Congenital diarrheal disorders (CDD) comprise > 50 monogenic entities featuring chronic diarrhea of early-onset, including defects in nutrient and electrolyte absorption, enterocyte polarization, enteroendocrine cell differentiation, and epithelial integrity. Diarrhea is also a predominant symptom in many immunodeficiencies, congenital disorders of glycosylation, and in some defects of the vesicular sorting and transporting machinery. We set out to identify the etiology of an intractable diarrhea in 2 consanguineous families by whole-exome sequencing, and identified two novel AP1S1 mutations, c.269T>C (p.Leu90Pro) and c.346G>A (p.Glu116Lys). AP1S1 encodes the small subunit of the adaptor protein 1 complex (AP-1), which plays roles in clathrin coat-assembly and trafficking between trans-Golgi network, endosomes and the plasma membrane. An AP1S1 knock-out (KO) of a CaCo2 intestinal cell line was generated to characterize intestinal AP1S1 deficiency as well as identified mutations by stable expression in KO background. Morphology and prototype transporter protein distribution were comparable between parental and KO cells. We observed altered localization of tight-junction proteins ZO-1 and claudin 3, decreased transepithelial electrical resistance and an increased dextran permeability of the CaCo2-AP1S1-KO monolayer. In addition, lumen formation in 3D cultures of these cells was abnormal. Re-expression of wild-type AP1S1 in CaCo2-AP1S1-KO cells reverted these abnormalities, while expression of AP1S1 containing either missense mutation did not. Our data indicate that loss of AP1S1 function causes an intestinal epithelial barrier defect, and that AP1S1 mutations can cause a non-syndromic form of congenital diarrhea, whereas 2 reported truncating AP1S1 mutations caused MEDNIK syndrome, characterized by mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratodermia.

中文翻译：

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AP1S1错义突变通过上皮屏障缺陷引起先天性肠病。

先天性腹泻疾病（CDD）包括> 50种单基因实体，其特征是早发性慢性腹泻，包括营养和电解质吸收缺陷，肠细胞极化，肠内分泌细胞分化和上皮完整性。在许多免疫缺陷，先天性糖基化疾病以及囊泡分选和运输机械的某些缺陷中，腹泻也是主要症状。我们着手通过全外显子组测序鉴定2个近亲家族中顽固性腹泻的病因，并鉴定出两个新的AP1S1突变，即c.269T> C（p.Leu90Pro）和c.346G> A（p.Glu116Lys）。AP1S1编码衔接蛋白1复合物（AP-1）的一个小亚基，它在网格蛋白外套组装和反式高尔基体网络，内体和质膜之间的运输中发挥作用。生成CaCo2肠道细胞系的AP1S1敲除（KO），以表征肠道AP1S1缺乏以及通过在KO背景中稳定表达鉴定出的突变。亲代细胞和KO细胞之间的形态和原型转运蛋白分布相当。我们观察到紧密连接蛋白ZO-1和claudin 3的定位改变，跨上皮电阻降低和CaCo2-AP1S1-KO单层葡聚糖渗透性增加。另外，这些细胞的3D培养物中的管腔形成异常。在CaCo2-AP1S1-KO细胞中野生型AP1S1的重新表达可逆转这些异常，而含有任何一种错义突变的AP1S1的表达均不会。我们的数据表明，AP1S1功能丧失导致肠道上皮屏障缺陷，