Whole Exome Sequencing of Medullary Thyroid Carcinomas did not identify oncogenic drivers alternative to RET and RAS genes

About 60% of sporadic Medullary Thyroid Carcinomas (MTC) harbor somatic mutations in RET and RAS genes while roughly 40% of cases are still orphan of an oncogenic driver. Aim of this study was to investigate putative novel genetic alterations leading to pathogenesis of MTC from Whole Exome Sequencing (WES) data obtained in RET+ and RET- cases. Methods: WES analysis was performed on 6 sporadic MTC cases (2 RET+, 4 RET-) using an Illumina platform. Analyzing the list of Single Nucleotide Variations (SNV) shared by the RET- cases, we studied the gene CNOT1 presenting different SNV in the 4 RET- cases; among these SNV we chose a SNP, the R299Q, with a Minor Allele Frequency (MAF) ≤ 0.1. We first performed a validation panel of 83 sporadic MTC cases and 94 healthy controls. A larger case-control study was then performed on 874 MTC cases and 896 healthy controls. Genotyping was performed using a TaqMan SNP Assay (Life Technologies) on genomic DNA extracted from the blood of the patients. We also analyzed the presence of mutations in genes belonging to a panel of 344 oncogene panel list (NuGEN). We chose 7 SNV not present in dbSNP database as somatic mutation candidates: RB1 (R787Q), MSH2 (A189T), HIF1A (K334R), PCNA (V102I), FES (R764Q), THBS1 (G26A), NSD1 (P702S). These SNV were studied by direct sequencing on genomic DNA extracted from tumoral tissue and blood in order to evaluate their somatic or germinal nature. Results: On a first step of validation, we found that the frequency of the mutated allele (allele A: CGA/CAA) of the CNOT1 R299Q in our total series of 89 MTC cases was significantly higher than in the 94 healthy controls (0.045 vs 0.011, χ2=4.04, p=0.04); despite this, further validation on a larger case-control panel did not confirm the significant difference (0.0215 vs 0.0189, χ2=0.12, p=0.729). The validation obtained in the tissue and blood DNA of patient harboring the 7 oncogene SNV, revealed their germinal nature and they were not further studied. Conclusions: Despite the great potentiality of WES, in sporadic MTC cases it was unable to find any oncogenic driver alternative to RET and RAS. Nevertheless, the huge amount of data generated by WES will be further analyzed to complete the study