Genotyping R1336X and Eliminating the Pseudogene Amplification in Type 3 von Willebrand Disease Patients

Among inherited bleeding disorders, von Willebrand disease (VWD) is the most common disease worldwide. Type 3, the most severe form, is more prevalent in developing countries primarily due to consanguineous marriages. The enormous size of the von Willebrand factor gene, different kinds of mutations, and a partial unprocessed pseudogene make variation detection more difficult. To distinguish the VWF gene and the pseudogene sequence and prevent pseudogene amplification, it is necessary to utilize a suitable method. In this study, 42 unrelated VWD type 3 patients and 22 normal controls were included. Tetra-primer ARMS-PCR was applied for genotyping the single nucleotide variation, R1336X located on exon 28, which overlaps the pseudogene. The High percentage of heterozygotes (17% in VWD patients and 13% in controls) raised the suspicion of concomitant amplification of the VWF gene and it's pseudogene due to similarity in their primer-binding site sequence. To overcome this issue, we applied a two-step method using touch-down PCR followed by a tetra-primer ARMS-PCR. All cases with the heterozygous pattern on the first tetra-primer ARMS PCR were found to be normal homozygotes. In type 3 VWD patients genotyping is essential in decreasing the incidence of the disease, but the presence of a pseudogene with 97% homology makes it difficult. Applying appropriate methods that combine touch-down PCR and tetra-primer ARMS-PCR in terms of reliability, sensitivity, and accuracy can help to eliminate the pseudogene effect and diagnose more affected individuals.