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Tumor Discovery SNPs rs9929218 and rs6983267 in Kurdish CRC

Table 1. The clinicopathological features of CRC patients in confidence intervals (CIs) calculated to estimate the
the study strength of associations.
For categorical variables such as sex, tumor grade,
Variable Classification n (%) TNM stage, perineural invasion, vascular invasion, and
Sex Male 151 (56.5)
tumor location, Chi-square tests were used to compare
Female 139 (43.5) distributions between wild-type and mutant SNP groups.
Age Median 69 (43 – 88) In cases where expected frequencies in any category were
Duke’s stage A 39 (10) below 5, Fisher’s exact test was used as an alternative.
B 97 (31.7) Continuous variables, including tumor size and age, were
C 126 (43.4) assessed using the Mann–Whitney U-test, as these data did
D 28 (7.9) not follow a normal distribution. To adjust for potential
Vascular invasion V0 149 (47.9) confounders, including age, sex, tumor grade, and TNM
staging, logistic regression analysis was applied. All
V1 101 (31.3) statistical tests were two-tailed, and p<0.05 was considered
V2 40 (10.6) statistically significant.
Nodal stage N0 57 (51.3)
N1 150 (34.8) 3. Results
N2 83 (13.7) 3.1. SSP-PCR and genotyping strategy
Tumor stage T1 48 (16.5) Genotyping was performed using real-time PCR followed
T2 37 (12.7) by melt curve analysis, allowing the differentiation between
T3 138 (47.5) wild-type and mutant alleles based on distinct melting
T4 67 (23.1) temperatures. Homozygous wild-type and homozygous
Abbreviation: CRC: Colorectal cancer. mutant samples exhibited single melting peaks, while
heterozygous samples displayed two distinct peaks
were purified using the QIAquick PCR Purification Kit representing both alleles (Figures 1 and 2). To validate the
(Qiagen, Netherlands) following the manufacturer’s accuracy of the PCR-based genotyping approach, a subset of
protocol. Sequencing was performed directly using the six samples – two homozygous wild-type, two homozygous
corresponding PCR primers. The resulting chromatograms mutant, and two heterozygous – was selected for Sanger
were analyzed using Chromas Lite software (v2.01, sequencing. The sequencing results were consistent with
Technelysium Pty Ltd, Australia) and sequence alignment the initial genotyping, confirming the reliability of the
was conducted using the Basic Local Alignment Search PCR-based approach in detecting these SNPs.
5
Tool (BLAST) to compare the sequences with wild-type
reference sequences. All primer sequences are listed in 3.2. Association of rs9929218 and rs6983267 SNPs
Table 2. The PCR reaction was performed in a final volume with CRC
of 25 µL, containing 1× HotShot master mix (Cadama Analysis of genotype distributions revealed a higher
Medical, UK), 250 nM of each primer, and 20 ng template prevalence of rs9929218 (CDH1) and rs6983267 (8q24)
DNA. Thermal cycling conditions were as follows: initial among CRC patients compared to healthy controls
denaturation at 95°C for 5 min, followed by 40 cycles of (Table 3). However, increased prevalence alone does
95°C for 10 s, 55°C for 30 s, and 72°C for 10 s. not establish an independent association, even when

2.4. Statistical analysis statistically significant in univariate analyses. To address
this, we applied both univariate analysis (Chi-square and
All statistical analyses were conducted using SPSS (v.26, Fisher’s exact tests) and multivariate logistic regression
IBM Corporation, US). The Hardy-Weinberg equilibrium adjusting for age, sex, tumor grade, and TNM stage. The
(HWE) was assessed in the control group using the Chi- logistic regression analysis did not confirm either SNP
square test to determine whether genotype distributions as an independent risk factor for CRC, suggesting that
deviated from expected proportions. Associations the observed associations may be influenced by other
between rs9929218 in CDH1 and rs6983267 in 8q24 confounding clinicopathological variables.
with CRC risk were evaluated using Chi-square tests
and Fisher’s exact tests, with odds ratios (ORs) and 95% 3.3. Association of SNPs with CRC risk
To determine whether rs9929218 and rs6983267
5 http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi independently contribute to CRC risk, logistic regression

Volume 4 Issue 2 (2025) 84 doi: 10.36922/TD025110021