Quantitative reverse-transcriptase–polymerase-chain-reaction, cytokine, protein, and gene-expression analyses were performed according to standard procedures and are described in the Supplementary Appendix , available with the full text of this article at NEJM.org. Constructs of mutated TMEM173 (V147L, N154S, V155M, and V155R) and nonmutated TMEM173 were transfected into a STING-negative cell line (HEK293T cells) and stimulated with the STING ligand cyclic guanosine monophosphate–adenosine monophosphate (cGAMP [3′3′-cGAMP, Invivogen]).
When possible, we obtained blood and tissue samples from the study participants to assess activation and cell death of peripheral-blood cells. Tissue blocks from skin biopsies (in five patients), samples from lung biopsies (in two), and slides of a sample from a previous muscle biopsy (in one) were obtained and analyzed. Dermal fibroblast lines were obtained from two patients, four healthy controls, and three controls with the CANDLE syndrome. Primary endothelial cells were stimulated with the STING ligand cGAMP.
CD4 T cells and CD19 B cells from Patients 4 and 6 were treated for 4 hours with one of three Janus kinase (JAK) inhibitors — tofacitinib (1 μM), ruxolitinib (100 nM), or baricitinib (200 nM) — to assess their ability to block phosphorylation of the signal transducers and activators of transcription 1 (STAT1) and 3 (STAT3). Fibroblasts from Patient 1 and healthy controls were stimulated with 500 ng of cGAMP per milliliter and were also treated with 0.1 or 1.0 μM tofacitinib. We assayed the suppression of the gene encoding interferon-β (IFNB1) and other interferon-induced genes (CXCL10, MX1, and OAS3). Additional details are provided in theSupplementary Appendix .
When possible, we obtained blood and tissue samples from the study participants to assess activation and cell death of peripheral-blood cells. Tissue blocks from skin biopsies (in five patients), samples from lung biopsies (in two), and slides of a sample from a previous muscle biopsy (in one) were obtained and analyzed. Dermal fibroblast lines were obtained from two patients, four healthy controls, and three controls with the CANDLE syndrome. Primary endothelial cells were stimulated with the STING ligand cGAMP.
CD4 T cells and CD19 B cells from Patients 4 and 6 were treated for 4 hours with one of three Janus kinase (JAK) inhibitors — tofacitinib (1 μM), ruxolitinib (100 nM), or baricitinib (200 nM) — to assess their ability to block phosphorylation of the signal transducers and activators of transcription 1 (STAT1) and 3 (STAT3). Fibroblasts from Patient 1 and healthy controls were stimulated with 500 ng of cGAMP per milliliter and were also treated with 0.1 or 1.0 μM tofacitinib. We assayed the suppression of the gene encoding interferon-β (IFNB1) and other interferon-induced genes (CXCL10, MX1, and OAS3). Additional details are provided in the
