Disease: Von Willebrand Disease (VWD)

Patient(s): Found in 2 families

Variant: VWF NM_000552.5: c.2311A>G, p.(M771V Homozygous variant in exon 18 (VWF D' domain; 8 residues down from proteolytic VWFpp furin cleavage site)

Family: In family 1 there are 4 homozygous patients (2 male and 2 female), and one heterozygous patient (1 female). The affected females are denoted as person 1 and person 4 and the affected males are person 2 and person 3. There are three WT family members (1 female and 2 male), grandparents of these members are of unknown genotype including a daughter of an affected female and a WT male. Note here that in the family a p.R2663P variant has co-segregated with the above-mentioned variant but is not suspected to be the pathogenic driver of resulting bleeding tendency.

In family 2 the parents of the homozygous affected male are of unknown genotype. The affected male is denoted as person 7.

Phenotypes: Person 1- nose bleed, skin bleed, GI bleeding, oral cavity bleeds, Menorrhagia, muscle bleeding, and joint bleeding. Receives on-demand treatment for bleeding.

Person 2-Nose bleed, skin bleed, bleeding from small wounds, oral cavity bleeds, bleeding after tooth extraction, joint bleeding. Received prophylactic treatment, reduced to on-demand treatment after a few years.

Person 3-Nose bleed, skin bleed, oral cavity bleeds, bleeding after tooth extraction, muscle bleeding. Receives on-demand treatment for bleeding phenotype.

Person 4- Nose bleed, bleeding from small wounds, oral cavity bleed, bleeding after tooth extraction, joint bleeding. Received prophylactic treatment that was increased after her menarche.

Person 7- Nose bleed, oral cavity bleeds, bleeding after surgery or trauma, joint bleeding. Previously on prophylaxis, now managing bleeding with on-demand treatment.

Note that both the p.R2663P co-segregated variant and p.M771V variant are reported in NCBI dbSNP database but functional effect not yet established.

NGS confirmed the genotype of all study participants.

Disease: Von-willebrand Disorder Type 3

Patient: 26 yo, female

Variant: VWF NM_000552.5 c:997+118 T>G g.(6073501 A>C), homozygous, intronic

Phenotypes: No detectable VWF in plasma, early onset bleeding complications, epistaxis, easy bruising, bleeding following injury, menorrhagia, iron-deficient anemia

Note: underwent prophylaxis replacement therapy, on-demand antihemorrhagic treatments, oral contraceptives, and replacement therapy.

Family: not mentioned

Predictions:

VEP SpliceAI tool predicted variant likely deleterious (delta score 0.95)

Used Polyphen-2 and SIFT which determined pathogenic likelihood.

Neural Network Splicing, Alternative Splice Site Predictor, plug-in MaxEnt(For 5' donor site) of Human Splicing Finder all concur this variant can create a new donor splice site in intron 8. Contains premature stop codon and susceptible to NMD.

Functional work:

qRT-PCR performed to identify levels of VWF in IP-derived endothelial cells.

histochemical immunostaining for IP-derived endothelial cells confirm no VWF production, only a residual amount present. Suggests leaky mutation.

performed RNA sequencing to assess co-regulated gene networks

Disease: Von-willebrand Disorder Type 2M

Patient1: 13 YO male

Patient2: 16 YO female

Note they are siblings

Variant: VWF NM_000552.5: c.5192C>T p.(Ser1731Leu), heterozygous variant, in A3 domain in exon 30

Phenotype patient 1: several bleedings after tosillectomy, recurrent epistaxis, decreased VWF:CB ratio, decreased VWF:CB/VWF:Ag ratio

Phenotype patient 2: menorrhagia, intermittent gum bleeding, easy bruising, decreased VWF:CB ratio, decreased VWF:CB/VWF:Ag ratio

For both patients: VWF:CB ratio corresponds to collagen type 1, multimer analysis was normal, VWF:Ag, VWF:Ac, and factor VIII activity was normal.

Family: Not listed

Present in dbSNP (rs764077750)

Present in gnomAD, rare MAF (ALL: 0.0012%)

Predictions:

SIFT- predicted deleterious (Score 0.01, median 3.34)

MutationTaster- Predicted disease causing (probability score 1)

PolyPhen2- Predicted probably damaging (Score 0.983)

CADD score- 26.5

Authors mention similar AA substitutions at the p.1731 position which leads to reduced binding of VWF to collagen in other patients.

Authors also cite a functional human cell experiment in COS7 cell line which resulted in functional VWF defect.

Authors conclude this novel variant as likely pathogenic.

