Case: IP #23 in type 2N VWD sub-cohort

DiseaseAssertion: type 2N/1 VWD

FamilyInfo:

ParentalGenotype(s):

CasePresentingHPOs: HP:0032370 - blood group A

CaseHPOFreeRext: VWF:Ag [IU/dL] = 40; VWF:Ac [IU/dL] = 54; FVIII:C [IU/dL] = 50

CaseNOTHPOs:

CaseNOTHPOFreeText:

CasePreviousTesting:

GenotypingMethod: Sequence variations in the VWF were identified through direct sequencing, while the detection of large rearrangements utilized the multiplex ligation-dependent probe amplification (MLPA) technique or assessed copy number variations (CNVs). Sanger sequencing methodology was applied for VWF analysis in patients investigated prior to 2016 (approximately 14% of IPs), whereas next-generation sequencing (NGS) was employed for the analysis of VWF in patients inspected from 2017 onwards (about 86% of IPs). In both strategies, the entire VWF, encompassing all 52 exons, intron/exon boundaries, and promoter regions, was analyzed.

PreviouslyPublished:

Gene: VWF

Variant: c.2561G>A p.Arg854Gln

HGVS:

ClinVarID: 296

CAID:

gnomAD: 0.004956 (8000 in 1614214) 26 homozygotes https://gnomad.broadinstitute.org/variant/12-6034812-C-T?dataset=gnomad_r4

MultipleGeneVariants:

Gene: VWF

Variant: c.4751A>G p.Tyr1584Cys

ClinVarID: 310

gnomAD: 0.003442 (5555 in 1613678) 15 homozygotes https://gnomad.broadinstitute.org/variant/12-6018667-T-C?dataset=gnomad_r4

Case UZ098: 41 year old female (row 99 in supplemental table)

DiseaseAssertion: type 1 VWD

FamilyInfo:

ParentalGenotype(s):

CasePresentingHPOs: HP:0032370

CaseHPOFreeRext: vWF:Ag (%) = 30.1; vWF:GPIbR (%) = 25.8; Act/Ag ratio = 0.86; FVIII:C (%) = 61.5; Platelets (x10^9/L) = 377

CaseNOTHPOs:

CaseNOTHPOFreeText:

CasePreviousTesting: The Thrombosis-Hemostasis gene panel test used whole exome sequencing and included 99 diagnostic-grade genes (table 1 in PMID:36696193)

GenotypingMethod: WES using the coagulation subpanel of the UZ Leuven Thrombosis-Haemostasis gene panel test. WES was performed on the sequencing platform NovaSeq 6000 (Illumina), and Alissa Interpret software v5.2-5.4 (Agilent) was used for variant interpretation. Assessment of copy number variation was validated within the bioinformatics pipeline. The American College of Medical Genetics and Genomics and the Association of Molecular Pathology guidelines were used to classify variants as pathogenic (P), likely pathogenic (LP), a variant of uncertain significance (VUS), or (likely) benign (LB, B) variants

PreviouslyPublished:

Gene: VWF

Variant: c.4751A>G p.Tyr1584Cys

HGVS:

ClinVarID: 310

CAID:

gnomAD: 0.003442 (5555 in 1613678) 15 homozygotes https://gnomad.broadinstitute.org/variant/12-6018667-T-C?dataset=gnomad_r4

Case 17: 47 year old female, study ID UZ099 in supplemental table

DiseaseAssertion: acquired VWD presequencing, acquired VWD + type 1 VWD variant postsequencing

FamilyInfo:

ParentalGenotype(s):

CasePresentingHPOs: HP:0032370

CaseHPOFreeRext: vWF:Ag (%) = 2.6; vWF:GPIbR (%) = 2.2; Act/Ag ratio = 0.85; FVIII:C (%) = 7; Platelets (x10^9/L) = 325

CaseNOTHPOs:

CaseNOTHPOFreeText:

CasePreviousTesting: The Thrombosis-Hemostasis gene panel test used whole exome sequencing and included 99 diagnostic-grade genes (table 1 in PMID:36696193)

GenotypingMethod: WES using the coagulation subpanel of the UZ Leuven Thrombosis-Haemostasis gene panel test. WES was performed on the sequencing platform NovaSeq 6000 (Illumina), and Alissa Interpret software v5.2-5.4 (Agilent) was used for variant interpretation. Assessment of copy number variation was validated within the bioinformatics pipeline. The American College of Medical Genetics and Genomics and the Association of Molecular Pathology guidelines were used to classify variants as pathogenic (P), likely pathogenic (LP), a variant of uncertain significance (VUS), or (likely) benign (LB, B) variants

PreviouslyPublished:

Gene: VWF

Variant: c.4751A>G p.Tyr1584Cys

HGVS:

ClinVarID: 310

CAID:

gnomAD: 0.003442 (5555 in 1613678) 15 homozygotes https://gnomad.broadinstitute.org/variant/12-6018667-T-C?dataset=gnomad_r4

MultipleGeneVariants:

FGG variant

Variant: c.323C>G p.Ala108Gly

ClinVarID: 547969

gnomAD: 0.003031 (4885 in 1611606) 12 homozygotes https://gnomad.broadinstitute.org/variant/4-154611883-G-C?dataset=gnomad_r4

Case: Patient 51, Family Y (Table 1.), F/33, Afro-Caribbean

DiseaseAssertion: VWD1

FamilyInfo: NA

ParentalGenotype(s): NA

CasePresentingHPOs:

