Patient8: Male, Southern China

CasePresentingHPOs:

HPOsFreeText: IOPD

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo:

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000152.4(GAA):c.1411_1414delGAGA (p.Glu471Profs)

ClinVar: 188874, https://www.ncbi.nlm.nih.gov/clinvar/variation/188874/

gnomAD: 1.688e-05, http://exac.broadinstitute.org/variant/17-78083827-CGAGA-C

Patient19: Male, Southern China

CasePresentingHPOs:

HPOsFreeText: IOPD

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo:

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000152.4(GAA):c.1935C>A (p.Asp645Glu)

ClinVar: 4029, https://www.ncbi.nlm.nih.gov/clinvar/variation/4029/

gnomAD: 0.0001721, http://exac.broadinstitute.org/variant/17-78086721-C-A

Variant2: NM_000152.4(GAA):c.2815_2816delGT (p.Val939Leufs)

ClinVar: 371481, https://www.ncbi.nlm.nih.gov/clinvar/variation/371481/

gnomAD: 1.653e-05, http://exac.broadinstitute.org/variant/17-78093082-CTG-C

Case#: Patient 7, female

CasePresentingHPOs: HP:0003236, HP:0003325

CaseAddInfo: 53yo at onset, 55yo at diagnosis

VariantAllele1: NM_000152.4(GAA):c.-32-13T>G

ClinVar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

VariantAllele2: NM_000152.4(GAA):c.1927G>A (p.Gly643Arg)

ClinVar: 4023, https://www.ncbi.nlm.nih.gov/clinvar/variation/4023/

gnomAD: 5.884e-05, http://exac.broadinstitute.org/variant/17-78086713-G-A

Case#: Patient 13, female

CasePresentingHPOs: HP:0003236, HP:0003325

CaseAddInfo: 28yo at onset, 48yo at diagnosis

VariantAllele1: NM_000152.4(GAA):c.-32-13T>G

ClinVar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

VariantAllele2: NM_000152.4(GAA):c.2189+1G>T

ClinVar: 558223, https://www.ncbi.nlm.nih.gov/clinvar/variation/558223/

gnomAD: n/a

Case#: Patient 15, female

CasePresentingHPOs: HP:0003236, HP:0003325

CaseAddInfo: 42yo at onset, 52yo at diagnosis

VariantAllele1: NM_000152.4(GAA):c.-32-13T>G

ClinVar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

VariantAllele2: NM_000152.4(GAA):c.1927G>A (p.Gly643Arg)

ClinVar: 4023, https://www.ncbi.nlm.nih.gov/clinvar/variation/4023/

gnomAD: 5.884e-05, http://exac.broadinstitute.org/variant/17-78086713-G-A

Case: male, gestational age of 37 weeks and 4 days, birth weight = 3.640kg

CasePresentingHPOs: HP:0001639, HP:0030148, HP:0200128, HP:0001640, HP:0003236, HP:0002910, HP:0002788, HP:0001522, HP:0009051

HPOsFreeText: ventricular septal thickness = 11mm, left ventricular posterior wall thickness = 8-9mm (higher at apical level), CK = 4168 UI/L, glutamic oxaloacetic transaminase = 250 UI/L, normal brain and abdominal ultrasounds, GAA activity = 0.9 μmol/L/h, CRIM positive, treated with ERT and beta-blockers, subsarcolemmal vacuolization of the cardiac muscle, moderate vacuoles in skeletal and smooth muscle, neurons, and glial cells due to glycogen accumulation

CaseNotHPOs: HP:0006673, HP:0001941, HP:0001943

CaseAddInfo: diagnosis of HCM at 48hrs of life, non consanguineous Nigerian parents, no family history of heart disease, admitted to hospital at 2 months due to upper respiratory infection, death due to ventricular fibrillation during central line placement procedure

CasePreviousTesting: (2D) echocardiogram, EKG, laboratory tests, brain and abdominal ultrasounds, DBS

CaseGenotypingMethod: n/a

Variant: NM_000152.4(GAA):c.2560C>T (p.Arg854Ter)

ClinVar: 4034, https://www.ncbi.nlm.nih.gov/clinvar/variation/4034/

gnomAD: 0.0002146, http://exac.broadinstitute.org/variant/17-78092070-C-T

Patient10: IOPD Group, 46days old

CasePresentingHPOs:

HPOsFreeText: LDH = 425 U/L, CK = 221 U/L, ALT = 41 U/L, AST = 61 U/L, Mann-Whitney Test = 2

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo:

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant: NM_000152.4(GAA):c.1935C>A (p.Asp645Glu)

ClinVar: 4029, https://www.ncbi.nlm.nih.gov/clinvar/variation/4029/

gnomAD: 0.0001240, https://gnomad.broadinstitute.org/variant/17-78086721-C-A

Patient7: IOPD Group, 10days old

CasePresentingHPOs:

HPOsFreeText: LDH = 431 U/L, CK = 237 U/L, ALT = 23 U/L, AST = 58 U/L, Mann-Whitney Test = 2

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo:

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant: NM_000152.4(GAA):c.1935C>A (p.Asp645Glu)

ClinVar: 4029, https://www.ncbi.nlm.nih.gov/clinvar/variation/4029/

gnomAD: 0.0001240, https://gnomad.broadinstitute.org/variant/17-78086721-C-A

Patient16: Female, 49yo

CasePresentingHPOs: HP:0003077

HPOsFreeText: LOPD

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: 23yr disease duration, ERT lasted 119mo, 5yr follow-up MRI

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000152.4(GAA):c.-32-13T>G

Clinvar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

Variant2: NM_000152.4(GAA):c.307T>G (p.Cys103Gly)

ClinVar: 92483, https://www.ncbi.nlm.nih.gov/clinvar/variation/92483/

gnomAD: n/a

Patient13: Male, 61yo

CasePresentingHPOs: HP:0003077, HP:0000822

HPOsFreeText: LOPD

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: homozygous, 13yr disease duration, ERT lasted 29mo

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant: NM_000152.4(GAA):c.-32-13T>G

Clinvar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

CaseLOPD-4: 28 years diagnosis

HPOsFreeText: GAA activity 3.4 (nmol · h−1 · mg−1), Mean percentage of normal 4.7%

CaseNotHPOs: HP:0003236 (Increased serum CK)

GenotypingMethod: Newborn Screening Panels

Variant: NM_000152.4:c.-32-13T>G

ClinVar: 4027 http://www.ncbi.nlm.nih.gov/clinvar/variation/4027

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

CaseAddInfo: Late Onset Pompe Disease, Patient is homozygous for the above variant

CasePD/Pathogenic-4: 2 weeks diagnosis

HPOsFreeText: GAA activity 5.0 (nmol · h−1 · mg−1), Mean percentage of normal 7.0%

GenotypingMethod: Newborn Screening Panels

Variant1: NM_000152.4:c.1726G>A (p.Gly576Ser)

ClinVar: 92467 https://www.ncbi.nlm.nih.gov/clinvar/variation/92467/

gnomAD: East Asian 0.1427, Overall 0.01700 http://gnomad.broadinstitute.org/variant/17-78085871-G-A

Variant2: NM_000152.4:c.2065G>A (p.Glu689Lys)

ClinVar: 4030 https://www.ncbi.nlm.nih.gov/clinvar/variation/4030/

gnomAD: East Asian 0.2355, Overall 0.05524 http://gnomad.broadinstitute.org/variant/17-78087041-G-A

Variant3: NM_000152.4:c.752C>T (p.Ser251Leu)

ClinVar: 325781 https://www.ncbi.nlm.nih.gov/clinvar/variation/325781/

gnomAD: East Asian 2.760e-3, Overall 3.540e-4 http://gnomad.broadinstitute.org/variant/17-78081415-C-T

Variant4: NM_000152.4:c.761C>T (p.Ser254Leu)

ClinVar: 325782 https://www.ncbi.nlm.nih.gov/clinvar/variation/325782/

gnomAD: East Asian 2.759e-3, Overall 1.947e-4 http://gnomad.broadinstitute.org/variant/17-78081424-C-T

CaseAddInfo: Patient has one pseudodeficency allele and one pathogenic allele, Nomenclature claims Variant 1 and 2 are in cis, Nomenclature claims Variant 3 and 4 are in cis

CasePD/Pathogenic-3: 2 weeks diagnosis

HPOsFreeText: GAA activity 4.9 (nmol · h−1 · mg−1), Mean percentage of normal 6.9%

GenotypingMethod: Newborn Screening Panels

Variant1: NM_000152.4:c.1726G>A (p.Gly576Ser)

ClinVar: 92467 https://www.ncbi.nlm.nih.gov/clinvar/variation/92467/

gnomAD: East Asian 0.1427, Overall 0.01700 http://gnomad.broadinstitute.org/variant/17-78085871-G-A

Variant2: NM_000152.4:c.2065G>A (p.Glu689Lys)

ClinVar: 4030 https://www.ncbi.nlm.nih.gov/clinvar/variation/4030/

gnomAD: East Asian 0.2355, Overall 0.05524 http://gnomad.broadinstitute.org/variant/17-78087041-G-A

Variant3: NM_000152.4:c.752C>T (p.Ser251Leu)

ClinVar: 325781 https://www.ncbi.nlm.nih.gov/clinvar/variation/325781/

gnomAD: East Asian 2.760e-3, Overall 3.540e-4 http://gnomad.broadinstitute.org/variant/17-78081415-C-T

Variant4: NM_000152.4:c.761C>T (p.Ser254Leu)

ClinVar: 325782 https://www.ncbi.nlm.nih.gov/clinvar/variation/325782/

gnomAD: East Asian 2.759e-3, Overall 1.947e-4 http://gnomad.broadinstitute.org/variant/17-78081424-C-T

CaseAddInfo: Patient has one pseudodeficency allele and one pathogenic allele, Nomenclature claims Variant 1 and 2 are in cis, Nomenclature claims Variant 3 and 4 are in cis

Case43

HPOsFreeText: See Group Phenotypes

CasePreviousTesting: Sanger sequencing

CaseMethod1: Sanger sequencing and PCR Amplification

CaseMethod2: Whole Exome Sequencing (WES)

CaseGenotypingMethod: Patients were previously confirmed with Pompe disease by Sanger sequencing to capture the pathogenic variants. The process was repeated as part of the study taking care to amplify the coding regions and exon/intron boundaries to compare with the WES results afterwards.

Variant1: NM_000152.4:c.2815_2816delGT (p.Val939fs*78)

ClinVar: 371481 https://www.ncbi.nlm.nih.gov/clinvar/variation/371481/

gnomAD: East Asian 1.631e-4, Overall 1.194e-5 http://gnomad.broadinstitute.org/variant/17-78093082-CTG-C

Variant2: NM_000152.4:c.1935C>A (p.Asp645Glu)

CAID: 4029 https://www.ncbi.nlm.nih.gov/clinvar/variation/4029/

gnomAD: East Asian 1.729e-3, Overall 1.240e-4 http://gnomad.broadinstitute.org/variant/17-78086721-C-A

CaseAddInfo: Infantile Onset Pompe Disease

Case79

HPOsFreeText: See Group Phenotypes

CasePreviousTesting: Sanger sequencing

CaseMethod1: Sanger sequencing and PCR Amplification

CaseMethod2: Whole Exome Sequencing (WES)

CaseGenotypingMethod: Patients were previously confirmed with Pompe disease by Sanger sequencing to capture the pathogenic variants. The process was repeated as part of the study taking care to amplify the coding regions and exon/intron boundaries to compare with the WES results afterwards.

