Diversity of the Genes Implicated in Algerian Patients Affected by Usher Syndrome

12 Pages·2017·1.96 MB·English

Diversity of the Genes Implicated in Algerian Patients
Affected by Usher Syndrome
Samia Abdi, Amel Bahloul, Asma Behlouli, Jean-Pierre Hardelin, Mohamed
Makrelouf, Kamel Boudjelida, Malek Louha, Ahmed Cheknene, Rachid
Belouni, Yahia Rous, et al.
To cite this version:
Samia Abdi, Amel Bahloul, Asma Behlouli, Jean-Pierre Hardelin, Mohamed Makrelouf, et al.. Diver-
sity of the Genes Implicated in Algerian Patients Affected by Usher Syndrome. PLoS ONE, 2016, 11
(9), pp.e0161893. ￿10.1371/journal.pone.0161893￿. ￿hal-01388303￿
HAL Id: hal-01388303
https://hal.sorbonne-universite.fr/hal-01388303
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RESEARCHARTICLE
Diversity of the Genes Implicated in Algerian
Patients Affected by Usher Syndrome
SamiaAbdi1,2,3,AmelBahloul4,AsmaBehlouli1,5,Jean-PierreHardelin4,
MohamedMakrelouf1,KamelBoudjelida3,6,MalekLouha7,AhmedCheknene3,8,
RachidBelouni2,3,YahiaRous3,8,ZahidaMerad3,6,DjamelSelmane9,
MokhtarHasbelaoui10,CrystelBonnet11,AkilaZenati1,ChristinePetit4,11,12*
1 Laboratoiredebiochimiegénétique,Servicedebiologie-CHUdeBabElOued,Universitéd'Alger1,16
Alger,Algérie,2 Laboratoirecentraldebiologie,CHUFrantzFanon,09Blida,Algérie,3 Facultéde
médecine,UniversitéSaadDahleb,09Blida,Algérie,4 Unitédegénétiqueetphysiologiedel’audition,
INSERMUMRS1120,InstitutPasteur,75015,Paris,France,5 Facultédessciencesbiologiques,Université
dessciencesetdelatechnologieHouariBoumédiène,16Alger,Algérie,6 Serviced’ophtalmologie,CHU
FrantzFanon,09Blida,Algérie,7 Servicedebiochimieetdebiologiemoléculaire,HôpitalArmand
Trousseau,APHP,75012,Paris,France,8 Serviced’ORL,CHUFrantzFanon,09Blida,Algérie,9 Service
a11111 d’ORL,CHUBabelOued,16Alger,Algérie,10 Serviced’ORL,CHUTiziOuzou,15Tizi-Ouzou,Algérie,
11 INSERMUMRS1120,Institutdelavision,UniversitéPierreetMarieCurie,75005,Paris,France,
12 CollègedeFrance,75005,Paris,France
*[email protected]
Abstract
OPENACCESS
Citation:AbdiS,BahloulA,BehlouliA,HardelinJ-P,
Ushersyndrome(USH)isanautosomalrecessivedisordercharacterizedbyadualsensory
MakreloufM,BoudjelidaK,etal.(2016)Diversityof
impairmentaffectinghearingandvision.USHisclinicallyandgeneticallyheterogeneous.
theGenesImplicatedinAlgerianPatientsAffectedby
UsherSyndrome.PLoSONE11(9):e0161893. Tendifferentcausalgeneshavebeenreported.Westudiedthemolecularbasesofthedis-
doi:10.1371/journal.pone.0161893 easein18unrelatedAlgerianpatientsbytargeted-exomesequencing,andidentifiedthe
Editor:FilippoDelBene,InstitutCurie,FRANCE causalbiallelicmutationsinallofthem:16patientscarriedthemutationsatthehomozygous
stateand2atthecompoundheterozygousstate.Nineofthe17differentmutationsdetected
Received:April29,2016
inMYO7A(1of5mutations),CDH23(4of7mutations),PCDH15(1mutation),USH1C(1
Accepted:August12,2016
mutation),USH1G(1mutation),andUSH2A(1of2mutations),hadnotbeenpreviously
Published:September1,2016 reported.ThedeleteriousconsequencesofamissensemutationofCDH23(p.
