Mutational analysis of Factor XI and Von Willebrand Factor genes by high resolution melting analysis and direct DNA sequencing

Abstract

Continuous flow of blood is essential for life and its maintenance is an important task for the body. Hemostasis is the balance between the coagulants, factors that promote blood clot formation and anti-coagulants, factors that restricts the action of coagulants. As a result of their actions, abnormal blood loss after trauma is prevented as well as excessive clot formation in the vessels. Von Willebrand Factor (VWF) and Factor XI (FXI) are among several proteins maintaining this balance. The deficiency of vWF results in the most common congenital bleedings disorder; Von Willebrand Disease (vWD), though the estimation of prevalence is complicated due to the incomplete penetrance and extreme variability in clinical symptoms of mild Von Willebrand Disease (VWD). Disease manifests itself with mucosal and trauma related bleeding, and caused by the mutations in VWF gene. Mutations in Factor 11 (F11) gene results in Factor XI deficiency (Hemophilia C), symptoms of which are trauma or surgery related bleeding. The deficiency is common among Ashkenazi Jewish, but rare in other populations. Its diagnosis is also complicated due to incomplete penetrance and variable expressivity. The identification of the underlying molecular pathology is important for the management of both diseases. 23 kb long Factor 11 gene comprises 15 exons and molecular genetic analysis is feasible by direct DNA sequencing. Through such analysis, exons and the promoter region of the F11 in two FXI deficiency patients and their relatives revealed one recurrent (c. 1556 G>A) and one novel (c. 151 A>C) mutation. The first mutation is in the catalytic domain and created a premature stop codon, the second is in the “Apple 1” domain, probably affecting the secretory pathway of the protein. Molecular analysis of the VWF gene is complicated because of the length of the gene which comprises 52 exons and encodes 8,2 kb mRNA. Additionally, the gene, especially the longest exon, Exon 28, is highly polymorphic. Due to these facts, analysis of the gene by direct DNA sequencing is not manageable. Therefore it is important to employ a mutation detection system to quickly, efficiently and inexpensively identify all sequence variations in the VWF gene. High Resolution Melting Analysis (HRMA) is a recently developed mutation screening system that detects nucleotide changes through differences in melting plots of amplicons. A light cycler platform combines PCR amplification with HRMA in a closed tube system and assays 96 samples in approximately 90 minutes. It also allows simultaneous analysis of more than one amplicon in one run so the whole gene analysis can potentially be screened in a few runs with appropriately optimized conditions for a single patient. The sequence alteration detected by HRMA is then identified by DNA sequencing. For the molecular analysis of VWF gene in Turkish VWD patients, 38 amplicons of VWF gene out of 60 were optimized for HRMA. These 38 amplicons were grouped into seven amplification groups that have different thermal conditions. Among these, 24 amplicons were sequenced to confirm proper amplification of targeted regions. Patient screening was initiated in 10 VWD patients and an apparently healthy control for 12 amplicons. Although further investigations are required, the preliminary screening of VWD patients with HRMA is appropriate and promising.