6 Matching Annotations
  1. Last 7 days
    1. 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

    2. 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.

    1. 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

    2. 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.

    3. 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

    1. 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