Identification and characterization of disease genes for the bladder exstrophy-epispadias complex (BEEC)

膀胱外翻-尿道外翻复合（BEEC）代表了人类先天性肾脏和尿道异常的严重结局。严重程度包括最温和的形式，上睑肌；中间和最常见的缺陷形式，经典膀胱外翻（CBE）；最严重的形式是泄殖腔外翻（CE）。大约98%的患者被归类为无综合征。在大多数患者中，遗传基础似乎是多因素的，涉及遗传和环境风险因素。我们之前对208名CBE患者进行了全基因组关联研究（GWAS），发现ISL1与全基因组相关。我们还对8例CE患者进行了全外显子组测序（WES）。我们在SLC20A1中发现了一种新的新生突变，这种基因在小鼠胚胎中被敲除后类似于人类CE表型。406例BEEC患者SLC20A1的Sanger测序显示，在两名无关的CBE患者中，还有另外两种新的变异。一个是从头发生的，另一个是遗传自一个轻度患病的母亲，她的联合关节转移。通过对斑马鱼幼体（zfl）发育早期isl1和slc20a1a的全载体RNA原位杂交（WISH）分析，发现isl1和slc20a1a在发育中的原肾近端附近表达，为这两个基因在物种间调节尿路发育提供了进一步的证据。Slc20a1a morpholino -敲除（MO）实验显示，zfl的直肠膜变宽和开裂，对应于人CE/CBE的开放膀胱板。对于拟议的研究项目，现有的BEEC患者样本包括900多名患者，这是全球基因研究中最大的BEEC患者样本。该项目的目标是(i)通过纳入额外的557名CBE患者和至少2,817名种族匹配的对照组来扩展我们之前的GWAS，以确定额外的风险位点；（ii）通过对胚胎阶段E10.5 - E15.5的CBE相关泌尿生殖组织的小鼠转录组分析来补充我们的GWAS数据集。（iii）随访slc20a1a和isl1在zfl中的精确表达和功能特征，进行进一步的WISH分析，部分结合MO敲除及其挽救实验，以及瞬时和/或CRISPR/Cas9介导的突变侧结合和表达分析，特别是在zfl中的isl1位点内。（iv）在另外40例CE病例-亲本三人组中继续进行WES分析，以确定其他罕见的孟德尔病基因。进一步发现危险位点和突变及其对应的基因和细胞通路，将对阐明BEEC的病理生理学做出重要贡献。了解BEEC的细胞原因可能最终导致基因-环境相互作用的鉴定，从而可能在未来的胚胎BEEC易感时期防止这些相互作用。

The bladder exstrophy-epispadias complex (BEEC) represents the severe end of human congenital anomalies of the kidney and urinary tract. The severity-spectrum comprises the mildest form, epispadias; the intermediate and most common defect form, classic bladder exstrophy (CBE); and the most severe form, cloacal exstrophy (CE). About 98% of all patients are classified as nonsyndromic. In the majority of these patients the genetic basis appears to be multifactorial involving genetic and environmental risk factors. We previously employed genome-wide association studies (GWAS) in 208 CBE patients and found genome-wide association with ISL1. We also employed whole exome sequencing (WES) in eight CE patients. We identified a novel de novo mutation in SLC20A1, a gene that when knocked out in a mouse an embryo resembles the human CE phenotype. Sanger sequencing of SLC20A1 in 406 BEEC patients revealed two additional novel variants in two unrelated CBE patients. One occurred de novo the other was inherited from a mildly affected mother with diastasis of her symphysis. Whole mount RNA in situ hybridization (WISH) analysis of isl1 and slc20a1 in early developmental stages of zebrafish larvae (zfl) detected isl1 and slc20a1a expression close to the proximal region of the developing pronephros providing further evidence for both genes to regulate urinary tract development across species. Slc20a1a Morpholino-knock-down (MO) experiments in zfl showed widening and clefting of their proctodeum corresponding to an open bladder plate in human CE/CBE.For the proposed research project the available BEEC patient sample comprises over 900 patients representing the largest BEEC patient sample available for genetic studies worldwide. The aims of the proposed project are (i) the extension of our previous GWAS by including additional 557 CBE patients and at least 2,817 ethnically matched controls to identify additional risk loci, (ii) complementation of our GWAS data set by murine transcriptome analysis of CBE-relevant urogenital tissue during embryonic stages E10.5 - E15.5, (iii) follow up with the precise expression and functional characterization of slc20a1a and isl1 in zfl performing further WISH analysis partly combined with MO knock down and its rescue experiments as well as transient and/or CRISPR/Cas9 mediated mutation-side-binding and expression analysis especially within the isl1 locus in zfl and (iv) continuation with WES analysis in additional 40 CE case-parent trios, in order to identify additional rare mendelian disease genes. The identification of further risk loci and mutations and their corresponding genes and cellular pathways will make an important contribution in the elucidation of the pathophysiology of the BEEC. Knowledge about the cellular causes of the BEEC might ultimately lead to the identification of gene-environmental interactions that might allow preventing these interactions during the embryonic BEEC vulnerable time-period in the future.