HHS Public Access Author manuscript Hum Mutat. Author manuscript; available in PMC 2021 April 01. Author Manuscript Published in final edited form as: Hum Mutat. 2020 April ; 41(4): 837–849. doi:10.1002/humu.23975. Genetic and phenotypic spectrum associated with IFIH1 gain-of- function A full list of authors and affiliations appears at the end of the article. Abstract IFIH1 gain-of-function has been reported as a cause of a type I interferonopathy encompassing a Author Manuscript spectrum of autoinflammatory phenotypes including Aicardi–Goutières syndrome and Singleton Merten syndrome. Ascertaining patients through a European and North American collaboration, we set out to describe the molecular, clinical and interferon status of a cohort of individuals with pathogenic heterozygous mutations in IFIH1. We identified 74 individuals from 51 families segregating a total of 27 likely pathogenic mutations in IFIH1. Ten adult individuals, 13.5% of all mutation carriers, were clinically asymptomatic (with seven of these aged over 50 years). All mutations were associated with enhanced type I interferon signaling, including six variants (22%) which were predicted as benign according to multiple in silico pathogenicity programs. The identified mutations cluster close to the ATP binding region of the protein. These data confirm variable expression and nonpenetrance as important characteristics of the IFIH1 genotype, a consistent association with enhanced type I interferon signaling, and a common mutational mechanism involving increased RNA binding affinity or decreased efficiency of ATP hydrolysis Author Manuscript and filament disassembly rate. Keywords Aicardi–Goutières syndrome; IFIH1; MDA5; Singleton Merten syndrome; Type I interferonopathy 1∣ INTRODUCTION In 2014, heterozygous gain-of-function mutations in IFIH1 were reported to cause a spectrum of neuroimmune phenotypes including classical Aicardi–Goutières syndrome (AGS; Oda et al., 2014; Rice et al., 2014). IFIH1 encodes interferon-induced helicase C domain-containing protein 1 (IFHI1; also known as melanoma differentiation associated Author Manuscript This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. http://creativecommons.org/licenses/by/4.0/ Correspondence Sun Hur, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Program in Cellular and Molecular Medicine, Boston Children’s Hospital, MA 02115. Sun.Hur@childrens.harvard.edu; Yanick J. Crow, Centre for Genomic and Experimental Medicine, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, United Kingdom. yanickcrow@mac.com. CONFLICT OF INTERESTS Y. J. Crow has undertaken consultancy work with Biogen on behalf of the University of Edinburgh. DATA AVAILABILITY STATEMENT Data available on request due to privacy/ethical restrictions. Identified variants submitted to ClinVar (Submission ID: SUB6667166; Organization ID: 507341). Gillian I. Rice and Sehoon Park equally contributed to this study. Rice et al. Page 2 gene 5 protein: MDA5) which senses viral double-stranded (ds) RNA in the cytosol, leading Author Manuscript to the induction of a type I interferon-mediated antiviral response. Consequent to Mendelian determined gain-of-function, it is suggested that IFIH1 inappropriately senses self-derived nucleic acid as viral, leading to an autoinflammatory state classified as a type I interferonopathy (Ahmad et al., 2018; Crow & Manel, 2015). In 2015, a p.Arg822Gln substitution in IFIH1 was shown to cause Singleton Merten syndrome (SMS), an autosomal dominant trait variably characterized by a deforming arthropathy, abnormal tooth development and cardiac valve calcification, again in association with enhanced type I interferon signaling (Rutsch et al., 2015). Although it was initially considered that SMS was a distinct, mutation-specific disorder, subsequent reports indicate that SMS and the neuroinflammatory phenotypes seen in the context of IFIH1 gain-of-function constitute part of the same disease spectrum (Buers, Rice, Crow, & Rutsch, 2017; Bursztejn et al., 2015). Type I interferonopathy associated IFIH1 mutations are either absent from control databases, Author Manuscript or only present at very low frequency. However, we have noted previously that in silico algorithms are not always reliable in differentiating IFIH1 disease-causing variants from benign polymorphisms (Ruaud et al., 2018). Such difficulty in assigning molecular pathogenicity is compounded by marked variability in disease expression, sometimes even within the same family, and the observation of complete non-penetrance in certain pedigrees (Rice et al., 2014). Given this background, we considered it important to provide an update of our experience of sequencing individuals for pathogenic IFIH1 mutations associated with a type I interferonopathy state. In total, we describe molecular and clinical data relating to 74 individuals from 51 families, identifying 27 likely pathogenic mutations that cluster close to the ATP binding region of the protein. Our data confirm variable expression and nonpenetrance as important characteristics of these mutant genotypes, and the consistent association with enhanced type I interferon signaling as assessed by interferon-stimulated Author Manuscript gene (ISG) expression, referred to as the interferon score. 