Endocrine Abstracts

Introduction: By Next Generation Sequencing (NGS) our team identified in two families presenting a phenotype compatible with familial non-medullary thyroid carcinoma (Family C and Family R), two new potentially pathogenic germline mutations. Family C presented p.Gly106Arg mutation in the KCNB2 gene, that codifies a voltage-gated potassium channel (vgKCN). Since potassium efflux by the cell is a necessary condition for cellular homeostasis, vgKCN disruption can impact the function of other ion channels. Mice studies showed that KCNE2 disruption indirectly impairs sodium-iodide symporter (NIS) function, and therefore iodide uptake by the cell, resulting in hypothyroidism or goiter. Family R presented p.Gly486Arg mutation in the ubiquitin-specific protease (USP) USP42, known for regulating p53, cell cycle arrest and apoptosis. Evidence shows that deregulation of USPs may be involved in carcinogenesis.

Hypothesis: In family C, by indirect effect on NIS function, vgKCN mutations may increase predisposition to thyroid cancer and help explain why some patients do not respond to radioiodine (RAI) therapy. In family R, by deregulation of the deubiquitinating processes, USP42 may be involved in thyroid carcinoma development.

Methodology: In silico studies using two NGS databases (18 and 513 sporadic thyroid cancer patients, respectively) were conducted. Alterations in 59 genes were searched for copy-number variation, point mutations and other alterations. In vitro assays using Nthy-ori 3-1 and FRTL-5 cell lines are ongoing. siRNAs are being used to silence USP42 gene. CRISPR-RNP will be used to knockout USP42 gene and to knock-in clones that harbor the specific mutations of KCNB2 and USP42 found in both families. Morphologic and functional assays will be performed in the transformed cell clones. Finally, in vivo assays will be conducted in zebrafish to replicate in vitro results.

Results: In silico results showed that KCNA3, KCNH7, KCNS3 and USP39 genes were underexpressed while KCNC1, KCNC3, KCNG2, KCNH2, USP42, BRAF and KRAS were overexpressed. BRAF, RAS, TERT and USP mutations were also identified, as well as vgKCN alterations. No significant association was found between vgKCN and USP alterations regarding patient clinical-pathological characteristics. By siRNAs we successfully downregulated USP42 in human thyroid Nthy-ori 3-1 cell line, as verified by RT-PCR. Protein expression is currently being analysed by Western blot and cellular functional assays are being conducted.

Conclusions: If our hypotheses are verified, vgKCN and USPs mutations could represent new markers, and possibly pharmacological targets, both in familial and in sporadic thyroid cancer.