Copyright:©2016Abdietal.Thisisanopenaccess Asp1501Asn)andthein-framesinglecodondeletioninUSH1G(p.Ala397del)onthecorre-
articledistributedunderthetermsoftheCreative
spondingproteinswerepredictedfromthesolved3D-structuresofextracellularcadherin
CommonsAttributionLicense,whichpermits
(EC)domainsofcadherin-23andthesterilealphamotif(SAM)domainofUSH1G/sans,
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare respectively.Inaddition,wewereabletoshowthattheUSH1Gmutationislikelytoaffect
credited. thebindinginterfacebetweentheSAMdomainandUSH1C/harmonin.Thisshouldspurthe
DataAvailabilityStatement:Allrelevantdataare useof3D-structures,notonlyofisolatedproteindomains,butalsoofprotein-proteininterac-
withinthepaperanditsSupportingInformationfiles. tioninterfaces,topredictthefunctionalimpactofmutationsdetectedintheUSHgenes.
Funding:Thisworkwassupportedbygrantsfrom
theTassiliProject,theAlgeriangovernment,theBNP
ParibasFoundation,theERCgrant“Hairbundle”
(ERC-2011-AdG294570),ANR-15-RHUS-001
(LIGHT4DEAF),andtheFrenchLABEX
Introduction
LIFESENSESgrant[referenceANR-10-LABX-65].
Ushersyndrome(USH,MIM276900,MIM276905,MIM605472)isanautosomalrecessivedis-
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. orderthatassociatessensorineuralhearingimpairmentwithretinitispigmentosa,andinsome
PLOSONE|DOI:10.1371/journal.pone.0161893 September1,2016 1/11
UsherSyndromeinAlgeria
casesvestibulardysfunction.USHaccountsformorethanahalfofthecasesofinheriteddeaf-
blindness,anditsprevalencehasbeenestimatedatbetween1/6000and1/25000[1–4].USHis
clinicallyandgeneticallyheterogeneous[5,6].Threeclinicalsubtypescanbedistinguished.USH
oftype1(USH1)isthemostsevereform,characterizedbycongenitalseveretoprofounddeaf-
ness,vestibulardysfunction,andprepubertalonsetofthevisualloss[2,6,7].USH1accountsfor
30%-40%ofUSHcasesintheEuropeanpopulation[2,4].Sixcausalgeneshavebeenreported:
MYO7A[8],PCDH15[9],CDH23[10,11],USH1C[12,13],USH1G[14],andCIB2[15],encod-
ingtheactin-basedmotorproteinmyosinVIIa(USH1B),thetransmembraneproteinsproto-
cadherin-15(USH1F)andcadherin-23(USH1D),thesubmembranescaffoldproteinsharmonin
(USH1C)andsans(USH1G),andthecalcium-integrin-bindingproteinCIB2(USH1J),respec-
tively.USHoftype2(USH2)ismorefrequent,butusuallylessseverethanUSH1asitassociates
congenital,moderatetoseverehearingimpairmentwithvisuallossbeginninginthefirstorsec-
onddecadeoflife,withoutvestibulardysfunction.Threecausalgeneshavebeenidentified:
USH2A[16],ADGRV1[17],andWHRN/DFNB31[18],encodingthelargetransmembranepro-
teinsusherin(USH2A)andadhesionGprotein-coupledreceptorV1(USH2C),andthesub-
membranescaffoldproteinwhirlin(USH2D),respectively.Finally,USHoftype3(USH3)is
characterizedbyprogressivehearingloss,variableageofonsetofthevisualloss,andvariable
vestibulardysfunction.OnlyoneUSH3gene,CLRN1,encodingthetransmembraneprotein
clarin-1,hasbeenreported[19].USH1proteinshavebeenshowntointeractandtoforma
molecularcomplex[20].USH2proteinsalsoformamolecularcomplex[21,22].
