Panel A: A schematic diagram shows the experimental setup for assessing STAT3 activity in HEK cells transfected with IL23R variants. A scatter bar plot displays the percentage of relative luciferase activity (RLA) normalized under unstimulated and IL-23 stimulated conditions. The x-axis lists various IL23R variants, and the y-axis shows the percentage of RLA. Different colored dots represent negative controls, wild-type, loss-of-function (LOF) variants, and isomorphic variants from the general population. Panel B: A western blot image shows pSTAT3 levels after stimulation with IL-23 or IFN-alpha 2 in HEK cells transfected with IL23R variants. Below the blot, a scatter bar plot depicts the pSTAT3/STAT3 band density as a percentage of stimulated wild-type conditions. The x-axis lists the variants, and the y-axis shows the percentage of pSTAT3/STAT3. Panel C: A scatter plot shows the Combined Annotation-Dependent Depletion (CADD) score versus minor allele frequency (MAF) for homozygous IL23R LOF variants and missense variants. The x-axis represents MAF, and the y-axis represents the CADD score. Different colors and symbols indicate LOF variants, isomorphic variants, and hypomorphic variants. Panel D: A schematic diagram of the IL23R protein domains highlights the locations of coding and non-coding LOF and hypomorphic variants.
Functional impact of IL23R variants on IL-23–dependent STAT3 activity. (A) Schematic representation of the experiment and STAT3 activity induced in response to IL-23 (10 ng/ml) by the IL23R variants used for the transient transfection of HEK cells, assessed with the luciferase assay. White dots represent negative controls (NT, not transfected and EV, empty vector); green dots, WT condition; red dots, LOF variant; gray dots, homozygous variants from the general population. Each variant was transfected at least five times, and each data point is represented by a circle. (B) Western blot of pSTAT3 after stimulation with IL-23 (1 ng/ml) or IFN-α (1 ng/ml) in HEK cells transiently transfected with IL23R variants. The graph shows pSTAT3/STAT3 band density as a percentage of that in stimulated WT conditions. At least two independent experiments were performed. The blot is duplicated in Fig. S1 B. (C) CADD/MAF graph for the homozygous IL23R LOF variants previously described (red triangle) in the MSMD cohort and the missense variants (dots) from the general population and our in-house cohort. The four hypomorphic variants are presented in different colors, whereas the isomorphic variants are shown in gray. MSC, mutation significance cutoff with a 99% CI. CADD, combined annotation-dependent depletion. (D) Localization of coding and non-coding LOF (red) or hypomorphic (orange, yellow, blue, and violet) IL23R variants across IL-23R protein domains. Source data are available for this figure: SourceData F1.