A novel MSH2 mutation in a Chinese family with ...

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the age of 25 and right semi-colon at 37) who has two as yet unaffected children, the daughter (IV-1) aged 16 and the son. (IV-2) aged 8. One niece of the index ...
Int J Colorectal Dis DOI 10.1007/s00384-006-0253-z

ORIGINAL ARTICLE

A novel MSH2 mutation in a Chinese family with hereditary non-polyposis colorectal cancer Duo Zheng & Tiegang Li & Xiaoping Liu & Weixin Hu & Hanchun Chen & Yongjia Yang

Accepted: 30 November 2006 # Springer-Verlag 2007

Abstract Background and aims Hereditary non-polyposis colorectal cancer (HNPCC) is one of the most common hereditary colon cancer syndrome accounting for 1–5% of all colorectal cancer cases. Germline mutations in DNA mismatch repair (MMR) genes are associated with the clinical phenotype of HNPCC. Defects in the MSH2 gene may account for about 40% of HNPCC cases including nucleotide substitutions, deletions and insertions. Only a few mutations were reported in Chinese families. This study was performed to identify the disease-causing gene mutation(s) and conduct pre-symptomatic diagnosis in a Chinese family with HNPCC. Materials and methods Mutation analysis in MLH1 and MSH2 followed by pre-symptomatic diagnosis in MSH2 was performed on genomic DNA isolated from the family members. Results A novel duplication mutation of four nucleotides in exon 7 of MSH2 (MSH2: c.1216_1219dupCGAC) resulting in a premature stop 10 codons downstream in MSH2 (p.L407fsX417) was found. The mutation was associated with HNPCC and an asymptomatic carrier was found in the family.

Duo Zheng, Tiegang Li and Xiaoping Liu have contributed equally to this work. D. Zheng (*) : W. Hu : H. Chen : Y. Yang School of Biological Science and Technology, Central South University, Changsha, People’s Republic of China e-mail: [email protected] T. Li : X. Liu Department of Surgery, the Second Xiangya Hospital, Central South University, Changsha, People’s Republic of China

Conclusion HNPCC in a Chinese family is associated with a novel mutation in the MSH2 gene (MSH2: c.1216_12 19dupCGAC). Keywords Hereditary non-polyposis colorectal cancer . Mismatch repair gene . Germline mutation . Pre-symptomatic diagnosis

Introduction Hereditary non-polyposis colorectal cancer (HNPCC, MIM114500), also called Lynch syndrome, is an autosomal, dominantly inherited cancer syndrome accounting for approximately 1–5% of all colorectal cancer cases [1]. Affected individuals are at increased risk for developing colorectal cancer and extra-colonic carcinomas including cancers of the endometrium, ovaries, urothelium, stomach and other organs [2]. The genetic basis of HNPCC is related to germline mutations in mismatch repair (MMR) genes and the function of the MMR system is to replace and eliminate the single base mismatches and insertion–deletion loops. The functional inactivation of cellular DNA MMR leads to microsatellite instability (MSI) phenotype [3, 4] resulting in mutations of repeated sequences in various tumor-related genes [5]. Disease-causing germline mutations in four DNA MMR genes (MSH2 [6, 7], MLH1 [8, 9], MSH6 [10, 11] and PMS2 [12]) were described to date. The majority of the mutations occurred in MSH2 and MLH1. A review of the HNPCC mutation database revealed 448 different mutations that primarily involved MLH1 (50%), MSH2 (39%) and MSH6 (7%) and occurred in 748 families from all over the world [13]. However, only a few mutations were reported in Chinese families.

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The first gene identified to be associated with HNPCC was the MSH2 on chromosome 1p22–21 by both the candidate gene approach and positional cloning [6, 7]. MSH2 is composed of 16 exons encoding a protein of 935 amino acids. More than 200 mutations in MSH2 were found to be associated with HNPCC including nucleotide substitutions, deletions and insertions. Pathogenic mutations in MSH2 were scattered throughout all exons [2]. In this article, we report on a novel MSH2 gene mutation identified in a Chinese family affected with hereditary nonpolyposis colorectal cancer (HNPCC). This novel MSH2 gene mutation, which is characteristic of a 4-bp duplication in exon 7, disrupts the normal open reading frame and leads to a predicted premature truncated MSH2.

