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Genetic, Phenotypic, and Interferon BiomarkerStatus in ADAR1-Related Neurological DiseaseGillian I. Rice1 Naoki Kitabayashi2,3 Magalie Barth4 Tracy A. Briggs1,5 Annabel C.E. Burton6
Maria Luisa Carpanelli7 Alfredo M. Cerisola8 Cindy Colson9 Russell C. Dale10
Federica Rachele Danti11,12,13 Niklas Darin14 Begoña De Azua15 Valentina De Giorgis16
Christian G. L De Goede17 Isabelle Desguerre18 Corinne De Laet19 Atieh Eslahi20 Michael C. Fahey21
Penny Fallon22 Alex Fay23 Elisa Fazzi24 Mark P. Gorman25 Nirmala Rani Gowrinathan26
Marie Hully18 Manju A. Kurian11,12 Nicolas Leboucq27 Jean-Pierre S-M Lin28 Matthew A. Lines29
Soe S. Mar30 Reza Maroofian31 Laura Martí-Sanchez32 Gary McCullagh33 Majid Mojarrad20
Vinodh Narayanan34 Simona Orcesi16 Juan Dario Ortigoza-Escobar32 Belén Pérez-Dueñas32
Florence Petit9 Keri M. Ramsey34 Magnhild Rasmussen35 François Rivier36,37
Pilar Rodríguez-Pombo38 Agathe Roubertie36,39 Tommy I. Stödberg40 Mehran Beiraghi Toosi41
Annick Toutain42 Florence Uettwiller43,44 Nicole Ulrick45 Adeline Vanderver45 Amy Waldman45
John H. Livingston46 Yanick J. Crow1,2,3
1Division of Evolution and Genomic Sciences, Manchester AcademicHealth Science Centre, School of Biological Sciences, Faculty ofBiology, Medicine and Health, University of Manchester,Manchester, United Kingdom
2Laboratory of Neurogenetics and Neuroinflammation, INSERM UMR1163, Paris, France
3Sorbonne-Paris-Cité, Institut Imagine, Hôpital Necker EnfantsMalades, Assistance Publique-Hôpitaux de Paris, Paris DescartesUniversity, Paris, France
4Department of Genetics, CHU Angers, Angers, France5Manchester Centre for Genomic Medicine, Central ManchesterUniversity Hospitals NHS Foundation Trust, Manchester AcademicHealth Science Centre, St Mary’s Hospital, Manchester,United Kingdom
6Department of Paediatrics and Child Health, St George’sUniversity Hospitals NHS Foundation Trust, London,United Kingdom
7Department of Child Neurology and Psychiatry, A. ManzoniHospital, Lecco, Italy
8Department of Pediatric Neurology, Facultad de Medicina, UDELAR,Montevideo, Uruguay
9Clinique de Génétique, Hôpital Jeanne de Flandre, CHU Lille, Lille,France,
10 Institute for Neuroscience and Muscle Research, Children’s Hospitalat Westmead, University of Sydney, Sydney, Australia,
11Department of Developmental Neurosciences, Institute of ChildHealth, UCL, London, United Kingdom,
12Department of Neurology, Great Ormond Street Hospital, London,United Kingdom,
13Department of Paediatrics, Child Neurology and Psychiatry,Sapienza University, Rome, Italy,
14Department of Pediatrics, Institute of Clinical Sciences,Sahlgrenska University Hospital, University of Gothenburg,Gothenburg, Sweden,
Neuropediatrics 2017;48:166–184.
Address for correspondence Yanick J. Crow, MD, PhD, Laboratory ofNeurogenetics and Neuroinflammation, Institut Imagine, 3rd Floor,Room 309, 24 Boulevard du Montparnasse, 75015 Paris, France(e-mail: [email protected]).
15Department of Pediatrics, Hospital Son Llátzer, Palma de Mallorca,Spain,
16Child Neurology and Psychiatry Unit, C. Mondino NationalNeurological Institute, Pavia, Italy,
17Department of Paediatric Neurology, Royal Preston Hospital,Preston, United Kingdom,
18Department of Paediatric Neurology, Hôpital Necker-EnfantsMalades, AP-HP, Paris, France,
19Nutrition and metabolic Unit, Hôpital Universitaire des EnfantsReine Fabiola, Brussels, Belgium,
20Department of Medical Genetics, School of Medicine, MashhadUniversity of Medical Sciences, Mashhad, Iran,
21Department of Paediatrics, Monash University, Melbourne,Australia,
22Department of Paediatric Neurology, St George’s UniversityHospitals NHS Foundation Trust, London, United Kingdom,
23Department of Neurology, University of California, California, SanFrancisco, United States,
24Unit of Child Neurology and Psychiatry, Department of Clinical andExperimental Sciences, Civil Hospital, University of Brescia, Brescia,Italy,
25Department of Neurology, Boston Children’s Hospital, Boston,United States,
26Department of Neurology, Kaiser Permanente, Los Angeles,United States,
27Neuroradiologie, CHU de Montpellier, Montpellier, France,28General Neurology and Complex Motor Disorders Service, Evelina
Children’s Hospital, Guy’s & St Thomas’ NHS Foundation Trust,London, United Kingdom,
receivedJanuary 9, 2017accepted after revisionFebruary 21, 2017published onlineApril 10, 2017
© 2017 Georg Thieme Verlag KGStuttgart · New York
DOI https:/doi.org/10.1055/s-0037-1601449.ISSN 0174-304X.
Original Article166
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29Department of Pediatrics, University of Ottawa, Ottawa, Canada,30Department of Pediatric Neurology, St. Louis Children’s Hospital,
Washington University School of Medicine, St. Louis,United States,
31Medical Research, RILD Wellcome Wolfson Centre, Exeter MedicalSchool, Royal Devon and Exeter NHS Foundation Trust, Exeter,United Kingdom,
32Department of Child Neurology, Hospital Sant Joan de Déu,Esplugues de Llobregat, Catalonia, Spain,
33Department of Paediatric Neurology, Royal Manchester Children’sHospital, Manchester, United Kingdom
34Neurogenomics Division, Center for Rare Childhood Disorders, TGen –The Translational Genomics Research Institute, Phoenix, United States,
35Department of Clinical Neurosciences for Children, and Unit forCongenital and Hereditary Neuromuscular Disorders, OsloUniversity Hospital, Oslo, Norway,
36Department of Neuropédiatrie and CR Maladies Neuromusculaires,CHU de Montpellier, France,
37PhyMedExp, University of Montpellier, INSERM U1046, CNRS UMR9214, Montpellier, France,
38Centro de Diagnóstico de Enfermedades Moleculares, Centro deBiología Molecular Severo Ochoa, Universidad Autónoma Madrid,CIBERER, IDIPAZ, Madrid, Spain,
39 INSERM U1051, Institut des Neurosciences de Montpellier,Montpellier, France,
40Neuropediatric Unit, Karolinska University Hospital, Stockholm,Sweden,
41Department of Pediatric Neurology, Ghaem Medical Center, Schoolof Medicine, Mashhad University of Medical Sciences, Mashhad,Iran,
42Service de Génétique, CHU de Tours, Tours, France,43Pediatric Immunology-Hematology and Rheumatology Unit,
Institut Imagine, Hôpital Necker Enfants Malades, AssistancePublique-Hôpitaux de Paris, Paris, France,
44Department of Allergology and Clinical Immunology, CHRU Tours,Tours, France,
45Department of Pediatrics, Children’s Hospital of Philadelphia,Philadelphia, United States,
46Department of Paediatric Neurology, Leeds General Infirmary,Leeds, United Kingdom
Keywords
► Aicardi–Goutièressyndrome
► bilateral striatalnecrosis
► spastic paraparesis► dystonia► idiopathic basal
ganglia calcification
Abstract We investigated the genetic, phenotypic, and interferon status of 46 patients from 37families with neurological disease due to mutations in ADAR1. The clinicoradiologicalphenotype encompassed a spectrum of Aicardi–Goutières syndrome, isolated bilateralstriatal necrosis, spastic paraparesis with normal neuroimaging, a progressive spasticdystonic motor disorder, and adult-onset psychological difficulties with intracranialcalcification. Homozygous missense mutations were recorded in five families. Weobserved a p.Pro193Ala variant in the heterozygous state in 22 of 23 families withcompound heterozygous mutations. We also ascertained 11 cases from nine familieswith a p.Gly1007Arg dominant-negative mutation, which occurred de novo in fourpatients, and was inherited in three families in association with marked phenotypicvariability. In 50 of 52 samples from 34 patients, we identified amarked upregulation oftype I interferon-stimulated gene transcripts in peripheral blood, with a medianinterferon score of 16.99 (interquartile range [IQR]: 10.64–25.71) compared withcontrols (median: 0.93, IQR: 0.57–1.30). Thus, mutations in ADAR1 are associated witha variety of clinically distinct neurological phenotypes presenting from early infancy toadulthood, inherited either as an autosomal recessive or dominant trait. Testing for aninterferon signature in blood represents a useful biomarker in this context.
