A promoter polymorphism in the hMLH1 gene (-93G/A) associated with sporadic colorectal cancer
- Authors:
- Published online on: September 26, 2016 https://doi.org/10.3892/ol.2016.5188
- Pages: 4035-4040
Abstract
Introduction
Colorectal cancer (CRC) is a worldwide problem for public health, and is becoming more prevalent in Asian countries, particularly China (1). CRC is a prototypic model for the genetic basis of cancer. Alterations in the DNA mismatch repair (MMR) pathway have been causally linked to its etiology. The MMR pathway is one of the major DNA repair pathways; it plays an important role in repairing single-base mismatches and in insertion-deletion loops, which result from slippage during DNA replication (2,3). More and more MMR genes are being found to contain common single nucleotide polymorphisms (SNPs), which can predispose individuals to non-familial CRC with low to moderate penetrance (4–6). mutL homolog 1 (MLH1), which is located on chromosome 3p22.2, is a key component of the MMR system; it is involved in mismatch strand excision and subsequent repair (7), while recruiting other mismatch repair proteins to the mismatch sites to correct the errors during DNA replication (8,9). The MLH1-93G/A polymorphism (rs1800734) is located in the core promoter region, which is essential for maximum transcriptional activity. Polymorphism variants in this region are predicted to affect MLH1 protein expression (10,11). It was previously reported that the loss of MLH1 proteins expression had been associated with the susceptibility of several cancers. Based on these observations, particularly in view of the importance of MLH1 in colorectal carcinogenesis, we hypothesized that the polymorphism in the MLH1 gene may modulate the risk of CRC. Thus, the present matched case-control study was performed to investigate whether any associations exist between the −93G>A polymorphism and SCRC in China. In addition, immunohistochemical staining was used to measure the expression of MLH1 protein in cases with different alleles, which including CRC and normal control cases, in order to check the function of the −93G>A polymorphism.
Materials and methods
Approval and consent
The Institutional Review Boards of the Drum Tower Hospital (Nanjing, Jiangsu, China) approved the study and written informed consent was obtained from all participants.
Study population
The case-control study included 312 SCRC patients (age range, 19–89 years; mean age, 60.52±16.38 years; male/female, 193/119) and 300 normal healthy controls (age range, 23–78 years; mean age, 58.64±12.33 years; male/female, 169/131) recruited from Wuxi No. 2 People's Hospital (Wuxi, China) between January 2006 and October 2010. All patients were diagnosed with pathologically confirmed CRC and 284 patients received surgery. The 300 control subjects were randomly selected from the Center of Physical Examination (Wuxi No. 2 People's Hospital) from patients who had no history of malignancy during the same time period of the case recruitment. None of the subjects were blood-related. All the subjects were interviewed to obtain information on their sociodemographic characteristics, dietary habits, smoking and drinking status, and their individual and family history of cancer. SCRC cases and controls were matched for age, gender, and smoking and drinking history.
Genotyping analysis
The National Center for Biotechnology Information SNP database (http://www.ncbi.nlm.nih.gov/snp/) and related literature was searched to identify functional SNPs in human (h)MLH1. The criteria for SNP selection were as follows: i) A minor allele frequency of >0.05 in the Chinese population; ii) a genotype call rate of ≥95%; and iii) SNPs that have been closely associated with tumorigenesis. The MLH1-93G>A polymorphism (SNP rs1800734) was selected for genotyping. Genomic DNA was extracted from the peripheral blood samples of all subjects using a purification kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. The MLH1-93G>A polymorphism was genotyped using the TaqMan assay on an Applied Biosystems® 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The PCR cycling conditions were as follows: 50 cycles at 95°C for 10 min, 95°C for 15 sec and 60°C for 50 sec. The primer sequences were as follows: Forward, 5′-ACCCAGCAACCCACAGAGT-3′ and reverse, 5′-GTCTAGATGCTCAACGGAAGTG-3′.
