STAT1 and STAT3 are transcription factors activated by cytokines such as IFN-γ and IL-6, respectively. Germline variants in these genes are associated with distinct immune dysregulation syndromes, and functional validation is essential for variant interpretation.
We report two Argentinean pediatric patients with novel variants in STAT1 and STAT3. Patient 1, a 10-year-old male with chronic mucocutaneous candidiasis, persistent diarrhea, eczema, recurrent respiratory infections, failure to thrive, and oral candidiasis, carried a heterozygous STAT1 variant c.874G>A(p.Asp292Asn). Patient 2, a 12-year-old male, presented with splenomegaly, multiple adenopathies, leukopenia, thrombocytopenia, eosinophilia, hypogammaglobulinemia, and bone marrow hypoplasia. A lymph node biopsy showed reactive follicular hyperplasia. Genetic analysis revealed a heterozygous STAT3 variant c.1044A>C (p.Lys348Asn). Both variants arose de novo as confirmed by parental testing.
We used site-directed mutagenesis to introduce novel variants into FLAG-tagged STAT1 and STAT3 expression plasmids. HEK293T cells were transfected with WT or mutant constructs. STAT1 activity was assessed using a luciferase reporter activated by IFN-γ and STAT3 activity was measured via a STAT3-responsive GFP reporter upon IL-6 stimulation. Additionally, STAT1 phosphorylation levels upon IFN-γ stimulation were assessed on monocytes by flow cytometry.
The STAT1 p.Asp292Asn variant phosphorylation in patient monocytes was increased to approximately twice the level observed in healthy controls, confirming a gain-of-function (GOF) effect. The STAT3 p.Lys348Asn variant showed increased GFP reporter activity following IL-6 stimulation, suggesting a GOF effect. These functional hyperactivations were compatible with immune dysregulation phenotypes.
Our findings highlight the pathogenic role of the STAT1 and STAT3 variants in immune dysregulation syndromes. We were able to establish that both variants confer a GOF effect. Functional assays are critical for interpreting variants of uncertain significance and contribute to precise diagnosis and tailored therapeutic strategies in patients with suspected monogenic immune diseases.