Materials and methods Family data and DNA samples A Chinese family with hereditary non-polyposis colorectal cancer was collected from Hunan province. A complete family history was obtained and peripheral blood of seven individuals from the family was collected with informed written consents. Family members were clinically diagnosed at the Second Xiangya Hospital of Central South University. Four individuals were considered to be affected and three were unaffected. Fifty normal individuals with no personal or family history of cancer were sampled as controls. Genomic DNA was isolated from peripheral blood using standard phenol extraction protocol.

a primary sequence database and nucleotide +1 is the first nucleotide of the MSH2 CDS sequence (NCBI GenBank using Reference Sequence IDs NM_00249 and NM_000251 for MLH1 and MSH2, respectively). The reference sequences from UCSC (http://www.genome. ucsc.edu/) were used for mutation mapping of intronic regions of the MLH1 and MSH2 genes. The amino acid changes were deduced from nucleotide alterations in relation to a protein reference sequence from SWISSPROT, using ID: P40692 for MLH1 and ID: P43246 for MSH2.

Pre-symptomatic diagnosis Site-specific polymerase chain reaction was applied to 50 normal individuals and the family members including the two young family members who were 16 years old and 8 years old, respectively. At the mutated site of MSH2, modified forward primer with two additional duplicated nucleotides (CG) in the 3′ end was designed (5′-ACTTA CAAGATTGTTACCGACCG-3′). PCR amplification was conducted with the modified forward primer and a reverse primer located in the flanking exon 7 (5′-AATCTGAA TGTGTCCTAAGAGTG-3′) in a 25 μl reaction mixture (described previously) under the following conditions: initial denaturation at 94°C for 5 min; 35 cycles of 30 s at 94°C for denaturation, 30 s at 55°C for primer annealing, 30 s at 72°C for extension and final extension at 72°C for 10 min. The PCR products were analyzed by 1.5% agarose gel electrophoresis according to standard protocols.

Mutation detection in the family Results Mutation analysis was performed by complete sequencing of genomic DNA isolated from five adult family members. Primers were designed to amplify all exons and flanking intronic splicing sites of MLH1 and MSH2 from genomic DNA. PCRs were carried out using standard reaction mixtures of TaKaRa Taq™ (TaKaRa Biotechnology, Dalian, China). The PCR reaction mixture (25 μl) contained 300 ng of genomic DNA, 200 μM of each dNTP, 80 pmol each of forward and reverse primers and 0.5 U of TaKaRa Taq DNA polymerase. The PCR products were then purified using the Wizard SV Gel and PCR Clean-Up System according to the manufacturer’s instructions (Promega, Madison, WI, USA) and directly sequenced by ABI Prism 377XL DNA sequencer. Nucleotide sequences were compared with the MLH1 and MSH2 cDNA sequences. The identified DNA alterations were confirmed by sequencing from both DNA ends in two independent PCR products. Sequence variations were described in relation to the cDNA reference sequence from

Characteristic of the patients The Chinese family fulfills the Amsterdam criteria for hereditary non-polyposis colorectal cancer (HNPCC) (Fig. 1). There were 17 patients with colorectal carcinomas and/or other cancers in 3 successive generations and 13 of them were diagnosed before the age of 50. The index patient (II-3) is a 69-year-old man who was diagnosed with 3 asynchronous colorectal carcinomas (Fig. 2) (right semicolon at the age of 52, sigmoid at 61 and rectum at 68). He was treated with a right colectomy, sigmoidectomy and Dixon procedure, respectively. His daughter (III-8) developed endometrium cancer at the age of 31 and his son (III-7) developed asynchronous colorectal carcinomas (rectum at the age of 25 and right semi-colon at 37) who has two as yet unaffected children, the daughter (IV-1) aged 16 and the son (IV-2) aged 8. One niece of the index patient died from endometrium cancer at the age of 32 and two nephews died

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Fig. 1 A Chinese HNPCC family with a germline MSH2 genomic mutation. Squares: male, circles: female, open symbols: no tumor, solid symbols: patients with carcinomas, symbols with a diagonal: deceased. Letters indicate the site of cancer. Co: colon or rectum, Ga: stomach, Bo: bone, Br: brain, En: endometrium or uterus, Li: liver.

