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Heterozygous germline DNA mismatch repair gene mutations are typically associated with HNPCC. Here we report the case of a proband whose father was known for familial adenomatous polyposis. The number of polyps (less than ten) was not typical of polyposis; therefore, the diagnosis of HNPCC was entertained. Microsatellite instability analyses were performed on peripheral blood and biopsy of a right-sided dysplastic adenoma. The tumour tissue showed high-grade instability, and subsequently, immunohistochemistry showed that neither MSH2 nor MSH6 proteins were expressed in tumour cells. Prophylactic colectomy was performed, and an adenocarcinoma developing within the adenoma was diagnosed (pT1N0). Genomic DNA analysis revealed a novel mutation in MSH2 as a frameshift mutation in exon 7 (c.1,191_1,192dupG). Both parents of the proband were analyzed for MSH2 and APC mutations, and in the father, a truncating mutation in exon 15 of APC was identified as del3471-3473GAGA. This mutation was found to be present in the proband. His mother was found to bear the MSH2 exon 7 mutation. At follow-up, the proband was diagnosed with fundic, antral and duodenal adenomas (one fundic adenoma showed low-grade dysplasia). Several tubular rectal adenomas with low-grade dysplasia were excised. The patient later developed an intra-abdominal desmoid tumour.

In 10–30% of patients with classical familial adenomatous polyposis (FAP) and up to 90% of those with attenuated (

DNA sequences of mono-, di-, and trinucleotide repeats are prone to replication errors and thus constitute mutational hot spots. This is well illustrated by the occurrence of DNA microsatellite instability in tumors from patients affected by hereditary nonpolyposis colorectal cancer (HNPCC) resulting from a defect in a gene that participates in postreplicative DNA mismatch repair (MMR). We selected repeat sequences present within coding regions of four genes involved in either cell proliferation or promotion of apoptosis. These repeats consisted of (A)10 in the TGF beta RII, (G)8 in the BAX, (A)8 in the CASP1, and (CCA)7 in the APP genes. These repeats were analyzed in ten tumors from HNPCC patients carrying a germline pathogenic mutation in the MMR gene MLH1. For each tumor the rate of somatic replication errors was measured by sequencing 20-50 cloned PCR-amplified fragments. Substantial intertumor variations were observed in the pattern of repeat alterations, with error rates varying between 12% and 80% for TGF beta RII, 2% and 84% for BAX, 0% and 30% for CASP1, and 0% to 18% for APP. The BAX repeat error rate did not exceed 20% in nine of the ten tumors, in contrast to results from previous studies. High error rates in more than one gene in a same tumor suggested additive selective effects from inactivation of different pathways influencing tumorigenesis. Our methodology can contribute to define tumor characteristics and may, if applied to genes strongly involved in tumorigenesis, improve tumor classification and outcome prediction.

A pimeloyl-CoA synthetase from Pseudomonas mendocina 35 was purified and characterized, the DNA sequence determined, and the gene cloned into Escherichia coli to yield an active enzyme. The purified enzyme had a pH optimum of approximately 8.0, Km values of 0.49 mM for pimelic acid, 0.18 mM for CoA and 0.72 mM for ATP, a subunit Mr of approximately 80000 as determined by SDS/PAGE, and was found to be a tetramer by gel-filtration chromatography. The specific activity of the purified enzyme was 77.3 units/mg of protein. The enzyme was not absolutely specific for pimelic acid. The relative activity for adipic acid (C6) was 72% and for azaleic acid (C9) was 18% of that for pimelic acid (C7). The N-terminal amino acid was blocked to amino acid sequencing, but controlled proteolysis resulted in three peptide fragments for which amino acid sequences were obtained. An oligonucleotide gene probe corresponding to one of the amino acid sequences was synthesized and used to isolate the gene (pauA, pimelic acid-utilizing A) coding for pimeloyl-CoA synthetase. The pauA gene, which codes for a protein with a theoretical Mr of 74643, was then sequenced. The deduced amino acid sequence of the enzyme showed similarity to hypothetical proteins from Archaeoglobus fulgidus, Methanococcus jannaschii, Pyrococcus horikoshii, E. coli and Streptomyces coelicolor, and some limited similarity to microbial succinyl-CoA synthetases. The similarity with the protein from A. fulgidus was especially strong, thus indicating a function for this unidentified protein. The pauA gene was cloned into E. coli, where it was expressed and resulted in an active enzyme.

