Computational protocol: Common Genetic Variants of the Human Steroid 21-Hydroxylase Gene (CYP21A2) Are Related to Differences in Circulating Hormone Levels

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Protocol publication

[…] The gene copy numbers of C4 and CYP21A2, and the haplotypes of intron 2 and the 5′-part of exon 3 of CYP21A2 (the region will be henceforth denoted as ‘CYP21A2 intron 2′) were determined as we have previously described , , . Briefly, C4-type-specific quantitative PCRs (qPCR) , and CYP21-gene-specific qPCR were applied to determine gene copy numbers. Haplotypic or genotypic nested polymerase chain reaction (PCR) products of CYP21A2 were generated from the templates of allele-specific long-range PCRs , then CYP21A2 intron 2 was sequenced by SEQ_12F_v2 and SEQ_16R sequencing primers . In addition to the CAH mutations detected by the aforementioned methods, the six commonly occurring CAH mutations (I172N, exon 6 cluster, p.V281L, p.L307insT, p.Q318* and p.R356W) were also determined . Experimentally indeterminable haplotypic CYP21A2 intron 2 sequences were resolved by the PHASE software v2.1.1 , . A phylogenetic tree of the CYP21A2 intron 2 haplotype variants and Hardy-Weinberg equilibrium (HWE) were calculated by Arlequin v3.5 . Linkage disequilibrium (LD) was evaluated using DnaSP v5.10.01 , and was visualized by Haploview v4.2 . [...] To find the relationship between hormone levels and CYP21A2 intron 2 haplotypes, the haplotype tree of CYP21A2 intron 2 was scanned by TreeScan v1.0 . Traditional statistical analyses were performed with G*Power v3.1.3 , SPSS v20 (SPSS Inc., Chicago, IL, USA) and STATISTICA v8 (Statsoft inc. Tulsa, OK, USA) software. Briefly, the outliers of hormone data were filtered based on a two-sided standard deviation, and the normal distributions of hormone datasets were tested by the Kolmogorov-Smirnov test. The datasets passing the normality test were analysed by unpaired t-test and analysis of variance (ANOVA), and the datasets with non-normal distribution were examined by Mann-Whitney and Kruskal-Wallis tests. The genetic associations adjusted for gender, age, tumor diameter and body mass index (BMI) and correlations between steroid hormone levels and adjusting variables were calculated by multiple logistic regression analysis with cutoff values obtained by receiver operating characteristic (ROC) analysis. All tests were two-tailed. […]

Pipeline specifications

Software tools Arlequin, DnaSP, Haploview, G*Power, Statistica
Applications Miscellaneous, Phylogenetics, Population genetic analysis, GWAS
Organisms Homo sapiens
Diseases Adrenal Hyperplasia, Congenital, Cardiovascular Diseases
Chemicals Aldosterone, Cortodoxone, Hydrocortisone, Metyrapone, 17-alpha-Hydroxyprogesterone