Loss of the wild type MLH1 gene is a feature of hereditary nonpolyposis colorectal cancer.

The mechanism by which germline mutations of DNA mismatch repair genes cause susceptibility to tumour formation is not yet understood. Studies in vitro indicate that heterozygosity for these mutations, unlike homozygosity, does not affect mismatch repair. Surprisingly, no loss of heterozygosity at the predisposing loci has so far been described in hereditary nonpolyposis colorectal cancers. Here, we show that loss of heterozygosity (LOH) of markers within or adjacent to the MLH1 gene on chromosome 3p occurs nonrandomly in tumours from members of families in which the disease phenotype cosegregates with MLH1. In every informative case, the loss affects the wild type allele. These results suggest that DNA mismatch repair genes resemble tumour suppressor genes in that two hits are required to cause a phenotypic effect.

Founding mutations and Alu-mediated recombination in hereditary colon cancer.

By screening members of Finnish families displaying hereditary nonpolyposis colorectal cancer (HNPCC) for predisposing germline mutations in MSH2 and MLH1, we show that two mutations in MLH1 together account for 63% (19/30) of kindreds meeting international diagnostic criteria. Mutation 1, originally detected as a 165-base pair deletion in MLH1 cDNA comprising exon 16, was shown to consist of a 3.5-kilobase genomic deletion most likely resulting from Alu-mediated recombination. Mutation 2 destroys the splice acceptor site of exon 6. A simple diagnostic test based on polymerase chain reaction was designed for both mutations. Our results show that these two ancestral founding mutations account for a majority of Finnish HNPCC kindreds and represent the first report of Alu-mediated recombination causing a prevalent, dominantly inherited predisposition to cancer.

Mismatch repair defects in cancer.

Post-replicative mismatch repair in humans utilises the hMSH2, hMSH6, hMSH3, hMLH1 and hPMS2 genes and possibly the newly identified hMLH3 gene. Recently, a link has been established between hMSH6 mutations and 'atypical' hereditary non-polyposis colon cancer (HNPCC) with an increased incidence of endometrial cancers. To satisfy the need for a diagnostic test capable of differentiating between pathogenic mutations and polymorphisms, several functional assays that fulfil these criteria have been described. These should allow for better diagnosis of HNPCC.

Genomic instability in colorectal cancer: relationship to clinicopathological variables and family history.

Recent reports have suggested that one or more genes may cause replication errors (RER) during colorectal tumorigenesis. Additional alleles are seen in the tumors when analyzing random microsatellite loci. We have studied seven dinucleotide repeat loci, located on seven different chromosomes, by use of polymerase chain reaction amplification and denaturing polyacrylamide gel electrophoresis. We found that 16.5% (40 of 243) colorectal cancers showed RER at one or several loci (RER+). This includes 31% (4 of 13) among cases with a strong positive family history according to previously published criteria and 17% (35 of 207) among cases with no history of familial cancer. Interestingly, no significant association was found between RER+ tumors and a general familial clustering of cancer. Microsatellite instability was significantly associated with DNA diploid status of the tumor (P < 0.001), with the location of the tumor in the proximal colon (P < 0.001), and with poorly differentiated tumor phenotype (P < 0.001). Patients with RER+ at > or = 2 loci tumors had an increased survival (P = 0.05). We further analyzed 84 breast cancers and 86 male germ cell cancers using the same seven markers. None of the tumors were RER+, indicating that this phenomenon may be specific to certain types of tumors.

Microsatellite instability is associated with tumors that characterize the hereditary non-polyposis colorectal carcinoma syndrome.