Variant1: NM_000152.4:c.525delT (p.Glu176Argfs*45)

ClinVar: 4033 https://www.ncbi.nlm.nih.gov/clinvar/variation/4033/

gnomAD: European (Non-Finnish) 1.880e-4, Overall 9.959e-5 http://gnomad.broadinstitute.org/variant/17-78078909-CT-C

Variant2: NM_000152.4:c.-32-13T>G

ClinVar: 4027 https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

CaseAddInfo: Adult Late Onset Pompe Disease, 3 other patients have the same genotype

Case23

HPOsFreeText: See Group Phenotypes

CasePreviousTesting: Sanger sequencing

CaseMethod1: Sanger sequencing and PCR Amplification

CaseMethod2: Whole Exome Sequencing (WES)

CaseGenotypingMethod: Patients were previously confirmed with Pompe disease by Sanger sequencing to capture the pathogenic variants. The process was repeated as part of the study taking care to amplify the coding regions and exon/intron boundaries to compare with the WES results afterwards.

Variant1: NM_000152.4:c.2560C>T (p.Arg854*)

ClinVar: 4034 https://www.ncbi.nlm.nih.gov/clinvar/variation/4034/

gnomAD: African 1.891e-3, Overall 2.090e-4 http://gnomad.broadinstitute.org/variant/17-78092070-C-T

Variant2: NM_000152.4:c.2236T>C (p.Trp746Arg)

ClinVar: 499293 https://www.ncbi.nlm.nih.gov/clinvar/variation/499293/

gnomAD: Not in gnomAD

CaseAddInfo: Infantile Onset Pompe Disease

Case26

HPOsFreeText: See Group Phenotypes

CasePreviousTesting: Sanger sequencing

CaseMethod1: Sanger sequencing and PCR Amplification

CaseMethod2: Whole Exome Sequencing (WES)

CaseGenotypingMethod: Patients were previously confirmed with Pompe disease by Sanger sequencing to capture the pathogenic variants. The process was repeated as part of the study taking care to amplify the coding regions and exon/intron boundaries to compare with the WES results afterwards.

Variant code not given

Variant1: NM_000152.4: [del Exons 15–20]

ClinVar: N/A

gnomAD: N/A

Variant2: NM_000152.4:c.2012T>G (p.Met671Arg)

ClinVar: 92469 https://www.ncbi.nlm.nih.gov/clinvar/variation/92469/

gnomAD: Not in gnomAD

CaseAddInfo: Infantile Onset Pompe Disease

Case41

HPOsFreeText: See Group Phenotypes

CasePreviousTesting: Sanger sequencing

CaseMethod1: Sanger sequencing and PCR Amplification

CaseMethod2: Whole Exome Sequencing (WES)

CaseGenotypingMethod: Patients were previously confirmed with Pompe disease by Sanger sequencing to capture the pathogenic variants. The process was repeated as part of the study taking care to amplify the coding regions and exon/intron boundaries to compare with the WES results afterwards.

Variant: NM_000152.4:c.2481+102_2646+31del (p.Gly828_Asn882del) (exon 18 del)

ClinVar: 4031 https://www.ncbi.nlm.nih.gov/clinvar/variation/4031/

gnomAD: Not in gnomAD

CaseAddInfo: Infantile Onset Pompe Disease, Patient is homozygous for this variant

Patient1: the efficacy of AMOs 1, 2, and 3 were tested in cultured fibroblasts from this patient

Under basal conditions, the fibroblasts retained 20% enzymatic activity compared to WT cells; treatment with combination of the AMOs showed rescue of normally spliced GAA mRNA, this was confirmed with real-time qPCR

CasePresentingHPOs:

HPOsFreeText:

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo:

CasePMIDs:

CasePreviousTesting: n/a

CaseGenotypingMethod: n/a

Variant1: NM_000152.4(GAA):c.-32-13T>G

Clinvar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

Variant2: unknown

Case13: Female, 30 years report

HPOsFreeText: 7 years of ERT completed, 6 Minute Walking Test at 520 meters, Forced Vital Capacity at 50%

GenotypingMethod: GAA Gene Sequencing

Variant1: NM_000152.4:c.-32-13T>G

ClinVar: 4027 http://www.ncbi.nlm.nih.gov/clinvar/variation/4027

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

Variant2: NM_000152.4:c.525delT (p.Glu176Argfs*42)

ClinVar: 4033 https://www.ncbi.nlm.nih.gov/clinvar/variation/4033/

gnomAD: European (Non-Finnish) 1.880e-4, Overall 9.959e-5 http://gnomad.broadinstitute.org/variant/17-78078909-CT-C

CaseAddInfo: Late Onset Pompe Disease

Case36: Female, 69 years report

HPOsFreeText: ERT not started, 6 Minute Walking Test at 252 meters, Forced Vital Capacity at 87%

GenotypingMethod: GAA Gene Sequencing

Variant1: NM_000152.4:c.-32-13T>G

ClinVar: 4027 http://www.ncbi.nlm.nih.gov/clinvar/variation/4027

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

Variant2: NM_000152.4:c.854C>G (p.Pro285Arg)

ClinVar: 225114 https://www.ncbi.nlm.nih.gov/clinvar/variation/225114/

gnomAD: European (Non-Finnish) 1.148e-5, Overall 5.109e-6 http://gnomad.broadinstitute.org/variant/17-78081517-C-G

CaseAddInfo: Late Onset Pompe Disease

Case4: Male, 50 years report

HPOsFreeText: 9 years of ERT completed, 6 Minute Walking Test at 140 meters, Forced Vital Capacity at 34%

GenotypingMethod: GAA Gene Sequencing

Variant1: NM_000152.4:c.-32-13T>G

ClinVar: 4027 http://www.ncbi.nlm.nih.gov/clinvar/variation/4027

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

CaseAddInfo: Late Onset Pompe Disease, Variant is homozygous for this patient

Case24: Female, 48 years report

HPOsFreeText: 9 years of ERT completed, 6 Minute Walking Test at 180 meters, Forced Vital Capacity at 67%

GenotypingMethod: GAA Gene Sequencing

Variant1: NM_000152.4:c.-32-13T>G

ClinVar: 4027 http://www.ncbi.nlm.nih.gov/clinvar/variation/4027

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

Variant2: NM_000152.4:c.2481+102_2646+31del (p.Gly828_Asn882del) (exon 18 del)

ClinVar: 4031 https://www.ncbi.nlm.nih.gov/clinvar/variation/4031/

gnomAD: Not in gnomAD

CaseAddInfo: Late Onset Pompe Disease

Case14: Female, 69 years report

HPOsFreeText: 9 years of ERT completed, 6 Minute Walking Test at 200 meters, Forced Vital Capacity at 51%

GenotypingMethod: GAA Gene Sequencing

Variant1: NM_000152.4:c.-32-13T>G

ClinVar: 4027 http://www.ncbi.nlm.nih.gov/clinvar/variation/4027

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

Variant2: NM_000152.4:c.118C>T (p.Arg40Ter)

ClinVar: 426593 https://www.ncbi.nlm.nih.gov/clinvar/variation/426593/

gnomAD: South Asian 6.561e-5, Overall 1.428e-5 http://gnomad.broadinstitute.org/variant/17-78078503-C-T

CaseAddInfo: Late Onset Pompe Disease

Case3: Female, 72 years report

HPOsFreeText: 4 years of ERT completed, 6 Minute Walking Test at 264 meters, Forced Vital Capacity at 56%

GenotypingMethod: GAA Gene Sequencing

Variant1: NM_000152.4:c.-32-13T>G

ClinVar: 4027 http://www.ncbi.nlm.nih.gov/clinvar/variation/4027

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

Variant2: NM_000152.4:c.2481+102_2646+31del (p.Gly828_Asn882del) (exon 18 del)

ClinVar: 4031 https://www.ncbi.nlm.nih.gov/clinvar/variation/4031/

gnomAD: Not in gnomAD

CaseAddInfo: Late Onset Pompe Disease

Case4: Female, 40 years onset, 51 years diagnosis, Italy

HPOsFreeText: See Group Phenotypes, ERT started at 51, GAA Activity at ~7.5 pmol/min/mg before ERT and ~11 after

GroupGenotypingMethod: PCR Amplification and GAA Gene Sequencing

Variant1: NM_000152.4:c.-32-13T>G

ClinVar: 4027 https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

Variant2: NM_000152.4:c.525delT (p.Glu176Argfs*45)

ClinVar: 4033 https://www.ncbi.nlm.nih.gov/clinvar/variation/4033/

gnomAD: European (Non-Finnish) 1.880e-4, Overall 9.959e-5 http://gnomad.broadinstitute.org/variant/17-78078909-CT-C

CaseAddInfo: Late Onset Pompe Disease

Case6: Male, 25 years onset, 56 years diagnosis, Italy

HPOsFreeText: See Group Phenotypes, ERT started at 57, GAA Activity at ~10.5 pmol/min/mg before ERT and ~11 after

GroupGenotypingMethod: PCR Amplification and GAA Gene Sequencing

Variant1: NM_000152.4:c.-32-13T>G

ClinVar: 4027 https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

Variant2: NM_000152.4:c.1655T>C (p.Leu552Pro)

ClinVar: 279811 https://www.ncbi.nlm.nih.gov/clinvar/variation/279811/

gnomAD: European (Non-Finnish) 7.053e-5, Overall 3.188e-5 http://gnomad.broadinstitute.org/variant/17-78085800-T-C

CaseAddInfo: Late Onset Pompe Disease

Case16: Female, 37 years onset, 49 years diagnosis, Italy

HPOsFreeText: See Group Phenotypes, ERT started at 50, GAA Activity at ~4 pmol/min/mg before ERT and ~11 after

GroupGenotypingMethod: PCR Amplification and GAA Gene Sequencing

Variant1: NM_000152.4:c.-32-13T>G

ClinVar: 4027 https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 http://gnomad.broadinstitute.org/variant/17-78078341-T-G

Variant2: NM_000152.4:c.1465G>A (p.Asp489Asn)

ClinVar: 92465 https://www.ncbi.nlm.nih.gov/clinvar/variation/92465/

gnomAD: European (Finnish) 4.621e-5, Overall 1.195e-5 http://gnomad.broadinstitute.org/variant/17-78084553-G-A

CaseAddInfo: Late Onset Pompe Disease

Patient9: male, 10yo, juvenile onset

CasePresentingHPOs:

