Novel mutations in CYP21 gene in patients with 21-hydroxylase deficiency in Iran
Purpose: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common autosomal recessive disorders with the incidence rate of 1 in 10,000 births in its classical forms. The aim of this study was to design a two step mutational analysis to first detect the most common CYP21 gene mutations (del-CYP21, del-8bp, i2g, R356W, I172N, P30L, E6 cluster, Q318, V281L) using common molecular techniques and then detect the unknown mutations in our population using direct sequencing. Phenotype-genotype correlation in Iranian 21-hydroxylase deficient patients and prenatal diagnosis was performed.
Methods: Molecular analysis of the CYP21 gene for detection of its most common mutations was performed in 40 patients with classical CAH using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR), amplification-created restriction site (ACRS), allele specific-PCR (AS-PCR), semiquantitative-PCR (SQ-PCR) and direct sequencing.
Results: We characterized 90% of the CAH chromosomes. The most frequent mutations were del-CYP21 (25%), I172N (22%) and i2g (15%). Unlike other ethnic groups, there was no R356W mutation, however, a higher rate of del-8bp (10%) was found. We also found 6 complex alleles in our patients. Prenatal CYP21 gene analysis for 5 families resulted in the exact diagnosis and appropriate treatment. Phenotype-genotype correlation was observed. We used direct sequencing for samples with unknown mutation which resulted in detection of 2 novel mutations, F404S in exon 9 and a del-3bp in exon 1 (134-136 del.), in 3 patients with salt-wasting form of CAH.
Conclusion: Our genotyping approach allowed reliable and rapid diagnosis of CAH patients and prenatal diagnosis in Iranian population. The novel del-3bp in exon 1 seems to be a common mutation in our population which needs more investigation.