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Background: Spliceogenic variants in disease-causing genes are often presumed pathogenic since most induce frameshifts resulting in loss of function. However, it was recently shown in cancer predisposition genes that some may trigger in-frame anomalies that preserve function. Here, we addressed this question by using MSH2 , a DNA mismatch repair gene implicated in Lynch syndrome, as a model system., Methods: Eighteen MSH2 variants, mostly localised within canonical splice sites, were analysed by using minigene splicing assays. The impact of the resulting protein alterations was assessed in a methylation tolerance-based assay. Clinicopathological characteristics of variant carriers were collected., Results: Three in-frame RNA biotypes were identified based on variant-induced spliceogenic outcomes: exon skipping (E3, E4, E5 and E12), segmental exonic deletions (E7 and E15) and intronic retentions (I3, I6, I12 and I13). The 10 corresponding protein isoforms exhibit either large deletions (49-93 amino acids (aa)), small deletions (12 or 16 aa) or insertions (3-10 aa) within different functional domains. We showed that all these modifications abrogate MSH2 function, in agreement with the clinicopathological features of variant carriers., Conclusion: Altogether, these data demonstrate that MSH2 function is intolerant to in-frame indels caused by the spliceogenic variants analysed in this study, supporting their pathogenic nature. This work stresses the importance of combining complementary RNA and protein approaches to ensure accurate clinical interpretation of in-frame spliceogenic variants., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Constitutional mismatch repair-deficiency, due to biallelic mutations of MMR genes, results in a tumour spectrum characterized by leukaemias, lymphomas, brain tumours and adenocarcinomas of the gastro-intestinal tract, occurring mostly in childhood. We report here two families illustrating the phenotypic diversity associated with biallelic MMR mutations. In the first family, two siblings developed six malignancies including glioblastoma, lymphoblastic T cell lymphoma, rectal and small bowel adenocarcinoma with onset as early as 6 years of age. We showed that this dramatic clinical presentation was due to the presence of two complex genomic PMS2 deletions in each patient predicted to result into complete PMS2 inactivation. In the second family, the index case presented with an early form of Lynch syndrome with colorectal adenocarcinomas at ages 17 and 20 years, and urinary tract tumours at the age of 25 years. We identified in this patient two MSH6 mutations corresponding to a frameshift deletion and an in frame deletion. The latter was not predicted to result into complete inactivation of MSH6. These reports show that the clinical expression of biallelic MMR mutations depends on the biological impact of the second MMR mutation and that, in clinical practice, the presence of a second MMR mutation located in trans should also be considered in patients suspected to present a Lynch syndrome with an unusual early-onset of tumours.

Based on nationwide data from the French national cancer institute (INCa), we analyzed the evolution of cancer genetics consultations and testing over time, and the uptake of targeted tests in relatives of families with BRCA1/2 or MMR genes mutation. Genetic testing and consultations for familial high-risk individuals are exclusively funded and monitored by the INCa in France. All nationwide cancer genetics centers reported annually standardized parameters of activity from 2003 to 2011. The analysis included a total of 240,134 consultations and 134,652 genetic tests enabling to identify 32,494 mutation carriers. Referral for hereditary breast and ovarian cancer (HBOC) or colorectal cancer predisposition syndromes represented 59 % (141,639) and 23.2 % (55,698) consultations, respectively. From 2003 to 2011, we found a dramatic and steady increase of tests performed for BRCA1/2 (from 2,095 to 7,393 tests/year, P < 0.0001) but not for MMR genes (from 1,144 to 1,635/year, P = NS). The overall percentage of deleterious mutations identified in the probands tested was 13.8 and 20.9 % in HBOC and Lynch syndromes, respectively. Pooled analysis for BRCA1/2 and Lynch syndrome tests showed an inverse relationship between the percentage of mutation detected and the absolute number of tests performed over the time (overall Cochran-Armitage test for trend: P < 0.001). In families with BRCA1/2 or MMR identified mutations, there was an average number of 2.94 and 3.28 relatives performing targeted tests, respectively. This nationwide study shows a lack of referral and genetic testing in Lynch as compared to HBOC syndromes. Only a third of relatives of a proband with a predisposing mutation performed a targeted test. Enhanced information about benefit of genetic testing should be given to clinicians and patients for Lynch syndrome and relatives of a proband carrying an identified predisposing mutation.

