The role of PREPL in Hypotonia-Cystinuria syndrome; Genetic and immunocytochemical studies

Abstract

Hypotonia Cystinuria Syndrome is caused by recessive loss of SLC3A1 and PREPL. HCS patients exhibit cystinuria type I, neonatal hypotonia, and growth hormone deficiency. Previous study identified PREPL as an effector of AP-1 which is involved in vesicular trafficking, and loss of PREPL decreases vesicles secretion. We hypothesized PREPL mediates regulated secretion by interracting with AP-1. We attempt to investigate the pathophysiology of PREPL deficiency through immunocytochemical (ICC) and genetical studies. We optimized B01 anti-PREPL against human and mouse PREPL, and E9 anti-PREPL against human PREPL. Both antibodies were optimized by ICC and western blot. To monitor the effect of PREPL in vesicles trafficking by live cell imaging, we developed shRNA construct that targets mouse PREPL and confirmed phogrin-pHluorin expression by signals at 90 kDa in western blot. We also confirmed that loss of PREPL accumulates AP-1 in TGN, while the presence of PREPL induces AP-1 dispersal in the cytoplasm. In the genetics study, 8 patients were screened for novel deletions in PREPL, SLC3A1 and/or C2Orf34. Four were found to have deletion B, while the rest of the patients appear to have novel deletions. To conclude, we optimized antibodies against PREPL, and shRNA and phogrin-pHluorin construct to be used for further study. The data that PREPL causes AP-1 redistribution in TGN supports our hypothesis that PREPL is involved in vesicles secretion. The genetics study expanded the prevalence of HCS deletion in northern Europe.