2∣ MATERIALS AND METHODS 2.1 ∣ Subjects Patients were ascertained through direct contact and/or collaborating physicians across clinical research laboratories in the UK and France (Crow), the USA (Vanderver), and Italy (Orcesi). The study was approved by the Leeds (East) Research Ethics Committee (10/ H1307/132), the Comite de Protection des Personnes (ID-RCB/EUDRACT: 2014- A01017-40), IRB study protocol (Myelin Disorders Bioregistry Project: IRB# 14-011236) and the local ethics committee of the IRCCS Mondino Foundation, Pavia, Italy (3549/2009 of 30/9/2009 and 11/12/2009; n.20170035275 of 23/10/2017). Amino acid substitutions Author Manuscript were considered as pathogenic mutations when they were seen in the context of a neuroimmune/autoinflammatory state (including AGS, a spastic-dystonic syndrome, nonsyndromic spastic paraparesis or SMS), and when two or more of the following applied: observation of the same variant in an unrelated family; de novo occurrence; documented increase in ISG expression; in vitro data consistent with IFIH1 gain-of-function. Hum Mutat. Author manuscript; available in PMC 2021 April 01. Rice et al. Page 3 2.2 ∣ Mutational analysis Author Manuscript Mutations were identified on a variety of next-generation sequencing platforms. Where Sanger sequencing was undertaken, primers were designed to amplify the coding exons of IFH1, with mutation annotation based on the reference cDNA sequence NM_022168.2. Variants were assessed using the in silico programs SIFT (http://sift.jcvi.org), Polyphen2 (http://genetics.bwh.harvard.edu/pph2/), and CADD (https://cadd.gs.washington.edu), summarized in VarCards (http://varcards.biols.ac.cn/). Population allele frequencies were obtained from the gnomAD database (http://gnomad.broadinstitute.org). 2.3 ∣ Protein modeling Molecular graphics figures were generated with PyMOL (Schrödinger) using the PDB coordinates (4GL2). Author Manuscript 2.4 ∣ Interferon score Interferon scores were calculated on the basis of the expression of ISGs according to previously published protocols. In brief, this involved either a quantitative reverse transcription-polymerase chain reaction (qPCR) analysis using TaqMan probes (Crow laboratory: Rice et al., 2013), or testing on a Nanostring platform (Vanderver laboratory: Adang et al., 2018+). In the former, the relative abundance of IFI27 (Hs01086370_m1), IFI44L (Hs00199115_m1), IFIT1 (Hs00356631_g1), ISG15 (Hs00192713_m1), RSAD2 (Hs01057264_m1), and SIGLEC1 (Hs00988063_m1) transcripts was normalized to the expression levels of HPRT1 (Hs03929096_g1) and 18S (Hs999999001_s1). The median fold change of the six genes, compared to the median of 29 previously collected healthy controls, was then used to create an interferon score for each individual, with an abnormal interferon score being defined as greater than +2 standard deviations above the mean of the control Author Manuscript group that is 2.466. Alternatively, the copy number of mRNA transcripts of the six ISGs listed above, and four housekeeping genes (ALAS1, HPRT1, TBP, and TUBB), was quantified using a Nanostring nCounter™ Digital Analyzer. The raw copy number of mRNA transcripts of each ISG was standardized using the geometric mean of the four housekeeping genes for each individual, and the six-gene interferon signature for each individual calculated using the median of the Z scores, with the result considered positive if ≥1.96 (>98th centile; one tail analysis). 2.5 ∣ Interferon reporter assay The pFLAG-CMV4 plasmid encoding IFIH1 has been described elsewhere (Rice et al., 2014). Indicated mutations were introduced using Phusion HiFi DNA polymerase. HEK 293T cells (ATCC) were maintained in 48-well plates in DMEM (Cellgro) supplemented Author Manuscript with 10% fetal bovine serum and 1% L-glutamine. At 80% confluence, cells were cotransfected with pFLAGCMV4 plasmids encoding wild-type or mutant IFIH1 (5 ng, unless indicated otherwise), interferon β (IFNb) promoter-driven firefly luciferase reporter plasmid (100 ng), and a constitutively expressed Renilla luciferase reporter plasmid (pRL- TK, 10 ng), by using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s protocol. The medium was changed 6 hr after transfection, and cells were subsequently incubated for 18 hr with or without stimulation with poly(I-C) (500 ng; Hum Mutat. Author manuscript; available in PMC 2021 April 01. Rice et al. Page 4 InvivoGen) using Lipofectamine 2000. Cells were lysed with Passive Lysis Buffer Author Manuscript (Promega), and IFNb promoter activity was measured using a Dual-Luciferase Reporter Assay (Promega) and a Synergy 2 plate reader (BioTek). Firefly luciferase activity was normalized to Renilla luciferase activity Each experiment was performed in triplicate and data are presented as mean ± standard mean of error. Statistical significance was determined by two-tailed, unpaired Student’s t-test with *, **, and *** indicating p values <.05, <.01, and <.001, respectively. Expression levels of individual constructs were tested by western blot analysis. 3 ∣ RESULTS 3.1 ∣ Molecular data We collected data on 74 individuals from 51 families, identifying 27 distinct mutations in total (Figure 1; Table 1). Fourteen mutations were recorded in a single proband, seven in Author Manuscript more than one individual belonging to a single-family, and six in more than one family. Of these six recurrent mutations, the p.Arg720Gln, p.Arg779Cys, and p.Arg779His substitutions were observed most frequently (6, 8, and 10 times, respectively). Twenty-two mutations were recorded to have occurred de novo in at least one individual, whilst four mutations were only ascertained in familial cases demonstrating autosomal dominant transmission (two mutations, p.Ala489Thr and p.Gly495Arg, were transmitted from a father in whom the mutation arose de novo). Three mutations, p.Thr331Arg, p.Arg779Cys, and p.Arg779His, was documented to have occurred both de novo, in association with severe, AGS-like, neurological disease, and in families with transmission across two or more generations. For six putative mutations (p.Gly389Arg; p.Asn449Lys; p.Ile583Val; p.Ile803Phe; Author Manuscript p.Asp848Glu; p.Ile956Val), in silico predictions using both SIFT and Poyphen2 suggested that the substitutions were benign, with relatively poor evolutionary conservation (Figure S1). However, all of these variants were novel (i.e., not recorded in gnomAD), and assays of interferon signaling (ISG expression and in vitro testing) indicate that they represent pathogenic mutations conferring gain-of-function (Table S1; Figure S2). Of note, four of these variants were seen in the context of a spastic paraparesis phenotype with no or minimal cognitive impairment. Clinical nonpenetrance was observed in three of these families (the other three variants arising in the proband de novo). 