HPOsFreeText: LOPD, BMI = 14.7 / 15.2 / 15.8, WGMS = 0 / 0 / 0

CaseNotHPOs: HP:0002355

NotHPOsFreeText: did not require respiratory support

CaseAddInfo:

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000152.4(GAA):c.-32-13T>G

Clinvar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

Variant2: NM_000152.4(GAA):c.1561G>A (p.Glu521Lys)

ClinVar: 4022, https://www.ncbi.nlm.nih.gov/clinvar/variation/4022/

gnomAD: 0.000003980, http://gnomad.broadinstitute.org/variant/17-78084749-G-A

Patient3: female, 62yo, adult onset

CasePresentingHPOs:

HPOsFreeText: LOPD, BMI = 19.4;19.7, WGMS = 3;3

CaseNotHPOs: HP:0002355

NotHPOsFreeText: did not require respiratory support

CaseAddInfo:

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000152.4(GAA):c.-32-13T>G

Clinvar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

Variant2: c.2322_2323ins no ClinVar, no CAID

Patient8: female, 51yo, adult onset

CasePresentingHPOs:

HPOsFreeText: LOPD, BMI = 26.6 / 27.2 / n-a, required use of wheelchair, WGMS = 4 / 4 / n-a

CaseNotHPOs:

NotHPOsFreeText: did not require respiratory support

CaseAddInfo:

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000152.4(GAA):c.-32-13T>G

Clinvar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

Variant2: NM_000152.4(GAA):c.877G>A (p.Gly293Arg)

ClinVar: 4036, https://www.ncbi.nlm.nih.gov/clinvar/variation/4036/

gnomAD: 0.00001807, http://gnomad.broadinstitute.org/variant/17-78081617-G-A

Patient32: Male, Caucasian, first investigated at 15yo

CasePresentingHPOs: HP:0003677, HP:0002111, HP:0002875, HP:0003458

HPOsFreeText: CK < 10x increase, CK = 662 U/L. Reduced alpha-glucosidase activity.

CaseNotHPOs: HP:0003325, HP:0002093

CaseAddInfo: onset in childhood, confirmed Pompe Disease via Sanger Sequencing, 2 siblings also carried same variants and presented with reduced enzyme activity but were asymptomatic at time of study.

CasePreviousTesting: same as group, muscle biopsy, EMG

CaseGenotypingMethod: same as group

Variant1: NM_000152.4(GAA):c.525delT (p.Glu176Argfs)

ClinVar: 4033, https://www.ncbi.nlm.nih.gov/clinvar/variation/4033/

gnomAD: 7.041e-05, http://exac.broadinstitute.org/variant/17-78078909-CT-C

Variant2: NM_000152.4(GAA):c.-32-13T>G

Clinvar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

NM_000152.4(GAA):c.-32-13T>G

Subject III-9: Female

CasePresentingHPOs:

HPOsFreeText: DBS neutral/acid = 36.0

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: asymptomatic, asserted carrier status

CasePMIDs:

CasePreviousTesting: DBS

CaseGenotypingMethod: GAA Genotyping

Variant: NM_000152.4(GAA):c.2560C>T (p.Arg854Ter)

ClinVar: 4034, https://www.ncbi.nlm.nih.gov/clinvar/variation/4034/

gnomAD: 0.0002090, http://gnomad.broadinstitute.org/variant/17-78092070-C-T

Patient1: 40yo

CasePresentingHPOs:

HPOsFreeText: HyperCKemia, GAA = 4%, BMI = 25.8, normal CTA, temporo-insular lacunes

CaseNotHPOs:

NotHPOsFreeText: no cardiovascular risk factor

CaseAddInfo: 6yr disease duration

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant: NM_000152.4(GAA):c.-32-13T>G

Clinvar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

Patient15: 53yo

CasePresentingHPOs: HP:0002315, HP:0004887, HP:0000822, HP:0003141, HP:0002870, HP:0020038

HPOsFreeText: hyperCKema, morning headaches, GAA = 13%, BMI = 29.6, right ventricular artery dominant, Normal CT

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: 5yr disease duration, homozygous variants in trans

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant: NM_000152.4(GAA):c.-32-13T>G

Clinvar: 4027, https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: 0.003582, http://exac.broadinstitute.org/variant/17-78078341-T-G

Patient3: Male, 1.2mo at ERT start, 5.5yo at study end

CasePresentingHPOs:

HPOsFreeText: treated with 20mg/kg/2wks, patient had 27 infusion associated reactions

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo:

CasePMIDs:

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000152.4(GAA):c.1933G>T (p.Asp645Tyr)

ClinVar: 556386, https://www.ncbi.nlm.nih.gov/clinvar/variation/556386/

gnomAD: 0.000008250, http://gnomad.broadinstitute.org/variant/17-78086719-G-T

Variant2: NM_000152.4(GAA):c.525delT (p.Glu176Argfs)

ClinVar: 4033, https://www.ncbi.nlm.nih.gov/clinvar/variation/4033/

gnomAD: 0.00009959, http://gnomad.broadinstitute.org/variant/17-78078909-CT-C

Patient 2 - homozygous

Variant: NM_000152.4(GAA):c.2560C>T (p.Arg854Ter)

ClinVar: 4034, https://www.ncbi.nlm.nih.gov/clinvar/variation/4034/#clinical-assertions

gnomAD: 0.0002090, http://gnomad.broadinstitute.org/variant/17-78092070-C-T

Patient20: Female

CasePresentingHPOs: HP:0004887, HP:0011471, HP:0001270, HP:0002546, HP:0000365

HPOsFreeText: CRIM positive, Anti-rh GGA antibodies maximum titer = 1:1.600, tracheostomy, partial heart normalization, moderate periventricular white matter abnormalities (hypomyelination)

CaseNotHPOs: HP:0002421, HP:0001371

NotHPOsFreeText: infusion associated reaction

CaseAddInfo: homozygous variant in trans, symtom onset and diagnosis at 4yo, ERT at 5yo

CasePreviousTesting: same as group

CaseGenotypingMethod: same as group

Variant: NM_000152.4(GAA):c.1564C>G (p.Pro522Ala)

ClinVar: 371277, https://www.ncbi.nlm.nih.gov/clinvar/variation/371277/

gnomAD: n/a

CaseOPTPD-4: Male, Taiwan, Birth diagnosis

HPOsFreeText: GAA (MS/MS) enzyme activity at 0.72 umol/hr/L

GenotypingMethod: GAA Gene Sequencing and PCR Amplification

Variant1: NM_000152.4:c.1726G>A (p.Gly576Ser)

ClinVar: 92467 https://www.ncbi.nlm.nih.gov/clinvar/variation/92467/

gnomAD: East Asian 0.1427, Overall 0.01700 http://gnomad.broadinstitute.org/variant/17-78085871-G-A

Variant2: NM_000152.4:c.752C>T (p.Ser251Leu)

ClinVar: 325781 https://www.ncbi.nlm.nih.gov/clinvar/variation/325781/

gnomAD: East Asian 2.760e-3, Overall 3.540e-4 http://gnomad.broadinstitute.org/variant/17-78081415-C-T

Variant3: NM_000152.4:c.761C>T (p.Ser254Leu)

ClinVar: 325782 https://www.ncbi.nlm.nih.gov/clinvar/variation/325782/

gnomAD: East Asian 2.759e-3, Overall 1.947e-4 http://gnomad.broadinstitute.org/variant/17-78081424-C-T

CaseAddInfo: One Pompe Mutation allele and Two Pseudodeficency Mutation alleles, Nomenclature indicates patient is homozygous for Variant 1 and Variants 2 and 3 are in cis with each other and only one copy of 1

CaseIP-3: Female, Taiwan, Birth diagnosis

HPOsFreeText: 4MU enzyme activity at 0.42, MS/MS ennzyme activity at 0.10

GenotypingMethod: GAA Gene Sequencing and PCR Amplification

Variant1: NM_000152.4:c.1726G>A (p.Gly576Ser)

ClinVar: 92467 https://www.ncbi.nlm.nih.gov/clinvar/variation/92467/

gnomAD: East Asian 0.1427, Overall 0.01700 http://gnomad.broadinstitute.org/variant/17-78085871-G-A

Variant2: NM_000152.4:c.1935C>A (p.Asp645Glu)

ClinVar: 4029 https://www.ncbi.nlm.nih.gov/clinvar/variation/4029/

gnomAD: East Asian 1.729e-3, Overall 1.240e-4 http://gnomad.broadinstitute.org/variant/17-78086721-C-A

CaseAddInfo: Infantile Onset Pompe Disease, Nomenclature indicates Variant 1 and 2 are in cis and that the patient is homozygous for this allele

CaseOPOPD-53: Female, Taiwan, Birth diagnosis

HPOsFreeText: GAA (MS/MS) enzyme activity at 0.80 umol/hr/L

GenotypingMethod: GAA Gene Sequencing and PCR Amplification

Variant1: NM_000152.4:c.1726G>A (p.Gly576Ser)

ClinVar: 92467 https://www.ncbi.nlm.nih.gov/clinvar/variation/92467/

gnomAD: East Asian 0.1427, Overall 0.01700 http://gnomad.broadinstitute.org/variant/17-78085871-G-A

Variant2: NM_000152.4:c.1935C>A (p.Asp645Glu)

ClinVar: 4029 https://www.ncbi.nlm.nih.gov/clinvar/variation/4029/

gnomAD: East Asian 1.729e-3, Overall 1.240e-4 http://gnomad.broadinstitute.org/variant/17-78086721-C-A

CaseAddInfo: One Pompe Mutation allele and One Pseudodeficency Mutation allele, 3 other people have this genotype

Case5HPOsFreeText: 10 mg/kg GAA treatment, GAA activity at 0.6 pmol/punch/hr, GAA activity at 6% lower limit of normal

CaseAddInfo: Late Onset Pompe Disease

CaseGenotypingMethod: Method not described, only that the diagnosis was "confirmed … based on 2 GAA gene mutations"

Variant1: NM_000152.4:c.-32-13T>G

ClinVar: 4027 https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 https://gnomad.broadinstitute.org/variant/17-78078341-T-G

Unknown variant, likely a typo

Variant2: NM_000152.4:c.193G>T

ClinVar:

gnomAD:

CaseAddInfo:

Case9HPOsFreeText: 20 mg/kg GAA treatment, GAA activity at 0.6 pmol/punch/hr, GAA activity at 5.5% lower limit of normal

CaseAddInfo: Late Onset Pompe Disease

CaseGenotypingMethod: Method not described, only that the diagnosis was "confirmed … based on 2 GAA gene mutations"

Variant: NM_000152.4:c.-32-13T>G

ClinVar: 4027 https://www.ncbi.nlm.nih.gov/clinvar/variation/4027/

gnomAD: Ashkenazi Jewish 5.541e-3, Overall 3.401e-3 https://gnomad.broadinstitute.org/variant/17-78078341-T-G

CaseAddInfo: Patient is homozygous for this variant

Patient7:

CasePresentingHPOs:

HPOsFreeText: classic IOPD

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo: homozygous

CasePMIDs:

CasePreviousTesting:

CaseGenotypingMethod: method not described

Variant: NM_000152.4(GAA):c.1124G>T (p.Arg375Leu)

ClinVar: 283230, https://www.ncbi.nlm.nih.gov/clinvar/variation/283230/

gnomAD: 0.000008050, https://gnomad.broadinstitute.org/variant/17-78082336-G-T

NM_004994:c.1174+6->A

Unable to identify variant for following reasons: 1) NM_004994 is for MMP9 not ITGA2B 2) c.1174 is not a exon boundary in ITGA2B Mane transcript 3) ->A is not standard notation, is this a substitution to A of whatever is normally there or is it an insertion of A or something else?

