P0071MOLECULAR ALTERATIONS AND THE SEARCH FOR GENETIC MODIFIERS IN BRANCHIOOTORENAL SYNDROME

Abstract Background and Aims Branchiootorenal (BOR) syndrome (MIM# 113650) is an exceedingly rare disorder of autosomal-dominant inheritance that describes the combination of branchial anomalies, structurally defective ears with associated hearing loss, and congenital anomalies of the kidney and urinary tract (CAKUT). Pathogenic variants in the developmentally important gene EYA1 have been found to underlie most instances of BOR syndrome. Given the high inter- and intrafamilial variability of phenotypic traits in BOR syndrome, renal involvement varies significantly. However, distinct somatic, genetic, or environmental factors underlying this phenomenon remain elusive. Moreover, the precise role of EYA1 in renal development and pathogenesis of BOR related kidney disease remains ill-defined. Here, we sought to investigate molecular alterations as well as genetic modifiers of renal involvement in a family segregating a previously reported (Rodríguez-Soriano et al. Pediatr Nephrol 2001) heterozygous canonical splice site variant in EYA1 (c.1698 + 1G>A, NM_000503.5). Remarkably, structural kidney anomalies led to end-stage renal disease in a two-year old boy while his father exhibited normal renal function. Method For analysis of the splice site EYA1 variant, cDNA was obtained from primary patient fibroblasts. Immunofluorescence microscopy was employed to assess the intracellular distribution of EYA1 in both patient and control fibroblasts. Semiquantitative data on cellular EYA1 abundance were obtained from immunoblotting of whole protein extracts from patient and control fibroblast cultures. As for the investigation of genetic modifiers of renal involvement in BOR syndrome, identification of candidate genes was based upon whole exome sequencing data in all four family members (the parental couple and both affected children). Analysis of variants was restricted to de novo variants and those inherited from the non-affected mother. Results The splice site EYA1 variant that was assumed to be disease-causing turned out to be pathogenic. Mutant transcript variants result from exon skipping, leading to the translation of an aberrant C-terminus. This affects the highly conserved C-terminal EYA domain that is crucial for interaction with several transcription factors (e.g., from the SIX family). In line with these findings, our imaging data demonstrate a reduced nuclear enrichment of EYA1 in the mutant case, suggesting an impaired nuclear translocation process. Immunoblotting showed an increased EYA1 abundance in mutant fibroblasts, possibly indicating compensation of gene dose effects. Data from immunofluorescence microscopy and immunoblotting are preliminary. Finally, our search for genetic modifiers of renal involvement in BOR syndrome yielded a promising candidate variant in CYP51A1 in both affected children and their mother. CYP51A1 is an essential enzyme of cholesterol synthesis and has been shown to be indispensable for embryonic development. Conclusion Here, we sought to define the molecular basis of BOR syndrome in a family segregating a canonical splice site variant in EYA1. Analysis of the splice site demonstrates pathogenicity of this very variant. Further in vitro functional analyses show reduced nuclear localization of EYA1 in mutant fibroblasts, albeit we detected an increase in EYA1 abundance. Our findings are compatible with the notion that the interaction of mutant EYA1 with various transcription factors in the cytosol is impaired, preventing nuclear translocation of the protein complex. We propose that an increase in EYA1 abundance in mutant cells constitutes a compensatory mechanism. In searching for genetic modifiers of renal involvement in BOR syndrome, we identified a candidate variant in CYP51A1. Both CYP51A1 and EYA1 are developmentally important and concurrence of pathogenic variants in both genes may impair normal renal development.