3.2 ∣ Clinical phenotype Consistent with previous data, we observed a spectrum of phenotypes in our cohort, encompassing classical AGS, less easily defined rapid neuroregression, a spastic-dystonic Author Manuscript syndrome, spastic paraparesis, SMS, and clinical nonpenetrance (Figure 2; Table 2; Table S2). A single individual, AGS2222, experienced neonatal hepatitis and then developed chronic fibrotic liver disease in the absence of any other clinical features (note that this same variant was seen in another proband, AGS735, presenting with neuroregression at age 1 year). Unequivocal episodes of rapid neuroregression were noted in at least 20 patients, in seven of whom an acute loss of skills occurred after the age of 1 year on a background of completely normal development. Recognition/onset of symptoms was frequently later in Hum Mutat. Author manuscript; available in PMC 2021 April 01. Rice et al. Page 5 patients with a spastic paraparesis phenotype, with one patient experiencing the development Author Manuscript of lower limb spasticity beginning at 13 years of age (AGS531_P4). Six symptomatic patients were recorded to have died. Five of these individuals demonstrated a severe AGS phenotype with features obvious at, or soon after, birth that is indicating prenatal onset. One further deceased patient presented with neuroregression at age 15 months, and died suddenly of a cardiorespiratory arrest at 16 years of age, with pulmonary hypertension documented on postmortem examination. Ten individuals were reported as asymptomatic mutation carriers, across five mutations (p.Gly389Arg, p.Arg779Cys, p.Arg779His p.Asp848Glu, and p.Ile956Val), with seven aged over 50 years. 3.3 ∣ Interferon status Where tested, all mutations (i.e., 26 of 27) were associated with increased expression of ISGs in peripheral blood (Table 1). Samples were unavailable for the single patient carrying Author Manuscript the p.Glu773Gln substitution. This variant is not recorded in gnomAD, occurring de novo in the context of a phenotype compatible with IFIH1 upregulation, and conferring a gain-of- function in our in vitro assay (Figure S2). Considering all (51) mutation-positive individuals tested for ISG expression in the Crow laboratory (given that a direct comparison of results across laboratories is not possible), 109 of 117 values were positive (Table S3; Figure S3). Only one clinically symptomatic patient (AGS2154_1) demonstrated a negative interferon signature (on two of three occasions tested). The phenotype, in this case, was unusual; a child with white matter disease confined to the right cerebral hemisphere on MRI and no abnormal neurological signs on examination, having presented at age 8 years with headaches. We leave open the possibility that these two normal results, and three normal results from his mother, might be due to technical artifact, given that the samples had been stored for many months before testing. Sixteen samples from seven clinically nonpenetrant Author Manuscript subjects exhibited an upregulation of interferon signaling, with two asymptomatic mutation carriers demonstrating normal interferon signatures (each tested on three occasions). 3.4 ∣ Modeling of IFIH1 gain-of-function mutations Modeling of the 27 mutations described here showed that most residues cluster near the ATP binding site within the helicase domain (Figure 3). Three mutations, p.Ileu583Val, p.Ileu956Val, and p.Leu979Trp were the only residues not situated in the cluster (colored cyan; only p.Ileu583Val and p.Leu979Val are shown since residue p.Ileu956 is disordered in the crystal structure). Within this main cluster, residues can be further categorized into three groups: those at the ATP binding pocket (magenta spheres), those in the double stranded RNA (dsRNA) binding surface (colored blue) and those not directly involved in either ATP or RNA binding (colored green). Three published mutations (p.Leu372phe; p.Ala452Thr; Author Manuscript p.Glu813Asp; Table S4) not ascertained in our cohort are also located within the main cluster (colored orange), further supporting the importance of this region in the regulation of IFIH1 signaling activity. 4∣ DISCUSSION Here we present data on 74 individuals, 41 previously unreported, from 51 families, with a putative gain-of-function mutation in IFIH1. Consistent with previous descriptions, we Hum Mutat. Author manuscript; available in PMC 2021 April 01. Rice et al. Page 6 observed a spectrum of phenotypes, encompassing AGS, spastic-dystonia, spastic Author Manuscript paraparesis, SMS and clinical nonpenetrance. Phenotypic variability was common, both in the context of familial inheritance and mutations seen recurrently across families so that no obvious genotype–phenotype correlations could be ascertained. Acute regression was noted in almost one-third of symptomatic mutation carriers, occurring after the age of 1 year in seven patients demonstrating completely normal development to that time. Beyond acute regression, a slower onset of disease, and subsequent progression, was seen in patients demonstrating a spastic paraparesis phenotype. Together with the observation of clinical nonpenetrance (10:13.5% of 74 mutation-positive individuals in our series), with seven individuals identified to be apparently disease-free beyond the age of 50 years, these data suggest the importance of additive genetic factors and/or environmental triggers in determining phenotypic status. Although we did not formally record neuroimaging features in our cohort, white matter disease and intracranial calcification were Author Manuscript observed frequently. Such imaging characteristics can be seen in the absence of overt neurological signs (see Bursztejn et al., 2015 and de Carvalho et al., 2017). Conversely, significant neurological disease, most typically spastic paraparesis, can occur in the context of normal brain and spinal imaging (e.g., the father in family AGS524). Clinically manifest extraneurological illness was uncommon in our series, but there appears to be a real association between IFIH1 gain-of-function and lupus-like illness, autoimmune hepatitis, and hypothyroidism. Furthermore, psoriatic-like skin disease is a well-recognized