tlr3 deficiency in herpes simplex encephalitis: high allelic heterogeneity and recurrence risk

ARTICLES

Hye Kyung Lim MScMikko Seppaumlnen MDTimo Hautala MDMichael J Ciancanelli

PhDYuval Itan PhDFabien G Lafaille PhDWilliam Dell MScLazaro Lorenzo MScMinji Byun PhDElodie Pauwels PhDYlva Roumlnnelid MScXin Cai MScSoraya Boucherit MDEmmanuelle Jouanguy

PhDAnders Paetau MDPierre Lebon MDFlore Rozenberg MD

PhDMarc Tardieu MDLaurent Abel MD PhDAlisan Yildiran MDAnne Vergison MDReina Roivainen MDAmos Etzioni MDPentti J Tienari MDJean-Laurent Casanova

MD PhDShen-Ying Zhang MD

PhD

Correspondence toDr Casanovacasanovarockefellereduor Dr Zhangshzh289rockefelleredu

Editorial page 1882

Supplemental dataat Neurologyorg

TLR3 deficiency in herpes simplexencephalitisHigh allelic heterogeneity and recurrence risk

ABSTRACT

Objective To determine the proportion of children with herpes simplex encephalitis (HSE) display-ing TLR3 deficiency the extent of TLR3 allelic heterogeneity and the specific clinical features ofTLR3 deficiency

Methods We determined the sequence of all exons of TLR3 in 110 of the 120 patients with HSEenrolled in our study who do not carry any of the previously described HSE-predisposing muta-tions of TLR3 pathway genes (TLR3 UNC93B1 TRIF TRAF3 and TBK1) All the new mutantTLR3 alleles detected were characterized experimentally in-depth to establish the causal rela-tionship between the genotype and phenotype

Results In addition to the 3 previously reported TLR3-deficient patients from the same cohort 6other children or young adults with HSE carry 1 of 5 unique or extremely rare (minor allele fre-quency0001) missense TLR3 alleles Two alleles (M374T D592N) heterozygous in 3 patientsare not deleterious in vitro The other 3 are deleterious via different mechanisms G743D1R811Iand L360P heterozygous in 2 patients are loss-of-function due to low levels of expression andlack of cleavage respectively and R867Q homozygous in 1 patient is hypomorphic The 3 pa-tientsrsquo fibroblasts display impaired TLR3 responses and enhanced herpes simplex virus 1 suscep-tibility Overall TLR3 deficiency is therefore found in 6 (5) of the 120 patients studied There ishigh allelic heterogeneity with 3 forms of autosomal dominant partial defect by negative domi-nance or haploinsufficiency and 2 forms of autosomal recessive defect with complete or partialdeficiency Finally 4 (66) of the 6 TLR3-deficient patients had at least 1 late relapse of HSEwhereas relapse occurred in only 12 (10) of the total cohort of 120 patients

Conclusions Childhood-onset HSE is due to TLR3 deficiency in a traceable fraction of patients inparticular the ones with HSE recurrence Mutations in TLR3 and TLR3 pathway genes should besearched and experimentally studied in children with HSE and patients with proven TLR3 defi-ciency should be followed carefully Neurologyreg 2014831888ndash1897

GLOSSARYAD 5 autosomal dominant AR 5 autosomal recessive CEPH-HGDP 5 Centre drsquoEtude du Polymorphisme HumainndashHumanGenome Diversity Project dbSNP 5 Single Nucleotide Polymorphism database DN 5 dominant negative ds 5 double-stranded ECD 5 ectodomain GFP 5 green fluorescent protein HA 5 hemagglutinin HSE 5 herpes simplex encephalitisHSV-15 herpes simplex virus 1 IFN5 interferon iPSC5 induced pluripotent stem cell poly(IC)5 polyinosinepolycytidylicacid TIR 5 Tollinterleukin-1 receptor VSV 5 vesicular stomatitis virus WES 5 whole-exome sequencing WT 5 wild-type

TLR3 is one of the most highly conserved TLRs in humans that have evolved under the strongestpurifying selection1 TLR3 recognizes double-stranded RNA (dsRNA) a by-product producedduring the viral replication of most viruses including herpes simplex virus 1 (HSV-1)2 The mostcommon known clinical consequence of human TLR3 deficiency is childhood herpes simplexencephalitis (HSE) Childhood HSE is a rare life-threatening complication of primary infectionwith HSV-1 a common neurotropic dsDNA virus that is innocuous in most children3 HSE is themost common form of sporadic viral encephalitis in Western countries45 The pathogenesis of HSEhad long remained unclear Our recent studies have demonstrated that HSEmay result from single-gene inborn errors of TLR3-mediated immunity in some children6 with homozygous or

These authors contributed equally to this work

Authorsrsquo affiliations are listed at the end of the article

Go to Neurologyorg for full disclosures Funding information and disclosures deemed relevant by the authors if any are provided at the end of the article

1888 copy 2014 American Academy of Neurology

heterozygous mutations of a TLR3 pathwaygene (TLR3 UNC93B1 TRIF TRAF3 andTBK1)6 TLR3 is expressed on CNS-residentcells that are permissive for HSV-1 infection7

High susceptibility to HSV-1 infection inpatient-specific induced pluripotent stemcell (iPSC)ndashderived UNC-93B- and TLR3-deficient neurons and oligodendrocytes has beendemonstrated recently7 Impaired CNSndashintrin-sic TLR3ndashdependent interferon (IFN)-ab andIFN-l immunity to HSV-1 may thereforeunderlie HSE in children with TLR3 pathwaydeficiencies7 However only 10 of the 120 chil-dren or young adults with HSE studied by ourgroup to date have been found to carry muta-tions affecting the TLR3 pathway Two haveautosomal dominant (AD) partial TLR3 defi-ciency8 and a third has autosomal recessive(AR) complete TLR3 deficiency9 We investi-gated the morbid allelic diversity at the TLR3locus and the proportion of patients with HSEcarrying TLR3mutations by sequencing TLR3in the remaining 110 patients We describenew forms of TLR3 deficiency in humanpatients with early age-onset recurrent HSE