ThespectrumofgenesandmutationsinvolvedinAlgerianUSHpatientsisstillpoorly
definedbecauseonlyafewAlgerianpatientshavebeenstudiedsofar[23,24].Weanalyzedthe
molecularbasesofUSHin18unrelatedAlgerianpatients,16ofthembeingaffectedbyUSH1
andtheothertwobyUSH2,bytargeted-exomesequencingofthetenidentifiedUSHgenes.
PatientsandMethods
Ethicsstatement
ThisstudywasapprovedbythelocalethicscommitteeoftheCHUBabEl-Oued,inaccordance
withthe"Loisanitairen°85–05du16février1985"applicableinAlgeria.Thisstudywascon-
ductedaccordingtotheprinciplesofthedeclarationofHelsinki.Patientswereanonymized,
andthecorrespondingcodewasconservedinaconfidentialfile.Writtenconsentforgenetic
testingwasobtainedfromtheadultparticipantsandfromtheparentsofminorparticipants.
Patients
EighteenunrelatedAlgerianUSHpatients(agerangingfrom19monthsto54yearsold)from
theENTdepartmentsofCHUBlidaHospital(15patients),CHUBabElOuedinAlgiers(one
patient),andCHUTiziOuzou(2patients)wereincludedinthisstudy,togetherwiththeirclin-
icallyunaffectedparents.Mostpatients(14outof18)wereborntoparentsknowntobecon-
sanguineous.Clinicalhistorywastakenforeachpatient.Thepatientsunderwentaudiograms,
ocularfundusautofluorescenceimaging,andelectroretinogram.Allpatientshadbilateral,
moderatetoprofounddeafness,andthe12patientsagedatleastfiveyearsalsohadreduced
visualfieldandnightvision.AllUSH1patientshaddelayedonsetofindependentwalking
(>18months)andvestibulardysfunction.Theparentsofallpatientshadnormalhearingand
noretinaldegenerationonfundusexamination.
Targeted-exomesequencing
GenomicDNAwasextractedfromperipheralbloodusingstandardprocedures.Targeted-
exomesequencingandbioinformaticsanalysiswereperformedaspreviouslydescribed[25].
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UsherSyndromeinAlgeria
Toamplifyandsequenceallcodingexonsofthe10USHgenesidentifiedsofar(i.e.,atotalof
366exons),specificprimersweredesignedusingPrimer3(http://bioinfo.ut.ee/primer3-0.4.0/);
thesequencesoftheseoligonucleotidesarelistedinS1Table.SequenceswererunonABI3730
DNAanalyzer,andcomparedwithGenbankreferencesequences(http://www.ncbi.nlm.nih.
gov/genbank/)usingABIPrismSeqscape2.1.
ThesegregationofthemutationsidentifiedinthepatientswasstudiedbySangersequencing
ofthecorrespondingDNAfragmentsintheparentsandotherrelatives,wheneveravailable.
TheSortingIntolerantfromTolerant(SIFT),MutationTaster,andPolyPhen-2softwarepro-
gramswereusedtopredicttheimpactofallaminoacidsubstitutionsontheproteinstructure
andfunction.TheNNSplice,ESEfinder,MaxEntScan,GeneSplicer,andHumanSplicing
Finderprogramswereusedtopredictabnormalsplicesites.
Referencesequencesformutationnomenclatureandexonnumbering
Inthisarticle,thenomenclatureofallsequencevariantsandexonnumberingrefertothefol-
lowinggenomicandcDNAreferencesequences(NG_andNM_NCBIaccessionnumbers,
respectively):MYO7A[NG_009086.1,NM_000260.3];USH1C[NG_011883.1,NM_153676.3];
CDH23[NG_008835.1,NM_022124.5];PCDH15[NG_009191.1,NM_033056.3];USH1G
[NG_007882.1,NM_173477.4];USH2A[NG_009497.1,NM_206933.2].