Introduction
Adenosine deaminases acting on RNA (ADARs) catalyze thehydrolytic deamination of adenosine to inosine in double-stranded RNA, and thereby potentially alter the informationcontent and structure of cellular RNAs.1ADAR1 is encoded bya single-copy gene that maps to human chromosome 1q21and is present in two main isoforms in mammalian cells. Inmice, a loss of ADAR1 activity leads to a dramatic upregula-tion of interferon-stimulated gene (ISG) expression, which isdependent on the editing activity of ADAR1 and specific tothe interferon-inducible full-length p150 isoform of theprotein.2–4
In 2012, we reported mutations in ADAR1 to cause aphenotype consistent with the infantile encephalopathyAicardi–Goutières syndrome (AGS), and demonstrated that,
similar to the ADAR1 null mouse, the mutant genotype wasassociatedwith anupregulationof type I interferon signaling.5
Further to this, in 2014, we described both bilateral striatalnecrosis (BSN), sometimes occurring after a trivial childhoodinfection, and otherwise nonsyndromic, slowly progressivespastic paraparesis associatedwith normal intellect occur dueto ADAR1 dysfunction, again in associationwith the enhancedexpression of type I interferon-induced gene transcripts.6–8
These data indicate that neurological disease can occurthrough inappropriate inductionof the innate immunesystemby self-derived nucleic acids.
Here, we present an update of our experience of screeningfor ADAR1 mutations, describing the clinical, radiological,molecular, and interferon biomarker characteristics of acohort of 46 patients from 37 families with neurologicaldysfunction due to mutations in ADAR1.
Neuropediatrics Vol. 48 No. 3/2017
Neurological Disease due to ADAR1 Dysfunction Rice et al. 167
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Materials and Methods
Patients and MethodsWe ascertained clinical and molecular data through directcontact and/or via collaborating physicians. The study wasapproved by the Leeds (East) Research Ethics Committee(reference number 10/H1307/132), and the Comité de Pro-tection des Personnes (ID-RCB/EUDRACT: 2014-A01017-40).
A diagnosis of AGSwas suggested by characteristic clinicaland neuroimaging features including cerebral atrophy, whitematter disease, and intracranial calcification.9 BSN wasdiagnosed in the context of an acute or subacute onset of adystonic/rigid motor disorder associated with magnetic res-onance imaging features of bilateral striatal signal changewith or without swelling. Spastic paraparesis/tetraparesisand spastic dystonia were diagnosed according to clinicalsigns, in the presence of either normal neuroimaging or mildnonspecific changes sometimes including calcification of thebasal ganglia. Assessment of the motor and communicationstatus of patients over the age of 1 year was made using theGross Motor Function Classification System (GMFCS),10 theManual Ability Classification System (MACS),11 and theCommunication Function Classification System (CFCS).12
Mutational AnalysisPrimers were designed to amplify the coding exons of ADAR1(►Supplementary Table S1, online-only). Purified polymer-ase chain reaction (PCR) amplification products weresequenced using BigDye terminator chemistry and an ABI3130 DNA sequencer. Mutation description is based on thereference cDNA sequence NM_001111.4, with nucleotidenumbering beginning from the first A in the initiating ATGcodon. Variants were assessed using the in silico programsSIFT (http://sift.jcvi.org) and Polyphen2 (http://genetics.bwh.harvard.edu/pph2/), and population allele frequenciesobtained from the ExAC (http://exac.broadinstitute.org) andgnomAD (http://gnomad.broadinstitute.org) databases.
Interferon ScoreWhole blood was collected into PAXgene tubes, total RNAextracted using a PreAnalytix RNA isolation kit and RNAconcentration assessed using a spectrophotometer (FLUOs-tar Omega, Labtech). Quantitative reverse transcription PCR(qPCR) analysis was performed using the TaqMan UniversalPCR Master Mix (Applied Biosystems), and cDNA derivedfrom 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 rel-ative abundance of each target transcript was normalized tothe expression level of HPRT1 (Hs03929096_g1) and 18S(Hs999999001_s1), and assessed with the Applied Biosys-tems StepOne Software v2.1 and DataAssist Software v.3.01.For each of the six probes, individual data were expressedrelative to a single calibrator. Relative quantification is equalto 2�ΔΔCt that is, the normalized fold change relative to thecontrol data. The median fold change of the 6 genes com-pared with the median of 29 previously collected healthy
controls is used to create an interferon score for eachindividual, with an abnormal interferon score being definedas greater thanþ2 standard deviations above themean of thecontrol group, that is, 2.466.
Results
Molecular DataWe collected data on 46 patients from 37 families of pan-ethnic origin with either biallelic mutations in ADAR1 (28families) or the single known dominant-negativemutation p.Gly1007Arg (nine families) (►Table 1; ►Fig. 1). In fourfamilies, the p.Gly1007A mutation was considered to haveoccurred de novo, while in three families, inheritance wasconfirmed or inferred (two paternal half-siblings born to anunaffected father unavailable for testing), with somaticmosaicism recorded in one case. In two families, inheritancecould not be determined becauseDNA fromboth parentswasnot available. We observed three distinct homozygous mu-tations in five families (two families each sharing the samemutation), in four of which the parents were knowinglyrelated. All of these mutations were missense. Of 23 familieswith compound heterozygous mutations, 22 carried the p.Pro193Ala mutation on one allele. In 13 of 22 familiessegregating this p.Pro193Ala substitution, the second mo-lecular lesion was a null or splicing variant.
Clinical Radiological PhenotypeClinical radiological characteristics of all patients are sum-marized in ►Table 2, and characteristic radiological appear-ances are summarized in►Fig. 2. Median age of disease onsetwas 14 months (range: birth–30 years). We observed 21 and25 affected females and males, respectively. Although spas-ticity and dystonia were common features present in themajority of patients, clinically and radiologically distinctphenotypes could be defined, including classical AGS (15patients), BSN (16 patients), apparently isolated spasticparaparesis (1 patient)/tetraparesis (2 patients), and a pro-gressive spastic dystonic motor disorder (7 patients). In twoof these latter cases, the initial presentation was of isolatedlower limb spasticity, with a dystonic component and in-volvement of the upper limbs only becoming evident severalyears later. Four patients demonstrated radiological featuresof both AGS and BSN. The mother of a child with an AGSpresentationwas diagnosed at the age of 30 yearswith subtlepsychological features and marked intracranial calcification.We identified three patients with significant neurologicaldisease (a spastic/dystonic phenotype) in the absence ofchanges on brain imaging at presentation.
