Pathogenic variations in and contribute to congenital hypothyroidism due to dyshormonogenesis by regulating the Notch signalling pathway.

Background: In several countries, thyroid dyshormonogenesis is more common than thyroid dysgenesis in patients with congenital hypothyroidism (CH). However, known pathogenic genes are limited to those directly involved in hormone biosynthesis. The aetiology and pathogenesis of thyroid dyshormonogenesis remain unknown in many patients.

Methods: To identify additional candidate pathogenetic genes, we performed next-generation sequencing in 538 patients with CH and then confirmed the functions of the identified genes in vitro using HEK293T and Nthy-ori 3.1 cells, and in vivo using zebrafish and mouse model organisms.

Results: We identified one pathogenic variant and two pathogenic variants that downregulated canonical Notch signalling in three patients with CH. Zebrafish and mice treated with N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester, a γ-secretase inhibitor exhibited clinical manifestations of hypothyroidism and thyroid dyshormonogenesis. Through organoid culture of primary mouse thyroid cells and transcriptome sequencing, we demonstrated that Notch signalling within thyroid cells directly affects thyroid hormone biosynthesis rather than follicular formation. Additionally, these three variants blocked the expression of genes associated with thyroid hormone biosynthesis, which was restored by expression. The variant exerted a dominant-negative effect on both the canonical pathway and thyroid hormone biosynthesis. also regulated hormone biosynthesis through the expression of , the target gene of the non-canonical pathway.

Conclusions: This study identified three mastermind-like family gene variants in CH and revealed that both canonical and non-canonical Notch signalling affected thyroid hormone biosynthesis.