PMC6503099_01
Inheritance and functional impact of STAT1 variant. (A) T437N family pedigree. (B) Immunoblot assay to assess phosphorylation of STAT1 in T437N as well as T437I (known GOF), R274Q (known GOF) and Y701C (LOF). (C) Transiently expressed WT or mutant STAT1 (R274Q and T437I, both GOF, and Y701C, LOF) with IRF1 reporter plasmids into STAT1 null cell line (U3C cells). Cells were stimulated with IFN-gamma at varied concentrations for 16 h and IRF1 transcriptional activity was then measured with a luciferase assay. (D) Measurement of the effect of IFN-alpha and IFN-gamma stimulation on the rate of STAT1 de-phosphorylation in NK cells, T cells and monocytes from patient with STAT1-T437N mutation.
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F1
The two affected siblings have two additional siblings who are both being followed closely clinically and have not had evidence of lymphoma (Figure 1A). STAT1-null U3C cells and measured the alteration in phosphorylation of STAT1 (pSTAT1) with IFN-gamma stimulation using an immunoblot (Figure 1B). Specifically, we compared mock transfected (STAT1 negative) U3C cells, U3C cells transfected with wildtype STAT1 (WT) and U3C cells transfected with patient derived variants of STAT1. Transfected cells were stimulated with IFN-gamma at varied concentrations for 16 h and transcriptional activity of the STAT1 downstream pathway member IRF1 was measured using a luciferase assay (Figure 1C). T437N patient vs. healthy donor NK cells, T cells and monocytes (Figure 1D). This demonstrated a delay in STAT1 dephosphorylation in the patient samples as expected in STAT1 GOF.