Original Article

Mutations in ADAR1, IFIH1, and RNASEH2B Presenting As Spastic Paraplegia Yanick J. Crow1,2,3 Maha S. Zaki4 Mohamed S. Abdel-Hamid5 Ghada Abdel-Salam4 Odile Boespflug-Tanguy6,7 Nuno J. V. Cordeiro8 Joseph G. Gleeson9 Nirmala Rani Gowrinathan10 Vincent Laugel11 Florence Renaldo12,13,14 Diana Rodriguez15 John H. Livingston16 Gillian I. Rice3 1 INSERM UMR 1163, Laboratory of Neurogenetics and

Neuroinflammation, Institut Imagine, Paris, France 2 Paris Descartes–Sorbonne Paris Cité University, Paris, France 3 Manchester Academic Health Science Centre, University of Manchester, Genetic Medicine, Manchester, United Kingdom 4 Division of Human Genetics and Genome Research, Department of Clinical Genetics, National Research Center, Cairo, Egypt 5 Division of Human Genetics and Genome Research, Department of Medical Genetics, National Research Center, Cairo, Egypt 6 National Reference Center for Rare Diseases “leukodystrophies,” INSERM U676, Université Paris Diderot, Sorbonne Paris Cité Université, Paris, France 7 Pediatric Neurology and Metabolic Disease Service, Hôpital Robert Debré, Paris, France 8 Department of Paediatrics, Rainbow House NHS Ayrshire & Arran, Irvine, Scotland, United Kingdom 9 Department of Neurosciences, University of California, San Diego, La Jolla, California, United States 10 Kaiser Permanente Los Angeles Medical Center, Los Angeles, California, United States 11 Pediatric Neurology, Strasbourg–Hautepierre University Hospital, Avenue Moliere, Strasbourg, France 12 Centre de Référence des Leucodystrophies, Service de Neuropédiatrie et Maladies Métaboliques; Hôpital Robert Debré, APHP, Paris, France 13 Centre de Référence des maladies Neurogénétiques de l’enfant à l’adulte, Service de Neuropédiatrie, Hôpital Armand Trousseau, APHP, Paris, France 14 UMR 1141; Physiopathologie et neuroprotection des atteintes du cerveau en développement/Pathophysiology and neuroprotection of perinatal brain lesions; Université Paris Diderot, Paris, France 15 Service de Neuropédiatrie, Hôpital Armand Trousseau, Paris, France 16 Department of Paediatric Neurology, F Floor Martin Wing, Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom

Address for correspondence Yanick. J. Crow, MD, Institut Imagine, 24 Boulevard Du Montparnasse, 75015, Paris, France (e-mail: [email protected]).

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Neuropediatrics 2014;45:386–391.

received May 22, 2014 accepted July 28, 2014 published online September 22, 2014

© 2014 Georg Thieme Verlag KG Stuttgart · New York

DOI http://dx.doi.org/ 10.1055/s-0034-1389161. ISSN 0174-304X.

Abstract

Keywords

► Aicardi–Goutières syndrome ► spastic paraparesis ► RNASEH2B ► ADAR1 ► IFIH1 ► type I interferon ► type I interferonopathy

Crow et al.

Background Hereditary spastic paraplegia is a neurodegenerative phenotype characterized by a progressive loss of corticospinal motor tract function. In a majority of affected individuals the pathogenesis remains undetermined. Methods We identified a series of patients with a phenotype of nonsyndromic spastic paraplegia in whom no diagnosis had been reached before exome sequencing. We measured the expression of interferon stimulated genes (ISGs) in peripheral blood from these patients. Results Five patients from four families with previously unexplained spastic paraplegia were identified with mutations in either ADAR1 (one patient), IFIH1 (one patient), or RNASEH2B (three patients from two families). All patients were developmentally normal before the onset of features beginning in the second year of life. All patients remain of normal intellect. Four patients demonstrated normal neuroimaging, while a single patient had features of nonspecific dysmyelination. The patients with ADAR1 and IFIH1related disease showed a robust interferon signature. The patients with mutations in RNASEH2B demonstrated no (two patients) or a minimal (one patient) upregulation of ISGs compared with controls. Conclusions Mutations in ADAR1, IFIH1, and RNASEH2B can cause a phenotype of spastic paraplegia with normal neuroimaging, or in association with nonspecific dysmyelination. Although the presence of an interferon signature can be helpful in interpreting the significance of gene variants in this context, patients with pathogenic mutations in RNASEH2B may demonstrate no upregulation of ISGs in peripheral blood. However, it remains possible that type I interferons act as a neurotoxin in the context of all genotypes.

