The new missense G376V-TDP-43 variant induces late-onset distal myopathy but not amyotrophic lateral sclerosis.

TDP-43-positive inclusions in neurons are a hallmark of several neurodegenerative diseases including familial amyotrophic lateral sclerosis (fALS) caused by pathogenic TARDBP variants as well as more common non-Mendelian sporadic ALS (sALS). Here we report a G376V-TDP-43 missense variant in the C-terminal prion-like domain of the protein in two French families affected by an autosomal dominant myopathy but not fulfilling diagnostic criteria for ALS. Patients from both families presented with progressive weakness and atrophy of distal muscles, starting in their 5th-7th decade. Muscle biopsies revealed a degenerative myopathy characterized by accumulation of rimmed (autophagic) vacuoles, disruption of sarcomere integrity and severe myofibrillar disorganization. The G376 V variant altered a highly conserved amino acid residue and was absent in databases on human genome variation. Variant pathogenicity was supported by in silico analyses and functional studies. The G376 V mutant increased the formation of cytoplasmic TDP-43 condensates in cell culture models, promoted assembly into high molecular weight oligomers and aggregates in vitro, and altered morphology of TDP-43 condensates arising from phase separation. Moreover, the variant led to the formation of cytoplasmic TDP-43 condensates in patient-derived myoblasts and induced abnormal mRNA splicing in patient muscle tissue. The identification of individuals with TDP-43-related myopathy but not ALS implies that TARDBP missense variants may have more pleiotropic effects than previously anticipated and support a primary role for TDP-43 in skeletal muscle pathophysiology. We propose to include TARDBP screening in the genetic work-up of patients with late-onset distal myopathy. Further research is warranted to examine the precise pathogenic mechanisms of TARDBP variants causing either a neurodegenerative or myopathic phenotype.

SLC35A2 deficiency reduces protein levels of core 1 ß-1,3-galactosyltransferase 1 (C1GalT1) and its chaperone Cosmc and affects their subcellular localization.

Nucleotide sugar transporters (NSTs) are multitransmembrane proteins, localized in the Golgi apparatus and/or endoplasmic reticulum, which provide glycosylation enzymes with their substrates. It has been demonstrated that NSTs may form complexes with functionally related glycosyltransferases, especially in the N-glycosylation pathway. However, potential interactions of NSTs with enzymes mediating the biosynthesis of mucin-type O-glycans have not been addressed to date. Here we report that UDP-galactose transporter (UGT; SLC35A2) associates with core 1 ß-1,3-galactosyltransferase 1 (C1GalT1; T-synthase). This provides the first example of an interaction between an enzyme that acts exclusively in the O-glycosylation pathway and an NST. We also found that SLC35A2 associated with the C1GalT1-specific chaperone Cosmc, and that the endogenous Cosmc was localized in both the endoplasmic reticulum and Golgi apparatus of wild-type HEK293T cells. Furthermore, in SLC35A2-deficient cells protein levels of C1GalT1 and Cosmc were decreased and their Golgi localization was less pronounced. Finally, we identified SLC35A2 as a novel molecular target for the antifungal agent itraconazole. Based on our findings we propose that NSTs may contribute to the stabilization of their interaction partners and help them to achieve target localization in the cell, most likely by facilitating their assembly into larger functional units.

Novel Insights into Selected Disease-Causing Mutations within the SLC35A1 Gene Encoding the CMP-Sialic Acid Transporter.

Congenital disorders of glycosylation (CDG) are a group of rare genetic and metabolic diseases caused by alterations in glycosylation pathways. Five patients bearing CDG-causing mutations in the SLC35A1 gene encoding the CMP-sialic acid transporter (CST) have been reported to date. In this study we examined how specific mutations in the SLC35A1 gene affect the protein's properties in two previously described SLC35A1-CDG cases: one caused by a substitution (Q101H) and another involving a compound heterozygous mutation (T156R/E196K). The effects of single mutations and the combination of T156R and E196K mutations on the CST's functionality was examined separately in CST-deficient HEK293T cells. As shown by microscopic studies, none of the CDG-causing mutations affected the protein's proper localization in the Golgi apparatus. Cellular glycophenotypes were characterized using lectins, structural assignment of N- and O-glycans and analysis of glycolipids. Single Q101H, T156R and E196K mutants were able to partially restore sialylation in CST-deficient cells, and the deleterious effect of a single T156R or E196K mutation on the CST functionality was strongly enhanced upon their combination. We also revealed differences in the ability of CST variants to form dimers. The results of this study improve our understanding of the molecular background of SLC35A1-CDG cases.