[Methylation of the factor IX gene--a basic reason for the mutation causing hemophilia B]

The analysis of 750 mutations in the gene of blood coagulation factor IX has been made for 806 patients with haemophilia B. It has been found that 40% of all point mutations take place in 11 "hot spots", i.e., methylated CG sites, and are CG-->TG or CA transitions. Mechanism proposed explains the high rate of these transitions by m5C deamination during replicative DNA methylation and by mistakes of G/T-repair. This is why mutations constantly occur de novo in the factor IX gene, and haemophilia B maintains on a high level. Asymmetry of C-->T and G-->A transitions is found in some CG sites at different DNA chains, and is related to occurring "missense" mutations, which usually escape from the detection. Summing up these substitutions, CG methylation of the factor IX gene contributes to 50% of all point mutations. Thus, mutations in the CG sites take place in overall 48 times more frequently than in other sites of the gene. It has been found that at least 35 new CG sites are a result of sporadic mutations, and methylation and mutations of these sites may cause 14% of substitutions in the factor IX gene. The "hot spots" for T-->C transitions in codon of Ile397 can be a result both of "founder effect" and of recessive mutations in such CG site in mother's parents. It is shown that methylated CTCG sites, as well as G/T-repair mistakes, can be potential sources of 5.4% of mutations in the gene. Thus, from 50 to 70% of all mutations may be the result of the gene methylation in human genome. The analysis of duplex frequencies in the factor IX gene has shown that the loss of 60 CG sites and the accumulation of 8% of mutations may be a result of "fossil" CG methylation. The remained 20 CG sites are located in the codons of those amino acids which positions are more critical for the factor IX activity. It has been proposed that the most radical way in haemophilia B therapy can be a protection of the factor IX gene from methylation in the human genome.