Computational protocol: Investigation of CD28 Gene Polymorphisms in Patients with Sporadic Breast Cancer in a Chinese Han Population in Northeast China

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Protocol publication

[…] SNPs were selected using the HapMap database. Thirteen haplotype-tagging SNPs [rs1879877, rs3181097, rs3181100, rs2140148, rs1181388, rs10932017, rs4673259, rs3769684, rs3116486 (replaced by rs3116487 in this study with r2 = 1), rs3116494, rs3116496, rs12693993, and rs3769686] from the CD28 gene were selected from the HapMap database using Haploview software 4.1, with pair-wise r2<0.8 for each SNP pair and minor allele frequencies >5%. In addition to the SNPs mentioned above, the SNP rs35593994, on which the HapMap database has no information, was selected in this study because it has been reported to be potentially functional. All selected SNPs were validated using extensive database searches (, In total, 14 SNPs were selected to span the entire CD28 gene region. [...] The deviation from Hardy-Weinberg equilibrium (HWE) was determined using a goodness-of-fit chi-squared test to compare the observed genotype frequencies with the expected frequencies, from the healthy controls. The polymorphisms were excluded if they deviated from HWE, or if missing data composed more than 10% of the total data. Using different models of inheritance (additive, dominant, recessive), the genotype frequencies of the subjects were analyzed using the chi-squared test and Fisher’s exact test. Estimated odds ratio (OR) and 95% confidence interval (CI) were assessed using logistic regression with Plink software. To determine the significance with corrections for multiple testing biases, we ran 10,000 permutations using Haploview to determine the P value. All data were analyzed using SPSS (version 17.0), Plink (version 1.07) (, and Haploview (version 4.1) ( The threshold for significance was P<0.05, and the relative risks associated with haplotypes were estimated as odds ratios (ORs), with 95% confidence intervals (CIs).Haplotype patterns were generated using the algorithm described in Haploview , which constructs haplotypes based on the D’ measure of linkage disequilibrium and a LOD score as a measure of significance. The pair-wise linkage disequilibrium for a genotyped SNP was confirmed using r2 values , . SNPs with a minor allele frequency of less than 5% were excluded in our haplotype constructions. Haplotype block definitions were based on the method of Gabriel et al. , with confidence limits for strong LD (upper, 0.85; lower, 0.70), and confidence interval maximums for strong recombination (upper, 0.85) and strong LD (upper, at least 0.8) in informative comparisons. […]

Pipeline specifications

Software tools Haploview, PLINK, SPSS
Applications Miscellaneous, GWAS
Organisms Homo sapiens
Diseases Breast Neoplasms, Neoplasms
Chemicals Estrogens, Progesterone