Immunohistochemical staining for MLH1
CRC tissue samples were collected from 60 CRC patients who underwent surgery. Normal colon tissue samples were collected from 56 outpatients via colonic biopsies during colonoscopy examination. Immunohistochemistry was performed using the avidin-biotin-peroxidase complex method (12). Immunohistochemical staining was performed on the 4-µm thick paraffin-embedded tissues, which were mounted on positively-charged slides. The tissue sections were incubated with primary mouse monoclonal antibody against MLH1 (dilution, 1:50; cat. no. ab14206; Abcam, CA, USA) overnight at 4°C, followed by the biotin-conjugated goat anti-mouse IgG H&L secondary antibody (dilution, 1:1,000; cat. no. ab6788; Abcam) for 30 min at 37°C. Images were captured using an Olympus-BX50F4 microscope (Olympus, Tokyo, Japan) and quantitatively analyzed using the Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). The normal colorectal tissues were used as positive controls for MLH1 staining. Negative controls were run without the primary antibody.
Statistical analysis
Standard χ2 tests were used to determine the differences in allelic and genotypic frequencies between SCRC patients and control subjects in the case-control study. Student's t-test was used to compare MLH1 expression between the G and A alleles. Allele and genotype proportions were tested for Hardy-Weinberg equilibrium. The genotype data were further stratified by gender, age, smoking history, alcohol intake, tumor location and size, differentiation, Duke's stage (13), and lymphatic and distant metastasis status of CRC. The statistical tests were analyzed by SPSS 16.0 system software (SPSS Inc., Chicago, IL, USA) and a two-tailed significance level of P<0.05 was used.
Results
Table I shows the allele and genotype distributions for SCRC patients and controls. The allele and genotype proportions were in Hardy-Weinberg equilibrium (P=0.099 and P=0.418, respectively). Meanwhile, the allele and genotype frequencies between the SCRC patients and controls were compared, and no significant differences were observed in either the allele (P=0.879) or genotype (P=0.219) frequencies. When the CRC patients were stratified by gender, age, smoking history, alcohol intake, tumor location and size, differentiation, Duke's stage, and lymphatic and distant metastasis, no association was found between the rs1800734 polymorphism and the clinical variables of gender, age, smoking, alcohol intake, tumor location and size, differentiation or distant metastasis in SCRC patients (all P>0.05). However, stratifying the samples by Duke's stage and lymphatic metastasis, significant differences were found in the allele (χ2 test, P=0.004) and genotype (χ2 test, P=0.015) frequencies (Tables II and III). In addition, there were also a significant association between the allele (χ2 test, P=0.010) and genotype (χ2 test, P=0.031) frequencies and lymphatic metastasis (Tables II and III). The frequency of the A/A genotype and allele A was higher in Duke's stage C+D SCRC patients than in Duke's stage A+B SCRC patients. The frequency of the A/A genotype and allele A was also higher in the SCRC patients with lymphatic metastasis than in the SCRC patients without lymphatic metastasis.
 | Table I.Distribution of alleles and genotypes for MLH1-93G/A (single nucleotide polymorphism rs1800734) in sporadic colorectal cancer patients and controls. |
| Table II.Stratified analyses between the human mutL homolog 1 (rs1800734) genotypes and the sporadic CRC risk. |
| Table III.Allellic distribution of human mutL homolog 1–93G/A (single nucleotide polymorphism rs1800734) with regard to Duke's stage and lymphatic metastasis status among 284 sporadic colorectal cancer cases. |
Immunohistochemical staining for MLH1 was evaluated in 60 SCRC patients (G/G vs. G/A vs. A/A: 17 vs. 26 vs. 17) and 56 normal controls (G/G vs. G/A vs. A/A: 18 vs. 22 vs. 17) (Fig. 1). MLH1 expression levels with the different alleles were compared between the CRC patients and the normal controls, as well as between Duke's stage A+B and C+D SCRC patients (Fig. 2). MLH1 expression was significantly higher in the normal controls, in Duke's stage A+B patients and in G allele SCRC patients than in SCRC patients (P=0.029), Duke's stage C+D patients (P=0.001) and G allele SCRC patients (P=0.018), respectively. By contrast, there was no statistically significant difference in hMLH1 expression for the A and G alleles in the normal controls (P=0.965).
Discussion
Several studies have confirmed that hMLH1 plays an important role in CRC, while SNPs of mismatch repair genes are believed to provide important information for the diagnosis of CRC (14). However, to the best of our knowledge, few studies on the association between SNPs of MMR genes and sporadic CRC (SCRC) in China are available. Thus, in the present study, it was proposed that the SNP of the hMLH1 gene was linked to CRC.
