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The frequency, origin, and phenotypic expression of a germline MSH2 gene mutation previously identified in seven kindreds with hereditary non-polyposis cancer syndrome (HNPCC) was investigated. The mutation (A-->T at nt943+3) disrupts the 3' splice site of exon 5 leading to the deletion of this exon from MSH2 mRNA and represents the only frequent MSH2 mutation so far reported. Although this mutation was initially detected in four of 33 colorectal cancer families analysed from eastern England, more extensive analysis has reduced the frequency to four of 52 (8%) English HNPCC kindreds analysed. In contrast, the MSH2 mutation was identified in 10 of 20 (50%) separately identified colorectal families from Newfoundland. To investigate the origin of this mutation in colorectal cancer families from England (n=4), Newfoundland (n=10), and the United States (n=3), haplotype analysis using microsatellite markers linked to MSH2 was performed. Within the English and US families there was little evidence for a recent common origin of the MSH2 splice site mutation in most families. In contrast, a common haplotype was identified at the two flanking markers (CA5 and D2S288) in eight of the Newfoundland families. These findings suggested a founder effect within Newfoundland similar to that reported by others for two MLH1 mutations in Finnish HNPCC families. We calculated age related risks of all, colorectal, endometrial, and ovarian cancers in nt943+3 A-->T MSH2 mutation carriers (n=76) for all patients and for men and women separately. For both sexes combined, the penetrances at age 60 years for all cancers and for colorectal cancer were 0.86 and 0.57, respectively. The risk of colorectal cancer was significantly higher (p<0.01) in males than females (0.63 v 0.30 and 0.84 v 0.44 at ages 50 and 60 years, respectively). For females there was a high risk of endometrial cancer (0.5 at age 60 years) and premenopausal ovarian cancer (0.2 at 50 years). These intersex differences in colorectal cancer risks have implications for screening programmes and for attempts to identify colorectal cancer susceptibility modifiers.

Hereditary non-polyposis colorectal cancer syndrome (HNPCC) is often considered to be the most common form of inherited colorectal cancer, although its precise incidence is unknown. The clinical diagnosis of HNPCC relies on a combination of family history and young age of onset of colorectal cancer, but as many familial aggregations of colorectal cancer do not fulfil the strict diagnostic criteria, HNPCC might be underdiagnosed. The majority of HNPCC families have germline mutations in mismatch repair (MMR) genes, such as MSH2 or MLH1, so that HNPCC cancers characteristically exhibit DNA replication errors (RERs) at microsatellite loci. Although an RER positive phenotype in tumours can also result from somatic mutations in an MMR gene, the prevalence of RER + tumours should provide a maximum estimate of the incidence of germline MMR gene mutations in patients with early onset and familial colorectal cancer. We investigated colorectal cancers for RERs from (1) a population based study of 33 patients with colorectal cancer aged 45 years or less, (2) 65 kindreds with familial colorectal cancer which only partially fulfilled the criteria for the diagnosis of HNPCC, and (3) 18 cancers from 12 HNPCC kindreds. Seven of 33 patients (21%) with colorectal cancer aged 45 years or less had an RER + cancer, with only two of these having a clear family history of HNPCC. A greater proportion of RER + tumours (5/7) occurred proximal to the splenic flexure than RER - tumours (4/26; chi2 = 6.14, p < 0.025). RERs were detected in all 18 cancers from HNPCC patients but in only six of 65 non-HNPCC familial colorectal cancer kindreds (9%; chi2 = 52.2, p < 0.0005). These findings suggest that most cancers in patients diagnosed at 45 years of age or less and familial aggregations of colorectal cancer which do not fulfil HNPCC diagnostic criteria do not have germline mutations in MSH2 and MLH1. Hence population screening for germline mutations in these genes is unlikely to be an efficient strategy for identifying people at high risk of developing colorectal cancer.

Desmoid tumours are generally very rare but occur about 100 times more frequently in the colorectal cancer predisposition syndrome familial adenomatous polyposis (MIM 175100), being represented in about 10% of patients. In addition to desmoid disease occurring in familial adenomatous polyposis (FAP) there exist familial infiltrative fibromatosis (MIM 135290) kindreds where there is no evidence of FAP. Previously we have described a kindred with familial infiltrative fibromatosis (FIF) in which desmoid tumours were associated with nonpolyposis colorectal cancer. FAP is caused by mutations in the APC gene and various genotype-phenotype relationships have been defined including reports that colorectal polyposis is less severe with mutations 5' to codon 157 and that the risk of desmoid tumours is high in FAP patients with APC gene mutations between codons 1444 and 1598. There is relatively little information on the phenotype of APC gene mutations 3' to codon 1598; however, one large family has been reported with a mutation at codon 1987 which presents with a highly variable phenotype which includes desmoid disease. We screened our original FIF kindred and three further families with a similar phenotype for mutations in the APC gene. A 4 bp frameshift deletion in codon 1962 was identified in the original FIF kindred and two further apparently unrelated families. Haplotype analysis suggests a common origin for the APC mutation in all three families. Affected individuals had no evidence of congenital hypertrophy of the retinal pigment epithelium. Colorectal polyposis was variable, and most affected patients had either none or a few late onset polyps. These findings demonstrate (i) that FAP and FIF are allelic, and (ii) that APC gene mutations which truncate the APC protein distal to the beta-catenin binding domain are associated with desmoid tumours, absent CHRPE and variable but attenuated polyposis expression.