A total of 25 patients were considered to have demon-strated normal development prior to disease onset, in 18 ofwhom there was a history of either vaccination (4 patients)or a notable infectious episode (14 patients) in the periodshortly preceding the development of clinical signs(►Fig. 3A). Several patients experienced a rapid onset ofdystonia/spasticity and loss of skills, with two patients beingadmitted to intensive care due to severe dystonic crisis.Others exhibited amore slowly progressive onset over weeks
Neuropediatrics Vol. 48 No. 3/2017
Neurological Disease due to ADAR1 Dysfunction Rice et al.168
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Table
1Family
structure,
ethn
icity,
andmolec
ular
data
ofasce
rtaine
dAD
AR1mutation-po
sitive
cases
AGS
number
Individu
als
tested
Consang
uinity
Ethnicity
cDNA
Protein
Alle
lic
status
Inhe
ritance
SIFT
Polyphen
2CADD
Phred
ExAc
freq
uen
cy
gnomAD
freq
uen
cy
AGS0
813A
,M,F
No
White
Europea
n
c.57
7C>G
p.Pro1
93Ala
Het
Materna
llyinhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.26
75G>A
p.Arg892
His
Het
Paternally
inhe
rited
Deleterious
0.01
Prob
ably
damag
ing
1.00
0
35Nov
el1/25
2010
AGS0
931A
,M,F
No
Italian
c.57
7C>G
p.Pro1
93Ala
Het
Paternally
inhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.26
08G>A
p.Ala87
0Thr
Het
Materna
llyinhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
34Nov
elNov
el
AGS1
072A
,M,F
Yes
Pakistan
ic.33
37G>C
p.Asp111
3His
Hom
Both
parentshe
tDeleterious
0.02
Prob
ably
damag
ing
1.00
0
33Nov
elNov
el
AGS1
501A
,M,F
No
Brazilian
c.30
19G>A
p.Gly100
7Arg
Het
Deno
vo(paternity
confi
rmed
)
Deleterious
0Prob
ably
damag
ing
1.00
0
34Nov
elNov
el
AGS2
191A
Yes
Pakistan
ic.33
35A>T
p.Tyr111
2Phe
Hom
Not
tested
Tolerated0.17
Prob
ably
damag
ing
1.00
0
33Nov
elNov
el
AGS2
281A
,M,F
No
Indian
c.29
97G>T
p.Lys9
99Asn
Hom
Both
parentshe
tDeleterious
0.03
Prob
ably
damag
ing
1.00
0
34Nov
elNov
el
AGS2
511A
,M,F
No
White
Europea
n
c.57
7C>G
p.Pro1
93Ala
Het
Materna
llyinhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.26
15T>
Cp.Ile
872
Thr
Het
Paternally
inhe
rited
Deleterious
0.01
Prob
ably
damag
ing
1.00
0
26.9
1/12
1342
1/25
2270
AGS3
271A
,M,F
No
Italian/
Hispan
ic
c.57
7C>G
p.Pro1
93Ala
Het
Materna
llyinhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.10
76_1
080d
elp.Lys3
59Argfs
� 14
Het
Paternally
inhe
rited
Fram
eshift
Fram
eshift
Fram
e-
shift
Nov
elNov
el
AGS4
302A
,M,F
No
Span
ish
c.57
7C>G
p.Pro1
93Ala
Het
Materna
llyinhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.26
75G>A
p.Arg892
His
Het
Paternally
inhe
rited
Deleterious
0.01
Prob
ably
damag
ing
1.00
0
35Nov
el1/25
2010
AGS4
741A
,M,F
No
White
Europea
n
c.30
19G>A
p.Gly100
7Arg
Het
Deno
vo(paternity
confi
rmed
)
Deleterious
0Prob
ably
damag
ing
1.00
0
34Nov
elNov
el
AGS5
302A
,M
No
White
Europea
n
c.30
19G>A
p.Gly100
7Arg
Het
Presumed
inhe
rited
from
asym
ptom
atic
Father
Deleterious
0Prob
ably
damag
ing
1.00
0
34Nov
elNov
el
AGS5
501A
,M,F
No
White
Europea
n
c.57
7C>G
p.Pro1
93Ala
Het
Paternally
inhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.25
65_2
568d
elp.Asn
857A
lafs
� 17
Het
Materna
llyinhe
rited
Fram
eshift
Fram
eshift
Fram
e-
shift
Nov
el1/30
224
AGS5
671A
,M,F
No
c.51
8A>G
p.Asn
173S
erHet
Paternally
inhe
rited
N/A
24.3
34/121
366
(Con
tinue
d)
Neuropediatrics Vol. 48 No. 3/2017
Neurological Disease due to ADAR1 Dysfunction Rice et al. 16
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Table
1(Con
tinue
d)
AGS
num
ber
Individuals
tested
Con
sangu
inity
Ethnicity
cDNA
Protein
Alle
lic
status
Inhe
ritance
SIFT
Polyph
en2
CADD
Phred
ExAc
freq
uen
cy
gnomAD
freq
uenc
y
Greek
/
Leban
ese
Prob
ably
damag
ing
0.99
9
144/28
2658
1
hom
c.25
15de
lp.Th
r839
Profs�6
Het
Materna
llyinhe
rited
Fram
eshift
Fram
eshift
Fram
e-
shift
Nov
elNov
el
AGS5
821A
No
White
Europe
an
c.57
7C>G
p.Pro1
93Ala
Het
Not
know
nDeleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.26
47_2
648d
upp.Val88
4Serfs
� 12
Het
Not
know
nFram
eshift
Fram
eshift
Fram
e-
shift
Nov
elNov
el
AGS6
632A
,M,F
No
White
Europe
an
c.57
7C>G
p.Pro1
93Ala
Het
Paternally
inhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.16
30C>T
p.Arg544
�Het
Materna
llyinhe
rited
Stop
Stop
Stop
Nov
el2/25
2366
AGS6
791A
No
White
Europe
an
c.57
7C>G
p.Pro1
93Ala
Het
Not
know
nDeleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.35
56A>G
p.Lys1
186G
luHet
Not
know
nTo
lerated0.11
Prob
ably
damag
ing
0.99
9
31Nov
elNov
el
AGS6
991A
,M,F
No
White
Europe
an
c.30
19G>A
p.Gly100
7Arg
Het
Deno
vo(gen
otyp
ing
notun
dertake
n)
Deleterious
0Prob
ably
damag
ing
1.00
0
34Nov
elNov
el
AGS7
031A
No
Asian
/
White
Europe
an
c.57
7C>G
p.Pro1
93Ala
Het
Not
know
nDeleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.31
00A>G
p.Met10
34Val
Het
Not
know
nDeleterious
0.03
Possibly
damag
ing
0.76
0
25.8
Nov
elNov
el
AGS7
201A
,M,F
No
White
Europe
an
c.57
7C>G
p.Pro1
93Ala
Het
Materna
llyinhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.22
50de
lp.Gly751
Aspfs
� 42
Het
Deno
vo(gen
otyp
ing
notun
dertake
n)
Fram
eshift
Fram
eshift
Fram
e-
shift
Nov
elNov
el
AGS7
591A
,M,F
No
White
Europe
an
c.57
7C>G
p.Pro1
93Ala
Het
Paternally
inhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.29
02G>A
p.Asp968
Asn
Het
Materna
llyinhe
rited
Tolerated0.06
Prob
ably
damag
ing
1.00
0
34Nov
elNov
el
AGS7
651A
No
White
Europe
an
c.57
7C>G
p.Pro1
93Ala
Het
Not
know
nDeleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.55
6C>T
p.Gln18
6�Het
Not
know
nStop
Stop
Stop
Nov
elNov
el
AGS7
881A
,M,F
No
White
Europe
an
c.57
7C>G
p.Pro1
93Ala
Het
Materna
llyinhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.13
86_1
390d
elp.Asp462
Glufs
� 2Het
Deno
vo(paternity
confirm
ed)
Fram
eshift
Fram
eshift
Fram
e-
shift
Nov
elNov
el
Neuropediatrics Vol. 48 No. 3/2017
Neurological Disease due to ADAR1 Dysfunction Rice et al.170
Dow
nloa
ded
by: J
ohns
Hop
kins
Uni
vers
ity. C
opyr
ight
ed m
ater
ial.
Table
1(Con
tinue
d)
AGS
numbe
r
Individu
als
tested
Consang
uinity
Ethnicity
cDNA
Protein
Alle
lic
status
Inhe
ritance
SIFT
Polyphen
2CADD
Phred
ExAc
freq
uenc
y
gnomAD
freq
uen
cy
AGS8
101A
,MA,F
No
White
Europea
n
c.30
19G>A
p.Gly100
7Arg
Het
Inhe
ritedfrom
symp-
tomatic
mothe
r
Deleterious
0Prob
ably
damag
ing
1.00
0
34Nov
elNov
el
AGS9
431A
,M,F
No
North
African
c.30
19G>A
p.Gly100
7Arg
Het
Deno
vo(gen
otyp
ing
notun
dertake
n)
Deleterious
0Prob
ably
damag
ing
1.00
0
34Nov
elNov
el
AGS1
115
1A,M
,FYe
sPe
rsian
c.29
97G>T
p.Lys9
99Asn
Hom
Both
parentshe
tDeleterious
0.03
Prob
ably
damag
ing
1.00
0
34Nov
elNov
el
AGS1
170
1ANo
Asian
c.57
7C>G
p.Pro1
93Ala
Het
Not
know
nDeleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.31
00A>G
p.Met10
34Val
Het
Not
know
nDeleterious
0.03
Possibly
damag
ing
0.76
0
25.8
Nov
elNov
el
AGS1
315
2A,M
,F
(mos
aic)
No
White
Europea
n
c.30
19G>A
p.Gly100
7Arg
Het
Father
mos
aic
Deleterious
0Prob
ably
damag
ing
1.00
0
34Nov
elNov
el
AGS1
456
1ANo
White
Europea
n
c.57
7C>G
p.Pro1
93Ala
Het
Not
know
nDeleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.30
20–3
C>G
Splicing
Het
Not
know
nSp
licing
Splicing
Splicing
Nov
elNov
el
AGS1
507
1A,M
,FNo
Asian
/
White
Europea
n
c.57
7C>G
p.Pro1
93Ala
Het
Materna
llyinhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.27
63–2
A>G
Splicing
Het
Paternally
inhe
rited
Splic
ing
Splicing
Splicing
Nov
elNov
el
AGS1
537
1ANo
White
Europea
n
c.30
19G>A
p.Gly100
7Arg
Het
Not
know
nDeleterious
0Prob
ably
damag
ing
1.00
0
34Nov
elNov
el
AGS1
542
2A,M
,FYe
sAsian
c.33
35A>T
p.Tyr111
2Phe
Hom
Both
parentshe
tTo
lerated0.17
Prob
ably
damag
ing
1.00
0
33Nov
elNov
el
AGS1
824
1ANo
White
Europea
n
c.57
7C>G
p.Pro1
93Ala
Het
Paternally
inhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.10
84_1
085d
elp.Arg362
Aspfs
� 12
Het
Materna
llyinhe
rited
Fram
eshift
Fram
eshift
Fram
e-
shift
Nov
elNov
el
AGS1
980
1ANo
White
Europea
n
c.57
7C>G
p.Pro1
93Ala
Het
Not
know
nDeleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.21
30du
pCp.Asn
711G
lnfs
� 33
Het
Not
know
nFram
eshift
Fram
eshift
Fram
e-
shift
Nov
elNov
el
AGS1
989
1A,M
,FNo
South
American
c.57
7C>G
p.Pro1
93Ala
Het
Paternally
inhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.21
87_2
198d
e-
linsG
T
p.Gly730
Cysfs�60
Het
Materna
llyinhe
rited
Fram
eshift
Fram
eshift
Fram
e-
shift
Nov
elNov
el
AGS2
007
1ANo
White
Europea
n
c.57
7C>G
p.Pro1
93Ala
Het
Not
know
nDeleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
(Con
tinu
ed)
Neuropediatrics Vol. 48 No. 3/2017
Neurological Disease due to ADAR1 Dysfunction Rice et al. 171
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ohns
Hop
kins
Uni
vers
ity. C
opyr
ight
ed m
ater
ial.