Microsatellite instability implying multiple replication errors (RER+ phenotype) characterizes a proportion of colorectal carcinomas, particularly those from patients with the hereditary non-polyposis colorectal carcinoma syndrome. We studied the incidence of microsatellite instability in more than 500 sporadic tumors representing 6 different types of cancer. Apart from colorectal carcinoma [see the paper by Lothe et al. (Cancer Res., 53:5849-5852, 1993)] the RER+ phenotype was found in 18% (6 of 33) of gastric carcinomas and 22% (4 of 18) of endometrial carcinomas. In contrast, no evidence of this abnormality was detected in cancers of the lung (N = 85), breast (N = 84), and testis (N = 86). Importantly, the first three cancers, as opposed to the latter three, are characteristic of the hereditary non-polyposis colorectal carcinoma syndrome. These findings suggest that the cancers belonging to the hereditary non-polyposis colorectal carcinoma tumor spectrum may have essential pathogenetic steps in common, including a tendency to multiple replication errors.

Lack of MSH2 and MSH6 characterizes endometrial but not colon carcinomas in hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer syndrome is associated with an inherited predisposition to primarily colorectal cancer (CRC) and endometrial cancer (EC); however, the biological basis of the organ involvement remains unknown. As an attempt to explore whether the expression levels of MLH1, MSH2, and MSH6 may play a role, we used immunohistochemistry to study 42 ECs and 35 CRCs from patients carrying the same predisposing mutations. Among MSH2 mutation carriers, MLH1 was expressed in both tumor types, whereas MSH2 and, in many cases, also MSH6, were absent. Remarkably, among MLH1 mutation carriers, 54% of ECs (21 of 39), but none of the CRCs (0 of 32), lacked the MSH2 and/or MSH6 protein in addition to lacking MLH1 protein expression. These results demonstrate a marked difference between hereditary nonpolyposis colorectal cancer-related CRCs and ECs and suggest that the development of the latter tumors is selectively associated with the MSH2/MSH6 protein complex deficiency.

Life-time risk of different cancers in hereditary non-polyposis colorectal cancer (HNPCC) syndrome.

Identification of hereditary non-polyposis colorectal cancer (HNPCC) indicates theoretical life-time risks of 50% for the descendants of an affected family member and of 100% for the true gene carriers. However, besides colorectal cancer (CRC), many other cancer types and sites are also involved, which gives reason to evaluate the magnitude of risk for various other cancer types. A detailed pedigree analysis of 40 families with HNPCC identified 414 patients affected with cancer. A Kaplan-Meier life-table analysis for the cumulative risk of various cancers was performed on the basis of the 293 putative gene carriers who had adequate clinical and histological documentation of their tumors. Cumulative risks were highest for colorectal (78%) and endometrial cancers (43%, women only), followed by gastric, biliary tract, urinary tract and ovarian cancers (19-9%). For the other probably HNPCC-related cancer types, such as small bowel carcinoma and brain tumors, the life-time risk was only 1%. The risk of any metachronous cancer reached 90% after treatment of CRC and 75% after endometrial cancer; the second tumor was most often a new CRC or endometrial cancer. CRC remains the most important cancer type in the HNPCC syndrome but does not develop in all gene carriers. This makes the decision of possible prophylactic colectomy for test-detected gene carriers difficult. Of the many other cancer types involved, at least endometrial cancer is common enough to necessitate a specific surveillance program.

Identification of hMutLbeta, a heterodimer of hMLH1 and hPMS1.

hMLH1 and hPMS2 function in postreplicative mismatch repair in the form of a heterodimer referred to as hMutLalpha. Tumors or cell lines lacking this factor display mutator phenotypes and microsatellite instability, and mutations in the hMLH1 and hPMS2 genes predispose to hereditary non-polyposis colon cancer. A third MutL homologue, hPMS1, has also been reported to be mutated in one cancer-prone kindred, but the protein encoded by this locus has so far remained without function. We now show that hPMS1 is expressed in human cells and that it interacts with hMLH1 with high affinity to form the heterodimer hMutLbeta. Recombinant hMutLalpha and hMutLbeta, expressed in the baculovirus system, were tested for their activity in an in vitro mismatch repair assay. While hMutLalpha could fully complement extracts of mismatch repair-deficient cell lines lacking hMLH1 or hPMS2, hMutLbeta failed to do so with any of the different substrates tested in this assay. The involvement of the latter factor in postreplicative mismatch repair thus remains to be demonstrated.

