Mutation analysis of the CYP21A2 gene in the Iranian population

Abstract

Background: Defects in the CYP21A2 gene cause steroid 21-hydroxylase deficiency, which is the most frequent cause of congenital adrenal hyperplasia. Forty four affected families were investigated to identify the mutation spectrum of the CYP21A2 gene.

Methods: Families were subjected to clinical, biochemical, and molecular analyses. Allele-specific polymerase chain reaction amplification was used for eight common mutations followed by dosage analysis to exclude CYP21A2 deletions.

Results: The most frequent mutations detected were gene deletions and chimera (31.8%). Other mutation frequencies were as follows: Q318X, 15.9%; I2G, 14.8%; I172N, 5.8%; gene duplication, 5.7%; R356W, 8%; and E6 cluster mutations, 2.3%. Direct sequencing of the CYP21A2 gene revealed R316X, P453S, c.484insT, and a change at the start codon. Different modules carried by patients were classified into five different haplotypes. The genotype phenotype correlation (positive predictive value) for group null, A, B, and C were 92.3%, 85.7%, 100%, and 0, respectively.

Conclusions: Methods used will be helpful for carrier detection and antenatal diagnosis, especially with inclusion of the multiplex ligation probe dependent amplification technique, which is easier for routine tests in comparison with other methods. Mutation frequencies indicate that Iranians are possible descendants of Asians and Europeans.

FIG. 1.

Position of multiplex ligation probe dependent amplification probes on CYP21A2 and CYP21A1P gene for indicating the haplotypes, gene duplications, gene deletions, and gene conversions. Probe 1 (1) is positioned at 5′CYP21A1P pseudogene at -316 to -264 of regulatory region; this position includes -306G>C, -295T>C, -294A>C, -283A>G, and -281T>G pseudo-derived mutations, which reduce the activity to 50% (Zhang et al., 2009). Probe 2 (2) is located on the complementary sequence of exon 10 of CYP21A1P, which is the opposite strand comprising TNXA gene. Probe 3 (3) shows the C4B gene on exon 19. Probe 4 (4) depicted 18nt upstream of 5′untranslated region of CYP21A2 gene; this site is positioned in -147 to -90 of the regulatory region of CYP21A2, which includes a protein binding site. Variants of -126C>T, -113G>A, -110T>C, and -103A>G are positioned in this probe, which would influence transcriptional activity by fivefold lower than CYP21A2 gene.

FIG. 2.

A scheme representing different haplotypes found in the present study. The black box indicates CYP21A1P pseudogene, and the white box shows CYP21A2 gene. (a) haplotype A: A bimodule form of RCCX comprising a CYP21A1P and a CYP21A2 gene, (b) haplotype C: a trimodule with two CYP21A1P pseudogene formed from unequal crossing over events of C4 gene (c) haplotype D: complete CYP21A2 gene conversion to CYP21A1P, (d) haplotype D CYP21A1P-like gene: partial gene conversion of CYP21A2 to CYP21A1P, (e) haplotype E: a CYP21A1P gene due to deletion of TNXA-RP2-C4B-CYP21A2-TNXB, (f) haplotype E indicated as CYP21A1P-like gene: CYP21A1P-CYP21A2 chimers resulting from gene deletions of CYP21A1P-TNXA-RP2-C4B-CYP21A2, (g) haplotype F: a trimodule with two CYP21A2 genes resulting from TNXA-TNXB chimer.