• medullary thyroid cancer
• ret gene

Medullary thyroid carcinoma (MTC) originates from neural crest-derived parafollicular C cells and accounts for about 7% of cancers affecting the thyroid. It occurs in both sporadic and hereditary forms. The latter accounts for approximately 25% of all MTC cases and can occur as part of Multiple Endocrine Neoplasia syndromes (MEN), 2A and 2B type, or as isolated disease in the familiar medullary carcinoma (FMTC). Germline activating RET mutations are found in 95-98% of hereditary MTC while somatic RET mutations are present in 25-40% of sporadic MTC. Activating point mutations in the RAS genes (mainly H- and K-RAS) are present in another 11% of sporadic cases and they are mutually exclusive with RET mutations. About 2% of hereditary and 50% of sporadic forms, however, are still orphans of driver genetic alterations.
The introduction of next-generation sequencing (NGS) techniques is a valuable tool for discovering new mutations involved in cancer physiopathology.
Aim of this project has been to validate and analyze new single nucleotide variations (SNVs), previously identified by whole exome sequencing (WES) performed in 3 patients affected by sporadic MTC and 1 patient affected by FMTC. All cases were negative for known RET and RAS mutations.
After an appropriate filtering of the data obtained from WES, we have obtained a list of 57 mutations in 57 genes. These were all missense mutations in heterozygous form never reported or, if present in single nucleotide polymorphisms (SNPs) databases, such as dbSNP, ExAC, 1000genomes, with a minimum allele frequency (MAF) lower than 0.01. The SNVs were present in genes potentially involved in carcinogenesis and showed a high pathogenicity score evidenced by in silico analysis.
All SNVs obtained by NGS were validated by Sanger sequencing of genomic DNA extracted from blood of the patient harboring the SNV itself in order to assess the germline origin, as well as to confirm the presence of the SNV. The results obtained confirmed the presence of 53/57 (92.9%) germline SNVs while the remaining 4/57 (7.1%) represented false positives.
Given the germline origin of all mutations, we focused on the 28 SNVs present in the patient affected by FMTC and we analyzed them also in two other family members: one affected and one healthy, in order to assess the possible segregation of these SNVs with the disease. This analysis has shown two rare SNPs: rs372939895 in the KIAA1522 gene and rs760417150 in the REM1 gene that were present in the two affected patients but not in the healthy one. Considering these data, we hypothesized a predisposing role of these SNP in the pathogenesis of MTC and we investigated their presence in the genomic DNA of 871 patients affected by sporadic MTC by specific TaqMan SNP Genotyping Custom assays, comparing the data with healthy controls in public databases, but we have detected no additional cases.
In conclusion, in this study we validated 92.9% of 57 SNVs obtained by exome sequencing of 4 cases of MTC: all SNVs were germline. Two SNPs on two genes, KIAA1522 and REM1, segregating with the disease in a family affected by FMTC, were analyzed in additional 871 cases of MTC but no additional cases were reported. We can hypothesize that these two rare polymorphisms may represent "private" mutations, that could be restricted to this single family, predisposing to MTC.
Functional studies of the two SNP able to assess the transforming ability and the real role in this family represent the future development of this study.