This is a cohort of 12 Iraqi-Jewish Glanzmann thrombasthenia patients

Gene: ITGA2B

Variant: NM_000419.3:c.409-2_419del

ClinVar: 2889

CAID: CA10575471

gnomAD: N/A

PMID: 14508803

Gene: ITGA2B and ITGB3

HGNC: 6138 and 6156

InsufficientEvidence: This article mentions GT patients however no variants in ITGA2B and/or ITGB3 are mentioned in these patients.

In this study the goal was to determine what the common subtype of GT was: The expression of GPIIb/IIIa on the platelet surface was assessed in 10 patients with Glanzmann thrombasthenia and their families by flow cytometry to determine the common subtype in North Indians

Supplementary Tables 1 and 2 combined information:

Case19: GT19, Female, 32 years old

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: Not available per Table 1.

ParentalGenotype: Parents are consanguineous, no other genotype information available

CasePresentingHPOs: HP:0000421, HP:0000225, HP:0000790, HP:0000132

(Epistaxis, gingival bleeding, hematuria, menorrhagia)

CaseHPOFreeText: Post traumatic hemorrhage

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Showed <5% of αIIbβ3 expression measured by flow cytometry

PlateletAggregation: Platelet aggregation showed no response to ADP and collagen but normal response to ristocetin

CasePreviousTesting: All exons from ITGA2B and ITGB3 genes were amplified by PCR using intronic primers. DNA sequencing was determined by Sanger sequencing.

Gene: ITGA2B

Variant: NM_000419.3:c.1234G>A (p.Gly412Arg)

LegacyVariant: N/A

ClinVar: 381748

CAiD: CA8603182 (p.Gly412Arg)

gnomAD: MAF in Latino population: 0.0001453 https://gnomad.broadinstitute.org/variant/17-42458406-C-T

Case#: GT3 male, 17 years old

Disease: Glazmann's Thrombasthenia

CasePresentingHPOs: (found in appendix) HP:0001892, HP:0000421, HP:0000225, HP:0000967, HP:0000979, HP:0031364, HP:0006298

ExpressionEvidence: Flow Cytometry of platelet αIIbβ3: 0%CD41, .43% CD61

FamilyInfo: Positive consanguinity

GenotypingMethod: Sanger DNA sequencing to identify causative mutations in each case

ClinVar: 1691476

CAID: CA400030171

Variant: NM_000212.3:c.1641C>G

GnomAD: Not Found

Case30: Patient CM

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: HP:0000421, HP:0000225, HP:0000132

(Epistaxis, gingival bleeding, menorrhagia)

CaseHPOFreeText: N/A

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: N/A

PlateletAggregation: N/A

CasePreviousTesting: N/A

Gene: ITGB3

Variant: NM_000212.2:c.718C>T (p.Arg240Trp)

LegacyVariant: N/A

ClinVar: 13555

CAiD: CA123228

gnomAD: MAF in the East Asian population is 0.00005437 https://gnomad.broadinstitute.org/variant/17-45363729-C-T

PreviouslyPublished: Reference 48 and 49

Case32: Patient SH

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: N/A

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: 20% GPIIIa and GPIIb surface expression

PlateletAggregation: N/A

CasePreviousTesting: N/A

Gene: ITGB3

Variant: NM_000212.2:c.725G>A (p.Arg242Gln)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA291224896

gnomAD: N/A

PreviouslyPublished: Reference number 5 in this article

Supplementary Table S1 & S2 Proband and Variant Information:

Case15: GT18, Male, 20, French

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: HP:0031364, HP:0000978

(Ecchymosis, easy bruising)

CaseHPOFreeText: N/A

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry showed 10% expression of αIIbβ3 (See Supplementary Table 1)

PlateletAggregation: Residual platelet aggregation (See Supplementary Table 1)

CasePreviousTesting: Sanger sequencing of ITGA2B and ITGB3

Gene: ITGA2B

Variant: NM_000419.3:c.1201G>T (pGly401Cys) andNM_000419.3:c.2800G>T (p.Val934Phe)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA399804280 and CA290946484

gnomAD: N/A

Supplementary Table S1 & S2 Proband and Variant Information:

Case31: GT35a, Female, 18 years old, USA

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: Affected sister who is 35 b in this study (See Supplementary Table S1)

ParentalGenotype: N/A

CasePresentingHPOs: HP:0000421, HP:0002239

(Epistaxis, GI bleeding)

CaseHPOFreeText: N/A

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry showed <5% expression of αIIbβ3 (See Supplementary Table 1)

PlateletAggregation: Absent platelet aggregation (See Supplementary Table 1)

CasePreviousTesting: Sanger sequencing of ITGA2B and ITGB3

Gene: ITGA2B and ITGB3

Variant: ITGB3: NM_000212.2:c.392G>C (p.Arg131Pro) and NM_000212.2:c.777+1G>A ITGA2B: NM_000419.3:c.2264G>A (p.Arg755Gln)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA8622954, CA8623036, CA8602771

gnomAD: MAF in the African population is 0.00006152 http://gnomad.broadinstitute.org/variant/17-45361839-G-C, MAF in the Latino population is 0.0006774 http://gnomad.broadinstitute.org/variant/17-45363789-G-A, MAF in the Latino population is 0.0001903 http://gnomad.broadinstitute.org/variant/17-42454380-C-T

Supplementary Table S1 & S2 Proband and Variant Information:

Case53: GT53, Male, 16 years old, Argentenian/Brazilian

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: HP:0000421

(Epistaxis)

CaseHPOFreeText: Hematomas

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry showed <5% expression of αIIbβ3 (See Supplementary Table 1)

PlateletAggregation: Absent platelet aggregation (See Supplementary Table 1)

CasePreviousTesting: Sanger sequencing of ITGA2B and ITGB3

Gene: ITGB3

Variant: NM_000212.2:c.356G>A (p.Gly201Ser) and NM_000212.2:c.1697G>A (p.Arg119Gln)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA8622925 and CA400032338

gnomAD: MAF in the African population is http://gnomad.broadinstitute.org/variant/17-45360910-G-A

Supplementary Table S1 & S2 Proband and Variant Information:

Case46: GT45, Male, 86 years old, French

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: HP:0000421, HP:0000225, HP:0002239

(Epistaxis, gingival bleeding, GI bleeding)

CaseHPOFreeText: N/A

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry showed <5% expression of αIIbβ3 (See Supplementary Table 1)

PlateletAggregation: Absent platelet aggregation (See Supplementary Table 1)

CasePreviousTesting: Sanger sequencing of ITGA2B and ITGB3

Gene: ITGA2B

Variant: NM_000419.3:c.551_626del (p.Arg184_Gln208del) and LR deletion

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA915940373

gnomAD: N/A

Supplementary Table S1 & S2 Proband and Variant Information:

Case45: GT44, Female, 10 years old, French

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: N/A

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry showed <5% expression of αIIbβ3 (See Supplementary Table 1)

PlateletAggregation: Absent platelet aggregation (See Supplementary Table 1)

CasePreviousTesting: Sanger sequencing of ITGA2B and ITGB3

Gene: ITGA2B

Variant: NM_000419.3:c.1357C>T (p.Arg453Ter)

LegacyVariant: N/A

ClinVar: 631773

CAiD: CA399803529

gnomAD: N/A

Supplementary Table S1 & S2 Proband and Variant Information:

Case48: GT48, Male, 10 years old, French

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: HP:0031364, HP:0000421, HP:0001934

(Ecchymosis, epistaxis, persistent bleeding after trauma)

CaseHPOFreeText: Frequent hematomas

CaseNotHPOs: N/A

CaseNotHPOsFreeText: No serious episode of spontaneous bleeding

ProteinExpression: Flow cytometry showed <5% expression of αIIbβ3 (See Supplementary Table 1)

PlateletAggregation: N/A (Not available for this proband see Supp Table S1)

CasePreviousTesting: Sanger sequencing of ITGA2B and ITGB3

Gene: ITGB3

Variant: NM_000212.2:c.1094A>G (p.Asn365Ter) and NM_000212.2:c.1522del (p.Gln508Serfs*161)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA400026292 and CA772541579

gnomAD: N/A

Supplementary Table S1 & S2 Proband and Variant Information:

Case23: GT26, Male, 45, French

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: HP:0000421, HP:0000978

(Epistaxis, easy bruising)

CaseHPOFreeText: N/A

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry showed <5% expression of αIIbβ3 (See Supplementary Table 1)

PlateletAggregation: Much reduced platelet aggregation (See Supplementary Table 1)