The genotype distribution of the SNP MLH1-93G/A (rs1800734) has shown differences among varying ethnic populations. The frequency of polymorphism −93G/G was found to be higher than other polymorphisms in European and North-American populations (15). However, in the present study, the G/A frequency in normal controls and CRC cases was observed to be higher than others. This was in agreement with other studies on Asian populations (16,17). These discrepancies may be explained by genetic variation in the different ethnic groups of the various study populations.
It is notable that the MLH1-93G/A variant has been associated with several cancers. For example, the MLH1-93A allele has been positively associated with the risk of developing MMR-deficient CRC, particularly CRC with somatic loss of MLH1 protein expression (18), and the risk of microsatellite instability (MSI)-positive colon cancer (19). A previous study also suggested that the −93A allele was associated with an increased risk of MSI-high, but not microsatellite-stable, colorectal tumors (19). It was also reported that the −93A allele of the MLH1 gene was associated with the risk for squamous cell lung cancers in Korean patients with a gene-smoking interaction (16). In the present study, it was found that the MLH1-93G/A variant was not associated with the risk of SCRC overall, which is consistent with the results of a study by Campbell et al (15). However, the frequency of the A allele was significantly higher in Duke's stage C+D SCRC patients and SCRC patients with lymphatic metastasis, compared with Duke's stage A+B SCRC patients and those without lymphatic metastasis, respectively. Furthermore, MLH1 expression was lower in the Duke's stage C+D SCRC patients, and the MLH1 expression was lower for the A-allele than for the G-allele in the CRC patients. Considering these findings, the MLH1-93G/A polymorphism did not appear to affect MLH1 expression in the normal controls. However, it may play a role in the expression of MLH1 following SCRC formation, and it may be associated with the tumor progression of CRC.
The molecular mechanisms responsible for the involvement of MLH1 in CRC progression remain unclear. The MLH1-93G/A polymorphism is located in the MLH1 CpG island, at −93 nucleotides from the transcription start site in the core promoter region (10). There are two transcription binding sites, nuclear factor for interleukin-6 expression and GT-IIB trihelix transcription factor, harbored in this region, which are required for maximal transcriptional activity (10). Based on this knowledge, it is possible that the −93 A allele is susceptible to MLH1 abnormal methylation and gene silencing as a result of altered transcription factor binding. As aforementioned, polymorphism in this region is predicted to regulate MLH1 protein expression. We suggest that the −93G to A transition could plausibly reduce MLH1 gene transcription and expression by altering its epigenetic status, thereby reducing the DNA repair capability. A study by Chen et al showed an association between the MLH1 −93A allele and the methylation of the MLH1 promoter in CRC and endometrial cancer (20). Recent studies have suggested that site-specific repressors of transcription may recruit DNA methyltransferases (21,22).
In conclusion, in the present study, an association was found between the MLH1-93A allele variant and the elevated risk of Duke's stage C+D CRC. Furthermore, the A-allele may be a repressive factor for the transcriptional activity of MLH1, and thereby affect MLH1 expression. However, the manner via which the variation affects the risk for epigenetic silencing and has a possible affect on the progression of CRC remains undetermined. In view of this, further studies and larger sample sizes are required to confirm these findings.
Acknowledgements
This study was supported by a grant from the Project of Wuxi Science and Technology Bureau (grant no. CSE01N1217).
