Molecular and clinical profile of VWD in a large cohort of Chinese population: application of next generation sequencing and CNVplex® technique

Abstract

Von Willebrand disease (VWD), the most common inherited bleeding disorder, is characterised by a variable bleeding tendency, heterogeneous laboratory phenotype and race specific distribution of mutations. The present study aimed to determine the correlation of genotype and phenotype in 200 Chinese individuals from 90 unrelated families with VWD. Next generation sequencing (NGS) of the whole coding VWF, copy number analysis of VWF by CNVplex technique as well as a comprehensive phenotypic assessment were carried out in all index patients (IPs). We identified putative mutations in all IPs except five mild type 1 (85/90, 94.4 %). In total, 98 different mutations were detected, 62 (63.3 %) of which were reported for the first time (23 missense mutations, 1 regulatory mutation, 12 splice site mutations and 26 null mutations). Mutations p.Ser1506Leu and p.Arg1374His/Cys/Ser were the most frequent mutations in 2A (33 % of cases) and 2M VWD (67 % of cases), respectively. In addition, mutation p.Arg816Trp was detected repeatedly in type 2N patients, while mutation p.Arg854Gln, extremely common in Caucasians, was not found in our cohort. Thirty-three patients had two or more putative mutations. Unlike most cases of type 1 and type 2 VWD, which were transmitted dominantly, we presented seven severe type 1, two type 2A and one type 2M with autosomal recessive inheritance. Here the phenotypic data of patients with novel mutations will certainly contribute to the better understanding of the molecular genetics of VWF-related phenotypes.

Phenotype and genotype of FXIII deficiency in two unrelated probands: identification of a novel F13A1 large deletion mediated by complex rearrangement

“…Peripheral blood was collected from the affected individuals and related family members. Platelet-poor plasma (PPP) and platelet-rich plasma (PRP) were prepared as previously described [19]. Coagulation screen tests including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB) in PPP were performed on a CA7000 automatic coagulometer (Sysmex, Kobe, Japan) as routine protocols.…”

“…Platelet aggregation in PRP with ristocetin at final concentrations of 0.5 mg/ml and 1.2 mg/ml or adenosine diphosphate (ADP) at 2umol/L was performed on a Chrono-log aggregometer model 560 (Chrono-log Corporation, Harvertown, USA). VWF antigen (VWF:Ag), VWF activity (VWF:Ac) and FVIII activity (FVIII:C) were also assayed as preciously described [19]. ABO-blood-type was detected as routine protocols.…”

“…CNVs of F13A1 and F13B were analyzed by the CNVplex® technique (Genesky Biotechnologies, Shanghai, China), a high-throughput multiplex CNV detection method [19]. Using synthetic oligonucleotides, 63 target-specific probe pairs were selected including 39 pairs in F13A1 with five pairs on the 5′ UTR, two pairs on each of 15 exons and four pairs on the 3′ UTR, and 24 pairs covering all 12 exons with two pairs on each of the exons in F13B (Additional file 1: Table S1).…”

Update on Molecular Testing in von Willebrand Disease

“…51 In addition, a different NGS approach based on multiplex PCRs was applied to a Chinese cohort (200 patients). 52 These reports demonstrated the utility of NGS in VWD diagnostics and highlighted the need to progress toward methods that sequence the whole coding region of VWF employing a common strategy for all VWD types. NGS-based methods appear 47 cost-effective for large cohorts of VWD patients, but it can also be applied to individual samples using multigene panel approaches (discussed later).…”

Carrier Screening of SMA in North China

“…If both partners were de ned as SMA carriers, the genetic counseling was provided for invasive prenatal genetic diagnostic testing of the fetus. The copy number of SMN1 and SMN2 genes of amniotic uid sample were explored by CNVplex® (a technique for high-throughput detection of sub-chromosomal copy number aberrations) as described before [10]. The homozygous deletion SMN1 of fetus result was de ned as true positive upon postnatal genetic diagnostic con rmation or clinical follow-up results.…”