- Last 7 days
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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
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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.
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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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
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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.
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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
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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
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