METHODS Patients Inclusion criteria were (1) age between

3 months and 15 years at the time of the first episode of HSE or

young adults developing HSE due to primary HSV-1 infection

and (2) clinically (signs of meningoencephalitis) radiologically

(detectable lesions on cerebral CT scan or MRI) and

virologically (CSF PCR positive for HSV-1 or detectable HSV-

1-specific antibodies in serum and CSF) confirmed HSE

Clinical case reports on the 6 patients carrying novel mutations

in TLR3 are provided on the Neurologyreg Web site at

Neurologyorg

Standard protocol approvals registrations and patientconsents Informed consent was obtained from patients who

were followed up in their home country according to local regu-

lation All experiments were performed at Rockefeller University

in the United States and Institut National de la Santeacute et de la

Recherche Meacutedicale in France under local regulations and the

institutional review board approvals of each institution

Molecular genetics Genomic DNA was extracted from leuko-

cytes and primary fibroblasts The exons of TLR3 were amplified

by PCR and sequenced with the BigDye Terminator Cycle

Sequencing Kit (Applied Biosystems Foster City CA) Whole

exome sequencing was performed as previously described7

Cell culture and stable transfections Human primary and

SV40-immortalized fibroblasts were cultured as previously

described8 The TLR3-deficient P21 fibrosarcoma cell line was

provided by Douglas W Leaman10 The vector encoding the

C-terminally hemagglutinin (HA)ndashtagged pUNO-hTLR3 was

purchased from InvivoGen (San Diego CA) Mutants of the

TLR3 gene were generated by site-directed mutagenesis SV40-

fibroblasts and P21 cells were transfected in the presence of

X-tremeGENE9 Reagent (Roche South San Francisco CA)

Blasticidin (Invitrogen Carlsbad CA 5 mgmL) was added on

media for selection

Immunoblots Equal amounts of total cell protein extracts from

each sample were subjected to immunoprecipitation with a goat

antihuman TLR3 antibody directed against the human TLR3 ec-

todomain (RampD Systems Minneapolis MN) The immunoblot

procedure was performed as previously described9 Anti-TLR3

anti-HA (InvivoGen) and anti-GAPDH (Sigma-Aldrich St

Louis MO) were used

TLR3 agonist stimulation and HSV-1 infection Polyino-sinepolycytidylic acid [poly(IC)] a TLR-3 agonist was used at

various concentrations as indicated Cells were infected with HSV-1

(strain KOS-1) at an MOI of 1 or HSV-1-GFP (strain KOS)11 at

various MOI Cells and supernatants were harvested and cytokine

production at mRNA or protein level were determined by qPCR or

ELISA respectively as previously described9 The green fluorescent

protein (GFP) fluorescence of the HSV-1-GFP-infected samples was

quantified at various time points For assays of cell protection upon

viral infection cells were treated with IFN-a2b (Intron A Schering-

Plough Kenilworth NJ) at a concentration of 104 IUmL for 18

hours before infection

RESULTS Five new TLR3 mutant alleles in 6 unrelated

patients with HSE Using Sanger sequencing we deter-mined the sequence of all exons of TLR3 in 110 patientswith HSE enrolled in our study who do not carry any ofthe previously described HSE-associated mutations ofTLR3 pathway genes (TLR3 UNC93B1 TRIFTRAF3 and TBK1) In 6 unrelated patients weidentified 5 rare missense mutations and one of thesepatients had 2 missense mutations (figure 1A table 1figure e-1A supplementary text e-1) Four ofthe 5 mutant allelesmdashG743D1R811I D592NM374T and L360Pmdashhave not been reported inpublic databases including the Single NucleotidePolymorphism database (dbSNP) 1000 genomeswhich contains 1128 human whole exomes and theExome Variant Server which contains 6503 humanexomes They were not found in our in-house whole-exome sequencing (WES) database which containsdata for 1098 patients Furthermore they were notfound in the Centre drsquoEtude du PolymorphismeHumainndashHuman Genome Diversity Project (CEPH-HGDP) panel which contains DNA samples from1050 healthy individuals from 51 differentpopulations from around the world12 The fifthmutant allele R867Q was reported in dbSNP(rs199768900) with a minor allele frequency of00009 and 3 DNA samples from the CEPH-HGDP DNA panel are heterozygous for the R867Qmutation which was however homozygous in P6We further performed WES in the 6 patientsinvestigated here and found no mutations in otheressential TLR3 pathway genes nor any homozygousHSE-relevant mutation in patient 1 or patient 2 whowere born to consanguineous parents (supplementarytext e-2 table e-1 table e-2) The TLR3 mutations in

Neurology 83 November 18 2014 1889

the 6 patients therefore appeared to be the most likelyHSE-predisposing mutations present

In silico study of the mutations In silico study of the 5TLR3 mutants showed that the L360 G743 R811and R867 residues were strictly conserved in all 32species studied whereas the M374 and D592 resi-dues were not conserved (figure e-1B) As such theL360P G743D R811I and R867Q mutations werepredicted to be highly damaging by both Polymor-phism Phenotyping v2 (PolyPhen-2)13 and SortingIntolerant From Tolerant (SIFT)14 whereas theM374T and D592N mutations were predicted to bebenign The TLR3 protein structure is represented infigure 1B The ectodomain (ECD) of TLR3 is essentialfor ligand binding-triggeredmultimerization15ndash22 and theL360P M374T and D592N mutations are located inthe ECD of TLR3 The linker region bridges thetransmembrane domain and the Tollinterleukin-1 receptor (TIR) domain and the G743D mutationis located within The TIR domain is essential for therecruitment of TRIF the only known adaptor ofTLR3 and thus for downstream signaling2324 TheR811I and R867Q mutations are located in the TIRdomain Collectively the TLR3mutations identifiedparticularly L360P G743D R811I and R867Qare likely to result in a loss of TLR3 function by

various molecular mechanisms underlying AR orAD TLR3 deficiency

Expression of the mutant TLR3 alleles in a TLR3-

deficient cell lineWe investigated the functional impactof the 5 TLR3 mutants by generating P21 cell linesstably transfected with constructs encoding C-terminallyHA-tagged wild-type (WT) or mutated TLR3 proteinsP21 is a TLR3-deficient fibrosarcoma cell line that doesnot express functional TLR3 and does not respondto extracellular stimulation with poly(IC)10 Similarlevels of TLR3 mRNA were detected in P21 cellstransfected with the WT and mutant TLR3 allelesbut not in cells with and without mock vectortransfection (figure 2A) The WT TLR3 proteinswere detected at 2 molecular weights (figure 2B)mdashabout 130 kDa and about 70 kDamdashcorresponding tothe uncleaved full-length protein and the cleaved C-terminal part of the TLR3 protein2526 The D592NM374T and R867Q TLR3 proteins were produced insimilar amounts to the WT TLR3 and displayedthe same cleavage pattern The same expressionpattern was also observed with the G743D R811Iand G743D1R811I alleles whereas the G743Dand R811I alleles were expressed at very low levelsand the G743D1R811I allele was particularlyweakly expressed (figure 2B) Interestingly the