Several studies have reported that, in Lynch syndrome resulting from mutations of the mismatch repair (MMR) genes, a CA repeat ≤17 within the IGF1 promoter, SNPs within the xenobiotic metabolizing enzyme gene CYP1A1 and SNPs on 8q23.3 and 11q23.1 modify colorectal cancer (CRC) risk in MMR mutation carriers. We analysed the impact of these polymorphisms on CRC risk in 748 French MMR mutation carriers derived from 359 families. We also analysed the effect of the Novel 1 SNP (18q21), which has recently been shown to increase CRC risk in the general population. We observed a significant difference in the CRC-free survival time between males and females, between MSH2 and MSH6 mutation carriers and between MLH1 and MSH6, indicating that this series is representative of Lynch syndrome. In contrast, the univariate log-rank test, as well as multivariate Cox model analysis controlling for familial aggregation and mutated MMR gene, year of birth and gender showed that the polymorphic alleles tested were not associated with a significant CRC risk increase, neither on the entire sample nor among males and females. This discrepancy with previous reports might be explained both by the genetic heterogeneity between the different populations analysed and the allelic heterogeneity of the MMR mutations. We conclude that genotyping of these polymorphisms is not useful to evaluate CRC risk in MMR mutation carriers and to optimize their clinical follow-up.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal disorder caused by mutations in DNA mismatch repair (MMR) genes. Tumors of the HNPCC-spectrum are associated with microsatellite instability (MSI) and loss of MMR protein expression. Lymphomas are not considered to be HNPCC-related tumors. We report and analyze a case of an HNPCC patient with three colorectal cancers and a B-cell non-Hodgkin lymphoma. Quantitative multiplex PCR of short fluorescent fragments detected a novel MSH2 rearrangement involving exons 9 and 10, which proved to be the pathogenic cause of the disease in the family. Tumor tissues including the lymphoma showed MSI and loss of MSH2 expression. Multiplex ligation-dependent probe amplification analysis revealed a somatic loss of the wild-type MSH2 allele in the lymphoma. These results support the fact that the total loss of a MMR gene can lead to lymphomagenesis, as seen in biallelic MMR-deficient families and knockout mice. Moreover, this is the first report of a B-cell non-Hodgkin lymphoma with a loss of the MSH2 protein expression, linked to a heterozygous germline MSH2 mutation in an HNPCC family., ((c) 2008 Wiley-Liss, Inc.)

Numerous reports have highlighted the contribution of MSH2 and MLH1 genomic deletions to hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch's syndrome, but genomic duplications of these genes have been rarely reported. Using quantitative multiplex PCR of short fluorescent fragments (QMPSF), 962 and 611 index cases were, respectively, screened for MSH2 and MLH1 genomic rearrangements. This allowed us to detect, in 11 families, seven MSH2 duplications affecting exons 1-2-3, exons 4-5-6, exon 7, exons 7-8, exons 9-10, exon 11, and exon 15, and three MLH1 duplications affecting exons 2-3, exon 4 and exons 6-7-8. All duplications were confirmed by an independent method. The contribution of genomic duplications of MSH2 and MLH1 to HNPCC can therefore be estimated approximately to 1% of the HNPCC cases. Although this frequency is much lower than that of genomic deletions, the presence of MSH2 or MLH1 genomic duplications should be considered in HNPCC families without detectable point mutations.

