Association of rs 1042522 Polymorphism with Increased Risk of Prostate Adenocarcinoma in the Pakistani Population and its HuGE Review

Cancer is a global public health concern due to its fatality, disease burden and potential for increased incidence (Acikgoz and Ergor, 2013; Sahin, 2013, Tan and Polat, 2013). Evolution of cells towards cancerous one involves complex multi step genetic and epigenetic aberrations, conferring selective advantages. Regardless of the substantial research efforts made over the past decades, molecular perceptive of cancer is still a major challeng, (Rivlin et al., 2011). Despite the vast diversity among the genes responsible for tumorigenesis, TP53, encoding a hub protein standout as a key player in stress responses, preservation of genomic stability and tumor suppression (Reinhardt and Schumacher, 2012; Xu et al., 2012). It is also a prime regulator of multiple signaling pathways (Levine and Oren, 2009) and has a rigid correlation with almost all kinds of human malignancies. Prostate adenocarcinoma is one of the most commonly diagnosed cancers and a leading cause of cancer mortality


Introduction
Cancer is a global public health concern due to its fatality, disease burden and potential for increased incidence (Acikgoz and Ergor, 2013;Sahin, 2013, Tan andPolat, 2013).Evolution of cells towards cancerous one involves complex multi step genetic and epigenetic aberrations, conferring selective advantages.Regardless of the substantial research efforts made over the past decades, molecular perceptive of cancer is still a major challeng, (Rivlin et al., 2011).Despite the vast diversity among the genes responsible for tumorigenesis, TP53, encoding a hub protein standout as a key player in stress responses, preservation of genomic stability and tumor suppression (Reinhardt and Schumacher, 2012;Xu et al., 2012).It is also a prime regulator of multiple signaling pathways (Levine and Oren, 2009) and has a rigid correlation with almost all kinds of human malignancies.
Prostate adenocarcinoma is one of the most commonly diagnosed cancers and a leading cause of cancer mortality reference to different ethnic groups but unfortunately, no literature is available on the subject of its allelic frequencies and its association with PCa in Pakistani population.Critical review of the relevant literature on rs1042522 polymorphism from diverse ethnic groups provoked us to investigate the status of rs1042522 polymorphism in PCa patients and its association with increased risk in Pakistani population.

Approval of the study
This study was approved by the Ethics Committee and Institutional Review Committee of the Institute of Biomedical and Genetic Engineering (IB&GE), Islamabad, Pakistan.All the samples were collected along with the signed informed consents from the donors participating in this study.

Sampling
Peripheral blood samples included 146 PCa cases and 107 controls of the same ethnic and age groups were collected from distinct random individuals of Pakistan with informed signed consent.Clinical characteristics of the subjects including Gleason grade, PSA level and diagnosis of prostatitis were obtained from their medical records.The PCa patients were between 45 to 90 years of age with mean age of 71.01±12.05,which was significantly greater than the controls (age ranges from 38-85 with mean age of 66.08±12.26)(Table 1).It was also observed that all the controls had PSA levels smaller 4.0 ng/ml and normal digital rectal exams.Individuals with benign prostatic hyperplasia were not considered in this study.Genomic DNA was extracted using the standard pheno-chloroform method.

Genotyping
Primers were designed for TP53 exon 4 by using the online resource of Primer3 web version 3.0.0(http:// primer 3.wi.mit.edu).PCR was carried out to amplify the DNA sequence including p53 codon 72 by using (Forward) 5'TGCTCTTTTCACCCATCTAC3' and (Reverse) 5'ACTTCAATGCCTGGCCGTAT3' primers.The detection of rs1042522 polymorphism was carried through RFLP to discriminate the C from G allele.The purified amplified fragments were digested with restriction enzyme BstU1 (Fermentas, Vilnius, Lithuania) at 57°C for 16hrs.The digested fragments were electrophoresed on 4% agarose gel and then visualized under UV light after ethidium bromide staining.The expected fragment sizes were, the Pro uncut allele had a band of 353bp, the Arg allele which is recognized by BstU1 was digested into two fragments of 212 and 141bp while the heterozygote had all the three bands.For quality control, 25% samples were repeated and >95% concordance was recorded.After PCR-RFLP, the samples having Pro/Arg or Arg/Arg bands were sequenced through ABI genetic analyzer for further confirmation and validation of the observed results.

Statistical methods
The statistical analysis for the case-control study was carried out with the help of online available tools.Differences in subjects were compared through Fisher's exact test.Hardy-Weinberg equilibrium (HWE) with chisquare was calculated by comparing the expected genotype frequencies to the observed frequencies.Unconditional logistic regression was used for the calculation of odd ratios (OR) with 95% confidence intervals to estimate the affect of the SNP presence on disease risk.Twotailed p-values ≤0.05 was hypothesized to be statistically significant.

