Computational protocol: Association of type 2 diabetes mellitus genes in polycystic ovary syndrome aetiology among women from southern India

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Protocol publication

[…] Sampling criteria: Case and control groups: As part of the larger project entitled, “Identification of susceptibility genes associated with PCOS among Indian women” undertaken by Molecular Anthropology Group of the Biological Anthropology Unit, Indian Statistical Institute, Hyderabad, India, 250 women with PCOS and 299 normal healthy controls were enrolled in the study during 2008-2009. Due to resource constraints, only 210 of the 299 controls and 248 of 250 cases were genotyped for the present study. The patients were recruited consecutively from the Gynecology Clinic of the Osmania General Hospital and Anu Infertility Clinic in Hyderabad as per the Rotterdam criteria. According to these criteria any two of the following three conditions need to be fulfilled for the inclusion: (i) presence of clinical and/or biochemical signs of hyperandrogenism, (ii) infrequent periods with intermenstrual interval of more than 35 days, and (iii) polycystic ovaries [an ovary with the ultrasound appearance of more than 10 subcapsular follicles (<10 mm in diameter) in the presence of prominent ovarian stroma was considered polycystic]. Patients with hyperprolactinaemia, thyroid and adrenal diseases, 21-hydroxylase deficiency and androgen-secreting tumours were excluded from the study. The ethnically matched normal controls with no history of treatment for fertility, with normal menstrual cycles every 25-32 days and with no signs of clinical hyperandrogenism (hirsutism, acne and alopecia) were recruited from the family planning centre of the Osmania General Hospital, Hyderabad and from the general population in the same city, (covering different residential complexes, government offices, academic institutions and software companies). Both cases and controls represented similar ethnic and linguistic backgrounds.Intravenous blood samples (5 ml) were collected from both patients and controls. Informed written consent was obtained from each participant before their enrolment in the study. The Indian Statistical Institute Review Committee for Protection of Research Risks to Humans specifically approved this project and the study protocol.Genotyping of T2DM genes: DNA was extracted using phenol–chloroform method. Fifteen well-replicated SNPs from nine T2DM genes identified through candidate and GWASs were selected. The SNPs included were rs7903146, rs11196205 and rs12255372 (TCF7L2), rs4402960 and rs1470579 (IGF2BP2), rs13266634 (SLC30A8), rs1111875 and rs7923837 (HHEX), rs7754840 and rs7756992 (CDKAL1), rs10811661 (CDKN2A/B), rs1801278 (IRS1), rs3792267 and rs5030952 (CAPN10) and rs1801282 (PPARG). Genotyping of the 15 SNPs was performed on Sequenom MassARRAY platform (Sequenom Inc., San Diego, CA, USA) at the Centre for Genomics Application (TCGA), Delhi, India. The raw data files generated by MassARRAY Sequenom were analyzed for the intensity peaks of calibrant to ascertain the quality of the data. An overall call rate of >90 per cent was maintained. For every 96 samples (a quadrant of Sequenom chip), four samples were duplicated and the call rates were checked for concordance. The calls in the negative control (no DNA) were also monitored in all the runs. The genotype call rate for individual SNPs ranged from 96-99 per cent. Of the 248 cases, only one sample was removed with no call for all the SNPs while in the controls all the samples worked. For the interaction analysis, only samples with complete genotype information were considered for all the nine SNPs.Statistical analysis: Allele and genotype frequencies were calculated using SPSS Statistical Software (SPSS Inc., version 20.0, SPSS, Chicago, USA). Logistic regression analysis of the alleles and genotypes was carried out for all the SNPs individually. Bonferroni correction for multiple testing was carried out by setting P = 0.003 as threshold for significance. Linkage disequilibrium (LD) plots were estimated using Haploview software (version 4.1, Broad Institute, Cambridge, MA), and THESIAS software (version 3.1, INSERM U525, Paris, France) was used to generate haplotype frequencies and logistic regression analysis of the haplotypes. Univariate and multivariate logistic regression analyses and gene–environment interaction were carried out using EpiCalc package of R program version 3.0 (R Foundation,, Gene–Gene interaction was carried out using multifactor dimensionality reduction (MDR) software and permutation tool (University of Pennsylvania, Philadelphia, USA). The power of the study was calculated for individual SNPs using G*Power software (version 3.1, Universitat Dusseldorf, Germany). […]

Pipeline specifications

Software tools Haploview, THESIAS, G*Power
Applications Miscellaneous, GWAS
Organisms Homo sapiens
Diseases Diabetes Mellitus, Endocrine System Diseases, Polycystic Ovary Syndrome