Background: Preventive programs for individuals who have high lifetime risks of colorectal cancer may reduce disease morbidity and mortality. Thus, it is important to identify the factors that are associated with hereditary colorectal cancer and to monitor the effects of tailored surveillance. In particular, patients with Lynch syndrome, hereditary nonpolyposis colorectal cancer (HNPCC), have an increased risk to develop colorectal cancer at an early age. The syndrome is explained by germline mutations in DNA mismatch repair (MMR) genes, and there is a need for diagnostic tools to preselect patients for genetic testing to diagnose those with HNPCC., Methods: Patients (n = 112) from 285 families who were counseled between 1990 and 2005 at a clinic for patients at high risk for HNPCC were selected for screening to detect mutations in MMR genes MLH1, MSH2, MSH6, and PMS2 based on family history, microsatellite instability (MSI), and immunohistochemical analysis of MMR protein expression. Tumors were also screened for BRAF V600E mutations; patients with the mutation were considered as non-HNPCC., Results: Among the 112 patients who were selected for screening, 69 had germline MMR mutations (58 pathogenic and 11 of unknown biologic relevance). Sixteen of the 69 mutations (23%) were missense mutations. Among patients with MSI-positive tumors, pathogenic MMR mutations were found in 38 of 43 (88%) of patients in families who met Amsterdam criteria and in 13 of 22 (59%) of patients in families who did not. Among patients with MSI-negative tumors, pathogenic MMR mutations were found in 5 of 17 (29%) of families meeting Amsterdam criteria and in 1 of 30 (3%) of non-Amsterdam families with one patient younger than age 50 years. In three patients with MSI-negative tumors who had pathogenic mutations in MLH1 or MSH6, immunohistochemistry showed loss of the mutated protein., Conclusion: Our findings suggest that missense MMR gene mutations are common in HNPCC and that germline MMR mutations are also found in patients with MSI-negative tumors.

MSH2 rearrangements are involved in approximately 10% of hereditary non-polyposis colorectal cancer (HNPCC) families, and in most of the rearrangements, exon 1 is deleted. We scanned by quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF) 200 kb of genomic sequences upstream of the MSH2 transcription initiation site in 21 HNPCC families with exon 1 deletions. This QMPSF scan revealed 12 distinct 5' breakpoints located up to 200 kb upstream of the MSH2 transcription initiation site. Sequencing analysis of the rearranged allele in 17 families revealed that most of the deletions (15/17) resulted from homologous Alu-mediated recombination. QMPSF and sequencing analysis in these 21 families led us to detect the presence of 20 distinct 5' breakpoints. In 14 out of 15 Alu-mediated recombinations, we found, either within the identical region in which the recombination had probably occurred or in its vicinity, the 26-bp Alu core sequence containing the recombinogenic Chi-like motif. Compared to the equivalent regions of other human genes, the MSH2 upstream region was found to contain a high density of Alu repeats (30% within 228 kb and 43% within 50 kb), most of which belong to the old Alu S subfamilies. In conclusion, this study demonstrates the heterogeneity of the breakpoints within the MSH2 upstream region and reveals the remarkable density of recombinogenic Alu sequences in this region.

Aim of the Study: To detect hereditary non-polyposis colorectal cancer (HNPCC) patients with a strategy combining clinical selection (patient age at onset of cancer less than 50 years or family history of HNPCC tumors) and microsatellite instability typing plus immunohistochemistry, leading to mismatch repair (MMR) germline mutation analysis., Methods: Tumors were screened for microsatellite instability (MSI) and for hmlh1 and hmsh2 immunohistochemical expression. Germline mutation analysis was performed to search for MLH1 and MSH2 mutations in patients with MSI-High and MSI-Low tumors., Results: 197 adenocarcinomas were studied: 164 patients were< or =50 years old, 33 were older than 50 years but had a family history of HNPCC tumors. Fifty tumors (25.4%) were MSI-High, 10 were MSI-Low (5.1%), and 130 were MS-Stable (66%). MSI typing was inconclusive in 7 (3.5%). Immunohistochemistry screening was performed on 165 tumors: sensitivity was 63.6%, specificity was 99%. Germline mutation analysis was performed in 33/60 MSI-High or Low tumors: 23 mutations were noted (70% of the tested patients)., Conclusion: This proposed strategy of determining microsatellite instability in young colorectal cancer patients or in patients with a family history of HNPCC tumors led to an increased frequency in the detection of MMR germline mutations.

