Clinical, Biochemical, and Molecular Genetic Characterization of Two Patients with Congenital Hypothyroidism Harboring Novel Compound Heterozygous Variants in the Thyroglobulin Gene

Context Thyroglobulin (TG) is the most abundant glycoprotein secreted by thyrocytes into the follicular lumen, serving as the essential substrate for thyroid hormone biosynthesis. The clinical spectrum of TG defects is highly heterogeneous, ranging from euthyroid states to mild or severe permanent hypothyroidism. The aim of this study was to identify and characterize novel TG variants to advance our understanding of the molecular mechanisms underlying thyroid dyshormonogenesis. Methods Two patients from unrelated, non-consanguineous families with impaired TG synthesis were investigated, both undergoing comprehensive clinical, biochemical, and imaging evaluations. Genetic testing included DNA sequencing, genotyping, and bioinformatics analyses. Main results Molecular analysis revealed two previously unreported TG variants—NM_003235.5:c.1375C>T (NP_003226.4:p.(Gln459Ter)) and NM_003235.5:c.5509_5518delAAAGACACAG (NP_003226.4:p.(Lys1837CysfsTer12))—alongside two known variants, NM_003235.5:c.378C>A (NP_003226.4:p.(Tyr126Ter)) and NM_003235.5:c.7880A>G (NP_003226.4:p.(Asp2627Gly)). A frameshift variant, combined with a clinically relevant missense variant in the ChEL domain (NP_003226.4:p.(Lys1837CysfsTer12)/ NP_003226.4:p.(Asp2627Gly)), was identified in one patient with congenital hypothyroidism (CH) who presented with normal serum TG levels. In contrast, the second patient, with classic goitrous CH and reduced serum TG levels, carried two nonsense variants introducing premature stop codons (NP_003226.4:p.(Tyr126Ter)/NP_003226.4(p.Gln459Ter)), resulting in truncated proteins and severely impaired hormone synthesis. The deleterious impact predicted by amino acid analysis software, together with strict evolutionary conservation and three-dimensional modeling of NP_003226.4:p.(Asp2627Gly) in the ChEL domain strongly supports a pivotal role of this residue in maintaining TG structural integrity. Conclusion The systematic exhaustive analysis of two novel variants, together with two previously reported TG variants, using molecular and bioinformatics tools, broadens the spectrum of deleterious TG variants and provides deeper insights into the etiology of CH.