CasePreviousTesting: Sanger sequencing of ITGA2B and ITGB3

Gene: ITGA2B

Variant: NM_000419.3:c.1073G>A (p.Arg358His)

LegacyVariant: N/A

ClinVar: 2896

CAiD: CA115839

gnomAD: N/A

Supplementary Table S1:

Case28: GT33a, USA/Laos, gender and age not specified or reported

DiseaseAssertion: Glanzmann thrombasthenia type I

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: All patients had a life-long history of mucocutaneous bleeding

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: All patients in this study met the clinical and platelet function requirements for GT-- this includes an absent or strongly reduced expression of alphaIIbBeta3 in platelets

PlateletAggregation: All patients in this study met the clinical and platelet function requirements for GT-- this includes absent or severely reduced platelet aggregation with at least three physiological agonists

CasePreviousTesting: Sanger sequencing was performed. Sanger sequencing ensured coverage of all exons and splice sties of the ITGA2B and ITGB3 genes and extended 300-500 bp into the introns as well as including the UTR. They sequenced 416 bp upstream of the ITGA2B promoter and 463 bp upstream of the ITGB3 promoter. In total they sequenced 51.2% of the ITGA2B gene and 19.4% of the larger ITGB3 gene

Gene: ITGB3

Variant: NM_000212.2:c.262C>T (p.Arg88Ter) and NM_000212.2:c.2113del (p.Ley705Cysfs*4)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA400020703 and CA8623443

gnomAD: MAF in the Other population is 0.0001385 for (p.Arg88Ter) https://gnomad.broadinstitute.org/variant/17-45360816-C-T and MAF in the East Asian population is 0.0001087 for (p.Ley705Cysfs*4) https://gnomad.broadinstitute.org/variant/17-45380183-TC-T

Supplementary Table S1:

Case6: GT7, French, gender and age not specified

DiseaseAssertion: Glanzmann thrombasthenia type I

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: All patients had a life-long history of mucocutaneous bleeding

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: All patients in this study met the clinical and platelet function requirements for GT-- this includes an absent or strongly reduced expression of alphaIIbBeta3 in platelets

PlateletAggregation: All patients in this study met the clinical and platelet function requirements for GT-- this includes absent or severely reduced platelet aggregation with at least three physiological agonists

CasePreviousTesting: Sanger sequencing was performed. Sanger sequencing ensured coverage of all exons and splice sties of the ITGA2B and ITGB3 genes and extended 300-500 bp into the introns as well as including the UTR. They sequenced 416 bp upstream of the ITGA2B promoter and 463 bp upstream of the ITGB3 promoter. In total they sequenced 51.2% of the ITGA2B gene and 19.4% of the larger ITGB3 gene

Gene: ITGB3

Variant: NM_000212.2:c.187C>T (p.Arg63Cys) and NM_000212.2:c.505C>T (p.Arg169Ter)

LegacyVariant: N/A

ClinVar: 631774

CAiD: CA8622892 and CA291224658

gnomAD: MAF in the East Asian population is 0.00005012 https://gnomad.broadinstitute.org/variant/17-45360741-C-T

Supplementary Table S1:

Case55: GT65, French, gender and age not specified

DiseaseAssertion: Glanzmann thrombasthenia type I

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: All patients had a life-long history of mucocutaneous bleeding

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: All patients in this study met the clinical and platelet function requirements for GT-- this includes an absent or strongly reduced expression of alphaIIbBeta3 in platelets

PlateletAggregation: All patients in this study met the clinical and platelet function requirements for GT-- this includes absent or severely reduced platelet aggregation with at least three physiological agonists

CasePreviousTesting: Sanger sequencing was performed. Sanger sequencing ensured coverage of all exons and splice sties of the ITGA2B and ITGB3 genes and extended 300-500 bp into the introns as well as including the UTR. They sequenced 416 bp upstream of the ITGA2B promoter and 463 bp upstream of the ITGB3 promoter. In total they sequenced 51.2% of the ITGA2B gene and 19.4% of the larger ITGB3 gene

Gene: ITGA2B

Variant: NM_000419.3:c.574+1G>A and NM_000419.3:c.1946+3G>T

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA399805691 and CA626121071

gnomAD: MAF in the African population is 0.000 https://gnomad.broadinstitute.org/variant/17-42456008-C-A

Supplementary Table S1:

Case60: GT70, French/Arab, gender and ethnicity are not specified

DiseaseAssertion: Glanzmann thrombasthenia type I

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: All patients had a life-long history of mucocutaneous bleeding

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: All patients in this study met the clinical and platelet function requirements for GT-- this includes an absent or strongly reduced expression of alphaIIbBeta3 in platelets

PlateletAggregation: All patients in this study met the clinical and platelet function requirements for GT-- this includes absent or severely reduced platelet aggregation with at least three physiological agonists

CasePreviousTesting: Sanger sequencing was performed. Sanger sequencing ensured coverage of all exons and splice sties of the ITGA2B and ITGB3 genes and extended 300-500 bp into the introns as well as including the UTR. They sequenced 416 bp upstream of the ITGA2B promoter and 463 bp upstream of the ITGB3 promoter. In total they sequenced 51.2% of the ITGA2B gene and 19.4% of the larger ITGB3 gene

Gene: ITGB3

Variant: M_000212.2:c.613_614+2del

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA915940309

gnomAD: N/A

Supplementary Table S1:

Case13: GT14, French, gender age and ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia type I

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: All patients had a life-long history of mucocutaneous bleeding

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: All patients in this study met the clinical and platelet function requirements for GT-- this includes an absent or strongly reduced expression of alphaIIbBeta3 in platelets

PlateletAggregation: All patients in this study met the clinical and platelet function requirements for GT-- this includes absent or severely reduced platelet aggregation with at least three physiological agonists

CasePreviousTesting: Sanger sequencing was performed. Sanger sequencing ensured coverage of all exons and splice sties of the ITGA2B and ITGB3 genes and extended 300-500 bp into the introns as well as including the UTR. They sequenced 416 bp upstream of the ITGA2B promoter and 463 bp upstream of the ITGB3 promoter. In total they sequenced 51.2% of the ITGA2B gene and 19.4% of the larger ITGB3 gene

Gene: ITGA2B

Variant: NM_000419.3:c.423G>T (p.Trp141Cys) and NM_000419.3:c.2902del (p.Tyr968Metfs*163)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA399806022 and CA915940332

gnomAD: N/A

Supplementary Table S1:

Case9: French/Italian, gender and age not specified

DiseaseAssertion: Glanzmann thrombasthenia type I

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: All patients had a life-long history of mucocutaneous bleeding

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: All patients in this study met the clinical and platelet function requirements for GT-- this includes an absent or strongly reduced expression of alphaIIbBeta3 in platelets

PlateletAggregation: All patients in this study met the clinical and platelet function requirements for GT-- this includes absent or severely reduced platelet aggregation with at least three physiological agonists

CasePreviousTesting: Sanger sequencing was performed. Sanger sequencing ensured coverage of all exons and splice sties of the ITGA2B and ITGB3 genes and extended 300-500 bp into the introns as well as including the UTR. They sequenced 416 bp upstream of the ITGA2B promoter and 463 bp upstream of the ITGB3 promoter. In total they sequenced 51.2% of the ITGA2B gene and 19.4% of the larger ITGB3 gene

Gene: ITGA2B

Variant: NM_000419.3:c.1440-13_1440-1del (p.Arg480Serfs*106) and NM_000419.3:c.3077G>A (p.Arg1026Gln)

LegacyVariant: N/A

ClinVar: 50232

CAiD: CA913184940 and CA10575572

gnomAD: MAF in the European (non-Finnish) population is 0.000008801 https://gnomad.broadinstitute.org/variant/17-42449775-C-T for (p.Arg1026Gln)

Case#: 13 year old, male

Disease: Glazmann's Thrombasthenia

CasePresentingHPOs: HP:0000967, HP:0000978, HP:0004406, HP:0000225, HP:0006298, HP:0001892, HP:0002239

PlateletAgreggation: Defective response to ADP, Adrenaline, Collagen, Arachidonic acid. Normal response to Ristocetin.

GenotypingMethod: Genomic DNA was extracted from peripheral blood leukocytes for mutational analysis of the αIIb and β3 genes by DNA sequencing

Allele 1:

ClinVar: 996212

CAID: CA399803786

Variant: NM_000419.5:c.1229C>G

GnomAD: European non-Finnish at rate of 0.0000089411 (1/111846)

Allele 2:

ClinVar: 225393

CAID: CA290947544

Variant: NM_000419.5:c.2333A>C

GnomAD: East Asian population at rate of 0.0001087 (2/18394)

ExpressionEvidence: Flow Cytometry of platelet integrin: 92.9% B3 expression, 2.1% A2B expression

Case1: Female, 21 years old, Japanese (Named Osaka-12 in this study)

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: The substitution was inherited from her father. The molecular genetic defect inherited from her mother remains determined.

