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

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: 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: platelet-type von Willebrand disease (PT-VWD)

Patient: 14 yo, Male

Variant: GP1BA NM_000173.7:c.389G>A p.(Arg127Gln), Heterozygous, Gain-of-Function (GOF)

Located in LRR5 domain of GP1BA

Family: Mother did not refer any bleeding symptoms (variant absent in mother) Father not available for collection of clinical history or platelet function testing