Computational protocol: DEFB1 polymorphisms are involved in susceptibility to humanpapillomavirus infection in Brazilian gynaecological patients

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Protocol publication

[…] We enrolled 356 women at the Oswaldo Cruz University Hospital and Clinical Hospital in the state of Pernambuco, northeastern Brazil. The study included 154 HPV-infected women (mean age, 36.3 years; range, 16-70 years): 40 women with normal cytology (no lesions), 48 women with cervical intraepithelial neoplasia (CIN) grade I, 28 women with CIN II, 25 women with CIN III and 13 women with cervical cancer.The control group consisted of 202 women (mean age, 36.5 years; range, 18-65 years) with normal cytology and HPV-negative status [healthy controls (HCs)]; no information was available concerning the possible exposure of the HCs to HPV. All of the women were from the same geographical area (Recife metropolitan region), were HIV-negative and were not being treated with immunosuppressive medication.We evaluated the genomic ancestries of the patients and controls following the methodology of , with an in-house modification consisting of the genotyping of 12 genetic ancestry markers (, unpublished observations). The cases and controls presented the following similar distributions of genomic contribution: approximately 60% contribution from European, 23% from African and 17% from Amerindian ancestral populations. DNA isolation and HPV typing - Genomic DNA was extracted from cervical swabs using the DNeasy Blood and Tissue Kit (Qiagen Inc, USA) in accordance with the manufacturer’s manual.HPV typing was performed by polymerase chain reaction (PCR)-based amplification of the viral L1 gene fragment using the MY09/11 degenerate primers (, ). DEFB1 genotyping - The three DEFB1 polymorphisms in the 5’UTR and the two in the 3’UTR were genotyped using Taqman (Life Technologies, USA) allele-specific fluorescent probes C__11636795_20 (g-52G>A), C__11636794_10 (g-44C>G), C__11636793_20 (g-20G>A), C___8845558_10 (c.*5G>A) and C___8845559_10 (c.*87A>G) on the ABI 7500 SDS real-time PCR platform (Life Technologies). The genotyping results were checked by direct sequencing of 50 randomly chosen amplicons and a 100% concordance rate was achieved. Statistical analysis - The allele and genotype frequencies of the DEFB1 polymorphisms were calculated by direct gene counting; the haplotype frequencies and linkage disequilibrium (LD) were computed using Arlequin software v.3.5 () and Haploview (.Fisher’s exact test was used to test the differences between the proportions of the study groups (comparing the allele, the genotype in general, the dominant models and the haplotype frequencies using 2 x 2 and 3 x 2 contingency tables, as appropriate) and the odds ratios (ORs) and 95% confidence intervals (CIs) for the comparisons were calculated. Adjustments for multiple tests were performed using several correction methods (). R software v.3.0.0 () was employed for the statistical analyses. Research ethics - The study procedures were performed in accordance with the ethical standards of the Declaration of Helsinki. The Federal University of Pernambuco (Recife, Brazil) and the University of Pernambuco Research Ethical committees approved this study (protocol CAAE 03606212.7.0000.5208, HUOC/PROCAPE 64/2010). Written informed consent was obtained from all of the patients and controls. […]

Pipeline specifications

Software tools Arlequin, Haploview
Applications Population genetic analysis, GWAS
Organisms Homo sapiens, Human papillomavirus
Diseases Urogenital Abnormalities, Papillomavirus Infections