NM_002203:c.185+8T>C

Unable to identify variant for following reasons: 1) NM_002203 is for ITGA2 not ITGA2B 2) c.185 is not at an exon boundary in ITGA2B Mane transcript

NM_002203:c.1600G>A

Unable to identify variant for following reasons: 1) NM_002203 is for ITGA2 not ITGA2B 2) c.1600 in all ITGA2B transcripts is a T not a G in allele registry

Case1874(T.C.) Female, 5yo diagnosis, Irish

CasePresentingHPOs HP:0010864, HP:0000939, HP:0002942, HP:0000766, HP:0000276, HP:0000275, HP:0000218, HP:0000689, HP:0002857, HP:0001166, HP:0001634, HP:0001263, HP:0000750, HP:0002240, HP:0002353

(Severe intellectual disability, Osteoporosis, Thoracic kyphosis, Sternal anomaly, Long face, Thin face, High palate, Malocclusion, Genu valgum, Arachnodactyly, Lens dislocation, Mitral valve prolapse, Delayed milestones, Speech delay, Enlarged liver, EEG abnormality)

HPOsFreeText Patient had severe intellectual disability. Her spine was normal, but long bones were osteoporotic. Upon exam at 5yo, she had mild kyphosis of upper thoracic spine, mild sternal depression, marked genu valgum, and arachnodactyly. Patient was also myopic with bilateral lens dislocation. She had prominent recurrent malar flush, long and thin face, high-arched palate, malocclusion and mitral valve prolapse.

(PMID: 20329304): Patient was born at term after normal pregnancy weighing 3.4kg. Normal neonatal course, but with delayed milestones. Speech at 5yo was monosyllabic , but verbal comprehension was good. No convulsions at this time. She had a palpable liver, 2cm below margin, but had normal consistence. She had an abnormal EEG with non-specific diffuse changes and excess slow-wave activity. Patient's IQ was 36.

CaseNotHPOs HP:0001250, HP:0002086, HP:0000707

(Seizures, Respiratory abnormality, Neurologic abnormality)

NotHPOsFreeText n/a

CasePreviousTesting Plasma homocysteine was 70μM pretreatment. Pyroxidine treatment reduced those levels to trace amounts. In patient fibroblasts, no CBS protein was detect on Western blot and CBS assay detected no activity. (PMID: 20329304): "Full hemogram, routine urine analysis, liver function tests, serum ion and iron-binding capacity, plasma magnesium, serum calcium, inorganic phosphate, fasting blood sugars, blood urea nitrogen, and bone marrow examination were all normal". Heavy homocysteine spot shown on chromatogram of urinary amino acids.

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod Created a bacteria expression system in E.coli by substituting one of three fragments of (PCR amplified)patient cDNA into WT CBS sequence already subcloned in expression plasmids. Bacteria were screened based on which ones did not have CBS activity. Those that did not have activity were thought to have LOF variants in CBS. Plasmids containing the inactivating fragments, ones cut with NcoI/XmaI, were sequenced.

Variant 1. NM_000071.2:c.233C>G (p.Pro78Arg);2. NM_000071.2:c.306G>C (p.Lys102Asn) - 1&2 in cis

ClinVar 1. 188988;2. 188989

gnomAD 1. MAF=0.0001631(Other), no homozygotes, 2. MAF=0.0001630(Other), no homozygotes

CaseAddInfo n/a

CasePMIDs 20329304

Case# HCU-102-3, 8yo report, Iran, Sistani

CasePresentingHPOs HP:0001083, HP:0003235, HP:0000924

(Ectopia lentis, Increased methionine in blood, Skeletal abnormality)

HPOsFreeText skeletal abnormality/elongated long bone, increased urinary cysteine, elevated plasma methionine

CaseNotHPOs HP:0001249, HP:0002970, HP:0001010, HP:0000501

(Intellectual disability, Genu varum, Hypopigmentation, Glaucoma)

NotHPOsFreeText n/a

CasePreviousTesting plasma methionine 108μm/l(normal range 6-40μm/l)

CaseMethod1 Sanger

CaseMethod2 n/a

CaseGenotypingMethod Sanger sequenced exon 3 of the CBS gene

Variant NM_000071.2(CBS):c.346G>A (p.Gly116Arg), homozygous

ClinVar 188787

gnomAD MAF=0.00002892(Latino), no homozygotes

CaseAddInfo from the province of Sistan and Baluchestan. no known consanguinity although the patient is homozygous for a founder variant

CasePMIDs n/a

FamilyLabel HCU-103

Family#PresentingHPOs HP:0001249

(Intellectual disability)

HPOsFreeText elevated plasma homocysteine, elevated urinary cysteine

FamilyNotHPOs HP:0001083, HP:0002970, HP:0001010, HP:0000501, HP:0000924

(Ectopia lentis, Genu varum, Hypopigmentation, Glaucoma, Skeletal abnormalities)

NotHPOsFreeText skeletal abnormality/elongated long bones

CountryOrigin Iran

Ethnicity Sistani

Race n/a

Family#PreviousTesting tested for homocysteine in plasma and for cysteine in urine

FamilyMethod1 Sanger

FamilyMethod2 n/a

FamilyGenotypingMethod Sanger sequenced exon 3 of the CBS gene

ProbandLabel HCU-103-4

Variant NM_000071.2(CBS):c.346G>A (p.Gly116Arg), homozygous

ClinVar 188787

gnomAD MAF=0.00002892(Latino), no homozygotes

FamilyAddInfo from the province of Sistan and Baluchestan. no known consanguinity although the affected individuals are homozygous for a founder variant

FamilyPMIDs n/a

Case# HCU-101-3, 10yo report, Iran, Sistani

CasePresentingHPOs HP:0001249, HP:0001083, HP:0002970, HP:0001634, HP:0003235, HP:0000924

(Intellectual disability, Ectopia lentis, Genu varum, Mitral valve prolapse, Increased methionine in blood, Skeletal abnormality)

HPOsFreeText skeletal abnormality/elongated long bones, elevated, elevated plasma homocysteine, elevated urinary cysteine, elevated plasma methionine

CaseNotHPOs HP:0001010, HP:0000501

(Hypopigmentation, Glaucoma)

NotHPOsFreeText n/a

CasePreviousTesting Plasma homocysteine 330μm/l(upper normal limit 15μm/l), plasma methionine 142μm/l(normal range 6-40μm/l)

CaseMethod1 Sanger

CaseMethod2 n/a

CaseGenotypingMethod Sanger sequenced exon 3 of the CBS gene

Variant NM_000071.2(CBS):c.346G>A (p.Gly116Arg), homozygous

ClinVar 188787

gnomAD MAF=0.00002892(Latino), no homozygotes

CaseAddInfo from the province of Sistan and Baluchestan. no known consanguinity although the patient is homozygous for a founder variant

CasePMIDs n/a

Case2: Female

CasePresentingHPOs: HP:0000252, HP:0001270, HP:0000750, HP:0001252 (Microcephaly, Motor delay, Delayed speech and language development, Muscular hypotonia)

HPOsFreeText: N/A

CaseNotHPOs: HP:0000407 (Sensorineural hearing impairment)

NotHPOsFreeText: N/A

CaseAddInfo: Parents are non-consanguineous, Compound heterozyosity was confirmed in trans via parental testing

CasePreviousTesting: At 2 yrs levels were at 2-oxoadipate, 520–970 mmol/mol creatinine; 2-hydroxyadipate, 100–150 mmol/mol creatinine; 2-aminoadipate, elevated; Moderate protein restriction did not influence disease course; IQ of 87

CaseGenotypingMethod: After confirmation of the DHTKD1 mutations in Individual 1, Sanger sequencing confirmed compound heterozygous mutations in the same gene here.

Variant1: NM_018706.7(DHTKD1):c.1228C>T (p.Arg410Ter)

ClinVar: 39565 https://www.ncbi.nlm.nih.gov/clinvar/variation/39565/

gnomAD: 0.00005437, https://gnomad.broadinstitute.org/variant/10-12136140-C-T

Variant2: NM_018706.5(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

NonPatientCell: EFO:0002529 (INS-1), EFO:0000856 (islet of Langerhans), CL:0000182 (Hepatocyte)

GeneFunction: GO:0004353 (glutamate dehydrogenase [NAD(P)+] activity)

GeneAlteration: Recombinant adenoviruses encoding WT human GLUD1 and the S498L variant were generated via an expression system, with the human GLUD1 gene being amplified and sub-cloned into a plasmid with a promoter. Digestion using EcoRV was used to discriminate between the wild-type and the mutant with sequencing verifying each step of the cloning. This adenovirus was amplified in HEK-293 cells and INS-1E β-cells were transduced with it. Mouse and human hepatocytes were isolated and seeded before being transduced with the same adenoviruses. The transfection efficency was confirmed via immunoblotting.

FunctionalEvidence: The enzymatic activity was evaluated in both wild-type and mutant mice, with both showing very low activity without glutamate concentrations and the mutant showing higher activity when glutamate was increased. The reductive direction using α-ketoglutarate as substrate was also tested with similar increased results for the mutant. Sensitivity of both lines to the reaction activator (ADP) and inhibitor (GTP) levels were tested, showing that the mutant was more sensitive to the ADP activator and slightly reduced for the GTP inhibitor (particularly in the presence of ADP). Mitochondiral function in wid-type and mutant cells was assessed, demonstrating that glucose response from mutant and WT cells was preserved but that glutamine stimulation abnormally generates ATP in mutant cells.Calcium stimulation was also activated by both glutamine and glucose in mutant cells while only being affected by glucose in WT. Studies in mutant and WT-transfected mouse and human islets showed increased insulin and ammonia production in mutants from stimulation through this responsive glutamine sensitivity. All of this supports GLUD1's pathogenic mechanism, specifically causing a hyperctive mutant protein that responds to glutamine levels rather than simply glucose.

