Pathogenicity Classification of TARDBP Variants of Uncertain Significance: An Integrative Clinical Characterization and Functional Validation.

TAR DNA binding protein (TARDBP) is one of the major causative genes of amyotrophic lateral sclerosis (ALS), which drives disease progression through both gain-of-toxicity (GOT) and loss-of-function (LOF) mechanisms. The mutant TDP-43 exhibits aberrant nucleocytoplasmic distribution and forms cytotoxic hyperphosphorylated aggregates, a process that can be robustly recapitulated in vitro. Thus, functional assays in cell lines serve as a reliable metric for the pathogenicity classification of TARDBP variants. In this study, we performed in vitro experiments to classify the pathogenicity of 28 TARDBP variants of uncertain significance (VUS) among the 172 previously reported TARDBP variants. 22 of these VUS were determined to be functionally abnormal, of which 12 could be further classified as likely pathogenic (LP) variants according to American College of Medical Genetics (ACMG) and the ClinGen Sequence Variant Interpretation (SVI) Working Group guidelines. We also summarized the clinical characteristics of 35 ALS patients carrying 12 variants in the TARDBP gene. Pathogenic missense variants were predominantly clustered in the C-terminal domain (CTD) of TARDBP. Variants in TARDBP exon 6 may lead to an earlier age at onset. ALS caused by TARDBP mutations exhibits marked phenotypic heterogeneity, along with incomplete penetrance in carriers. Patient-derived primary skin fibroblasts serve as a feasible cellular model for the functional assessment of variant pathogenicity. Our findings expand the TARDBP mutation spectrum, and provides a preliminary basis for preclinical research on TARDBP-targeted therapies for ALS.