To Improve the characterization of the variability and the genotype-phenotype correlation in patients with Multiple Endocrine Neoplasia type 2 (MEN2) and Medullary Thyroid Carcinoma "apparently sporadic" using Next Generation sequencing (NGS)

Abstract

Multiple Endocrine Neoplasia type 2 (MEN2) was one of the first hereditary syndromes which they identified a genetic and molecular origin. It was possible due to the high phenotypic penetrance in families associated with the manifest clinically diagnosed diseases such as medullary thyroid cancer (MTC) and pheochromocytoma (PHEO).For families with MEN2 and MTC that present clinical heterogeneity, a more extensive genetic investigation is justified using next-generation sequencing, instead of the usual search for a single gene (Sanger technique) in the RET gene, by the following factors:a) The possibility of discovering and understanding alternative mechanisms that modulate the phenotype in addition to the already known oncogenicity of pathogenic variants of the RET; b) the perception of greater or lesser severity of the disease depending on these other involved genetic factors; c) advice to family members who present these genetic events that modify the phenotype, whether protective or aggravating; d) the characterization of temporal genetic heterogeneity as in the study of the genetic profile for tumor recurrence and, spatially, for the investigation of different locations to assess tumor evolution and possible resistance to treatment; e) the chance of discovering new therapeutic targets, depending on the identification of other signaling pathways or associated genetic factors.When we analyze large cohorts, such as the one obtained by the BrasMEN Consortium, we observe clinical heterogeneity between families with the same pathogenic variant in RET or even between affected individuals in the same family. One explanation for this heterogeneity (reduced penetrance and varied expressivity) is the association with germline or somatic genetic variants in the RET oncogene or tumor suppressor genes that may act as phenotype modifiers. Therefore, evaluating RET by traditional means using Sanger sequencing on the "hot-spot" region would not allow the determination of other modulatory variants in practice since it is in exon 5, an area usually not included in routine diagnostic sequencing.When studying hereditary cases with phenotypic variability, it will also be necessary to investigate further the disease mechanism in patients with "apparently" sporadic TCM, that is, those who do not present the germline variant of RET but who show suggestive clinical and laboratory characteristics of hereditary origin, such as the onset of the disease at a young age or with a histological tumor lesion showing multicentricity; with this biological behavior, it is conjectured that there is another genetic event that justifies the earlier and multifocal appearance of this "apparently" sporadic neoplasm.The advances in knowledge of MTC and MEN2 obtained so far were based on classical sequencing using the single-gene Sanger technique, which is still the sequencing strategy recommended by the MEN2 diagnostic guidelines. However, sequencing using the Sanger technique has limitations and becomes a costly tool when we consider sequencing many exons or multiple genes simultaneously. Therefore, this subproject intends to use new large-scale sequencing techniques that allow the simultaneous investigation of several genes and even the complete genome in MEN2 and sporadic MTC, which can help us to understand both the individual susceptibility to the disease as well as create more optimized genetic diagnosis flow that brings an adequate cost-effectiveness ratio.Thus, this project will select affected family members and "apparently sporadic" patients who present more significant clinical variability related to tumor aggressiveness, including the presence of regional or distant metastasis and other risk factors, with the aim of the objective of identifying additional genetic variants that may explain the correlation with phenotypic variability.