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

• Applicants: Dr. Phillip Grote; Privatdozent Dr. Michael Knapp; Professor Dr. Benjamin Odermatt; Professor Dr. Heiko Reutter
• Subject Area: Human Genetics
• Term: from 2012 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 228821268

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.

• DFG Programme: Research Grants