FAVariants: NM_005271.5(GLUD1):c.1493C>T (p.Ser498Leu)

ClinVar: 16122, https://www.ncbi.nlm.nih.gov/clinvar/variation/16122/

CaseBII-1: N/A

CasePresentingHPOs: N/A

HPOsFreeText: Pretreatment phenylalanine level at 890 umol/L

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband for Family B

Variant1: NM_000277.3(PAH):c.1222C>T (p.Arg408Trp)

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

gnomAD: 0.001766, https://gnomad.broadinstitute.org/variant/12-102840493-G-A?dataset=gnomad_r3

Variant2: NM_000277.3(PAH):c.1241A>G (p.Tyr414Cys)

ClinVar: 593 https://www.ncbi.nlm.nih.gov/clinvar/variation/593/

gnomAD: 0.0005264, https://gnomad.broadinstitute.org/variant/12-102840474-T-C?dataset=gnomad_r3

CaseEII-2: N/A

CasePresentingHPOs: N/A

HPOsFreeText: Pretreatment phenylalanine level at 320 umol/L

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: N/A

Variant1: NM_000277.3(PAH):c.1222C>T (p.Arg408Trp)

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

gnomAD: 0.001766, https://gnomad.broadinstitute.org/variant/12-102840493-G-A?dataset=gnomad_r3

Variant2: NM_000277.3(PAH):c.533A>G (p.Glu178Gly)

ClinVar: 92746 https://www.ncbi.nlm.nih.gov/clinvar/variation/92746/

gnomAD: 0.0001473, https://gnomad.broadinstitute.org/variant/12-103249087-T-C

CaseCI-1: N/A

CasePresentingHPOs: N/A

HPOsFreeText: Pretreatment phenylalanine level at 365 umol/L

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: N/A

Variant1: NM_000277.3(PAH):c.194T>C (p.Ile65Thr)

ClinVar: 636 https://www.ncbi.nlm.nih.gov/clinvar/variation/636/

gnomAD: 0.0005574, https://gnomad.broadinstitute.org/variant/12-102894893-A-G?dataset=gnomad_r3

Variant2: NM_000277.3(PAH):c.261C>A (p.Ser87Arg)

ClinVar: 583 https://www.ncbi.nlm.nih.gov/clinvar/variation/583/

gnomAD: 0.00009294, https://gnomad.broadinstitute.org/variant/12-102894826-G-T?dataset=gnomad_r3

CaseDII-3: N/A

CasePresentingHPOs: N/A

HPOsFreeText: Pretreatment phenylalanine level at 725 umol/L

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: N/A

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

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

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

Variant2: NM_000277.3(PAH):c.782G>C (p.Arg261Pro)

ClinVar: 102832 https://www.ncbi.nlm.nih.gov/clinvar/variation/102832/

gnomAD: Not in gnomAD

CaseFZ8: Male, United States, 3 yrs report

CasePresentingHPOs: HP:0001511, HP:0008897, HP:0001999, HP:0000973, HP:0100678, HP:0000963, HP:0010648, HP:0001388, HP:0001374, HP:0001181, HP:0000938, HP:0000270, HP:0025356, HP:0001252, HP:0000518, HP:0000325, HP:0011220, HP:0000337, HP:0000411, HP:0000369, HP:0100790, HP:0005116, HP:0000717, HP:0008872, HP:0000486, HP:0500163, HP:0003572, HP:0500139 (Intrauterine growth retardation, Postnatal growth retardation, Abnormal facial shape, Cutis laxa, Premature skin wrinkling, Thin skin, Dermal translucency, Joint laxity, Congenital hip dislocation, Adducted thumb, Osteopenia, Delayed cranial suture closure, Psychomotor retardation, Muscular hypotonia, Cataract, Triangular face, Prominent forehead, Broad forehead, Protruding ear, Low-set ears, Hernias, Arterial tortuosity, Autism, Feeding difficulties in infancy, Strabismus, Hypoornithinemia, Low plasma citrulline, Hypoprolinemia)

HPOsFreeText: Typical facial gestalt

CaseNotHPOs: HP:0002645, HP:0012443, HP:0001762, HP:0002650, HP:0000252, HP:0001348 (Wormian bones, Abnormality of brain morphology, Talipes equinovarus, Scoliosis, Microcephaly, Brisk reflexes)

NotHPOsFreeText: N/A

CaseAddInfo: Variant is confirmed de novo in this case with one other confirmed and one non-confirmed case with the same variant and similar presentation, No family history

CasePreviousTesting: N/A

CaseGenotypingMethod: ALDH18A1 and PYRC1 were investigated by Sanger sequencing, with the latter revealing no abnormalities and the former showing a heterozygous change at the Arg138 residue.Investigation revealed no further variants in the coding regions. Familial sequencing revealed the inheritance was de novo with no variants in the parents.

Variant: NM_002860.4(ALDH18A1):c.413G>A (p.Arg138Gln)

ClinVar: 217260 https://www.ncbi.nlm.nih.gov/clinvar/variation/217260/

gnomAD: Not in gnomAD

FamilyLabel: Family 1

Family1PresentingHPOs: HP:0001987, HP:0001942, HP:0003128, HP:0002912 (hyperammonemia, metabolic acidosis, lactic acidosis, methylmalonic acidemia)

NotHPOsFreeText: normal plasma total homocysteine

CountryOrigin: China

Ethnicity: Chinese

ProbandLabel: Case35, Case36

Family1 (Level 2)

FamilyPreviousTesting: tandem mass spectrometry for blood levels, gas chromatography-mass spectrometry for urine, NBS

FamilyMethod1: PCR

FamilyGenotypingMethod: genomic DNA extracted from peripheral blood leukocytes from patients and their parents. Exonic ad flanking intronic regions of the MUT gene were amplified through PCR. PCR products were sequenced directly using forward and reverse primers on ABI 3700 sequencer. Mutation analysis using MUT reference NM_000255.3. Nucleotide numbering reflects cDNA numbering (initiation codon= codon1). All detected mutations were confirmed in the patients' parents. Novel variants were screened in 100 alleles from 50 control children. Novel variants were considered pathogenic when predicted to be deleterious by two algorithms.

FamilyAddInfo: counted in group annotation also. identical twins. mut- and mut0 mutation together may explain relatively good clinical condition.

Case: K.A./ BIODEF 264, male, Caucasian, 1 day

CasePresentingHPOs:  HP:0008297, HP:0040206, HP:0040210, HP:0001252

HPOText: Plasma Phe level of 290umol/L at 3 days old, 853 umol/L at 6 days old, and 1997 umol/L at 13 days old. Urinary pterins indicated high neopterin/biopterin ratio and increased levels of primapterin. At 1 month old, neopterin and biopterin levels in normal range. At one month old showed mild hypotonia (extermities and trunk), but at 2.5 years Phe diet stopped and patient developed normally.

CasePreviousTesting: n/a

CaseGenotypingMethod: Direct nucleotide-sequence determination of PCR products from blood samples was performed for the coding exons (2-4) of PCBD1.

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

ClinVar ID: 16799

No-stars, 1 submission from OMIM.

Homozygous, non-LOF.

gnomAD: MAF (Euro-NF)= 2.2291E-4 (29/126606) hets only. No homozygotes recorded

http://gnomad.broadinstitute.org/variant/10-72643730-G-A

AddCaseInfo: The only individual in the family diagnosed with HyperPhe.

VariantEvidence: The mutant Q97X protein was expressed in E.Coli to determine the solubility of the mutant enzyme and the activity. The mQ97X fragment was mostly found in the insoluble fraction of the supernatant. The remaining fraction obtained in the soluble fraction did retain some activity in vitro. Interestingly, when more Q97X was obtained in the soluble fraction (by growing the E.coli at 20 degrees C), the Km (Michaelis constant) was 2-3 times higher than wildtype (text only, no figures to compare).

Patient part of the BIODEF database: PMID:8830181

Case: I.B./ BIODEF 273, male, Ashkenazi Jewish, 3 days

CasePresentingHPOs: HP:0040210, HP:0008297,

HPOText: Plasma Phe levels were 240 umol/L at 3 days old, and 1845 umol/L at 14 days old. A low-Phe diet caused a rapid decline of Phe level to normal within 36 hours. An oral BH4–loading test (10 mg/kg body weight) resulted in a decrease of plasma Phe from 540 μmol/liter to 234 μmol/liter and then to 196 μmol/liter within 4 and 8 h, respectively. At 1 month, pterin and neurotransmitter in the CSF were normal. Starting a 2 month of age the patient was administered a low Phe diet and BH4 for 4 months. After the 4 months, the BH4 was discontinued, and Phe levels are between 120-300 umol/L at 5 years. Patient is developing normally.

CaseNOTHPOs: n/a

CasePreviousTesting: n/a

CaseGenotypingMethod: Direct nucleotide-sequence determination of PCR products from blood samples was performed for the coding exons (2-4) of PCBD1.

Variant: NM_000281.3(PCBD1):c.259G>T (p.Glu87Ter) 

ClinVar ID: 16795

No star, one submission for OMIM

Homozygous

gnomAD: MAF (Ashkenazi Jewish)= 1.183E-3 (12/10146) in heterozygous only. No homozygous reported.

http://gnomad.broadinstitute.org/variant/10-72643763-C-A

AddCaseInfo: The proband's older sister (J.B., 18 . months older) has the same genotype (homozygous for the T79I mutation), but was never noted as having HyperPhe, and was never treated. She has developed "normally." This family is recorded as Family B in the paper.

VariantEvidence: The mutant E86X/E87X protein was expressed in E.Coli to determine the solubility of the mutant enzyme and the activity. The mutant E86X/E87X fragment was mostly found in the insoluble fraction of the supernatant. The remaining fraction obtained in the soluble fraction did retain some enzyme activity in vitro. Interestingly, when more E86X/E87X was obtained in the soluble fraction (by growing the E.coli at 20 degrees C), the Km (Michaelis constant) was similar to wildtype levels (text only, no figures to compare).

Patient part of the BIODEF database: PMID:8830181

Case: J.S./ BIODEF 211, female, Hispanic, 3 days

CasePresentingHPOs: HP:0008297, HP:0001252, HP:0001270

HPOText: Phe levels were 9 umol/L on day 1, 756 ummol/L on day 28. Primapterinuria was diagnosed on day 8. Following oral BH4 administration (2 mg/kg/d), neopterin normalized, and biopterin as well as primapterin increased about eight-fold; the same results were observed when dihydrobiopterin and sepiapterin were administered. At 8.5 years old, on a normal diet, the patient is developing normally with no HyperPhe.

CasePreviousTesting: n/a

CaseGenotypingMethod: Direct nucleotide-sequence determination of PCR products from blood samples was performed for the coding exons (2-4) of PCBD1.

Variant: NM_000281.3(PCBD1):c.236C>T (p.Thr79Ile)

ClinVar ID: 16797

No stars, 1 submission for OMIM.

Homozygous

gnomAD: n/a

AddCaseInfo: Family history (Family S) of HyperPhe, as the patients older brother, T.S./ BIODEF212, who is 1.5 years older, showed the same biochemical pattern and is now considered normal and normally developing.

At 8.5 years old, on a normal diet, the patient J.S. is developing normally with no HyperPhe.

