First large-scale screening of Notch biallelic variants implicates novel candidate genes in congenital hypothyroidism

Context: Congenital hypothyroidism (CH) is a major preventable cause of developmental and cognitive impairment. Current genetic testing identifies causative variants for only half of cases, leaving the majority without a molecular diagnosis. Although animal studies implicate Notch signaling in thyroid development, its role in human CH remains unexplored. Objective: To investigate whether Notch pathway variants contribute to genetically unexplained CH. Design: Genetic screening using whole-exome sequencing with zebrafish functional validation. Setting: Endocrinology outpatient clinic at Shanghai Ninth People's Hospital. Patients or other participants: 781 unrelated Chinese patients with CH. After identifying variants in 21 known causative genes in 364 patients, the remaining 417 unsolved cases were screened for variants in 77 Notch signaling pathway genes. Main outcome measure(s): Identification of biallelic Notch pathway variants; functional validation through zebrafish morpholino knockdown experiments assessing thyroid morphology and hormone production; clinical phenotype including thyroid function parameters, thyroid morphology, and levothyroxine dosage requirements; comparison with 267 DUOX2 patients. Results: We identified biallelic variants in 11 Notch pathway genes (NEURL1, CNTN6, NOTCH3, DTX1, DVL1, DTX2, ATXN1, SPEN, CTBP2, NCOR2, MAMLD1) in 21 patients. Zebrafish knockdown provided functional support for the potential pathogenic role of these genes, showing reduced thyroglobulin expression, abnormal morphology, elevated tsh levels, and decreased T4. Notch-variant patients showed higher initial FT4 but required significantly higher levothyroxine doses than DUOX2 patients. Conclusions: Notch pathway variants may contribute to approximately 5% of genetically unexplained CH. Variants across multiple pathway components collectively impair thyroid function. These findings suggest a potential expansion of CH genetic architecture and suggest that Notch-variant patients may require enhanced hormone replacement despite milder initial presentation, warranting pathway-specific screening.