Figure 1 Five novel TLR3 mutant alleles in 6 unrelated patients with herpes simplex encephalitis

(A) Family pedigrees with allele segregation in the 6 families The patients are indicated in black Healthy TLR3 wild-typerelatives of patients 1 and 5 and heterozygous parents of patient 6 are shown in white Heterozygous carriers of the patient1 and 5 mutation and a homozygous sibling of patient 6 are indicated by bold vertical lines in each pedigree respectivelyThe other family members of patients 2 3 and 4 were not tested (B) Schematic diagram of the human TLR3 gene with thepreviously reported (blue) and new (red) mutations indicated at the corresponding location of each mutation The codingexons are numbered with Roman numerals and delimited by a vertical bar The regions corresponding to the leader sequence(L) leucine-rich repeats (LRR) transmembrane domain (TM) linker region (LR) and Tollinterleukin-1 receptor (TIR) domainare shaded in light gray and are delimited by dark gray lines The 2 LRRs with an insertion are indicated by asterisks

1890 Neurology 83 November 18 2014

L360P TLR3 appeared to be uncleaved as wedetected the 130 kDa protein but not the 70 kDaproduct (figure 2B) This suggests that the L360Pmutation which is located in the immediate vicinityof the TLR3 cleavage site (residues 323ndash356) mayrender the mutant protein resistant to cleavage bylysosomal cathepsins2526

Function of the TLR3 mutants expressed in a TLR3-

deficient cell line We then studied the poly(IC)response in P21 cells transfected with the WT or amutant TLR3 Unlike WT or the D592N orM374T mutant TLR3 alleles the G743D1R811Iand L360P alleles could not confer poly(IC) respon-siveness in P21 cells as measured by IFNL1 mRNAproduction89 and the G743D R811I and R867Qallele only marginally confer a poly(IC) response (fig-ure 2C) IFNL1 mRNA was induced to comparablelevels (figure 2D) in all cells after infection with amutant of vesicular stomatitis virus (VSV M51R) apotent inducer of IFN in many human cells27 Thusconsistent with in silico predictions the D592N andM374T TLR3 proteins functioned normally in ouroverexpression system whereas the G743D1R811I(and both G743 and R811I individually) L360Pand R867Q TLR3 proteins were dysfunctionalThe function of the L360P TLR3 was severelyimpaired probably because the L360P mutant couldnot be cleaved and the primary signaling protein wasnot formed2526 The G743D1R811I TLR3 was pro-duced in only small amounts and its function wasimpaired The R867Q mutant was produced in nor-mal amounts but was hypomorphic in terms of

function Thus 3 of the 5 novel TLR3 mutantshad severely impaired functions

Severely impaired poly(IC) responses in fibroblasts from

patients 1 5 and 6 Human dermal fibroblasts display aTLR3-dependent response to extracellular stimulationwith poly(IC)8 The production of IFNB IFNL1and IL6 mRNA was almost abolished in heterozygousG743D1R811I fibroblasts (patient 1) and severelyimpaired in L360P heterozygous (patient 5) and inR867Q homozygous fibroblasts (patient 6) (figure3A) The level of mRNA induction for IFNB IFNL1and IL6 in D592N and M374T heterozygousfibroblasts (patients 2 3 and 4) was similar to that ofthe healthy control cells (figure 3A) As a controlmRNA for IFNB IFNL1 and IL6 was induced tosimilar levels in the fibroblasts of all patients andcontrols after infection with VSV M51R (figure 3B)These results were further confirmed by measuring thesecretion of the IFN-b IFN-l and IL-6 proteins(figure e-1C) The impaired response to poly(IC) wasfurther rescued in TLR3 G743D1R811I heterozygousfibroblasts from patient 1 and R867Q homozygousfibroblasts from patient 6 and partially rescued inheterozygous L360P fibroblasts from patient 5 whenan exogenous WT TLR3 allele was stably expressed inthese cells as shown by the mRNA and proteinproduction of IFN-l (figures 3C and e-1D)Overexpression of WT TLR3 was confirmed byRT-qPCR in patient 1 5 and 6 fibroblasts (figure3D) Thus these results validated our hypothesis thatthe heterozygous G743D1R811I and L360P alleleswere responsible for AD partial TLR3 deficiency in

Table 1 TLR3 pathway deficiencies in human patients with HSE

Gene MutationsInheritancemodel Defect Molecular mechanism No of HSE episodes

Ages at which HSEoccurred References

TLR3 p P554S AD Partial Negative dominance 2 5 y 65 y 68

TLR3 p P554S AD Partial Negative dominance 1 5 mo 68

TLR3 p P554SE746X AR Complete Compound heterozygous (2null alleles)

1 8 y 69

TLR3 p G743D1R811I AD Partial Haploinsufficiency 2 8 mo 35 y This article(patient 1)

TLR3 p L360P AD Partial Negative dominance 3 25 y 22 y 28 y This article(patient 5)

TLR3 p R867Q AR Partial Homozygosity of1 hypomorphic allele

1 followed byldquosmolderingrdquo HSE

From 24 yearsonwards

This article(patient 6)

UNC93B1 c 1034del4 AR Complete Homozygosity of 1 null allele 3 11 mo 14 mo 35 y 6

