Abstract：
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.
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.
We identified one pathogenic MAML2 variant and two pathogenic MAMLD1 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 HES1 expression. The MAML2 variant exerted a dominant-negative effect on both the canonical pathway and thyroid hormone biosynthesis. MAMLD1 also regulated hormone biosynthesis through the expression of HES3, the target gene of the non-canonical pathway.
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.
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.
We identified one pathogenic MAML2 variant and two pathogenic MAMLD1 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 HES1 expression. The MAML2 variant exerted a dominant-negative effect on both the canonical pathway and thyroid hormone biosynthesis. MAMLD1 also regulated hormone biosynthesis through the expression of HES3, the target gene of the non-canonical pathway.
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.
摘要：
背景：在几个国家，在先天性甲状腺功能减退症（CH）患者中，甲状腺发育不良比甲状腺发育不良更常见。然而,已知的致病基因仅限于那些直接参与激素生物合成的基因。在许多患者中，甲状腺功能异常的病因和发病机理仍然未知。
方法：为了确定其他候选致病基因，我们在538例CH患者中进行了下一代测序，然后使用HEK293T和Nthy-ori3.1细胞在体外证实了鉴定基因的功能，并在体内使用斑马鱼和小鼠模型生物。
结果：我们在3例CH患者中发现了一个致病性MAML2变异体和两个致病性MAMLD1变异体，它们下调了典型Notch信号。斑马鱼和用N-[N-（3,5-二氟苯乙酰基）-1-丙氨酰]-S-苯基甘氨酸叔丁基酯治疗的小鼠，γ-分泌酶抑制剂表现出甲状腺功能减退和甲状腺功能异常的临床表现。通过原代小鼠甲状腺细胞的类器官培养和转录组测序，我们证明,甲状腺细胞内的Notch信号直接影响甲状腺激素的生物合成,而不是卵泡的形成.此外,这三个变异体阻断了甲状腺激素生物合成相关基因的表达,通过HES1表达恢复。MAML2变体对规范途径和甲状腺激素生物合成均具有显性负效应。MAMLD1还通过HES3的表达来调节激素的生物合成，HES3是非经典途径的靶基因。
结论：这项研究在CH中确定了三个类似于策划者的家族基因变异，并揭示了规范和非规范的Notch信号传导都会影响甲状腺激素的生物合成。
方法：为了确定其他候选致病基因，我们在538例CH患者中进行了下一代测序，然后使用HEK293T和Nthy-ori3.1细胞在体外证实了鉴定基因的功能，并在体内使用斑马鱼和小鼠模型生物。
结果：我们在3例CH患者中发现了一个致病性MAML2变异体和两个致病性MAMLD1变异体，它们下调了典型Notch信号。斑马鱼和用N-[N-（3,5-二氟苯乙酰基）-1-丙氨酰]-S-苯基甘氨酸叔丁基酯治疗的小鼠，γ-分泌酶抑制剂表现出甲状腺功能减退和甲状腺功能异常的临床表现。通过原代小鼠甲状腺细胞的类器官培养和转录组测序，我们证明,甲状腺细胞内的Notch信号直接影响甲状腺激素的生物合成,而不是卵泡的形成.此外,这三个变异体阻断了甲状腺激素生物合成相关基因的表达,通过HES1表达恢复。MAML2变体对规范途径和甲状腺激素生物合成均具有显性负效应。MAMLD1还通过HES3的表达来调节激素的生物合成，HES3是非经典途径的靶基因。
结论：这项研究在CH中确定了三个类似于策划者的家族基因变异，并揭示了规范和非规范的Notch信号传导都会影响甲状腺激素的生物合成。
