A ONECUT1 regulatory, non-coding region in pancreatic development and diabetes [RNA-seq]

In a patient with permanent neonatal syndromic diabetes clinically similar to cases with ONECUT1 biallelic mutations, we identified a disease-causing deletion located upstream of ONECUT1. Through genetic, genomic and functional studies we identified a crucial regulatory region acting as an enhancer of ONECUT1 specifically during pancreatic development. This enhancer region contains a low-frequency variant showing strong association with type 2 diabetes and other glycemic traits, thus extending the contribution of this region to common forms of diabetes. Clinical relevance is provided by experimentally tailored therapy options for patients carrying ONECUT1 coding or regulatory mutations. Overall design: RNA-sequencing (RNA-seq) of pancreatic progenitors (PP) differentiated from hESCs (HUES8). The following genotypes were generated with CRISPR/Cas9 gene editing and used for RNA-seq: Wildtype (WT), ONECUT1 knockouts (heterozygous and homozygous), 108 kb deletion of Patient 1 (Pat1-del; heterozygous and homozygous), heterozygous Pat1-del in heterozygous ONECUT1 deleted clones (orientation of deletions in cis or trans).

针对1例临床表型与ONECUT1基因（ONECUT1）双等位基因突变病例相似的永久新生儿综合征性糖尿病患者，我们鉴定出了位于ONECUT1基因上游的致病缺失变异。通过遗传、基因组学与功能实验研究，我们发现了一个关键调控区域，其在胰腺发育过程中特异性充当ONECUT1基因的增强子。该增强子区域包含一个低频变异位点，该位点与2型糖尿病及其他血糖性状存在强关联，由此将该区域的致病关联范围拓展至常见型糖尿病。针对携带ONECUT1基因编码区或调控区突变的患者，经实验验证的个体化治疗方案进一步明确了本研究的临床相关性。
实验整体设计：对由人胚胎干细胞（human embryonic stem cells, hESCs，HUES8株）分化得到的胰腺祖细胞（pancreatic progenitors, PP）进行RNA测序（RNA-sequencing, RNA-seq）。通过CRISPR/Cas9基因编辑构建了以下基因型样本并用于本次RNA-seq实验：野生型（Wildtype, WT）、ONECUT1基因敲除型（杂合与纯合）、患者1的108kb缺失型（Pat1-del；杂合与纯合），以及同时携带杂合ONECUT1基因缺失与杂合Pat1-del的克隆（需明确缺失的顺式/反式取向）。