Disease-Causing STAT3 Variants Can Be Discriminated by a Functional Flow Cytometry Test

BackgroundHeterozygous STAT3 protein variants acting by dominant-negative (DN), gain-of-function (GOF), or haploinsufficiency (HI) mechanisms have been associated with different clinical/immunological phenotypes. However, the biological impact of STAT3 protein variants is complex to determine, and it generally requires vector generation, transfection, and multiple iterations for definition.ObjectiveTo establish a single, reliable test to functionally characterize putative disease-causing STAT3 protein variants in patient primary cells.MethodsWe recruited patients carrying previously published/validated STAT3-DN (n = 13), STAT3-GOF (n = 4), and STAT3-HI (n = 5) variants. We functionally evaluated the IL-10-mediated/STAT3-dependent inhibition of LPS-induced TNF-a production by peripheral blood mononuclear cell (PBMC) monocytes using flow cytometry to assess the biological impact of STAT3 protein variants. Optimization and threshold definition were performed by bootstrapping and k-fold cross-validation of the receiver operating characteristic (ROC)-area under the curve (AUC).ResultsThe inhibition ratio of TNF-a production was significantly diminished in STAT3-DN (−3.44-fold, p < 0.0001) and STAT3-HI patient samples (−2.28-fold, p < 0.0001), while significantly augmented in STAT3-GOF patient samples (+1.47-fold, p = 0.002) when compared to healthy controls (HC, n = 16). Optimization of the combinations of IL-10 concentrations discriminated between HC from STAT3-DN (ROC-AUC = 1) and STAT3-GOF (ROC-AUC = 0.89). Further test optimization allowed discrimination of STAT3-HI variants when compared to HC (ROC-AUC = 1) and to STAT3-DN (ROC-AUC = 0.92).ConclusionDetermination of the IL-10-mediated, STAT3-dependent TNF-α inhibition after LPS stimulation ratio in PBMC monocytes using flow cytometry is a sensitive and specific method to functionally assess and discriminate STAT3-DN, -GOF, and -HI variants. This test can be used in research and clinical laboratory settings.