Loss-of-function variants in the CAPN1 activator CD99L2 cause X-linked spastic ataxia

Most patients with a rare movement disorder (MD) do not receive a molecular diagnosis, and the underlying genetic variants and mediating genes remain elusive. Here, we evaluate the diagnostic accuracy of conventional and next-generation sequencing-based genetic testing strategies in a cohort of 2,811 individuals with ataxia, spastic paraplegia and dystonia. Exome sequencing establishes genetic diagnoses in 19.3% of cases, and specificity of phenotypic features and age at testing are positive predictors. Genome analysis 'beyond the exome' increases the diagnostic yield by 7.5%, mostly due to the improved detection of structural variants and repeat expansions. Unsolved cases are included in the Solve-RD cohort and subjected to gene-burden analysis, providing evidence for loss-of-function variants in X-chromosomal CD99L2 causing spastic ataxia. Cellular studies show that the transmembrane protein CD99L2 occurs mainly in a ubiquitinated form and serves as an activating interactor of the calcium-dependent protease CAPN1. Ablation of cytoplasmic or extracellular domains of CD99L2 leads to its intracellular mislocalization and abrogation of its interplay with CAPN1. Transcriptome analysis in CD99L2 patient-derived fibroblasts reveals synaptic function-specific disturbances. Impaired CAPN1 activation and dysregulation of downstream neuronal pathways constitute the likely molecular cause for neurodegeneration.