STAT1 gain-of-function (GOF) mutations leading to defective Th17 development are the most common genetic cause of chronic mucocutaneous candidiasis (CMC). We have identified enrichment of the STAT1 V266I variant of unknown significance (VUS) in our cohort of patients with Common Variable Immunodeficiency (CVID). We aim to define the functional impact of this mutation.
Cell lines expressing wild-type STAT1, STAT1-V266I and the described GOF CMC-associated mutation STAT1-A267V were generated. Levels of basal and post-IFN-α stimulus of phosphorylated STAT-1 (pSTAT-1) and total STAT-1 were measured by flow cytometry in cell lines with and without ruxolitinib and in primary cells from patients and controls (healthy, other CVID and known STAT1 GOF). In cell lines, STAT1 translocation to the nucleus was assessed by confocal microscopy. IFN-induced expression of the STAT-1-dependent cytokine, CXCL10, was evaluated in primary cells.
We demonstrate that the V266I STAT-1 VUS confers GOF as shown by increased levels of pSTAT1 and expression of CXCL10 compared with healthy and CVID disease controls. Similarly, cell lines expressing STAT1-V266I exhibited increased pSTAT1 and resistance to ruxolitinib treatment compared with wild-type STAT1 but less than seen in cells expressing STAT1-A267V. In comparison with STAT1-A267V, a clear defect of dephosphorylation was not seen in cell lines expressing STAT1-V266I but enhanced nuclear translocation after IFN-α stimulation was seen for both.
The V226I STAT1 VUS, enriched in our cohort of CVID patients, confers GOF suggesting contribution to disease susceptibility or pathogenesis.