AllthemutationsidentifiedweredepositedintheLeidenOpenVariationDatabase(http://
www.lovd.nl/3.0/home).
Mutationmodeling
ThestructuralconsequencesofthetwonovelmissensemutationsinCDH23andofthein-
framesinglecodondeletioninUSH1Gwereanalyzedonthe3D-structuremodelsofthecad-
herin-23EC1-EC2&protocadherin-15EC1-EC2(PDB4APX)andsansSAM-PBM&harmo-
ninNter-PDZ1(PDB3K1R)molecularcomplexes,respectively.ThePyMOLsoftware
programwasusedforcartoonrepresentations.
ResultsandDiscussion
Inacohortof180Algeriandeafpatients,werecruited18unrelatedUSHpatients(16USH1
and2USH2patients)onthebasisofassociatedvestibulardysfunction(includingdelayed
onsetofindependentwalkinginall16USH1patients)and/orreducednightvision.Thepres-
enceofaretinaldysfunctionwasconfirmedbyelectroretinogramanalysis.Bytargeted-exome
sequencingoftheknownUSHgenes,wefoundbiallelicmutationsinallthepatients.Sixteen
patientscarriedmutationsatthehomozygousstate,andtwopatientsatthecompoundhetero-
zygousstate.Atotalof17differentpathogenicorpresumablypathogenicmutationswereiden-
tifiedinfiveUSH1genes(MYO7A,CDH23,PCDH15,USH1C,andUSH1G)andinUSH2A:4
nonsense,1frameshift,3splice-site,and6missensemutations,asynonymousmutationpre-
dictedtocreateaspliceacceptorsite,anin-framedeletionofonecodon,andalargeintragenic
deletion(Table1,Fig1,andS1Fig).
Ninemutationshavepreviouslybeenreported(seeTable1).Thep.Arg212Hisandp.
Gly163ArgmissensemutationsinMYO7A(seeTable2)havealsobeenfoundinBelgian,
Dutch,andAmericanpatients[26]andinAlgerianandTurkishpatients[27],respectively.
Thec.2283-1G>TsplicesitemutationinMYO7A,firstdescribedinanAlgerianpatient[27],
seemstobefrequentinMaghrebianpopulations[28].WefoundthismutationinthreeAlge-
rianpatients,atthehomozygousandcompoundheterozygousstates.Theothersplicesite
mutationinMYO7A(c.470+1G>A)hasbeenreportedinTunisianpatients[29].Thep.
Gln2113(cid:1),c.6829+1G>A,andp.Gly291ArgmutationsinCDH23(seeTable2)havebeen
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UsherSyndromeinAlgeria
Table1. ListofthemutationsfoundinUSHgenes.
Patient Age(years) Gene Allele1 Allele2
ALG01U06S310 8 MYO7A c.5428A>T(p.Lys1810*) c.5428A>T(p.Lys1810*)
ALG01U03S170 14 MYO7A c.635G>A(p.Arg212His)[26] c.635G>A(p.Arg212His)[26]
ALG01U0576051 4 MYO7A c.2283-1G>T[27] c.2283-1G>T[27]
ALG01U04A67100 18 MYO7A c.2283-1G>T[27] c.2283-1G>T[27]
ALG01U0274761 3 MYO7A c.487G>A(p.Gly163Arg)[27] c.2283-1G>T[27]
ALG01U0194950 10 MYO7A c.470+1G>A[29] c.470+1G>A[29]
ALG01U0774920 5 USH1C c.778G>T(p.Glu260*) c.778G>T(p.Glu260*)
ALG01U0892201 3 USH1C c.778G>T(p.Glu260*) c.778G>T(p.Glu260*)
ALG01U0974821 3 CDH23 c.115T>C(p.Tyr39His) c.115T>C(p.Tyr39His)
ALG01U1275791 10 CDH23 c.5850T>A(p.Ser1950Ser) c.5850T>A(p.Ser1950Ser)
ALG01U1476321 <2(19 CDH23 c.6829+1G>A[31] c.6829+1G>A[31]
months)
ALG01U1376351 54 CDH23 c.6337C>T(p.Gln2113*)[30] c.6337C>T(p.Gln2113*)[30]
ALG01U1076470 6 CDH23 c.871G>A(p.Gly291Arg)[32] c.871G>A(p.Gly291Arg)[32]
ALG01U11A64230 7 CDH23 c.1084C>T(p.Gln362*) c.4501G>A(p.Asp1501Asn)
ALG01U1574950 17 PCDH15 c.(876+29089)_(1590+3491)del(p. c.(876+29089)_(1590+3491)del(p.