or months. Definite clinical progression beyond the initialpresentation was recorded in 16 cases. Nine patients aredeceased, between the ages of 10months and 19 years, six ofwhom had early-onset disease consistent with AGS.
An assessment of gross motor function, manual ability,and communication status at last contact was made in 45patients, of whom 27 were recorded to have none of anypurposeful gross motor, hand and communication function(score of 5 on all three scales) (►Fig. 3B). Five patients wereable to walk with no or some support (GMFCS I–III). Elevenpatients were capable of effective sender and receiver com-munication (CFCS I–III). Although formal testing was notundertaken, seven patients were considered to have normalintellectual function.
Five patients were reported to demonstrate hypo/hyper-pigmentation consistent with dyschromatosis symmetricahereditaria (DSH) 1, and two patients were described withchilblain-like vasculitic lesions. Four patients were docu-mentedwith autoimmune hemolytic anemia. Glaucomawasnot recorded in any patient.
Interferon StatusWe derived 52 interferon scores from 34 patients, 50 ofwhichwere abnormal, with amedian interferon score acrossthe group of 16.99 (interquartile range [IQR]: 10.64–25.71)compared with controls (median: 0.93, IQR: 0.57–1.30)(►Fig. 4). Positive scores were observed up to 25 years afterdisease onset. We also tested 20 interferon scores from 16parental carriers of a recessive mutation in ADAR1. Twosamples from seven parents heterozygous for the recurrentp.Pro193Ala mutation demonstrated a positive interferonscore, versus six samples from nine parents carrying adifferent mutation (►Supplementary Fig. S1, online-only).
Discussion
In 2012, ADAR1 mutations were described in the context ofthe early-onset encephalopathy AGS, associated with thepresence of intracranial calcification, white matter disease,and severe developmental delay.5 Subsequently, in 2014,mutations in ADAR1 were also shown to underlie cases ofapparently nonsyndromic BSN, and of isolated spastic para-paresis with normal neuroimaging.6,7 Here, we confirmthese associations, thus emphasizing the need to considerADAR1-related disease in several distinct clinical scenariostriggering different investigative algorithms. Furthermore,we now describe a patient with a dominant-negative muta-tion in ADAR1 demonstrating an adult-onset phenotypeevocative of “idiopathic” basal ganglia calcification charac-terized by intracranial calcification and subtle psychologicaldisturbance. Our clinical and radiological findings highlightthe propensity of ADAR1-related disease to incur basalganglia dysfunction, and the value of basal ganglia calcifica-tion, frequently only appreciated on computed tomography,as a diagnostic indicator. In general, mutations in ADAR1should be considered in the context of a motor disordercharacterized by spasticity and dystonia. The onset of diseasecan occur after a period of normal development, sometimesTa
ble
1(Con
tinue
d)
AGS
num
ber
Individu
als
tested
Cons
angu
inity
Ethn
icity
cDNA
Protein
Alle
lic
status
Inheritan
ceSIFT
Polyph
en2
CADD
Phred
ExAc
freq
uen
cy
gnomAD
freq
uen
cy
c.98
2C>T
p.Arg328
�Het
Not
know
nStop
Stop
Stop
Nov
el1/25
2210
AGS2
009
1A,M
,FNo
White
Europea
n
c.57
7C>G
p.Pro1
93Ala
Het
Paternally
inhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
23.9
260/
1214
02
602/28
2636
1
hom
c.27
46C>T
p.Arg916
Trp
Het
Materna
llyinhe
rited
Deleterious
0Prob
ably
damag
ing
1.00
0
35Nov
elNov
el
AGS2
010
1A,M
No
Hispan
icc.30
19G>A
p.Gly100
7Arg
Het
MWT,
Fno
ttested
Deleterious
0Prob
ably
damag
ing
1.00
0
34Nov
elNov
el
Abb
reviations
:A,affected
;F,father;Het,he
terozygo
us;Hom
,ho
moz
ygou
s;M,mothe
r;MA,mothe
raffected
;WT,
wild
type
.Note:
Nuc
leotidenu
mberingba
sedon
tran
script
ADAR
1NM_0
0111
1.4.ExAcbrow
serBe
tave
rsionacce
ssed
onOctob
er28
,20
16(http://exac.broad
institute.org),gn
omAD
brow
serβve
rsionacce
ssed
onOctob
er28
,20
16(http://gn
omad
.broad
institute.org).
Neuropediatrics Vol. 48 No. 3/2017
Neurological Disease due to ADAR1 Dysfunction Rice et al.172
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ohns
Hop
kins
Uni
vers
ity. C
opyr
ight
ed m
ater
ial.
associated with a rapid loss of skills, or a much slowerprogression over many years. Assessments using the GMFCS,MACS, and CFCS rating scales indicate that disease outcomein the cases thatwehave ascertained is frequently severe. It isof note that we observed cases with completely preservedintellect þ/� normal neuroimaging in the face of significantmotor disability.
Our own research focus is biased toward the ascertain-ment of pediatric disease. However, Tojo et al described afemale patient with the dominant-negative p.Gly1007Argmutation, presenting at the age of 17 years with gait distur-bance and dystonic posturing of the legs, who experiencedintellectual deterioration from 21 years of age, and becamewheelchair bound a year later.13 Together with our observa-tion of an adult female whose clinical phenotype onlybecame evident at the age of 30 years, it is clear that lateronset disease can occur due to ADAR1 deficiency. This lattercase also illustrates the significant intrafamilial variabilitywhich can be seen in association with ADAR1 dysfunction,the mother presenting in adulthood with subtle psychologi-cal disturbance, while her son experienced a devastatingearly-onset encephalopathy.
ADAR1-related neurological disease can be inherited aseither an autosomal recessive or autosomal dominant trait.We observed homozygosity for amissensemutation infive of28 families segregating recessive disease. As previouslysuggested, the absence of patients with homozygous nullmutations indicates that, as for the ADAR1 null mouse,complete loss of ADAR1 protein activity is likely embryoniclethal.5 Our molecular data reveal a remarkably high fre-quency of the p.Pro193Ala substitution, seen in 22 of 23families with compound heterozygous molecular lesions inADAR1. This mutation, which is recorded on 602 of 282,636alleles in the gnomAD database, was not observed in thehomozygous state in our cohort. That this variant was seen incombinationwith a nullmutation in 13 families suggests thathomozygosity for the p.Pro193Ala allele leads to a milder,
later onset, or distinct phenotype not ascertained here, ormay not be associated with disease. Perhaps of note, thegnomAD database includes one individual homozygous forthis mutation. Finally, our molecular data highlight thedominant-negative p.Gly1007Argmutation, which can occurde novo, or be inherited with variable expression and/ornonpenetrance at least into mid-adult life. The proximity ofGly1007 to the backbone of its RNA ligand, and the possibili-ty for an arginine residue to make polyvalent interactionsthere suggests a mechanism whereby Arg1007 might bindmore tightly to RNA and thus act as a competitive inhibitor ofwild-type protein, while being itself catalytically inactive.14
In keeping with thismodel, we previously demonstrated thata plasmid expressing Gly1007Arg showed stronger inhibi-tion of wild-type ADAR1 than equivalent amounts of aplasmid expressing catalytic inactive ADAR1.5
More than 130 different ADAR1 mutations have beendocumented in patients with DSH, an autosomal-dominantdisorder characterized by the childhood onset of hypopig-mented and hyperpigmented macules on the face and dorsalaspects of the extremities.15 DSH has only very rarely beenreported outside of Japan and China, and evenwithin identi-fied families, a marked variability in expression is wellrecognized. In our series, five patients were noted to dem-onstrate pigmentary lesions consistent with DSH. The fre-quent observation of stop and frameshift variants in DSHindicates haploinsufficiency as the likely molecular patholo-gy, consistent with the recent confirmation of our previoussuggestion that two individuals with DSH would be at one infour risk of a pregnancy with ADAR1-related neurologicaldisease.16
Loss-of-function mutations in ADAR1 have been classifiedwithin the so-called type I interferonopathy grouping, anovel set of inborn errors of immunity where it is proposedthat an upregulation of type I interferon signaling is centralto disease pathogenesis.17,18 The AGS phenotype can arisedue to mutations in any one of seven genotypes within this
Fig. 1 Schematic of ADAR1 gene showing mutations (according to protein nomenclature) ascertained in the present study. Missense andnonsense mutations are annotated above and below, respectively. Numbers in brackets indicate the number of families in which each mutationwas observed. †Indicates mutation acting as a dominant negative.