VariantEvidence: The T78I/T79I variant was retained completely within the insoluble fraction of the E.Coli lysate in which the recombinant proteins were produced. When the soluble fraction was examined for PCBD1 activity, no activity was observed. The authors note that the absence of activity could be due to the absence of the protein in the soluble fraction alone, instead of being indicative of the mutation causing a loss of enzyme activity. Only if the mutant recombinant protein could be retained in the soluble fraction can the true enzyme activity be assessed.

Patient part of the BIODEF database: PMID:8830181

Case: B.E./ BIODEF 272, male, 3 weeks

CasePresentingHPOs: HP:0008297, HP:0001252, HP:0001270

HPOText: Neonatal (3 days old) plasma screen showed Phe levels at 183 umol/L, which is below the normal cutoff. Repeat screen was only performed at 3 weeks old in which the Phe level was shown to be 1600 umol/L. Peak Phe level was at 3.5 weeks and was 2589 umol/L. Child had considerable Phe tolerance ( ~100mg/kg). Initial neopterin-to-biopterin ratio of 15. A BH4-loading test (6 mg/kg) at age 6 wk showed only a slight response (Phe at 0 h, 731 μmol/liter; at 3 h, 612 μmol/liter; and at 6 h, 502 μmol/liter). A week later, at a BH4 dose of 22 mg/kg, there was a definite response (Phe at 0 h, 773 μmol/liter; at 3 h, 517 μmol/liter; and at 6 h, 155 μmol/liter). CSF neurotransmitter concentrations were normal, suggesting a peripheral deficiency. At 4 months of age, the BH4 was discontinued, no low Phe diet, and the Phe levels have remained normal. at 3.5 years, patient developing normally.

CaseNOTHPOs:

CasePreviousTesting: n/a

CaseGenotypingMethod: Direct nucleotide-sequence determination of PCR products from blood samples was performed for the coding exons (2-4) of PCBD1.

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

ClinVar ID: 16799

No star, one submission for OMIM

Homozygous

gnomAD: MAF (Euro-NF)= 2.2291E-4 (29/126606) hets only. No homozygotes recorded

AddCaseInfo: Family E in the article, and the proband had an older sibling that also carried the homozygous mutation in PCBD1, she was not noted as having increased Phe plasma levels, nor was ever treated with BH4 or low-Phe diet.

VariantEvidence: The mutant Q97X protein was expressed in E.Coli to determine the solubility of the mutant enzyme and the activity. The mQ97X fragment was mostly found in the insoluble fraction of the supernatant. The remaining fraction obtained in the soluble fraction did retain some activity in vitro. Interestingly, when more Q97X was obtained in the soluble fraction (by growing the E.coli at 20 degrees C), the Km (Michaelis constant) was 2-3 times higher than wildtype (text only, no figures to compare).

Patient part of the BIODEF database: PMID:8830181

Case#: DBT-2

CasePresentingHPOs: Clinical information not available

Variant: NM_001918.3(DBT):c.75_76delAT (p.Cys26Trpfs) (homozygous)

ClinVar: 11950

gnomAD: Ashkenazi Jewish 0.0002959, Overall 0.00006863

VariantEvidence: Predicted LOF.

Case# PA7

Variant NM_000709.3(BCKDHA):c.905A>C (p.Asp302Ala)

ClinVar 93377

VariantEvidence none

Case# PA8

Variant NM_000709.3(BCKDHA):c.940C>T (p.Arg314Ter)

ClinVar 224072

gnomAD 0.000008958(European (Non-Finnish)), 0.000004064(Overall)

Case54 Male, 7.3yo report, Ethnicity: Turkish

CasePresentingHPOs HP:0003571, HP:0008872, HP:0002013, HP:0008947, HP:0002789, HP:0002045

(propionicacidemia, feeding difficulties in infancy, vomiting, infantile hypotonia, tachypnea, hypothermia)

HPOsFreeText feeding difficulties, vomiting, hypotonia, tachypnea, hypothermia(3do). 55 IQ on Culture Free Test

CaseNotHPOs n/a

CasePreviousTesting Diagnosis by selective metabolic screen. Patient fibroblasts had 1.5% PCC activity of control

Variant NM_000532.4(PCCB):c.990dupT (p.Glu331Terfs), homozygous

ClinVar 167423

gnomAD MAF= 0.001001(African), no homozygotes

CaseAddInfo parental consanguinity

CasePMIDs n/a

Case34 female, 5.5yo report, Ethnicity: German

CasePresentingHPOs HP:0003571, HP:0012523, HP:0001531, HP:0002791, HP:0001944

(propionicacidemia, oral aversion, failure to thrive in infancy, bradypnea, dehydration)

HPOsFreeText feeding refusal, failure to thrive, bradypnea, dehydration(2do). 55 IQ on Culture Free Test

CaseNotHPOs n/a

CasePreviousTesting Diagnosed on newborn screen. PCC activity in patient fibroblasts was 0.9% of control

Variant NM_000532.4:c.483dup (p.Gly162TrpfsTer?), homozygous

ClinVar n/a

CAID to register

gnomAD not in gnomAD

CaseAddInfo no parental consanguinity

CasePMIDs n/a

FamilyBJG_F2: Malaysia

FamilyPresentingHPOs: N/A

HPOsFreeText: N/A

FamilyNotHPOs: N/A

NotHPOsFreeText: N/A

FamilyGenotypingMethod: See Group

FamilyPreviousTesting: See Group

FamilyAddInfo: Proband is confirmed homozygous for this variant

Variant: NM_006721.4(ADK):c.89G>A (p.Gly30Glu)

ClinVar: 29605, https://www.ncbi.nlm.nih.gov/clinvar/variation/29605/

gnomAD: Not in gnomAD

Affected: 2

Unaffected: 0

Segregations: 1

AddSegregationInfo: Not enough info

Method 1: Sanger

GenotypingMethod: Coding regions as well as exon–intron boundaries PCCA and PCCB amplified by PCR then analyzed with bidirectional sequencing. Variants were blasted against references genomes PCCA (NM_000282.3) and PCCB (NM_000532.4).

CaseF-15 female, 1yo onset, Ethnicity: North Indian, Hindu

CasePresentingHPOs HP:0006846, HP:0001254, HP:0011968, HP:0002098, HP:0002013, HP:0001942, HP:0001943, HP:0003571

(Acute encephalopathy, Lethargy, Poor feeding, Respiratory distress, Vomiting, Metabolic acidosis, Hypoglycemia, Propionicacidemia)

HPOsFreeText Ketones present, acycarnitines 6.2(normal <3.6nmol/mL), died after acute crisis(1yo)

CaseNotHPOs n/a

CasePreviousTesting Also screened for variants in PCCA

Variant NM_000532.4(PCCB):c.493C>T (p.Arg165Trp)

ClinVar 217894

gnomAD MAF= 0.00001456(European(non-Finnish), no homozygotes

CaseAddInfo nonconsanguineous, no abnormality in family history noted

CasePMIDs n/a

Case1: Male,

CasePresentingHPOs:

HPOsFreeText:

CaseNotHPOs:

NotHPOsFreeText:

CaseAddInfo:

CasePreviousTesting:

CaseGenotypingMethod:

Variant1: NM_018706.5(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

Variant2:

ClinVar:

gnomAD:

Case4 Female, 9yo report, USA, Lumbee

CasePresentingHPOs HP:0003150, HP:0011471, HP:0002540, HP:0001344, HP:0100309, HP:0012751, HP:0200147, HP:0100952

(Glutaricaciduria, Gastronomy tube feeding in infancy, Non-ambulatory, Nonverbal, Subdural haematoma, Abnormal basal ganglia MRI signal intensity, Neuronal loss in basal ganglia, Enlarged sylvian fissure)

HPOsFreeText Urine glutaric acid was highly elevated and 3-hydroxyglutaric acid was elevated. Patient presented with acute metabolic decompensation at 6mo. After decompensation, brain MRI found subdural hematomas, atrophied temporal lobes with widened Sylvian fissures, and elevated signal intensity in basal ganglia. Follow-up report studies noted volume loss in basal ganglia. Patient was diet restricted in lysine and tryptophan with per individual adjustments to maintain normal plasma amino acid levels. Treatment also included supplemental carnitine and riboflavin.

CaseNotHPOs n/a

NotHPOsFreeText n/a

CasePreviousTesting n/a

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod PCR and sanger sequencing was performed on exon 10 of the GCDH gene.

Variant NM_000159.3(GCDH):c.1240G>A (p.Glu414Lys), homozygous

ClinVar 167133

gnomAD MAF=0.0002017(African), no homozygotes

CaseAddInfo nonconsanguineous

CasePMIDs n/a

Case3 Male, 5yo report, USA, Lumbee

CasePresentingHPOs HP:0003150

(Glutaricaciduria)

HPOsFreeText Patient had elevated glutaric acid and small 3-hydroxyglutaric acid in urine. Patient feeds by mouth. Patient was diet restricted in lysine and tryptophan with per individual adjustments to maintain normal plasma amino acid levels. Treatment also included supplemental carnitine and riboflavin.

CaseNotHPOs HP:0002540, HP:0001344, HP:0011471

(Non-ambulatory, Nonverbal)

NotHPOsFreeText Patient did not present with acute metabolic decompensation.

CasePreviousTesting Patient's NBS detected glutarylcarnitine of 1.07μM(cutoff at >0.38). GCD enzyme activity was 0%.

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod PCR and sanger sequencing was performed on exon 10 of the GCDH gene.

Variant NM_000159.3(GCDH):c.1240G>A (p.Glu414Lys), homozygous

ClinVar 167133

gnomAD MAF=0.0002017(African), no homozygotes

CaseAddInfo nonconsanguineous

CasePMIDs n/a

FamilyPA2: Italy

FamilyPresentingHPOs: HP:0000519, HP:0002020, HP:0002572, HP:0002313, HP:0003693, HP:0002036, HP:0001761, HP:0003487, HP:0009830, HP:0001288, HP:0001348 (Developmental cataract, Gastroesophageal reflux, Episodic vomiting, Spastic paraparesis, Distal amyotrophy, Hiatus hernia, Pes cavus, Babinski sign, Peripheral neuropathy, Gait disturbance, Brisk reflexes)

HPOsFreeText: N/A

FamilyNotHPOs: N/A

NotHPOsFreeText: N/A

FamilyGenotypingMethod: ALDH18A1 was directly sequenced upon investigation for a causal role in SPG9, due to being within the linked region 10q23.3-q24.2, not having been discarded as a candidate gene, and mutations having been known to cause cutis laxa. Previous haplotype analysis also confirmed the linkage to this specific genomic region. This revealed one mutation in each pedigree that strictly co-segregated with the disease. Exome NGS sequencing within the interval showed only two variants were unknown in ExAC with the MYOF substitution unlikely to be disease causing due to poor conservation.

