Polymorphism of Gly39Glu (c.116G>A) hMSH6 is associated with sporadic colorectal cancer development in the Polish population: Preliminary results

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Introduction
Colorectal cancer (CRC) is the most common gastrointestinal malignancy.Only in the USA, around 13,449 new cases are expected to be diagnosed with colorectal malignancy with the estimated mortality of about 49,190 in 2016. 1 In the past decade, there has been a significant general decrease in the incidence of CRC associated mortality observed in Western countries. 2On the other hand, incidence rates are increasing among adults younger than 50 years of age and in certain countries with historically low prevalence of CRC, e.g., Eastern Europe. 1 Although often viewed as a single disease, CRC more accurately represents a constellation of heterogeneous subtypes that result from different combinations of genetic events and epigenetic alterations. 3Although the majority of CRCs are the results of chromosomal instability, approx.15% of cancers develop via an alternative pathway characterized by defective function of the DNA mismatch repair (MMR) system. 4The MMR system recognizes and corrects mismatched bases and small insertion-deletion loops generated during DNA replication.Highly conserved series of proteins, including MSH2, MSH6, MSH3, MLH1, PMS2, PCNA, and EXO1 are involved in the MMR system, which operates functionally, forming 2 heterodimers: hMSH2-hMSH6 and hMSH2-hMSH3.The recognition of mismatches and insertion-deletion loops are carried by these 2 heterodimers, respectively.This process is coordinated by another heterodimer formed out of MLH1 and PMS2.Inefficient function of the MMR system leads to the accumulation of mutations, resulting from the inability to repair single nucleotide DNA mismatches, particularly in highly repeated sequences known as microsatellite instability (MSI).Germline mutations in MMR genes or hypermethylation of the MLH1 promoter lead to a deficient MMR system (MMR-D) and a high degree of MSI in genome, resulting in the development of a variety of human cancers, e.g., endometrial or gastric cancer.MMR-D accounts for 15% of all CRCs, with 3% being associated with hereditary non-polyposis colorectal carcinoma (HNPCC), and the remaining 12% arise sporadically. 5Although the prevalence could vary between populations, it is clear that the MSI phenotype represents a clinically meaningful proportion of CRCs.On the other hand, lots of CRCs are sporadic and have a proficient MMR system with low-frequency MSI.It is, however, widely hypothesized that the heritable nature of CRC might be associated with the co-inheritance of multiple low-risk variants that may also further interact with environmental factors. 6,7This hypothesis was supported by the identification of single-nucleotide polymorphisms (SNPs) localized in different genomic regions that influence the risk of CRC. 8 SNPs of MMR genes may alter the gene and protein expression pattern, thus modifying its efficacy and therefore the risk of developing CRC.The risk of CRC associated with each of the vari-ants is individually low, but the combined effect of these variants could significantly contribute to disease development, especially given the high prevalence of these variants in the general population. 9MSH6 Gly39Glu (116G>A) polymorphism (rs1042821) first described by Nicolaides, et al. has been investigated for the role in its development of both HNPCC and sporadic CRCs. 10 The inactivating mutations of MSH6 in yeast and human tumor cell lines are associated with an impaired ability to repair singlebase mispairs and small insertion-deletion loops but not large insertion-deletion loops. 11This indicates that hMSH6 mutations are prone to be associated with a MSI-L phenotype than a MSI-H phenotype, which is more typical to sporadic CRC.MSI-L colorectal tumors have been proposed as possible candidates for hMSH6 mutations by Wu. 12 Berndt and Campbell reported an association between MSH6 Gly39Glu (116G>A) polymorphism and the development of rectal and colon cancer. 13,14However, the results of further studies were conflicting. 11,15These reports prompted us to investigate a common polymorphism in the MSH6 gene of MMR system and their role in the susceptibility to sporadic colorectal cancer in the population of our region.

