Computational protocol: A missense polymorphism (rs11895564, Ala380Thr) of integrin alpha 6 is associated with the development and progression of papillary thyroid carcinoma in Korean population

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Protocol publication

[…] We enrolled 104 PTC patients (29 males and 75 females) and 318 control subjects (105 males and 213 females). PTC patients were selected among participants who visit at the Departments of Surgery and Otolaryngology-Head and Neck Surgery. Subjects with nodular hyperplasia, anaplastic carcinoma, follicular carcinoma, double primary of PTC and follicular carcinoma, and follicular variant of PTC were excluded. PTC was confirmed by pathologic examinations. Controls were recruited from healthy participants through a general health check-up program. Subjects with thyroid disease, cancers, and any severe diseases were excluded. Informed consent was obtained from all subjects. This study was conducted in accordance with the guidelines of the Helsinki Declaration. Patients were divided into subgroups in accordance to the size (<1 cm and ≥1 cm), number (unifocality and multifocality), location (one lobe and both lobes), extrathyroidal invasion (present and absent), lymph node metastasis (present and absent), and angiolymphatic invasion (present and absent).For the selection of ITGA6 SNPs, we searched the promoter and coding regions of the ITGA6 gene in the SNP database of the National Center for Biotechnology Information (, BUILD 132). The SNPs with unknown heterozygosity or heterozygosity below 0.1 and unknown minor allele frequency (MAF) or MAF below 0.1 were excluded. Out of 14 promoter SNPs, there were 3 unknown heterozygosity, 2 heterozygosity below 0.1, 1 unknown MAF, and 7 MAF below 0.1. Among 12 missense SNPs, SNPs with unknown heterozygosity or heterozygosity below 0.1 were 9 and SNPs with unknown MAF were 2. Finally, one promoter SNP (rs2141698, -1687A/G) and one missense SNP (rs11895564, Ala380Thr) were selected.Genomic DNA was extracted from peripheral blood using DNA Isolation Kit for Cells and Tissues (Roche Diagnostics Co., Indianapolis, IN, USA) and genotyping of each SNP was determined using direct sequencing (Macrogen Inc., Seoul, Korea). Polymerase chain reactions (PCRs) were conducted with the following primers: for rs2141698 (sense, 5'-GTTAACAGCTGGTGTGACATGG-3'; antisense, 5'-TTAGCCTTTTCCCTTCGTGTAA-3'; product size, 324 bp) and for rs11895564 (sense, 5'-GGAGCCCCACAGTATTTTGATA-3'; antisense, 5'-TAGTTTCTCCCATGTTGGTCAGG-3'; product size, 348 bp). PCR comprised 40 cycles at 94℃ for 30 seconds, 58℃ for 30 seconds, 72℃ for 30 seconds, and 1 cycle at 72℃ for 5 minutes for the final reaction. The PCR products were sequenced using the ABI PRISM 3730XL analyzer (Applied Biosystems, Foster City, CA, USA) and the sequencing data were analyzed by the SeqManII software (DNASTAR, Madison, WI, USA).Hardy-Weinberg equilibrium (HWE) for each SNP in PTC patients and control subjects was estimated by the chi-square test. SNPStats (, SNPAnalyzer Pro (Istech Inc., Goyang, Korea), and Helixtree (Golden Helix, Bozeman, MT, USA) were performed to obtain odds ratios (ORs), 95% confidence intervals (CIs), and P-values. Multiple logistic regression analysis and the Fisher's exact test were conducted using the following models: codominant1 (major allele homozygotes vs. heterozygotes), codominant2 (major allele homozygotes vs. minor allele homozygotes), dominant (major allele homozygotes vs. heterozygotes + minor allele homozygotes), recessive (major allele homozygotes + heterozygotes vs. minor allele homozygotes), and log-additive (major allele homozygotes vs. heterozygotes vs. minor allele homozygotes). Age and gender as covariates were adjusted to obtain statistical significance. Haploview ver. 4.2 (Daly Lab, Cambridge, MA, USA) was used to determine the linkage disequilibrium (LD) block and haplotypes between two SNPs. The data was also analyzed using SPSS ver. 18.0 (SPSS Inc., Chicago, IL, USA). The statistical significance level was set at P < 0.05. […]

Pipeline specifications

Software tools snpStats, HelixTree, Haploview
Application GWAS
Organisms Homo sapiens