feature of the SMS phenotype. As recently described (Adang et al., 2018), two patients included here were diagnosed with pulmonary hypertension, a feature which was not searched for in most patients and may be under-recognized. Author Manuscript We observed a strong association of mutation status with an enhanced expression of ISGs, with 109 of 117 samples from 51 patients being positive in the experience of one laboratory. A similar conclusion can be drawn from in vitro testing. As such, upregulated interferon signaling represents a reliable biomarker of IFIH1 gain-of-function, and can serve as an indicator of variant pathogenicity where doubt exists as to the significance of a molecular lesion. This is important given that we show here that in silico algorithms do not always accurately predict pathogenicity (involving 22% of the mutations that we recorded). Where tested, clinical nonpenetrance was also associated with a persistent upregulation of interferon signaling, with only two of nine such individuals nonpenetrant on ISG testing in blood. Whether these individuals demonstrate fluctuations in ISG expression is not known at this time. Despite documented clinical nonpenetrance in some cases, all putative IFIH1 gain-of- Author Manuscript function substitutions are rare, with only two of the 30 discrete mutations described here and in previous reports recorded in gnomAD. Furthermore, all ascertained type I interferonopathy associated mutations are missense variants, likely conferring increased sensitivity to a self-derived nucleic acid. Although premature termination mutations in the helicase domain are seen in control populations as common polymorphisms, none has been associated with a type I interferonopathy phenotype, further supporting the role of nucleic acid binding by the helicase domain in disease pathogenesis. Substitutions of the arginine Hum Mutat. Author manuscript; available in PMC 2021 April 01. Rice et al. Page 7 residues at positions 720 and 779 were seen in six and 19 probands, respectively, in our Author Manuscript series. Given the focus of our laboratories on pediatric neurological disease, our data are likely to subject to ascertainment bias. Indeed, although only observed once by us, the p.Arg822Gln mutation has been reported in an additional five pedigrees demonstrating a classical SMS phenotype (Pettersson et al., 2017; Rutsch et al., 2015). IFIH1 is a member of the retinoic acid-inducible gene I (RIG-I) receptor family (del Toro Duany, Wu, & Hur, 2015). Recognition of cytoplasmic viral dsRNA by IFIH1 induces filament assembly along the dsRNA axis, with the helicase domains and C terminal domain responsible for RNA recognition. Filament formation then induces oligomerization of the tandem CARD domains (2CARD) of IFIH1, leading to the interaction with mitochondrial MAVS and subsequent induction of interferon and other proinflammatory cytokines. IFIH1 filament stability is intrinsically regulated by ATP hydrolysis, which is stimulated upon dsRNA binding. Mutations that impair ATP hydrolysis generally increase filament stability Author Manuscript and, often, but not always, confer gain-of-function signaling activity. The clustering of mutations that we ascertained, and of a further three unique published mutations, near the ATP binding region likely highlights common mechanisms, perhaps increasing RNA binding affinity or decreasing the efficiency of ATP hydrolysis and the rate of filament disassembly. Summarizing, IFIH1 gain-of-function is associated with a spectrum of phenotypes, occurring due to de novo mutations or transmitted as an autosomal dominant trait. Testing for an interferon signature in blood represents a useful biomarker in this context, which can aid in the interpretation of identified sequence variants. Supplementary Material Author Manuscript Refer to Web version on PubMed Central for supplementary material. Authors Gillian I. Rice1, Sehoon Park2,3, Francesco Gavazzi4, Laura A. Adang4, Loveline A. Ayuk5, Lien Van Eyck6, Luis Seabra6, Christophe Barrea7, Roberta Battini8,9, Alexandre Belot10,11, Stefan Berg12, Thierry Billette de Villemeur13, Annette E. Bley14, Lubov Blumkin15,16, Odile Boespflug-Tanguy17,18, Tracy A. Briggs1,19, Elise Brimble20, Russell C. Dale21, Niklas Darin22,23, François-Guillaume Debray24, Valentina De Giorgis25, Jonas Denecke14, Diane Doummar26, Gunilla Drake af Hagelsrum27, Despina Eleftheriou28, Margherita Estienne29, Elisa Fazzi30,31, François Feillet32, Jessica Galli30,31, Nicholas Hartog33, Julie Harvengt34, Bénédicte Heron35, Delphine Heron36, Diedre A. Kelly37, Dorit Lev16,38, Virginie Levrat39, John Author Manuscript H. Livingston40, Itxaso Marti41, Cyril Mignot42, Fanny Mochel43, Marie-Christine Nougues44, Ilena Oppermann14, Belén Pérez-Dueñas45, Bernt Popp46, Mathieu P. Rodero6, Diana Rodriguez47,48, Veronica Saletti49, Cia Sharpe50, Davide Tonduti51, Gayatri Vadlamani40, Keith Van Haren20, Miguel Tomas Vila52, Julie Vogt53, Evangeline Wassmer54, Arnaud Wiedemann32, Callum J. Wilson55, Ayelet Zerem15,16, Christiane Zweier46, Sameer M. Zuberi56,57, Simona Orcesi25,58, Adeline L. Vanderver4, Sun Hur2,3, Yanick J. Crow6,59,60 Hum Mutat. Author manuscript; available in PMC 2021 April 01. Rice et al. Page 8 Affiliations Author Manuscript 1Division of Evolution and Genomic Sciences, Faculty of Biology, Medicine and Health, School of Biological Sciences, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom 2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 3Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, Massachusetts 4Division of Neurology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 5Paediatric Department, Dumfries and Galloway Royal Infirmary, Cargenbridge, United Kingdom 6Laboratory of Neurogenetics and Neuroinflammation, Institut Imagine, Paris, France 7Department of Neuropaediatrics, CHU & University of Liège, Liege, Belgium 8Department Clinical and Experimental Medicine, University of Pisa, Pisa, Italy 9IRCCS Fondazione Stella Maris, Pisa, Italy 10Université de Lyon, INSERM U1111, Author Manuscript CIRI, Lyon, France 11Centre