Functional study of ROBO2 missense mutation identified in patients with congenital anomalies of the kidney and urinary tract (CAKUT)
Thao, Tou Sue
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BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) is in the family of structural renal tract birth defects. CAKUT is the major cause of chronic kidney disease and renal failure in children and adults <40 years of age. ROBO2 is a receptor for the SLIT2 ligand. ROBO2/SLIT2 signaling has been shown to play important roles in neuronal migration and in early renal tract development. Our laboratory has recently identified ROBO2 as a novel CAKUT-causing gene. So far, total 26 ROBO2 mutations have been identified in patients with CAKUT. However, most of these mutations are missense amino acid substitutions and their functional significances are unclear, although they are predicted to be disease-causing by several bioinformatics prediction software. OBJECTIVE: To clarify uncertainties and confusions in the CAKUT field regarding the causality for ROBO2 missense mutations, we performed functional analysis of a ROBO2 missense mutation p.G114W (c.340G>T) that was identified in a CAKUT family. This p.G114W ROBO2 mutation is located in the first Ig domain of the ROBO2 extracellular region. The ROBO2 first Ig domain is the binding site for ligand SLIT2 that is required for ROBO2-SLIT2 signaling chemorepulsive activity in neuronal migration. We hypothesize that this p.G114W ROBO2 mutation would disrupt the SLIT2-ROBO2 binding and compromise its chemorepulsive activity in a sensitive functional neuronal migration assay. METHODS: Site directed mutagenesis was used to introduce c.340G>T point mutation into a ROBO2 cDNA fusion construct that contains the first Ig domain. Point mutation was verified using Sanger sequencing. Mutant ROBO2 cDNA and wildtype control constructs were purified using Qiagen Midiprep kit and transfected in HEK cells via calcium phosphate co-precipitation. The conditioned medium (CM) containing ROBO2 fusion proteins were analyzed by Western Blot. Neuronal migration assays were performed using postnatal anterior subventricular zone (SVZa) tissue explants that were isolated from postnatal day 1 to 5 (P1-5) Sprague-Dawley rat brain. RESULTS: By Sanger sequencing, we verified the c.340G>T point mutation in the ROBO2 cDNA fusion construct. By GFP fluorescence and Western blot analysis, we found abundant expressions of ROBO2 fusion protein in the conditioned medium of transfected HEK cells. In SVZa neuronal migration assays, we found that, when compared to the wild type fusion protein, the mutant ROBO2 fusion protein with the p.G114W amino acid substitution lost its function to block SLIT2-medicated inhibition of neuronal migration at both 50% conditioned medium and 100% conditioned medium concentrations. CONCLUSION: Our results show that ROBO2 p.G114W is a loss-of-function mutation disrupting normal SLIT2-ROBO2 chemorepulsive activity on SVZa neuronal migration, suggesting that the presence of this missense mutation compromises SLIT2-ROBO2 signaling and contributes to the development of CAKUT.