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

Tests performed: Haemostatic tests ristocetin-induced platelet aggregation plasma VWF antigen VWF risocetin cofactor VWF FVIII-binding capacity VWF multimers and FVIII activity DDAVP test Concentration timecourses

sequencing from genomic DNA performed and reported in 2011

cDNA analysis

Real-time-PCR analysis

long PCR

Ellman assay (quantifying sulfhydryls)

Dynamic light scattering measurements

circular dichroism

ADAMTS13 proteolysis assay

ADAMTS13-VWF binding assay

Patient phenotypes listed: mild bleeding history normal platelet number increased mean platelet volume increased RIPA (les than typical PT-VWD) enhanced binding of VWF to platelets (assessed by flow cytometry)

Has functional and HXMS data to support classification to pathogenic

4 in silico prediction softwares classify as benign

Mentions according to ACMG guidelines classify variant as VUS initially

later reclassified 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

Variant present in gnomAD database(rs749454966)

8 allele in gnomAD but with low frequency exomes allele frequency = 0.0000201 genomes allele frequency = 0.0000956 total allele frequency = 0.00002851