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. 2013 Nov 20;8(11):e81194.
doi: 10.1371/journal.pone.0081194. eCollection 2013.

Integrative analysis of hereditary nonpolyposis colorectal cancer: the contribution of allele-specific expression and other assays to diagnostic algorithms

Laura De Lellis  1 , Gitana Maria Aceto, Maria Cristina Curia, Teresa Catalano, Sandra Mammarella, Serena Veschi, Fabiana Fantini, Pasquale Battista, Vittoria Stigliano, Luca Messerini, Cristina Mareni, Paola Sala, Lucio Bertario, Paolo Radice, Alessandro Cama
Affiliations

Integrative analysis of hereditary nonpolyposis colorectal cancer: the contribution of allele-specific expression and other assays to diagnostic algorithms

Laura De Lellis et al. PLoS One. .

Abstract

The identification of germline variants predisposing to hereditary nonpolyposis colorectal cancer (HNPCC) is crucial for clinical management of carriers, but several probands remain negative for such variants or bear variants of uncertain significance (VUS). Here we describe the results of integrative molecular analyses in 132 HNPCC patients providing evidences for improved genetic testing of HNPCC with traditional or next generation methods. Patients were screened for: germline allele-specific expression (ASE), nucleotide variants, rearrangements and promoter methylation of mismatch repair (MMR) genes; germline EPCAM rearrangements; tumor microsatellite instability (MSI) and immunohistochemical (IHC) MMR protein expression. Probands negative for pathogenic variants of MMR genes were screened for germline APC and MUTYH sequence variants. Most germline defects identified were sequence variants and rearrangements of MMR genes. Remarkably, altered germline ASE of MMR genes was detected in 8/22 (36.5%) probands analyzed, including 3 cases negative at other screenings. Moreover, ASE provided evidence for the pathogenic role and guided the characterization of a VUS shared by 2 additional probands. No germline MMR gene promoter methylation was observed and only one EPCAM rearrangement was detected. In several cases, tumor IHC and MSI diverged from germline screening results. Notably, APC or biallelic MUTYH germline defects were identified in 2/19 probands negative for pathogenic variants of MMR genes. Our results show that ASE complements gDNA-based analyses in the identification of MMR defects and in the characterization of VUS affecting gene expression, increasing the number of germline alterations detected. An appreciable fraction of probands negative for MMR gene variants harbors APC or MUTYH variants. These results indicate that germline ASE analysis and screening for APC and MUTYH defects should be included in HNPCC diagnostic algorithms.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ASE values observed.
ASE values between the 2 dashed lines correspond to less than twofold imbalances in allelic ratios (see Methods).
Figure 2
Figure 2. Results of integrative germline analyses performed in this study.
VUS included in-frame deletions, sequence variants with uncertain effect on splicing and missense variants. Evidence of a pathogenic, possibly pathogenic defect in 28 probands with VUS derived from previous studies (see Table S5). In one additional case (360#2916) availability of RNA allowed ASE analysis that helped to characterize a VUS shared also by another proband (986#3487, see Results). ASE analysis was conducted in 22 individuals, including 12 with ascertained germline defect that are not shown in the figure. APC and MUTYH screening was conducted in a subset of patients negative for MMR defects (see Methods). Inclusion of ASE analysis and screening for APC and MUTYH sequence variants in the integrative analyses increased the number of probands with germline alterations detected.
See this image and copyright information in PMC

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The study was supported by funds from the Italian Ministry of Education, University and Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.