Original article
Large genomic aberrations in MSH2 and MLH1 genes are frequent in Chinese colorectal cancer

https://doi.org/10.1016/j.cancergencyto.2004.12.008Get rights and content

Abstract

Hereditary nonpolyposis colorectal cancer is caused by inactivating mutations in the genes of the DNA mismatch repair (MMR) system. Studies have shown that large-fragment aberrations in MMR genes are responsible for a considerable proportion of hereditary colorectal cancer (CRC), but it has been rarely reported in Chinese patients. Here we used multiplex ligation-dependent probe amplification to analyze the genomic rearrangements of 45 Chinese hereditary CRC families, 20 young-age CRC patients (onset of CRC at younger than 50 years and no family history), and 13 patients with sporadic CRC diagnosed at age 50 years or older. Overall, we found 9 (13.8%) large genomic deletions or duplications: 7 out of 45 CRC patients with family history and 2 out of 20 young CRC patients. In all alterations, five genomic deletions were uncovered in the MSH2 gene, as well as one deletion and three duplications in the MLH1 gene. Furthermore, two of the duplications unveiled in this study may have more than a four-copy increase of the exon showing duplication in MLH1. The results indicate that genomic aberrations, large-fragment deletions and duplications, in both MSH2 and MLH1 genes play a role in the pathogenesis of Chinese CRC patients with a family history, as reported in western populations. Moreover, the genomic aberrations in these genes might also be a frequent cause of CRC at a young age in China.

Introduction

Hereditary nonpolyposis colorectal cancer (HNPCC; Mendelian Inheritance in Man [MIM] no. 114500) is an autosomal dominant disease characterized clinically by early onset of colorectal cancer (CRC) and other associated tumors [1], [2]. HNPCC is caused by inactivating mutations in the genes of the DNA mismatch repair system (MMR genes), at least four of which have been identified — MSH2, MLH1, PMS2, and MSH6 [3]. So far, most of the germline mutations in HNPCC families have been found in the MSH2 (MIM no. 120435) and MLH1 (MIM no. 120436) genes [4].

A large number of germline mutations, mainly small deletions or insertions leading to frameshifts, splice site alterations, and nonsense and missense mutations have been uncovered by conventional methods such as denaturing high-performance liquid chromatography (DHPLC), heteroduplex analysis, denaturing gradient gel electrophoresis (DGGE), single-strand conformational polymorphism (SSCP), and direct DNA sequencing [5], [6], [7], [8]. Large genomic aberrations, deletions, or duplications of exons, however, cannot be detected by these techniques. Nevertheless, detection of large genomic variants is important in genetic testing in HNPCC because they account for up to 30% of all pathogenic mutations in MSH2 and MLH1 genes [9], [10].

There are several methods for detecting large genomic deletions or duplications, such as Southern blotting, real-time polymerase chain reaction (PCR), protein truncation test (PTT), and semiquantitative multiplex PCR assay [11], [12], [13], [14], [15]. Nevertheless, it is difficult to use them conventionally in a laboratory because they are time consuming and have a low sample throughput. Schouten et al. [16] recently proposed a novel method, multiplex ligation-dependent probe amplification (MLPA), for relative quantification of DNA sequences in high throughput. With this method, it is possible to check all the exons of the MSH2 and MLH1 genes for deletions or duplications in one reaction. Because only one pair of PCR primers is used, MLPA reactions result in a reproducible gel pattern with fragments ranging from 130 to 490 base pairs. Comparison of the gel patterns from suspected HNPCC patients with those from control samples will uncover an aberrant copy number in the screening sequences [10], [17].

Until now, most of the mutational data on HNPCC are based on western subjects. The frequency of large genomic rearrangements of MMR genes in Chinese HNPCC is still unknown. Although the incidence of colorectal cancer ranks fifth among all cancers in China, which is much lower than that in western populations, Chinese CRC is characterized by onset of disease at an earlier age (mean age about 10 years younger than that in western cases) [18]. The mechanism behind this is still unknown.

In this work, we checked the germline genomic deletion or duplication in MSH2 and MLH1 genes to investigate the frequency of large genomic rearrangements of MMR genes in different groups of Chinese patients suspected of having HNPCC.

Section snippets

Patients

Screening for deletions and duplications in MSH2 and MLH1 genes was performed in three groups of Chinese patients. The first group contained 45 unrelated familial CRC patients, of whom 21 fulfill the Amsterdam criteria (AC+ for HNPCC), and 24 (family history [FH]) have a familial clustering of CRC but fail to meet the AC (AC). The second group consisted of 20 young-age CRC patients (age at diagnosis younger than 50 years and without a family history of HNPCC-related cancers). The third group

MLPA analysis of the negative and positive controls

Before screening the genomic DNA of suspected HNPCC, young CRC, and sporadic CRC patients, we tested the reproducibility of MLPA with DNA samples from healthy controls and from patients with known exon deletions in MSH2 and MLH1 genes. No genomic aberrations of the MSH2 and MLH1 genes were found in 22 normal individuals, and the known deletions of the 4 positive controls in MSH2 and MLH1 genes were reproducibly unveiled by MLPA assay (patient 281, MSH2 EX1_6del; patient 325, MLH1 EX7_10del;

Discussion

Several studies have shown that genomic deletions and duplications in MSH2 and MLH1 are a frequent cause of HNPCC [9], [10], [17]. It has been suggested that investigations for large genomic aberrations should be included in routine HNPCC mutation screening protocols. However, it is almost impossible to uncover exon deletions or duplications at the genomic DNA level with the traditional mutation detection methods such as heteroduplex analysis, DGGE, SSCP, or DHPLC [5], [6], [7], [8]. Southern

Acknowledgments

Dr. J.P. Schouten, MRC-Holland corp, kindly provided the MLPA P003 kit. The study was supported by the Health Department, Jiangsu Province, P.R.China (H0207, RC 200207).

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