Endocrine Abstracts

Introduction: Forkhead box E1 (FOXE1) gene encodes a transcription factor crucial for thyroid morphogenesis, differentiation, and function. We previously found evidence of the involvement of a rare germline FOXE1 variant in familial non-medullary thyroid carcinoma (FNMTC) etiology. FNMTC most common subtype is papillary thyroid carcinoma (PTC), and family members frequently present thyroid follicular nodular disease (FND). Germline FOXE1 mutations have also been associated to congenital hypothyroidism due to thyroid dysgenesis, thyroid ectopy, and cleft palate (CP). Struma ovarii is a teratoma, containing mostly ectopic thyroid tissue. Among malignant struma ovarii (MSO), the PTC type is the most common. Due to its rarity, the genetic basis of MSO remains poorly understood, although its somatic alterations resemble those arising in eutopic thyroid cancer (TC). Several polymorphisms in the FOXE1 gene regulatory regions, affecting expression levels, have been associated with increased susceptibility to CP, PTC and TC aggressiveness. In this study, two families were identified: F1) proband with MSO (PTC) and FND, mother asymptomatic, and maternal grandmother with FND; and F2) proband with follicular variant PTC (fvPTC) and septate uterus, sister, mother and maternal uncle with CP, and maternal grandmother with hypothyroidism.

Aim: To clarify the role of FOXE1 in CP, MSO and TC in these families.

Methods: The FOXE1 gene, including the promoter region, was analysed through Sanger sequencing, in probands’ leucocyte DNAs. Immunohistochemistry (IHC) and qRT-PCR analyses of patients’ tumours and adjacent normal tissues were undertaken. MSO’s somatic alterations were assessed by next-generation sequencing (NGS). Luciferase assays using plasmids with wild-type FOXE1 promoter or variants were performed in cervix uteri carcinoma (SiHa) and normal thyroid (PCCL3) cells.

Results: We identified two unreported germline heterozygous variants in the FOXE1 promoter: F1: c.-522G>C, detected in the proband and her mother; F2: c.9C>T, segregating in the proband, her sister and mother. Remaining relatives were not assessed yet. Both variants are described with very low frequency in population databases and were absent in healthy Portuguese population controls. IHC and qRT-PCR analyses of probands’ tumours revealed lower FOXE1 expression levels than in normal adjacent thyroid tissues. Luciferase assays showed significantly higher gene expression of the c.-522G>C FOXE1 variant, compared to wild-type, in SiHa (P<0.001) and PCCL3 (P < 0.05) cell models. MSO NGS analysis unveiled a likely pathogenic somatic variant in BRAF p.(Gly469Ala). Studies for c.9C>T are ongoing.

Conclusions: The present data suggest that germline promotor variants, particularly c.-522G>C, may account for deregulation of FOXE1 expression, influencing thyroid precursor cells migration, and initiation/progression of neoplasms; BRAF somatic activation may then lead to MSO development, as commonly observed in TC.