Molecular genetic studies of Haemophilia A in Maltese patients

Abstract

Introduction: Haemophilia A (HA) is an X-linked bleeding disorder caused by diverse mutations in the human coagulation factor VIII (FVIII) gene. The factor VIII gene (F8) comprises of 26 exons and spans 186kb. F8 has 3 copies of an A domain of 330 to 380 amino acids, a B domain of about 925 amino acids, and 2 C domains of about 160 amino acids. The domains are arranged A1-A2-B-A3-C1-C2. Occurring in around 1:30,000 this is considered to be a rare disorder. Diagnosis in Malta has been largely dependent on haematological and coagulation laboratory tests rather than direct identification of mutations. We have analysed DNA from 18 Maltese patients including some carriers with HA for their FVIII gene defects. Almost all the male patients had severe (FVIII:C <1%) HA with the exception of one having a moderate (FVIII:C 3%) HA. Methods: These patients were first screened for the commonest occurring mutations i.e. intron 22 and intron 1 inversions. Inversion negative samples were screened for mutations by whole exome sequencing and confirmed by DNA sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). In total, 8 different mutations were identified in 18 patients, 2 of which are novel mutations. Results: These included 4 patients with intron 22 inversions, 3 patients with an amino acid missense mutation for the c.5399G>A (p.Arg1800His) and c.3780C>G (p.Asp1260Glu), 3 patients with a duplication frameshift c.4825dupA, 2 different types of large deletions affecting 3 patients, a novel nonsense mutation in one patient and a novel indel mutation in 2 other patients. 2 Individuals in the study had a normal F8 sequence and are not carriers of HA. Conclusion: This project lays the foundations for a more useful and better genetic counselling and will also aid in choosing the appropriate method for the treatment of patients. The data also show that the genetics of rare blood disorders in Malta remains a very important field for the discovery of new genetic variants previosly unreported elsewhere. Disclosure: The research work disclosed in this publication is partially funded by the Endeavour Scholarship Scheme (Malta). Scholarships are part- financed by the European Union - European Social Fund (ESF) - Operational Programme II – Cohesion Policy 2014-2020