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

膀胱外翻-尿道上裂复合体 (BEEC) 疾病基因的鉴定和表征

• 批准号: 228821268
• 负责人: Dr. Phillip Grote
• 金额: --
• 依托单位: Georg-Speyer-Haus, Institut für Tumorbiologie und experimentelle Therapie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/228821268?language=en
• 关键词: Identification; characterization; disease; genes; bladder

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.

膀胱外翻-尿道上裂综合征（BEEC）代表了人类先天性肾脏和泌尿道异常的严重结局。严重程度谱包括最轻的形式，尿道上裂;中间和最常见的缺陷形式，经典膀胱外翻（CBE）;和最严重的形式，泄殖腔外翻（CE）。大约98%的患者被归类为非综合征型。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。我们之前在208例CBE患者中进行了全基因组关联研究（GWAS），发现了与ISL 1的全基因组关联。我们还在8例CE患者中采用了全外显子组测序（WES）。我们在SLC 20 A1中发现了一种新的从头突变，该基因在小鼠胚胎中被敲除时类似于人类CE表型。对406名BEEC患者的SLC 20 A1进行桑格测序，发现两名不相关的CBE患者中存在另外两种新的变异。一个是从头发生的，另一个是遗传自一个轻度受影响的母亲与她的联合退化。在早期发育阶段的斑马鱼幼虫（zfl）的isl 1和slc 20 a1的整体安装RNA原位杂交（WISH）分析检测到isl 1和slc 20 a1 a的表达接近近端区域的发展原肾提供进一步的证据，这两个基因调节跨物种的尿路发育。在zfl中的Slc 20 a1 a吗啉代敲低（MO）实验显示其直肠的加宽和裂开，对应于人类CE/CBE中的开放膀胱板。对于所提出的研究项目，可用的BEEC患者样本包括超过900名患者，代表了全球可用于遗传研究的最大BEEC患者样本。该项目的目的是（i）通过纳入额外的557名CBE患者和至少2，817名种族匹配的对照来扩展我们先前的GWAS，以确定额外的风险基因座，（ii）通过胚胎期E10.5 - E15.5期间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）跟踪slc 20 a1 a和isll在zfl中的精确表达和功能表征，进行进一步的WISH分析，部分地结合MO敲低及其拯救实验以及瞬时和/或CRISPR/Cas9介导的突变侧-结合和表达分析，特别是在zfl中的isl 1基因座内，以及（iv）在另外40个CE病例-亲本三人组中继续WES分析，以鉴定另外的罕见孟德尔疾病基因。进一步的风险基因座和突变及其相应的基因和细胞通路的鉴定将在阐明BEEC的病理生理学方面做出重要贡献。关于BEEC的细胞原因的知识可能最终导致识别基因-环境相互作用，这可能允许在未来的胚胎BEEC脆弱时期防止这些相互作用。

项目成果

Candidate gene association study implicates p63 in the etiology of nonsyndromic bladder-exstrophy-epispadias complex.

• DOI: 10.1002/bdra.23161
• 发表时间: 2013-12
• 期刊: Birth defects research. Part A, Clinical and molecular teratology
• 作者: L. Qi;mei Wang;Garima Yagnik;M. Mattheisen;J. Gearhart;Y. Lakshmanan;A. Ebert;W. Rösch;M. Ludwig;Markus Draaken;H. Reutter;S. Boyadjiev

Bladder exstrophy-epispadias complex and triple-X syndrome: incidental finding or causality?

膀胱外翻-尿道上裂复合体和 Triple-X 综合征：偶然发现还是因果关系？

• DOI: 10.1002/bdra.23299
• 发表时间: 2014
• 期刊: Birth defects research. Part A, Clinical and molecular teratology
• 作者: Ramaekers P;Loeys B;von Lowtzow C;Reutter H;Jacquemyn Y;Leroy Y;Colpaert C;Parizel M
• 通讯作者: Parizel M