References
|
Fengju S, Guanglin W and Kexin C: Incidence of colon cancer in Tianjin, China, 1981ߝ2000. Asia Pac J Public Health. 17:22–25. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
de la Chapelle A: Genetic predisposition to colorectal cancer. Nat Rev Cancer. 4:769–780. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Kunkel TA and Erie DA: DNA mismatch repair. Annu Rev Biochem. 74:681–710. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Lipkin SM, Rozek LS, Rennert G, Yang W, Chen PC, Hacia J, Hunt N, Shin B, Fodor S, Kokoris M, et al: The MLH1 D132H variant is associated with susceptibility to sporadic colorectal cancer. Nat Genet. 36:694–699. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Fearnhead NS, Wilding JL, Winney B, Tonks S, Bartlett S, Bicknell DC, Tomlinson IP, Mortensen NJ and Bodmer WF: Multiple rare variants in different genes account for multifactorial inherited susceptibility to colorectal adenomas. Proc Natl Acad Sci USA. 101:15992–15997. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Tomlinson IP, Webb E, Carvajal-Carmona L, Broderick P, Howarth K, Pittman AM, Spain S, Lubbe S, Walther A, Sullivan K, et al: A genome-wide association study identifies colorectal cancer susceptibility loci on chromosomes 10p14 and 8q23.3. Nat Genet. 40:623–630. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Christmann M, Tomicic MT, Roos WP and Kaina B: Mechanisms of human DNA repair: An update. Toxicology. 193:3–34. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Lynch HT and de la Chapelle A: Hereditary colorectal cancer. N Engl J Med. 348:919–932. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Hampel H, Frankel WL, Martin E, Arnold M, Khanduja K, Kuebler P, Nakagawa H, Sotamaa K, Prior TW, Westman J, et al: Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer). N Engl J Med. 352:1851–1860. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Ito E, Yanagisawa Y, Iwahashi Y, Suzuki Y, Nagasaki H, Akiyama Y, Sugano S, Yuasa Y and Maruyama K: A core promoter and a frequent single-nucleotide polymorphism of the mismatch repair gene hMLH1. Biochem Biophys Res Commun. 256:488–494. 1999. View Article : Google Scholar : PubMed/NCBI | |
|
Arita M, Zhong X, Min Z, Hemmi H and Shimatake H: Multiple sites required for expression in 5′-flanking region of the hMLH1 gene. Gene. 306:57–65. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Simpkins SB, Bocker T, Swisher EM, Mutch DG, Gersell DJ, Kovatich AJ, Palazzo JP, Fishel R and Goodfellow PJ: MLH1 promoter methylation and gene silencing is the primary cause of microsatellite instability in sporadic endometrial cancers. Hum Mol Genet. 8:661–666. 1999. View Article : Google Scholar : PubMed/NCBI | |
|
Zou XP, Dai WJ and Cao J: CDH1 promoter polymorphism (−347G->GA) is a possible prognostic factor in sporadic colorectal cancer. World J Gastroenterol. 15:5340–5345. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Mei Q, Yan HL, Ding FX, Xue G, Huang JJ, Wang YZ and Sun SH: Single-nucleotide polymorphisms of mismatch repair genes in healthy Chinese individuals and sporadic colorectal cancer patients. Cancer Genet Cytogenet. 171:17–23. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Campbell PT, Curtin K, Ulrich CM, Samowitz WS, Bigler J, Velicer CM, Caan B, Potter JD and Slattery ML: Mismatch repair polymorphisms and risk of colon cancer, tumour microsatellite instability and interactions with lifestyle factors. Gut. 58:661–667. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Park SH, Lee GY, Jeon HS, Lee SJ, Kim KM, Jang SS, Kim CH, Lee WK, Kam S, Park RW, et al: -93G->A polymorphism of hMLH1 and risk of primary lung cancer. Int J Cancer. 112:678–682. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Lo YL, Hsiao CF, Jou YS, Chang GC, Tsai YH, Su WC, Chen KY, Chen YM, Huang MS, Hsieh WS, et al: Polymorphisms of MLH1 and MSH2 genes and the risk of lung cancer among never smokers. Lung Cancer. 72:280–286. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Allan JM, Shorto J, Adlard J, Bury J, Coggins R, George R, Katory M, Quirke P, Richman S, Scott D, et al: MLH1-93G>A promoter polymorphism and risk of mismatch repair deficient colorectal cancer. Int J Cancer. 123:2456–2459. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Raptis S, Mrkonjic M, Green RC, Pethe VV, Monga N, Chan YM, Daftary D, Dicks E, Younghusband BH, Parfrey PS, et al: MLH1-93G>A promoter polymorphism and the risk of microsatellite-unstable colorectal cancer. J Natl Cancer Inst. 99:463–474. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Chen H, Taylor NP, Sotamaa KM, Mutch DG, Powell MA, Schmidt AP, Feng S, Hampel HL, de la Chapelle A and Goodfellow PJ: Evidence for heritable predisposition to epigenetic silencing of MLH1. Int J Cancer. 120:1684–1688. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Brenner C, Deplus R, Didelot C, Loriot A, Viré E, De Smet C, Gutierrez A, Danovi D, Bernard D, Boon T, et al: Myc represses transcription through recruitment of DNA methyltransferase corepressor. EMBO J. 24:336–346. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Brenner C and Fuks F: DNA methyltransferases: Facts, clues, mysteries. Curr Top Microbiol Immunol. 301:45–66. 2006.PubMed/NCBI |
