Functional tests of biallelic variants identified in the control cohort. (A) HEK293T cells were transfected with MDA5 variants, the firefly luciferase gene regulated by IFN-β, and the constitutively expressed Renilla luciferase gene for 24 h and were then stimulated with 2 μg/ml poly(I:C) with Lipofectamine. Firefly luciferase activities were first normalized against Renilla luciferase activity and then against WT values. The known LOF variant K365E served as an LOF control. EV, empty vector. Experiments were repeated three times. Error bars indicate the SD of repeats. (B) HEK293T cells were transfected with MDA5 variants, and protein levels were assessed by Western blotting. (C) HEK293T cells were transfected with IRF7 variants, the firefly luciferase gene regulated by IFN-β, and the constitutively expressed Renilla luciferase gene for 24 h and were then stimulated with Sendai viruses or left unstimulated. Firefly luciferase activity was first normalized against Renilla luciferase activity and then against unstimulated EV. Two known LOF variants, F410V and Q421X, served as LOF controls. Experiments were repeated three times. Error bars indicate the SD of repeats. (D) HEK293T cells were transfected with IRF7 variants, and protein levels were assessed by Western blotting.