Introduction The hereditary spastic paraplegias (HSPs) are a group of genetically heterogeneous neurodegenerative disorders with a prevalence of between 3 and 10 per 100,000 individuals.1 Hallmark features are axonal degeneration and progressive lower limb spasticity resulting from a loss of corticospinal tract function. HSP is classified into two broad categories on the basis of the presence, complicated HSP, or absence, uncomplicated HSP, of additional clinical features such as intellectual disability, seizures, ataxia, peripheral neuropathy, skin abnormalities, and visual defects. The condition displays several distinct modes of transmission, including autosomal dominant, autosomal recessive, and X-linked inheritance. Currently, a significant number of patients with an HSP phenotype remain without a molecular diagnosis.2 Aicardi–Goutières syndrome (AGS) is an inflammatory disease particularly affecting the brain and the skin, occurring due to mutations in any of the genes encoding the DNA exonuclease TREX1, the three nonallelic components of the RNase H2 endonuclease complex, the deoxynucleoside triphosphate triphosphohydrolase SAMHD1, the doublestranded RNA editing enzyme ADAR1, and the double-stranded cytosolic RNA sensor IFIH1/MDA5.3–7 Although the majority of recognized patients conform to one of two fairly stereotyped “classical” phenotypes with onset in the first year of life, there is now an extensive literature8–11 reporting a broader spectrum of disease presentation, progression, and outcome. In all the cases, it is considered likely that the

pathology underlying AGS relates to an inappropriate induction of a type I interferon-mediated immune response,12 and that type I interferons act as a neurotoxin.13–15 Here, we show that mutations in the AGS-associated genes ADAR1, IFIH1, and RNASEH2B can cause a clinical phenotype of spastic paraplegia in the absence of other distinguishing features.

Methods Patients Patients were ascertained through our own clinical practice and through international clinical collaborations. Exome, and confirmatory Sanger, sequencing was performed before inclusion in this report.

Interferon-Stimulated Gene Assessment and Interferon Score Methods for the assessment of the expression of a panel of interferon-stimulated genes (ISGs) have been described previously. Briefly, total RNA was extracted from whole blood using a PAXgene (PreAnalytix, Hombrechtikon, Switzerland) RNA isolation kit. Quantitative reverse transcription polymerase chain reaction (qPCR) analysis was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems, Paisley, United Kingdom), and cDNA derived from 40 ng total RNA. Using TaqMan probes for IFI27 (Hs01086370_m1), IFI44L (Hs00199115_m1), IFIT1 (Hs00356631_g1), ISG15 (Hs00192713_m1), RSAD2 (Hs01057264_m1), and SIGLEC1 (Hs00988063_m1), the Neuropediatrics

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Mutations in ADAR1, IFIH1, and RNASEH2B Presenting As Spastic Paraplegia

Mutations in ADAR1, IFIH1, and RNASEH2B Presenting As Spastic Paraplegia relative abundance of each target transcript was normalized to the expression level of HPRT1 (Hs03929096_g1) and 18S (Hs999999001_s1), and assessed with the Applied Biosystems StepOne Software v2.1 and DataAssist Software v.3.01 (Applied Biosystems, Paisley, United Kingdom). The median fold change of the six ISGs, when compared with the median of 29 healthy controls combined, was used to create an interferon score for each patient. The respiratory quotient is equal to 2
ΔΔCt, that is, the normalized fold change relative to a control. When a patient was assayed on more than one occasion, the data for repeat measurements were combined to calculate a mean value. The mean interferon score of the controls plus two standard deviations (SDs) above the mean (þ 2 SD) was calculated. Scores above this value (> 2.466) were designated as positive. The study was approved by a U. K. Multicentre Research Ethics Committee (reference number 04:MRE00/19).