Neuropediatrics Vol. 48 No. 3/2017
Neurological Disease due to ADAR1 Dysfunction Rice et al. 173
Dow
nloa
ded
by: J
ohns
Hop
kins
Uni
vers
ity. C
opyr
ight
ed m
ater
ial.
Table
2Clin
ical
andradiolog
ical
data
relating
toasce
rtaine
dAD
AR1mutation-po
sitive
cases
AGSnu
m-
ber
Individu
alSe
xDev
elop-
men
tal
status
prior
toons
et
Possible
trigge
r
Ageat
in-
itial
asce
rtain-
men
t
Featuresat
presen
ta-
tion
Curren
tag
e/
ageat
death
(cau
sewere
know
n)
Prog
res-
sive
course
Statusat
last
con-
tact
Neu
roim
a-
ging
Interferon
scores
(age
,de
ci-
malized
years)
GMFC
SMACS
CFC
SSu
mmary
AGS0
81P1
FDelay
edNo
5mo
DD,dy
sto-
nia,
irritability
Diedag
ed17
y
Yes
SDTwith
seve
reID
Cha
racter-
isticof
AGS
24.267
(14.53
);
53.356
(15.01
);
45.676
(15.78
)
VV
VAGS
P2F
Delay
edNo
5mo
DD,dy
sto-
nia,
irrita-
bility,
mi-
croc
epha
ly
Diedag
ed23
mo
Yes
SDTwith
seve
reID
Cha
racter-
isticof
AGS
NT
VV
VAGS
P3M
Diagno
sed
atbirth
No
Neo
natal
RaisedCSF
IFNat
birth
withtran
-
sien
t
thrombo
cy-
tope
niaan
d
petech
iae
9y
Not
obvious
SDTwith
seve
reID
Cha
racter-
isticof
AGS
37.822
(4.82);
21.590
(5.28)
VV
VAGS
AGS0
93P1
MDelay
edNo
1mo
DD,irrita-
bility,
slee
p
andfeed
ing
disturba
nce
20y
Not
obvious
SDTwith
seve
reID
Cha
racter-
isticof
AGS
andBS
N
25.608
(15.26
);
46.665
(16.59
)
VV
VAGS/BS
N
AGS1
07P1
FDelay
edNo
<7mo
DD,dy
sto-
nia,
irrita-
bility,
mi-
croc
epha
ly
Diedag
ed19
y
Not
obvious
SDTwith
seve
reID;
AIHA
Cha
racter-
isticof
AGS
64.22
(15.26
)
VV
VAGS
P2F
Delay
edNo
Neo
natal
DD,dy
sto-
nia,
irrita-
bility,
mi-
croc
epha
ly
14y
Not
obvious
SDTwith
seve
reID;
AIHA
Cha
racter-
isticof
AGS
NT
VV
VAGS
AGS1
50P1
FMild
delay
No
18mo
Loss
of
head
con-
trol,sitting
andsp
eech
15y
No
SDTwith
someID
Somewhite
matterdis-
ease
and
calcifica-
tion
ofGP
14.69
(10.88
)
VV
VAGS
AGS2
19P1
MDelay
edNo
<6mo
DD,po
or
head
control
Diedag
ed6y
Not
obvious
SDTwith
seve
reID;
AIHA
Cha
racter-
isticof
AGS
NT
VV
VAGS
AGS2
28P1
FDelay
edNo
Pren
atal
IUGR,
Diedag
ed10
mo
Not
obvious
SDTwith
seve
reID
Cha
racter-
isticof
AGS
NT
<1year
<1year
<1year
AGS
Neuropediatrics Vol. 48 No. 3/2017
Neurological Disease due to ADAR1 Dysfunction Rice et al.174
Dow
nloa
ded
by: J
ohns
Hop
kins
Uni
vers
ity. C
opyr
ight
ed m
ater
ial.
Table
2(Con
tinue
d)
AGSnu
m-
ber
Individual
Sex
Dev
elop
-
men
tal
status
prior
toon
set
Possible
trigger
Age
atin-
itial
ascertain-
men
t
Features
at
presen
ta-
tion
Curren
tag
e/
ageat
death
(cau
sewere
known)
Progres
-
sive
course
Status
at
last
con-
tact
Neu
roim
a-
ging
Interferon
scores
(age
,de
ci-
malized
years)
GMFC
SMACS
CFC
SSu
mmary
thrombo
cy-
tope
nia,
HSM
AGS2
51P1
FNorm
alVaricella
infection
9mo
Loss
ofskills
over
afew
wee
ks
12y
Not
obviou
s
SDTwith
someID;C
B
BSN
28.367
(8.1);
12.301
(9.27)
VV
IVBS
N
AGS3
27P1
MDelayed
Possible
vi-
ralinfection
(otitis
med
ia)
8mo
DD,e
n-
cepha
lop-
athy
,
irritability
8y
Not
obviou
s
SDTwith
seve
reID;
DSH
AGSwith
featuresof
BSN
23.382
(4.07);
9.40
2
(8.52)
VV
VAGS/BS
N
AGS4
30P1
FDelayed
No
<2mo
DD,d
ysto-
nia,
irrita-
bility,
mi-
croc
epha
ly
Diedag
ed6y
Unc
ertain
SDTwith
seve
reID
Charac
ter-
isticof
AGS
8.29
6
(4.75);
21.538
(5.53)
VV
VAGS
P2F
Delayed
No
<2mo
DD,d
ysto-
nia,
irrita-
bility,
mi-
croc
epha
ly
9y
Not
obviou
s
SDTwith
seve
reID
Charac
ter-
isticof
AGS
12.444
(4.75);
14.306
(5.53)
VV
VAGS
AGS4
74P1
MNorm
alVaccina
tion
4mo
Nystagm
us,
gros
san
d
fine
motor
delay
8y
Yes,
with
worsen
ing
resp
iratory
func
tion
andov
erall
neurologi-
cald
eterio-
ration
SDTwith
seve
reID
Charac
ter-
isticof
AGS
20.961
(5.42);
32.319
(5.88);
49.463
(6.02)
VV
VAGS
AGS5
30P1
FNorm
alNo
5y
Suba
cute
loss
ofskills
beco
ming
rigidov
era
fewmon
ths
17y
Yes
SDTwith
someun
-
derstand
-
ing
BSN
12.502
(13.41
)
VV
VBS
N
P2F
Norm
alNo
1y
Suba
cute
loss
ofskills
beco
ming
rigidov
era
fewmon
ths
29y
Yes
SDTwith
someun
-
derstand
-
ing
BSN
23.385
(26.21
)
VIV
IVBS
N
AGS5
50P1
MNorm
alD
&V
16mo
Sudd
en-on-
setmotor
regression
Diedag
ed9y
(pne
umon
ia)
Yes
SDTwith
some
BSN
6.42
9
(8.39)
VV
VBS
N (Con
tinue
d)
Neuropediatrics Vol. 48 No. 3/2017
Neurological Disease due to ADAR1 Dysfunction Rice et al. 175
Dow
nloa
ded
by: J
ohns
Hop
kins
Uni
vers
ity. C
opyr
ight
ed m
ater
ial.