Structure and function analysis
Nucleotide alteration can significantly affect the structure and function of a gene product.Associating these alterations with phenotypic characters is one of the important areas of research (Waheed et al., 2012).Sequence of Human p53 protein with accession number P04637 was retrieved from the UniProt (www.uniprot.org) database for detailed structure based assessment.Proteins naturally exist in complex folded structures rather than as linear polypeptides.These high order 3D conformations define their biological functions.For a detail insight, three-dimensional structure of human p53 was predicted through Modeller 9.11 (Eswar et al., 2006), refined through ModRefiner (Xu and Zhang, 2011) for quality enhancement and was validated for quality assurance through PROCHECK (Laskowski et al., 1993) and APOLLO (Wang et al., 2011).
The confirmed polymorphism, Pro72Arg was substituted in the native sequence of p53 using MUTATE_ MODEL, available as a Python script at http://salilab.org/modeller/wiki/Mutate_model, to get the altered protein for investigating structural and functional deviations.The mutant model was compared against the native in 3D through PDBeFOLD (http://www.ebi.ac.uk/msd-srv/ ssm) for structural similarities.Amino acid substitution can cause physiochemical differences, which in turn can affect the protein interactions, therefore physiochemical properties were predicted through ProtParam (http:// au.expasy.org/tools/protparam.html).SIFT (Ng and Henikoff, 2003), PolyPhen2 (Adzhubei et al., 2010), Mutation Assessor (Reva et al., 2011) and PROVEAN (Choi et al., 2012) were used for the analysis of functional impact of altered residue due to polymorphism.

Meta analysis
It was observed in the critical review about the topic that, some of the published articles reported non-significant association of TP53 P72R polymorphism and increased risk of PCa.We therefore carried out a systematic review and meta-analysis of the previous available results of TP53 P72R polymorphism in association with PCa from different countries to explore the notorious available data.This detailed study will help to further our knowledge about the diseases development and the biological phenomenon enhancing the risk of PCa which in turn will be used as cancer diagnostics and therapeutics.
The data searched was carried out through different databases like Pubmed, Science direct, Human genetic mutation database, Chinese biomedical literature and Google scholar from 1999 to 2013.The keywords DOI:http://dx.doi.org/10.7314/APJCP.2014.15.9.3973 rs1042522 Polymorphism and its HuGE Review in Pakistani Population used to search relevant studies were TP53 P72R polymorphism OR TP53 Pro72Arg polymorphism OR TP53 single nucleotide polymorphism OR TP53 codon 72 polymorphism OR rs1042522 and prostate cancer OR PCa OR prostate adenocarcinoma.Full text papers were retrieved wherever possible and the selection criteria for research articles were then narrowed down by keeping some of the important parameters constant and publications incorporating the full desired information were included in the Meta analysis.All the publications were manually examined for inclusion according to the following parameters, i) only case-control studies were selected; ii) having full information of Author/s; ii) Year of publication with country and population under study; iii) Total number of cases in the study; iv) cases genotypes; v) Total number of controls and; vi) Genotypes of the controls.A flow diagram was constructed for the collected data included in the Meta-analysis according to the PRISMA statement (Figure 4).The data was carefully taken as whole exact results from each eligible study.The genotypes of cases and controls were analyzed and the Meta analysis was performed for the total included data.Both the fixed and random effect model along with 95% CI was used to measure the genetic association and also to calculate the pooled effect estimates.
Although we have set very precise and strict inclusion criteria, there still exists heterogeneity due to a number of potential factors like the study design etc.To assess this inter study heterogeneity in a more precise fashion, I-squared, Cochran's Q and Chi2-p statistic was used to quantitatively evaluate the proportion of the total variation due to heterogeneity.The Forest plot was used for the graphical representations of the meta-analysis results while funnel plot for the publication biasness.Forest plots and funnel plots were constructed on the basis of different genotypic models.

