Expression studies of missense mutations p.D141Y, p.C275S located in the propeptide of von Willebrand factor in patients with type 3 von Willebrand disease

Abstract

Missense mutations are not considered a common cause of type 3 von Willebrand's disease (VWD), the most severe defect of von Willebrand factor (VWF) characterized by undetectable levels of this protein in plasma and platelets. Nevertheless, several missense mutations have been identified in these patients. In this study, we report the cases of two Italian patients with type 3 VWD, both compound heterozygotes for different missense mutations and null alleles, p.D141Y/c.2016_2019del and p.C275S/p.W222X. We performed in vitro expression studies of the candidate missense mutations, both located in the D1 domain of VWF propeptide, to confirm their link with the disease and to understand the mechanisms of type 3 VWD responsible in these patients. Mutant and wild-type (WT) expression vectors were used for transient transfection and co-transfection studies in COS-7 cells. Single construct transfections of both missense mutations showed a strongly reduced but detectable secretion of recombinant (r)VWFs (approximately 15% of WT), with essentially only dimers being visualized on multimeric analysis. As expected, expression of a single construct of either mutation with the WT, showed mildly reduced secretion (approximately 40% of WT) and a full set of multimers. These expression studies indicate that the two amino acids D141 and C275 are key residues in the tertiary structure of the VWF propeptide. Their replacement with a tyrosine and a serine, respectively, might compromise propeptide folding, affecting both its intracellular survival and its capacity to mediate multimerization. Co-expression of hybrid rVWFs confirmed the recessive inheritance pattern of these missense mutations.

Molecular characterization, recombinant protein expression, and mRNA analysis of type 3 von Willebrand disease: Studies of an Italian cohort of 10 patients

“…HRM analysis was then carried out for 25 exons (number 4,6,8,9,11,12,16,17,19,20,21,23,25,30,34,35,36,38,39,40,41,44,46,48,51) in all patients. This is a mutation scanning technique that monitors the progressive change in fluorescence caused by the release of an intercalating DNA dye from a DNA duplex as it is denatured with marginal increases in temperature [18].…”

“…(c.6634T>C), and pSV-VWFH-C2325S (c.6973T>A) were generated by site-directed mutagenesis (QuikChange 1 II XL Site-Directed Mutagenesis kit; Stratagene, Cedar Creek, TX), using as template the expression vector pSV-VWFH [11] and specifically designed oligonucleotides [20].…”

“…COS-7 cells were grown at full confluence into a six-well cell culture plate and were transfected with the appropriated amount of expression vector [wild-type (WT), mutant and both to generate mutant/WT recombinant VWF hybrids] using jetPEI TM transfection reagent (PolyPlus-transfection, Euroclone, Pero, Italy) as previously reported [20]. After 72 h of incubation, conditioned media and cell lysates were harvested and collected [11], recombinant VWF (rVWF) antigen levels were quantified and were expressed as mean values ± standard deviation. rVWF multimeric structure was analyzed by electrophoresis under nonreducing conditions in low-resolution gels as described before [11].…”

“…PAXgene TM Blood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland) were used to isolate total RNA from patients whole blood. FVIII coagulant (FVIII:C) and VWF antigen (VWF:Ag) levels were evaluated as described earlier [11]. Patients were diagnosed with VWD3 when plasma VWF:Ag was lower than 1 IU/dL.…”

“…Nucleotides are numbered from the first adenine (11) in the ATG initiation codon and amino acids are numbered from 1 to 2813, starting from the first methionine [15]. NM_000552.3 was used as a reference for nucleotide change whereas NP_000543.2 was used as protein reference sequence.…”