based DNA sequencing

Variant: NM_000257.4(MYH7):c.788T>C (p.Ile263Thr)

ClinVar: 43106 https://www.ncbi.nlm.nih.gov/clinvar/variation/43106/

CAID: n/a

gnomAD: 0.000008792 European (non-Finnish) https://gnomad.broadinstitute.org/variant/14-23900635-A-G

NonHumanModel: Mouse (Mus musculus)

GeneAlteration: Two approaches were used to create this model. The first imitated the slow fiber-specific expression of TPM3 by linking the coding region for αTm_slow(Met9Arg) to a human troponin I slow gene that drives expression specifically in these fibers via EcoRI-PstI digest and ligation into a linearized construct. The second, HSA–αTm_slow(Met9Arg), expressed the mutation in fast fibers by ligating a SmaI-BamHI cassette containing the intron and 3′-UTR of SV40 small t antigen directly downstream of the mutant cDNA sequence followed by blunt-end ligation of the HSA promoter into the EcoRV site upstream of the sequence before linearizing into a construct. These constructs were injected into fertilized eggs and screened by southern blot analysis to verify the successful insertion.

ModelPhenotype: HP:0003798, HP:0100303, HP:0100301, HP:0003557, HP:0001324, HP:0011807 (Nemaline bodies, Muscle fiber cytoplasmic bodies, Muscle fiber tubular aggregates, Increased variability in muscle fiber diameter, Muscle weakness, Type 1 muscle fiber atrophy)

ModelFreeText: Disorganized sarcomeres, Increased slow/fast oxidative fibers

HumanPhenotype: HP:0003798, HP:0003557, HP:0001324, HP:0011807 (Nemaline bodies, Increased variability in muscle fiber diameter, Muscle weakness, Type 1 muscle fiber atrophy)

HumanFreeText: Disorganized sarcomeres

ModelEvidence: The Met9Arg mouse models not only clearly recapitulate the phenotypes of nemaline/other myopathies but also provide evidence to suggest possible pathogenic mechanisms of the mutated allele in vivo. Mice expressing the mutated allele display nemaline rods and clear fiber-type disproportion, both of which are hallmarks for their respective diseases. They also displayed progressive muscle weakness starting around 5-6 months of age, a phenotype that matches the childhood-onset muscle weakness and other progressive symptoms common in TPM3-related congenital myopathy patients. Another classic phenotype, atrophy of Type I muscle fibers with a corresponding hypotrophy of Type II fibers was also present in the mice. This may be related to the increased number of slow/fast oxidative fibers in the mutant mice leading to the disruption of normal muscle fiber maturation. The mice also displayed some cytoplasmic bodies and tubular aggregates; these have been observed in varying functional studies and similar features have also been observed in humans.

ModelVariant: NM_152263.4(TPM3):c.26T>G (p.Met9Arg)

ClinVar: 12446 https://www.ncbi.nlm.nih.gov/clinvar/variation/12446/

CaseA009-IV-1 male, unknown age, Han Chinese

CasePresentingHPOs HP:0003251, HP:0012868, HP:0012207

(male infertility, sperm tail anomaly, reduced sperm motility)

HPOsFreeText Table S1: Sperm volume: 2.1mL, sperm concentration: 34.5million/mL, progressive motility: 0.2%, motility: 7.7%, normal spermatozoa: 6.5%, absent flagella: 30.0%, short flagella: 41.5%, coiled flagella: 18.5%, angulation: 2.5%, irregular caliber: 1.0%. Semen volume and sperm concentration were normal to the standards.

CaseNotHPOs n/a

CasePreviousTesting Ejaculation disorder, erectile dysfunction, and orchiatrophy not detected. Chromosomal karyotypes were normal (46; XY) and no deletion detected in the human Y chromosome. In addition, primary ciliary dyskinesia-related symptoms of sinusitis, bronchitis, pneumonia, and otitis media were also excluded.

Variant NM_144668.5:c.799C>T(p.Arg267Ter)

ClinVar n/a

CAID CA6840411

gnomAD Maximum population minor allele frequency in gnomAD = 0.00005903 (East Asian); no homozygotes

CaseAddInfo parents unavailable for testing

CasePMIDs n/a

Case# B12, Male, UK

CasePresentingHPOs HP:0010872

CasePreviousTesting re-sequenicing of genome

CaseGenotypingMethod genotyped for five microsatellite markers spanning TMEM43 on chromosome 3p25

Variant NM_024334.2(TMEM43):c.934C>T (p.Arg312Trp)

ClinVar 36873

gnomAD 0.03553 link