Pathogenic Effect of <i>TP73</i> Gene Variants in People With Amyotrophic Lateral Sclerosis.

Abstract

To identify novel disease associated loci for amyotrophic lateral sclerosis (ALS), we used sequencing data and performed in vitro and in vivo experiments to demonstrate pathogenicity of mutations identified in <i>TP73</i>. We analyzed exome sequences of 87 patients with sporadic ALS and 324 controls, with confirmatory sequencing in independent ALS cohorts of >2,800 patients. For the top hit, <i>TP73</i>, a regulator of apoptosis and differentiation and a binding partner and homolog of the tumor suppressor gene <i>TP53</i>, we assayed mutation effects using in vitro and in vivo experiments. C2C12 myoblast differentiation assays, characterization of myotube appearance, and immunoprecipitation of p53-p73 complexes were performed in vitro. In vivo, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 targeting of zebrafish <i>tp73</i> to assay motor neuron number and axon morphology. Four heterozygous rare, nonsynonymous mutations in <i>TP73</i> were identified in our sporadic ALS cohort. In independent ALS cohorts, we identified an additional 19 rare, deleterious variants in <i>TP73</i>. Patient <i>TP73</i> mutations caused abnormal differentiation and increased apoptosis in the myoblast differentiation assay, with abnormal myotube appearance. Immunoprecipitation of mutant ΔN-p73 demonstrated that patient mutations hinder the ability of ΔN-p73 to bind p53. CRISPR/Cas9 knockout of <i>tp73</i> in zebrafish led to impaired motor neuron development and abnormal axonal morphology, concordant with ALS pathology. Together, these results strongly suggest that variants in <i>TP73</i> correlate with risk for ALS and indicate a role for apoptosis in ALS disease pathology.