Results
A total of 212 samples including 120 PCa cases and 92 controls were analyzed through PCR-RFLP.The amplified fragments of TP53 containing the exon-4, when digested with BstU1 restriction enzyme followed by electrophoresis, containing the homozygous pro allele produced a single band of 353bp, homozygous arg allele produced two fragments of 212 and 141 bp while the heterozygous samples produced three bands of 353, 212 and 141 bp.A representative pattern of PCR-RFLP and sequencing is depicted in Figure 1 while Table 1 is representing the clinical features and Table 2 is presenting of the cases and controls.
It has been clear from the research that biological function of a protein is usually correlated with its sequence and so any change in the sequence will impose changes in its biological function by means of changing the high order conformations.Human p53 structural variation due to Pro72Arg polymorphism present in our samples was therefore investigated in the present study and it was found that the substitution is in the proline rich domain, responsible for Interaction with HRMT1L2 and WWOX.
The biochemical differences, nature and location of        amino acid substitution can affect the protein in various ways and is therefore important to determine whether it can alter the protein function.Isoelectric focusing point (pI) and charge is important for the solubility and interaction of a protein (Nandi et al., 2005;Khaldi and Shields, 2011).Theoretical pI of the p53 proteins were predicted by using Bioinformatics tools and were observed 6.34 and 6.47 for the native and polymorphic respectively.
A total of 250 relevant journal papers associated with TP53 Pro72Arg polymorphism and prostate cancer risk were retrieved in due time allocated for data retrieval.Only 12 studies were fulfilling our required criterion as mentioned above and thus data was taken out of these in addition to our's study for the Meta analysis.The studies included in the analysis consisted of a total of 1686 PCa cases and 1888 controls.The included studies were carried out in different populations in different countries (Table 4).

Discussion
Prostate adenocarcinoma is globally a leading cause of cancer related mortality (Ricks-Santi et al., 2012), but unfortunately the molecular mechanisms underlying its development and progression still remains poorly understood like other cancers (Boyd et al., 2012).To date, the mechanisms for increased risk of PCa in Pakistani population have not yet elucidated.We genotyped the Pro72Arg SNP (rs1042522) of tumor suppressor gene TP53 in a PCa case-control study of men at the Institute of In the study samples, controls were tended to be younger than cases with age range from 40-70 for controls and 40-85 for cases respectively.Similarly PSA level was also comparatively lower in the controls.The distribution of three genotypes namely, pro/pro, pro/arg and arg/arg were observed 18.49%, 69.18% and 12.33% in PCa patients and 14.95%, 26.17% and 58.88% in controls respectively.Highly significant differences were observed in the distribution of genotypes between patients and controls (df=2, p=0.00).The allele frequencies of patients and controls were fitted in the Hardy-Weinberg Equilibrium with frequencies of 0.78 (Controls) and 0.27 (patients).Logistic regression was used to evaluate the association between Pro72Arg polymorphism and prostate adenocarcinoma.Highly significant association was revealed by all the genetic models [allelic contrast (C vs G): OR=2.90, 95% CI: 1.2-4.23;p=0.0000;CC vs GG: OR=5.91, 95% CI: 2.63-13.28;p=0.000;CC vs GC: OR=0.47, 95% CI: 0.22-0.99,p=0.046;GC vs GG: OR=12.63,95% CI: 6.46-24.69,p=0.000;Dominant model (OR=10.18,95% CI: 5.45-19.04,p=0.000) except the Recessive model: (OR=1.29,95% CI: 0.66-2.54,p=0.000 and log additive model: OR=1.24, 95% CI: 0.64-2.41,p=0.53].Based on the results, it is apparent that the genotypes are in significant association with increased risk due to the increasing number of polymorphic alleles. Knowledge of protein structure provides an insight into its interactions (Aydin et al., 2011), which define the protein's biological role and functions (Cheng et al., 2005).Residues substitutions by any mean can affect the protein high order structure which determines protein functions.Human p53 structural variation due to Pro72Arg polymorphism present in our samples that showed statistically significant association was therefore investigated in the present study.Pro72Arg substitution is in the proline rich domain, responsible for Interaction with HRMT1L2 and WWOX.The substitution is replacing a non-polar residue with a positively charged residue which is bigger and less hydrophobic than the wild type which might lead to bumps and changes in hydrophobicity and lead to loss of hydrophobic interactions, either in the core or surface of the protein (Figure 2).3D structures are more conserved than sequence (Capriotti et al., 2010), thus native and polymorphic structures were aligned in 3D which actually produces a measure to assess the level of similarity of the aligned structures and RMSD value of 0.00A0 was observed (Figure -3).The biochemical differences, nature and location of amino acid substitution can affect the protein in various ways and is therefore important to determine whether it can alter the protein function.Isoelectric focusing point (pI) and charge is important for the solubility and interaction of a protein (Nandi et al., 2005;Khaldi and Shields, 2011).It is clear from the research that, pI of a protein can vary due to insertions, deletions, substitutions and the ecology of the organism (Kiraga et al., 2007).Theoretical pI of the p53 proteins were predicted by using Bioinformatics tools and were observed 6.34 and 6.47 for the native and polymorphic respectively.
Theoretical pI of the polymorphic p53 protein was higher than the native by 0.14, which is equivalent to 1-2 net positive charge increase per molecule.Differences were also observed in the aliphatic index and GRAVY.The substitution Pro72Arg was predicted to be TOLERATED with a score of 0.43 by SIFT, regarded as benign by PolyPhen2 with scores of 0.000 (sensitivity: 1.00 and specificity: 0.00), neutral by mutation assessor and PROVEAN with a PROVEAN score of-0.230.
The results showed that, the Arg coding G allele was extremely significantly associated with the disease prevalence in our sampled population.To the best of our knowledge, this is a pioneer study to test the association of this very common polymorphism of TP53 and PCa risk in Pakistani population.Regression analysis was specifically performed on the data by using different models to critically pinpoint the alleles association with PCa risk.Extremely significant associations were revealed by all the studied models between the SNP and PCa risk.
The TP53 Pro72Arg SNP has found to be related with changes in the efficiency and function of TP53 product (Murphy, 2006;Shi et al., 2009;Whibley et al., 2009).The pro coding C allele is responsible for an enhanced transcriptional transactivation which thus induces elevated cell-cycle arrest in G1 (Thut et al., 1995) while the G which is responsible for arg residue promotes induction of apoptosis (Dumont et al., 2003).It has also been observed that, the Arg residue, which is due to G allele, denatures at high temperatures and is comparatively less stable thermodynamically (Khoo et al., 2009) and has been hypothesized to be under selective pressure (Kiraga et al., 2007).The thermodynamically stable C allele responsible for coding Pro residue promotes the induction of TP53 for repair against oxidative damage in populations living in hot climates (Khoo et al., 2009).It has been proposed that C allele could provide selective advantage in populations living in comparatively colder areas around the globe by reducing implantation failure, as in the case of Pakistani population, providing better ability for the induction of cell-cycle arrest (Bensaad et al., 2006).Depending upon the ethnicity of the population studied, G allele responsible for coding Arg residue have been found to be extremely associated with increased risk of prostate adenocarcinoma.
A large number of case-control studies have already been conducted to assess the association TP53 Pro72Arg polymorphisms and increased risk of different cancers including PCa in humans.The results of all the reported studies are inconsistent, a comprehensive meta-analysis was therefore included in this study to provide further insights and to further explore this debated area.Majority of the previous publications reported the polymorphism non-significantly associated with PCa risk, while others found it significantly associated.The meta-analysis will help to evaluate the potential association of the polymorphism and increased PCa risk and to achieve more reliable conclusion.The results of meta-analysis showed statistically significant heterogeneity among the included studies which may be due to different potential factors.Careful investigation of the results in this study reflects that population under study is the most important contributing factor for this heterogeneity which is due to DOI:http://dx.doi.org/10.7314/APJCP.2014.15.9.3973 rs1042522 Polymorphism and its HuGE Review in Pakistani Population the differential genotypes distribution of TP53 Pro72Arg polymorphisms among different populations.
In conclusion, we examined the TP53 Pro72Arg polymorphism and the association of PCa risk in a casecontrol study of Pakistani population.It was observed that the TP53 CGC to CCC polymorphism in exon-4 is associated with the increased risk of PCa which was also confirmed through the meta-analysis of the published data.It is therefore recommended that TP53 Pro72Arg polymorphisms may be a potential biomarker for PCa.It can thus be concluded that depending upon the environment, nature favors the selection of one allele over the other during the course of evolution.On the basis of this systematic study, it is hoped that differences in allelic frequencies in different populations associated with a large number of health problems can provide a valuable foundation for unhiding the mechanisms of complex diseases like PCa and others.