Results Patient 1 (F699). This male child was born to nonconsanguineous parents of Hispanic descent. There was no family history of note. The pregnancy was normal, and the child was delivered vaginally at full term weighing 3.5 kg. He demonstrated normal early development, walking, and saying his first words at 12 months of age (►Supplementary Table S1 online-only). At the age of 2 years, the child began to trip and fall more frequently than before, and was noted to be walking on his tiptoes. Before this point, he was able to run, squat and get up, and had a normal gait. Now, at the age of 5 years, he is unable to walk independently. He can cruise very short distances (Gross Motor Function Classification System [GMFCS] III-IV). He has no weakness of his arms or hands, no difficulties with chewing, swallowing, and talking, and no bowel or bladder problems. His cognition is unaffected. The examination is unremarkable except for signs of spasticity in the lower limbs, with increased tone, reduced power, bilateral ankle clonus, an up-going plantar reflex on the right, and a spastic diplegic gait. Extensive testing of blood and cerebrospinal fluid (CSF) was normal, and magnetic resonance imaging (MRI) of the

Crow et al.

brain and spine was normal on two occasions (at the age of 3 years 9 months, and at 5 years). Nerve conduction study and electromyography were normal. Exome sequencing, followed by confirmatory Sanger sequencing of the child and his parents, identified a de novo heterozygous c.3019G > A (p.Gly1007Arg) mutation in ADAR1. This is a mutation that has been seen recurrently in patients with a phenotype of classical AGS, bilateral striatal necrosis, and dyschromatosis symmetrica hereditaria with dystonia, and for which functional studies suggest a dominant gain of function (►Table 1; ►Supplementary Table S2 (online-only). An interferon score of 16.8 was recorded at the age of 4.91 years (►Fig. 1). Patient 2 (F524). This male was born to nonconsanguineous white British parents. The birth and perinatal period were uneventful, and he acquired all of his early milestones appropriately—sitting at age of 6 months and walking independently before his first birthday. At the age of 2 years, it was noted that he was toe-walking, and he started to fall more than previously. By the age of 3 years, he had been given a diagnosis of cerebral palsy, and between the ages of 4 and 15 years, he underwent multiple tendon lengthening operations. His disorder has been very slowly progressive, so that he can now only walk with the aid of sticks (GMFCS II). At the age of 33 years, he demonstrates significant lower limb spasticity, with no involvement of the upper limbs. He is cognitively fully intact, and has experienced no other problems with his health. At this time, he had a borderline positive ANA result (titer of 1 in 160), with normal anti-DNA, anti-Ro, La, Sm, RNP, Scl-70, Jo-1, centromere, and ANCA antibody titers. Cardiolipin Ab (IgG) and (IgM), rheumatoid factor, erythrocyte sedimentation rate and C-reactive protein were also normal. He had a normal cranial and spinal MRI at the age of 29 years. Exome sequencing revealed a de novo heterozygous c.1483G > A (p.Gly495Arg) mutation in IFIH1. Microsatellite analysis of parental samples confirmed paternity and maternity. Functional studies suggest that this mutation confers a dominant gain of function. He demonstrated a persistently elevated interferon score on five occasions tested over an

Table 1 Ancestry, family members tested, consanguinity status, and sequence alterations in patients with uncomplicated spastic paraplegia Patient

Ancestry

Tested

Consanguinity

Gene

Nucleotide alteration

Amino Acid alteration

1 (F699)

European American

1A, M, F

No

ADAR1

c.3019G > A (Het)(de novo)

p.Gly1007Arg (Het)(de novo)

2 (F524)

White British

1A, M, F

No

IFIH1

c.1483G > A (Het)(de novo)

p.Gly495Arg (Het)(de novo)

3 (F739)a

Egyptian

2A, M, F

Yes

RNASEH2B

c.529G > A (Hom)

p.Ala177Thr (Hom)

a

Egyptian

2A, M, F

Yes

RNASEH2B

c.529G > A (Hom)

p.Ala177Thr (Hom)

North African

1A, M, F

No

RNASEH2B

c.529G > A (Hom)

p.Ala177Thr (Hom)

4 (F739) 5 (F768)

Abbreviations: A, affected; F, father; Hom, homozygous; Het, heterozygous; M ¼ mother. a Patients 3 and 4 are siblings. Note: ADAR1 accession number NM_001111.4. IFIH1 accession number NM_022168.2. RNASEH2B accession number NM_024570.3. Project (ESP) Exome Variant Server: http://evs.gs.washington.edu/EVS/. Neuropediatrics

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Fig. 1 Interferon score in all patients and controls calculated from the median fold change in relative quantification value for a panel of six interferon-stimulated genes. For patients with repeat samples, the mean combined measurement is shown. The horizontal lines show the median interferon score in patients and controls. Negative scores are those less than 2,466 (mean of control interferon score plus two standard deviation [SD] of mean—in white) and positive scores are 2,466 or greater (in black).

