The results of Southern blot analysis for detection of an inversion in intron 22, were obtained after radiography. Hybridization signals of 21.5, 16.0 and 14.0 kb fragments were displayed when normal DNA was analyzed; 20.0, 17.5 and 14.0 kb fragments when DNA with an inversion type 1 was analyzed, and 20.0, 16.0 and 15.5 kb fragments when DNA with the inversion type 2 was analyzed (Fig. 1). The inversion in the fVIII gene was identified in 19 out of 50 patients, all with a severe form of the disease. The incidence of the intron 22 inversion in hemophilia A patients from the Republic of Macedonia was 38% (19/50) of all screened patients, or 67% (19/29) of patients with a severe form of hemophilia A. Among 19 inversions found in hemophilia A patients from the Republic of Macedonia, 18 (94.7%) had the distal inversion type 1, while one patient had the rare form of inversion, type 3A. None of the hemophilia A patients from the Republic of Macedonia had the proximal inversion type 2. The incidence and type of inversion among our patients of different ethnic groups is shown in Table 1. The mothers of 15 cases with inver­sions were also studied, and all were found to be heterozy­gotes for the inversion.

The patients who were excluded for the presence of an inversion in intron 22, were further analyzed for the pres­ence of single base substitutions and small deletions/ insertions in the coding regions of the fVIII gene. A total of 11 different point mutations were found in 15 unrelated hemophilia A patients (Table 2). Four of these sequence changes were novel single base substitutions [ATG®AGG (Met®Arg), exon 1, codon 19; GCT®CCT (Ala®Pro), exon 3, codon 78; TGC®GGC (Cys®Gly), exon 24, codon 2174, and TAT®GAT (Tyr®Asp), exon 25, codon 2256]; while seven identified point mutations [GAA® TAA (Glu®Term), exon 7, codon 272; TAT®TGT (Tyr® Cys), exon 10, codon 473; CGG®TGG (Arg®Trp), codon 527, exon 11; GCC®ACC (Ala®Tyr), exon 14, codon 704; CGC®TGC (Arg®Cys), exon 14, codon 1689; CGA®TGA (Arg®Term), exon 14, codon 1696; CGC® TGC (Arg®Cys), exon 23, codon 2159] had already been reported in the hemophilia A database. The mutation Arg2159® Cys appears to be frequent among hemophilia A patients from the Republic of Macedonia, since the same mutation was found in four unrelated patients with a mild form of the disease.

Table 1. Incidence and type of inversion in intron 22 of the factor VIII gene among 50 studied patients of different ethnic origins from the Republic of Macedonia