MSH6 missense mutations are often associated with no or low cancer susceptibility.

Mismatch repair (MMR) deficiency in tumours from patients with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome is mainly caused by mutations in the MLH1, MSH2, and MSH6 genes. A major challenge in the clinical management of patients with suspected HNPCC is the frequent occurrence of missense mutations in MSH6. These can be considered neither deleterious nor clinically innocent a priori. To assess their significance we studied five novel MSH6 missense mutations in six patients derived from a series of consecutive endometrial and colorectal cancer patients selected for study after their tumours were determined to be microsatellite unstable. We tested each mutated protein for heterodimerisation with MSH2 and for in vitro MMR capability. Four mutations (R128L, P623L, K728T, G881K+S) showed no impairment of these functions while the fifth (E1193K) displayed marked impairment of both functions. These results, taken together with our previous similar findings concerning six other missense mutations in MSH6, allow us to conclude that many or most missense changes in MSH6 likely are clinically innocent, whereas some missense changes such as E1193K, which lead to impaired MMR, are likely to be clinically significant, but have low penetrance.

Missense and nonsense mutations in codon 659 of MLH1 cause aberrant splicing of messenger RNA in HNPCC kindreds.

Germline mutations that give rise to premature termination codons in mRNAs have frequently been associated with aberrant processing of the nascent transcripts. This can take the form either of nonsense-mediated mRNA decay or of aberrant splicing of the pre-mRNA. In a family affected by hereditary nonpolyposis colorectal cancer, a two-nucleotide deletion in codon 659, which introduces a frameshift and a new stop codon in exon 17 of the DNA mismatch repair gene MLH1, has been reported to lead to skipping of the exon. We now report that this phenomenon occurs also when there are missense or nonsense mutations in this codon. Our results thus suggest that in aberrant splicing the nature of the mutation may be less important than its position within the exon. These findings are of importance to mutation interpretation, as they imply that aberrant splicing could be associated even with silent mutations that do not lead to amino acid substitutions. Genes Chromosomes Cancer 26:372-375, 1999.

Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer.

Recent studies have shown that a locus responsible for hereditary nonpolyposis colorectal cancer (HNPCC) is on chromosome 2p and that tumors developing in these patients contain alterations in microsatellite sequences (RER+ phenotype). We have used chromosome microdissection to obtain highly polymorphic markers from chromosome 2p16. These and other markers were ordered in a panel of somatic cell hybrids and used to define a 0.8 Mb interval containing the HNPCC locus. Candidate genes were then mapped, and one was found to lie within the 0.8 Mb interval. We identified this candidate by virtue of its homology to mutS mismatch repair genes. cDNA clones were obtained and the sequence used to detect germline mutations, including those producing termination codons, in HNPCC kindreds. Somatic as well as germline mutations of the gene were identified in RER+ tumor cells. This mutS homolog is therefore likely to be responsible for HNPCC.

Close linkage to chromosome 3p and conservation of ancestral founding haplotype in hereditary nonpolyposis colorectal cancer families.

A susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC) was recently shown to be due to mutations in the MSH2 gene on chromosome 2p. A second susceptibility locus has been mapped to chromosome 3p in two families. The present report describes the results of a genetic study of Finnish HNPCC kindreds. Of 18 apparently unrelated families living in different parts of the country, 11 could be genealogically traced to a common ancestry dating at least 13 generations back in a small geographic area. Linkage studies were possible in 9 families, revealing conclusive or probable linkage to markers on 3p in 8. Five of these were among those having shared ancestry. The location of the gene was refined by a linkage study comprising 12 marker loci. By analysis of recombinations in such families, the HNPCC locus could be assigned to the 1-centimorgan interval between marker loci D3S1561 and D3S1298. A haplotype encompassing 10 centimorgans around the HNPCC locus was conserved in five of the pedigrees with shared ancestry and present in 2 further families in which linkage analysis was not possible. Our results suggest the presence of a widespread single ancestral founding mutation. Moreover, the map position of the 3p gene for HNPCC susceptibility was greatly refined.

