Molecular Genetics Analysis of Von Williebrand Disease: Studies in Cohort Pakistani Patients

Introduction: Von Willebrand disease (VWD) type III is the second most common inherited bleeding disorder in Pakistan. It is caused by severe quantitative deficiency of plasma Von Willebrand factor (VWF). Absence of VWF increases the severity of disease and is caused by homozygous/ compound heterozygous mutations in Von Willebrand factor gene.

Objective: The aim of study is to characterize molecular genetics of Von Willebrand type III in Pakistani population.

Setting: NIBD & BMT Karachi, Chughtais lab, Children's Hospital Lahore, PAEC Islamabad and HMC Peshawar.

Material And Method: In this cohort study of Von Willebrand disease (VWD) type III, Blood samples of 48 unrelated patients of type III VWD were collected in National Institute Of Blood Disease & Bone Marrow Transplant (NIBD) Karachi, Chughtais lab, Children's Hospital Lahore, PAEC Islamabad and HMC Peshawar. Genomic DNA was extracted from peripheral blood by QIAamp DNA Blood mini kit (Qiagen) and Exon specific PCR was done for VWF gene and Direct gene sequenced on automated ABI-3130 Genetic Analyzer (Applied Biosystems). Sequence Variations in VWF were checked on ISTH-SSC VWD homepage (http://www.vwf.group.shef.ac.uk/), Ensemble genome browser (http://www.ensembl.org/), VWFdb hemobase and biobase biological database (http://www.hgmd.cf.ac.uk/ac/index.php). We have adopted the nomenclature for numbering amino acids of Human genome variation society (HGVS). Statistics analysis was done SPSSv17.

Results: Sequence analysis detected mutations in 46 (95.83%) out of 48 samples. VWD type III has led to identification of 28 cases (60.8%) homozygous and 18 (39.1%) were compound heterozygous. We have indentified total 31 Mutations distributed as 17 missense mutations (54%), 7 nonsense mutations (22%) 2 small deletions (6%) , 2 insertion mutations (6%) and 3 splice site mutations (9%).19 of these were newly mutations in this cohort study.. Further method multiplex ligation dependent probe amplification (MLPA) is needed for detection of large deletions in two patients. Nonsense c.3931C>T, p.Q1311*was founder mutation in Pakistani patients.

Conclusion: In cohort study, missense mutations are detected as common in among patients and most of the mutations identified in this cohort were homozygous due to Consanguinity in the family of the patients.