AIM: To analyze the clinical characteristics of Chinese hereditary nonpolyposis colorectal cancer (HNPCC) families and to screen the germline mutations of human mismatch repair genes hMLH1 and hMSH2 in the probands.

METHODS: Thirty-one independent Chinese HNPCC families were collected in Zhejiang Province. All of them met Chinese HNPCC criteria. Clinical data about patient gender, site of colorectal cancer, age of onset, history of multiple colorectal cancer, associated extracolonic cancer were recorded. PCR and denaturing high performance liquid chromatography (DHPLC) were employed to screen the mutations. Sequencing analysis was used to find out the exact mutation site and characteristics of the samples showing abnormal DHPLC profiles.

RESULTS: One hundred and thirty-six malignant neoplasms were found in 107 patients including 14 multiple cancers．One hundred and six of the 136 neoplasms (77.9%) were diagnosed as colorectal cancer, with an average age of onset at 48.57 ± 29.00 years. Gastric cancer was the most common extracolonic cancer (10.3%) in these families. Twenty-three different sequence variations in hMLHl and hMSH2 genes were detected in these 17 families. Fifteen sequence variations were located in the exons, including 5 SNPs, 3 silent mutations, 3 missense mutations, 2 nonsense mutations and 2 frameshift mutations. The latter seven mutations seemed to be pathogenic.

CONCLUSION: Germline mutations of hMLH1 and hMSH2 genes are identified in about one-third HNPCC kindreds fulfilling Chinese HNPCC criteria. Chinese HNPCC families have some particular clinical characteristics, such as a left-sided predominance, less synchronous or metachronous colorectal cancer, and frequent occurrence of gastric cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominantly-inherited cancer susceptibility syndrome. It is estimated that HNPCC may account for 5%-10% of the total colorectal cancers (CRC) worldwide[1]. Clinically, HNPCC families are diagnosed based on the Amsterdam criteria. Although the Amsterdam criteria unify the diagnosis of HNPCC worldwide, they are too rigid to exclude extracolonic cancers associated with HNPCC. New criteria and guidelines have been proposed, such as the Japanese criteria, suspected HNPCC criteria. Based on the suspected HNPCC criteria and the specific characteristics of the tumor spectrum in Chinese population, the Chinese Hereditary Colorectal Cancer Collaboration has established the criteria for Chinese HNPCC families[2].

Six genes (hMSH2, hMLH1, hPMS1, hPMS2, hMSH6/GTBP and hMLH3) involved in DNA mismatch repair have been proven to be closely related with the development of HNPCC. hMLH1 and hMSH2 genes are thought to be the main genes responsible for HNPCC, because more than 90% detected germline mutations in HNPCC families are located in these two genes[3]. Denaturing high-performance liquid chromatography (DHPLC) is a mutation pre-screening method[4]. The major advantage of DHPLC is the low cost and the high speed of analysis. Therefore, in this study, we analyzed the clinical characteristics of our 31 Chinese HNPCC families registered in our cancer institute, and screened the germline mutations of hMLH1 and hMSH2 genes by DHPLC and DNA sequencing.

HNPCC is characterized by dominant right colon localization, early age of onset, high prevalence of synchronous and metachronous tumors, and certain extracolonic cancers[14]. In our study, patients with Chinese HNPCC developed CRC at an average age of 48.57 ± 29.00 years. As compared to the onset of sporadic colorectal malignancies at the age approximately 60 years, the onset of HNPCC was much earlier, similar to that in Western countries. In the present study, synchronous and metachronous colorectal cancers only accounted for 8.5% of all CRCs, lower than those in Western countries. The reason is still unclear. Left-sided colorectal cancers accounted for 60.6% (40/66) of all CRCs. This result might be due to the high incidence of left-sided colon cancer (including rectal cancer) in Chinese population. Gastric cancer was the most frequent extracolonic cancer in our 31 families, while endometrial cancer was the most common in Western countries. The reason might be due to the high incidence of gastric cancer in Chinese population. Liu et al[15] have reported similar results found in their cohort of Chinese HNPCC families. These characteristics might truly reflect the specific clinical phenotype of Chinese HNPCC.

