Splicing analysis of 24 potential spliceogenic variants in MMR genes and clinical interpretation based on refined ACMG/AMP criteria.

Lynch syndrome (LS) is a common hereditary cancer syndrome caused by heterozygous germline pathogenic variants in DNA mismatch repair (MMR) genes. Splicing defect constitutes one of the major mechanisms for MMR gene inactivation. Using RT-PCR based RNA analysis, we investigated 24 potential spliceogenic variants in MMR genes and determined their pathogenicity based on refined splicing-related American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria. Aberrant transcripts were confirmed in 19 variants and 17 of which were classified as pathogenic including 11 located outside of canonical splice sites. Most of these variants were previously reported in LS patients without mRNA splicing assessment. Thus, our study provides crucial evidence for pathogenicity determination, allowing for appropriate clinical follow-up. We also found that computational predictions were globally well correlated with RNA analysis results and the use of both SPiP and SpliceAI software appeared more efficient for splicing defect prediction.

PMS2 or PMS2CL? Characterization of variants detected in the 3' of the PMS2 gene.

PMS2 germline pathogenic variants are one of the major causes for Lynch syndrome and constitutional mismatch repair deficiencies. Variant identification in the 3' region of this gene is complicated by the presence of the pseudogene PMS2CL which shares a high sequence homology with PMS2. Consequently, short-fragment screening strategies (NGS, Sanger) may fail to discriminate variant's gene localization. Using a comprehensive analysis strategy, we assessed 42 NGS-detected variants in 76 patients and found 32 localized on PMS2 while 6 on PMS2CL. Interestingly, four variants were detected in either of them in different patients. Clinical phenotype was well correlated to genotype, making it very helpful in variant assessment. Our findings emphasize the necessity of more specific complementary analyses to confirm the gene origin of each variant detected in different individuals in order to avoid variant misinterpretation. In addition, we characterized two PMS2 genomic alterations involving Alu-mediated tandem duplication and gene conversion. Those mechanisms seemed to be particularly favored in PMS2 which contribute to frequent genomic rearrangements in the 3' region of the gene.

From variant of unknown significance to likely pathogenic: Characterization and pathogenicity determination of a large genomic deletion in the MLH1 gene.

BACKGROUND: The MLH1 gene is one of the DNA mismatch repair genes (MMR), implicated in Lynch syndrome (LS), an autosomal dominant hereditary tumor susceptibility disease. The advent of next-generation sequencing (NGS) technologies has accelerated the diagnosis of inherited diseases and increased the percentage of diagnosis of inherited cancers. However, some complex genomic alterations require the combination of several analytical strategies to allow correct biological interpretations. Here, we describe a novel MLH1 deletion and its pathogenicity determination in a patient suspected of LS. METHODS: The index case was a French 73-year-old man diagnosed with colorectal cancer displaying microsatellite instability and the loss of MLH1 and PMS2 expression. NGS analysis was used as the primary method for MMR genes screening. Long-range PCR and reverse transcriptase polymerase chain reaction (RT-PCR) were used for breakpoints and pathogenicity determinations. RESULTS: A large genomic deletion was detected which removed the last six nucleotides of MLH1 exon 11 together with a large part of intron 11. It was initially considered as a variant of unknown significance (VUS). Genomic breakpoints were subsequently characterized defining the deletion as c.1033_1039-248del. Further RNA analysis demonstrated that this variant activated a cryptic donor splice site at the 5' of the breakpoint, leading to a premature truncated protein: p.Thr345Alafs*13. CONCLUSION: Our finding suggested that although NGS technologies have increased variant detection yield, combined approaches were still needed for complex variant characterization and pathogenicity assessment.

A PMS2 non-canonical splicing site variant leads to aberrant splicing in a patient suspected for lynch syndrome.

The PMS2 gene is one of the DNA mismatch repair genes (MMR) implicated in Lynch syndrome (LS). A subset of PMS2 pathogenic variants (PVs) are splice variants mostly affecting canonical GT/AG splicing sequences. However, the majority of the intronic variants outside canonical splice sites remained as variants of unknown significance, even though some of them would alter the splicing process. In this report, we describe the analysis of such an intronic variant (c.251-5T > C) detected in an 82-year-old patient diagnosed with endometrial cancer displaying microsatellite instability and the loss of PMS2 expression displayed. RNA analysis demonstrated that this variant lead to the complete exon 4 skipping, resulting in the synthesis of a truncated protein. This finding shows the relevance of functional RNA analysis in the non-canonical intronic variant assessment and the importance of systematic evaluation of MSI/loss of expression of MMR genes for LS screening in patients with endometrial cancers.

Acquired somatic MMR deficiency is a major cause of MSI tumor in patients suspected for "Lynch-like syndrome" including young patients.

Patients with tumors displaying high microsatellite instability (MSI-H) but no germline MMR inactivation are suspected for Lynch-like syndrome (LLS). To explore the involvement of acquired somatic MMR alteration as a cause, we screened 113 patient tumor samples for MMR gene variations and loss of heterozygosity. Somatic MMR alterations were found in 85.8% of patients including "double hits" in 63.7% of patients, mainly diagnosed with colon and endometrial cancers. Interestingly, 37.5% of them were under the age of 50, and seven patients were under 30. Somatic alterations were mainly attributed to the MLH1, MSH2 genes, likely reflecting the functional importance of these key MMR genes. Pathogenic variants co-existed in other cancer genes in particular the APC gene displaying a characteristic MMR deficiency-related "mutational signature", indicating that it may be inactivated owing to MMR deficiency. We speculated that APC inactivation could trigger an accelerated malignant transformation underlying early-onset cancers. Our findings provide further insight into the mechanisms underlying LLS, somatic MMR inactivation being a major cause for early-onset LLS through pathways differing from those involved in late-onset sporadic cases.

