FOXE1 susceptibility polymorphisms and functional characterization of rare DUOX2 variants provide new insights into congenital hypothyroidism with thyroid dysgenesis.

Thyroid dysgenesis (TD) is the main cause of congenital hypothyroidism (CH). However, a definitive genetic cause is confirmed in only 2-5% of patients, since pathogenic mutations are rare. This study was conducted to advance the understanding of TD genetic basis by investigating genetic alterations in the exomes of affected individuals. A total of 71 TD patients were included; 35 underwent whole-exome sequencing (WES), while the remaining 36 patients and 128 Brazilian non-TD controls were genotyped by Sanger sequencing. Data from the Online Archive of Brazilian Mutations, 1000 Genomes Project were used for comparative analyses. WES revealed a high frequency of FOXE1 polymorphisms and five rare heterozygous DUOX2 variants previously identified in CH patients: p.F966SfsX29 (pathogenic); p.R516H (likely pathogenic), p.T423I and p.N1170T (variant of uncertain significance, VUS) and p.A1123T (likely benign) according to ACMG criteria. Patients with DUOX2 variants also harbored FOXE1 polymorphisms. Regarding population-wide risk, functional FOXE1 rs1867277 allele A and the rs71369530 14Ala allele were associated with TD, individually, and in combination in the Brazilian cohort, not previously reported. Functional analysis of DUOX2 variants showed disruption of epithelial-mesenchymal transition-related gene expression without affecting 3D spheroid formation; only the p.F966SfsX29 variant reduced hydrogen peroxide generation. In silico protein homology-based modeling revealed altered structures of all DUOX2-variants-DUOXA2 complex, including hydrogen bond position changes. In conclusion, our findings confirm two FOXE1 polymorphisms as TD risk factors, show that their combined inheritance increases disease risk, and reveal that rare DUOX2 variants co-occur with FOXE1 risk polymorphisms, supporting a polygenic model.