18-month period (15.2, 32.3 years; 11.5, 32.8 years; 17.9, 33.1 years; 29.3, 33.62 years; and 9.9, 33.9 years). Patients 3 and 4 (F739_1 and F739_2). These female siblings were born to nonconsanguineous Egyptian parents. There was no family history of note. The pregnancies were normal, with each child delivered vaginally at full term weighing over 3 kg. Both children demonstrated normal early development, walking at 12 months of age, and saying their first words at the same age. An abnormal gait was noted in patient 3, the older sister to patient 4, soon after she started walking. By the age of 18 months, she was falling repeatedly. She developed a scissoring gait over the next 6 months. Patient 4 had a normal gait until the age of 2 years. From this point, she began to fall recurrently, and developed a scissoring gait like her sister. Both children are of normal intellect, with normal head sizes. The children are still ambulant at the ages of 11 years and 7 years (GMFCS I). Neurological evaluation of the upper limbs demonstrated some mild increase in tone with brisk reflexes. The lower limbs were weak (muscle power grade 4), and hypertonic with brisk reflexes, ankle and patellar clonus, and a positive Babinski sign. Cerebellar function, sensation, speech, and cranial nerve examination were normal except in patient 3, who was recognized to have a previously undetected blindness of the left eye with features of optic nerve atrophy on fundal examination. Brain and spine MRI, hearing tests, electromyography, nerve conduction, and metabolic screening were all normal. Brain computed tomography (CT) performed at the ages of 11 years (patient 3) and 7 years (patient 4) was normal with no basal ganglia calcification. Exome sequencing, followed by confirmatory Sanger sequencing, identified a homozygous

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c.529G > A (p.Ala177Thr) mutation in RNASEH2B. Both the parents were heterozygous for this variant. This amino acid substitution represents the most frequent mutation seen in patients with RNASEH2B-associated AGS. An interferon score of 1.3 (within the normal range of controls) and of 3.0 (normal < 2.46) was recorded in the sisters at the respective ages 10.66 and 7.16 years. Patient 5 (F768). This male child was born to nonconsanguineous North African parents. There was no family history of note. The pregnancy was complicated by gestational diabetes and the child was delivered by caesarean section at full term weighing 4.2 kg. He demonstrated normal early motor development, walking at 14 months of age. He said his first words at the age of 24 months. His motor development was considered to be normal until 21 months of age when he was noted to fall more than previously. Now, at the age of 5 years, his condition is stable with upper motor neuron signs in the lower limbs, including increased reflexes and bilateral up-going plantars with clonus. He runs with a degree of scissoring (GMFCS I). He is normocephalic, his language is within normal limits and he is considered to be of normal intellect. A cranial CT scan at age 22 months was normal. Cranial MRI 1 month later, and again at 3 years of age, demonstrated diffuse, nonspecific high signal on T2-weighted imaging with some dilatation of the lateral ventricles. Extensive investigations including immunologic and metabolic testing, peripheral neurophysiology, and ophthalmologic examination were normal. Exome sequencing, followed by confirmatory Sanger sequencing, identified a homozygous c.529G > A (p. Ala177Thr) mutation in RNASEH2B. Both the parents were heterozygous for this variant. CSF analysis revealed a lymphocytosis (26 white cells/mm3). Interferon activity was below the limits of detection in both blood and CSF. An interferon score of 1.9, within the range of normal controls, was recorded at age 5.69 years.

