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Functional annotation of colorectal cancer susceptibility loci identifies MLH1 rs1800734 associated with MSI patients
  1. Gaoxiang Ma1,2,
  2. Yuqiu Ge1,2,
  3. Dongying Gu3,
  4. Mulong Du1,2,
  5. Haiyan Chu1,2,
  6. Jinfei Chen3,
  7. Zhengdong Zhang1,2,
  8. Meilin Wang1,2
  1. 1Department of Environmental Genomics, Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing Medical University, Nanjing, China
  2. 2Department of Genetic Toxicology, The Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, China
  3. 3Department of Oncology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China
  1. Correspondence to Dr Meilin Wang, Department of Environmental Genomics, School of Public Health, Nanjing Medical University, 101 Longmian Road, Nanjing, Nanjing 211166, China; mwang{at}

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Recently, we read the article by Ma et al with great interest. They conducted a comprehensive meta-analysis that nominated 62 variants in 50 candidate genes with high level of cumulative evidence for genetic susceptibility to colorectal cancer (CRC).1 Interestingly, the authors found that 10 variants in 7 genes were graded strong and moderate association with CRC risk. However, functional annotations of these variants remain largely unknown. To investigate their functional relevance, we annotated the variants and their high related single nucleotide polymorphisms (SNPs) (linkage disequilibrium, r2>0.9) using publicly available The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus datasets.

After exclusion of the variants with minor allele frequency <0.05, a total of four SNPs (rs1801155, rs1569686, rs1800734 and rs2736100) were included in further analysis (table 1). We then evaluated the effect of risk alleles on gene epigenetic and expression alterations in CRC tumour tissues from TCGA. Three SNPs (rs1569686, rs1800734 and rs2736100) were identified as methylation quantitative trait loci (meQTL) in the 500 kb upstream and downstream from the lead SNP (table 1). For example, the result revealed that the TERT rs2736100 can alter methylation status of cg01603247 located on CLPTM1L gene (table 1). MLH1 rs1800734 (G/A, active promoter) was both meQTL and eQTL for MLH1. The rs1800734 A allele was associated with promoter hypermethylation and decreased expression of MLH1. The meQTL analysis showed that the rs1800734 A allele changed methylation level of nine CpG sites, which all were located on the promoter of MLH1 (p<1.0×10−5) (table 1). Furthermore, the β value of the nine CpG sites between each other was all significantly correlated in a positive direction (see online supplementary figure S1). Among them the cg04841293 explained the most effect by principal component analysis.

Table 1

Functional annotation of four SNPs with high level of evidence for CRC susceptibility

Emerging studies suggested that the mismatch repair gene MLH1 had a low expression in CRC tissues, and its promoter was methylated,2 which were confirmed by our results (see online supplementary figure S2). A recent study identified that mutation or epigenetic silencing of MLH1 resulted in microsatellite instability (MSI) among patients with CRC.3 Furthermore, Campbell et al4 found that the MLH1 rs1800734 was associated with risk of MSI-positive CRC. Here, we found that the patients with the rs1800734 A allele had an aberrant hypermethylation level in the MLH1 promoter and lower MLH1 expression than those with G allele (figure 1). Moreover, the MLH1 was methylated and expression silence in the tumour tissues compared with paired normal tissues (see online supplementary figures S2A–C). The association between the methylation and expression level of MLH1 in CRC tumour tissues was significantly correlated in a negative direction (r=−0.58, p=0.121). When HCT116 cells were treated with 5-aza-2′-deoxycytidine, the methylation level of MLH1 promoter was decreased (see online supplementary figure S2D). Intriguingly, compared with the microsatellite stable patients, the MSI patients showed lower MLH1 expression and higher methylation level for MLH1 promoter in CRC tissues (see online supplementary figure S2E,F). Taken together, we propose that the rs1800734 A allele may lead to MSI in the genome by altering the promoter methylation and the expression level of MLH1, thus contributing to CRC development.

Figure 1

The meQTL and eQTL analysis for MLH1 rs1800734 in colorectal tumour tissues. (A) The association between the lead variants and CpG methylation probe within 1 Mb based on TCGA data set (n=88). For each CpG methylation probe, the region was defined as being less than 500 kb upstream and downstream from the lead SNP. The result showed the sites that were less than 1×10−5. The box plots show the distribution of the methylation levels in each genotype category with error bars representing the 25% and 75% quartiles. (B) The eQTL analysis between rs1800734 genotypes and MLH1mRNA expression in colorectal tumour tissues (n=415). The p values were obtained using linear regression. meQTL, methylation quantitative trait loci; SNP, single nucleotide polymorphism; TCGA, The Cancer Genome Atlas.

In conclusion, we throw light on functionality in CRC tissues for associations between these key variants and CRC susceptibility, and show the detailed mechanism that the MLH1 rs1800734 participated in the colorectal carcinogenesis among MSI patients.


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  • GM, YG and DG contributed equally to this work. ZZ and MW co-supervised this work.

  • Twitter Follow Yuqiu Ge at @gutjnl-2015-311299

  • Contributors ZZ and MW are co-responders for this paper. Study concept and design: MW and GM. Acquisition of data: GM and YG. Analysis and interpretation of data: MD, DG and HC. Drafting of the manuscript: GM and YG. Critical revision of the manuscript for important intellectual content: ZZ, MW and JC. Statistical analysis: GM and YG. Obtained funding: ZZ and MW. Approval of the final version of the manuscript: MW and ZZ.

  • Funding This study was partly supported by National Natural Science Foundation of China (81373091, 81230068 and 81201570), Distinguished Young Scholars of Nanjing (JQX13005) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine).

  • Competing interests None declared.

  • Patient consent Obtained.

  • Provenance and peer review Not commissioned; internally peer reviewed.

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