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ObjectiveTo 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 inTP73.MethodsWe 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,TP73, a regulator of apoptosis and differentiation and a binding partner and homolog of the tumor suppressor geneTP53, 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 zebrafishtp73to assay motor neuron number and axon morphology.ResultsFour heterozygous rare, nonsynonymous mutations inTP73were identified in our sporadic ALS cohort. In independent ALS cohorts, we identified an additional 19 rare, deleterious variants inTP73. PatientTP73mutations 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 oftp73in zebrafish led to impaired motor neuron development and abnormal axonal morphology, concordant with ALS pathology.ConclusionTogether, these results strongly suggest that variants inTP73correlate with risk for ALS and indicate a role for apoptosis in ALS disease pathology.

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.