Copy number variation analysis identifies novel CAKUT candidate genes in children with a solitary functioning kidney

Abstract

Copy number variations associate with different developmental phenotypes and represent a major cause of congenital anomalies of the kidney and urinary tract (CAKUT). Because rare pathogenic copy number variations are often large and contain multiple genes, identification of the underlying genetic drivers has proven to be difficult. Here we studied the role of rare copy number variations in 80 patients from the KIMONO study cohort for which pathogenic mutations in three genes commonly implicated in CAKUT were excluded. In total, 13 known or novel genomic imbalances in 11 of 80 patients were absent or extremely rare in 23,362 population controls. To identify the most likely genetic drivers for the CAKUT phenotype underlying these rare copy number variations, we used a systematic in silico approach based on frequency in a large data set of controls, annotation with publicly available databases for developmental diseases, tolerance and haploinsufficiency scores, and gene expression profile in the developing kidney and urinary tract. Five novel candidate genes for CAKUT were identified that showed specific expression in the human and mouse developing urinary tract. Among these genes, DLG1 and KIF12 are likely novel susceptibility genes for CAKUT in humans. Thus, there is a significant role of genomic imbalance in the determination of kidney developmental phenotypes. Additionally, we defined a systematic strategy to identify genetic drivers underlying rare copy number variations.

Figure 1. From CNVs to candidate genes for CAKUT

All identified CNVs (N) were included in the analysis. For all novel, rare CNVs, deletions and duplications that showed significant overlap to pathogenic or uncertain pathogenic CNVs in public databases were included (see Supplementary Figure 1 and Supplementary Table 6). After annotation of gene content (n), genes that displayed rare truncating variants (deletions) and rare missense variants (duplications) in the Exome Variant Sever Database (http://evs.gs.washington.edu/EVS) were selected. We then assessed haploinsufficiency (HI-)LOD-scores and residual variation intolerance (RVI) scores for the prioritized genes (threshold values: HI LOD ≥2 and/or RVI-score <10^th percentile) and included the prioritized genes within single gene CNVs as well as those genes that are implicated in renal disease. One gene met >1 threshold value for inclusion (DLG1). Gene expression profiles in the developing mouse kidney for all high-priority genes were evaluated by using GUDMAP (http://www.gudmap.org) and Genepaint (http://www.genepaint.org/) databases. Finally, we performed immunofluorescence studies in an E14.5 mouse kidney. By using this systematic bioinformatic approach, we prioritized 5 candidate genes for CAKUT. CAKUT, congenital anomalies of the kidney and urinary tract; CNV, copy number variation and LOD, logarithm of the odds. Web-resources: Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources Consortium (DECIPHER; http://decipher.sanger.ac.uk/); International Standards For Cytogenomic Arrays Consortium (ISCA; https://www.iscaconsortium.org/).

Figure 2. Expression of KIF12 and DLG1 in the developing human kidney

Transversal section through lumbosacral part of human embryo (6^th week of development): a) within the nephrogenic zone (nz), KIF12 is weakly expressed in the developing nephron (renal vesicle – rv) and negative in the metanephric mesenchyme (mm). KIF12 is strongly expressed (arrows) in the UB stalk and UB-derived structures, such as the epithelium of collecting ducts (Cd), while the surrounding mesenchyme is negative; b) DAPI nuclear staining; c) merge of a and b; negative isotype control. d) DLG1 is weakly or not expressed in the developing nephron (renal vesicle – rv, metanephric cup - mc) and negative in the metanephric mesenchyme (mm), while it is moderately expressed (arrows) in the epithelium of collecting ducts (Cd); e) DAPI nuclear staining; f) merge of d and f; negative isotype control. Immunostaining of Kif12 and Dlg1, magnification ×40, scale bar 25Xm.