CasePresentingHPOs: N/A

CaseHPOFreeText: Moderate mucocutaneous bleeding in childhood

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry showed revealed a significant reduction in the expression of αIIbβ3 on Osaka-12 platelets

PlateletAggregation: Absent platelet aggregation in response to ADP, epinephrine, and collagen but a normal response to ristocetin

CasePreviousTesting: To identify the molecular genetic defect for the proband of interest, all coding regions of ITGA2B and ITGB3 cDNA amplified by RT-PCR were analyzed

Gene: ITGA2B

Variant: NM_000419.3:c.520T>C (p.Tyr174His)

LegacyVariant: (p.Tyr143His)

ClinVar: N/A

CAiD: CA399805805

gnomAD: N/A

Case23: (GT-27) Male, age or ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: Proband GT28 is related to this proband GT 27; however, no DNA was available for testing for GT28.

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Bleeding severity: Moderate (See Supplementary Table 3)

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry performed and CD41 and CD61 were observed (See Supplementary Table 3)

PlateletAggregation: Minimal information (See Supplementary Table 3)

CasePreviousTesting: DNA was extracted from peripheral blood samples from patients and family members, and Sanger sequencing and PCR was performed. Samples were processed for targeted sequencing using the available SHGp heme panel of 393 genes.

Gene: ITGB3

Variant: NM_000212.2:c.1835G>A

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA400032647

gnomAD: N/A

Case47: (GT-58) Female, age or ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Bleeding severity: Severe (See Supplementary Table 3)

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry performed and CD41 and CD61 were observed (See Supplementary Table 3)

PlateletAggregation: Minimal information (See Supplementary Table 3)

CasePreviousTesting: DNA was extracted from peripheral blood samples from patients and family members, and Sanger sequencing and PCR was performed. Samples were processed for targeted sequencing using the available SHGp heme panel of 393 genes.

Gene: ITGB3

Variant: NM_000212.2:c.1543del (p.Ser513fs)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA500651429

gnomAD: N/A

Case30: (GT-35) Female, age or ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: Proband GT31-GT34 are related

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Bleeding severity: Severe (See Supplementary Table 3)

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry performed and CD41 and CD61 were observed (See Supplementary Table 3)

PlateletAggregation: Minimal information (See Supplementary Table 3)

CasePreviousTesting: DNA was extracted from peripheral blood samples from patients and family members, and Sanger sequencing and PCR was performed. Samples were processed for targeted sequencing using the available SHGp heme panel of 393 genes.

Gene: ITGA2B

Variant: NM_000419.3:c.1879-2A>G

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA290949031

gnomAD: N/A

Case8: (GT-8) Female, 10 years old, French

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Mucocutaneous bleeding

CaseNotHPOs: N/A

ProteinExpression: <5% surface expression levels of αIIbβ3

Platelet Aggregation: Absent platelet aggregation to all agonists (no mention for which agonists or response to ristocetin)

CasePreviousTesting: All the information we are given is "mutation screening of the ITGA2B and ITGB3 genes" the method for sequencing etc. was not stated

Gene: ITGA2B

Variant: NM_000419.3:c.682C>T (p.Gln228Ter) and NM_000419.3:c.1750C>T (p.Arg584Ter)

LegacyVariant: (p.Gln197Ter) and (p.Arg558Ter) (The paper gives legacy and current nomenclature)

ClinVar: 2892 (p.Arg584Ter)

CAiD: CA399805421 (p.Gln228Ter) CA115834 (p.Arg584Ter)

gnomAD MAF in the East Asian population is 0.0002176 https://gnomad.broadinstitute.org/variant/17-42457372-G-A

Case10: (M2) Female, 4 years old, French Gypsy

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Mucocutaneous bleeding

CaseNotHPOs: N/A

ProteinExpression: Failed to express alphaIIbbeta3 on the surface of their platelets

Platelet Aggregation: Absent platelet aggregation to all agonists (no mention for which agonists or response to ristocetin)

CasePreviousTesting: All the information we are given is "mutation screening of the ITGA2B and ITGB3 genes" the method for sequencing etc. was not stated

Gene: ITGA2B

Variant: NM_000419.3:c.1544+1G>A

LegacyVariant: N/A

ClinVar: 2895

CAiD: CA10575473

gnomAD N/A

Case3: (GT3) Male, 82 years old, French

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Mucocutaneous bleeding

CaseNotHPOs: N/A

ProteinExpression: 8-10% surface expression levels of αIIbβ3

Platelet Aggregation: Absent platelet aggregation to all agonists (no mention for which agonists or response to ristocetin)

CasePreviousTesting: All the information we are given is "mutation screening of the ITGA2B and ITGB3 genes" the method for sequencing etc. was not stated

Gene: ITGA2B

Variant: NM_000419.3:c.2348+5G>C

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA290947484

gnomAD N/A

Case1: Male, age not specified, ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: See Fig 3

ParentalGenotype: Both mutations were found to be heterozygous in all family members that includes parents

CasePresentingHPOs:

(Gum bleeding, ecchymosis, epistaxis)

CaseHPOFreeText: N/A

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry showed that the surface expression of GPIIb/IIIa in patients was 0% and both parents also showed reduced expression as well.

PlateletAggregation: Platelet aggregation in response to all the agonists except risocetin was completely absent in the patient

CasePreviousTesting: Mutation screening by conformational sensitive gel electrophoresis was performed in all the 30 exons of ITGA2B and 15 exons of ITGB3 gene in this patient

Gene: ITGA2B

Variant: NM_000419.3:c.1028T>C (p.Leu343Pro) and a promoter variant g.951G>A

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA399804619 and need to ask about second variant--made note in google doc to ask

gnomAD: N/A

PMID: 23300803

Genes: ITGA2B and ITGB3

HGNC: 6138 an d 6156

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

Case1: Female, 35 day old, ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: The proband’s mother and father carry heterozygous c.512delC and c.1686delG mutations, respectively. Other parameters for parents are shown in Table 1.

CasePresentingHPOs: HP:0031364, HP:0000967

(Ecchymosis, petechiae)

CaseHPOFreeText: Large parietal and cervical cephalohaematoma, severe anemia

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry of platelet surface antigens revealed the absence of the αIIbβ3 complex.

PlateletAggregation: Prolonged closure time at PFA-100, absent ADP, adrenaline, collagen induced platelet aggregation, and ristocetin induced platelet aggregation at the lower limit of normal

CasePreviousTesting: Sequencing of ITGA2B and ITGB3 genes showed that proband is compound het.

Gene: ITGA2B

Variant: Can not conclusively determine if c.481delC and c.1654delG are the variants intended by the authors based on the available information.

LegacyVariant: N/A

ClinVar: N/A

CAiD: N/A

gnomAD: N/A

PMID: 9694297

Gene: ITGA2B and/or ITGB3

HGNC: 6138 and 6156

InsufficientEvidence: This mentions a Glanzmann thrombasthenia patient; however, no variants in ITGA2B and/or ITGB3 are mentioned in this patient; therefore, we have insufficient evidence

Case5: Male, age or ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Severe bleeding

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: N/A

PlateletAggregation: Minimal information, not specific enough

CasePreviousTesting: Sanger sequencing identified disease-causing variants in 18 out of 20 patients (Probands 6 and 14 excluded) with istoantibodies to αIIbβ3. The mutations were evenly distributed between ITGB3 and ITGA2B.