Discussion AGS is a genetically determined disorder, most particularly affecting the central nervous system and the skin, characterized by the inappropriate induction of a type I interferonmediated immune response. In a significant minority of patients with AGS problems are recognized at birth, that is, the disease process begins in utero16. Over time, severe neurological dysfunction manifests as progressive microcephaly, spasticity, dystonia, psychomotor retardation and, in approximately 17% of cases, death by the age of 5 years. Typical neuroradiological features include intracranial calcification, marked white matter changes and significant brain atrophy.17 More frequently, a later-onset presentation of AGS is seen, in some cases occurring after several months of normal development. This form of the disease may be associated with a lesser degree of neurological dysfunction, albeit still severe in the majority of cases. Although most patients with AGS conform to the relatively stereotyped phenotypes described earlier, there is Neuropediatrics

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Mutations in ADAR1, IFIH1, and RNASEH2B Presenting As Spastic Paraplegia

Mutations in ADAR1, IFIH1, and RNASEH2B Presenting As Spastic Paraplegia now an extensive literature reporting a broader spectrum of disease presentation, progression, and outcome. Thus, neurological dysfunction is not always marked, nor indeed necessarily present at all; microcephaly is not invariable; onset is not always in the first year of life; and intracranial calcification and white matter changes are not inevitable. Importantly, disparity in the clinical phenotype can be seen even within the same family, 10 thereby, highlighting the role of modifying factors. Furthermore, distinct neurological phenotypes have been observed in the context of context of SAMHD1and ADAR1-mutations of intracranial large vessel disease (intracranial stenosis, in some cases reminiscent of moyamoya syndrome, and aneurysms) 8 and bilateral striatal necrosis, respectively. 11 Here, we describe five patients with mutations in three of the seven known AGS-related genes (►Table 1; (►Supplementary Table S1 online-only), who conform to a phenotype of nonsyndromic spastic paraplegia. In four of these patients, brain and spinal imaging were completely normal, in one patient at the age of 29 years, while a further patient demonstrated nonspecific patchy demyelination. The disease phenotype began in the second year of life in all the cases. Slow progression was clear in the oldest patient (patient 2) we describe (with a dominant mutation in IFIH1). All patients are of normal intellect, with normal head growth, an absence of chilblain lesions, and no other features of note (except for probably unrelated unilateral optic nerve atrophy in patient 3). Despite the absence of the more characteristic features of “classical” AGS, the mutations identified in these patients are very likely pathogenic, with the mutations seen in patients 1 and 3—patient 5 having been reported in multiple individuals with a phenotype typical of AGS. Indeed, we have observed several other cases of AGS, including a single child with TREX1 mutations, several cases with mutations in RNASEH2B, and one child with mutations in SAMHD1, where the clinical phenotype is dominated by a spastic paraplegia. These patients are not included here only because they demonstrate additional features, for example, predominant chilblains, intellectual compromise, or intracranial calcifications. We have previously reported an interferon signature as a reliable biomarker for AGS, present in 75% of patients with RNASEH2B mutations, and in almost 100% of patients with mutations in any of the other AGS-related genes tested at any age.12 There is strong evidence that interferon is a neurotoxin, and we consider it likely that inappropriate exposure to type I interferons is relevant to the AGS-related neurological phenotype. The absence of an interferon signature in blood, or of increased interferon activity in the CSF (patient 5), in two of three RNASEH2B mutated patients, with a minimal increase in the third (patient 3) described here, does not rule out this possibility, as toxicity might relate to local cellular factors or changes in exposure over time (►Fig. 1). With the integration of new sequencing technologies into standard clinical practice, we predict that the spectrum of phenotypes associated with mutations in the AGS-related Neuropediatrics

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genes will broaden further. These observations beg the question as to whether such cases should actually be referred to as AGS. Irrespective of questions of nosology, it is probable that these phenotypes likely relate to a common pathology, and might therefore potentially benefit from similar anti-interferon/anti-inflammatory therapeutic strategies.18

Acknowledgments We sincerely thank the patients and their families included in this research. We also thank all other clinicians who have contributed patients not included here. This work was funded through the European Union’s Seventh Framework Program (FP7/2007–2013) under grant agreement 241779, the European Research Council (GA 309449: Fellowship to Y.J.C), and a state subsidy managed by the National Research Agency (France) under the “Investments for the Future” program bearing the reference ANR-10IAHU-01. This article is dedicated to the memory of Dr. John L. Tolmie

Funding European Union’s Seventh Framework Program (FP7/ 2007–2013) under grant agreement 241779. European Research Council (GA 309449: Fellowship to Y.J.C). National Research Agency (France) under the “Investments for the Future” program (ANR-10-IAHU-01). Financial Disclosure None of the authors have any financial disclosures to report. Author Contributions Y.J.C collated and reviewed the clinical data. G.I.R. performed quantitative PCR analysis. Y.J.C wrote the article with the assistance of G.I.R. All other authors supplied clinical data. All authors critically reviewed the article and agreed to its publication.

