Gain-of-function mutations in IFIH1 cause a spectrum of human disease phenotypes associated with upregulated type I interferon signaling


The type I interferon system is integral to human antiviral immunity. However, inappropriate stimulation or defective negative regulation of this system can lead to inflammatory disease. We sought to determine the molecular basis of genetically uncharacterized cases of the type I interferonopathy Aicardi-Goutières syndrome and of other undefined neurological and immunological phenotypes also demonstrating an upregulated type I interferon response. We found that heterozygous mutations in the cytosolic double-stranded RNA receptor gene IFIH1 (also called MDA5) cause a spectrum of neuroimmunological features consistently associated with an enhanced interferon state. Cellular and biochemical assays indicate that these mutations confer gain of function such that mutant IFIH1 binds RNA more avidly, leading to increased baseline and ligand-induced interferon signaling. Our results demonstrate that aberrant sensing of nucleic acids can cause immune upregulation.

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Figure 1: Schematic of the human IFIH1 gene.
Figure 2: Quantitative RT-PCR (qPCR) of a panel of six ISGs in whole blood measured in IFIH1 mutation–positive probands and mutation-negative relatives and interferon scores in mutation-positive individuals, mutation-negative relatives and controls.
Figure 3: IFIH1 mutants activate the interferon signaling pathway more efficiently than wild-type IFIH1.
Figure 4: IFIH1 mutants form filaments.

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NCBI Reference Sequence

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We sincerely thank the participating families for the use of genetic samples and clinical information and all clinicians who contributed samples and data not included in this manuscript. We thank D. Chase for proofreading the manuscript. We thank G. Notarangelo for helpful discussion. Y.d.T.D. holds a Novartis Foundation postdoctoral fellowship. S.H. holds a Pew scholarship and Career Development award from Boston Children's Hospital. Y.J.C. acknowledges the Manchester Biomedical Research Centre, Manchester Academic Health Sciences Centre, the Greater Manchester Comprehensive Local Research Network, the Great Ormond Street Hospital Children's Charity, the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement 241779 and the European Research Council (GA 309449). The authors would like to thank the NHLBI GO ESP and its ongoing studies, which produced and provided exome variant calls for comparison: the Lung GO Sequencing Project (HL-102923), the Women's Health Initiative (WHI) Sequencing Project (HL-102924), the Broad GO Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL-102926) and the Heart GO Sequencing Project (HL-103010).

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B.H.A., J.O. and S.G.W. performed exome sequencing. E.M.J., G.I.R. and Y.J.C. performed exome data analysis. G.I.R. performed quantitative PCR analysis and Sanger sequencing with assistance from E.M.J. and G.M.A.F. G.M.A.F. and B.H.A. performed genotyping analysis with assistance from G.I.R. Y.d.T.D. performed IFIH1 protein studies. S.H. performed modeling studies. Y.J.C. and S.H. designed and supervised the project and wrote the manuscript, with support from G.I.R. G.A., B.B.-M., E.M.B., R.B., M.W.B., M. Casarano, M. Chouchane, R.C., A.E.C., N.J.V.C., R.C.D., J.E.D., L.D.W., I.D., L.F., E.F., B.I., L.L., A.R.L., P.L., C.L., J.H.L., C.M.L., M.M.M., A.M.-P., I.B.M., M.P.M., C.M., S.O., P.P.P., E.R., R.A.R., D.R., E.S., C.S., M.S., J.L.T., A.V., C.V., J.P.V., K.W., R.N.W., L.A.W. and S.M.Z. identified affected individuals or assisted with related clinical and laboratory studies.

Corresponding authors

Correspondence to Sun Hur or Yanick J Crow.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Families with mutations in IFIH1.

Family number is shown above each pedigree. Sequence chromatograms are given where available. IFIH1 protein status is stated if known, with WT denoting wild type. De novo mutations are annotated in parentheses. Uncolored shapes represent unaffected individuals. Black-filled shapes represent clinically affected individuals. Gray-filled shapes represent clinically asymptomatic mutation-positive individuals. A diagonal line indicates deceased status. IS denotes interferon score (for participants with repeat samples, the mean combined measurement is given).

Supplementary Figure 2 CLUSTAL Omega alignment of IFIH1 homologs.

IFIH1 homologs were identified on Ensembl and aligned using CLUSTAL Omega. Amino acids altered by IFIH1 mutations are highlighted by a red box. H.Sapiens, Homo sapiens (ENSP00000263642); P.troglodytes, Pan troglodytes (ENSPTRG00000012582); M.mulatta, Macaca mulatta (ENSMMUG00000003202); R.Norvegicus, Rattus norvegicus (ENSRNOG00000006227); M.Musculus, Mus musculus (ENSMUSG00000026896); C.Familiaris, Canis familiaris (ENSCAFG00000010438); B.Taurus, Bos Taurus (ENSBTAG00000008142); G.gallus, Gallus gallus (ENSGALG00000011089); X.Tropicalis, Xenopus tropicalis (ENSXETG00000013176); T.nigroviridis, Tetraodon nigroviridis (ENSTNIG00000016500); D.Rerio, Danio rerio (ENSDARG00000018553). Homology to the human IFIH1 reference sequence (ENSP00000263642): human–P. troglodytes, 100%; human–M. mulatta, 98%; human–B. torus, 84%; human–C. familiaris, 83%; human–M. musculus, 80%; human–R. norvegicus, 80%; human–G. gallus, 56%; human–X. tropicalis, 55%; human–D. rerio, 49%; human–T. nigroviridis, 44%.

Supplementary Figure 3 CLUSTAL Omega alignment of RNA helicases.

RNA helicases were identified on Ensembl and aligned using CLUSTAL Omega. Amino acids altered by IFIH1 mutations are highlighted by a red box. IFIH1 (ENSP00000263642), DDX58 (RIG-I) (ENSP00000369213), DHX58 (LGP2) (ENSP00000251642). Homology of human IFIH1 (reference sequence ENSP00000263642) to DDX58 (RIG-I) and DHX58 (LGP2) is 31% and 41%, respectively.

Supplementary Figure 4 Individual ISG transcript levels in IFIH1 mutation–positive individuals, their IFIH1 mutation–negative relatives and a cohort of 29 controls.

qPCR of a panel of six ISGs in whole blood measured in 5 families with mutations in IFIH1 and 29 healthy controls. RQ is equal to 2−ΔΔCt, with –ΔΔCt ± s.d. (i.e., the normalized fold change relative to a calibrator). Each value is derived from three technical replicates. For ease of presentation, mutation-negative relatives (n = 13) have been combined with the control cohort (M-NR+C) in this figure. Horizontal red bars show the median RQ value for each probe in each group. For individuals with repeat samples (biological replicates), all measurements are shown. Data were analyzed by one-way ANOVA using Dunnett's multiple-comparison test. ***P < 0.0001. Source data

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Rice, G., del Toro Duany, Y., Jenkinson, E. et al. Gain-of-function mutations in IFIH1 cause a spectrum of human disease phenotypes associated with upregulated type I interferon signaling. Nat Genet 46, 503–509 (2014).

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