br Table br Correlation of PARK promoter hypermethylation with clinical
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.
3.5. In-silico transcription factor binding site prediction
TFBS analysis by PROMO predicted that the presence of minor at both loci results in gain of Pax-5 and p53 binding sites. While AP-2alphaA binding site was lost for rs2276201and for rs9347683 C/ EBPbeta, NF-1 and ENKTF-1 Wortmannin were lost.
In recent times, a number of SNPs have been identified within PARK2 that are possible risk factors for PD, delayed neuropsychological
and typhoid (Ali et al., 2006a; Kay et al., 2010; Cummings et al., 2011; Pankratz et al., 2011; Liang et al., 2013). Furthermore, diseases like autism spectrum disorder and progressive supra nuclear palsy has also been associated with PARK2 mutations (Ros et al., 2008; Scheuerle and Wilson, 2013). Till date no study has evaluated the association of PARK2 SNPs with cancer. In this study we measured the genotype frequencies of two PARK2 promoter polymorphisms rs2276201and rs9347683 and found their significant association with colorectal cancer. Diﬀerent genotypes and genetic models of rs2276201 SNP showed statistically significant risk for CRC. For rs9347683 SNP only heterozygous genotype and dominant genetic model showed statisti-cally significant risk. Although we did not performed any in-vitro ex-periment to directly show the eﬀect of these SNPs on PARK2 to ex-pression, but since promoter SNPs are known to modulation expression (Pal et al., 2011). We can safely assume that these two polymorphisms can potentially modulate expression of PARK2 and contribute in cancer development. Very interestingly in the haplotype analysis we found that simultaneous presence of both minor alleles at their respective loci provided significant risk while the presence of both major allele pro-vided significant protection for CRC. These two polymorphisms are present roughly 500 bases apart on PARK2 promoter, still in LD analysis we didn't found any linkage between these two positions. We know that recombination rates for two loci that are < 1000 bases apart are usually low thus no linkage between these two polymorphisms in this region represents an unusual scenario. Previously SNP rs2276201 has been associated with leprosy susceptibility factors, Hepatitis C Virus infec-tion and its progression to cirrhosis, hepatocellular carcinoma and in ovarian Cancer patients of Southern Brazil (de Leseleuc et al., 2013; Al-Qahtani et al., 2016; Klimczak et al., 2016), therefore involvement of this polymorphism in regulating expression of PARK2 expression under diﬀerent diseases seems quite evident. r> Increased methylation of PARK2 promoter have been reported for acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) but not in colon cancer cells (Agirre et al., 2006). Our group