International de Recherche en Infectiologie, CIRI, Inserm, U1111, École Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Université de Lyon, Lyon, France 12Pediatric Immunology and Rheumatology, The Queen Silvia Children’s Hospital, Goteborg, Sweden 13Neuropédiatrie, Centre de référence Neurogénétique, Hôpital Trousseau, Sorbonne Université, Paris, France 14University Children’s Hospital, University Medical Center Hamburg Eppendorf, Hamburg, Germany 15Pediatric Neurology Unit, Metabolic Neurogenetic Service, Wolfson Medical Center, Holon, Israel 16Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel 17Génétique Médicale, Université Paris Diderot, Paris, France 18Service de Neuropédiatrie et des Maladies Métaboliques, Centre de Référence Maladies Rares “Leucodystrophies”, Hopital Robert Debré, Paris, France 19Manchester Centre for Genomic Medicine, St Author Manuscript Mary’s Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom 20Department of Neurology, Stanford University School of Medicine, Stanford, California 21Faculty of Medicine and Health, Kids Neuroscience Centre, Brain and Mind Centre, Children’s Hospital at Westmead, University of Sydney, Sydney, Australia 22Department of Pediatrics, Institute of Clinical Sciences, Sahlgrenska University Hospital, University of Gothenburg, Gothenburg, Sweden 23The Queen Silvia Children’s Hospital, Sahlgrenska University Hospital, Gothenburg, Sweden 24Metabolic Unit, Department of Medical Genetics, CHU & University of Liège, Gembloux, Belgium 25Child Neurology and Psychiatry Unit, IRCCS Mondino Foundation, Pavia, Italy 26GHUEP, département de neuropédiatrie, Centre de référence neurogénétique mouvement anormaux de l'enfant, Hôpital Armand Trousseau, Paris, France 27Pediatric Neurology, The Queen Silvia Author Manuscript Children’s Hospital, Goteborg, Sweden 28Paediatric Rheumatology, ARUK Centre for Adolescent Rheumatology, Institute of Child Health, University College London (UCL) Great Ormond Street Hospital, London, United Kingdom 29U.O. Neuropsichiatria Infantile, Fondazione IRCCS, Istituto Neurologico Carlo Besta, Milan, Italy 30Unit of Child Neurology and Psichiatry, ASST Spedali Civili of Brescia, Brescia, Italy 31Department of Experimental and Clinical Sciences, University of Brescia, Brescia, Italy 32Service de Médecine Infantile, Centre de Référence des Hum Mutat. Author manuscript; available in PMC 2021 April 01. Rice et al. Page 9 Author Manuscript maladies métaboliques de Nancy, CHU Brabois Enfants, Unité INSERM NGERE U1256, Nancy, France 33Department of Allergy/Immunology, Spectrum Health Helen Devos Children’s Hospital, Michigan State University College of Human Medicine, East Lansing, Michigan 34Department of Medical Genetics, CHU & University of Liège, Gembloux, Belgium 35Service de Neuropédiatrie, Centre Référence des Maladies Lysosomales, Hôpital Trousseau, Paris, France 36UF Génétique Médicale et Centre de Référence “Déficiences Intellectuelles”, Groupe Hospitalier Pitié- Salpêtrière, Paris, France 37The Liver Unit, Birmingham Women’s and Children’s Hospital NHS Foundation Trust, Birmingham, United Kingdom 38Metabolic Neurogenetic Service, Wolfson Medical Center, The Rina Mor Institute of Medical Genetics, Holon, Israel 39Service de pédiatrie, Centre Hospitalier Annecy Genevois, Pringy, France 40Department of Paediatric Neurology, Leeds General Infirmary, Author Manuscript Leeds, United Kingdom 41Pediatric Neurology, Hospital Universitario Donostia, Universidad del País Vasco UPV-EHU, San Sebastian, Spain 42Departement de Génétique & Centre de Référence Déficience Intellectuelle de cause rare, GH Pitié- Sapêtrière, Paris, France 43Institut du Cerveau et de la Moelle épinière, INSERM U 1127, Sorbonne Universités, Paris, France 44Service de Neuropédiatrie, GHUEP, Hôpital Armand Trousseau, APHP, Paris, France 45Pediatric Neurology Research Group, Hospital Vall d'Hebron—Research Institute (VHIR), Universitat Autonoma de Barcelona, Barcelona, Spain 46Institute of Human Genetics, Friedrich-Alexander- Universität Erlangen-Nürnberg (FAU), Erlangen, Germany 47GRC n°19, pathologies Congénitales du Cervelet-LeucoDystrophies, CRMR maladies neurogénétiques, Sorbonne Université, Paris, France 48Service de Neuropédiatrie, Hôpital Trousseau, Groupe Hospitalier HUEP, Inserm U1141, Paris, France 49Developmental Neurology Author Manuscript Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy 50Paediatric Neurology, Starship Children’s Hospital, Auckland, New Zealand 51Pediatric Neurology Unit, V. Buzzi Children’s Hospital, Milan, Italy 52Neuropediatría, Hospital Universitari i Pôlitecnic La Fe, Valencia, Spain 53West Midlands Regional Clinical Genetics Service and Birmingham Health Partners, Birmingham Women’s and Children’s Hospitals NHS Foundation Trust, Birmingham, United Kingdom 54Department of Paediatric Neurology, Birmingham Women’s and Children’s Hospitals NHS Foundation Trust, Birmingham, United Kingdom 55National Metabolic Service, Starship Children’s Hospital, Auckland, New Zealand 56Paediatric Neurosciences Research Group, Royal Hospital for Children, Glasgow, United Kingdom 57School of Medicine, University of Glasgow, Glasgow, United Kingdom 58Department of Brain and Behavioural Sciences, University of Pavia, Pavia, Italy Author Manuscript 59Sorbonne-Paris-Cité, Institut Imagine, Paris Descartes University, Paris, France 60Centre for Genomic and Experimental Medicine, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom ACKNOWLEDGMENTS Yanick J. Crow acknowledges The University of Maryland Brain and Tissue Bank of the NIH NeuroBioBank. Yanick J. Crow acknowledges the European Research Council (786142-E-T1IFNs), a state subsidy managed by the National Research Agency (France) under the “Investments for the Future” program bearing the reference ANR-10- Hum Mutat. Author manuscript; available in PMC 2021 April 01. Rice et al. Page 10 IAHU-01 and the MSDAvenir fund (DEVO-DECODE Project). Tracy A. Briggs acknowledges the National Institute for Health Research (NIHR; NIHR Transitional Research Fellowship, TRF-2016-09-002; with the views Author Manuscript expressed were