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Kidney Week

Abstract: TH-PO707

A Mutation Identified in Patients with Congenital Anomalies of the Kidney and Urinary Tract Interrupts ROBO2-SLIT2 Association and Signaling Pathway

Session Information

Category: Genetic Diseases of the Kidney

  • 1002 Genetic Diseases of the Kidney: Non-Cystic

Authors

  • Fan, Xueping, Boston University, Boston, Massachusetts, United States
  • Thao, Tou Sue, Boston University School of Medicine, Boston, Massachusetts, United States
  • Lu, Weining, Boston University Medical Center, Boston, Massachusetts, United States
Background

Congenital anomalies of the kidney and urinary tract (CAKUT) is a major cause of kidney failure in children and young adults. ROBO2 is a receptor for ligand SLIT2. ROBO2 and SLIT2 plays key roles in early ureteric bud outgrowth during kidney development and nervous system formation. There are approximately 30 ROBO2 missense mutations identified in CAKUT patients. However, functional significances of these mutations are remained to be characterized. To elucidate if these mutations would structurally disrupt ROBO2-SLIT2 interaction and functionally compromise its signaling pathway, we have analyzed a missense mutation of ROBO2, p.G98W (c.292G>T), located in the ROBO2 first Ig domain that serves as the ligand binding site for SLIT2.

Methods

Site-directed mutagenesis was used to introduce c.292G>T point mutation into a human ROBO2 cDNA. Calcium phosphate transfection strategy was used to express ROBO2 in HEK cells. Protein expression was confirmed by Western blot analysis and ROBO2-SLIT2 interaction was analyzed using the ForteBio Bio-Layer Interferometry (BLI) system Octet RED96e. Postnatal anterior subventricular zone (SVZa) neuronal migration assay was performed to examine the functional impact of the mutation on ROBO2-SLIT2 signaling pathway.

Results

BLI binding assay showed a positive interaction between ROBO2 and SLIT2, however, the p.G98W mutation in ROBO2 first Ig domain hinders this binding compared with its wild type control. Functional assay indicates that the extracellular portion of ROBO2, a 153 residue peptide containing the binding domain for SLIT2, is able to block ROBO2-SLIT2 signaling pathway that repels neuronal migration. In contrast, after the p.G98W mutation was introduced, the mutated ROBO2 peptide reduces this functional blocking activity on neuronal migration.

Conclusion

Our results provide molecular mechanistic evidence that p.G98W in ROBO2 first Ig domain interrupts the ROBO2-SLIT2 receptor-ligand association, which may in turn disrupt their signaling pathway during early kidney development and result in CAKUT phenotypes in patients carrying this mutation.

Funding

  • NIDDK Support