Hemophilia A (HA), the deficiency of coagulation factor VIII (FVIII), is the most common, sex-linked inherited bleeding disorder. The disease affects one in every 5,000 male births [1]. The FVIII gene is 186 kb in length and has 26 exons. Approximately 280 different mutations (deletional types) have been identified in this gene, which maps to the Xq28 locus (). The disease is caused by a wide range of heterogeneous mutations in the FVIII gene and leads to a partial or total deficiency of FVIII protein activity [2,3]. In this study, we have examined 61 unrelated patients with HA (37 severe types, 17 moderate types and seven mild types) to identify the incidence of intron 1-FVIII gene inversion in the West Anatolian population.

Peripheral blood samples (2 mL) were collected with EDTA as anticoagulant from 61 unrelated patients with HA. The FVIII clotting activity was analyzed by the one-stage clotting assay and clinical criteria of Eyster et al. [4], were used to determine disease severity. Total genomic DNA was extracted from peripheral blood leucocytes by the modified proteinase K method [5]. A detailed family history of the disease and the inhibitor status were also obtained. We performed two amplifications under polymerase chain reaction (PCR) conditions as suggested by Bagnall et al. [3]. One reaction contained tree primers: int1h-2, int1h2R and 9F. These PCRs are able to differentiate wild-type alleles (1300 and 1900 bp) from patients with inversions and patients with inversions from carriers. The amplified segments were readily separated on a 2% agorose gel.

We have examined 61 unrelated patients with HA (37 severe types, 17 moderate types and seven mild types; median age 26 (age range 3 to 58). In this study, an inhibitor was detected in 12 (19.6%) patients, arthropathy was detected in 45 (73.7%) patients and family history was detected in 37 (59.6%) patients (Table 1). According to the result of DNA analysis of 61 HA patients, intron 1 inversion of the FVIII gene was not found. A mutant DNA sample was obtained by Dr. Cemal Un (Bo—aziçi University, Istanbul, Turkey) from a patient and used as a positive control in every amplification (Fig. 1).

Thus far, the intron 1 inversion of the FVIII gene has not been investigated in West Anatolian HA patients, whereas this inversion has been detected in Italian (1.7-5%), in Spanish (5%), in the Czech Republic (4.3%), in the UK (1.8-3.8%), in Iranian (1.5%) and in Indian (3.8%) HA patients [6-11]. Although the number of HA patients is low, the results of this study provide the first data from the West Anatolian Turkish population. Further studies will be required to determine the intron 1 inversion of the FVIII gene in larger populations.