Material and methods
Test DNA was isolated from peripheral blood samples collected from 128 unrelated patients with confirmed CRC.Only patients with a resectable disease were enrolled into the study.Blood samples for the analysis were taken on the day of admission in both groups of patients.A detailed characteristic of the patients with the CRC is shown in Table 1.The control group consisted of 189 patients hospitalized in the same surgical ward without any medical history of cancer, inflammatory diseases and diabetes.The reason for hospitalization was mostly benign proctological disease or hernia.The analyzed and control groups members were age-and sex-matched.

Detection of hMSH6 gene mutation
The DNA for genotyping was isolated from the blood samples of CRC patients using a commercial kit QIAamp DNA Blood Mini Kit for isolating high-molecular-weight DNA (Qiagen).Detection of Gly39Glu (c.116G>A ) polymorphisms of the hMSH6 gene was carried out by RFLP-PCR analysis.Primers used for amplifications of the analyzed region are shown in Table 2. PCR products were generated using, in each reaction, a total volume of 20 µL containing 10 pM of each primer, 1 U Taq DNA polymerase (Qiagen), 0.2 mm of each dNTP, 1.5 mM MgCl 2 , and 50 mM KCl, 20 mM Tris-HCl (pH = 8.4), and 50 ng of genomic DNA.PCR amplification was performed in MultiGene TC9600-G termocycler (Labnet International, Inc., Edison, USA) under the following conditions: initial denaturation at 1 cycle at 95°C for 5 min and amplification of 35 cycles (30 s at 95°C, 30 s at 55°C, and 30 s at 72°C), followed by a final cycle of 7 min at 72°C; 8 µl of PCR products was diluted in 2 µl of denaturation buffer (95% formamide, 0.05% bromophenol blue, and 0.05% xylene cyanol) and was separated on a 1.5% polyacrylamid gel at 10 V/cm for 1 h at room temperature.The final products were visualized by ethidium bromide staining.

Statistical analysis
The statistical analysis was conducted using STATISTI-CA software v. 13 PL (StatSoft, Inc.).The resulting number of each genotype was compared with the expected value based on the Hardy-Weinberg equilibrium.The significance of differences between the frequencies of alleles and genotypes between the groups was assessed using the χ 2 test and the Fisher exact test.Odds ratio (OR) with corresponding confidence interval 95% (CI 95%) were calculated during multivariate regression analysis.

Results
Distributions of genotypes and alleles in the CRC and control group are presented in Table 3. G allele has been used in our study as a reference in calculations.According to the Hardy-Weinberg χ 2 analysis, the distribution of genotypes and alleles in CRC group and the control group was not consistent with H-W equilibrium (χ 2 = 0.07, p = 0.786 and χ 2 = 0.34, p = 0.558, respectively).No significant difference in alleles or genotypes distribution was observed between age and sex groups (<60, >60, females, males).Risk of CRC was approximately 2-fold higher for the G/A genotype (OR 1.57; 95% CI: 1.04-2.38;p = 0.032) and A allele (OR 1.65; 95% CI: 1.01-2.69;p = 0.44).When comparing the prevalence of genotypes with clinical staging, genotype G/A and A allele were significantly less frequent in stage III-IV than in I (OR 0.3409; 95% CI: 0.124-0.939;p = 0. 0375, and OR 0.4462; 95% CI: 0.201-0.991;p = 0.044, respectively) (Table 4).

