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Genotyping R1336X and Eliminating the Pseudogene Amplification in Type 3 von Willebrand Disease Patients
Mahmoud Khatib1, Azam Bolhassani2,3, Zahra Noormohammadi1
1Department of Biology, Faculty of Converging Sciences and Technologies, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Insights
Von Willebrand disease (VWD) type 3 diagnosis is challenging due to a pseudogene. A combined touch-down PCR and tetra-primer ARMS-PCR method improves accuracy for identifying affected individuals.
Area of Science:
- Genetics
- Molecular Biology
- Hematology
Background:
- Von Willebrand disease (VWD) is the most common inherited bleeding disorder globally.
- Type 3 VWD, the most severe form, presents diagnostic challenges due to the large von Willebrand factor (VWF) gene, numerous mutations, and a highly homologous pseudogene.
- Pseudogene amplification can interfere with accurate genetic variation detection.
Purpose of the Study:
- To develop and validate a reliable method for genotyping VWD type 3 patients, overcoming pseudogene interference.
- To accurately distinguish between the VWF gene and its pseudogene sequence to prevent amplification bias.
Main Methods:
- A cohort of 42 unrelated VWD type 3 patients and 22 normal controls were analyzed.
- Initial genotyping utilized tetra-primer ARMS-PCR for the R1336X single nucleotide variation on exon 28.
- A two-step approach combining touch-down PCR followed by tetra-primer ARMS-PCR was implemented to address pseudogene amplification issues.
Main Results:
- High heterozygote rates observed with initial tetra-primer ARMS-PCR suggested pseudogene co-amplification.
- The two-step method successfully differentiated VWF gene amplification from pseudogene amplification.
- Cases initially appearing heterozygous were reclassified as normal homozygotes, indicating the pseudogene's confounding effect.
Conclusions:
- Accurate genotyping in VWD type 3 is crucial for disease management and incidence reduction.
- The presence of a highly homologous VWF pseudogene complicates genetic analysis.
- Combining touch-down PCR with tetra-primer ARMS-PCR offers a reliable, sensitive, and accurate strategy to eliminate pseudogene effects and improve VWD diagnosis.
Abstract:
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.