Intensity-dependent constitutional MLH1 promoter methylation leads to early onset of colorectal cancer by affecting both alleles.

Constitutional epimutation is one of the causes for MLH1 gene inactivation associated with hereditary non-polyposis colon cancer (HNPCC) syndrome. Here we investigate MLH1 promoter hypermethylation in 110 sporadic early-onset colorectal cancer patients. Variable levels of hypermethylation were detected in 55 patients (50%). Importantly a reduced MLH1 gene expression was found in patients with high-level methylation, with the association of microsatellite instability (MSI) in their tumor cells. Such high-level methylation accounts for 7.4% of all patients included in this study. Furthermore, we found that in one case constitutional methylation affected both alleles, indicating a post-zygotic methylation dysregulation. Our findings suggest that constitutional epimutation is a mechanism underlying early-onset colorectal cancer, although it is involved in only a small proportion of patients, who require appropriate surveillance. Our findings provide further insight into the role of aberrant constitutional methylation in colon carcinogenesis and raise the question of whether prevalent low-level methylation constitutes a potential risk factor for cancer development.

Novel biallelic mutations in MSH6 and PMS2 genes: gene conversion as a likely cause of PMS2 gene inactivation.

Since the first report by our group in 1999, more than 20 unrelated biallelic mutations in DNA mismatch repair genes (MMR) have been identified. In the present report, we describe two novel cases: one carrying compound heterozygous mutations in the MSH6 gene; and the other, compound heterozygous mutations in the PMS2 gene. Interestingly, the inactivation of one PMS2 allele was likely caused by gene conversion. Although gene conversion has been suggested to be a mutation mechanism underlying PMS2 inactivation, this is the first report of its involvement in a pathogenic mutation. The clinical features of biallelic mutation carriers were similar to other previously described patients, with the presence of café-au-lait spots (CALS), early onset of brain tumors, and colorectal neoplasia. Our data provide further evidence of the existence, although rare, of a distinct recessively inherited syndrome on the basis of MMR constitutional inactivation. The identification of this syndrome should be useful for genetic counseling, especially in families with atypical hereditary nonpolyposis colon cancer (HNPCC) associated with childhood cancers, and for the clinical surveillance of these mutation carriers.

Systematic mRNA analysis for the effect of MLH1 and MSH2 missense and silent mutations on aberrant splicing.

A substantial proportion of MLH1 and MSH2 gene mutations in hereditary nonpolyposis colon cancer syndrome (HNPCC) families are characterized by nucleotide substitutions, either within the coding sequence (missense or silent mutations) or in introns. The question of whether these mutations affect the normal function of encoding mismatch DNA repair proteins and thus lead to the predisposition to cancer is determinant in genetic testing. Recent studies have suggested that some nucleotide substitutions can induce aberrant splicing by disrupting cis-transcription elements such as exonic enhancers (ESEs). ESE disruption has been proposed to be the mechanism that underlies the presumed pathological missense mutations identified in HNPCC families. To investigate the prevalence of aberrant splicing resulting from nucleotide substitutions, and its relevance to predicted ESEs, we conducted a systematic RNA screening of a series of 60 patients who carried unrelated exonic or intronic mutations in MLH1 or MSH2 genes. Aberrant splicing was found in 15 cases, five of which were associated with exonic mutations. We evaluated the link between those splicing mutations and predicted putative ESEs by using the computational tools ESEfinder and RESCUE-ESE. Our study shows that the algorithm-based ESE prediction cannot be definitely correlated to experimental observations from RNA screening. By using minigene constructs and in vitro transcription assay, we demonstrated that nucleotide substitutions are the direct cause of the splicing defect. This is the first systematic screening for the effect of missense and silent mutations on splicing in HNPCC patients. The pathogenic splicing mutations identified in this study will contribute to the assessment of "unclassified variants" in genetic counseling. Our results also suggest that one must use caution when determining the pathogenic effect of a missense or silent mutation using ESE prediction algorithms. Analysis at the RNA level is therefore necessary.

Neurofibromatosis type 1 gene as a mutational target in a mismatch repair-deficient cell type.

DNA mismatch repair (MMR) is the process by which incorrectly paired DNA nucleotides are recognized and repaired. A germline mutation in one of the genes involved in the process may be responsible for a dominantly inherited cancer syndrome, hereditary nonpolyposis colon cancer. Cancer progression in predisposed individuals results from the somatic inactivation of the normal copy of the MMR gene, leading to a mutator phenotype affecting preferentially repeat sequences (microsatellite instability, MSI). Recently, we identified children with a constitutional deficiency of MMR activity attributable to a mutation in the h MLH1 gene. These children exhibited a constitutional genetic instability associated with clinical features of de novo neurofibromatosis type 1 (NF1) and early onset of extracolonic cancer. Based on these observations, we hypothesized that somatic NF1 gene mutation was a frequent and possibly early event in MMR-deficient cells. To test this hypothesis, we screened for NF1 mutations in cancer cells. Genetic alterations were identified in five out of ten tumor cell lines with MSI, whereas five MMR-proficient tumor cell lines expressed a wild-type NF1 gene. Somatic NF1 mutations were also detected in two primary tumors exhibiting an MSI phenotype. Finally, a 35-bp deletion in the murine Nf1 coding region was identified in mlh1-/- mouse embryonic fibroblasts. These observations demonstrate that the NF1 gene is a mutational target of MMR deficiency and suggest that its inactivation is an important step of the malignant progression of MMR-deficient cells.