UNC93B1 c 781 GA AR Complete Homozygosity of 1 null allele 2 5 y 17 y 6

TRIF p R141X AR Complete Homozygosity of 1 null allele 1 2 y 6

TRIF P S186L AD Partial Negative dominance 1 21 mo 6

TRAF3 P R118W AD Partial Negative dominance 1 4 y 6

TBK1 p G159A AD Partial Negative dominance 1 7 y 6

TBK1 p D50A AD Partial Haploinsufficiency 1 11 mo 6

Abbreviations AD 5 autosomal dominant AR 5 autosomal recessive HSE 5 herpes simplex encephalitis


patients 1 and 5 respectively and that the homozygousR867Q allele was responsible for AR partial TLR3deficiency in patient 6

Three TLR3 mutants cause TLR3 deficiency by different

mechanisms TLR3 multimerizes after binding dsRNA

and several TLR3 mutations affecting the ECDincluding the P554S mutation that we previously iden-tified in 3 patients with HSE89 are dominant negative(DN)17ndash19 The loss-of-function uncleavable L360PTLR3 could also be DN Control fibroblasts stablytransfected with the L360P TLR3 allele lost their

Figure 2 Expression and function of the mutant TLR3 alleles

(A) TLR3mRNA levels were determined by quantitative reverse transcription PCR (RT-qPCR) in P21 TLR3-deficient fibrosarcoma cells with or without trans-fection with various TLR3 alleles (WT TLR3 G743D R811I G743D1R811I D592N M374T L360P R867Q E746Xmutant TLR3) or a mock vector GUSwas included for normalization (B) TLR3 expression as assessed by immunoblotting (IB) after immunoprecipitation (IP) in P21 TLR3-deficient fibrosarcomacells not transfected (P21) or stably transfected with wild-type (WT) or mutant TLR3 or mock vector with an anti-TLR3 N-terminal (N) antibody and anantihemagglutinin C-terminal tag antibody Ins12 served as an uncleavable form lacking the entire LRR12 insertion and 346Cterm served as a C-terminalcleaved fragment as previously established and characterized2627 The experiment shown is representative of 6 experiments performed Glyceraldehyde3-phosphate dehydrogenase (GAPDH) was used as an internal expression control for immunoblotting (C) IFNL1mRNA induction without stimulation (NS) orafter 2 and 4 hours of stimulation with 25 mgmL polyinosinepolycytidylic acid [poly(IC)] as assessed by RT-qPCR in P21 TLR3-deficient fibrosarcoma cellsnot transfected (P21) or transfected with WT TLR3 G743D R811I G743D1R811I D592N M374T L360P R867Q or E746X mutant TLR3 or mockvector The E746X mutant served as a loss-of-function control All transfections generated stable cell lines GAPDH was included for normalization (D)IFNL1mRNA induction without stimulation (NS) or after 16 hours of stimulation with vesicular stomatitis virus (VSV) M51R at amultiplicity of infection (MOI)of 1 Mean values 6 SD were calculated from 2 (A D) or 3 (C) independent experiments


ability to respond to poly(IC) in terms of IFNB andIFNL1 mRNA production whereas transfection withthe WT and G743D1R811I alleles had no suchimpact (figure 4A) These results were furtherconfirmed by measuring the production of IFN-band IFN-l proteins in those cells (figure 4B) Theexpression of exogenous TLR3 was confirmed at themRNA level (figure 4C) The loss-of-expressionG743D1R811I TLR3 allele had no detectable DN

effect in fibroblasts from healthy controls suggestingthat this allele underlies AD partial TLR3 deficiency(in patient 1) by haploinsufficiency Thus 3 novelTLR3 mutant alleles (including a double-mutatedallele) define 3 novel forms of partial TLR3deficiency in 3 patients the uncleavable L360Pmutant causes AD TLR3 deficiency due to a DNmechanism differing from that of P554S TheG743D1R811I mutant protein caused AD TLR3

Figure 3 Impaired poly(IC) responses in SV40 fibroblasts from patients 1 5 and 6 and rescue of the poly(IC) phenotype by wild-type TLR3 inSV40 fibroblasts from patients 1 and 6

(A) IFNB IFNL1 and IL6mRNA levels in SV40 fibroblasts from a control (C1) patients 1ndash6 and a TLR322 patient not stimulated (NS) or stimulated for 24 and 6 hours with 25 mgmL polyinosinepolycytidylic acid [poly(IC)] GUS was included for normalization (B) IFNB IFNL1 and IL6mRNA induction withoutstimulation (NS) or after 16 hours of stimulation with vesicular stomatitis virus (VSV) M51R at amultiplicity of infection (MOI) of 1 (C) IFNL1mRNA inductionwithout stimulation (NS) after 4 hours of stimulation with 25 mgmL poly(IC) or after 16 hours of stimulation with VSVM51R Fibroblasts from patients 1 5and 6 were left untransfected mock-transfected (mock) or transfected with a vector encoding hemagglutininndashtagged wild-type (WT) TLR3 (D) TLR3 mRNAlevels were assessed by quantitative reverse transcription PCR Mean values 6 SD were calculated from 3 (A B) or 2 (C D) independent experiments


Figure 4 Dominant-negative effect of P5 allele and susceptibility of the patientsrsquo fibroblasts to herpes simplex virus 1

(A) The induction of mRNA for IFNB and IFNL1was assessed by quantitative reverse transcription PCR (RT-qPCR) in the absence of stimulation (NS) or after4 hours of stimulation with 25 mgmL polyinosinepolycytidylic acid [poly(IC)] in SV40 fibroblasts from a healthy control transfected with an empty vector(C-mock) or with various TLR3 alleles and in cells from a TLR322 patient (B) Production of interferon (IFN)-b and IFN-l in the absence of stimulation (NS)after 24 hours of stimulation with 25 mgmL poly(IC) in the presence of lipofectamine [poly(IC)1L] or without lipofectamine [poly(IC)] or lipofectamine alone(L) as assessed by ELISA (C) The production of TLR3 mRNA was assessed by RT-qPCR in SV40 fibroblasts from healthy controls stably transfected withvarious TLR3 alleles (wild-type [WT] TLR3 G743D1R811I L360P mutant TLR3) or a mock vector GUS was included for normalization (D) Induction ofmRNA for IFNB IFNL1 and IFIT2 in the absence of stimulation (NS) or after 24 hours of stimulation with herpes simplex virus 1 (HSV-1) at an multiplicity ofinfection (MOI) of 1 in SV40 fibroblasts from3 healthy controls the patients (1 5 and 6) a TLR322 patient and a NEMO22 patient (E) HSV-1 replication

