Functionally important regions of the factor IX gene have a low rate of polymorphism and a high rate of mutation in the dinucleotide CpG.

We have recently described genomic amplification with transcript sequencing (GAWTS), a three-step procedure that allows direct genomic sequencing. By GAWTS more than 100,000 bp of sequence have been generated from eight regions of the factor IX gene, which include the putative promoter region, the coding region, and the splice junctions. All eight regions were examined in 20 unrelated normal individuals of defined ethnicity and subsequently in 22 hemophiliacs in different families. The following three major conclusions emerge: (1) The rate of polymorphism in these eight regions of functional significance has been measured in an X-linked gene, and it is about one-third of the average rate observed for intronic and intergenic sequences on the X chromosome. The rate is low enough that the causative mutation should be the only sequence change seen in the overwhelming majority of hemophiliacs. (2) Transitions of CpG account for 31% (5/16) of the distinct mutations and for 38% (5/13) of the single-base changes. The rate of transitions at CpG is elevated by an estimated 77-fold, presumably owing to lack of repair of thymidine generated by the spontaneous deamination of 5-methylcytidine. (3) High-quality, reproducible sequence data can be obtained on a time scale that makes direct carrier testing and prenatal diagnosis feasible.