Mismatch repair genes on chromosomes 2p and 3p account for a major share of hereditary nonpolyposis colorectal cancer families evaluable by linkage.

Two susceptibility loci for hereditary nonpolyposis colorectal cancer (HNPCC) have been identified, and each contains a mismatch repair gene: MSH2 on chromosome 2p and MLH1 on chromosome 3p. We studied the involvement of these loci in 13 large HNPCC kindreds originating from three different continents. Six families showed close linkage to the 2p locus, and a heritable mutation of the MSH2 gene was subsequently found in four. The 2p-linked kindreds included a family characterized by the lack of extracolonic manifestations (Lynch I syndrome), as well as two families with cutaneous manifestations typical of the Muir-Torre syndrome. Four families showed evidence for linkage to the 3p locus, and a heritable mutation of the MLH1 gene was later detected in three. One 3p-linked kindred was of Amerindian origin. Of the remaining three families studied for linkage, one showed lod scores compatible with exclusion of both MSH2 and MLH1, while lod scores obtained in the other two families suggested exclusion of one HNPCC locus (MSH2 or MLH1) but were uninformative for markers flanking the other locus. Our results suggest that mismatch repair genes on 2p and 3p account for a major share of HNPCC in kindreds that can be evaluated by linkage analysis.

Tolerance of human MSH2+/- lymphoblastoid cells to the methylating agent temozolomide.

Members of hereditary nonpolyposis colon cancer (HNPCC) families harboring heterozygous germline mutations in the DNA mismatch repair genes hMSH2 or hMLH1 present with tumors generally two to three decades earlier than individuals with nonfamilial sporadic colon cancer. We searched for phenotypic features that might predispose heterozygous cells from HNPCC kindreds to malignant transformation. hMSH2(+/-) lymphoblastoid cell lines were found to be on average about 4-fold more tolerant than wild-type cells to killing by the methylating agent temozolomide, a phenotype that is invariably linked with impairment of the mismatch repair system. This finding was associated with an average 2-fold decrease of the steady-state level of hMSH2 protein in hMSH2(+/-) cell lines. In contrast, hMLH1(+/-) heterozygous cells were indistinguishable from normal controls in these assays. Thus, despite the fact that HNPCC families harboring mutations in hMSH2 or hMLH1 cannot be distinguished clinically, the early stages of the carcinogenic process in hMSH2 and hMLH1 mutation carriers may be different. Should hMSH2(+/-) colonocytes and lymphoblasts harbor a similar phenotype, the increased tolerance of the former to DNA-damaging agents present in the human colon may play a key role in the initiation of the carcinogenic process.

MSH2 and MLH1 mutations in sporadic replication error-positive colorectal carcinoma as assessed by two-dimensional DNA electrophoresis.

Replication errors (RER) are frequently seen in both sporadic and hereditary forms of colorectal cancer. In hereditary nonpolyposis colorectal cancer (HNPCC), RER is associated with defects in DNA mismatch repair genes. Two of these genes, MSH2 and MLH1, account for a major share of this cancer syndrome. In order to assess the role of these genes in sporadic RER+ colorectal carcinoma, we have carried out a mutation analysis of MSH2 and MLH1 by two-dimensional (2-D) DNA electrophoresis, including heteroduplexing and separation in a denaturing gradient. All exons were amplified using multiplex PCR and were separated on the basis of both size and base pair composition under a single set of experimental conditions. Exons showing a spot position different from normal were sequenced. In screening 33 unselected, sporadic RER+ colorectal tumors, a germline mutation accompanied by loss of heterozygosity in tumor tissue was found in two patients. They were among the 4 patients out of the 33 screened that were diagnosed before the age of 50 years. In 8 of the remaining 31 tumors (26%), presence of somatic mutations (9 in total) could be demonstrated. While suggesting involvement of other genes in a substantial part of sporadic RER+ colorectal carcinomas, our results also demonstrate a clear role of MSH2 and MLH1 in these sporadic tumors and show that young sporadic RER+ colorectal carcinoma patients have a high probability of germline mutations. This has important implications for genetic testing and management of young colorectal cancer patients and their families.