Continued


deficiency due to haploinsufficiency Finally thehypomorphic R867Q TLR3 allele resulted in apartial form of AR TLR3 deficiency Overall alongwith the 2 previously published forms of TLR3deficiency89 there are now 5 different forms ofhuman TLR3 deficiency (table 1)

Enhanced susceptibility to HSV-1 in the patientsrsquo

fibroblasts A similar HSE-related cellular phenotypehas been observed in all the 5 previously reportedgenetic etiologies of HSE with mutations in TLR3UNC93B1 TRIF TRAF3 and TBK1 in a total of 10patients (table 1) Virus-induced IFN-b and IFN-lproduction was abnormally weak in fibroblasts frompatients with TLR3 pathway deficiencies followinginfection with HSV-1 and VSV and the impairmentof IFN production in turn leads to enhanced viralreplication and enhanced cell death in patientsrsquofibroblasts89 Like fibroblasts from the AR completeTLR3-deficient patient9 fibroblasts from patients 15 and 6 displayed impaired mRNA production ofIFNB IFNL1 and IFN-inducible genes such asIFIT2 specifically after infection with HSV-1while the same cells were able to produce highlevels of IFNB IFNL1 and IFIT2 mRNA afterinfection with VSV M51R (figure 4D) Likefibroblasts from a patient with AR complete TLR3deficiency and a patient with AR complete STAT1deficiency928 fibroblasts from patients 1 5 and 6displayed higher levels of HSV-1 replication thancells from healthy controls (figure 4E) When cellswere treated with IFN-a2b 16 hours before viralinfection rescue of the HSV-1 phenotype wasobserved in fibroblasts from patients 1 5 and 6and the AR TLR3-deficient patient but not inAR STAT1-deficient cells which have impairedresponses to IFN-a -b and -l29 (figure 4E) Byinference this fibroblast phenotype may account forthe molecular pathogenesis of HSE in CNS-residentcells in the 3 patients with novel forms of inbornerrors of TLR3 immunity as recently shown foriPSC-derived neurons and oligodendrocytes fromother UNC-93B- and TLR3-deficient patients7

DISCUSSION We report 3 novel forms of TLR3deficiency in 3 patients with recurrent HSE Togetherwith the TLR3-deficient patients previouslyreported89 this brings the total number of TLR3-deficient patients identified among the 120 HSEpatients evaluated by our group to date to 6 (5)Our studies provide compelling evidence that inbornerrors of CNS-intrinsic TLR3 immunity may

underlie the pathogenesis of HSE in the course ofprimary HSV-1 infection at least in some childrenThere appears to be genetic heterogeneity at thepopulation level (1 gene per patient multiplemorbid genes in the cohort) but physiopathologichomogeneity at the cellular level (the TLR3-IFNpathway being the core morbid axis which isrepresented by so far 13 patients [11 of the HSEpatients studied] with TLR3 pathway deficiencies)Not only is there locus heterogeneity there is alsoallelic heterogeneity because 5 types of TLR3defects have been found in 6 patients 2 forms ofAR TLR3 deficiency either complete or partial 2types of AD TLR3 deficiency by DN mechanismsdepending on whether TLR3 is excessively cleaved ornot cleaved at all and 1 type of AD TLR3 deficiency byhaploinsufficiency (table 1) The clinical penetrance ofTLR3 deficiencies as well as that of other human TLR3pathway deficiencies (UNC-93B TRIF TBK1) isincomplete for HSE6 Diverse factors may becausative of the incomplete penetrance of humanTLR3 pathway deficiencies including environmentalfactors pathogen-related (viral infection load andvirus strain) or pathogen-unrelated (another infection)factors or host factors including genetic (modifiers) orepigenetic (age at infection) factors In any event ourfinding that a small albeit sizeable fraction of childrenwith HSE (5) carry morbid mutations in TLR3suggests that this is a core morbid gene defining acore morbid pathway WES and whole-genomesequencing in children with HSE will be instrumentalfor testing whether the TLR3 signaling pathway ismutated in most children with HSE

Despite the incomplete clinical penetrance of theTLR3 pathway deficiencies in HSE a high propor-tion of TLR3-deficient patients with HSE had recur-rent HSE with late relapse Overall HSE recurrenceis rare being reported in only about 10 of affectedchildren43031 It may occur early (before 18 months)or late (after 18 months) reflecting different mecha-nisms Late relapses of HSE are particularly rare andtheir underlying mechanism remains unclear30 The 3patients with TLR3 deficiency reported in this studylike one of the 3 previously reported TLR3-deficientpatients6 had recurrent HSE with intervals of about2 to 26 years between HSE episodes (table 1) Laterelapses of HSE have occurred in 4 of the 6 (67)TLR3-deficient patients identified to date Interest-ingly among the other 8 patients with HSE withother deficiencies of the TLR3 pathway both ARUNC-93B-deficient patients had also presented laterelapses of HSE Thus in total 6 patients with TLR3

quantified by green fluorescent protein (GFP) measurement in SV-40 fibroblasts from 3 healthy controls the patients a TLR322 patient and a STAT122patient 24 hours after HSV-1 GFP infection at MOI of 01 1 and 10 with (lower panel) or without (upper panel) 16 hours of pretreatment with IFN-a2b Thedata shown are representative of 3 (A D E) or 2 (B C) independent experiments


pathway deficiencies from a total of 13 such patients(4615) had recurrent HSE HSE therefore seemsto recur more frequently in patients with inborn er-rors of TLR3 immunitymdashan observation that re-quires confirmation in a larger number of patientswith inborn errors of TLR3 immunity We can spec-ulate that TLR3 deficiency is associated with ineffi-cient virus control in the brain leading to incompleteviral latency in the CNS itself32ndash34 in turn leading to ahigh rate of HSE recurrence due to virus reactivationAdditional clinical and CNS cellular studies arerequired to test this hypothesis Meanwhile our find-ings suggest that children with HSE due to TLR3 defi-ciency should be carefully followed up given the risk ofrelapse and the threshold for antiviral treatment shouldbe low in such patients Moreover IFN-a2b in addi-tion to acyclovir might improve the prognosis of HSEif given early in the course of infection

