Novel Compound Heterozygous STK4 Variants Associated with Combined Immunodeficiency, Impaired Thymic Output, and Altered FOXO1-BACH2 Signaling

Abstract

Serine/threonine kinase 4 (STK4) deficiency is a rare combined immunodeficiency (CID). This study aims to analyze the clinical and immunological characteristics of a child carrying novel compound heterozygous STK4 variants and to explore potential alterations in FOXO1-BACH2 signaling associated with impaired T-cell development and regulatory T cell (Treg) homeostasis. We collected the patient’s clinical data and performed genetic diagnosis using whole-exome sequencing followed by Sanger validation. T cell receptor (TCR) repertoire diversity was analyzed, and peripheral blood lymphocyte subsets and their functions were comprehensively evaluated via flow cytometry. Single-cell RNA sequencing was used to identify transcriptional alterations in major immune-cell populations. We then used Stk4 global knockout mice and an STK4 -knockdown cellular model, together with Western blotting, quantitative real-time PCR, and chromatin immunoprecipitation-qPCR, to examine the potential relationship among STK4 deficiency, FOXO1-related signaling, BACH2 expression, and Treg-associated immune alterations. The patient carried compound heterozygous STK4 variants (c.733C > T, and c.771dupT) and presented with a CID phenotype, characterized by recurrent infections, naïve T-cell lymphopenia, reduced recent thymic emigrants, restricted TCR repertoire diversity, and altered Treg homeostasis. BACH2 expression was reduced in the patient and in STK4-deficient experimental models, suggesting a potential regulatory association between STK4 deficiency and BACH2 downregulation. ChIP-qPCR in total thymocytes showed FOXO1 enrichment at the Bach2 promoter region, supporting a potential FOXO1-BACH2 transcriptional relationship. This study identifies novel pathogenic STK4 variants and expands the critical and immunological spectrum of STK4 deficiency. The most prominent immunological features were impaired thymic output, naïve T-cell depletion, and restricted TCR repertoire diversity. Altered FOXO1-BACH2 signaling may contribute, at least in part, to impaired Treg homeostasis.