br The variables were colorectal cancer positivity of
The variables were colorectal cancer, positivity of gene variants, ethnicity, age and gender. The population of Lorestan province includes Lur and Lak ethnicities. We have previously
2.5. Data source and measurements
From each participant, 2 ml of peripheral blood was taken in EDTA containing tubes. The genomic DNA was extracted with salting-out method. After quality and quantity evaluation of the extracted DNA the samples were at -20°C till the time of test. Polymerase chain reaction with sequence specific primers (PCR-SSP) was used to genotyping the samples using KIR-type and EPITOP-type (BAG, Lich, Germany). These processes were done in immunology laboratory of Lorestan University of Medical Sciences under technical and ethical supervision of the university. The genotyping method of KIR was based on haplotypes of centromeric and telomeric ends. These haplotypes included centromeric A (cA), centromeric B (cB), centromeric X (cX), telomeric A (tA), telomeric B (tB) and telomeric X (tX). Haplotype- and genotyping process were done using a researcher designed Excel file. Along with each 1469284-79-4 B/X score was reported.
Management data software of our hospital was used to assess patients' information. In their electronic documents we assessed to their pathology and surgery reports. The final approve of their eligibility for this study was done by our gastroenterologist coauthor.
The potential source of bias could be ethnicity purity (population stratification) and family relationship of the participants because of traditional background of the culture of our region. This bias was controlled through a complete history taking using dialect of this region.
2.7. Statistical Methods
In order to find disease association of each gene, haplotype or genetic condition, 2 by 2 table was used. Odds ratio (OR) with 95% confidence interval (CI) were reported as the extremity of association. The significance of each association was evaluated with chi-square test (Pearson and Yate's correction) at alpha =0.05 as the significance level. Bonferroni's correction was used for multiple comparison. Effect of B/X score on prediction of colorectal cancer was investigated using logistic regression analysis. In order to compare the results with other previous studies, funnel plot and cluster analysis was used. Analyses were performed using STATA software version 14 (StataCorp LLC, US). Evaluation of Hardy Weinberg equilibrium was applicable for HLA-C1 -C2 as a biallelic gene .
A number of 50 colorectal cancer patients were imported to our study based on the inclusion and exclusion criteria mentioned above. This part was approved by our gastroenterologist clinical diagnosis and registry-based information of the hospital. All the participants were attempt for genotyping and all of them were successful.
All the 150 participants were Lur and resident of Lorestan province of Iran. The gender ratio was equal in both groups and the age range was 40-70 and 35-60 in the case and control groups respectively. There was no missing participant.
The gel-based PCR analysis was done in 27 vials for each participants (figure 1). Based on this analysis, the frameworks 2DL4, 3DL2 and 3DL3 was observed in all 150 participants. In addition, pseudogenes 3DP1 and 2DP1 was observed in 150 and 147 participants respectively. 2DL1 was observed in 98% of the patients and 98% of the controls. The other genes had different distributions among cases and controls (table 1).
Among the genotypes of KIR, 22 genotypes were found in this population. Like other populations, the genotype AA (cAcA-tAtA) was the most frequent one observed in 20% of the cases and 29% of the controls. Other genotypes of KIR and genotypes of HLA are shown as well (figures 2 and 3).
3.4.1. Single gene and interaction analysis
Among the single gene inferential analyses of KIR and HLA, only the association of KIR2DS5 was statistically significant (Pearson chi-square P =0.0206; Yate's corrected chi-square P =0.0323; OR =2.25 [1.12-4.49 95% CI]). After applying Bonferroni's correction, this association would not be remained significant (table 1). No significant association was observed for neither KIR nor HLA genotypes (figures 2 and 3). Among the KIR-HLA interactions, no significant association was observed as well (table 2).
Among the haplotype inferential analyses, telomeric B haplotype was more frequent in the cases (52% vs 35%) (Pearson chi-square P =0.0457; Yate's corrected chi-square P =0.0685). No significant association was observed for other haplotypes (table 3). In addition, no significant predictive association was found B/X score of KIR genotypes, of course with a positive trend (figure 4).