PMID: 19687512

Gene: VWF

HGNCID: 12726

Molecular findings:

Severe bleeding phenotype in homozygotes, alleviated bleeding phenotype in heterozygotes.

Difficult to classify based on the phenotype and plasma levels.

p.M771V variant shows elevated proVWF to mature VWF ratio, reduced levels of cleaved VWFpp.

p.M771V has potential to negatively influence VWFpp cleavage to contribute to decreased VWF processing in endothelial cells.

Other variants near p.M771 locus reported to disrupt the cleavage by furin. (Variants are at R763 and R760 respectively).

Other researchers have come across the p.M771 variant and reported it as well.

functional studies performed in cord blood derived ECFCs through adenine base editing and overexpressed in HEK293 cells.

Utilized Immunocytochemistry staining and confocal analysis

ELISA to test for secreted VWF in ECFCs

Multimer assay and densitometry graphs to view VWF multimer patterns

Western blotting to view proteolytic processing of VWF and VWFpp

Genomic sequencing to verify mutations.

Authors here used SpCas9-mediated base editing to mimic patient variant if primary ECFCs are not available or are difficult to culture to assist in improved characterization in a true endothelial background.

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

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

this assay involves a thiol intermediate, so it will be interfered by thiol containing compounds, or compounds that react with thiol group. But common HTRF will not be affected.

Assay guidance

a very good sumamry of the difference between Pull-down and IP assays. Pros and cons of both methods are described.

How does this assay work?

Daley, J. (n.d.). Millions of Rapid COVID-19 Antigen Tests May Help Fill the Testing Gap. Scientific American. Retrieved September 30, 2020, from https://www.scientificamerican.com/article/millions-of-rapid-covid-19-antigen-tests-may-help-fill-the-testing-gap/

WHO. (n.d.) Coronavirus disease (COVID-19) technical guidance: Laboratory testing for 2019-nCoV in humans. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/laboratory-guidance

This drinking water assay contains a membrane (0.45 µm) filtration step to remove bacteria from the water sample prior to enrichment on selective and differential media. The bacteria CFUs are quantified directly on the filter following a lengthy enrichment