Identification of mutations in the F9 gene of Maltese Haemophilia B patients

Abstract

Haemophilia B is a disease characterized by spontaneous or prolonged haemorrhagic bleeding due to Factor IX (FIX) deficiency. The factor IX gene (F9) lies on the long arm of the X chromosome at Xq27.1 and the entire sequence of 33 kb is known. It contains eight exons (1-8) encoding six major domains that make up the FIX protein. There are currently 1095 unique variants in the F9 gene that are collected in a database. In Malta, the disease is relatively rare and in this study 11 patients were tested by molecular techniques to characterize their mutations. Diagnosis in Malta has been largely dependent on haematological and coagulation laboratory tests rather than direct identification of mutations. DNA was extracted from each sample and amplified by polymerase chain reaction (PCR) using primers from each exon of the F9 gene. DNA sequencing was then performed for correct genotyping, which will test the presence of mutations. These were compared with reference DNA from healthy individuals. For any large deletions, multiplex ligation-dependent probe amplification (MLPA) was performed. Four patients were classified as severe, while the rest were mild to moderate form of the disease, due to enough circulating FIX protein. 1 gross deletion, 2 missense mutations and a single nucleotide deletion were identified. The single nucleotide deletion (FIX Malta I) was found to be a novel mutation affecting the transcription factor binding sites in the promoter area. This confirms a high heterogeneity of molecular defects leading to Haemophilia B in Malta. These mutations may contribute for more precise identification of the structure–function relationship and understanding the nature of the FIX molecule. Future testing will target the accurate genotyping of the rest of coagulation factor deficiencies in Malta.