Comprehensive constitutional genetic and epigenetic characterization of Lynch-like individuals [FFPE]

Background: In ~50% of Lynch syndrome (LS)-suspected patients (also called Lynch-like syndrome, LLS), the causal mechanism for cancer predisposition remains unknown. Our aim was to elucidate the constitutional basis of mismatch repair (MMR) deficiency in LLS patients throughout a comprehensive genetic and epigenetic analysis. Methods: One hundred and fifteen LLS patients harboring MMR deficient tumors and no pathogenic germline MMR gene mutations were included in this study. Mutational analysis of 26 colorectal cancer associated genes was performed by using a customized multigene panel and massively parallel sequencing. Pathogenicity of MMR variants was assessed by mRNA analysis and multifactorial likelihood calculations. Genome-wide methylome analysis was perfomed by using the Infinium HumanMethylation450K BeadChip. Results: The multigene panel analysis revealed the presence of two MMR gene truncating mutations not found in previous analysis. Of a total of 15 MMR variants of unknown significance identified, five - present in 6 unrelated individuals- were reclassified as pathogenic. In addition, 13 predicted deleterious variants in other CRC-predisposing genes (MSH3, MUTYH, POLD1, APC, EPCAM, BUB1, FAN1, EXO1 or PSM1) were found in 12 probands. Methylome analysis detected one constitutional MLH1 epimutation in an individual diagnosed with CRC at age 42, but no additional differentially methylated regions were identified in LLS compared to LS patients or healthy individuals. Conclusions: In conclusion, the use of an ad-hoc designed gene panel combined with pathogenicity assessment of variants allows the identification of deleterious MMR mutations as well as new LLS candidate genes. Moreover, constitutional epimutations in non-LS-associated genes are not responsible for the MMR-deficient phenotype observed in LLS patients. A total of 115 Caucasian Lynch-like syndrome patients harboring MMR deficient tumors MMR loss of expression and/or microsatellite instability (MSI) were analyzed by Infinium Human Methylation 450k Beadchip. From all included individuals, DNA from blood was collected. FFPE blocks of normal colorectal mucosa and colorectal cancer tissue were also included when available. The current subset includes only FFPE samples. Blood samples can be found in the other associated dataset from Damaso et al. Clinical data abbreviations: microsatellite instability (MSI+=Microsatellite Instabillity; MSS=Microsatellite stable; NP=Not performed; NV=Not Valuable) BRAF status (WT=wildtype; NP=Not performed)