Disorder studied: Type 1 von Willebrand disease (T1-VWD).

Type of study: Translational

Model organism: Mouse (inbred strains) Obtained from Jackson Laboratory

Analyses:

VWF plasma protein quantitation (ELISA)

Hertiability calculations

PCR genotyping

QTL analysis

Allele-specific primer extension analysis

Results:

Identified new modifier of VWF known as (Mvwf5). Also found two loci unliked to Vwf known as (Mvwf6-7)

Mice with this variant displayed statistically significant decrease in VWF levels, recapitulating the decreasing patterns displayed in humans.

However, another strain of inbred mice with a different mutation did not show an age-dependent decrease in VWF. Suggests strain-specific differences in regulation of VWF levels over time.

Mvwf5 is a cis-regulatory variant altering Vwf mRNA expression.

This is a natural variant of the Vwf allele among inbred strains of mice. Found this variant causes elevation in steady-state levels of Vwf mRNA.

Authors state findings show equivalent of of type 1 VWD is remarkably common in mice and humans. ALso state the Mvwf1 analysis in wild mouse populations suggest this locus is under selective pressure.

Of the 5 potential modifier loci identified, 3 display conservation of synteny with potential human modifier loci.

Disease: Platelet-type Von-willebrand Disorder (PT-VWD)

Patient: 17 yo, male, adopted

Variant: GP1BA NM_000173.7: c:580C>T p.(P.Leu194Phe), Heterozygous, gain-of-function

Phenotypes: moderate bleeding phenotype, ISTH-BAT bleeding score of 3, recurrent epistaxis, easy bruising, mild thrombocytopenia

Family: Adopted, no other family history mentioned, segregation studies not performed.

Genetic analysis performed: found variant in GP1BA, results obtained by sanger sequencing.

Variant present in gnomAD(rs368111193): low allele frequency, contradictory classifications

Variant is not present in ClinVar, LOVD, or HGMD databases

According to this paper, ACMG guidelines classified this variant as a VUS.

This paper entered it into Clinvar (var ID 1693270)

Disease: N/A, variant present in F12 gene

Patient: 36 yo, Female, Saudi descent

Variant:F12 NC_000005.9:g.176,830,269 G>A; p.Gly506Asp Homozygous mutation, exon 12 Located in peptidase S1 domain of F12

Family:

Consanguineous family history (parents first-degree cousins)

No family history of bleeding or thrombosis

Phenotypes:

Significantly high activated partial thromboplastin time

No history of bleeding during deliveries or tooth extractions

No history of thrombosis or skin manifestations

On no medications, physical examination unremarkable

Factor assays and VWF tests within normal ranges except Factor XII (Severely deficient)

variant is proposed to be deleterious but there is insufficient evidence to support this claim.

Disease: mild haemophilia A, influencing VWF levels

Patient: 20 yo, Female

Variant1: F8 NM_000132.3: c.1127T>G: p. Val376Gly (Exon 8, current clinvar interpretation not available)

Variant 2: F8 NM_000132.3: c.3780C>G: p. Asp1260Glu (Exon 14, current ClinVar interpretation is benign)

Variant 3: VWF NM_000552.5: c.1415A>G:p.His484Arg (Exon 13, current ClinVar interpretation is Benign/likely Benign)

Variant 4: VWF NM_000552.5: c.2365A>G:p.Thr789Ala (Exon 18, current ClinVar interpretation is Benign/ likely Benign)

Variant 5: VWF NM_000552.5: c.2771G>A:p.Arg924Gln (Exon 21, current ClinVar interpretation is conflicting interpretations of pathogenicity (VUS-3)(Benign-4)(Likely benign-1))

Variant 6: VWF NM_000552.5: c.4141A>G:p.Thr1381Ala (Exon 28, current ClinVar interpretation is Benign/ Likely Benign)

Variant 7: VWF NM_000552.5: c.6532G>T:p.Ala2178Ser (Exon 37, Conflicting interpretations of pathogenicity: (VUS-1) (Likely Benign-1))

Variant 8: F5 NM_000130.5: c.2773A>G:p.Lys925Glu (Exon 13, current ClinVar interpretation is Benign/Likely Benign)

Variant 9: F5 NM_000130.5: c.2594A>G:p.His865Arg (Exon 13, current ClinVar interpretation is Benign/Likely Benign)

Variant 10: F5 NM_000130.5: c.2573A>G:p.Lys858Arg (Exon 13, Conflicting interpretations of pathogenicity: (VUS-1) (Benign-2)(Likely Benign-1))

Variant 11: F5 NM_000130.5: c.5290A>G:p.Met1764Val (Exon 16, Conflicting interpretations of pathogenicity: (VUS-1) (Benign-2)(Likely Benign-1))

Variant 12: F13A1 NM_000129.4: c.103G>T:p.Val35Leu (Exon 2, Conflicting interpretations of pathogenicity: (VUS-1) (Benign-3))

Variant notes: All are heterozygous

Both variants in F8 are linked to reports associated with haemophilia, though second variant is considered benign.