AUTHOR AFFILIATIONSFrom the St Giles Laboratory of Human Genetics of Infectious Diseases

(HKL MJC YI FGL WD MB EP YR EJ LA J-LC

S-YZ) Rockefeller Branch The Rockefeller University New York NY

the Laboratory of Human Genetics of Infectious Diseases (HKL LL

XC SB EJ LA J-LC S-YZ) Necker Branch INSERM

U1163 Paris Paris Descartes University (HKL XC SB EJ LA

J-LC S-YZ) Sorbonne Paris Cite Imagine Institute France the

Immunodeficiency Unit Division of Infectious Diseases Department of

Medicine (MS) and the Department of Neurology (RR PJT)

Helsinki University Central Hospital the Department of Internal Medi-

cine (TH) Oulu University Hospital the Department of Pathology

(AP) University of Helsinki and HUSLAB Finland Virology (PL

FR) Cochin-Saint-Vincent de Paul Hospital Paris Descartes University

Pediatric Neurology (MT) Bicecirctre Hospital Paris Sud University

France the Departments of Pediatric Immunology-Allergy and Pediatric

Hematology (AY) School of Medicine Ondokuz Mayis University Sam-

sun Turkey Brussels Free University and Infectious Diseases Unit (AV)

Hocircpital Universitaire des Enfants Reine Fabiola Belgium Meyer Child-

renrsquos Hospital (AE) Haifa Israel Molecular Neurology (RR PJT)

Research Programs Unit Biomedicum University of Helsinki Finland

the Pediatric Immuno-Hematology Unit (J-LC) Necker Hospital Assis-

tance Publique-Hocircpitaux de Paris Necker Hospital France and Howard

Hughes Medical Institute (J-LC) New York NY

AUTHOR CONTRIBUTIONSHKL performed the experiments analyzed the data and wrote the paper

MS performed the experiments contributed patient samples and collected

clinical data TH performed the experiments contributed patient samples

and collected clinical data MJC performed the experiments YI analyzed

the data FGL performed the experiments WD performed the experi-

ments LL performed the experiments MB performed the experiments

EP performed the experiments YR performed the experiments XC per-

formed the experiments SB performed the experiments EJ performed the

experiments AP performed the experiments contributed patient samples

and collected clinical data PL performed the experiments contributed

patient samples and collected clinical data FR performed the experiments

contributed patient samples and collected clinical data MT performed the

experiments contributed patient samples and collected clinical data LA

analyzed the data AY performed the experiments contributed patient sam-

ples and collected clinical data AV performed the experiments contributed

patient samples and collected clinical data RR performed the experiments

contributed patient samples and collected clinical data AE performed the

experiments contributed patient samples and collected clinical data PJT

performed the experiments contributed patient samples and collected clin-

ical data JLC supervised the research and wrote the paper SYZ super-

vised the research and wrote the paper

ACKNOWLEDGMENTThe authors thank the patients and their families for participating in this

study and the past and present members of the Laboratory of Human

Genetics of Infectious Diseases for discussions and technical bioinfor-

matics or administrative assistance

STUDY FUNDINGSupported by the National Center for Advancing Translational Sciences

