Development and validation of a method for assessing STAT1 protein phosphorylation as a diagnostic tool for patients with defects in the conforming gene
AbstractIntroduction. Autosomal dominant STAT1-GOF (gain-of-function) pathogenic variants lead to combined immunodeficiency with CMC and autoimmune disorders. Heterozygous STAT1-GOF-mutation caused an enhanced activation with delayed STAT1 dephosphorylation. STAT1 depending cytokines affect negative regulation of gene transcription in naïve T cells, resulting in maturation of CD4+ lymphocytes into Th1 phenotype rather than Th17 phenotype. Besides that there are autosomal-dominant and autosomal-recessive variants which lead to loss of a STAT1 protein function (loss-of-function, LOF). Next-generation sequencing (NGS) has facilitated detection of novel variants. However, the biological relevance of novel mutations is often not clear and must be confirmed by immunological analysis.
The aim of this study was to develop and validate a method for assessing STAT1 protein phosphorylation.
Material and methods. The study included 17 patients with STAT1 pathogenic variants (STAT1-GOF n = 16 and STAT1-LOF n = 1), 7 patients with the variants of uncertain significance in the gene STAT1 and a cohort of healthy donors (n = 50). After the mononuclear fraction isolation, the cells were stimulated and STAT1 phosphorylation was evaluated by flow cytometry.
Results. We obtained reference values by comparing a cohort of STAT1-GOF patients with a healthy donors. The references were used to assess the variants of uncertain significance in the gene STAT1.
Conclusion. The method turned out to be sensitive and specific for detecting STAT1-GOF and STAT1-LOF genetic variants.
Keywords:STAT1; GOF; LOF; Inborn Errors of Immunity (IEI); flow cytometry; candidiasis; Th17-lymphocytes
For citation: Fadeeva M.S., Sozonova T.A., Bogdanova D.V., Alexenko M.Yu., Kulakovskaya E.A., Vedmedskaya V. A., Lodoeva O.B., Rodina Yu.A., Shcherbina A.Yu., Pershin D.E. Development and validation of a method for assessing STAT1 protein phosphorylation as a diagnostic tool for patients with defects in the conforming gene. Immunologiya. 2024; 45 (3): 329–42. DOI: https://doi.org/10.33029/1816-2134-2024-45-3-329-342 (in Russian)
Funding. The study had no sponsor support.
Conflict of interests. Authors declare no conflict of interests.
Authors’ contribution. Authors contributed equally to the writing of the article.
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