br In silico transcription factor binding site prediction br
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 binding sites 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 Z-Gly-Gly-Arg-AMC acetate 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
previously reported increased hypermethylation (29%) of PARK2 pro-moter in cervical cancer tumor grade III + IV but the diﬀerence ob-served was not statistically significant (Naseem et al., 2017). Here in colorectal cancer we found significant diﬀerence in PARK2 promoter hypermethylation similar to what was observed in ALL and CML by others. A correspondingly reduced expression in tumor tissues com-pared to normal tissue was observed in Western blotting. Reduction in expression of putative tumor suppressor PARK2 in aggressive tumor grades III and IV authenticates the role of PARK2 as a tumor suppressor. It makes all the sense that decreased expression of diﬀerent tumor suppressors will eventually lead to aggressive behaviour of tumor cells.
In-silico transcription binding analysis also revealed that Pax-5 and P53 transcription factors binds to PARK2 promoter due to presence of minor allele. Since PARK2 is considered a putative tumor suppressor it seems like PARK2 may be involved in a cascade of events where dif-ferent tumor suppressor transcription factors binds to PARK2 promoter under diﬀerent genomic background to regulate its expression. P53 has been shown to regulate PARK2 expression and together both of them regulate antioxidant defence, glucose metabolism and Warburg eﬀect (Zhang et al., 2011). To the best of our knowledge, we are reporting for the first time, a case control study on these PARK2 promoter variants taken together along with in-silico analysis and stratified analysis for possible correlation with patients' clinical parameters in susceptibility to colorectal cancer.
In conclusion, our study revealed a significant association of rs2276201 with colorectal cancer risk, as well as its interaction with one of the clinical parameters revealing its contribution to colorectal cancer susceptibility among North Indian population. Although, any immediate clinical implications of the present study are unlikely pos-sible, however, such studies may play a key role in elucidating the biological mechanism that emphasize the role of colorectal tumor heterogeneity, which may ultimately lead to improved treatment and prevention. Such studies may help reveal the functional mechanisms for the role of PARK2 SNPs in colorectal cancer. More experimental studies of larger sample size and expression studies are necessary to explore and confirm the role of these variants in increasing colorectal cancer risk, particularly from India, that will help in better understanding the genetic heterogeneity in complex diseases like colorectal cancer.