Table
2(Con
tinue
d)
AGSnu
m-
ber
Individu
alSe
xDev
elop
-
men
tal
status
prior
toon
set
Possible
trigger
Age
atin-
itial
ascertain-
men
t
Features
at
presen
ta-
tion
Curren
tag
e/
ageat
death
(cau
sewere
known)
Progres
-
sive
course
Status
at
last
con-
tact
Neu
roim
a-
ging
Interferon
scores
(age
,de
ci-
malized
years)
GMFC
SMACS
CFC
SSu
mmary
under-
stan
ding
AGS5
67P1
MMild
delay
Bronc
hioli-
tis
9mo
Sudd
enon
-
setmotor
regression
6y
Yes
SDTwith
mod
erate
ID;DSH
BSN
36.387
(2.81)
VV
VBS
N
AGS5
82P1
MNorm
alNo
14mo
Loss
ofskills
Diedag
ed10
y
Yes
SDTwith
mod
erate
ID
BSN
NT
VV
VBS
N
AGS6
63P1
MNorm
alURTI
11mo
Sudd
enon
-
setmotor
regression
12y
No
SDTmod
er-
ateID
BSN
NT
IVIII
IIIBS
N
P2M
Norm
alURTI
11mon
ths
Sudd
enon
-
setmotor
regression
Diedag
e18
years
Not
obviou
s
SDT
BSN
38.13
(17.53
)
VV
IVBS
N
AGS6
79P1
FNorm
alUnspec
ified
viral
infection
18mo
Sudd
enon
-
setmotor
regression
4y
Yes,
then
some
reco
very
Dystonic
gaitan
d
clum
sy
hand
fing
er
mov
e-
men
ts;in-
telle
ctua
lly
norm
al
BSN
3.80
2
(1.66)
IIIII
IIIBS
N
AGS6
99P1
MNorm
alNo
2y
Falling
8y
Yes
Major
LL
spasticity;
intelle
ctua
l-
lyno
rmal
Norm
al16
.833
(4.91)
III
ISP
AGS7
03P1
MMild
delay
No
2y
Loss
ofskills
over
afew
wee
ks
11y
Yes
SDTwith
seve
reID
Initially
structurally
norm
al
MRI;BG
calcifica
-
tion
noted
2years
later
20.427
(8.44);
29.817
(8.44)
VIV
IIISD
T
AGS7
20P1
FNorm
alUnspec
ified
viral
infection
18mo
Rapidloss
ofskills
9y
No
SDT;
intel-
lectua
lly
norm
al;
DSH
BSN
12.057
(6.90)
VV
IIIBS
N
AGS7
59P1
FNorm
alURTI
14mo
Motor
re-
gression
6y
No
SDT;
intel-
lectua
lly
norm
al
Calcifica-
tion
of
11.048
(4.09);
IIIII
IIISD
T
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ater
ial.
Table
2(Con
tinue
d)
AGSnu
m-
ber
Individu
alSe
xDev
elop
-
men
tal
status
prior
toons
et
Possible
trigger
Age
atin-
itial
ascertain-
men
t
Features
at
presen
ta-
tion
Curren
tag
e/
ageat
death
(cau
sewere
known)
Progres
-
sive
course
Status
at
last
con-
tact
Neu
roim
a-
ging
Interferon
scores
(age
,de
ci-
malized
years)
GMFC
SMACS
CFC
SSu
mmary
andspee
ch
arrest
caud
ate
and
putamen
18.633
(4.53)
AGS7
65P1
FNorm
alURTI
11mo
Rapidloss
ofskills
7y
No
SDTwith
someID
BGsign
al
chan
ges
andatro-
phywith
subc
ortical
hypo
myeli-
nation
NT
IVV
IVSD
T
AGS7
88P1
FNorm
alURTI/m
en-
ingitisC
vaccination
15mo
Acu
tere-
gression,
dyston
ia,
extra-py
ra-
midal
mov
e-
men
ts,oro-
facial
dyskinesia
3y
Not
obviou
s
SDTwith
severe
ID
BSN
1.99
(1.29);
4.59
(2.46)
VV
VBS
N
AGS8
10P1
(son
toP2
)M
Mild
delay
URTI
12mo
Rapidpsy-
chom
otor
regression
,
axialh
ypo-
tonia,
spas-
ticdy
ston
ic
tetrap
are-
sis
9y
Not
obviou
s
SDTwith
severe
ID
Charac
ter-
isticof
AGS
40.571
(7.13);
14.851
(7.27)
VV
VAGS/BS
N
P2(m
othe
rto
P1)
FNorm
alNo
30y
Pain,fa-
tigu
e,an
xi-
ety,
slee
p
prob
lems
35y
Possibly
Norm
al
clinical
ex-
amination;
subtle
psy-
cholog
ical
difficu
lties
Norm
alex
-
cept
forBG
,
WM,an
d
Cbcalcifi-
cation
25.743
(33.34
);
12.836
(33.48
)
II
IICCwith
psychiatric
features
AGS9
43P1
MNorm
alVaccination
22mo
SP13
yYe
s,de
vel-
oping
asym
metric
dyston
iaof
uppe
rlim
bs
7yafter
initialp
re-
sentation
SDT;
intel-
lectua
lly
norm
al
Someco
rti-
cala
trop
hy
withBG
and
WM
calcifi-
cation
24.753
(11.75
);
15.074
(12.11
)
IIII
ISP
beco
m-
ingSD
T
withpre-
served
intelle
ct
(Con
tinue
d)
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opyr
ight
ed m
ater
ial.
Table
2(Con
tinue
d)
AGSnu
m-
ber
Individu
alSe
xDev
elop-
men
tal
status
prior
toons
et
Possible
trigge
r
Ageat
in-
itial
asce
rtain-
men
t
Featuresat
presen
ta-
tion
Curren
tag
e/
ageat
death
(cau
sewere
know
n)
Prog
res-
sive
course
Statusat
last
con-
tact
Neu
roim
a-
ging
Interferon
scores
(age
,de
ci-
malized
years)
GMFC
SMACS
CFC
SSu
mmary
AGS1
115
P1M
Unk
now
nNo
4mo
Hyp
oton
ia
and
dyston
ia
2y
Not
obvious
SDTwith
severe
ID
Cha
racter-
isticof
AGS
NT
VV
VAGS
AGS1
170
P1F
Norm
alURTI
9mo
Acu
tere-
gression
withdy
sto-
niane
cessi-
tating
ICU
admission
2y
Not
obvious
SDTwith
severe
ID
Initialb
ilat-
eral
high
sign
alan
d
swellin
gof
BGpro-
gressing
to
extensive
WM
and
cortical
at-
roph
yan
d
seve
rely
atroph
ied
putamina
(noCT)
17.627
(0.84);
1.15
8
(0.90);
3.57
8
(1.23)
VV
VAGS/BS
N
AGS1
315
P1(brother
to
P2,so
nof
P3)
MNorm
alNo
2.5y
STwith
norm
al
intelle
ct
6y
Fluc
tua-
tion
s
ST;intelle
c-
tually
norm
al;
Norm
alMRI
(at2years)
andCT(at4
years)
10.506
(5.53)
IVII
IST
P2(brother
to
P1,so
nof
P3)
MDelay
edNo
DD
obviou
s
by1y
STan
d
spee
ch
delay
4y
Yes,ag
e2.5
yep
isod
eof
definite
regression
STwithse-
vere
ID
MRIn
or-
mal,B
Gan
d
PVcalcifica-
tion
onCT
17.147
(3.06)
IVIV
IVST
P3(Fathe
rto
P1an
dP2
;
mos
aic)
MAlw
ays
norm
al
NR
Alw
ays
norm
al
Alw
ays
norm
al
31y
No
Norm
alNoim
aging
2.69
2
(30.18
)
II
INorm
al
AGS1
456
P1M
Norm
alOtitis
med
ia
15mo
Lethargy
,
dyston
ia,
globa
l
regression
17y
Yes,
with
interm
it-
tent
flares
ofen
ceph
a-
lopa
thyan
d
slow
lypro-
gressive
dystonia
SDTwith
severe
ID;
DSH
Mild
hyper-
intensityof
theBG
(no
CT)
9.06
3
(16.68
)
VV
VSD
T
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ater
ial.