CaseHPOFreeRext: BAT = 5, FVIII:C (IU/dL) = NA, VWF:Ag (IU/dL) = 30, VWF:Act (IU/dL) = 31

CaseNOTHPOs:

CaseNOTHPOFreeText: Blood group O+

CasePreviousTesting:

GenotypingMethod: All samples were analyzed by captured-based high throughput sequencing which was performed with a custom-design of the VWF gene provided by Twist BioScience (San Francisco, USA). It comprised all 52 exons, intronic flanking regions, promoter and 3’UTR region of the VWF gene. Libraries were prepared by using the Twist NGS target enrichment solution (Twist BioScience) according to the manufacturer protocol. Samples were analyzed on a NovaSeq 6000 sequencer (Illumina) with a median read depth of 2500X by using the 300-cycle S1 NovaSeq 6000seq reagent kit with 150-bp paired-end reads. The raw data were analyzed with Mutect21 and Vardict2 tools. The inhouse NGSreport software (CHU Lille) was used for data visualization, sequences check and conservation of variants with high quality metrics. In some cases, patients genomic DNA was screened for sequence variations (large deletions, insertions and duplications) by Multiplex Ligation-dependent Probe Amplification (MCR Holland).

PreviouslyPublished: NA

Gene: VWF

Variant: c.4751A > G, p.(Tyr1584Cys)

HGVS: NM_000552.5:c.4751A>G (p.Tyr1584Cys)

ClinVarID: 310

CAID: CA114162

gnomAD: MAF of 0.004105 (483/1179782 alleles) in European/Non-Finnish population https://gnomad.broadinstitute.org/variant/chr12-6018667-T-C?dataset=gnomad_r4

Case: Proband (II-1), male, 56yo, Italian

DiseaseAssertion: VWD1 and Hemophilia A

FamilyInfo: Pedigree in Fig. 1 - Father had VWD1 and Mother had Hemophilia A - parents have normal or higher VWF levels, and normal FVIII/VWF:Ag ratios, as well as a normal bleeding score; 2 daughters carry F8 variant

ParentalGenotype(s): Paternal = NM_000552.4(VWF):c.4751A>G (p.Tyr1584Cys); Maternal = NM_000132.3(F8):c.2167G>A (p.Ala723Thr)

CasePresentingHPOs: HP:0012147, HP:0030129, HP:0030130, HP:0001892, HP:0000421, HP:0000978, HP:0004846, HP:0000225, HP:0005261, HP:0040242

(Reduced quantity of VWF, Impaired ristocetin cofactor assay activity, Impaired von Willebrand factor collagen binding activity, abnormal bleeding, epistaxis, Bruising susceptibility, Prolonged bleeding after surgery, bleeding gums, joint hemorrhage, muscle hemorrhage)

CaseHPOFreeText: blood group = 0, aPTT = 0.56sec, FVIII = 9.8 IU/dL, VWF:Ag = 43.2 IU/dL, VWF:CB = 35.6 IU/dL, VWF:Rco = 42.1 IU/dL, VWF:FVIIIB = 41.3 IU/dL, Platelet VWF:Ag = 58.2 IU/dL, bleeding score = 27

CaseNOTHPOs: HP:0011871

(Impaired RIPA)

CaseNOTHPOFreeText:

CasePreviousTesting:

GenotypingMethod: PCR and direct sequencing of VWF and FVIII

PreviouslyPublished: NA

Gene: VWF

Variant: c.4751A>G (p.Tyr1584Cys)

HGVS: NM_000552.4(VWF):c.4751A>G (p.Tyr1584Cys)

ClinVarID: 310; https://www.ncbi.nlm.nih.gov/clinvar/variation/310/

CAID: NA

gnomAD: MAF = 0.004024 (519/128972 alleles, 4 homozygotes) in European (non-Finnish) population

MultipleGeneVariants:

Gene: F8

Variant: c.2167G>A (p.Ala723Thr)

HGVS: NM_000132.3(F8):c.2167G>A (p.Ala723Thr)

ClinVarID: 10246; https://www.ncbi.nlm.nih.gov/clinvar/variation/10246/

CAid: NA

gnomAD: MAF = 0.00005257 (1/19022 alleles, 1 hemizygote) in South Asian population

Case#: 01028, Age 8, Male, Blood type O+( Table S3)

Disease Assertion: VWD 1

FamilyInfo: Not listed

Parental Genotype: Not listed

CasePresentingHPOs: N/A

CaseHPOFreeText: Bleeding score 7, VWF:Ag: 21, VWF:RCo: 33, FVIII:C: 34, VWF:CB: 30

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

Protein Expression: N/A

CasePreviousTesting: 48,48 Access-Array amplification. NGS Molecular analysis was performed. Sanger sequencing was used to validate selected mutations.

Gene: VWF

Variant: NM_000552.4:c.4751A>G (p.Tyr1584Cys) and NM_000552.3:c.5312-19A>C

ClinVar: 310 and 806794

ClinVar1

ClinVar2

CAID: CA114162 and CA6402297 CAID1 CAID2

gnomAD: MAF of 0.004024 (519/128972 alleles) in the European (non-Finnish) population (CA114162). MAF of 0.002507 (26/10370 alleles) in the Ashkenazi Jewish population.

gnomAD1

gnomAD2