Numbers under the symbols indicate the age of diagnosis as affected or unaffected. Numbers above the symbols indicate the age at death. Plus sign indicates the presence of germline mutation; minus sign indicates the absence of germline mutation. The arrow indicates the proband

from stomach or liver cancer at the age of 43 or 31, respectively.

10 codons downstream (p.L407fsX417). The patient is heterozygote for this mutation and thus his normal tissue carries both mutant and wild type alleles of the MSH2 gene. This mutation was identified in all the affected members (II-3, III-1, III-2, III-7), but not from the unaffected family member (III-5) or 50 normal controls (data not shown) indicating that the 4-bp genomic mutation of MSH2 is associated with the HNPCC in the family. These data expand the spectrum of MSH2 mutations causing HNPCC.

Germline MSH2 mutation Four DNA MMR genes (MLH1, MSH2, MSH6 and PMS2) were found to be associated with HNPCC and about 90% of mutations affect the MLH1 and MSH2 genes. Therefore, our initial genetic study of this family was focused on MLH1 and MSH2. To identify the disease-causing mutation, we sequenced all exons and intron–exon boundaries of MLH1 and MSH2 in the family. No mutation was found in MLH1. A heterozygous duplication of 4 nucleotides (CGAC) from nucleotide 1,216 to nucleotide 1,219 in MSH2 (MSH2: c.1216_1219dupCGAC) was detected in the index patient (II-3) (Fig. 3). The 4-bp duplication, which, to our knowledge was not previously reported, results in a frameshift mutation from codon 407 and a premature stop

Pre-symptomatic diagnosis Site-specific PCR was applied using the modified forward primer with mutated nucleotides and the reverse primer located in the flanking exon 7. The specific products of about 200 bp were amplified in the genomic DNA from all affected family members (II-3, III-1, III-2, III-7), but not from the unaffected one (III-5) (Fig. 4) or normal individuals (data not shown). These data indicated that the

Fig. 2 Pathological feature of the proband (II-3) diagnosed as rectum cancer at the age of 68. The tumor was differentiated with clear glandular duct structure, infiltrating to the muscular layer and tunica serosa. A lot of lymphocytes and plasma cells infiltrated around the tumor cells

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a

Discussion

b

c Normal

TGT TAC CGA CTC TAT CAG GGT Cys Tyr Arg Leu Tyr Gln Gly

Mutant

TGT TAC CGA CCG ACT CTA TCA GGG T Cys Tyr Arg Pro Thr Leu Ser Gly Fig. 3 A 4-bp duplication in MSH2 associated with HNPCC in a Chinese family. a Sequence analysis of normal individual (wild type). b Sequence analysis of the proband (II-3) showing a heterozygous duplication of four nucleotides (c.1216_1219dupCGAC). c The mutation results in a frameshift mutation from codon 407 and a premature stop 10 codons downstream (p.L407fsX417)

method is highly specific and it has the potential for effective pre-symptomatic diagnosis. One young family member (IV-1) showed the same PCR product as the affected members (Fig. 4) suggesting that she is a HNPCC carrier with the same disease-causing mutation in the family. No 200-bp PCR product was amplified from another young family member (IV-2) (Fig. 4) indicating that he is normal. The results of the pre-symptomatic diagnosis on the two young family members were confirmed by sequencing exon 7 of MSH2 as described above.

