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

Area of Science:

• Genomics
• Molecular Biology
• Clinical Genetics

Background:

• Genetic defects in the von Willebrand factor (VWF) gene cause von Willebrand disease (VWD).
• Accurate identification of VWF variants is essential for diagnosing and managing VWD.
• The VWF gene's large size and the presence of a pseudogene complicate genetic analysis using traditional sequencing methods.

Purpose of the Study:

• To develop and validate a long-read sequencing method utilizing Oxford Nanopore Technology (ONT) for comprehensive VWF gene analysis.
• To overcome the challenges associated with sequencing the complex VWF gene and its pseudogene (VWFP1).

Main Methods:

• Genomic DNA from healthy donors and VWD patients was analyzed.
• Long-range polymerase chain reaction (PCR) generated 21 amplicons covering the entire VWF gene.
• ONT nanopore sequencing was performed, and data were analyzed using Clair3, Longshot, and Sniffles software.
• Candidate variants were confirmed using Sanger sequencing and haplotyping.

Main Results:

• ONT nanopore sequencing successfully sequenced the entire VWF gene.
• Over 200 variants were identified per patient sample.
• A rare missense variant (p.(Gln2442His)) was identified in one patient.
• A deletion initially identified was confirmed as a long-range PCR artifact, highlighting a methodological consideration.

Conclusions:

• ONT nanopore sequencing provides an effective solution for identifying variants within the VWF gene.
• This long-read sequencing approach has the potential to enhance the diagnostic capabilities for VWF disorders.