Technique of PCR-ACRS for the detection of CYP21 gene mutations / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a rapid method of detecting CYP21 gene mutations.</p><p><b>METHODS</b>Fifty Chinese patients with 21-hydroxylase deficiency and some of their families were investigated. Blood samples were obtained for extraction of peripheral blood lymphocytes. A search for restriction sites discriminating between the morbid and the normal in CYP21 gene was made by the computer program DNAssist. PCR-based amplication-created restriction site(PCR-ACRS) was performed at I172N and R356W which are not natural recognition sequence. In addition, I172N and R356W were analysed in five families which conform to the applicability of PCR-ACRS.</p><p><b>RESULTS</b>In 50 identified 21-hydroxylase deficient Chinese patients, 21 were found to have I172 N (3 were homozygote, 18 were heterozygote); 8 were found to have R356W, all of them were heterozygote. By analysing the families, the findings were consistent with the characteristics of autosomal recessive genetic deficiency.</p><p><b>CONCLUSION</b>Analysis of CYP21 gene point mutations using PCR-ACRS is relatively simple, accurate and feasible.</p>

CYP21 gene point mutations study in 21-hydroxylase deficiency patients / 中华儿科杂志

<p><b>OBJECTIVE</b>The major cause of congenital adrenal hyperplasia (CAH) is 21-hydroxylase deficiency, which accounts for 90% - 95% of all cases in most populations. This study was conducted to characterize the molecular basis of the 21-hydroxylase deficiency and to obtain the spectrum of the CYP21 gene mutations in a group of Chinese patients, and analyze the relationship of genotype and phenotype.</p><p><b>METHODS</b>To detect the distribution of gene mutations in Chinese population samples from 52 patients with 21-hydroxylase deficiency from 51 families were collected, including two parents samples in 30 patients and one parents sample in 10 patients. Blood samples were obtained for extraction of peripheral blood lymphocytes. Polymerase chain reaction (PCR) followed by nesed PCR were used to study the 21-hydroxylase gene (CYP21) mutations. The primary PCR amplified two overlapping CYP21-specific DNA fragments, The product of the nested PCR which used products from the primary PCR was analysed by restriction fragment length polymorphism (RFLP) or amplification-created restriction site (ACRS). All patients were studied by 6 mutations, including P30L, I2g (intron 2 nt 656 c/a-->g splice mutation), E3Delta8nt (exon 3 codon111-codon113 8 bp deletion), I172N, V281L and Q318X.</p><p><b>RESULTS</b>Through analysis of 52 patients with 21-hydroxylase deficiency, in 5 patients no mutations were detected, in 17 patients only one mutated allele could be characterized, two different mutations were identified in 21 patients, three mutations were detected in 2 patients. Totally, in 73% of alleles the genotypes could be detected. The most common mutation was I2g, which present on 31% affected alleles, then followed by I172N, Q318X, V281L, P30L, E3Delta8nt, accounting for 23%, 14%, 9%, 3%, 2% of all identified mutations respectively, which included multiple mutations accounting for 6%. The most frequent molecular defects of the salt-wasting form were the I2g (45.7%), Q318X (26%). Of the simple virilizing form, the dominant mutations were I172N (40.7%) and I2g (18.5%).</p><p><b>CONCLUSION</b>Six different mutations were examined in this study, and the detected mutations accounted for 73% affected alleles, in which I2g and I172N were the most common mutations (accounting for 54%). Correlation between genotypes and phenotypes was compatible with the reported data. Two rounds of PCR followed by RFLP or ACRS analysis may provide important information for genetic counseling and for prenatal diagnosis.</p>