Germline mutations in four human mismatch repair genes (MSH2, MLH1, PMS1, and PMS2) have been reported to cause hereditary non-polyposis colon cancer syndrome (HNPCC). The identification of germline mutations in HNPCC kindreds allows precise diagnosis and accurate predictive testing. To investigate further the genetic epidemiology of HNPCC and the nature and frequency of germline mutations in this disorder, we studied 17 English HNPCC kindreds for germline mutations in MSH2 and MLH1. A previous genetic linkage study had suggested that most English HNPCC families will have mutations in one of these genes. Mutation analysis was performed in a three step process. (1) mRNA extracted from lymphoblastoid cell lines was analysed for gross rearrangements, (2) the in vitro transcription-translation (IVTT) assay was then performed to detect protein truncating mutations, and (3) partial cDNA sequencing of MSH2 or MLH1 was undertaken in families (n = 6) linked to MSH2 or MLH1 but without a detectable mutation. Seven different germline mutations were identified in eight of 17 (47%) kindreds (five in MSH2 and three in MLH1). In three cases there was a deletion of a single exon in MSH2 mRNA, three mutations resulted in a truncated protein product, and two missense mutations were identified by direct sequencing. Six mutations were novel. No precise correlation between genotype and phenotype was observed, although a MSH2 missense (Thr905Arg) mutation was associated with a susceptibility to multiple colorectal polyps. Age related risks for colorectal and uterine cancer were similar for MSH2 and MLH1 mutations.

Familial adenomatous polyposis coli is caused by constitutional mutations in the APC gene. The hallmark of familial adenomatous polyposis coli is the presence of numerous (> 100) colorectal polyps, but mutations in the 5' end of the APC gene have been associated with familial colorectal cancer without florid polyposis. Although familial adenomatous polyposis coli accounts for only a minority of familial colorectal cancer cases, we hypothesised that APC mutations which were not associated with florid polyposis might make a significant contribution to nonpolyposis familial colorectal cancer. To investigate this possibility, we analysed 40 unrelated patients with familial colorectal cancer without classical familial adenomatous polyposis coli for mutations in exons 1 to 6 (codons 1 to 243) of the APC gene. No mutations were detected, but a C-->T polymorphism at nucleotide 333 (Arg-->Trp at codon 99) was identified. No 5' APC mutations were detected in two patients with desmoid tumours and a family history of colorectal cancer and polyps. We conclude that mutations in exons 1 to 6 of the APC gene are infrequent in patients with familial colorectal cancer who do not have many colorectal polyps.

Hereditary Non-polyposis Colon Cancer Syndrome (HNPCC) is the most common cause of familial colorectal cancer. Molecular genetic studies of HNPCC have shown evidence of locus heterogeneity, and mutations in four genes (hMSH2, hMLH1, hPMS1, and hPMS2) which encode components of the mismatch enzyme repair system may cause HNPCC. To determine the extent and nature of locus heterogeneity in HNPCC, we performed genetic linkage studies in 14 HNPCC families from eastern and north-western England. Linkage to hMLH1 was excluded in six families, each of which were likely to be linked to hMSH2 (lod score > 1.0 in each family and total lod score for all six families = 7.64). Linkage to hMSH2 was excluded in three families, each of which were likely to be linked to hMLH1 (lod score > 1.0 in each family and total lod score at hMLH1 for all three families = 3.93). In the remaining five families linkage to hMSH2 or hMLH1 could not be excluded. These results confirm locus heterogeneity in HNPCC and suggest that, in the population studied, most large families with HNPCC will have mutations in hMSH2 or hMLH1. We did not detect any correlation between clinical phenotype and the genetic linkage results, but a Muir-Torre syndrome family excluded from linkage to hMLH1 was likely to be linked to hMSH2 and showed microsatellite instability in a tumour from an affected relative.

Whilst p53 aberrations have been documented in numerous malignancies, reports of alterations to the deleted in colorectal cancer (dcc) gene are infrequent, and studies investigating the status of both genes in the same colon tumour are rare. In this study we have analysed a panel of 35 pairs of normal and neoplastic human colorectal tissues for abnormalities in these tumour-suppressor genes. In contrast to previous studies we have found only a low incidence of mutations and deletions. p53 point mutations were identified in 8/35 tumours (22%). All were G.C to A.T transitions, with 7/8 occurring at CpG dinucleotides. p53 allelic loss was detected in 4/11 informative cases (36%). Although not quite attaining statistical significance, p53 alteration correlated with the adenoma/carcinoma transition. Gross dcc alterations were identified by Southern blotting in 7/35 (20%) tumours. Microsatellite analysis using two markers, one within and one proximal to the dcc gene, detected a low frequency of deletion overall (41% informative cases). 18q/dcc aberrations were associated with the progression of early to late carcinoma, rather than with increasing adenoma size, as has been previously reported. Both p53 alterations and dcc deletions were detected at a higher frequency in distal tumours than in proximal malignancies. Two tumours exhibiting microsatellite instability in both markers were each of proximal origin.