Table
2(Con
tinue
d)
AGSnum
-
ber
Individu
alSe
xDev
elop-
men
tal
status
prior
toon
set
Possible
trigge
r
Ageat
in-
itial
asce
rtain-
men
t
Featuresat
presen
ta-
tion
Curren
tag
e/
ageat
death
(cau
sewere
know
n)
Prog
res-
sive
course
Statusat
last
con-
tact
Neu
roim
a-
ging
Interferon
scores
(age
,de
ci-
malized
years)
GMFC
SMACS
CFC
SSu
mmary
AGS1
507
P1M
Mod
erate
delay
No
13mo
Develop-
men
tala
r-
rest
with
onsetof
gene
ralized
dyston
ia
9y
Yes,
epi-
sode
of
definite
re-
gressionat
age4years
SDTwith
someID;
DSH
BSN
8.29
3
(8.56)
VV
VBS
N
AGS1
537
P1F
Delayed
No
15mo
Motor
delay
withspastic
tetrap
are-
sis
11y
No
SDTwith
someID;
AIHA
BGca
lcifi-
cation
(CT);
norm
alMRI
atag
e10
years
12.865
(10.33
)
VIV
IIISD
T
AGS1
542
P1M
Norm
alNo
21mo
Rapidly
prog
ressive
SP
7y
Yes,
with
prog
ressive
invo
lve-
men
tof
UL
andspastic
dyston
ia
SDTwith
someID
Norm
al(no
CT)
12.24
(6.38);
18.051
(6.43)
IVIII
IVSP
beco
m-
ingSD
T
P2M
Like
ly
delaye
d
No
14mo
Onset
of
dyston
ia
andloss
of
skills
19mo
No
SDTwith
seve
reID
Noim
aging
7.03
1
(2.17)
VV
VClin
ically
AGS-lik
e
(but
no
imag
ing)
AGS1
824
P1M
Norm
alUnspe
cified
viral
infection
11mo
Acu
tere-
gression
withdy
sto-
niane
cessi-
tating
ICU
admission
5y
No
SDTwith
seve
reID;
CB
BSN
with
BGca
lcifi-
cation
8.71
3
(5.55)
VV
VBS
N
AGS1
980
P1M
Norm
alFebrile
illne
ss
14mo
Left
hemi-
paresiswith
loss
ofam
-
bulation
2y
Yes,
from
uni-to
bi-
lateral;
howev
er,
someskills
(e.g.,craw
l-
ing,
pulling
tostan
d)
subs
e-
quen
tly
reac
quired
SDTwith
someID
BSN(noCT)
NT
IVIV
IIIBS
N (Con
tinue
d)
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Neurological Disease due to ADAR1 Dysfunction Rice et al. 17
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opyr
ight
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ater
ial.
Table
2(Con
tinue
d)
AGSnu
m-
ber
Individu
alSe
xDev
elop
-
men
tal
status
prior
toons
et
Possible
trigger
Age
atin-
itial
asce
rtain-
men
t
Featuresat
presen
ta-
tion
Curren
tag
e/
ageat
death
(cau
sewere
known)
Prog
res-
sive
course
Status
at
last
con-
tact
Neu
roim
a-
ging
Interferon
scores
(age
,de
ci-
malized
years)
GMFC
SMACS
CFC
SSu
mmary
AGS1
989
P1M
Norm
alOtitis
med
ia
12mo
Trem
oran
d
rapidloss
of
skills
4y
No
SDTwith
severe
ID
BSN
with
BGcalcifi-
cation
NT
VV
VBS
N
AGS2
007
P1M
Possible
mild
delay
Febrile
re-
spiratory
illness
15mo
Dev
elop
-
men
talre-
gression
withloss
of
craw
ling
andothe
r
skills
3y
No
SDTwith
severe
ID
Charac
ter-
isticof
AGS
NT
VV
VAGS
AGS2
009
P1F
Norm
alMMRan
d
varice
lla
vaccination
13mo
Dev
elop
-
men
talre-
gression
withloss
of
skills
6y
No
SDTwith
severe
ID
BSN
with
BGcalcifi-
cation
NT
IVIV
IVBS
N
AGS2
010
P1F
Norm
alVaccina
tion
6mo
Lost
alla
c-
quired
skills
over
6mo
period
9y
No
SDTwith
severe
ID
Somewhite
matterdis-
ease
and
calcifica-
tion
ofGP
NT
VV
VAGS
Abb
reviations
:AGS,
Aicardi–G
outières
synd
rome;AIHA,autoimmun
ehe
molytican
emia;B
G,b
asalga
nglia
;BSN
,bila
teralstriatalnec
rosis;CB,
chilb
lains;CFC
S,Com
mun
icationFu
nction
Classification
System
;CSF,
cerebrospina
lfluid;C
T,co
mputed
tomograp
hy;D
D,d
evelop
men
tald
elay
;DSH
,dysch
romatos
issymmetrica
hereditaria;D
&V,d
iarrhe
aan
dvo
miting;G
MFC
S,Gross
Motor
Func
tionClassification
System
;GP,
glob
uspa
llidu
s;HSM
,hep
atosplen
omeg
aly;
ICC,intracran
ialcalcification
;ICU,inten
sive
care
unit;ID,intellectua
ldisab
ility;IFN
,interferon;
IUGR,intrauterinegrow
thretardation;
LL,low
erlim
b;M
ACS,
Man
ual
AbilityClassification
System
;MRI,mag
neticresona
nceim
aging;N
R,no
treleva
nt;N
T,no
ttested
;PV,p
eriven
tricular;S
D,spa
sticdy
ston
ia;S
DT,
spasticdy
ston
ictetrap
aresis;S
P,spasticpa
rapa
resis;
ST,spa
stic
tetrap
aresis;UL,
uppe
rlim
b;URT
I,up
perrespiratorytrac
tinfection;
WM,white
matter.
Note:
AGS1
315_
P3(different
shad
ing)isno
tinclud
edin
thepa
tien
tda
taan
alysisbe
caus
eof
mosaic
status
;disabilityscales
wereno
tcalculated
forAGS2
28be
causeof
age<
1year
atlast
contac
t.
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grouping (AGS1–7: TREX1, RNASEH2A, RNASEH2B, RNASEH2C,SAMHD1, ADAR1, and IFIH1), and apparently isolated spasticparaparesis has been reported in patientsmutated in three ofthese genes (RNASEH2B, ADAR1, and IFIH1). In contrast, in anoverviewof 374 patients from299 familieswithmutations inAGS1–7, BSN, the most frequently ascertained phenotype inthe current series, was only recorded in the context ofADAR1-related disease, suggesting discrete factors relevantto gene/protein expression and disease mechanism conse-quent upon ADAR1 dysfunction.19 Also possibly reflective ofthis apparent specificity, in comparison to other genotypes,is the frequency of clinical progression, and the low risk ofdeveloping glaucoma and chilblain-like lesions (since werecorded no examples of the former and only two cases ofthe latter in our cohort).
The consistent finding of a positive interferon signature inperipheral blood in the series of patients reported hereindicates the potential utility of this biomarker as a screeningtest for ADAR1-related disease, for the interpretation ofADAR1 genetic sequence variants of uncertain significance,and in the possible monitoring of treatment efficacy as anti-interferon therapies is developed.20,21 We emphasize that
the interferon signature remains elevated many years afterdisease onset, providing evidence of ongoing pathology.ADAR1 is expressed throughout the brain including the basalganglia (http://www.brain-map.org), and it has been shownthat a loss of ADAR1 renders cells more susceptible toapoptosis following stress, including infection.22 We cannotrule out the possibility that the occurrence of fevers prior tofrank neurological regression represents a prodrome in somecases. However, a history of vaccination or an apparentlydiscrete infectious episode in several patients considered tobe completely developmentally normal prior to diseaseonset, of whom 12 demonstrated BSN on neuroimaging,raises the possibility that the acute degeneration of striataltissue seen in many patients with ADAR1 mutations mightrelate to a rapid induction of apoptosis triggered by viralinfection/metabolic stress. Beyond this possibility, there isstrong evidence that interferon is a neurotoxin,23–27 and weconsider it likely that inappropriate and chronic exposure totype I interferons may be directly relevant to the ADAR1-related neurological phenotypes described here, perhapsinduced by dsRNA species which are normally edited byADAR1, thereby rendering them as immunology inert/
Fig. 2 Characteristic neuroradiological features of ADAR1-related disease. Images (A) and (D) are axial T2 images of AGS251, presenting at9 months of age with bilateral striatal necrosis following varicella zoster infection, showing characteristic high signal and swelling of head ofcaudate and putamen (A). (D) Follow-up at 35 months shows persisting signal change and shrinkage of caudate and putamen. Images (B) and (E)are from AGS150, a 10-year-old child presenting with an Aicardi–Goutières syndrome phenotype. (B) T2 axial MR shows cerebral atrophy withmildly increased signal in white matter. (E) CT shows dense bilateral globus pallidus calcification. Image (C) is of a patient presenting with anAicardi–Goutières syndrome phenotype (AGS810_P1). (C) T2 axial MR at 5 years shows marked cerebral atrophy, white matter high signal, andsignal change and shrinkage of the putamen. (F) CT scan of his mother (AGS810_P2) aged 34 years shows dense calcification of globus pallidus,head of caudate, and deep frontal white matter. Her MR (not shown) was normal. CT, computed tomography; MR, magnetic resonance.
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A B
Fig. 3 Age at presentation and associated disability. (A) Age at presentation in patients developing disease after a period of clearly normaldevelopment. (B) Assessment of gross motor function, manual ability, and communication status in living patients with mutations in ADAR1 over1 year of age.
A B
Fig. 4 Interferon score data in ADAR1-mutated patients and controls. Summary of interferon score data (A) in ADAR1-mutated patients andcontrols and (B) in ADAR1-mutated patients by age. Circles indicate results above þ2 SD of the mean of 29 controls (¼ 2.466, considered“positive”). Solid horizontal lines indicate median value of ADAR1-mutated and control groups. Dotted line indicates positive/negative boundary(2.466) of interferon score.