In 10-30% of patients with classical familial adenomatous polyposis (FAP) and up to 90% of those with attenuated (100 colorectal adenomas; AFAP) polyposis, no pathogenic germline mutation in the adenomatous polyposis coli (APC) gene can be identified (APC mutation-negative). Recently, biallelic mutations in the base excision repair gene MYH have been shown to predispose to a multiple adenoma and carcinoma phenotype. This study aimed to (i) assess the MYH mutation carrier frequency among Swiss APC mutation-negative patients and (ii) identify phenotypic differences between MYH mutation carriers and APC/MYH mutation-negative polyposis patients. Seventy-nine unrelated APC mutation-negative Swiss patients with either classical (n=18) or attenuated (n=61) polyposis were screened for germline mutations in MYH by dHPLC and direct genomic DNA sequencing. Overall, 7 (8.9%) biallelic and 9 (11.4%) monoallelic MYH germline mutation carriers were identified. Among patients with a family history compatible with autosomal recessive inheritance (n=45), 1 (10.0%) out of 10 classical polyposis and 6 (17.1%) out of 35 attenuated polyposis patients carried biallelic MYH alterations, 2 of which represent novel gene variants (p.R171Q and p.R231H). Colorectal cancer was significantly (p0.007) more frequent in biallelic mutation carriers (71.4%) compared with that of monoallelic and MYH mutation-negative polyposis patients (0 and 13.8%, respectively). On the basis of our findings and earlier reports, MYH mutation screening should be considered if all of the following criteria are fulfilled: (i) presence of classical or attenuated polyposis coli, (ii) absence of a pathogenic APC mutation, and (iii) a family history compatible with an autosomal recessive mode of inheritance.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal, dominantly inherited, colorectal cancer (CRC) predisposition syndrome caused by germline mutations in DNA mismatch repair (MMR) genes, predominantly MLH1 and MSH2. Thus far, only limited data exist on the occurrence of genetic anticipation in HNPCC, i.e. the earlier age at diagnosis of CRC in successive generations. Performing nonparametric distribution-free statistical analyses, we investigated 55 parent-child pairs who had been diagnosed with CRC and who came from 21 Swiss HNPCC families with characterised MMR germline mutation (15 in MLH1 and 6 in MSH2). The overall median age at diagnosis was 43 years, with an interquartile range (IQR) of 14 and incidence ages ranging from 18 to 62 years. Descendants of HNPCC patients (median age at diagnosis 39 years, IQR=12) were found to be diagnosed with CRC significantly earlier than their parents (47 years, IQR=10), with the median of the paired age difference amounting to 8 years (IQR=15; P

Background: The mismatch repair gene, MLH1, appears to occur as two main haplotypes at least in white populations. These are referred to as A and G types with reference to the A/G polymorphism at IVS14-19. On the basis of preliminary experimental data, we hypothesised that deviations from the expected frequency of these two haplotypes could exist in carriers of disease associated MLH1 germline mutations. Methods: We assembled a series (n=119) of germline MLH1 mutation carriers in whom phase between the haplotype and the mutation had been conclusively established. Controls, without cancer, were obtained from each contributing centre. Cases and controls were genotyped for the polymorphism in IVS14. Results: Overall, 66 of 119 MLH1 mutations occurred on a G haplotype (55.5%), compared with 315 G haplotypes on 804 control chromosomes (39.2%, p=0.001). The odds ratio (OR) of a mutation occurring on a G rather than an A haplotype was 1.93 (95% CI 1.29 to 2.91). When we compared the haplotype frequencies in mutation bearing chromosomes carried by people of different nationalities with those seen in pooled controls, all groups showed a ratio of A/G haplotypes that was skewed towards G, except the Dutch group. On further analysis of the type of each mutation, it was notable that, compared with control frequencies, deletion and substitution mutations were preferentially represented on the G haplotype (p=0.003 and 0.005, respectively). Conclusion: We have found that disease associated mutations in MLH1 appear to occur more often on one of only two known ancient haplotypes. The underlying reason for this observation is obscure, but it is tempting to suggest a possible role of either distant regulatory sequences or of chromatin structure influencing access to DNA sequence. Alternatively, differential behaviour of otherwise similar haplotypes should be considered as prime areas for further study.

While searching for germline mutations in MLH1 and MSH2 mismatch repair genes in patients affected with hereditary non-polyposis colorectal cancer (HNPCC), we have observed that human chromosome 3 carries two main haplotypes of the housekeeping gene MLH1 . This so called caretaker gene acts as a major guardian of the genome,1 and cells in which MLH1 is inactivated develop a characteristic mutator phenotype, as a result of a default in post-replicative DNA mismatch repair.2 3 In humans, the protein encoded by MLH1 forms at least two dimeric factors with either PMS2 or MLH3 .4-6 Germline defects in MLH1 account for a number of sporadic epithelial cancers and for the majority of cases of HNPCC,7 which is the most common form of all familial cancers along with breast cancer. In addition to its role in DNA editing, the multifunctional MLH1 gene is thought to participate in mitotic and meiotic recombination,8 as well as in apoptosis.9 In E coli , evidence has accumulated to suggest that MLH1 MutL homologue may behave as a “molecular matchmaker” by acting as a chaperone, which facilitates the conformational changes required to assemble a DNA repair proficient complex from its individual components.10 In yeast, studies on segregation data of all genes known to participate in mismatch repair have shown that the MLH1 gene plays a predominant role in promoting crossing over,11 and at least in mice, the MLH1 protein appears to be a component of the late replication nodules that probably prevent non-homologous genetic recombination between homeologous sequences.8 During chromosome pairing in meiosis I, the protein foci allow even better mapping of crossover events and interference distances than using chiasmata.12 Our data suggest that two major haplotypes spanning at least 55 kb of the MLH1 gene, completely …