Pathogenic effect of TP73 Gene Variants in People With Amyotrophic Lateral Sclerosis.

OBJECTIVE: To identify novel disease associated loci for amyotrophic lateral sclerosis (ALS), we utilized sequencing data and performed in vitro and in vivo experiments to demonstrate pathogenicity of mutations identified in TP73. METHODS: We analyzed exome sequences of 87 sporadic ALS patients and 324 controls, with confirmatory sequencing in independent ALS cohorts of >2,800 patients. For the top hit, TP73, a regulator of apoptosis, differentiation, and a binding partner as well as homolog of the tumor suppressor gene TP53, 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 perform in vitro. In vivo, we used CRISPR/Cas9 targeting of zebrafish tp73 to assay motor neuron number and axon morphology. RESULTS: Five heterozygous rare, nonsynonymous mutations in TP73 were identified in our sporadic ALS cohort. In independent ALS cohorts, we identified an additional 19 rare, deleterious variants in TP73. Patient TP73 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 ΔN-p73's ability to bind p53. CRISPR/Cas9 knockout of tp73 in zebrafish led to impaired motor neuron development and abnormal axonal morphology, concordant with ALS pathology. CONCLUSION: Together, these results strongly suggest that variants in TP73 correlate with risk for ALS and indicate a novel role for apoptosis in ALS disease pathology.

Purification of Glycosylphosphatidylinositol-Anchored Mucins from Trypanosoma cruzi Trypomastigotes and Synthesis of α-Gal-Containing Neoglycoproteins: Application as Biomarkers for Reliable Diagnosis and Early Assessment of Chemotherapeutic Outcomes of Chagas Disease.

Chagas disease (ChD), caused by the protozoan parasite Trypanosoma cruzi, affects millions of people worldwide. Chemotherapy is restricted to two drugs, which are partially effective and may cause severe side effects, leading to cessation of treatment in a significant number of patients. Currently, there are no biomarkers to assess therapeutic efficacy of these drugs in the chronic stage. Moreover, no preventive or therapeutic vaccines are available. In this chapter, we describe the purification of Trypanosoma cruzi trypomastigote-derived glycosylphosphatidylinositol (GPI)-anchored mucins (tGPI-mucins) for their use as antigens for the reliable primary or confirmatory diagnosis and as prognostic biomarkers for early assessment of cure following ChD chemotherapy. We also describe, as an example, the synthesis of a potential tGPI-mucin-derived α-Gal-terminating glycan and its coupling to a carrier protein for use as diagnostic and prognostic biomarker in ChD.