Background: Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below 30 years of age. Many monogenic forms have been discovered mainly due to comprehensive genetic testing like exome sequencing (ES). However, disease-causing variants in known disease-associated genes still only explain a proportion of cases. Aim of this study was to unravel the underlying molecular mechanism of syndromic CAKUT in a multiplex Arab and a Turkish family with presumed autosomal recessive inheritance. Methods and Results: ES in the index individuals revealed two different rare homozygous variants in FOXD2, a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arab and a missense variant in the Turkish family with family segregation patterns consistent with autosomal-recessive inheritance. CRISPR/Cas9-derived Foxd2 knock-out (KO) mice presented with bilateral dilated renal pelvis accompanied by renal papilla atrophy while extrarenal features included mandibular, ophthalmologic, and behavioral anomalies, recapitulating the phenotype of humans with FOXD2 dysfunction. To study the pathomechanism of FOXD2-dysfunction-mediated developmental renal defects, in a complementary approach, we generated CRISPR/Cas9-mediated KO of Foxd2 in ureteric-bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important in renal/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a cell identity shift towards a stromal cell identity. Histology of Foxd2 KO mouse kidneys confirmed increased fibrosis. Further, GWAS data (genome-wide association studies) suggests that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Conclusions: In summary, our data implicate that FOXD2 dysfunction is a very rare cause of autosomal recessive syndromic CAKUT and suggest disturbances of the PAX2-WNT4 cell signaling axis contribute to this phenotype.

The authors have declared no competing interest.

This work was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) and the Technical University of Munich (TUM) in the framework of the Open Access Publishing Program and by the Scientific Research Projects Coordination Unit of Istanbul University Cerrahpasa (No TOA 2021 35349). JH received funding from the DFG (HO 2583 8 3). MS acknowledges funding via the Radboudumc Hypatia tenure track funding scheme and the ERC starting grant TREATCilia (grant agreement no. 716344), and YL, SH, MW, MS, AK, and SA acknowledge funding from the DFG Project ID 431984000, SFB 1453 and Excellence Initiative CIBSS, EXC 2189, Project ID 390939984. GMC was supported by the German Federal Ministry of Education and Research (Infrafrontier grant 01KX1012 to MHdA) and German Center for Diabetes Research (DZD) (MHdA). CB holds a part-time faculty appointment at the University of Freiburg in addition to his engagement with the Medizinische Genetik Mainz and his employment with the Limbach Group for which he heads and manages Limbach Genetics GmbH. His labs receive support from the DFG (BE 3910/8 1, BE 3910 9 1 and Collaborative Research Center SFB 1453 (Project ID: 431984000) and the Federal Ministry of Education and Research (BMBF, 01GM1903I and 01GM1903G). F.H. was supported by a grant from the National Institutes of Health (DK068306).

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