METHYLATION OF THE FACTOR-IX GENE IS THE MAIN SOURCE OF MUTATIONS CAUSING HEMOPHILIA-B

A total of 750 mutations in the human coagulation factor IX gene in 806 patients with hemophilia B were analyzed. It was found that 40% of all point mutations occur in 11 ''hot spots,'' which are CG-methylation sites where *CG-->TG or CA substitutions take place. A mechanism is proposed which explains the high frequency of such transitions by 5mC deamination during the replicative DNA methylation and by misrepair of G . T pairs. Such processes may be the main source of mutations in this gene, which repeatedly occur de novo and support the incidence of hemophilia B at a high level. Asymmetry of C-->T and G-->A transitions in some CG sites was found in complementary DNA strands, resulting in ''silent'' mutations which usually escape detection. When such substitutions are taken into account, it becomes evident that CG methylation of the factor IX gene generates up to 50% of all point mutations; this is why the rate of mutations at CG sites is 48-fold higher than at any Other position of the gene. As a result of such mutations, at least 35 new CG sites originate sporadically in the gene. Methylation and mutation of these sites may cause up to 14% of all the observed point substitutions in the factor IX gene. The origin of T-->C transition ''hot spot'' in the Ile-397 codon may be explained not only by the ''founder effect'' but also by recessive mutations in such a CG site in the maternal ancestors. It was calculated that *C(C)(T)G methylation sites as well as some alterations of G/T repair potentially may cause up to 5.4% of mutations in the gene. Summing up, 50-70% of all the mutations in the human factor IX gene may occur through methylation. Analysis of doubler frequencies in the factor IX gene showed that as a result of ''fossil'' methylation approximately 60 CG sites Vanished and 8% of *CG-->TG+CA substitutions were accumulated. The remaining 20 CG sites are located in the codons of the amino acids which are crucial for the factor IX activity. One may assume that one of the methods of treating hemophilia B could be protection of the gene in the human genome from methylation.