those of the author and not necessarily those of the NHS, the NIHR or the Department of Health). Adeline L. Vanderver is supported by the Kamens endowed chair for Translational Neurotherapeutics and the Myelin Disorders Bioregistry Project. Adeline L. Vanderver and Laura A. Adang acknowledge the CURE Pennsylvania Frontiers in Leukodystrophy grant and U01HD082806. Laura A. Adang also acknowledges the National Center for Advancing Translational Sciences of the National Institutes of Health under award number KL2TR001879. Lien Van Eyck received funding from Research Foundation Flanders (FWO). Funding information Public Health Research Programme, Grant/Award Number: TRF-2016-09-002; National Center for Advancing Translational Sciences of the National Institutes of Health, Grant/Award Number: KL2TR001879; CURE Pennsylvania Frontiers in Leukodystrophy, Grant/Award Number: U01HD082806; H2020 European Research Council, Grant/Award Number: 786142-E-T1IFNs; Agence Nationale de la Recherche, Grant/Award Number: ANR-10-IAHU-01; MSDAvenir fund, Grant/Award Number: DEVO-DECODE Project; NIHR Transitional Research Fellowship, Grant/Award Number: TRF-2016-09-002 REFERENCES Author Manuscript Adang LA, Frank DB, Gilani A, Takanohashi A, Ulrick N, Collins A, & Vanderver AL (2018). Aicardi goutieres syndrome is associated with pulmonary hypertension. 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Further evidence for specific IFIH1 mutation as a cause of Singleton-Merten syndrome Hum Mutat. Author manuscript; available in PMC 2021 April 01. Rice et al. Page 11 with phenotypic heterogeneity. American Journal of Medical Genetics. Part A, 173(5), 1396–1399. 10.1002/ajmg.a.38214 [PubMed: 28319323] Author Manuscript Popp B, Ekici AB, Thiel CT, Hoyer J, Wiesener A, Kraus C, … Zweier C (2017). Exome Pool-Seq in neurodevelopmental disorders. European Journal Human Genetics, 25(12), 1364–1376. Rice GI, Forte GM, Szynkiewicz M, Chase DS, Aeby A, Abdel-Hamid MS, & Crow YJ (2013). Assessment of interferon-related biomarkers in Aicardi-Goutieres syndrome associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR: A case- control study. Lancet Neurology, 12(12), 1159–1169. 10.1016/S1474-4422(13)70258-8 [PubMed: 24183309] Rice GI, Del Toro Duany Y, Jenkinson EM, Forte GM, Anderson BH, Ariaudo G, & Crow YJ (2014). Gain-of-function mutations in IFIH1 cause a spectrum of human disease phenotypes associated with upregulated type I interferon signaling. Nature Genetics, 46(5), 503–509. 10.1038/ng.2933 [PubMed: 24686847] Ruaud L, Rice GI, Cabrol C, Piard J, Rodero M, van Eyk L, & Van Maldergem L (2018). Autosomal- dominant early-onset spastic paraparesis with brain calcification due to IFIH1 gain-of-function. Human Mutation, 39(8), 1076–1080. 10.1002/humu.23554 [PubMed: 29782060] Author Manuscript Rutsch F, MacDougall M, Lu C, Buers I, Mamaeva O, Nitschke Y, & Hennekam RC (2015). A specific IFIH1 gain-of-function mutation causes Singleton-Merten syndrome. American Journal of Human Genetics, 96(2), 275–282. 10.1016/j.ajhg.2014.12.014 [PubMed: 25620204] Author Manuscript Author Manuscript Hum Mutat. Author manuscript; available in PMC 2021 April 01. Rice et al. Page 12 Author Manuscript Author Manuscript FIGURE 1. Schematic showing the positions of protein domains and their amino acid boundaries within the 1,025-residue IFIH1 protein. The 27 mutations ascertained in the present study are annotated, with the numbers in brackets indicating the number of families in which each mutation was observed. Three previously published mutations (p.Leu372Phe; p.Ala452Thr; p.Glu813Asp), not ascertained in our series, are also denoted (below the cartoon). CARD, caspase activation recruitment domain; Hel, helicase domain, where Hel1 and Hel2 are the two conserved core helicase domains and Hel2i is an insertion domain that is conserved in the RIG-I-like helicase family; P, pincer or bridge region connecting Hel2 to the C-terminal domain (CTD) involved in binding double stranded RNA Author Manuscript Author Manuscript Hum Mutat. Author manuscript; available in PMC 2021 April 01. Rice et al. Page 13 Author Manuscript FIGURE 2. Overview of phenotypes observed in the IFIH1-mutation-positive cohort. Classification of 68 of 74 individuals according to phenotype. For clarity, six individuals displaying Author Manuscript characteristics difficult to classify were omitted from this analysis Author Manuscript Author Manuscript Hum Mutat. Author manuscript; available in PMC 2021 April 01. Rice et al. Page 14 Author Manuscript Author Manuscript FIGURE 3. Mutation mapping. Structure of human IFIH1 (4GL2) in complex with double stranded RNA (dsRNA; blue stick model in the center). Only the RNA binding domain (helicase domain and C-terminal domain, CTD) are included in the crystal structure. Note that the Author Manuscript helicase domain consists of Hel1, Hel2i, and Hel2. Mutations are indicated by spheres using the following color code: residues in the ATP binding pocket (magenta), residues in the dsRNA binding surface (blue), residues within the main cluster but not directly involved in RNA binding or ATP binding (green), residues outside the main cluster (cyan), and residues previously reported by others but not in our cohort (orange). We considered all 27 mutations reported here plus three previously published mutations (p.Leu372Phe; p.Ala452Thr; p.Glu813Asp) not ascertained in our series. Residues p.Arg822, p.Arg824, and p.Ile956 are not shown because they are disordered in the crystal structure, but are expected to be located in the ATP binding (p.Arg822 and p.Arg824) and RNA binding (p.Ile956) pockets Author Manuscript Hum Mutat. Author manuscript; available in PMC 2021 April 01. Author Manuscript Author Manuscript Author Manuscript Author Manuscript TABLE 1 Details of individual IFIH1 mutations identified in the families included in the present data set Families (de novo Rice et al. inheritance; or, Associated number of symptomatic phenotypes (‘/’ and non- within Upregulation penetrant individuals family)(‘;’ of Assessment by where between interferon interferon CADD Var- cDNA change