A molecular mechanism of intrahepatic cholestasis in osteo-oto-hepato-enteric syndrome

BACKGROUND: Intrahepatic cholestasis is a hallmark of the rare osteo-oto-hepato-enteric (O2HE) syndrome. O2HE is caused by pathogenic variants in UNC45A. UNC45A encodes a co-chaperone that coordinates the proper folding and function of target proteins. The molecular basis of cholestasis in O2HE has not been demonstrated. Notably, the clinical phenotype overlaps with other familial cholestatic disorders involving impaired localization of the bile salt export pump (BSEP/ABCB11). METHODS: To investigate a mechanistic link between UNC45A dysfunction and BSEP localization, we used immunofluorescence microscopy on liver biopsies from patients, CRISPR-Cas9-engineered male hepatocyte cell lines, site-directed mutagenesis and co-immunoprecipitation assays. We focused on the recurrent c.292C>T/p.(Arg98Trp) variant. RESULTS: Liver tissue of a patient carrying the UNC45A-c.292C>T/p.(Arg98Trp) variant displayed a zonated canalicular BSEP expression defect. MYO5B, a known regulator of canalicular trafficking, was identified as an UNC45A client. UNC45A-p.(Arg98Trp) caused BSEP to accumulate in MYO5B+/RAB11A+ endosomes that failed to reach the canalicular membrane. Mutant UNC45A showed a weakened interaction with HSP70 chaperones. Of these, HSPA1A showed a zonated expression in the liver, inversely correlating with the zonated BSEP defect in the patient. Paracetamol-mediated increase in HSPA1A abundance restored MYO5B+/RAB11A+ endosome positioning and canalicular BSEP localization. CONCLUSION: A weakened UNC45A-p.(Arg98Trp)-HSP70 interaction disrupts BSEP trafficking through impaired MYO5B function and zonated HSPA1A availability. These results connect O2HE with MYO5B-associated cholestasis and suggests HSP70-targeted therapies to restore canalicular BSEP expression.