PB0355 - Genetic analysis using long-read sequencing to overcome the difficulties in VWF gene

Background: Abnormalities of von Willebrand factor (VWF) caused by genetic mutations would result in von Willebrand disease (VWD). In acquired von Willebrand syndrome (AVWS), the contribution of variants in the VWF gene to the risk of disease onset remains unclear. The identification of causative variants in VWF is important for the diagnosis, classification, and clinical management of these VWF disorders. However, given the length (175 kb) and complexity of the VWF gene, which contains 52 exons with multiple repetitive sequences, and the presence of the pseudogene VWFP1, Sanger sequencing or short-read next-generation sequencing often faces difficulties.

Aims: To establish a long-read sequencing method using Oxford Nanopore Technology (ONT) to overcome the difficulties in VWF gene analysis.

Methods: Genetic analysis was performed using DNA samples from three patients registered as VWD or AVWS in the National Cerebral and Cardiovascular Center Biobank. Specific primers were designed and optimized to amplify ~15-kb PCR amplicons covering the entire VWF, avoiding unwanted amplification due to repetitive sequences or VWFP1. All amplicons were subjected to DNA library preparation. ONT data were then analyzed using dedicated software, including Clair3 and Longshot as variant callers, and the 54KJPN database and ClinVar for variant details. Identified candidate variants were verified by Sanger sequencing.

Results: Twenty-one 12~15-kb amplicons were produced and used for ONT sequencing (Figure 1). The entire VWF gene including introns was successfully read and more than 200 variants were called in each sample. Although no pathogenic variant was found in the VWD sample, a rare missense variant was identified in the AVWS sample, which may pose some risk. Some artifacts were also detected that deserve attention when performing this method (Table 1).

Conclusion(s): Here, we present an optimal solution using ONT sequencing to identify causative variants in VWF, which may improve the diagnosis of VWD.