Thirty Novel Mutations in the Factor VIII Gene of Indian Patients with Haemophilia A

Abstract number: P0212

G¹ Jayandharan, RV¹ Shaji, SC² Nair, A¹ Viswabandya, V¹ Mathews, B¹ George, M¹ Chandy, A¹ Srivastava

¹¹Department of Haematology, Christian Medical College, Vellore, India ²²Department of Clinical Pathology, Christian Medical College, Vellore, India

Haemophilia A (HA) is an X-linked bleeding disorder caused by diverse mutations in the human coagulation factor (F) VIII gene. We describe 30 novel mutations in patients with severe (n= 26), moderate (n= 1) and mild (n= 3) HA. All these patients belong to a previously described (Haemophilia 2004; 10 (S3) 15 PO 17) cohort of 109 patients and were negative for the common intron 1 and 22 inversions. Screening of the FVIII gene was performed using a multiplex PCR and conformation sensitive gel electrophoresis strategy followed by sequencing. Twenty non-missense (3 gross deletions, 3 splice site defects, 6 nonsense, 8 frame shift mutations) and 10 missense mutations were identified. We have predicted their molecular consequence by studying the nature of mutation, the evolutionary conservation of mutated aminoacid and by molecular modeling. The novel partial deletions (delexon2–5, delpromoter-exon5, delexon 2–9) remove the coding sequence (35–55 Kb) for the major part of the FVIII protein A1 and A2 domains, thereby manifesting as severe phenotype. Pre-mRNA splicing was possibly affected in the 3 severe HA patients presenting with splice site mutations (IVS 15 (+6) T > G, IVS 10 (-2) A > G, IVS 1(-26) A > T). In patients with nonsense mutations (Lys1581Stop, Trp1835Stop, Trp1535Stop, Gln1502Stop, Tyr1571Stop and Gln2013Stop) and frame-shifts (Gly864FS, Lys948FS, Val1504FS, Thr233FS, Pro1392FS, Leu1227FS and Gln1686FS) the severe phenotype is a consequence of premature termination of translation. Of the 10 missense mutations, 6 (Phe31Ser, Gly102Val, Cys528Tyr, Glu1751Lys, Phe1775Pro, Cys2169His) were associated with severe HA. Moderate HA (FVIII: C-2%) was seen in a patient with Ser104Pro mutation. Mild HA phenotype was seen in patients with Glu143Lys (FVIII: C 26%), Arg2147Gly (FVIII: C 6.2%) and Thr2253Pro (FVIII: C 5.4%) mutations. Further characterization of some of these mutations is in progress.