Fig. 4 PCR amplification of the mutated fragment in exon 7 of MSH2. PCR products were from symptomatic (A: II-3, B: III-7, F: III2, G: III-1), asymptomatic (E: III-5) and two pre-symptomatic family members (C: IV-1, D: IV-2). The mutated products were detected in the genomic DNA of all patients present, but not in that of the unaffected member. One of the two young members (IV-1) presented a mutant band suggesting that she is a HNPCC carrier

HNPCC is an autosomal, dominantly inherited disease associated with marked increase in cancer susceptibility. It is characterized by a familial predisposition to early onset colorectal carcinoma and extra-colonic cancers of the gastrointestinal, urological and female reproductive tracts. Diagnosis of classical HNPCC is based on strict international criteria, namely, the Amsterdam criteria I or II (ACI or ACII) [14, 15] or the less-stringent criteria referred to as the Bethesda guidelines (BG) [16]. The Amsterdam criteria is: three or more relatives affected by colorectal cancer, one a first degree relative of the other two; two or more generations affected; one or more colorectal cancers presenting before 50 years of age; exclusion of hereditary polyposis syndromes. By clinical diagnose and genetic counseling, the family chosen for this study meets the Amsterdam criteria for HNPCC. There were 17 patients with colorectal carcinomas or other cancers in three successive generations and 13 of them were diagnosed before the age of 50. The proband was diagnosed with 3 asynchronous colorectal carcinomas (right semi-colon at the age of 52, sigmoid at 61 and rectum at 68), his mother died from colon cancer and five of his siblings were affected by cancers. He has three children, one daughter with endometrium cancer and a son with asynchronous colorectal carcinomas (rectum at the age of 25 and right semi-colon at 37). The DNA mismatch repair (MMR) system is essential for the maintenance of genomic stability. All main functions of the MMR system including the correction of biosynthetic errors, DNA damage surveillance and prevention of recombination between non-identical sequences serve this important purpose. Failure to accomplish these functions may result in cancer. Inherited defects in DNA MMR genes, such as MLH1 [MIM# 120436], MSH2 [MIM# 120435], MSH6 [MIM# 600678] and PMS2 [MIM# 600259] underlie HNPCC, one of the most prevalent cancer syndromes in humans. In this study, a novel 4-bp duplication in MSH2 (c.1216_1219dupCGAC) was found in a Chinese family with HNPCC. To our knowledge, this is the first report of the duplication of 4 nucleotides (CGAC) occurring at exon 7 from nucleotide 1,216 to nucleotide 1,219 in MSH2 (nucleotide +1 is the first nucleotide of the MSH2 CDS sequence). The mutation co-segregates with the HNPCC phenotype in patients, but not with the unaffected family members and 50 controls, which strongly supports that this mutation may be the cause of HNPCC in this family. MSH2 is involved in post-replication mismatch repair by binding specifically to DNA containing mismatched nucleotides and thus providing a target for the excision repair processes. Mutations of MSH2 causing HNPCC

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commonly target domains required for protein–protein or protein–DNA interactions. The novel mutation described here is located in exon 7 of MSH2 and it results in a frameshift mutation from codon 407 and hence a truncated protein of 417 aa with 10 abnormal amino acids before the premature stop (p.L407fsX417). Almost at the same site of MSH2 gene, a CGA to TGA transition in codon 406 was found in a kindred with HNPCC resulting in change of arginine to stop codon [7]. The truncated products from these mutations lack evolutionarily conserved domains required for interaction with either MSH3 or MSH6 and DNA binding. Clinical diagnosis of HNPCC patients is difficult due to non-distinct pre-symptomatic warnings. When the diseasecausing mutation is detected in the family, a satisfactory explanation can be provided for an accumulation of tumors at a young age and often of untimely death. More importantly, pre-symptomatic testing of disease-causing mutation becomes available to all family members, which is effective for the prediction and early detection of tumors. Thus, treatment can be administrated at an early stage to reduce mortality among at risk family members. In addition, relatives not at risk can avoid uncertainty and needlessly intensive surveillance. In this report, polymerase chain reaction with modified primer was applied to the presymptomatic diagnosis. The PCR-based mutation specific testing is a simple and efficient method, which can be used for pre-symptomatic molecular diagnosis on all members, especially young children of the family and screening of HNPCC patients for the same germline mutation. Acknowledgements We would like to thank the family who participated in this study. This work was supported in part by a grant from the National Natural Science Foundation of China (NSFC) (Duo Zheng, grant no. 30270735).

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