Experimental and Computational Analysis of Newly Identified Pathogenic Mutations in the Creatine Transporter SLC6A8

Abstract: Creatine is an essential metabolite for the storage and rapid supply of energy in muscle and nerve cells. In humans, impaired metabolism, transport, and distribution of creatine throughout tissues can cause varying forms of mental disability, also known as creatine deficiency syndrome (CDS). So far, 80 mutations in the creatine transporter (SLC6A8) have been associated to CDS. To better understand the effect of human genetic variants on the physiology of SLC6A8 and their possible impact on CDS, we studied 30 missense variants including 15 variants of unknown significance, two of which are reported here for the first time. We expressed these variants in HEK293 cells and explored their subcellular localization and transport activity. We also applied computational methods to predict variant effect and estimate site-specific changes in thermodynamic stability. To explore variants that might have a differential effect on the transporter’s conformers along the transport cycle, we constructed homology models of the inward facing, and outward facing conformations. In addition, we used mass-spectrometry to study proteins that interact with wild type SLC6A8 and five selected variants in HEK293 cells. In silico models of the protein complexes revealed how two variants impact the interaction interface of SLC6A8 with other proteins and how pathogenic variants lead to an enrichment of ER protein partners. Overall, our integrated analysis disambiguates the pathogenicity of 15 variants of unknown significance revealing diverse mechanisms of pathogenicity, including two previously unreported variants obtained from patients suffering from the creatine deficiency syndrome.

Parent Summary: This study investigates how specific genetic changes in the creatine transporter gene (SLC6A8) affect its function and contribute to creatine deficiency syndrome. Researchers examined 30 mutations, including 15 with previously unclear significance and two newly identified in patients. By testing these mutations in cells and using computational modeling, they assessed how each mutation affects transporter function, structure, and interaction with other proteins. Some mutations disrupted how the transported assembled or changed how it interacted with other proteins in cells. This study helps clarify which mutations affect the transporter enough to cause disease and shows that different mutations can disrupt the transporter through distinct mechanisms, improving our understanding of CTD and guiding future diagnostic and therapeutic efforts.

Link to article: https://www.sciencedirect.com/science/article/pii/S0022283623004941?via%3Dihub

PubMed:  https://pubmed.ncbi.nlm.nih.gov/38070861/

Authors: Evandro Ferrada, Tabea Wiedmer, Wen-An Wang, Fabian Frommelt, Barbara Steurer, Christoph Klimek, Sabrina Lindinger, Tanja Osthushenrich, Andrea Garofoli, Silvia Brocchetti, Samuel Bradberry, Jiahui Huang, Aidan MacNamara, Lia Scarabottolo, Gerhard F. Ecker, Anders Malarstig, and Giulio Superti-Furga

Key Terms: CTD, Mutation Study, In vitro, Basic Science, Clinical Study, Pediatric Patient, Male Patient