(NCATS) NIH Clinical and Translational Science Award (CTSA) pro-

gram grant UL1TR000043 NIH grant 5R01AI088364 the Rockefeller

University INSERM Paris Descartes University the ANR (French

National Agency for Research) the St Giles Foundation the Thrasher

Research Fund the European Research Council (grant ERC-2010-

AdG-268777) Helsinki University Central Hospital and the Finnish

Academy YI was supported by the AXA Research Fund FL by the

New York Stem Cell Foundation and MB by the Charles H Revson

Foundation The plasmids containing Ins12 or 346Cterm mutant

TLR3 were a gift from Dr Serge Lebecque

DISCLOSUREThe authors report no disclosures relevant to the manuscript Go to

Neurologyorg for full disclosures

Received January 7 2014 Accepted in final form June 23 2014

REFERENCES1 Barreiro LB Ben-Ali M Quach H et al Evolutionary

dynamics of human toll-like receptors and their different

contributions to host defense PLoS Genet 20095

e1000562

2 Jacobs BL Langland JO When two strands are better

than one the mediators and modulators of the cellular

responses to double-stranded RNA Virology 1996219

339ndash349

3 Whitley RJ Kimberlin DW Herpes simplex encephalitis

children and adolescents Semin Pediatr Infect Dis 2005

1617ndash23

4 Abel L Plancoulaine S Jouanguy E et al Age-dependent

Mendelian predisposition to herpes simplex virus type

1 encephalitis in childhood J Pediatr 2010157623ndash629

5 De Tiege X Rozenberg F Heron B The spectrum of

herpes simplex encephalitis in children Eur J Paediatr

Neurol 20081272ndash81

6 Zhang SY Herman M Ciancanelli MJ et al TLR3

immunity to infection in mice and humans Curr Opin

Immunol 20132519ndash33

7 Lafaille FG Pessach IM Zhang SY et al Impaired intrin-

sic immunity to HSV-1 in human iPSC-derived TLR3-

deficient CNS cells Nature 2012491769ndash773

8 Zhang SY Jouanguy E Ugolini S et al TLR3 deficiency

in patients with herpes simplex encephalitis Science 2007

3171522ndash1527

9 Guo Y Audry M Ciancanelli M et al Herpes simplex

virus encephalitis in a patient with complete TLR3 defi-

ciency TLR3 is otherwise redundant in protective immu-

nity J Exp Med 20112082083ndash2098

10 Sun Y Leaman DW Ectopic expression of toll-like recep-

tor-3 (TLR-3) overcomes the double-stranded RNA

(dsRNA) signaling defects of P21 cells J Interferon Cyto-

kine Res 200424350ndash361

11 Desai P Person S Incorporation of the green fluorescent

protein into the herpes simplex virus type 1 capsid J Virol

1998727563ndash7568

12 Cann HM de Toma C Cazes L et al A human genome

diversity cell line panel Science 2002296261ndash262


13 Adzhubei I Schmidt S Peshkin L et al A method and

server for predicting damaging missense mutations Nat

Methods 20107248ndash249

14 Kumar P Henikoff S Ng PC Predicting the effects

of coding non-synonymous variants on protein function

using the SIFT algorithm Nat Protoc 200941073ndash

1081

15 Choe J Kelker MS Wilson IA Crystal structure of human

toll-like receptor 3 (TLR3) ectodomain Science 2005

309581ndash585

16 Bell JK Botos I Hall PR et al The molecular structure of

the Toll-like receptor 3 ligand-binding domain Proc Natl

Acad Sci U S A 200510210976ndash10980

17 Bell JK Askins J Hall PR Davies DR Segal DM The

dsRNA binding site of human Toll-like receptor 3 Proc

Natl Acad Sci U S A 20061038792ndash8797

18 de Bouteiller O Merck E Hasan UA et al Recognition of

double-stranded RNA by human toll-like receptor 3 and

downstream receptor signaling requires multimerization

and an acidic pH J Biol Chem 200528038133ndash38145

19 Ranjith-Kumar CT Miller W Xiong J et al Biochemical

and functional analyses of the human Toll-like receptor 3

ectodomain J Biol Chem 20072827668ndash7678

20 Takada E Okahira S Sasai M Funami K Seya T

Matsumoto M C-terminal LRRs of human Toll-like

receptor 3 control receptor dimerization and signal trans-

mission Mol Immunol 2007443633ndash3640

21 Liu L Botos I Wang Y et al Structural basis of toll-like

receptor 3 signaling with double-stranded RNA Science

2008320379ndash381

22 Botos I Segal DM Davies DR The structural biology of

Toll-like receptors Structure 201119447ndash459

23 Oshiumi H Matsumoto M Funami K Akazawa T

Seya T TICAM-1 an adaptor molecule that participates

in Toll-like receptor 3-mediated interferon-beta induction

Nat Immunol 20034161ndash167

24 Yamamoto M Sato S Hemmi H et al Role of adaptor

TRIF in the MyD88-independent toll-like receptor signal-

ing pathway Science 2003301640ndash643

25 Toscano F Estornes Y Virard F et al Cleavedassociated

TLR3 represents the primary form of the signaling recep-

tor J Immunol 2013190764ndash773

26 Garcia-Cattaneo A Gobert FX Muller M et al Cleavage of

Toll-like receptor 3 by cathepsins B and H is essential for

signaling Proc Natl Acad Sci U S A 20121099053ndash9058

27 Stojdl DF Lichty BD tenOever BR et al VSV strains

with defects in their ability to shutdown innate immunity

are potent systemic anti-cancer agents Cancer Cell 2003

4263ndash275

28 Chapgier A Wynn RF Jouanguy E et al Human com-

plete Stat-1 deficiency is associated with defective type I

and II IFN responses in vitro but immunity to some low

virulence viruses in vivo J Immunol 20061765078ndash

5083

29 Chapgier A Kong XF Boisson-Dupuis S et al A partial

form of recessive STAT1 deficiency in humans J Clin

Invest 20091191502ndash1514

30 Spiegel R Miron D Yodko H Lumelsky D Habib A

Horovitz Y Late relapse of herpes simplex virus encepha-

litis in a child due to reactivation of latent virus clinico-

pathological report and review J Child Neurol 200823

344ndash348

31 Valencia I Miles DK Melvin J et al Relapse of herpes

encephalitis after acyclovir therapy report of two new cases

and review of the literature Neuropediatrics 200435

371ndash376

32 Asenbauer B McEntagart M King MD Gallagher P

Burke M Farrell MA Chronic active destructive herpes

simplex encephalitis with recovery of viral DNA 12 years

after disease onset Neuropediatrics 199829120ndash123

33 Nicoll JA Love S Kinrade E Distribution of herpes sim-

plex virus DNA in the brains of human long-term survi-

vors of encephalitis Neurosci Lett 1993157215ndash218

34 Lellouch-Tubiana A Fohlen M Robain O Rozenberg F

Immunocytochemical characterization of long-term persis-

tent immune activation in human brain after herpes sim-

plex encephalitis Neuropathol Appl Neurobiol 200026

285ndash294

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DOI 101212WNL00000000000009992014831888-1897 Published Online before print October 22 2014Neurology Hye Kyung Lim Mikko Seppaumlnen Timo Hautala et al

recurrence riskTLR3 deficiency in herpes simplex encephalitis High allelic heterogeneity and

This information is current as of October 22 2014

ServicesUpdated Information amp

httpwwwneurologyorgcontent83211888fullhtmlincluding high resolution figures can be found at

Supplementary Material

000999DC2htmlhttpwwwneurologyorgcontentsuppl20141022WNL0000000000

000999DC1htmlhttpwwwneurologyorgcontentsuppl20141022WNL0000000000Supplementary material can be found at

References httpwwwneurologyorgcontent83211888fullhtmlref-list-1

This article cites 34 articles 14 of which you can access for free at

Citations httpwwwneurologyorgcontent83211888fullhtmlotherarticles

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rights reserved Print ISSN 0028-3878 Online ISSN 1526-632X1951 it is now a weekly with 48 issues per year Copyright copy 2014 American Academy of Neurology All

reg is the official journal of the American Academy of Neurology Published continuously sinceNeurology

heterozygous mutations of a TLR3 pathwaygene (TLR3 UNC93B1 TRIF TRAF3 andTBK1)6 TLR3 is expressed on CNS-residentcells that are permissive for HSV-1 infection7

High susceptibility to HSV-1 infection inpatient-specific induced pluripotent stemcell (iPSC)ndashderived UNC-93B- and TLR3-deficient neurons and oligodendrocytes has beendemonstrated recently7 Impaired CNSndashintrin-sic TLR3ndashdependent interferon (IFN)-ab andIFN-l immunity to HSV-1 may thereforeunderlie HSE in children with TLR3 pathwaydeficiencies7 However only 10 of the 120 chil-dren or young adults with HSE studied by ourgroup to date have been found to carry muta-tions affecting the TLR3 pathway Two haveautosomal dominant (AD) partial TLR3 defi-ciency8 and a third has autosomal recessive(AR) complete TLR3 deficiency9 We investi-gated the morbid allelic diversity at the TLR3locus and the proportion of patients with HSEcarrying TLR3mutations by sequencing TLR3in the remaining 110 patients We describenew forms of TLR3 deficiency in humanpatients with early age-onset recurrent HSE

