Inversions of the factor VIII gene in Slovenian patients with severe haemophilia A

To the Editor: Haemophilia A is a chromosome X-linked hereditary bleeding disorder, affecting approximately one male in five to ten thousand Caucasians. It is caused by deficient factor VIII (FVIII). The human factor VIII was cloned in 1984 by Gitschier et al. It comprises 186 000 base pairs and maps to the long arm of chromosome X at position Xq28. The gene has 26 exons producing an mRNA transcript approximately 9 kb in length (1). The 22nd intron of the FVIII gene was shown to be unusual in many respects (2, 3). It is the largest intron in the gene, comprising 32 kb. It contains a CpG island about 10 kb downstream of exon 22, which appears to serve as a bi-directional promoter for two genes, referred to as factor VIII-associated genes A (F8A) and B (F8B). F8A is completely contained within intron 22 but is arranged and coded in the reverse direction. Two additional copies of F8A have been found approximately 400 kb telomeric of FVIII gene. F8B is transcribed in the same direction and shares exons 23-26 with the FVIII gene. The telomeric F8A copies are implicated in almost half of severe haemophilia A cases (4), causing an intrachromosomal inversion defect in these patients. The translation of mRNA in these patients terminates shortly after exon 22, rendering patients devoid of any factor VIII protein. Depending on the copy of F8A involved in the inversion, the resulting altered pattern, of BclI digested genomic DNA, can be classified into proximal (type A) and distal (type B). The unusual intrachromosomal inversion defect is hypothesised to be due to the intragenic and extragenic copies of gene A, the proximity of this region to the telomere, and its location on the long arm of the X chrosomosome (5) . In this letter we are reporting results of the FVIII intron 22 inversion analysis in severe haemophilia A patients of the Slovenian population of about 2 million inhabitants. Thirty-nine severe haemophilia A patients attending the Haemophilia Centre, Paediatric Clinic of Ljubljana, were included in the present study. Thirty-seven patients were severe cases with FVIII:C< 1 IU/ml. Two patients (HA32 and HA86) were borderline cases with FVII1:C 1.2-1.3 IU/ml. Coagulation assays were performed with the modified APTT method (Activated Partial Thromboplastin Time Method, IL Test Kit 97570-10, IL). Seven patients have factor VIII inhibitor antibodies according to the Bethesda assay (6). Samples of peripheral blood were collected in test tubes containing EDTA. DNA was isolated with the standard salting-out procedure. 10 pg of DNA were digested with 60 units of BclI overnight at 50 "C and electrophoresed on 0.7% 1 cm thick agarose gels at 30 V for 20 h. The gel was depurinated for 10 min with 0.25 M HC1, denatured in 0.5 M NaOH, 1.5 M NaCl for 20 min, neutralised in 0.5 M Tris-HCl pH 7.5, 3 M NaCl for 30 min and blotted overnight onto Hybond N+ membrane (Amersham) using l0XSSC buffer. The membrane was prehybridized for 1 h and hybridized overnight at 42 "C in 50% formamide, with the 0.9 kb EcoRI/SacI fragment from plasmid p482.6 used as a probe, radiolabeled with 32P using random hexamers (Amersham). The membrane was washed for 20 min in 2XSSC, 0.1% SDS at room temperature, then in OSXSSC, 0.05% SDS at 50 "C for an additional 35 min and finally washed in 2XSSC. Of the 39 patients included in the study 20 showed the presence of 21.5, 16 and 14 kb bands seen in normal DNA. Nineteen patients showed abnormal band patterns (Table 1). In 14 patients recombination with the distal gene F8A occurred, seen as 20, 17.5 and 14 kb band pattern after Southern blotting. This type of recombination is designated as inversion B. In 2 patients the inversion occurred with the proximal gene F8A, resulting in a band pattern of 20, 16 and 15.5 kb, designated as inversion type A. Three patients demonstrated unusual band patterns, designated as inversion X. Of the seven patients with inhibitors, four had the inversion of intron 22; three had the distal inversion (type B), one had the proximal inversion (type A) and three had a normal band pattern.