Gene: ITGB3

Variant: NM_000212.2:c.892C>G (p.Gln298Ter)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA400025097

gnomAD: N/A

PMID: 28299211

Gene: ITGA2B and ITGB3

HGNC: 6138 and 6156

InsufficientEvidence: This article mentions cases of patients with Glanzmann thrombasthenia and their phenotypes; however, no mention of variants in ITGB3 or ITGA2B

Case14: Patient ID N, Male, 7 years old, ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: HP:0000967, HP:0000979, HP:0000978, HP:0004406, HP:0000225

(Petechiae, purpura, easy bruising, recurrent epistaxis, gingival bleeding)

CaseHPOFreeText: Hemorrhage after dental, hemorrhage after trauma

CaseNotHPOs: HP:0002239, HP:0002239, HP:0000790, HP:0005261, HP:0002248

(GI bleeding, intracranial hemorrhage, hematuria, hemarthrosis, hematemesis)

CaseNotHPOsFreeText: Ear bleeding

ProteinExpression: N/A

PlateletAggregation: Defective response to ADP, adrenaline, collagen, arachidonic acid, and normal response to ristocetin

CasePreviousTesting: Exon PCR was used to amplify coding regions of the ITGA2B and ITGB3 genes. PCR products were purified and Sanger sequenced. Segregation using parental samples was undertaken when available

Gene: ITGB3

Variant: NM_000212.2:c.774T>A (p.Cys258Ter)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA400023704

gnomAD: N/A

Case5: Patient ID E, Female, 1 year old, ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: HP:0000978, HP:0000225

(Easy bruising, gingival bleeding)

CaseHPOFreeText: N/A

CaseNotHPOs: HP:0000967, HP:0000979, HP:0004406, HP:0002239, HP:0002239, HP:0000790, HP:0005261, HP:0002248

(Petechiae, purpura, recurrent epistaxis, GI bleeding, Intracranial hemorrhage, hematuria, hemarthrosis, hematemesis)

CaseNotHPOsFreeText: Hemorrhage after dental, hemorrhage after trauma, ear bleeding, hematoma

ProteinExpression: N/A

PlateletAggregation: Defective response to ADP, adrenaline, collagen, arachidonic acid, and normal response to ristocetin

CasePreviousTesting: Exon PCR was used to amplify a;; coding regions of the ITGA2B and ITGB3 genes. PCR products were purified and Sanger sequenced. Segregation using parental samples was undertaken when available.

Gene: ITGA2B

Variant: c.1460insAGGT (p.Ser477Gfs*111)

LegacyVariant: N/A

ClinVar: N/A

CAiD: Unregistered Variant

gnomAD: N/A

Case4: Gender or age not specified, Thai ethnicity

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: Parental information available in Table 2

ParentalGenotype: Father: p.Gln508Hisfs*3/WT and Mother: No mutations in the coding region

Note: No parental consanguinity

CasePresentingHPOs: N/A

CaseHPOFreeText: The article does not mention specific bleeding phenotypes for each of the 4 probands in the article, rather, there is a general statement that says: "The common presentations included recurrent epistaxis, bleeding in oral cavity, and easy-bruising spontaneously or after trauma"

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinEpression: General statement from the article that states that flow cytometry to assess the surface GPIIb/IIIa complex showed reduced expression for all probands.

PlateletAggregation: The platelet aggregation test was typically defective with all physiological agonists except ristocetin, for all patients.

CasePreviousTesting: Whole exome sequencing was performed on the 30 exons of ITGA2B and 15 exons in ITGB3. PCR and direct sequencing were then performed in the DNA samples of the patients and their available parents to verify the presence of the identified mutations.

Gene: ITGA2B

Variant: NM_000419.3:c.1524_1533del (p.Gln508Hisfs*3)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA915940265

gnomAD: N/A

Case26: GT33, Female, India, age of onset is 18 years old

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Patients were recruited as they presented consecutively from 1996 to 1998 for evaluation of a mucocutaneous bleeding disorder

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: N/A

PlateletAggregation: All patients examined had defective ADP, epinephrine, and collagen induced aggregation and normal ristocetin induced aggregation

CasePreviousTesting: Mutations were detected by the method of single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing and confirmation by restriction fragment length polymorphism (RFLP) assays. The sequences of the primers employed and the reaction conditions for PCR amplification of the exons, adjacent intronic regions, and 50 and 30 untranslated regions of the aIIb and b3 gene

Gene: ITGB3

Variant: NM_000212.2:c.428T>G (p.Leu143Trp)

LegacyVariant: N/A

ClinVar: 13567

CAiD: CA123252

gnomAD: MAF in the Other population is 0.0001629 https://gnomad.broadinstitute.org/variant/17-45361875-T-G)

Case24: GT25, Female, India, age of onset is 5 years old

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Patients were recruited as they presented consecutively from 1996 to 1998 for evaluation of a mucocutaneous bleeding disorder

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: N/A

PlateletAggregation: All patients examined had defective ADP, epinephrine, and collagen induced aggregation and normal ristocetin induced aggregation

CasePreviousTesting: Mutations were detected by the method of single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing and confirmation by restriction fragment length polymorphism (RFLP) assays. The sequences of the primers employed and the reaction conditions for PCR amplification of the exons, adjacent intronic regions, and 50 and 30 untranslated regions of the aIIb and b3 gene

Gene: ITGB3

Variant: NM_000212.2:c.921C>A (p.Tyr307Ter)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA400025209

gnomAD: N/A

PMID: 8126100

Gene: ITGA2B and ITGB3

HGNC: 6138 and 6156

NoEvidence: This article does not mention GT probands or variants in ITGA2B and ITGB3. In this study, researchers have examined the talin phosphorylation and proteolytic cleavage as possible mechanisms by which talin's subcellular distribution could be regulated in platelets.

Case1: (M.S) Female, 19 years old, Caucasian

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: The mutation has been inherited from proband's parents who are heterozygous for the same mutation the proband has.

CasePresentingHPOs: HP:0000978 (Bruising)

CaseHPOFreeText: Intracerebral hematoma

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: See Fig 4 for flow cytometry analysis of protein expression

PlateletAggregation: Absence of platelet aggregation in response to all agonists tested i.e. ADP, collagen, platelet activating factor acether thrombin, and arachidonic acid; however, ristocetin induced platelet aggregation was normal

CasePreviousTesting: Sequenced patient's GPIIIa exons encompasing a highlight conserved reion of GPIIIa.

Gene: ITGB3

Variant: NM_000212.2:c.718C>T (p.Arg240Trp)

LegacyVariant: (p.Arg214Trp)

ClinVar: 13555

CAiD: CA123228

gnomAD: MAF in the East Asian population is 0.00005437 http://gnomad.broadinstitute.org/variant/17-45363729-C-T

Case7: (GT26) Gender, age and ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Inclusion criteria were patients with a history of mucocutaneous bleeding (Figure 1 shows a bar diagram of all the clinical manifestations in the patient cohort in this article)

CaseNotHPOs: N/A

ProteinExpression: Platelet receptor alphaIIbbeta3 protein was analyzed by flow cytometry followed by western blotting

Platelet Aggregation: Inclusion criteria for probands was absent or reduced platelet aggregation with agonists such as ADP, adrenaline, arachidonic acid, and collagen (no mention of ristocetin here-- this article does not tell us the specific aggregation for each proband in the paper)

CasePreviousTesting: For mutation analysis of ITGA2B and ITGB3 genes, DNA extraction from whole blood, PCR, heteroduplex PCR followed by CSGE and DNA sequencing were performed. All 30 exons of ITGA2B and 15 exons of ITGB3, as well as their promoters, were analyzed by CSGE following heteroduplex PCR.

Gene: ITGA2B

Variant: NM_000419.3:c.641T>C (p.Leu214Pro)

LegacyVariant: N/A

ClinVar: 2901

CAiD: CA115848

gnomAD N/A

Case2: GT18, Gender, age, ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: 1 brother is heterozygous for the g.951G>A promoter variant and does not have the other Leu343Pro mutation and one brother is normal and does not have the NM_000419.3:c.2915dup mutation

ParentalGenotype: Father: homozygous for the g.951G>A promoter variant and the Leu343Pro Mother: is heterozyous for both those mutations and mother does not have NM_000419.3:c.2915dup while the father is heterozygous for NM_000419.3:c.2915dup

CasePresentingHPOs: N/A

CaseHPOFreeText: Inclusion criteria were patients with a history of mucocutaneous bleeding (Figure 1 shows a bar diagram of all the clinical manifestations in the patient cohort in this article)

CaseNotHPOs: N/A

ProteinExpression: Platelet receptor alphaIIbbeta3 protein was analyzed by flow cytometry followed by western blotting

Platelet Aggregation: Inclusion criteria for probands was absent or reduced platelet aggregation with agonists such as ADP, adrenaline, arachidonic acid, and collagen (no mention of ristocetin here-- this article does not tell us the specific aggregation for each proband in the paper)

CasePreviousTesting: For mutation analysis of ITGA2B and ITGB3 genes, DNA extraction from whole blood, PCR, heteroduplex PCR followed by CSGE and DNA sequencing were performed. All 30 exons of ITGA2B and 15 exons of ITGB3, as well as their promoters, were analyzed by CSGE following heteroduplex PCR.