Figure 1 .
Figure 1.Representative PCR-RFLP Pattern of p53 exon-4 Digested with BstU1.A) The Pro uncut allele had a single band of 353 bp, while the Arg allele recognized by BstU1 has restricted into two fragments of 212 and 141 bp.The heterozygotes have all three bands of 353, 212 and 141 bp; B) Sequence of TP53 exon 4 showing a common variant (rs1042522) at codon 72 (CGC to CCC)

Figure 2 .
Figure 2. Representation of A) Structures of Proline and Arginine residues; B) Partial diagram representing secondary structure comparison of native and polymorphic p53 proteins.The red window shows the amino acid substitution but has no affect on the structure; C) Partial diagram representing alignment of secondary structure elements of native and polymorphic p53 proteins.The red window shows that the polymorphism has no affect on the structure elements

Figure
Figure 4. Flow Diagram for the Collected Data Included in the Meta-Analysis According to the PRISMA Statement

Figure 5 .
Figure 5. Forest plot of the Association of TP53 Pro72Arg Polymorphism and Increased PCa Risk in Different Populations of the World According to Homozygous (CC vs GG) Model

Table 5 . Log Odd Ratios, Standard Error, Variance and p-value of the Studies Included in the Meta-analysis Under Allelic Contrast and Homozygous Genotypic Models
*LOR=Log odd ratio; SE=Standard error; Var=Varianc; p=p value