A relatively high frequency of germ-line genomic rearrangements in MLH1 and MSH2 has been reported among Lynch Syndrome (HNPCC) patients from different ethnic populations. To investigate the underlying molecular mechanisms, we characterized the DNA breakpoints of 11 germ-line deletions, six for MLH1 and five for MSH2. Distinct deletion patterns were found for the two genes. The five cases of MSH2 deletions result exclusively from intragenic unequal recombination mediated by repetitive Alu sequences. In contrast, five out of the six MLH1 deletions are due to recombinations involving sequences of no significant homology (P =0.015). A detailed analysis of the DNA breakpoints in the two genes, previously characterized by other groups, validated the observation that Alumediated unequal recombination is the main type of deletion in MSH2 (n = 34), but not in MLH1 (n = 21) (P <0.0001). Plotting the distribution of known DNA breakpoints among the introns of the two genes showed that, the highest breakpoint density is co-localized with the highest Alu density. Our study suggests that Alu is a promoting factor for the genomic recombinations in both MLH1 and MSH2, and the local Alu density may be involved in shaping the deletion pattern.

Background: A 3 bp deletion located at the 59 end of exon 3 of MLH1, resulting in deletion of exon 3 from RNA, was recently identified. Hypothesis: That this mutation disrupts an exon splicing enhancer (ESE) because it occurs in a purine-rich sequence previously identified as an ESE in other genes, and ESEs are often found in exons with splice signals that deviate from the consensus signals, as does the 39 splice signal in exon 3 of MLH1. Design: The 3 bp deletion and several other mutations were created by polymerase chain reaction mutagenesis and tested using an in vitro splicing assay. Both mutant and wild type exon 3 sequences were cloned into an exon trapping vector and transiently expressed in Cos-1 cells. Results: Analysis of the RNA indicates that the 3 bp deletion c.213_215delAGA (gi:28559089, NM_000249.2), a silent mutation c.216TRC, a missense mutation c.214GRC, and a nonsense mutation c.214GRT all cause varying degrees of exon skipping, suggesting the presence of an ESE at the 59 end of exon 3. These mutations are situated in a GAAGAT sequence 3 bp downstream from the start of exon 3. Conclusions:The results of the splicing assay suggest that inclusion of exon 3 in the mRNA is ESE dependent. The exon 3 ESE is not recognised by all available motif scoring matrices, highlighting the importance of RNA analysis in the detection of ESE disrupting mutations.

There has been interest in the literature in the possible existence of a gene that predisposes to both breast cancer (BC) and colorectal cancer (CRC). We describe the detailed characterisation of one kindred, MON1080, with 10 cases of BC or CRC invasive cancer among 26 first-, second-or third-degree relatives. Linkage analysis suggested that a mutation was present in BRCA2. DNA sequencing from III: 22 (diagnosed with lobular BC) identified a BRCA2 exon 3 542G4T (L105X) mutation. Her sister (III: 25) had BC and endometrial cancer and carries the same mutation. Following immunohistochemical and microsatellite instability studies, mutation analysis by protein truncation test, cDNA sequencing and quantitative real-time PCR revealed a deletion of MSH2 exon 8 in III: 25, confirming her as a double heterozygote for truncating mutations in both BRCA2 and MSH2. The exon 8 deletion was identified as a 14.9 kb deletion occurring between two Alu sequences. The breakpoint lies within a sequence of 45 bp that is identical in both Alu sequences. In this large BC/CRC kindred, MON1080, disease-causing truncating mutations are present in both MSH2 and BRCA2. There appeared to be no increased susceptibility to the development of colorectal tumours in BRCA2 mutation carriers or to the development of breast tumours in MSH2 mutation carriers. Additionally, two double heterozygotes did not appear to have a different phenotype than would be expected from the presence of a mutation in each gene alone. Keywords: MSH2; BRCA2; microsatellite instability; colorectal cancer; breast cancer; astrocytoma; multiple primary cancers BRCA1 and BRCA2 are the most important susceptibility genes for breast and ovarian cancer, and mutations in these two genes account for 480% of all kindreds with hereditary breast/ovarian cancer and for about 2 -3% of breast cancer (BC) cases overall. Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common form of hereditary colorectal cancer (CRC) (Lynch and de la Chapelle, 1999) and is responsible for 0.5 -3% of all cases of CRC. Mutations in the DNA repair genes MLH1 and MSH2 segregate in up to 90% of HNPCC pedigrees (Peltomaki, 2001).Several groups have studied the genetic relationship between breast and colorectal cancer, with varying findings. In one large Dutch family with a segregating BRCA1 mutation, there are several mutation carriers who have developed CRC. However, detailed analysis suggested that the BRCA1 mutation was not contributing to the CRCs observed. By contrast, in a large international study, Thompson et al (2002) observed a relative risk of 2.03 (Po0.001) for CRC in BRCA1 mutation carriers, compared with general population cancer incidence. Studies in HNPCC kindreds have shown no excess of BC, or have shown that when BC does occur, microsatellite instability (MSI), a hallmark of HNPCC-related cancer, is usually absent (Aarnio et al, 1995;Caluseriu et al, 2001). Interestingly, Borg et al reported a family with two missense mutations in MLH1 and a single truncating mutation in BRCA1. ...

Hereditary non-polyposis colorectal cancer (HNPCC) is a dominantly inherited cancer syndrome caused by a mutation in one of the mismatch repair genes, most frequently MLH1 or MSH2. The rate of mutation detection is influenced by many factors, including the diagnostic methods used. Large deletions, which occur frequently in MLH1 and MSH2, are not detected by exon-by-exon screening methods. Here, we describe three mutations in mismatch repair genes detected using a screening protocol that combines protein truncation test (PTT) analysis and multiplex ligation-dependent probe amplification (MLPA) with genomic and cDNA sequencing. Two of these mutations consist of large deletions in MLH1 that were detected by both MLPA and PTT but that would have been missed by genomic DNA sequencing. The third is a large deletion in MSH2 that could not be detected by PTT because of its location relative to the primers used to amplify the cDNA, or by sequencing. This mutation was detected by MLPA.