Phenotypes: History of bleeding (Heavy mentrual bleeding since menarche)(Treated with transdermal oestrogen and Levonorgestel), iron deficiency anaemia. High Janssen score for pictorial blood assessment. Gum bleeding lasting longer than 10 minutes(Treated with local application of tranexamic acid), recurrent nosebleeds, high score for ISTH and BAT assessments. Decrease in VWF:Ag ratio, VWF:CB ratio decreased, VWF: GPIbR ratio decreased

Family: Maternal grandfather possibly haemophiliac, mother asymptomatic

Disease: Von Willebrand Disease (VWD) type 1

Patient(s): 13 yo, female and 14 yo, female, both Italian

Variant: VWF NM_000552.5: c.820A>C p. (Thr274Pro)

Dominant negative effect

Heterozygous carrier

Variant located in the D1 domain on VWF

Phenotypes:

heterozygous carriers have no bleeding history

reduced VWF levels compatible with diagnosis of VWD type 1

increased FVIII:C/VWF:Ag ratio, suggests reduced VWF synthesis/secretion as possible phathophysiological mechanism

Normal VWFpp/VWF:Ag ratio

Modest alteration of multimeric pattern in plasma and platelet multimers

plasma VWF showed slight increase of LMWM and decrease of IMWM and HMWM

Platelet VWF showed quantitative decrease of IMWM, HMWM, and UL multimers

In silico analysis:

SIFT, ALIGN, GVD Polyphen 2.0, SNP&GO, Mutation Taster, Pmut all suggest damaging consequences.

PROVEAN and Effect suggest neutral effect

according to ACMG guidelines this variant was classified as pathogenic

Disease: Von-Willebrand Disorder

Patient 2 Variant(s):

VWF NM_000552.5: c.4135C>T p.(Arg1379Cys) Exon 28 VWF NM_000552.5: c.4130C>T p.(Ala1377Val) Exon 28 VWF NM_000552.5: c.3797C>T p.(Pro1266Leu) Exon 28 VWF NM_000552.5: c.3835G>A p.(Val1279Ile) Exon 28

Note: Pro1266Leu and Val1279Ile are in trans with Ala1377Val and Arg1379Cys

Note2: MAF of Ala1377Val is present in Exome Variant Server (<0.01) and 1000 Genomes database (<0.02), designated as rare variant, typically found in indiv with African ethnicity

Family: Relatives molecular analysis showed Arg1379 and Ala1377Val variants mentioned above were in cis

Patient 2 Phenotype: Mild bleeding symptom Nearly normal VWF:Ag value, reduced VWF:RCo value slight loss of HMWM with smear decreased/slightly decreased proteolysis Slightly reduced RIPA VWFpp/VWF:Ag ratio shows increased VWF clearance VWF platelet levels reduced reduced rBpIba binding in VWF plasma VWF:GPIbM values slightly increased

Note: Treated with desmopressin in case of minor surgeries or delivery

Note 2: Slightly decreased RIPA may be explained by presence of 2B New York Variant (Pro1266Leu) that mitigates RIPA assay in 2M phenotype.

In silico analysis available:

I-Mutant 3.0 states decrease in A1 domain stability for both mutations (Ala1377Val = -0.91, Arg1379Cys = -1.36)

PYMOL predicts Ala1377Val does not alter formation of hydrogen bonds with Arg1374 residue and water. Predicts substitution of ARG 1379 with a cysteine results in the loss of hydrogen bonds with Lys1407 and Lys1408, predicted change in secondary structure of A1 domain

Ala1377Val was previously reported in 3 other patients Publications:

Millar CM, Riddel AF, Mellors G, Yee TT. The spectrum of VWD type 2 phenotypes associated with A1 domain mutations posters. J Thromb Haemost 2009; 7: 531–2.

Logsdon BA, Dai JY, Auer PL et al. A variational Bayes discrete mixture test for rare variant association. Genet Epidemiol 2014; 38: 21–30.