METHODS Patients Inclusion criteria were (1) age between

3 months and 15 years at the time of the first episode of HSE or

young adults developing HSE due to primary HSV-1 infection

and (2) clinically (signs of meningoencephalitis) radiologically

(detectable lesions on cerebral CT scan or MRI) and

virologically (CSF PCR positive for HSV-1 or detectable HSV-

1-specific antibodies in serum and CSF) confirmed HSE

Clinical case reports on the 6 patients carrying novel mutations

in TLR3 are provided on the Neurologyreg Web site at

Neurologyorg

Standard protocol approvals registrations and patientconsents Informed consent was obtained from patients who

were followed up in their home country according to local regu-

lation All experiments were performed at Rockefeller University

in the United States and Institut National de la Santeacute et de la

Recherche Meacutedicale in France under local regulations and the

institutional review board approvals of each institution

Molecular genetics Genomic DNA was extracted from leuko-

cytes and primary fibroblasts The exons of TLR3 were amplified

by PCR and sequenced with the BigDye Terminator Cycle

Sequencing Kit (Applied Biosystems Foster City CA) Whole

exome sequencing was performed as previously described7

Cell culture and stable transfections Human primary and

SV40-immortalized fibroblasts were cultured as previously

described8 The TLR3-deficient P21 fibrosarcoma cell line was

provided by Douglas W Leaman10 The vector encoding the

C-terminally hemagglutinin (HA)ndashtagged pUNO-hTLR3 was

purchased from InvivoGen (San Diego CA) Mutants of the

TLR3 gene were generated by site-directed mutagenesis SV40-

fibroblasts and P21 cells were transfected in the presence of

X-tremeGENE9 Reagent (Roche South San Francisco CA)

Blasticidin (Invitrogen Carlsbad CA 5 mgmL) was added on

media for selection

Immunoblots Equal amounts of total cell protein extracts from

each sample were subjected to immunoprecipitation with a goat

antihuman TLR3 antibody directed against the human TLR3 ec-

todomain (RampD Systems Minneapolis MN) The immunoblot

procedure was performed as previously described9 Anti-TLR3

anti-HA (InvivoGen) and anti-GAPDH (Sigma-Aldrich St

Louis MO) were used

TLR3 agonist stimulation and HSV-1 infection Polyino-sinepolycytidylic acid [poly(IC)] a TLR-3 agonist was used at

various concentrations as indicated Cells were infected with HSV-1

(strain KOS-1) at an MOI of 1 or HSV-1-GFP (strain KOS)11 at

various MOI Cells and supernatants were harvested and cytokine

production at mRNA or protein level were determined by qPCR or

ELISA respectively as previously described9 The green fluorescent

protein (GFP) fluorescence of the HSV-1-GFP-infected samples was

quantified at various time points For assays of cell protection upon

viral infection cells were treated with IFN-a2b (Intron A Schering-

Plough Kenilworth NJ) at a concentration of 104 IUmL for 18

hours before infection

RESULTS Five new TLR3 mutant alleles in 6 unrelated

patients with HSE Using Sanger sequencing we deter-mined the sequence of all exons of TLR3 in 110 patientswith HSE enrolled in our study who do not carry any ofthe previously described HSE-associated mutations ofTLR3 pathway genes (TLR3 UNC93B1 TRIFTRAF3 and TBK1) In 6 unrelated patients weidentified 5 rare missense mutations and one of thesepatients had 2 missense mutations (figure 1A table 1figure e-1A supplementary text e-1) Four ofthe 5 mutant allelesmdashG743D1R811I D592NM374T and L360Pmdashhave not been reported inpublic databases including the Single NucleotidePolymorphism database (dbSNP) 1000 genomeswhich contains 1128 human whole exomes and theExome Variant Server which contains 6503 humanexomes They were not found in our in-house whole-exome sequencing (WES) database which containsdata for 1098 patients Furthermore they were notfound in the Centre drsquoEtude du PolymorphismeHumainndashHuman Genome Diversity Project (CEPH-HGDP) panel which contains DNA samples from1050 healthy individuals from 51 differentpopulations from around the world12 The fifthmutant allele R867Q was reported in dbSNP(rs199768900) with a minor allele frequency of00009 and 3 DNA samples from the CEPH-HGDP DNA panel are heterozygous for the R867Qmutation which was however homozygous in P6We further performed WES in the 6 patientsinvestigated here and found no mutations in otheressential TLR3 pathway genes nor any homozygousHSE-relevant mutation in patient 1 or patient 2 whowere born to consanguineous parents (supplementarytext e-2 table e-1 table e-2) The TLR3 mutations in






















1 8 y 69






























Continued












































































e1000562




339ndash349



1617ndash23















3171522ndash1527











1998727563ndash7568










1081



309581ndash585




















2008320379ndash381



















4263ndash275





5083








344ndash348




371ndash376












285ndash294

















Reprints
























1 8 y 69






























Continued












































































e1000562




339ndash349



1617ndash23















3171522ndash1527











1998727563ndash7568










1081



309581ndash585




















2008320379ndash381



















4263ndash275





5083








344ndash348




371ndash376












285ndash294

















Reprints


















Continued












































































e1000562




339ndash349



1617ndash23















3171522ndash1527











1998727563ndash7568










1081



309581ndash585




















2008320379ndash381



















4263ndash275





5083








344ndash348




371ndash376












285ndash294

















Reprints














































































e1000562




339ndash349



1617ndash23















3171522ndash1527











1998727563ndash7568










1081



309581ndash585




















2008320379ndash381



















4263ndash275





5083








344ndash348




371ndash376












285ndash294

















Reprints










1081



309581ndash585




















2008320379ndash381



















4263ndash275





5083








344ndash348




371ndash376












285ndash294

















Reprints


















Reprints




tlr3 deficiency in herpes simplex encephalitis: high allelic heterogeneity and recurrence risk

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