The p.Cys1281Tyr variant in the hinge module/flap region of thyroglobulin causes intracellular transport disorder and congenital hypothyroidism.

Congenital hypothyroidism (CH) due to thyroglobulin (TG) variants causes very low serum TG levels with normal or enlarged thyroid glands, depending on the severity of the defect, and with autosomal recessive inheritance. The purpose of this study was to functionally characterize p.Cys1281Tyr variant in the TG gene in order to increase our knowledge of the molecular mechanisms associated with CH. In order to find evidence that support the hypothesis that the p.Cys1281Tyr variant would affect the TG folding were performed amino acid prediction, 3D modeling and transient expression analysis in HEK293T cells. 18 of the 21″in silico" algorithms predict a deleterious effect of the p.Cys1281Tyr variant. The full-length 3D model p.Cys1281Tyr TG showed disulfide bond cleavage between the cysteines at positions 1249 and 1281 and rearrangement of the TG structure, while transient expression analysis indicated that p.Cys1281Tyr causes retention of the protein inside the cell. Consequently, these results show that this pathogenic variant makes it impossible for TG to fulfill its function in the biosynthesis process of thyroid hormones, causing CH. In conclusion, our results confirm the pathophysiological importance of misfolding of TG as a consequence of p.Cys1281Tyr variant located in the hinge module/flap region of TG.

The p.Pro2232Leu variant in the ChEL domain of thyroglobulin gene causes intracellular transport disorder and congenital hypothyroidism.

Thyroglobulin (TG), the predominant glycoprotein of the thyroid gland, functions as matrix protein in thyroid hormonegenesis. TG deficiency results in thyroid dyshormonogenesis. These variants produce a heterogeneous spectrum of congenital goitre, with an autosomal recessive mode of inheritance. The purpose of this study was to identify and functionally characterize new variants in the TG gene in order to increase the understanding of the molecular mechanisms responsible for thyroid dyshormonogenesis. A total of four patients from two non-consanguineous families with marked alteration of TG synthesis were studied. The two families were previously analysed in our laboratory, only one deleterious allele, in each one, was detected after sequencing the TG gene (c.2359 C > T [p.Arg787*], c.5560 G > T [p.Glu1854*]). These findings were confirmed in the present studies by Next-Generation Sequencing. The single nucleotide coding variants of the TG gene were then analyzed to predict the possible variant causing the disease. The p.Pro2232Leu (c.6695 C > T), identified in both families, showing a low frequency population in gnomAD v2.1.1 database and protein homology, amino acid prediction, and 3D modeling analysis predict a potential pathogenic effect of this variant. We also transiently express p.Pro2232Leu in a full-length rat TG cDNA clone and confirmed that this point variant was sufficient to cause intracellular retention of mutant TG in HEK293T cells. Consequently, each family carried a compound heterozygous for p.Arg787*/p.Pro2232Leu or p.Glu1854*/p.Pro2232Leu variants. In conclusion, our results confirm the pathophysiological importance of altered TG folding as a consequence of missense variants located in the ChEL domain of TG.