Predictive genetic testing for hereditary non-polyposis colorectal cancer: uptake and long-term satisfaction.

The aim of this prospective study was to assess the uptake of predictive genetic testing for hereditary non-polyposis colorectal cancer (HNPCC) and its associations with sociodemographic and other factors, and long-term satisfaction with taking the test. The test was offered to all high-risk members (n = 446) of 36 Finnish HNPCC families in which the mutation was known. The procedure comprised an educational counselling session, a period for reflection, and a test disclosure session. Data were collected by questionnaires sent before the educational counselling and 1 month and 1 year after the test disclosure. Of those eligible, 85% (n = 381) completed the first questionnaire study. Non-participation was more common among men living alone who had not participated in the clinical cancer surveillance programme. Of the 347 subjects who attended counselling, 334 (75% of all subjects) were actually tested. After logistic-regression analysis, the only significant factor predicting test acceptance proved to be employment status: those employed were more likely than others to accept the test (odds ratio = 2.25; 95% confidence intervals, 1.09 to 4.6 1). At follow-up, over 90% of the subjects were fully satisfied with the decision to take the test. In conclusion, acceptance of the test was considerably higher than in previously reported studies. We attribute this to our careful face-to-face individualized counselling, our health care system, and to attitudes of the Finnish population, which are generally favourable towards health care and disease prevention.

Epigenetic phenotypes distinguish microsatellite-stable and -unstable colorectal cancers.

Aberrant DNA methylation is a common phenomenon in human cancer, but its patterns, causes, and consequences are poorly defined. Promoter methylation of the DNA mismatch repair gene MutL homologue (MLH1) has been implicated in the subset of colorectal cancers that shows microsatellite instability (MSI). The present analysis of four MspI/HpaII sites at the MLH1 promoter region in a series of 89 sporadic colorectal cancers revealed two main methylation patterns that closely correlated with the MSI status of the tumors. These sites were hypermethylated in tumor tissue relative to normal mucosa in most MSI(+) cases (31/51, 61%). By contrast, in the majority of MSI(-) cases (20/38, 53%) the same sites showed methylation in normal mucosa and hypomethylation in tumor tissue. Hypermethylation displayed a direct correlation with increasing age and proximal location in the bowel and was accompanied by immunohistochemically documented loss of MLH1 protein both in tumors and in normal tissue. Similar patterns of methylation were observed in the promoter region of the calcitonin gene that does not have a known functional role in tumorigenesis. We propose a model of carcinogenesis where different epigenetic phenotypes distinguish the colonic mucosa in individuals who develop MSI(+) and MSI(-) tumors. These phenotypes may underlie the different developmental pathways that are known to occur in these tumors.

Pseudoautosomal DNA sequences in the pairing region of the human sex chromosomes.

A DNA probe from a human Y chromosome-derived cosmid detects a single-copy genomic DNA fragment which can appear in different allelic forms shared by both sex chromosomes. Variants at this DNA locus show an autosomal pattern of inheritance, undergo recombination with sexual phenotype and can therefore be described as 'pseudoautosomal'. Another probe from the same cosmid detects a sequence repeated 15-20 times per haploid genome. These repeats also appear pseudoautosomal and map exclusively to the short-arm terminal region of each sex chromosome.