Up to 30% of colorectal cancers (CRCs) have evidence of a familial component, and about 5% are thought to be due to inherited mutations in MMR (MisMatch Repair) genes. Lynch syndrome (LS) is characterized by a very early onset and lifetime risk estimated to 80%. The gold standard for LS diagnostic is complete Sanger sequencing, a very complex and expensive analysis. MMR mutations are detected in only 60% of criteria fulfilling families, while they are present in up to 20% of families not fulfilling these criteria and which are implicitly excluded from genetic counselling. Therefore, we propose an adapted algorithm, based on germline and tumor analysis, intended to increase molecular diagnostic efficiency and CRC casuistry coverage.
20 LS families were selected according to Amsterdam criteria, and one index case per family agreed to participate by signing informed consent. All coding regions and exon-intron boundaries of MSH2, MLH1 and MSH6 were screened by double strand Sanger sequencing. Sequence variants were interpreted by in-silico analysis. MLPA was performed for large genomic rearrangements. MMR protein expression in tumors was investigated by immunohistochemistry. Somatic tumor DNA was checked for Microsatellite instability (MSI), MLH1 promoter hypermethylation (PHM), as well as for BRAF V600E mutation.
Over 50% of our samples presented germline variants, the majority being benign. Four unclassified variants are altering splicing enhancers. One deleterious variant, but no recurrent MMR mutations were detected. No large genomic rearrangements were identified. IHC showed loss of MSH2 and MLH1 in several samples also presenting high MSI. No PHM was observed, but somatic BRAF V500E showed to be present in several samples. Data from germline analysis correctly correlated with somatic investigations.
This is the first complete molecular approach of LS in Romania. Our work has an important impact on reducing the cost and time of molecular diagnostic, include for diagnostic more patients than usually selected, perform a more efficient diagnostic with >80% mutation finding probability, provide evidence-based recommendations for oncogenetic diagnostic. Acknowledgement: PN-II-RU-TE-2014-4-2257.
Clinical trial identification
Legal entity responsible for the study
G. T. Popa University of Medicine and Pharmacy, Iasi, Romania
Executive Agency for Higher Education, Research, Development and Innovation Funding (UEFISCDI), Romania
All authors have declared no conflicts of interest.