FamilyPreviousTesting: N/A

FamilyAddInfo: Family shows autosomal dominant segregation and haplotyping, Proband is PA2:III-21

FamilyPMIDs: 9973297

Variant: NM_002860.4(ALDH18A1):c.755G>A (p.Arg252Gln)

ClinVar: 217117 https://www.ncbi.nlm.nih.gov/clinvar/variation/217117/

gnomAD: Not in gnomAD

Affected: 11

Unaffected: 15

Segregations: 17

pLOD: 4.83

AddSegregationInfo: Candidate gene sequencing

CaseAM7162: Female, 6 yrs report

CasePresentingHPOs: HP:0001992, HP:0002179, HP:0010864, HP:0008936, HP:0002509, HP:0000750, HP:0011968, HP:0008872, HP:0100247, HP:0002373, HP:0001347, HP:0011449 (Organic aciduria, Opisthotonus, Severe Intellectual disability, Muscular hypotonia of the trunk, Limb hypertonia, Delayed speech and language development, Feeding difficulties, Feeding difficulties in infancy, Recurrent singultus, Febrile seizure, Hyperreflexia, Knee clonus)

HPOsFreeText: Birth weight 2.86 kg, Cranial circumference is 49 cm (3rd percentile), weight 18.6 kg (25th percentile) and height 115 cm (50th percentile) at 6 yrs of age

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are first cousin consanguineous; Proband is confirmed homozygous for this variant via a QFM‐PCR assay for the dosage of ACY1 exons 12 and 13; Variant leads to skipping of Exon 13 and a resulting frameshift with a premature termination

CasePreviousTesting: Urinary organic acid analysis confirmed by enzyme assay showed elevated levels of N ‐acetylated derivatives of glutamic acid, leucine, valine and isoleucine; Several genetic investigations, such as karyotyping, ring 15 chromosome analysis and mutational analysis of MECP5 and STXBP1 genes, were performed and were found to be normal; MRI showed dilatation of the left temporal horn at the age of 13 months;

CaseGenotypingMethod: Genomic DNA was isolated and the ACY1 gene was PCR-amplified and sequenced. PCR analysis of retrotranscribed RNA was performed to analyze the effect of this variant on splicing.

Variant: NM_000666.3(ACY1):c.1001_1001+5del

ClinVar: 162019, https://www.ncbi.nlm.nih.gov/clinvar/variation/162019/

gnomAD: 0.00001758, https://gnomad.broadinstitute.org/variant/3-52022618-ATGTGAG-A

CaseC: Male, Childhood onset, Asian, China

CasePresentingHPOs: HP:0004923 (Hyperphenylalaninemia)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are confirmed heterozyogus and proband is homozygous for this mutation

CasePreviousTesting: "Classical Severe PKU" presence confirmed by Guthrie test and clinical criteria described in PMID:7006308, Plasma phenylalanine level in excess of 20 mg/dl, Homozygous for RFLP haplotype 4

CaseGenotypingMethod: PCR amplification of PAH exon three plus the flanking intronic regions was performed. The genomic DNA was cloned and sequenced to confirm the presence of the mutation. Haplotype analysis confirmed linkage disequillibrium with RFLP haplotype 4.

Variant: NM_000277.3(PAH):c.331C>T (p.Arg111Ter)

ClinVar: 581 https://www.ncbi.nlm.nih.gov/clinvar/variation/581/

gnomAD: 0.0001230, https://gnomad.broadinstitute.org/variant/12-103288534-G-A

CaseSE4: Male, Australia

CasePresentingHPOs: HP:0008353 (Neutral hyperaminoaciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

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

CasePreviousTesting: SLC1A5 had no causative variants detected.

CaseAddInfo: Asymptomatic except for hyperaminoaciduria

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

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

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

CaseSE7: Female, Australia

CasePresentingHPOs: HP:0008353 (Neutral hyperaminoaciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

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

CasePreviousTesting: SLC1A5 had no causative variants detected.

CaseAddInfo: Asymptomatic except for hyperaminoaciduria

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

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

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

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

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

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

Case2927: Female, 2 years diagnosis, Czech

CasePresentingHPOs: HP:0001270, HP:0001263, HP:0003153, HP:0012443, HP:0000486, HP:0012795, HP:0000639, HP:0000545, HP:0000646, HP:0002280, HP:0030890, HP:0000488, HP:0001744 (Motor delay, Global developmental delay, Cystathioninuria, Abnormality of brain morphology, Strabismus, Abnormality of the optic disc, Nystagmus, Myopia, Amblyopia, Enlarged cisterna magna, White matter hyperintensity, Retinopathy, Splenomegaly)

HPOsFreeText: Normal total homocysteine (7.1 μM/l; reference 5.1–13.9), Elevated cystathionine (6 μM/l; reference 0.04–0.34), Normal total cysteine (306 μmol/l; reference 203–369), Birth weight was 2400 g, Responsive to B6 treatment

CaseNotHPOs: HP:0002240, HP:0001977 (Hepatomegaly, Abnormal thrombosis)

NotHPOsFreeText: N/A

CaseAddInfo: Patient is homozygous for this mutation

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.

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

CaseAC013_II-1: Female, 7 yrs report, Turkey

CasePresentingHPOs: HP:0001992, HP:0001249, HP:0004322, HP:0001513, HP:0030838 (Organic aciduria, Intellectual disability, Short stature, Obesity, Hip pain)

HPOsFreeText: BMI 93rd percentile

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

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

CasePreviousTesting: Identified by the assessment of urinary organic acids by gas chromatography–mass spectrometry; Accumulation of N-acetylated amino acids was much less pronounced than in previously reported patients; Metabolic studies were initiated after the patient complained of pain in her left hip

CaseGenotypingMethod: Genomic DNA was extracted either from peripheral blood or EBV-transformed lymphocytes. PCR-amplification and bidirectional sequencing of all exons and exon-intron boundaries of ACY1 was performed.

Variant: NM_000666.3(ACY1):c.1133G>A (p.Arg378Gln)

ClinVar: 809490, https://www.ncbi.nlm.nih.gov/clinvar/variation/809490/

gnomAD: 0.003104, https://gnomad.broadinstitute.org/variant/3-52022997-G-A

Case39 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 16(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 77(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 1. NM_000159.3(GCDH):c.877G>A (p.Ala293Thr), 2. NM_000159.3(GCDH):c.764C>T (p.Ser255Leu), compound heterozygous

ClinVar 1. 2083, 2. 374435

gnomAD 1. MAF= 0.0004013(African), no homozygotes, 2. MAF= 0.00005437(East Asian), no homozygotes

CaseAddInfo Sibling (Case 40) presented with same enzyme activity, clinical onset and phenotype. Gluterate was 10mmol/mol creatinine and 3-OH-glutarate was 31mmol/mol creatinine.

CasePMIDs see notes on previous studies

Case30 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 25(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 571(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 1. Patient tested negative in glutarylcarnitine blood spot test, even under carnitine supplementation.

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The V400M variant creates an NlaIII restriction site. Thus, while the patient's DNA fragments would cleave, control fragments would not be cleaved. 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.1198G>A (p.Val400Met), 2. NM_000159.3(GCDH):c.1204C>T (p.Arg402Trp), compound heterozygous

ClinVar 1. 2088, 2. 2085

gnomAD 1. MAF= 0.0002769(Other), no homozygotes, 2. MAF= 0.0007566(European(Finnish)), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Case36 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 24(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 221(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 22. Patient tested negative in glutarylcarnitine blood spot test, even under carnitine supplementation.

CaseMethod1 SSCP

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified and SSCP screened 11 exons and intron boundaries of GCDH gene. Confirmed variants with Sanger sequencing and restriction digest. The V400M variant creates an NlaIII restriction site. Thus, while the patient's DNA fragments would cleave, control fragments would not be cleaved. Likewise, the R227P variant creates a NlaIV site. This would lead to cleaved patient DNA fragments and uncleaved control fragments.

Variant 1. NM_000159.3(GCDH):c.1198G>A (p.Val400Met), 2. NM_000159.3(GCDH):c.680G>C (p.Arg227Pro), compound heterozygous

ClinVar 1. 2088, 2. 2089

gnomAD 1. MAF= 0.0002769(Other), no homozygotes, 2. MAF= 0.0003566(European(non-Finnish)), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

each of these nucleotide positions is off by 36

so I came up with this:

NM_000159.3:c.387_388del(p.Glu129AspfsTer?)

register CAID

Mutalyzer

Case12 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 8188(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 3573(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 n/a

CasePMIDs n/a

Case19 sex unknown, Spain

CasePresentingHPOs HP:0001332

(Dystonia)

HPOsFreeText Clinical onset was "insidious," involving slowly progressive psychomotor delay, dystonic postures, lethargy, or macrocephaly. Phenotypes were "mild," owing to static or improving dystonia, with maintained ability to walk without assistance. 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 HP:0002540

(Non-ambulatory)

NotHPOsFreeText n/a

CasePreviousTesting GC-MS measured glutarate of 8955(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 2262(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 R372K variant destroys an MnlI restriction site. Thus, while the patient's DNA fragments would not cleave, control fragments would be cleaved.

Variant 1. NM_000159.3(GCDH):c.1115G>A (p.Arg372Lys), 2. NM_000159.3(GCDH):c.1173delG (p.Asn392Metfs), compound heterozygous

ClinVar 1. 557606, 2. 370106

gnomAD 1. not in gnomAD, 2. MAF= 0.00005646(Latino), no homozygotes

CaseAddInfo n/a

CasePMIDs see note on previous studies

Case8 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 1098(nv 2–10 mmol/mol creatinine) and 3-OH-glutarate 148(nv 2–14 mm/mol creatinine). In patient fibroblasts, %GCD activity was 6.

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 1. NM_000159.3(GCDH):c.877G>A (p.Ala293Thr), 2. NM_000159.3(GCDH):c.268G>A (p.Glu90Lys), compound heterozygous

ClinVar 1. 2083, 2. 459949

gnomAD 1. MAF=0.0004013(African), no homozygotes, 2. MAF= 0.00008011(African), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

FamilyInfo: *Family 2, Hispanic

FamilyHPOs: HP:0001987, HP:0500149, HP:0003572

FamilyNotHPOs: HP:0003218

FamilyGenotypingMethod: All seven exons including intron/exon boundaries of the NAGS gene were amplified and sequenced.

Variant: NM_153006.2(NAGS):c.1025delG (p.Arg342Profs)

ClinVarID: 2427

gnomAD: Absent

Proband: Family 2-Patient 1

Case#: Patient 1, male, died on DOL 4, from the Faroe islands

CaseHPOs: HP:0003811 (neonatal death)

CaseHPOsFreeText: At 16 hours old, began grunting, was jittery, and had tachypnea. At 48 hours, had increased jitteriness, hypertonia, hypothermia, and alkalosis. Coma and death at 4 days. In urine, undetectable citrulline, argininosuccinate, and orotic acid. Plasma amino acids showed extremely elevated glutamine and alanine suggestive of hyperammonemia. No ammonia levels were available.

CaseNotHPOs: HP:0003218 (Orotic aciduria)

CasePreviousTesting: Analysis of urea cycle enzymes on post mortem liver tissue: CPSI activity was 30.7 U (normal >12), basal NAGS activity was 10 U (normal >34), arginine stimulated NAGS activity was 20 U (normal >144).