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marking them as self.1,3,4,28 These observations highlight thepotential utility of treatments for ADAR1-related disease,which recent data suggest might be usefully targeted atantagonism of type I interferon signaling.29
Authors’ ContributionsJ.H.L. and Y.J.C. collated and reviewed all clinical andradiological data. G.I.R. performed quantitative PCR analy-sis, with assistance fromN.K.,M.B., T.A.B., A.C.E.B.,M.L.C., A.M.C., C.C.,R.C.D., F.R.D.,N.D., B.DeA.,V.DeG., C.G.E.L.DeG., I.D., C De L., A.E., M.C.F., P.F., A.F., E.F.,M.P.G., N.R.G., M.H.,M.A.K., N.L., J.-P.S.-M.L.,M.A.L., S.S.M., R.M., L.M.-S., G.M.,M.M., V.N., S.O., J.D.O.-E., B.P.-D., F.P., K.M.R.,M.R., F.R., P.R.-P., A.R., T.I.S., M.B.T., A.T., F.U., N.U., A.V., and A.W. provided clinicalsamples and critically reviewed clinical and immunologicalpatient data. Y.J.C. conceived the study andwrote the initialdraft with the assistance of G.I.R. All authors criticallyreviewed the article and agreed to its publication.
Financial DisclosureNone of the authors have any financial disclosure to report.
FundingY.J.C. acknowledges funding from the European ResearchCouncil (GA 309449: Fellowship to Y.J.C.), ERA-NET Neuron(MR/M501803/1), and a state subsidy managed by theNational Research Agency (France) under the “Investmentsfor the Future” (ANR-10-IAHU-01). T.A.B. acknowledgesfunding from the NIHR. V.N. and K.M.R. acknowledge theclinical support of the C4RCD Research Group.
AcknowledgmentsWe are grateful to the affected families for their involve-ment in our research, and to all clinicianswho contributedsamples and clinical data not included here. We thankMarie-Louise Frémond for critical reading of the article.We would like to thank the Exome Aggregation Consor-tium (ExAC), the Genome Aggregation Database (gno-mAD), and the groups that provided exome variant datafor comparison.
References1 Liddicoat BJ, Chalk AM, Walkley CR. ADAR1, inosine and the
immune sensing system: distinguishing self from non-self. WileyInterdiscip Rev RNA 2016;7(2):157–172
2 Hartner JC, Walkley CR, Lu J, Orkin SH. ADAR1 is essential for themaintenance of hematopoiesis and suppression of interferonsignaling. Nat Immunol 2009;10(1):109–115
3 Liddicoat BJ, Piskol R, Chalk AM, et al. RNA editing by ADAR1prevents MDA5 sensing of endogenous dsRNA as nonself. Science2015;349(6252):1115–1120
4 Pestal K, Funk CC, Snyder JM, Price ND, Treuting PM, Stetson DB.Isoforms of RNA-editing enzyme ADAR1 independently control
nucleic acid sensor MDA5-driven autoimmunity andmulti-organdevelopment. Immunity 2015;43(5):933–944
5 Rice GI, Kasher PR, Forte GM, et al. Mutations in ADAR1 causeAicardi-Goutières syndrome associated with a type I interferonsignature. Nat Genet 2012;44(11):1243–1248
6 Livingston JH, Lin JP, Dale RC, et al. A type I interferon signatureidentifies bilateral striatal necrosis due to mutations in ADAR1.J Med Genet 2014;51(2):76–82
7 Crow YJ, Zaki MS, Abdel-Hamid MS, et al. Mutations in ADAR1,IFIH1, and RNASEH2B presenting as spastic paraplegia. Neuro-pediatrics 2014;45(6):386–393
8 La Piana R, Uggetti C, Olivieri I, et al. Bilateral striatal necrosis intwo subjects with Aicardi-Goutières syndrome due to muta-tions in ADAR1 (AGS6). Am J Med Genet A 2014;164A(3):815–819
9 La Piana R, Uggetti C, Roncarolo F, et al. Neuroradiologic patternsand novel imaging findings in Aicardi-Goutières syndrome.Neurology 2016;86(1):28–35
10 Palisano R, Rosenbaum P, Walter S, Russell D, Wood E, Galuppi B.Development and reliability of a system to classify gross motorfunction in children with cerebral palsy. Dev Med Child Neurol1997;39(4):214–223
11 Eliasson AC, Krumlinde-Sundholm L, Rösblad B, et al. The ManualAbility Classification System (MACS) for children with cerebralpalsy: scale development and evidence of validity and reliability.Dev Med Child Neurol 2006;48(7):549–554
12 Hidecker MJ, Paneth N, Rosenbaum PL, et al. Developing andvalidating the Communication Function Classification System forindividuals with cerebral palsy. Dev Med Child Neurol 2011;53(8):704–710
13 Tojo K, Sekijima Y, Suzuki T, et al. Dystonia, mental deterioration,and dyschromatosis symmetrica hereditaria in a family withADAR1 mutation. Mov Disord 2006;21(9):1510–1513
14 Heale BS, Keegan LP, McGurk L, et al. Editing independent effectsof ADARs on the miRNA/siRNA pathways. EMBO J 2009;28(20):3145–3156
15 Hayashi M, Suzuki T. Dyschromatosis symmetrica hereditaria.J Dermatol 2013;40(5):336–343
16 Kono M, Matsumoto F, Suzuki Y, et al. Dyschromatosis symmetr-ica hereditaria and Aicardi-Goutières syndrome 6 are phenotypicvariants caused by ADAR1 mutations. J Invest Dermatol 2016;136(4):875–878
17 Crow YJ. Type I interferonopathies: a novel set of inborn errors ofimmunity. Ann N Y Acad Sci 2011;1238:91–98
18 Crow YJ, Manel N. Aicardi-Goutières syndrome and thetype I interferonopathies. Nat Rev Immunol 2015;15(7):429–440
19 CrowYJ, Chase DS, Lowenstein Schmidt J, et al. Characterization ofhuman disease phenotypes associated with mutations in TREX1,RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR, and IFIH1.Am J Med Genet A 2015;167A(2):296–312
20 Rice GI, Forte GM, Szynkiewicz M, et al. Assessment of interferon-related biomarkers in Aicardi-Goutières syndrome associatedwith mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C,SAMHD1, and ADAR: a case-control study. Lancet Neurol 2013;12(12):1159–1169
21 Rice GI, Melki I, FrémondML, et al. Assessment of type I interferonsignaling in pediatric inflammatory disease. J Clin Immunol 2017;37(2):123–132
22 Toth AM, Li Z, Cattaneo R, Samuel CE. RNA-specific adenosinedeaminase ADAR1 suppresses measles virus-induced apoptosisand activation of protein kinase PKR. J Biol Chem 2009;284(43):29350–29356
23 Kavanagh D, McGlasson S, Jury A, et al. Type I interferon causesthrombotic microangiopathy by a dose-dependent toxiceffect on the microvasculature. Blood 2016;128(24):2824–2833
Neuropediatrics Vol. 48 No. 3/2017
Neurological Disease due to ADAR1 Dysfunction Rice et al. 183
Dow
nloa
ded
by: J
ohns
Hop
kins
Uni
vers
ity. C
opyr
ight
ed m
ater
ial.
24 Akwa Y, Hassett DE, Eloranta ML, et al. Transgenic expression ofIFN-alpha in the central nervous system of mice protects againstlethal neurotropic viral infection but induces inflammation andneurodegeneration. J Immunol 1998;161(9):5016–5026
25 Campbell IL, Krucker T, Steffensen S, et al. Structural and func-tional neuropathology in transgenic mice with CNS expression ofIFN-alpha. Brain Res 1999;835(1):46–61
26 Barlow CF, Priebe CJ, Mulliken JB, et al. Spastic diplegia as acomplication of interferon Alfa-2a treatment of hemangiomas ofinfancy. J Pediatr 1998;132(3 Pt 1):527–530
27 Michaud AP, Bauman NM, Burke DK, Manaligod JM, Smith RJ.Spastic diplegia and othermotor disturbances in infants receivinginterferon-alpha. Laryngoscope 2004;114(7):1231–1236
28 Vitali P, Scadden AD. Double-stranded RNAs containing multipleIU pairs are sufficient to suppress interferon induction andapoptosis. Nat Struct Mol Biol 2010;17(9):1043–1050
29 Frémond ML, Rodero MP, Jeremiah N, et al. Efficacy of the Januskinase 1/2 inhibitor ruxolitinib in the treatment of vasculopathyassociated with TMEM173-activating mutations in 3 children.J Allergy Clin Immunol 2016;138(6):1752–1755
Neuropediatrics Vol. 48 No. 3/2017
Neurological Disease due to ADAR1 Dysfunction Rice et al.184
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nloa
ded
by: J
ohns
Hop
kins
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vers
ity. C
opyr
ight
ed m
ater
ial.