Final note: Authors suggest patients' 2M phenotype is due to the presence of Ala1377Val and Arg1379Cys together to create synergistic effect. Though difficult to discern with this specific patient having two other variants in addition to the two mentioned above.

Disease: Von-willebrand Disorder, type 1

Patient 1 Variant: VWF NM_000552.5: c.4135C>T p.(Arg1379Cys) Exon 28

Family: History not mentioned

Patient 1 phenotype: near normal VWF:Ag and WVF:RCo levels RIPA within normal range Platelet VWF levels were normal normal pattern for multimeric analysis of plasma Slightly reduced VWF levels

In silico predictions available:

I-Mutant 3.0 value = -1.36 PYMOL prediction = substitution of ARG 1379 with a cysteine results in the loss of hydrogen bonds with Lys1407 and Lys1408, predicted change in secondary structure of A1 domain

functional assays

Chemiluminescence assays (measuring binding capacity)

VWF-collagen binding assay (CBA)

Electrophoresis (Western blot)

Bidirectional direct sequencing of PCR products

Paternity test

PCR and restriction assays to detect SNVs

in vitro expression of recombinant WT and p.P1127S VWF variants in HEK293 cells

Platelet aggregation studies

DDAVP test

Binding assays

Proteolysis assays

in silico modeling

Disease: Von-willebrand Disorder

Patient: 21 yo, female, Italian descent

Variant: VWF NM_000552.5 c:C3379 > T p.(P1127S), homozygous

Heterozygous and Homozygous polymorphic variant in exon 25

Phenotypes: Bleeding Score System (BSS) = 3 minor bruising normal menstrual bleeding

Family: (father paternity confirmed) Father suffered from rectorrhagia for rectal polyps Mother (same variant, heterozygous) has heavy menstrual bleeding, epistaxis events up to age 30, BBS= 2

Present in dbSNP (rs139579968) MAF in European pop = 0.0001-0.0004

Present in gnomAD, said to be present in 2 transcripts in VWF 40 alleles are present

Predictions: listed with PolyPhen-2 and SIFT = probably damaging to protein expression/function

CADD (score =33) and REVEL(score = 0.748) suggest deleterious effect of pathogenic variant

I-TASSER showed large difference in 3D configuration of sequences differing by a single amino acid.

Variant: VWF NM_000552.5: c.7682T>A p.(Phe2561Tyr)

Suggested GOF mechanism

Located in exon 45 of VWF (C4 domain)

Present in gnomAD, suggested as likely benign/benign Found in exome and genome samples (Allele count is 69570, allele freq = 4.32e-2, homozygote count= 1725)

Note that the assessment here is primarily in white individuals

Functional outcomes:

Among patients with recurrent Myocardial Infarction the Tyr2561-VWF variant is more common than control, particularly in younger women. (Caveat, need a larger study as this result is suggestive)

functionally characterized Tyr2561-VWF compared with Phe2561-VWF in carriers of the respective variants by 3 static and 2 shear-based assays.

in one static assay, using isolated GPIIb/IIIa from platelets, found no enhanced binding for rTyr2561-VWF, but a slightly enhanced binding for rTyr2561-VWF coexpressed with rPhe2561-VWF. (Caveat, cannot confirm these results by using cell-based binding assay with constitutatively GPIIb/IIIa incorporated in the cell membrane)

In shear conditions measured by CPA, platelet aggregate size in blood of TYR2561 probands was significantly increased at all shear conditions compared to homozygous Phe2561 carriers. Using recombinant Tyr2561-VWF, confirmed specificity of the effect for VWF.

Recombinant variant causes marked decreae in critical shear rate for collective network formation of VWF and platelets to less than 50%. complementary evidence for GOF of Tyr2561-VWF under high-shear conditions. In accordance with clinical data.

Functional study methods:

WAVE DNA fragment analysis to screen for variant

VWF collagen binding activity

cone and plate analysis (CPA)

Microfluidics

static VWF-platelet receptor binding assay

protein expression in cultured HEK293 cell clones

Near and far-UV circular dichroism spectra

Disease: Von Willebrand Disease (VWD)

Patient: 18 yo, Male, heterozygote

Variant: VWF NM_000552.5: c.5456_5842del p.(R1819_C1948delinsS)

Was not present in gnomAD when searched

Dominant negative effect

Phenotypes:

lower collagen-binding capacity

History of bleeding (epistaxis)

gum bleeding

cutaneous bruises

ADAMTS13 resistant

Family: Mother, father, sister are asymptomatic

Suggested as de novo, no picture found in patient's relative of the deletion, loss of A3 loop