In this study, we screened mutations of the hMLH1 and hMSH2 genes in 31 Chinese HNPCC families by DHPLC and PCR as well as DNA sequencing. Twenty-three sequence abnormalities were detected in 17 probands, 15 in exons and 8 in introns. Base variants identified were compared with those described in the human gene mutation database (HGMD) (http://www.uwcm.ac.uk/uwcm/mg/hgmd0.html). Seven of the 15 sequence abnormalities including 3 missense mutations (H4, H11, H12), 2 nonsense mutations (H20, H27), and 2 frameshift mutations (H24, H29) in coding regions of the hMLH1 and hMSH2 genes might probably affect the protein functions. Detailed information of these mutations is listed in Table 1. Among them, mutations of H4, H11 and H20 have been reported before[8,9,12], while the other four mutations of H12, H27, H24 and H29 are novel. The mutation rate was 22.6% (7/31). Nearly half of the mutations were missense mutations. Three (H5, H16, H28) of the fifteen sequence abnormalities in coding sequences are novel silent mutations in exons 1, 7, 5 of the hMSH2 gene, respectively. The last five (H3, H7, H16, H21 and H22) of the fifteen sequence abnormalities in coding sequences are known as SNPs[7,10,11,13].

The missense mutation detected in sample H4 located at codon 67 in exon 2 of the hMLH1 gene, has been reported as a pathological mutation[8]. The missense mutation detected in sample H11 located at codon 629 in exon 12 of the hMSH2 gene has also been reported[9]. In sample H12, a novel missense mutation at codon 839 in exon 15 of the hMSH2 gene was first discovered in our study, which is located in the Muts V structural motif (peptides 619-854) of the hMSH2 gene and leads to amino acid change from histidine to arginine. In order to determine the significance of this mutation, 100 blood samples from cancer-free donors were tested under the same conditions and none of the 100 normal samples harbored the same base change as sample H12, suggesting that this base change is pathologic.

The nonsense mutation detected in sample H20 altered nucleotide 2250 (genomic DNA) from C to G at codon 750 in exon 19 of the hMLH1 gene, resulting in a stop codon instead of tyrosine, forming a truncated protein of MutL losing 6 peptides from 751 residue to 756 residue. This nonsense mutation has been reported by Syngal et al [12]. The other nonsense mutation in sample H27 altered nucleotide 2292 (genomic DNA) from G to A at codon 764 in exon 11 of the hMSH2 gene, leading to the substitution of a stop codon TGA for tryptophan condon TGG, forming a truncated protein of MutC structural motif (peptides 619-854) losing 170 peptides from 765 residue to 934 residue, suggesting that these nonsense mutations may have a causative effect on cancer predisposition and further studies are needed to examine their co-segregation with the phenotype.

Two framshift mutations detected in samples H24 and H29 have not been reported previously. In sample H24, the base A deleted at codon 555 in exon 11 of the hMSH2 gene led to a stop codon at codon 556. While in sample H29, the GT deleted at codon 531 in exon 14 of the hMLH1 gene led to a stop codon at codon 555. Both mutations produced truncated proteins with insufficient function.

Of the 23 base variants, 8 were located in intronic regions. Five of these 8 variants have been previously reported and known as SNPs (http://www.ncbi.nlm.nih.gov/), the significance of the other 3 remains uncertain.

DHPLC is an elegant, new method for mutation detection. It detects heteroduplex formation in renaturated PCR fragments possessing heterozygous sequence variations, and has been applied successfully to mutation screening of various genes[3]. In the past few years, we have searched successfully for mutations in hMLH1 and hMSH2 genes in 29 HNPCC families and 34 suspected HNPCC families with PCR-SSCP and DNA sequencing methods[16] . However, SSCP is inefficient in detecting mutations and misses 20% of point and frameshift mutations. Though DNA sequencing is supposed to be a screening method with a high sensitivity, it is time consuming and expensive. It was reported that the sensitivity of DHPLC for screening mutations of the hMLH1 and hMSH2 genes can reach 97% without false negatives[6]. Furthermore, the system is highly automatic, the running time per sample averages only 7 min. Because of these advantages of DHPLC, it enables a broad screening of HNPCC families for human mismatch repair gene alterations in routine analysis.