Background: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease caused by mutations in the CYP21A2 gene, which codes for steroid 21-hydroxylase. More than 90% of patients with CAH have mutations in CYP21A2 or have large deletions in the RCCX module on chromosome 6p21.3, which also includes the pseudogene CYP21A1P. Genotyping of CYP21A2 is required for diagnosis of CAH, but current genotyping methods, such as direct sequencing, allele-specific PCR amplification, or PCR amplification and restriction fragment length polymorphism (PCR-RFLP) still need further improvements to reduce test time and cost.
Methods: We developed a novel CAH mutation screening method based on allele-specific primer extension (ASPE), followed by bead-array hybridization, for the ten major point mutation sites and the 8 bp deletion in CYP21A2, and a long PCR assay to detect large deletions between CYP21A1P and CYP21A2. After the first long PCR amplification, a second short PCR amplification was adapted to increase the ASPE efficiency. The total genotyping procedure takes approximately 8 hours.
Results: Eighteen CAH patients and two controls were tested using the bead-array method. Homozygous or heterozygous large gene deletions and three point mutation sites were detected by this method, and most of the results were consistent with sequencing or PCR-RFLP analysis. Nine of the 18 patients had a large deletion in the RCCX module, which was not easily detected using the conventional genotyping method.
Conclusion: A novel CAH mutation screening method has been developed to detect ten point mutations and the 8 bp deletion in CYP21A2, as well as large deletions between CYP21A1P and CYP21A2. This novel genotyping strategy is superior to PCR-RFLP-based methods and equally as accurate as sequencing.