GENE CONVERSION IN MOUSE 21-HYDROXYLASE GENE. † 633

Congenital adrenal hyperplasia (CAH) is a common genetic disease affecting 1 in 10,000 newborns. CAH is caused by deficiency of 21-hydroxylase (CYP21B). The last exon of CYP21B is overlapped by Tenascin-X (TN-X) and both genes are duplicated in man and mouse. During cloning of a mouse TNX cDNA, we noted its 3′UTR was equally similar to the published cyp21A and B genes, suggesting a gene conversion event might have occurred in the mouse from which these sequences were derived. In humans, gene conversion events alter CYP21B gene sequences to those of the CYP21A gene and are the primary cause of CAH. To identify whether similar gene conversions occur in the mouse, we developed a two-step PCR method to identify a specific conversion in the 3′ UTR of cyp21A using mouse sperm genomic DNA. In the first step, cyp21A-specific primers were selected so that amplification occurs from both normal cyp21A alleles and converted cyp21A-B-A chimeric alleles. 1 to 10⁵ genomes were amplified in separate reactions. Amplification was optimized to produce single copy sensitivity. Products from this reaction were diluted 10,000-fold, and used as template for a second PCR with the same cyp21A-specific sense primer and an antisense primer specific for cyp21B. Using 1 to 10⁵ genomes of liver genomic DNA, no amplification was seen in the second PCR, showing that artifactual recombination during PCR does not occur under the conditions used. Sperm samples were analysed in pools of three; altogether, seven pools were tested. Each pool showed single-molecule sensitivity in the first PCR. Gene conversion was detected with a frequency of 1 in 10³ in one pool, 1 in 10⁴ genomes in four pools, while the remaining two pools showed no conversion in 10⁴ genomes. Because low frequency events are being detected in the second amplification, this variation is the expected result. These experiments show that gene conversion occurs in the murine cyp21 genes with a rate of 1 in 10⁴ meioses. This method will allow study of the molecular mechanism(s) of cyp21 gene conversion.