Integrative Analysis of Hereditary Nonpolyposis Colorectal Cancer: the Contribution of Allele-Specific Expression and Other Assays to Diagnostic Algorithms

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.

Functional interrogation of Lynch syndrome‐associatedMSH2missense variants via CRISPR‐Cas9 gene editing in human embryonic stem cells

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Carcinoma of the lower uterine segment diagnosed with Lynch syndrome based on MSH6 germline mutation: A case report

“…One of these cases was reported as small‐intestinal cancer with MSI‐L . In the other case, the results of MSI testing and detailed information on the cancer were unknown . The current report is the first to describe this mutation in Lynch‐syndrome‐related endometrial cancer.…”

Complementary molecular approaches reveal heterogeneous CDH1 germline defects in Italian patients with hereditary diffuse gastric cancer (HDGC) syndrome

“…By using this conservative approach, one patient only proved to carry a significant AI, pointing to the lowered expression of one CDH1 allele as the germline defect associated with HDGC. Constitutive silenced or lowered expression of one allele has been reported not only for CDH1 in HDGC (Pinheiro et al, 2010), but also for other cancer-susceptibility genes such as, for example, APC in familial adenomatous polyposis (Renkonen et al, 2005;Castellsagu e et al, 2010), mismatch repair genes in Hereditary Nonpolyposis Colorectal Cancer (Renkonen et al, 2003;De Lellis et al, 2013), and BRCA1 and BRCA2 genes in Hereditary Breast/Ovarian Cancer (Caux-Moncoutier et al 2009). These findings clearly demonstrate that methods to identify expression defects are needed to complement standard mutation screening.…”