Molecular pathology of haemophilia A in Turkish patients: identification of 36 independent mutations

Haemophilia A is an X-linked recessive bleeding,disorder caused by heteroge neous mutations in the factor VIII gene. In an attempt to reveal the molecu lar pathology of Turkish haemophilia A patients, the coding sequence of the gene, excluding a large portion of exon 14, was amplified from genomic DNA and subjected to denaturing gradient gel electrophoresis prior to DNA sequ encing. Fifty-nine haemophilia A patients were included in the study with s evere, moderate and mild phenotypes observed in 24, 15 and 16 patients, res pectively. Factor VIII activity and clinical phenotypes were not available for four patients. A total of 36 independent mutations were found, with a m utation detection efficacy of 61%. The mutations that were reported for the first time include 20 point mutations, one 8-bp insertion (TCAAGATA) in ex on 4 and one large deletion greater than 2.8 kb involving exon 14. The nove l point mutations were composed of three nonsense (Ser681-Ter, Cys2021Ter a nd Gln2113Ter), one splicing error (IVS-1G-->A), 15 missense mutations (Lys 48Asn; Leu-98Phe; Thr118Ala; Cys248Tyr; Glu456Lys; Asp560Ala; Tyr664Cys; Ph e679Leu; Gly691Trp; Asp1769His; Val1857Leu; Gly2026Gln; Arg2163-Pro; Asp228 8Ala; and Arg2304Leu) and a T deletion in exon 25 that caused a frameshift followed by a stop codon. All missense mutations except Val1857Leu, which m aintained a conserved nonpolar R group, occurred at amino acids conserved a mong four species and were most probably pathogenic. In addition, two seque nce changes (IVS3-9C-->T) and (Leu2230Leu) were also detected in patients c arrying Val1857Leu and Phe679Leu missense mutations, respectively. Identifi cation of mutation origins in eight sporadic cases revealed an equal sex ra tio of mutations.