Functional Assessment of a Novel ZAP70 Gene Mutation

Abstract

Zeta chain associated protein kinase 70 (ZAP-70) is a critical mediator of T cell receptor (TCR) signaling required for thymic selection and peripheral T cell activation. Defects in ZAP-70 result in combined immunodeficiency characterized by impaired T cell function and increased susceptibility to infections. This study investigates a pediatric patient presenting with recurrent infections and carrying a novel homozygous ZAP70 variant (c.1525G>A; p.Glu509Lys), classified as a variant of uncertain significance. To assess the functional impact of this variant, a comprehensive immunological analysis was performed, including flow cytometric immunophenotyping and T cell activation assays. Immunophenotyping revealed presence of a small population of CD8⁺ T cells, an unexpected finding in the context of classical ZAP-70 deficiency. Functional assays demonstrated impaired upregulation of activation markers and T cell proliferation following TCR stimulation, indicating defective T cell activation. These findings support a presumed hypomorphic effect of the identified variant, resulting in residual TCR signaling rather than complete loss of function. This study underscores the importance of functional validation in the interpretation of novel variants and expands the phenotypic spectrum of ZAP-70 deficiency.