Point mutations in the PDX1 transactivation domain impair human β-cell development and function (RNA-Seq). Point mutations in the PDX1 transactivation domain impair human β-cell development and function (RNA-Seq)

Missense mutations in coding region of PDX1 predispose to type-2 diabetes mellitus as well as cause MODY through largely unexplored mechanisms. Here, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying heterozygous missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/-). Using an in vitro β-cell differentiation protocol, we demonstrated that both PDX1P33T/+, PDX1C18R/+ and PDX1P33T/P33T, PDX1C18R/C18R mutations impair β-cell differentiation and function. Furthermore, PDX1+/- and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NEURONATIN, both involved in insulin synthesis and secretion. Our results reveal mechanistic details of how diabetes-associated PDX1 point mutations impair human pancreatic endocrine lineage formation and β-cell function and contribute to pre-disposition for diabetes. Overall design: We performed RNA-seq of control and isogenic PDX1 mutant cell lines at PP stage

PDX1编码区的错义突变易患2型糖尿病，同时可通过尚未完全阐明的机制引发青少年发病的成年型糖尿病（Maturity Onset Diabetes of the Young, MODY）。
本研究筛选了大规模糖尿病风险升高受试者队列，鉴定出2名糖耐量受损的个体，其PDX1编码区携带杂合错义突变，导致其反式激活结构域发生单个氨基酸替换（P33T、C18R）。
我们从携带杂合PDX1P33T/+、PDX1C18R/+突变的患者中诱导生成了诱导多能干细胞（induced pluripotent stem cells, iPSCs），并构建了携带纯合PDX1P33T/P33T、PDX1C18R/C18R突变以及杂合PDX1功能丧失突变（PDX1+/-）的同基因细胞系。
采用体外β细胞分化方案，我们证实PDX1P33T/+、PDX1C18R/+杂合突变与PDX1P33T/P33T、PDX1C18R/C18R纯合突变均会损伤β细胞的分化进程与功能。
进一步研究表明，PDX1+/-及PDX1P33T/P33T突变会降低胰腺祖细胞（pancreatic progenitors, PPs）的分化效率，其原因为PDX1结合基因的表达下调，这类基因包括转录因子MNX1、PDX1以及胰岛素抵抗基因CES1。
此外，在胰腺祖细胞中，PDX1P33T/+与PDX1P33T/P33T突变均会降低PDX1结合基因的表达水平，其中包括长链非编码RNA（long non-coding RNA）MEG3以及参与胰岛素合成与分泌的印记基因NEURONATIN。
本研究结果揭示了糖尿病相关PDX1点突变如何损伤人类胰腺内分泌谱系形成与β细胞功能，并增加糖尿病易感性的机制细节。
整体实验设计：我们对胰腺祖细胞阶段的对照组及同基因PDX1突变细胞系开展了RNA测序（RNA-seq）。