Protein change familial) families) signalling reporter assay gnomAD SIFT Polyphen2 score cards c.992C>G p.Thr331Arg AGS674 (de novo); AGS-SMS; SMS Yes Yes (de Carvalho et Novel Deleterious 0 Probably 29.7 22:23 AGS1972 (2;0) al., 2017) damaging 1.000 c.992C>T p.Thr331Ile AGS1938 (3;0) SMS Yes Yes (de Carvalho et Novel Deleterious 0 Probably 31 22:23 al., 2017) damaging 1.000 c.1009A>G p.Arg337Gly AGS237 (de novo) NR Yes Yes (Rice et al., Novel Tolerated 0.12 Probably 26.8 17:23 2014) damaging 1.000 c.1165G>A p.Gly389Arg AGS848 (2;1) AGS/SP/CNP Yes Yes (this paper) Novel Tolerated 0.88 Benign 0.108 5.325 01:23 c.1178A>T p.Asp393Val AGS626 (de novo) NR Yes Yes (Rice et al., Novel Deleterious Probably 28.6 16:23 2014) 0.01 damaging 0.998 c.1178A>C p.Asp393Ala AGS2586 (de novo) AGS Yes No Novel Deleterious Possibly 24.8 12:23 0.03 damaging 0.913 c.1331A>G p.Glu444Gly AGS2669 (de novo) AGS Yes Yes (this paper) Novel Deleterious 0 Probably 31 23.23 damaging 1 c.1347C>G p.Asn449Lys AGS1001 (de novo) SP Yes Yes (this paper) Novel Tolerated 0.64 Benign 0.163 13.91 03:23 c.1465G>A p.Ala489Thr a CLL/AGS- Yes Yes (Bursztejn et Novel Deleterious 0 Probably 32 21:23 AGS755 (3;0) SMS/SMS al., 2015) damaging 1.000 c.1483G>A p.Gly495Arg a SP-LLD/SP Yes Yes (Rice et al., Novel Deleterious Probably 23.3 14:23 AGS524 (2;0) 2014) 0.01 damaging 0.982 c.1747A>G p.Ile583Val AGS2369 (de novo) AGS Yes Yes (this paper) Novel Tolerated 0.48 Benign 0.00 0.573 5.23 Hum Mutat. Author manuscript; available in PMC 2021 April 01. c.2156C>T p.Ala719Val Hm_1 (de novo) AGS Yes No Novel Tolerated 0.07 Possibly 27.1 09:23 damaging 0.949 c.2159G>A p.Arg720Gln AGS102 (de novo); AGS; SP Yes Yes (Rice et al., Novel Deleterious 0 Probably 34 17:23 AGS647 (de novo); 2014) damaging 0.992 AGS1504 (de novo); AGS2422 (NPDT); AGS2548 (de novo); LD_0982.0 (de novo) c.2317G>C p.Glu773Gln AGS2399 (de novo) NR NA Yes (this paper) Novel Tolerated 0.27 Possibly 24.8 13:23 damaging 0.743 c.2335C>T p.Arg779Cys AGS376 (NPDT); AGS-LLD; SP- Yes Yes (Rice et al., Novel Deleterious Probably 34 21:23 AGS723 (NPDT); ICC; 2014) 0.01 damaging 1.000 AGS1004 (de novo); NR; unilateral AGS1156 (de novo); white matter Page 15 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Families (de novo inheritance; or, Associated number of symptomatic phenotypes (‘/’ and non- within Upregulation penetrant individuals family)(‘;’ of Assessment by where between interferon interferon CADD Var- Rice et al. cDNA change Protein change familial) families) signalling reporter assay gnomAD SIFT Polyphen2 score cards AGS2154 (1;1); AGS2180 disease/CNP; (de novo); AGS2507 (de AGS novo); LD_1030.0 (de novo) c.2336G>A p.Arg779His AGS163 (de novo); AGS; CNP; NR; Yes Yes (Rice et al., 1/244230 Tolerated 0.05 Probably 28.9 19:23 AGS259 (3;2); AGS1351 SP 2014) damaging 0.994 (de novo); AGS1509 (de novo); AGS2177 (1;2); Berg_1 (de novo); Orc_0098 (de novo); LD_1199.0 (de novo); LD_1381 (3;1); LD_1585.0 (de novo) c.2336G>T p.Arg779Leu LD_1067.0 (de novo) AGS Yes No Novel Tolerated 0.06 Probably 35 21:23 damaging 1.000 c.2342G>A p.Gly781Glu LD_0940.0 (de novo); NR; SP Yes No Novel Deleterious 0 Probably 32 19:23 LD_0943.0 (de novo) damaging 1.000 c.2404A>G p.Asn802Asp AGS2662 (de novo) NR Yes No Novel Tolerated 0.22 Probably 28.1 18:23 damaging 1.000 c.2407A>T p.Ile803Phe LD_1488.0 (de novo) AGS Yes Yes (this paper) Novel Tolerated 0.24 Benign 0.043 11.8 04:23 c.2465G>A p.Arg822Gln AGS1514 (de novo) SD-ICC Yes Yes (Rutsch et al., 6/244096 Deleterious 0 Probably 35 23:23 2015) damaging 1.000 c.2471G>A p.Arg824Lys AGS735 (de novo); NR; Isolated liver Yes No Novel Deleterious 0 Probably 34 22:23 AGS2222 (de novo) disease damaging 1.000 c.2486C>G p.Thr829Ser AGS1290 (2 siblings and AGS Yes No Novel Tolerated 0.73 Possibly 16.61 12:23 NPDT) damaging 0.512 Hum Mutat. Author manuscript; available in PMC 2021 April 01. c.2544T>G p.Asp848Glu AGS531 (3;2) SP-ICC/CNP Yes Yes (Ruaud et al., Novel Tolerated 0.4 Benign 0.004 10.08 02:23 2018) c.2561T>A p.Met854Lys AGS2081 (de novo) AGS/SMS Yes No Novel Deleterious 0 Probably 31 18:23 damaging 1.000 c.2866A>G p.Ile956Val AGS1430 (2;1) SP-ICC/CNP Yes Yes (this paper) Novel Tolerated 0.77 Benign 0.004 3.576 06:23 c.2936T>G p.Leu979Trp LD_1346.0 (de novo) AGS Yes Yes (this paper) Novel Deleterious Probably 26.6 16:23 0.01 damaging 1.000 Note: IFIH1 mutation annotation based on the reference complementary DNA sequence NM_022168.2. Abbreviations: AGS, Aicardi–Goutières syndrome; CLL, Chilblain-like lesions; CNP, clinical nonpenetrance; ICC, Intracranial calcification; LLD, Lupus-like disease; NPDT, no parental DNA testing; NR, neuro-regression; SD, spastic dystonia; SP, spastic paraparesis; SMS, Singleton Merten syndrome. a This mutation was shown to have been paternally inherited by the proband and to have occurred de novo in the proband’s father. Page 16 Author Manuscript Author Manuscript Author Manuscript Author Manuscript TABLE 2 Molecular and clinical data by family Inheritance (number Rice et al. of mutation-positive Previously Status at last contact Family Individual Sex cDNA Protein individuals) reported (reference) Clinical phenotype (age in years) AGS102 P1 M c.2159G>A p.Arg720Gln De novo Rice et al. (2014) AGS Deceased (2) AGS163 P1 M c.2336G>A p.Arg779His De novo Rice et al. (2014) AGS Alive (13) AGS237 P1 M c.1009A>G p.Arg337Gly De novo Rice et al. 2014; Neuroregression and SD starting at Deceased (16) (LD_0762) Adang et al., 2018 age 15 months AGS259 P1 M c.2336G>A p.Arg779His Familial (3) Rice et al. (2014) AGS Alive (13) P2 (father of P1) M Clinically nonpenetrant Alive (54) P3 (mother of P2) F Clinically nonpenetrant Deceased (84) AGS376 P1 M c.2335C>T p.Arg779Cys No parental testing Rice et al. (2014) AGS with LLD Deceased (3) AGS524 P1 F c.1483G>A p.Gly495Arg Familial (2)(shown to Rice et al. (2014); SP with LLD and AQP4 + TM Alive (10) have occurred de novo Hacohen et al. 2015; in P2) Crow et al. 2015; McLellan et al. 2018 P2 (father of P1) M Pure SP Alive (39) AGS531 P1 F c.2544T>G p.Asp848Glu Familial (5) Ruaud et al. (2018) SP with ICC Alive (13) P2 (brother of P1) M