References 1 Blackstone C. Cellular pathways of hereditary spastic paraplegia.

Annu Rev Neurosci 2012;35:25–47 2 Novarino G, Fenstermaker AG, Zaki MS, et al. Exome sequencing

links corticospinal motor neuron disease to common neurodegenerative disorders. Science 2014;343(6170):506–511 3 Crow YJ, Hayward BE, Parmar R, et al. Mutations in the gene encoding the 3′-5′ DNA exonuclease TREX1 cause Aicardi-Goutières syndrome at the AGS1 locus. Nat Genet 2006;38(8): 917–920 4 Crow YJ, Leitch A, Hayward BE, et al. Mutations in genes encoding ribonuclease H2 subunits cause Aicardi-Goutières syndrome and mimic congenital viral brain infection. Nat Genet 2006;38(8): 910–916 5 Rice GI, Bond J, Asipu A, et al. Mutations involved in AicardiGoutières syndrome implicate SAMHD1 as regulator of the innate immune response. Nat Genet 2009;41(7):829–832

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6 Rice GI, Kasher PR, Forte GM, et al. Mutations in ADAR1 cause

12 Rice GI, Forte GM, Szynkiewicz M, et al. Assessment of interferon-

Aicardi-Goutières syndrome associated with a type I interferon signature. Nat Genet 2012;44(11):1243–1248 Rice GI, del Toro Duany Y, Jenkinson EM, et al. Gain-of-function mutations in IFIH1 cause a spectrum of human disease phenotypes associated with upregulated type I interferon signaling. Nat Genet 2014;46(5):503–509 Ramesh V, Bernardi B, Stafa A, et al. Intracerebral large artery disease in Aicardi-Goutières syndrome implicates SAMHD1 in vascular homeostasis. Dev Med Child Neurol 2010;52(8): 725–732 Dale RC, Gornall H, Singh-Grewal D, Alcausin M, Rice GI, Crow YJ. Familial Aicardi-Goutières syndrome due to SAMHD1 mutations is associated with chronic arthropathy and contractures. Am J Med Genet A 2010;152A(4):938–942 Vogt J, Agrawal S, Ibrahim Z, et al. Striking intrafamilial phenotypic variability in Aicardi-Goutières syndrome associated with the recurrent Asian founder mutation in RNASEH2C. Am J Med Genet A 2013;161A(2):338–342 Livingston JH, Lin JP, Dale RC, et al. A type I interferon signature identifies bilateral striatal necrosis due to mutations in ADAR1. J Med Genet 2014;51(2):76–82

related biomarkers in Aicardi-Goutières syndrome associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR: a case-control study. Lancet Neurol 2013;12(12):1159–1169 Akwa Y, Hassett DE, Eloranta ML, et al. Transgenic expression of IFN-alpha in the central nervous system of mice protects against lethal neurotropic viral infection but induces inflammation and neurodegeneration. J Immunol 1998;161(9):5016–5026 Campbell IL, Krucker T, Steffensen S, et al. Structural and functional neuropathology in transgenic mice with CNS expression of IFNalpha. Brain Res 1999;835(1):46–61 Fritz-French C, Tyor W. Interferon-α (IFNα) neurotoxicity. Cytokine Growth Factor Rev 2012;23(1-2):7–14 Rice G, Patrick T, Parmar R, et al. Clinical and molecular phenotype of Aicardi-Goutieres syndrome. Am J Hum Genet 2007;81(4): 713–725 Uggetti C, La Piana R, Orcesi S, Egitto MG, Crow YJ, Fazzi E. AicardiGoutieres syndrome: neuroradiologic findings and follow-up. AJNR Am J Neuroradiol 2009;30(10):1971–1976 Crow YJ, Vanderver A, Orcesi S, Kuijpers TW, Rice GI. Therapies in Aicardi-Goutières syndrome. Clin Exp Immunol 2014; 175(1):1–8

