Comprehensive Constitutional Genetic and Epigenetic Characterization of Lynch-Like Individuals

Abstract

The causal mechanism for cancer predisposition in Lynch-like syndrome (LLS) remains unknown. Our aim was to elucidate the constitutional basis of mismatch repair (MMR) deficiency in LLS patients throughout a comprehensive (epi)genetic analysis. One hundred and fifteen LLS patients harboring MMR-deficient tumors and no germline MMR mutations were included. Mutational analysis of 26 colorectal cancer (CRC)-associated genes was performed. Pathogenicity of MMR variants was assessed by splicing and multifactorial likelihood analyses. Genome-wide methylome analysis was performed by the Infinium Human Methylation 450K Bead Chip. The multigene panel analysis revealed the presence of two MMR gene truncating mutations not previously found. Of a total of 15 additional MMR variants identified, five -present in 6 unrelated individuals- were reclassified as pathogenic. In addition, 13 predicted deleterious variants in other CRC-predisposing genes were found in 12 probands. Methylome analysis detected one constitutional MLH1 epimutation, but no additional differentially methylated regions were identified in LLS compared to LS patients or cancer-free individuals. In conclusion, the use of an ad-hoc designed gene panel combined with pathogenicity assessment of variants allowed the identification of deleterious MMR mutations as well as new LLS candidate causal genes. Constitutional epimutations in non-LS-associated genes are not responsible for LLS.

Keywords: Lynch syndrome; Lynch-like syndrome; cancer genes panel; epimutation; methylation; mismatch repair; next generation sequencing; variant of unknown significance.

Figure A1

Schematic workflow of the study design and the obtained results.

Figure 1

Pedigrees from patients reclassified as Lynch syndrome in the current study. Abbreviations: CRC, colorectal cancer; EC, endometrial cancer, PC, prostate cancer, GC, gastric cancer, OC, ovarian cancer, BM(UTP), brain metastasis from unknown primary tumor, KC, kidney cancer, TC, testis cancer, My, myeloma, MSI+, microsatellite instability, NV, No valuable, +, variant carrier, -, variant non carrier.

Figure 2

Identification of a new case of constitutional MLH1 epimutation. (A) Pedigree of case 7. Representation of mean β-values in blood DNA (B) and FFPE normal colorectal mucosa (C) from case 7 against MLH1 epimutation carriers, mutation-positive Lynch syndrome patients and healthy controls at differentially methylated region described for constitutional MLH1 epimutation carriers. Chromosome coordinates of CpG sites are graphed at axis of abscissa. The location of the CpG sites are not drawn to scale. CpG islands (CI) are represented as black rectangles and their shores in grey. Location of Deng’s promoter regions (DR) are indicated as white rectangles. Genes (G) including displayed CpG sites are represented as grey rectangles. Cytoband divisions (CB) are displayed as grey rectangles. Ensembl GRCh37 was taken as reference for gene coordinates.

Figure 3

Scatterplot of the normalized mean B-values obtained using the Infinium 450k Human Methylation array to identify differentially methylated CpG islands (A) and genes (B) in tumors from LLS cases (left) and LS controls (right). The transparency corresponds to point density. One % of the points in the sparsest populated plot regions are drawn explicitly. The colored points represent differentially methylated CpG islands and genes with an FDR adjusted p-values lower than 0,05. (C) Venn diagrams of the differentially methylated CpG islands (left) and CpG sites (right), which shown the overlapping of epigenetic changes during tumorigenesis in LLS cases (yellow) and LS controls (red).