Background: According to the international criteria for hereditary non-polyposis colorectal cancer (HNPCC) diagnostics, cancer patients with a family history or early onset of colorectal tumours showing high microsatellite instability (MSI-H) should receive genetic counselling and be offered testing for germline mutations in DNA repair genes, mainly MLH1 and MSH2. Recently, an oncogenic V600E hotspot mutation within BRAF, a kinase encoding gene from the RAS/RAF/MAPK pathway, has been found to be associated with sporadic MSI-H colon cancer, but its association with HNPCC remains to be further clarified., Methods: BRAF-V600E mutations were analysed by automatic sequencing in colorectal cancers from 206 sporadic cases with MSI-H and 111 HNPCC cases with known germline mutations in MLH1 and MSH2. In addition, 45 HNPCC cases showing abnormal immunostaining for MSH2 were also analysed., Results: The BRAF-V600E hotspot mutation was found in 40% (82/206) of the sporadic MSI-H tumours analysed but in none of the 111 tested HNPCC tumours or in the 45 cases showing abnormal MSH2 immunostaining., Conclusions: Detection of the V600E mutation in a colorectal MSI-H tumour argues against the presence of a germline mutation in either the MLH1 or MSH2 gene. Therefore, screening of these mismatch repair (MMR) genes can be avoided in cases positive for V600E if no other significant evidence, such as fulfilment of the strict Amsterdam criteria, suggests MMR associated HNPCC. In this context, mutation analysis of the BRAF hotspot is a reliable, fast, and low cost strategy which simplifies genetic testing for HNPCC.

To estimate the relative frequency of mismatch repair genes, rearrangements in hereditary nonpolyposis colorectal cancer (HNPCC) families without detectable mutations in MSH2 or MLH1, we have analyzed by multiplex PCR of short fluorescent fragments MSH2, MLH1, and MSH6 in 61 families, either fulfilling Amsterdam criteria or including cases of multiple primary cancers belonging to the HNPCC spectrum. We detected 13 different genomic rearrangements of MSH2 in 14 families (23%), whereas we found no rearrangement of MLH1 and MSH6. Analysis of 31 other families, partially meeting Amsterdam criteria, revealed no additional rearrangement of MSH2. All of the MSH2 rearrangements, except one, corresponded to genomic deletions involving one or several exons. In 8 of 13 families with a MSH2 genomic deletion, the MSH2 promoter was also deleted, and the 5' breakpoint was located either within or upstream the MSH2 gene. This study demonstrates the heterogeneity of MSH2 exonic and promoter rearrangements and shows that, in HNPCC families without detectable MSH2 or MLH1 point mutation, one must consider the presence of MSH2 genomic rearrangements before the involvement of other mismatch repair genes. The simplicity and rapidity of their detection, using fluorescent multiplex PCR, led us to recommend to begin the molecular analysis in HNPCC by screening for MSH2 rearrangements.

Large genomic deletions within the mismatch repair MLH1 and MSH2 genes have been identified in families with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, and their detection represents a technical problem. To facilitate their detection, we developed a simple semiquantitative procedure based on the multiplex PCR of short fluorescent fragments. This method allowed us to confirm in HNPCC families three known deletions of MLH1 or MSH2 and to detect in 19 HNPCC families, in which analysis of mismatch repair genes using classical methods had revealed no alteration, a deletion of exon 5 and a duplication of MSH2 involving exons 9 and 10. The presence of such duplications, the frequency of which is probably underestimated, must be considered in HNPCC families in which conventional screening methods have failed to detect mutations.

We analyzed the hMLH1 gene in 17 unrelated families with putative hereditary nonpolyposis colorectal cancer. The complete hMLH1 cDNA was amplified in one step, and after a second amplification, four overlapping segments were directly sequenced. We detected, in five families that did not meet the complete Amsterdam criteria, five alterations, including a double-base change resulting in a missense mutation (Lys-618-Ala), a splicing mutation affecting the intron 4 splice acceptor site, a 2-bp deletion at codon 726, a 7-bp deletion at codon 626, and a deletion of exons 13-16. The latter alteration was shown to result from a 22-kb genomic deletion due to a homologous recombination between Alu repeats located in introns 12 and 16. The detection of five germline hMLH1 mutations in five families that only partially fulfilled the Amsterdam criteria shows that these criteria do not allow the identification of all familial colorectal cancers due to mutations of the mismatch repair genes. The numerous Alu repeats present within the hMLH1 gene and the observation of large genomic deletions suggest that (a) Alu-mediated deletions might frequently be involved in hMLH1 inactivation, and (b) reverse transcription-PCR analysis, which allows the amplification of the entire coding region of the hMLH1 gene in one step, might be the most appropriate method for the detection of hMLH1 alterations.