7

8

9

10

11

13

14

15 16

17

18

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IFIH1

ADAR1 M

Gene

Sex

No. 6/2014

Slowly progressive over 3 y. GMFCS ¼ III-IV Can cruise short distances but cannot walk independently. No upper limb involvement. Intellect normal age 5 y Normal

Progression

Current developmental status

Imaging (age) 15.2 (32.3); 11.5 (32.8); 17.9 (33.1); 29.3 (33.62); 9.9 (33.9)

Normal (29 y)

Uses sticks to walk. No upper limb involvement. Intellect normal age 33 y

Slowly progressive over 31 y. GMFCS ¼ II

Noted that he was toe-walking, and falling more than previously

1.3 (10.66)

3.0 (7.16)

Normal

1.9 (5.69)

Nonspecific leukodystrophy

Can run, with a scissoring gait. No upper limb involvement. Intellect normal Ambulant at age 7 y. Walks with scissoring gait. No upper limb involvement. Intellect normal

Ambulant at age 11 y. Walks with scissoring gait. No upper limb involvement. Intellect normal Normal

Slowly progressive over 12 mo then stabilizing. GMFCS ¼ I

Falling more than previously

Normal

23 mo

Slowly progressive over 12 mo then apparently stabilizing. GMFCS ¼ I

Recurrent falls and scissoring gait

Normal

24 mo

Slowly progressive over 12 mo then apparently stabilizing. GMFCS ¼ I

Recurrent falls and scissoring gait

Normal

North African

M

RNASEH2B

Patient 5 (F768)

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Abbreviations: CSF, cerebrospinal fluid; GMFCS, Gross Motor Function Classification System (LeveI I ¼ Athletic mobility and managing stairs unaided; Level V ¼ no means of independent mobility); ISG, interferon stimulated genes; mo ¼ months; wk ¼ weeks; RQ, respiratory quotient y ¼ years; a Summary clinical and molecular details of patient 2 have been published previously. b Interferon score is calculated from the median fold change in RQ values for a panel of six ISGs measured in whole blood, compared with the median of 29 healthy controls. RQ is equal to 2-ΔΔCt, with -ΔΔCt  standard deviations, that is the normalized fold change relative to a calibrator.

Interferon score (age tested in y)

16.8 (4.91)

Falling more than previously, toe-walking, and loss of independent walking

Features at presentation

Normal

Egyptian

Sister to patient 3

F

RNASEH2B

Patient 4 (F739_2)

Mutations in ADAR1, IFIH1, and RNASEH2B Presenting As Spastic Paraplegia

b

Normal

Developmental status before onset

12 mo

24 mo

Age at presentation

22 mo

Egyptian

European American

Ancestry

White British

Sister to patient 4

F

RNASEH2B

Patient 3 (F739_1)

Note

M

Patient 2 (F524) a

Patient 1 (F699)

Patient

Supplementary Table S1 Clinical details of cases with features of spastic paraparesis and mutations in Aicardi–Goutières syndrome–related genes

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Supplementary Table S2 Summary evidence in favor of pathogenicity of the amino acid substitutions Gene

cDNA

Protein

SIFT

PolyPhen-2

EVS frequency

ADAR1

c.3019G>A

p.Gly1007Arg

Deleterious (0)

Probably damaging (1.0)

0/13006

IFIH1

c.1483G>A

p.Gly495Arg

Deleterious (0.01)

Probably damaging (0.982)

0/13006

RNASEH2B

c.529G>A

p.Ala177Thr

Tolerated (0.23)

Probably damaging (0.992)

26/12980

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Source: SIFT: http://sift.jcvi.org/www/SIFT_enst_submit.html and PolyPhen-2: http://genetics.bwh.harvard.edu/pph2/. SIFT score ranges from 0 to 1. The amino acid change is predicted to be damaging if the score is 0.05, and tolerated if the score is > 0.05. Note: PolyPhen2 score ranges from 0 to 1, with the threshold for probably damaging at 0.85. Align GVGD classes mutations into a spectrum (C0, C15, C25, C35, C45, C55, and C65) with C65 the most likely to interfere with function, and C0 the least likely EVS (NHLBI Exome Sequencing).

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