Gene: ITGA2B

Variant: NC_000017.11:g.44393555C>T (g.951G>A promoter variant) and NM_000419.3:c.1028T>C (p.Leu343Pro) and NM_000419.3:c.2915dup

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA913189168 (promoter variant) and CA399804619 (p.Leu343Pro) and CA8602531

gnomAD MAF in the East Asian population is 0.0001088 https://gnomad.broadinstitute.org/variant/17-42452054-C-CG for the NM_000419.3:c.2915dup variant

Case12: (GT39) Gender, age and ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Inclusion criteria were patients with a history of mucocutaneous bleeding (Figure 1 shows a bar diagram of all the clinical manifestations in the patient cohort in this article)

CaseNotHPOs: N/A

ProteinExpression: Platelet receptor alphaIIbbeta3 protein was analyzed by flow cytometry followed by western blotting

Platelet Aggregation: Inclusion criteria for probands was absent or reduced platelet aggregation with agonists such as ADP, adrenaline, arachidonic acid, and collagen (no mention of ristocetin here-- this article does not tell us the specific aggregation for each proband in the paper)

CasePreviousTesting: For mutation analysis of ITGA2B and ITGB3 genes, DNA extraction from whole blood, PCR, heteroduplex PCR followed by CSGE and DNA sequencing were performed. All 30 exons of ITGA2B and 15 exons of ITGB3, as well as their promoters, were analyzed by CSGE following heteroduplex PCR.

Gene: ITGB3

Variant: NM_000212.2:c.92G>A (p.Cys31Tyr)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA400031593

gnomAD N/A

Case1: Male. 2 years old, Gypsy

DiseaseAssertion: Glanzmann's thrombasthenia

FamilyInfo: See Fig 3 and 4 for family pedigree

ParentalGenotype: Parents are heterozygous for the variant of interest

CasePresentingHPOs: N/A

CaseHPOFreeText: Mucocutaneous hemorrhages and spontaneous bruising

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Protein expression results can be found in Table 1

PlateletAggregation: Platelets aggregated in response to agonists although they reacted positively to ristocetin (See Case History section for this sentence

CasePreviousTesting: Analyzed the amplification of products of all exons of GPII and their intronic flanking regions by single stranded conformational polymorphism analysis. All GPIIIa exons were also analyzed by SSCP and gave results identical to controls

Gene: ITGA2B

Variant: NM_000419.3:c.1063G>A (p.Glu355Lys)

LegacyVariant: N/A

ClinVar: 2899

CAiD: CA115844

gnomAD: MAF in the European (non-Finnish) population is 0.00001978 http://gnomad.broadinstitute.org/variant/17-42461008-C-T

Case2: GTP2, gender, age, or ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: N/A

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: N/A

PlateletAggregation: N/A

CasePreviousTesting: N/A

Gene: ITGA2B

Variant: NM_000419.3:c.818G>A (p.Gly273Asp)

LegacyVariant: N/A

ClinVar: 2894

CAiD: CA115837

gnomAD: N/A

Case3: GT-3, Turkish, gender or age not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Bleeding symptoms are severe and bleeding time is pathogenic (See Table 1) Abstract mentions patients' bleeding signs include epistaxis, mucocutaneous bleeding, haematomas, petechiae, GI bleeding and menorrhagia; however, not specified per proband.

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry was performed and expression is stated as pathogenic in Table 1

PlateletAggregation: Platelet aggregation was performed and is stated as pathogenic in Table 1; however, no specific agonists mentioned

CasePreviousTesting: All exons of ITGA2B and ITGB3 genes as well as surrounding intron regions were amplified by PCR using intronic primers as pre-viously described (12). The purified DNA fragments were then di-rectly sequenced.

Gene: ITGB3

Variant: NM_000419.3:c.310+3_310+6del

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA915940325

gnomAD: N/A

PMID: 18200593

Gene: ITGA2B and/or ITGB3

HGNC: 6138 and 6156

InsufficientEvidence: This article mentions a 27 year old proband with Glanzmann thrombasthenia; however, no variants in ITGA2B and/or ITGB3 were mentioned in this proband therefore we have insufficient evidence

Case1: (Patient L.W) Male, 55 years old, ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: Father passed away and mother has not yet been studied

CasePresentingHPOs: HP:0004406, HP:0006298, HP:0002239

(Recurrent epistaxis, prolonged bleeding following dental extraction, gastrointestinal bleeding,

CaseHPOFreeText: Pharyngeal bleeding

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

ProteinExpression: Surface expression levels found in Table 2

PlateletAggregation: Absent platelet aggregation tests in response to ADP but a normal initial response to ristocetin

CasePreviousTesting: Sequence analysis of GPIIb RNA was performed. Using 10 pairs of primers that hybridize to sequences specific for GPIIb cDNA, PCR products were generated by RT-PCR from total RNA of the patient and a normal control

Gene: ITGA2B

Variant: NM_000419.3:c.641T>C (p.Leu214Pro)

LegacyVariant: N/A

ClinVar: 2901

CAiD: CA115848

gnomAD: N/A

Case1: Female, 3 years old, Cypriot

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: Father and paternal uncle were also affected with same mutation (See Figure 3 for family pedigree)

ParentalGenotype: Father is homozygous for the mutation and the mother is heterozygous for the mutation (See Discussion)

CasePresentingHPOs: HP:0000225, HP:0000421, HP:0031364

(Gingival bleeding, epistaxis, ecchymosis)

CaseHPOFreeText: N/A

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry showed decreased surface expression of αIIb and β3 in patients' platelets

PlateletAggregation: Absent platelet aggregation in response to ADP, epineprhine, collagen but normal ristocetin induced platelet aggregation

CasePreviousTesting: SSCP analysis of the αIIb gene in the three patients revealed an altered pattern of migration of the DNA fragment containing exons 5 and 6 and their flanking intronic regions.

Gene: ITGA2B

Variant: NM_000419.3:c.605T>G (p.Phe202Cys)

LegacyVariant: (p.Phe171Cys) + 31= (p.Phe202Cys)

ClinVar: N/A

CAiD: CA399805605

gnomAD: N/A

Case1: GT-1, gender, age, ethnicity not specified

DiseaseAssertion: Glanzmann thrombasthenia

FamilyInfo: N/A

ParentalGenotype: N/A

CasePresentingHPOs: N/A

CaseHPOFreeText: Relatively mild bleeding syndrome and does not routinely require treatment except for iron supplementation

CaseNotHPOs: N/A

CaseNotHPOsFreeText: N/A

ProteinExpression: Flow cytometry showed that this patient lacked integrin αIIbβ3 expression

PlateletAggregation: Platelets failed to aggregate with physical agonists

CasePreviousTesting: N/A

Gene: ITGA2B

Variant: NM_000419.3:c.2933G>T (p.Gly978Val) and NM_000419.3:c.2994G>A (p.Trp998Ter)

LegacyVariant: N/A

ClinVar: N/A

CAiD: CA399790478 and CA399789759

gnomAD: N/A

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

Case1875(M.C.) Female, 22yo died, Irish

CasePresentingHPOs HP:0001263, HP:0001249, HP:0001250, HP:0002650, HP:0002625, HP:0005625, HP:0002857, HP:0000545, HP:0000276, HP:0000275, HP:0000218, HP:0000678, HP:0002878, HP:0001695, HP:0002240

(Developmental delay, Intellectual disability, Seizures, Scoliosis, Deep vein thrombosis, Osteoporosis of vertebrae, Genu valgum, Myopia, Long face, Narrow face, High palate, Crowded teeth, Respiratory failure, Cardiac arrest, Enlarged liver)

HPOsFreeText Diagnosed with homocystinuria after exam for developmental delay at 13mo. Patient had severe intellectual disability and seizures. At 14yo she had surgery for scoliosis, postsurgery she had a popliteal vein thrombosis. In addition to osteoporosis of the spine, she had genu valgum and myopia. The patient also had a long and narrow face, high-arched palate and crowded teeth. Respiratory failure and cardiac arrest at 22yo likely due to overuse of antiasthmatic medication.

(PMID:20329304): She was delivered normally at term, weighed 3kg, made uncomplicated neonatal progress, no history of convulsions,reached early milestones at expected times but did not sit up until 13mo. On report she was well fed and well developed, but with persistent shadow in right upper load on roentgenogram, enlarged liver of normal consistence, spleen was not palpable, negative neurological exam, mental age was 9mo. Aortogram was negative for vascular abnormalities in upper left chest. Skull and long bones were normal but osteoporosis of the spine was detected by roentgenogram.

CaseNotHPOs HP:0001083

(Lens dislocation)

NotHPOsFreeText no malar flush, lens displacement, or skeletal deformity on exam at 13mo.