Discussion
The existence of a link between human cancers and the MMR determines the relevance of investigations of the DNA mismatch repair system.The inheritance of variations in MMR genes may influence individual susceptibility to the development of colorectal cancer.While the presence of mutation in hMLH1 and hMSH2, leading to MSI-H CRCs is well described, this involves mostly tumors developing in HNPCC. 7,16For sporadic CRCs, where MSI-L or MSI stable genotype is observed, it was suggested that impairment in MMR system efficiency may arise from other genes, whose alternations lead to the complete loss of function of heterodimers. 17,18That is why hMSH6 was investigated for their possible role in tumor neogenesis in sporadic CRCs.hMSH6 form complex with hMSH2, that recognizes single-base mispairs and small (i.e., single-base) insertion-deletion loops. 19Mutations in MSH6 result in a partial loss of mismatch repair. 20uman tumor cell lines with MSH6 mutations exhibit MSI primarily in mononucleotide and not in dinucleotide repeats, which suggests its association rather with MSI-L CRCs.Parc, et al. identified 16 hMSH6 alterations. 11Fifteen of these were germ-line changes and 1 was a somatic change.Only 2 of the 15 germ-line alterations led to a change in the primary sequence of the hMSH6 protein: Gly39Glu and Leu395Val, both of which are reported to be polymorphisms. 10,21By now, few studies investigated the association between the MSH6 Gly39Glu variant and the risk of sporadic colorectal cancer. 13,14,21erndt, et al. reported that homozygosity for the MSH6 39Glu variant was associated with a more than triple risk of rectal cancer.However, no association was found between the MSH6 variant and colorectal cancer overall.Since the number of rectal cases in this study was small, the authors recommended interpreting the observed association for rectal cancer with caution. 13Campbell, et al. analyzed whether MSH6 (Gly39Glu) polymorphisms were associated with the risk of colon cancer in the data from 1,609 colon cancer cases and 1,972 controls.Male participants heterozygous (G/A) or homozygous (A/A) for the MSH6 variant were at 27% increased risk of colon cancer, while no associations were observed among females.On the other hand, further association was not observed with MSI-negative status of CRCs.Additionally, this study observed effect modification between MMR variants and lifestyle factors that contribute to higher CRC risk overall. 14his is the first report concerning polymorphism Gly-39Glu and the risk of colorectal cancer in the Polish population.In our study, GG genotype was more prevalent than the AA genotype.The presence of the MSH6 39Glu variant allele (A) was associated with the increased risk of colon cancer (OR 1.57; 95% CI: 1.04-2.38;p = 0.032) in the analyzed population.In contrast to the previous study, the homozygosity for A allele did not lead to a higher risk of CRC, but was observed in the cases of heterozygosity (OR 1.65; 95% CI: 1.01-2.69;p = 0.44).Similar results concerning higher concentration of hMSH6 A allele in CRC cases were obtained in the study by Kolender (23% in colorectal cancer cases as compared to 15% in controls), although no formal statistical comparisons were made. 21he presence of G/A genotype and A allele were more frequently detected in early stage tumors, which can suggest the possible MSI-H phenotype.Though MSI status was not checked in our study, the linkage between hMSH6 mutation and MSI status is now a matter of wide debate.The lack of a clear association with MSI positive or negative status raises many questions concerning the role of mutations in this gene on the development of MSI-L or MSI-H CRC, or broadly speaking in NHPCC and sporadic colorectal cancers.In comparisons to hMLH1 or hMLH2, the observed mutations in hMSH6 do not cause lesser gene expression in healthy and cancerous tissues. 22Plaschke reported that only 3 out of 146 patients with sporadic cancer exhibited abnormal hMSH6 expression, with 2 having germ-line mutations.However, possible missense mutations may be present that impair gene function without substantially affecting protein expression or resulting in the MSI-H phenotype. 15Wu et al. emphasized that an MSI-low phenotype cannot be considered an exclusion criterion for mutation testing of MMR genes in general. 12Still, we can suspect that a fair amount of sporadic cancers can arise in HNPCC-like families, in which, though not evident, the MSI-H phenotype and other MMR genes mutations may be present.The possible answers may also involve epigenetic or somatic changes whose impact on carcinogenesis is intensified by the presence of environmental factors such as smoking or a Western diet.4][25][26] However, for a better explanation of these phenomena further research is warranted.

Table 1 .
Primers used for RFLP-PCR

Table 2 .
Clinical and pathological characteristics of the analyzed group of patients with colorectal cancer

Table 3 .
Genotypes and alleles distribution of Gly39Glu (c.116G>A) polymorphism of hMSH6 gene in patients with colorectal cancer and in control group

Table 4 .
Genotypes and alleles distribution of Gly39Glu (c.116G>A) polymorphism of hMSH6 gene in patients with colorectal cancer according to staging by American Joint Committee on Cancer