Molecular characterisation of the CYP21 gene for congenital adrenal hyperplasia in Singapore

Abstract

Introduction and Aims: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder, resulting from a deficiency of the cytochrome P450 enzymes which are responsible for the synthesis of adrenal steroids. The most common form is 21-hydroxylase deficiency, which is encoded by the CYP21 gene, located on chromosome 6p21.3. This is a study of the epidemiology of 21-hydroxylase deficiency, and the molecular analysis of CYP21 gene mutations in Singapore. The specific aims were to determine the incidence of 21-hydroxylase deficiency in Singapore, to establish accurate screening techniques to study the CYP21 gene mutations, catalogue the spectrum of molecular variation at the CYP21 gene locus, identify novel mutations, determine genotype-phenotype correlations, and to apply this to prenatal diagnosis, prognostication and treatment. Methods: Retrospective clinical data over the past 21 years was extracted for the epidemiological studies. For the molecular studies, genomic DNA was isolated from peripheral leucocytes, followed by polymerase chain reaction (PCR) and sequencing on the ABI automated sequencer. Novel mutations were analysed to confirm their pathogenicity, using the methods of bacterial cloning, site-directed mutagenesis and transfection studies. For the prenatal studies, DNA was extracted from chorionic villus tissue, followed by PCR and sequencing. Results: The crude incidence of 21-hydroxylase deficiency in Singapore is 4.3 per 100,000 live births, with the carrier frequency of 1/77. In the Singapore cohort of 21-hydroxylase deficient patients, we identified twelve different mutations. The three most common mutations in Singapore are the Intron 2 splice site mutation (33.3 percent), the I172N mutation (21.2 percent) and the R356W mutation (19.9 percent). Two novel mutations were proven to be pathogenic: (1) L261P substitution in exon 7 (2) a duplication of 111 bp in exon 1 from codons 21-57. Prenatal diagnosis has been successfully performed in one Singaporean pedigree. Conclusions: The system for the molecular analysis of 21-hydroxylase deficiency has been established, together with the first catalogue of the common mutations in the CYP21 gene in Singapore. Two novel mutations have been characterized. Genotype-phenotype correlations have been analysed, with new and exciting potential therapeutic applications.