CasePreviousTesting Plasma homocysteine was 45μM 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". Strong positive on cyanide nitroprusside, heavy homocysteine spot 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, one cut with NcoI/XmaI and another cut with Bpu1102/BstX, were sequenced.

Variant 1. NM_000071.2:c.715G>A (p.Glu239Lys),

1. NM_000071.2:c.233C>G (p.Pro78Arg);3. NM_000071.2:c.306G>C (p.Lys102Asn) - 2&3 in cis, compound heterozygous

ClinVar 1. n/a, 2. 188988;3. 188989

CAID 1. CA410600501

gnomAD 1. not in gnomAD, 2. MAF=0.0001631(Other), no homozygotes, 3. MAF=0.0001630(Other), no homozygotes

CaseAddInfo n/a

CasePMIDs 20329304

FamilyLabel Family 1

Family1PresentingHPOs HP:0000924, HP:0000939

(Skeletal anomalies, Osteoporosis)

HPOsFreeText Patients diagnosed with homocystinuria after finding abnormally high homocysteine on urine sample exam.

FamilyNotHPOs n/a

NotHPOsFreeText n/a

CountryOrigin Ireland

Ethnicity n/a

Race n/a

Family1PreviousTesting Tested pretreatment plasma homocysteine in each patient. Pyroxidine treatment dramatically reduced plasma homocysteine in all patients. 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 was detected on chromatogram of urinary amino acids.

FamilyMethod1 PCR

FamilyMethod2 Sanger

FamilyGenotypingMethod 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 were sequenced.

ProbandLabel 1875

Variant 1. NM_000071.2:c.715G>A (p.Glu239Lys),

1. NM_000071.2:c.233C>G (p.Pro78Arg);3. NM_000071.2:c.306G>C (p.Lys102Asn) - 2&3 in cis, compound heterozygous

ClinVar 1. n/a, 2. 188988;3. 188989

CAID 1. CA410600501

gnomAD 1. not in gnomAD, 2. MAF=0.0001631(Other), no homozygotes, 3. MAF=0.0001630(Other), no homozygotes

FamilyAddInfo Father unavailable for study. Mother was clinically/biochemically healthy with an unexplained macrocytosis. Mother had absent CBS activity, but no homocystinuria symptoms and was heterozygous for the Glu239Lys variant. The variant was found in inactive bacteria carrying the BB segment. Presence of the variant was reinforced by amplifying, subcloning, then sequencing her genomic DNA of the region corresponding to BB.

FamilyPMIDs 20329304

Case1 (Level 1)

Case1: male, 5y (at time of study, born 1998), Greece, Gypsy (Roma)

CasePresentingHPOs: HP:0002587, HP:0008872, HP:0001662, HP:0003235 (projectile vomiting (at birth), feeding difficulties in infancy, bradycardia (infancy), hypermethioninemia)

HPOsFreeText: 3350 g birthweight, elevated serum aminmotransferases, mildly elevated ALT and AST at 2y, mild ferritin elevation 173 ng/ml, serum triglycerides 155 mg/dl, total cholesterol 248 mg/dl, aspartate transaminase 31 to 154 U/L, alanine transaminase 34-180 U/L, urine methionine 508-627 umol/L, methionine excretion 60 umol/mmol, plasma methionine 1005-1049 umol/L, plasma AdoMet 1643-2222 nmol/L, plasma cystathionine 740-900 nmol/L, plasma phosphatidylcholine 2622-2641 umol/L, plasma betaine 218-382 umol/L, plasma dimethylglycine 11.7-12.1 umol/L

CaseNotHPOs: HP:0002240 (hepatomegaly)

NotHPOsFreeText: normal apgar score, normal blood ammonia, normal lactate, normal tyrosine, , normal plasma N-methylglycine 1.4-1.6 umol/L, normal plasma AdoHcy 32-43 nmol/L, normal plasma tHcy 11.1-12.1 umol/L, normal plasma tCys 206-217 umol/L, normal plasma choline 10.2-14.0 umol/L

CaseAddInfo: Adopted at 9 mo, no family info,

CasePMIDs: n/a

Case1 (Level 2)

CasePreviousTesting: metabolite assays: tHcy- fluorescent polarization method; urinary organic acids- gas chromatography mass spectrometry; plasma methionine, tHcy, cystathionine, tCys, dimethylglycine, sarcosine- capillary gas chromatography

CaseMethod1: PCR

CaseGenotypingMethod: genomic DNA isolated from blood samples (affected patient and 10 healthy American controls). 1.9 kb 5'-fragment of GNMT gene prepared using PCR primers and 1.8 kb 3' fragment prepared using other primers. PCR fragments were purified. Sequencing done by dideoxy sequencing method. Plasmid pGE3 containing wild-type GNMT cDNA was used to prepare mutated human GNMT cDNA. Fragments of all exons and most introns and 300 nucelotides at 5' and 3' flanking ends were sequence using the GNMT sequence specified by GI 8671581 which found a single nucleotide substitution in AAC codon of exon 3:3415A>G.

Variant: NM_018960.5(GNMT):c.422A>G (p.Asn141Ser)

ClinVar: 218931

CAID: CA347847

gnomAD: not in gnomAD

CaseAddInfo: No additional point mutation was found in the coding regions of patients GNMT gene, so the paper concludes that either he is homozygous or he has a deletion on the other allele that overlaps 3415A>G change and prevented PCR amplification in region of question.

Biallelic

Case#: Hazelwood case, 43 yrs (report), male

CaseHPOs: HP:0003235, HP:0012185 (hypermethioninemia, carpal tunnel)

CaseHPOsFreeText: Elevated urine methionine; Halitosis due to elevated breath dimethylsulphide; mild aortic insufficiency “attributable to rheumatic fever”, Gilbert disease (based on transient hyperbilirubinemia)

CaseNotHPOsFreeText: Normal brain MRI with no evidence of demyelination (age 43 years). Developmentally, physically, and mentally normal. Liver histology was normal (age 31 years). Normal neurologic exam. Normal electrolytes and blood chemistry, normal cholesterol, normal AST, ALT, and LDH, normal testosterone.

CasePreviousTesting: 25–30-fold elevation of plasma methionine (716-941 uM; controls 15-40, mean 26). Normal plasma tyrosine, normal homocysteine. Urine methionine 352-578 umol/day (controls 39-225, mean 99). Urine transamination metabolites 174-254 mmol/mol creatinine (controls ~1.8); breath dimethylsulfide 2.52-5.86 nmol/L (controls 0.13-0.65, mean 0.34); 7% normal activity of hepatic methionine adenosyltransferase in liver; hepatic activities of cystathionine-beta-synthase, gamma-cystathionase, and betaine homocysteine methyltransferase were normal.

CaseMethod1: SSCP

CaseMethod2: Sequencing

CaseGenotypingMethod: All 9 exons and intron-exon junctions of MAT1A were amplified by PCR from genomic DNA. This was followed by SSCP analysis and sequence analysis of PCR products with abnormal mobility.

Variant: c.539insTG (homozygous)

ClinVar: NA

CAID: CA210326818

gnomAD: The highest population minor allele frequency = 0.00001759 (non-Finnish); no homozygotes in any population.

CaseAddInfo: Both parents are heterozygous for the variant. Possible consanguinity - the patient’s father’s maternal grandfather and his mother’s paternal grandmother shared the same name and had lived their entire lives in an isolated West Virginia community.

CasePMIDs: 3812486, 3339126

Points: 1.5 points. This individual is homozygous for a frameshift variant in MAT1A. The highest population minor allele frequency = 0.00001759 (non-Finnish); no homozygotes in any population. The score is reduced because only some, but not all, causes of hypermethioninemia have been ruled out.

Case1: Newborn, male, Hmong (China)

CasePresentingHPOs:

HPOFreeText: Delivered at 33 weeks of gestation as a result of preterm labor. Postnatally required care for respiratory distress and pneumonia. NBS revealed elevated C5 acylcarnitine. Treated with L-carnitine supplementation and discharged at 4 weeks. Normal development at 12 months.

CaseNotHPOs:

CasePreviousTesting: Plasma C5-acylcarnitine 0.7, 3.4 umol/L (ref <0.6); urine 2-MBG 1.8, 10.2, 13.4, 28.7 ug/mg creatinine (ref 0.3-7.5); urine IVG 0.1, 0.3, 0.4, 1.1 ug/mg creatinine (ref 0.3-14.3).

CaseGenotypingMethod: PCR and sequencing of entire gene.

Variant: NM_001609.3:c.1165A>G (p.Met389Val) (homozygous)

CAID: CA5730948

gnomAD: East Asian 0.0005513

VariantEvidence: PCR of cDNA using primers in exon 9 and exon 11 showed that exon 10 was missing. Analysis of intron exon junctions flanking exon 10 did not reveal any changes.

