br Table br Association of PARK
Association of PARK2 Promoter rs2276201, rs9347683 SNPs with clinicopathological, life style and environmental characteristics of colorectal cancer patients from North India.
Genotype(n) aOR P value Genotype(n) aOR P value
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Table 3 (continued)
Genotype(n) aOR P value Genotype(n) aOR P value
P < 0.00125, P values significant after Bonferroni coeﬃcient. aOR age adjusted odds ratio, CI confidence interval. P value and corresponding age-adjusted OR (aOR) with 95% Cls[aOR(95% CI)] by logistic regression analysis.
Association of tumor grade with alcohol and smoking.
Characteristics Tumor grade
P is the Chi square value and aOR age adjusted odds ratio, CI confidence in-terval.
Haplotype frequencies in all cases and controls.
Haplotypes Cases Controls P value Odds ratio [95% CI]
Loci chosen for the haplo-analysis are rs2276201 and rs9347683. OR odds ratio, CI confidence interval. P value and corresponding OR with 95% CI for Fisher's exact test.
There were no significant diﬀerences between rs2276201 and rs9347683 genotype distribution among patients in terms of diﬀerent clinicopathological parameters (Table 3). Sub-analysis of tumor grades with alcohol drinking 1.307 (0.725–2.357) and smoking 1.448 (0.791–2.652) suggested that both these parameters increases the risk of tumor grade worsening but statistical significance could not be ob-tained (Table 4).
We estimated the haplotypes of the PARK2 rs2276201, rs9347683 polymorphisms and compared their frequency distributions between the cases and controls. The D' and r2 values showed that these two polymorphisms were not in LD. The inferred haplotype distribution for the controls and cases and the colorectal cancer risk related to each haplotype are shown in Table 5. Although these two SNP's were not in LD but interestingly minor allele combination CC exhibited significant risk (OR: 1.618, 95% CI: 1.112–2.352, P = 0.011) for CRC development while the major allele combination TA showed significant protection (OR: 0.732, 95% CI: 0.543–0.987, P = 0.040) against CRC. Thus we conclude that individuals having minor AM251 of rs2276201 and rs9347683 SNPs in PARK2 promoter are at a higher risk of developing CRC when compared to individuals having major allele at the same positions.
Fig. 2. Representative MS-PCR gel picture showing promoter hypermethylation of PARK-2 in colorectal tumors. Where —T: tumor specimens, PC: positive control, NC: negative control; L: ladder (100 bp), M: methylation (178 bp band), and UM: unmethylation (160 bp band).
The genotype frequencies of PARK2 promoter SNPrs2276201 showed a notably statistical significant association (P = 0.047) with colorectal cancer susceptibility in North India population (Table 2) while no overall association (P = 0.113) was seen with rs9347683
3.3. PARK2 promoter methylation and its correlation with clinicopathological parameters
The representative gel for PARK-2 promoter methylation analysis is provided in Fig. 2. Each sample was analyzed with two primers: one primer specific for unmethylated allele while the other primer specific for methylated allele. PARK2 promoter hypermethylation was observed in 66 (33%) out of 200 tumor samples. Among diﬀerent clin-icopathological parameters statistically significant association of pro-moter methylation was observed only for tumor grades (I + II vs
III + IV) (Table 6). Where tumor grades III + IV showed significantly higher hypermethylation of PARK2 promoter (OR: 2.3, P = 0.019). No significant diﬀerence was observed for any other clinical parameter studied. Since PARK2 is a putative tumor suppressor we can safely conclude that PARK2 promoter hypermethylation is a critical event for tumor aggression.
Correlation of PARK-2 promoter hypermethylation with clinical parameters in colorectal cancer patients.
Characteristics Total cases(n) Methylated PARK-2 (%) Unmethylated PARK-2 (%) P value OR (95% CI)
OR: odds ratio; CI: confidence interval; WD: well diﬀerentiated; P value was calculated by Fisher's exact test.
Fig. 3. PARK2 protein expression status in the tumor and control samples. Western blot analysis depicting the status of endogenous levels of PARK2 protein (52 kDa) and loading control β-actin (37 kDa) in tumor and normal tissues.
3.4. PARK2 expression status in tumor and normal tissues
Western blot analysis was performed to measure the expression level of PARK2 in a representative set of CRC tumor and control tissue. Controls were taken from the periphery of the colorectal section with no malignancy. PARK2 level was significantly lower in the tumor samples as compared to the controls (Fig. 3). Thus promoter hy-permethylation observed in the earlier result is responsible for the re-duced expression of PARK2 in tumor tissues.