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: Tissue or DNA from the deceased infant of family 1 was unavailable; blood genomic DNA from both parents to search for a mutation in the NAGS gene. All seven exons including intron/exon boundaries of the NAGS gene were amplified and sequenced.

Variant: NM_153006.2(NAGS):c.971G>A (p.Trp324Ter) (homozygous) (based on parental results; both parents are heterozygous)

ClinVar: 2428

gnomAD: Absent

VariantEvidence: NA

Case17 4yo diagnosis, Anglo-Celtic

CasePresentingHPOs HP:0001083

(Lens dislocation)

HPOsFreeText 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 was not responsive to the treatment, plasma homocysteine levels went unchanged. This patient was given additional oral betaine(3g twice per day) with a low protein diet.

CaseNotHPOs HP:0001249

(Intellectual disability)

NotHPOsFreeText no other sequelae

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 NM_000071.2(CBS):c.919G>A (p.Gly307Ser), homozygous

ClinVar 117

gnomAD MAF= 0.00041539(Other), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

FamilyLabel Family 15

Family15PresentingHPOs n/a

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

FamilyNotHPOs n/a

NotHPOsFreeText n/a

CountryOrigin n/a

Ethnicity Anglo-Celtic

Race n/a

Family15PreviousTesting 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 18

Variant NM_000071.2(CBS):c.919G>A (p.Gly307Ser), homozygous

ClinVar 117

gnomAD MAF= 0.00041539(Other), no homozygotes

FamilyAddInfo n/a

FamilyPMIDs n/a

Case29 Male, 1yo diagnosis (Family 25)

CasePresentingHPOs HP:0001256, HP:0001263

(Mild intellectual disability, Developmental delay)

HPOsFreeText Diagnosed was on the basis of developmental delay. 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 no other sequelae

CasePreviousTesting n/a

Variant 1. NM_000071.2(CBS):c.536_553del18 (p.Asp179_Leu184del), 2. NM_000071.2(CBS):c.325T>C (p.Cys109Arg), comp het

ClinVar 1. 198388, 2. 212878

gnomAD 1. MAF=0.00002683(European(non-Finnish), no homozygotes, 2. MAF= 0.0001631(Other), no homozygotes

CaseAddInfo son of patient 30

CasePMIDs n/a

FamilyLabel Family 26

Family26PresentingHPOs HP:0001083

(Lens dislocation)

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

FamilyNotHPOs n/a

NotHPOsFreeText n/a

CountryOrigin n/a

Ethnicity Portugese

Race n/a

Family26PreviousTesting 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 31

Variant NM_000071.2(CBS):c.374G>A (p.Arg125Gln), homozygous

ClinVar 197625

gnomAD MAF= 0.00002326(European(non-Finnish)), no homozygotes

FamilyAddInfo n/a

FamilyPMIDs n/a

Case25 9yo diagnosis, Australia, Anglo-Celtic

CasePresentingHPOs HP:0001083, HP:0002342, HP:0000939, HP:0001263, HP:0001369

(Lens dislocation, Moderate intellectual disability, Osteoporosis, Developmental delay, Arthritis)

HPOsFreeText Patient had severe osteoporosis, juvenile arthritis and was diagnosed on the basis of dislocated lenses and developmental delay. 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. (PMID:9156316): Total homocysteine was 252μmol/l before and 34μmol/l after treatment of betaine, folic acid, and low-dose aspirin.

CaseMethod1 PCR

CaseMethod2 Sanger

CaseGenotypingMethod PCR amplified then direct sequenced the entire coding region of CBS. (PMID: 9156316): Restriction analysis confirmed the presence of two variants. The variant c.430G>A destroys a TaqI site. Thus, TaqI digestion yielded uncleaved 155bp fragments in both the proband and his father, while the WT was cleaved into 107 and 48bp fragments. The c.1316G>A variant likewise destroys a HapII site. The proband and his father had uncleaved 120bp fragments while the WT had 87 and 33bp fragments on HapII digestion. As both variants were detected in the proband, father, and two paternal aunts, c.1316G>A and c.430G>A were proposed to be in cis.

Variant 1. NM_000071.2:c.892dup (p.Gln298ProfsTer?), 2. NM_000071.2(CBS):c.430G>A (p.Glu144Lys); 3. NM_000071.2(CBS):c.1316G>A (p.Arg439Gln), comp het, 2&3 in cis

ClinVar 1. n/a, 2. 122; 3. 189088

CAID 1. register

gnomAD 1. MAF=0.00001777(European(Non-finnish)), no homozygotes 2. MAF=0.00009799(South Asian), no homozygotes, 3. MAF=0.00005341(African), no homozygotes

CaseAddInfo n/a

CasePMIDs 9156316

Case15 3yo diagnosis, Anglo-Celtic

CasePresentingHPOs HP:0001083, HP:0001256

(Lens dislocation, Mild intellectual disability)

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

CaseNotHPOs n/a

NotHPOsFreeText no other sequelae

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 NM_000071.2(CBS):c.919G>A (p.Gly307Ser), homozygous

ClinVar 117

gnomAD MAF= 0.00041539(Other), no homozygotes

CaseAddInfo n/a

CasePMIDs n/a

Case1

Variant: NM_013391.3:c.326A>G (p.His109Arg)

VariantEvidence: The above mutation was introduced into hDMGDH cDNA and both the wild-type and mutant sequences were expressed in E. coli, Saccharomyces cerevisiae, and other mammalian tissue culture samples. In the mutant E coli, no active enzyme could be detected. Similarly, no antigen could be detected in the other cells. The wild-type cells had detectable activity and much higher levels of mRNA than the mutants. Western blot of the cellular extracts showed that the levels of wild-type and mutant protein were similar, but the activity was undetectable.

CaseGM01781: Female, 18 years report, Caucasian, Europe

CasePresentingHPOs: HP:0003153 (Cystathioninuria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is a compound heterozygote for these variants, Variants were confirmed in trans by parental testing

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.

Variant1: 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

Variant2: NM_001902.5:c.718C>G (p.Gln240Glu)

ClinVar: 2940 https://www.ncbi.nlm.nih.gov/clinvar/variation/2940/

gnomAD: European (Non-Finnish) 1.759e-5, Overall 7.957e-6 https://gnomad.broadinstitute.org/variant/1-70896071-C-G

Case: Male, 5 days onset, Iran

CasePresentingHPOs: HP:0002572, HP:0004923 (Episodic vomiting, Hyperphenylalaninemia)

HPOsFreeText: Brown eyes

CaseNotHPOs: HP:0001249, HP:0032807 (Intellectual disability, Neonatal seizure)

NotHPOsFreeText: N/A

CaseAddInfo: Parents are consanguineous first cousins

CasePreviousTesting: Screened for HPAs by Guthrie test and on special diet

CaseGenotypingMethod: Genomic DNA was extracted from peripheral blood. PAH gene was screened but no mutations were found. BH4-metabolizing genes were screened via STR markers, with PCBD1 having a homozygous pattern. Direct sequencing revealed a homozygous termination codon extension.

Variant: NM_000281.4(PCBD1):c.313T>C (p.Ter105Gln)

ClinVar: 428616, https://www.ncbi.nlm.nih.gov/clinvar/variation/428616/

gnomAD: 0.00003301, https://gnomad.broadinstitute.org/variant/10-72643709-A-G

Case1: Newborn, Indian (consanguineous)

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 0.88 nM/ml (ref <0.8). 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.295C>T (p.Gln99Ter) (homozygous)

ClinVar: 281177

gnomAD: 0.00006537 South Asian

VariantEvidence: LOF

CaseAddInfo:

CasePMIDs:

Case#: Patient 2; English/Dutch/German/French

CaseHPOs: Clinical details in Weleber et al, 1982, “Gyrate atrophy of the choroid and retina… Birth defects: Original Article Series 18 (6) 219-230. Article has been requested.

CaseHPOsFreeText: Not responsive to Vitamin B6.

CaseNotHPOs:

CasePreviousTesting: OAT activity was nondetectable in the patient’s fibroblasts. Northern analysis detected no OAT mRNA. Western blot analysis of OAT protein in the patient's fibroblasts demonstrated an essentially negligible amount of OAT.

CaseMethod1: PCR

CaseMethod2: Sequence analysis

CaseGenotypingMethod: Truncated 5.0-kbp EcoRI OAT gene fragment in addition to the normal 5.8-kbp fragment on Southern blot analysis (Inana et al., 1988), suggesting heterozygosity for a deletion. Cloning and sequencing of the truncated fragment revealed a deletion of 1,072 base pairs. DGGE and sequence analysis of genomic DNA was carried out to identify the variant on the other allele.

Variant: Variant 1: A 1,072-bp deletion starting in intron 5, 172 bp upstream of exon 6, and ending in intron 6, 772 bp downstream of exon 6 Variant 2: NM_000274.3(OAT):c.627T>A (p.Tyr209Ter)

ClinVar: Variant 1: 174; Variant 2: 175

gnomAD: Variant 1: Not listed (but large deletion may not be picked up) Variant 2: Maximum population minor allele frequency = 0.00004645 (European non-Finnish); no homozygotes in any population.

Case: Citron-1/ BIODEF 270, male, 4 days

CasePresentingHPOs: HP:0008297

HyperPhe was noted, with a plasma Phe level of 700uM. Normal neopterin and biopterin levels were observed in the urine, but the presence of primapterin was found.

CaseNOTHPOs:

CasePreviousTesting: n/a

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

Variant 1: NM_000281.3(PCBD1):c.259G>T (p.Glu87Ter)

ClinVar ID 1: 16795

Variant 2: NM_000281.3(PCBD1):c.244T>C (p.Cys82Arg)

ClinVar ID 2: 16796

Compound heterozygous

gnomAD 1: MAF (Ash-Jews)= 1.183E-3, all hets no homozygous.

gnomAD 2: MAF (Euro-NF)= 2.690E-5 all hets, no homozygous noted. http://gnomad.broadinstitute.org/variant/10-72643778-A-G

AddCaseInfo: Parents were tested and found to have normal Phe levels. The father carriers the termination variant, E87X, the mother harbors the C82R, and both parents are heterozygous for their respective PCBD1 variants.

VariantEvidence:  For the Glu87Ter variant, Thony et al., PMID: 9585615 showed that the mutant E86X/E87X protein was expressed in E.Coli to determine the solubility of the mutant enzyme and the activity. The mutant E86X/E87X fragment was mostly found in the insoluble fraction of the supernatant. The remaining fraction obtained in the soluble fraction did retain some enzyme activity in vitro. Interestingly, when more E86X/E87X was obtained in the soluble fraction (by growing the E.coli at 20 degrees C), the Km (Michaelis constant) was similar to wildtype levels (text only, no figures to compare).

For the C82R/C81R variant was analyzed by Ayling et al., 2000 (PMID:10924272) and was found to still be retained in the soluble fraction in bacteria expressing the recombinant protein. Furthermore, enzyme assays show that the soluble C82RPCBD1 variant retained activity, albeit at 40% of the wildtype.