CaseAddInfo: Diagnosis confirmed by Western blot of fibroblasts which showed no SBCAD protein. Treatment discontinued without consequence.

CasePMIDs:

PMID: 12837870

Gene: ACADSB

Disease: 2-methylbutyrylglycinuria

MonDO: MONDO:0012392

InheritancePattern: AR

Case2: Newborn, Hmong (China)

CasePresentingHPOs:

HPOFreeText: Born after normal pregnancy, postnatal course normal except for moderately elevated C5-acylcarnitine found during NBS. Treatment with L-carnitine supplementation. Treatment was discontinued without consequence.

CaseNotHPOs:

CasePreviousTesting: Plasma C5-acylcarnitine 3.0, 3.2 umol/L (ref <0.6); urine 2-MBG 2.9, 15.5 ug/mg creatinine (ref 0.3-7.5); urine IVG 0.1, 0.6 ug/mg creatinine (ref 0.3-14.3).

CaseGenotypingMethod: PCR and sequencing of entire gene.

Variant: NM_001609.3:c.1165A>G (p.Met389Val) (homozygous)

CAID: CA5730948

gnomAD: East Asian 0.0005513

VariantEvidence: PCR of cDNA using primers in exon 9 and exon 11 showed that exon 10 was missing. Analysis of intron exon junctions flanking exon 10 did not reveal any changes.

CaseAddInfo: Diagnosis confirmed by Western blot of fibroblasts which showed no SBCAD protein.

CasePMIDs:

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

Case19: 1 mo (at biopsy), White

Case 19 (Level 2)

CasePreviousTesting: same as group. found 1.2 nmol/mg protein/18 h propionate incorporation, 1.0 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.655A>T (p.Asn219Tyr)

ClinVar: 1886

CAID: CA249731

gnomAD: 0.0001318 (European non-Finnish)

Variant2: NM_000255.3:c.1332+1delG

ClinVar: 554656

CAID: CA3846920

gnomAD: 0.000008828 (European non-Finnish)

CaseAddInfo: mut0 type, heterozygous mutations, unknown cis or trans

Case18: 2 mo (at biopsy), French Canadian

Case 18 (Level 2)

CasePreviousTesting: same as group. found 0.7 nmol/mg protein/18 h propionate incorporation, 0.7 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.655A>T (p.Asn219Tyr)

ClinVar: 1886

CAID: CA249731

gnomAD: 0.0001318 (European non-Finnish)

__

Variant2: NM_000255.3:c.1240delG (p.Glu414LysfsTer17)

ClinVar: n/a

CAID: CA3846936

gnomAD: 0.000008796 (European Non-Finnish)

CaseAddInfo: mut0 type. Variant 2 is novel. heterozygous mutations, unknown cis or trans.

Case17: 6 y (at biopsy), Black

Case 17 (Level 2)

CasePreviousTesting: same as group. found 0.6 nmol/mg protein/18 h propionate incorporation, 0.6 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.631_633delGAG (p.Glu211del)

ClinVar: n/a

CAID: CA913187321

gnomAD: n/a

Variant2: NM_000255.3:c.682C>T (p.Arg228Ter)

ClinVar: 203855

CAID: CA312778

gnomAD: 0.0002615 (Latino)

CaseAddInfo: mut0 type, heterozygous mutations. Variant 1 is novel. MUT mutations are in trans- demonstrated by RT-PCR of mRNA. Additional variant found in MCEE gene (NM_032601.3:c.428G>A, ClinVar: 336936, CAID: CA1702575, gnomAD: 0.08102 (Latino)) MCEE variant is unlikely to be associated with patient's MMA.

Case16: 5 days (at biopsy), Lao/Thai/Mexican/Portugese

Case 16 (Level 2)

CasePreviousTesting: same as group. found 0.6 nmol/mg protein/18 h propionate incorporation, 0.5 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.581C>T (p.Pro194Leu)

ClinVar: n/a

CAID: CA364404418

gnomAD: n/a

Variant2: NM_000255.3:c.682C>T (p.Arg228Ter)

ClinVar: 203855

CAID: CA312778

gnomAD: 0.0002615 (Latino)

CaseAddInfo: mut0 type, heterozygous mutation, unknown cis or trans

Case15: 3 wks (at biopsy), white

Case (Level 2)

CasePreviousTesting: same as group. found 0.5 nmol/mg protein/18 h propionate incorporation, 0.7 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.572C>A (p.Ala191Glu)

ClinVar: 203854

CAID: CA312777

gnomAD: 0.00006976 (European non-FInnish)

Variant2: NM_000255.3:c.754-2A>G

ClinVar: 552691

CAID: CA364402853

gnomAD: n/a

CaseAddInfo: variant 2 is a novel, splice site mutation. Heterozygous mutations, unknown cis or trans.

Case13: 21 y (at biopsy), English/Irish

Case (Level 2)

CasePreviousTesting: same as group. found 1.5 nmol/mg protein/18 h propionate incorporation, 1.5 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.572C>A (p.Ala191Glu)

ClinVar: 203854

CAID: CA312777

gnomAD: 0.00006976 (European non-FInnish)

Variant2: NM_000255.3:c.682C>T (p.Arg228Ter)

ClinVar: 203855

CAID: CA312778

gnomAD: 0.0002615 (Latino)

CaseAddInfo: mut0 type, heterozygous mutation, unknown cis or trans

Case12: 6y (at biopsy), no ethnicity recorded

Case (Level 2)

CasePreviousTesting: same as group. found 0.5 nmol/mg protein/18 h propionate incorporation, 1.3 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.556A>G (p.Met186Val)

ClinVar: 551460

CAID: CA3847093

gnomAD: 0.00001760 (European Non-Finnish)

Variant2: NM_000255.3:c.1106G>A (p.Arg369His)

ClinVar: 203846

CAID: CA312764

gnomAD: 0.0001504 (East Asian)

CaseAddInfo: mut- type, heterozygous mutations, unknown cis or trans

Case11: 6y (at biopsy), White/Hispanic

Case (Level 2)

CasePreviousTesting: same as group. found 2.6 nmol/mg protein/18 h propionate incorporation, 7.0 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.566A>T (p.Asn189Ille)

ClinVar: 222915

CAID: CA3847091

gnomAD: 0.000008804 (European Non-Finnish)

Variant2: NM_000255.3:c.655A>T (p.Asn219Tyr)

ClinVar: 1886

CAID: CA249731

gnomAD: 0.0001318 (European non-Finnish)

CaseAddInfo: Variant 1 is novel, unknown cis or trans. mut- type.

Case14: 3 mo (at biopsy), White/Australian

Case (Level 2)

CasePreviousTesting: same as group. found 0.5 nmol/mg protein/18 h propionate incorporation, 0.5 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.572C>A (p.Ala191Glu)

ClinVar: 203854

CAID: CA312777

gnomAD: 0.00006976 (European non-FInnish)

Variant2: NM_000255.3:c.682C>T (p.Arg228Ter)

ClinVar: 203855

CAID: CA312778

gnomAD: 0.0002615 (Latino)

CaseAddInfo: mut0 type, heterozygous mutation, unknown cis or trans

Case10: 6 days (at biopsy), Northern European/Filipino

Case (Level 2)

CasePreviousTesting: same as group. found 0.8 nmol/mg protein/18 h propionate incorporation, 1.2 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.322C>T (p.Arg108Cys)

ClinVar: 1887

CAID: CA249732

gnomAD: 0.0008393 (Latino)

Variant2: NM_000255.3:c.682C>T (p.Arg228Ter)

ClinVar: 203855

CAID: CA312778

gnomAD: 0.0002615 (Latino)

CaseAddInfo: mut0 type, heterozygous mutations, unknown cis or trans.

Case9: 46 days (at biopsy), Hispanic

Case (Level 2)

CasePreviousTesting: same as group. found 0.8 nmol/mg protein/18 h propionate incorporation, 1.2 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.322C>T (p.Arg108Cys)

ClinVar: 1887

CAID: CA249732

gnomAD: 0.0008393 (Latino)

Variant2: NM_000255.3:c.654A>C (p.Gln218His)

ClinVar: 551243

CAID: CA364404273

gnomAD: 0.000008809 (European non-Finnish)

CaseAddInfo: mut0 class, heterozygous mutations, unknown cis or trans.