Clinically nonpenetrant Alive (13) P3 (father of P1 and P2) M SP with ICC Alive (40) P4 (brother of P3) M SP with ICC Alive (38) P5 (father of P3 and P4) M Clinically non-penetrant Alive (66) AGS626 P1 M c.1178A>T p.Asp393Val De novo Rice et al. (2014) Neuroregression and SD starting at Alive (13) 13 months Hum Mutat. Author manuscript; available in PMC 2021 April 01. AGS647 P1 M c.2159G>A p.Arg720Gln De novo Rice et al. (2014) AGS Alive (2) AGS674 P1 M c.992C>G p.Thr331Arg De novo Unreported SP-SMS overlap Alive (14) AGS723 P1 F c.2335C>T p.Arg779Cys Mother negative; no Unreported SP with ICC Alive (19) paternal DNA AGS735 P1 M c.2471G>A p.Arg824Lys De novo Galli et al. 2018 Neuroregression and SD starting at Alive (19) 12 months AGS755 P1 M c.1465G>A p.Ala489Thr Familial (3) Bursztejn et al. (2015) CLL Alive (4) P2 (brother of P1) M AGS-SMS overlap Alive (3) P3 (father of P1 and P2) M SMS-like Alive (41) AGS848 P1 M c.1165G>A p.Gly389Arg Familial (3) Unreported AGS Alive (8) Page 17 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Inheritance (number of mutation-positive Previously Status at last contact Family Individual Sex cDNA Protein individuals) reported (reference) Clinical phenotype (age in years) P2 (father of P1) M SP Alive (42) Rice et al. P3 (maternal grandmother F Clinically nonpenetrant Alive (84) of P2) AGS1001 P1 M c.1347C>G p.Asn449Lys De novo Unreported SP Alive (19) AGS1004 P1 F c.2335C>T p.Arg779Cys De novo Unreported AGS (neuroregression with onset Alive (8) at age 8 months) AGS1156 P1 M c.2335C>T p.Arg779Cys De novo Kothur et al. 2018 AGS (neuroregression with onset Alive (5) at age 8 months) AGS1290 P1 M c.2486C>G p.Thr829Ser 2 affected (no parental Unreported AGS Alive (6) DNA) P2 (brother of P1) M AGS Alive (4) AGS1351 P1 F c.2336G>A p.Arg779His De novo Unreported AGS Deceased (2) AGS1430 P1 M c.2866A>G p.Ile956Val Familial (3) Unreported SP with ICC with onset at age 6 Alive (14) years P2 (father of P1) M SP with onset at age 2 years Alive (50) P3 (father of P2) M Clinically non-penetrant Alive (72) AGS1504 P1 F c.2159G>A p.Arg720Gln De novo Unreported AGS Alive (10) (LD_1175) AGS1509 P1 M c.2336G>A p.Arg779His De novo Unreported AGS Alive (8) AGS1514 P1 M c.2465G>A p.Arg822Gln De novo Buers et al. (2017) SD with ICC Alive (6) AGS1938 P1 F c.992C>T p.Thr331Ile Familial (3) de Carvalho et al. SMS Alive (18) (2017) P2 (mother of P1) F SMS Alive (45) Hum Mutat. Author manuscript; available in PMC 2021 April 01. P3 (sister of P2) F SMS Alive (27) AGS1972 P1 F c.992C>G p.Thr331Arg Familial (2) de Carvalho et al. SMS Alive (9) (2017) P2 (father of P1) M SMS Alive (47) AGS2081 P1 M c.2561T>A p.Met854Lys De novo Unreported SP-SMS overlap Alive (12) AGS2154 P1 M c.2335C>T p.Arg779Cys Familial (2) Unreported Unilateral white matter disease Alive (13) (LD_1240) with normal development P2 (mother of P1) F Clinically nonpenetrant Alive (40) AGS2177 P1 M c.2336G>A p.Arg779His Familial (3) Neuroregression and SD starting at Alive (29) age 12 months P2 (mother of P1) F Clinically nonpenetrant Alive (62) Page 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Inheritance (number of mutation-positive Previously Status at last contact Family Individual Sex cDNA Protein individuals) reported (reference) Clinical phenotype (age in years) P3 (sister of P1) F Clinically nonpenetrant Alive (33) Rice et al. AGS2180 P1 F c.2335C>T p.Arg779Cys De novo Unreported AGS Alive (4) AGS2222 P1 M c.2471G>A p.Arg824Lys De novo Unreported Isolated liver disease Alive (9) AGS2369 P1 M c.1747A>G p.Ile583Val De novo Unreported AGS Alive (10) AGS2399 P1 M c.2317G>C p.Glu773Gln De novo Unreported Neuroregression and SD starting at Alive (8) age 16 months AGS2422 P1 F c.2159G>A p.Arg720Gln No parental testing Unreported SP Alive (38) AGS2507 P1 F c.2335C>T p.Arg779Cys De novo Unreported AGS Alive (1) AGS2548 P1 M c.2159G>A p.Arg720Gln De novo Unreported AGS Alive (3) AGS2586 P1 M c.1178A>C p.Asp393Ala De novo Unreported AGS-like with frank regression at Alive (3) age 21 months AGS2662 P1 F c.2404A>G p.Asn802Asp De novo Unreported Neuroregression and SD starting at Alive (1) (LD_1640) age 11 months AGS2669 P1 M c.1331A>G p.Glu444Gly De novo Unreported AGS Deceased (0.5) Hm_1 P1 F c.2156C>T p.Ala719Val De novo Unreported AGS Alive (2) Berg_1 P1 F c.2336G>A p.Arg779His De novo Unreported Neuroregression and SD starting at Alive (7) age 9 months Orc_0098 P1 M c.2336G>A p.Arg779His De novo Unreported AGS Alive (4) LD_0940.0 P1 M c.2342G>A p.Gly781Glu De novo Unreported Neuroregression and SD starting at Alive (5) age 15 months LD_0943.0 P1 F c.2342G>A p.Gly781Glu De novo Unreported SP Alive (14) LD_0982.0 P1 M c.2159G>A p.Arg720Gln De novo Adang et al. (2018); AGS Alive (9) Case 2 Hum Mutat. Author manuscript; available in PMC 2021 April 01. LD_1030.0 P1 F c.2335C>T p.Arg779Cys De novo Unreported AGS Alive (5) LD_1067.0 P1 M c.2336G>T p.Arg779Leu De novo Unreported AGS Alive (8) LD_1199.0 P1 F c.2336G>A p.Arg779His De novo Unreported AGS Alive (4) LD_1346.0 P1 M c.2936T>G p.Leu979Trp De novo Adang et al. (2018); AGS Deceased (0.4) Case 3 LD_1381 (Hart) P1 F c.2336G>A p.Arg779His Familial (4) Unreported SP Alive (4) P2 (brother of P1) M SP Alive (3) P3 (father of P1 and P2) M SP Alive (32) P4 (father of P3) M Clinically nonpenetrant Alive (68) LD_1488.0 P1 F c.2407A>T p.Ile803Phe De novo Unreported AGS Alive (2) Page 19 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Inheritance (number of mutation-positive Previously Status at last contact Family Individual Sex cDNA Protein individuals) reported (reference) Clinical phenotype (age in years) LD_1585.0 P1 F c.2336G>A p.Arg779His De novo Unreported AGS Alive (5) Rice et al. Note: IFIH1 mutation annotation based on the reference complementary DNA sequence NM_022168.2. Abbreviations: AGS, Aicardi–Goutières syndrome; CLL, Chilblain-like lesions; F, Female; ICC, intracranial calcification; LLD, Lupus-like disease; M, Male; SD, spastic dystonia; SP, spastic paraparesis; SMS: Singleton Merten syndrome; TM, transverse myelitis Hum Mutat. Author manuscript; available in PMC 2021 April 01. Page 20