Abstract:
21-hydroxylase deficiency is the major cause of congenital adrenal hyperplasia (CAH), a disorder that results in impaired Cortisol biosynthesis. It is caused by mutations in the 21-hydroxylase gene that codes for the steroid 21-hydroxylase enzyme. The majority of these mutations have arisen as a result of recombination events between the active gene (CYP21B) and a highly homologous pseudogene (CYP21A). Gene deletions, caused by misalignment and unequal crossing over between the CYP21A and CYP21B genes, account for 20% of these mutations while the remainder are apparent gene conversions.
Previous studies have indicated that the unequal crossovers occur predominately in meiotic cells with a frequency of approximately 1 in 10 5 – 10 6 genomes, and that gene conversions occur in both meiotic and mitotic cells at a frequency of 1 in 10 3 – 10 5. The product of an unequal crossover is a chimeric gene (CYP21A/CYP21B) in which the 5' region corresponds to CYP21A and the 3' region corresponds to CYP21B. This research uses gene-specific PCR to analyse the frequency of unequal crossing over within the CYP21 gene in genomic DNA enriched by restriction enzyme digestion of genomic DNA and agarose gel electrophoresis, followed by excision of the appropriate CYP21A fragment (3.2 kb). This approach minimises artefacts caused by in vitro recombination during the PCR. Estimation of the number of chimeric genes in an enriched sample was by serial dilution to a null point (the point at which there is no amplifiable copy). The PCR assay was sensitive enough to detect a single copy of chimeric gene. Detection of rare chimeric genes in a high background of CYP21A and CYP21B required high numbers of PCR cycles, however, this generated artefacts caused by in vitro recombination. The enrichment for CYP21A and CYP21B solved this problem by eliminating non-specific target genes, thus minimising non-specific amplification. In this study, no de novo recombinations were detected using 10 7 copies of matched sperm or leukocyte DNA, suggesting that data from previous studies were likely to be erroneous due to PCR artefacts.