Case8: 12 days (at biopsy), White/Mexican

Case (Level 2)

CasePreviousTesting: same as group. found 0.8 nmol/mg protein/18 h propionate incorporation, 1.0 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.322C>T (p.Arg108Cys)

ClinVar: 1887

CAID: CA249732

gnomAD: 0.0008393 (Latino)

Variant2: NM_000255.3:c.654A>C (p.Gln218His)

ClinVar: 551243

CAID: CA364404273

gnomAD: 0.000008809 (European non-Finnish)

CaseAddInfo: mut0 class, heterozygous mutations, unknown cis or trans.

Case7: 10 days (at biopsy), White

Case (Level 2)

CasePreviousTesting: same as group. found 0.4 nmol/mg protein/18 h propionate incorporation, 0.5 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.146_147ins279

ClinVar: n/a

CAID: n/a- could not register

gnomAD: n/a

Variant2: NM_000255.3:c.372_374dupGA (p.Lys124_Asp125insGlu)

ClinVar: 203861

CAID: CA312788

gnomAD: 0.000008799

CaseAddInfo: mut0 class. heterozygous mutations. identified to be in trans through cDNA analysis. variant 1 is novel and consists of a 279 base pair insertion.

Case6 2 mo (at biopsy), White

Case (Level 2)

CasePreviousTesting: same as group. found 1.1 nmol/mg protein/18 h propionate incorporation, 3.0 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.129G>A (p.Trp43Ter)

ClinVar: 222908

CAID: CA10575886

gnomAD: n/a

Variant2: NM_000255.3:c.299A>G (p.Tyr100Cys)

ClinVar: 218991

CAID: CA347866

gnomAD: 0.000008793 (European non-Finnish)

CaseAddInfo: mut- class. heterozygous mutations. unknown cis or trans. variant 1 is novel.

Case5: 1y 9 mo (at biopsy), Korean

Case (Level 2)

CasePreviousTesting: same as group. found 0.7 nmol/mg protein/18 h propionate incorporation, 0.7 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.91C>T (p.Arg31Ter)

ClinVar: 92688

CAID: CA249743

gnomAD: 0.00008013 (African)

Variant2: NM_000255.3:c.349G>T (p.Glu117Ter)

ClinVar: 1882

CAID: CA115264

gnomAD: n/a

CaseAddInfo: mut0 class. heterozygous mutations. unknown cis or trans.

Case4: 2 y (at biopsy), Black

Case (Level 2)

CasePreviousTesting: same as group. found 2.1 nmol/mg protein/18 h propionate incorporation, 3.1 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.91C>T (p.Arg31Ter)

ClinVar: 92688

CAID: CA249743

gnomAD: 0.00008013 (African)

Variant2: NM_000255.3:c.323G>A (p.Arg108His)

ClinVar: 100707

CAID: CA235522

gnomAD: 0.00006152 (African)

CaseAddInfo: mut- class. heterozygous mutations. unknown cis or trans. Third variant in TCN2 gene (NM_000355.3:c.89T>G p.Leu30Arg, CAID: CA10184491, ClinVar: 529781, gnomAD: 0.01329 (African))

Case3: 19y (at biopsy), Irish/Scottish/Polish

Case (Level 2)

CasePreviousTesting: same as group. found 1.2 nmol/mg protein/18 h propionate incorporation, 1.2 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.29dupT (p.Leu10PhefsTer39)

ClinVar: 553287

CAID: CA567155988

gnomAD: 0.00003269 (South Asian)

Variant2: NM_000255.3:c.1658delT (p.Val553GlyfsTer17)

ClinVar: n/a

CAID: CA450397348

gnomAD: 0.000008808 (European non-Finnish)

CaseAddInfo: mut0 class. heterozygous mutations. variant 1 is novel. unknown cis or trans.

Case2: 1 mo (at biopsy), Palestinian

Case (Level 2)

CasePreviousTesting: same as group. found 1.5 nmol/mg protein/18 h propionate incorporation, 1.5 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.2T>C (p.Met1Thr)

ClinVar: 222903

CAID: CA10575890

gnomAD: n/a

Variant2: NM_000255.3:c.1630_1631delGGinsTA (p.Gly544Ter)

ClinVar: 203860

CAID: CA312786

gnomAD: 0.0001088 (East Asian)

CaseAddInfo: mut0 class. heterozygous mutations- unknown cis or trans. 3rd mutation in CD320 gene (NM_016579.3:c.658G>A p.Gly220Arg, ClinVar: 136684, CAID: CA289989, gnomAD: 0.3086 (African) )

Case1: 13 mo (at biopsy), no recorded ethnicity

Case (Level 2)

CasePreviousTesting: same as group. found 2.4 nmol/mg protein/18 h propionate incorporation, 4.7 nmol/mg/protein/18 h propionate incorporation with hydroxocobalamin supplementation

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.1A>G (p.M1?)

ClinVar: n/a

CAID: CA364406084

gnomAD: n/a

Variant2: NM_000255.3:c.2150G>T (p.Gly717Val)

ClinVar: 1881

CAID: CA249729

gnomAD: 0.0006810 (African)

CaseAddInfo: mut- class. heterozygous mutations. unknown cis or trans

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

Case#: 64 year old (report), female

CaseHPOsFreeText: At age 52 years, sudden difficulty with swallowing and speech, ophthalmoparesis with a vertical and horizontal eye movement disorder, dysesthesia of the hands with a quality of “pins and needles” and a glove-like distribution, as well as generalized areflexia. Diagnosed with Miller Fisher syndrome. Treatment with intravenous immunoglobulins led to remission of symptoms. Abdominal pain since childhood. At 63 years of age, she had generalized epileptic seizures with prolonged postictal confusion, persisting diarrhea, abdominal pain, renal failure, repeat episodes of lactic acidosis. Ventilated for >8 weeks due to massive metabolic acidosis and respiratory failure during seizure; tracheotomy, PEG. VitB6 supplement. Rehabilitation, and recovered after several months.

CaseNotHPOsFreeText: Muscle strength was normal. Abdominal computer tomography (CT), magnetic resonance imaging (MRI) and gastroscopy were normal. No signs of infection in CSF. CT and MRI of the brain as well as abdominal- and thorax-CT were normal initially then fat embolies in brain, pulmonary embolism, vertebral fractures. EMG was normal. No signs of malnutrition or liver disease (which can cause hyperprolinemia).

CasePreviousTesting: No evidence of porphyria on “laboratory tests”, or diseases of copper metabolism, lead poisoning, or adrenoleukodystrophy. Muscle biopsy showed nonspecific atrophy, but no evidence of mitochondrial disease. Serum proline 3085 μmol/l (reference 90-342 μmol/l), urine proline 46,531 μmol/g Crea (reference < 100 μmol/g Crea), CSF proline 104 mg/dl (reference < 6 mg/dl), urine Hydroxyproline 1395 μmol/g Crea (reference <100). Vitamin-B6 was decreased at 3.3 μg/l (reference > 4.9 μg/l). “targeted genetic analyses by Sanger sequencing revealed no pathogenic variant within the PRODH-gene (hyperprolinemia type I)”

CaseMethod1: PCR

CaseMethod2: Sequencing

CaseGenotypingMethod: “targeted genetic analyses by Sanger sequencing” for ALDH4A1 (no further details).

Variant: Compound heterozygous, confirmed in trans (the patient’s son only has one of the variants) 1) NM_003748.3:c.62+1G>A, 2) NM_003748.3:c.349G>C (p.Asp117His)

ClinVar: 1) N/A; 2) N/A

CAID: 1) CA338769667; 2) CA338760898

gnomAD: 1) Not in gnomAD; 2) Not in gnomAD

VariantEvidence: No functional evidence for the missense change. Score: This patient is an adult with hyperprolinemia who is compound heterozygous for two variants in ALDH4A1, c.62+1G>A and c.349G>C (p.Asp117His). Neither variant is in gnomAD. Variants in PRODH have been ruled out.

Case# Case 3, M, Chinese, 3day onset

CasePresentingHPOs HP:0008344, HP:0001954, HP:0005972, HP:0011968, HP:0008947

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.659del(p.D229fs)

CAID CA658820635

CaseAddInfo Mother was not tested but WES was used in the patient and father

Case39: Female, Chinese, 3.4y (end of study)

HPOsFreeText: 1y onset, poor neurological outcome

NotHPOsFreeText: not responsive to vitamin B12

Case39 (Level 2)

CasePreviousTesting: same as group

CaseMethod1: same as group

CaseMethod2: same as group

CaseGenotypingMethod: same as group

Variant1: NM_000255.3:c.1741C>T (p.Arg581Ter)

ClinVar: 554675

CAID: CA364395893

gnomAD: n/a

CaseAddInfo: one variant, not homozygous.