Screening Variants of Unknown Significance to Identify Pathogenic Variants of the Manganese Transporter SLC39A8

Project Summary/Abstract Manganese (Mn) is a trace element essential for life. As a cofactor of many enzymes, Mn is crucial for many biological processes in humans. On the other hand, excessive Mn causes neurological disorders, including dystonia and parkinsonism. Mn transporters play a central role in maintaining Mn homeostasis by controlling Mn flux across plasma and organellar membranes. Among them, a plasma membrane transporter belonging to the Zrt/Irt-like protein (ZIP) family, SLC39A8 (or ZIP8), dominates Mn reabsorption from the bile (a major route for Mn excretion). Loss-of-function (LOF) mutations of slc39a8 lead to systemic Mn deficiency and is associated with a rare and potentially lethal genetic disorder, SLC39A8-Congenital Disorders of Glycosylation (SLC39A8- CDG), due to the abolished activity of Mn-dependent galactosyltransferases. Till now, more than three hundred slc39a8 mutations found in the human population have been documented, but whether these genetic variations are pathogenic or benign is an unresolved problem due to lack of experimental data and poor performance of computational approaches for functional prediction. We hypothesize that many variants of unknown significance (VUS) have defects in Mn transport and cellular trafficking. To test this hypothesis, we propose to apply an ICP- MS-based transport assay and flow cytometry-based trafficking analysis to screen most of the missense variants of SLC39A8. In Aim 1, we will identify the SLC39A8 variants with impaired Mn transport function by developing and applying an improved cell-based transport assay with greatly increased throughput to screen a library consisting of nearly 300 variants. In Aim 2, we will identify the SLC39A8 variants with defects in expression and/or trafficking to cell surface by applying the established flow cytometry-based analysis to the SLC39A8 variants with impaired Mn transport activity. The success of this project will provide critical evidence for improved risk prediction and early treatment of the SLC39A8-CDG patients. Functional characterization of hundreds of variants will also provide valuable data to elucidate the molecular basis of SLC39A8-CDG and to better understand the transport and cellular trafficking mechanisms of this understudied metal transporter.