Glu293_Gln530del) Glu293_Gln530del)
ALG01U1663800 9 USH1G c.1188_1190del(p.Ala397del) c.1188_1190del(p.Ala397del)
ALG01U17SA41 26 USH2A c.2299delG(p.Glu767Serfs*21)[16] c.2299delG(p.Glu767Serfs*21)[16]
ALG01U1881900 18 USH2A c.15017C>T(p.Thr5006Met)[33] c.15017C>T(p.Thr5006Met)[33]
Novelmutationsareindicatedinbold.
doi:10.1371/journal.pone.0161893.t001
foundinanAmericanfamily[30],aFrenchfamily[31],andaSpanishfamily[32],respectively.
Thep.Thr5006MetmissensemutationinUSH2A(seeTable2)hasbeenreportedinFrench
andDanishpatients[33].Finally,thec.2299delGancestralmutationinUSH2A[16]accounts
for47.5%and15%ofthemutantallelesidentifiedinUSH2ApatientslivinginDenmarkand
inSpain,respectively[34,35],but,toourknowledge,hadnotyetbeenreportedinpatients
fromNorthAfrica.
Theothereightmutationshadnotbeenpreviouslyreported.Sixwerepresentatthehomo-
zygousstate(6USH1patients),andtwowerepresentatthecompoundheterozygousstatein
anUSH1patient(seeTable1).ThesemutationswereabsentinHapMap,1000genomes,
ExomeVariantServer,andDeafnessVariationdatabases,andintheLivesetdatabase(LSDB)
forUSHgenes,andtheywerenotdetectedin150Algeriancontrolindividuals.Thetwomis-
sensemutationsarelocatedinCDH23exon3(p.Tyr39His)andexon36(p.Asp1501Asn),
encodingthefirstand14thextracellularcadherin(EC)domainsofcadherin-23,respectively.
BothmutationswerepredictedtobepathogenicbyPolyPhen-2,SortingIntolerantfromToler-
ant(SIFT),andMutationTaster(seeTable2).
Thetwomostamino-terminalECrepeats(EC1+EC2)ofcadherin-23andprotocadherin-15
caninteracttoformanoverlapped,antiparallelheterodimerthatisbelievedtobethestructural
basisofapicalfibrouslinksjoininginnerearhaircells’stereocilia[36].TheTyr39residueof
cadherin-23belongstotheloopbetweenthe3 helixandAstrandofEC1,andislocatedat
10
thecadherin23-protocadherin15interactioninterface,buttheTyr39Hismutationdoesnot
appeartoperturbthehydrophobicenvironmentofthissurfaceaccordingtothestructural
modeloftheinteraction(Fig2A).However,wecannotexcludethepossibilitythatTyr39plays
animportantroleinanothermodeofcadherin-cadherininteractionthatmightalsobe
involvedinstereocilialinks,especiallysincethisresidueislocatedattheN-terminalendofthe
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Fig1.Schematicrepresentationofmyosin-VIIa(USH1B),harmonin(USH1C),cadherin-23(USH1D),protocadherin-15(USH1F),sans
(USH1G),andusherin(USH2A).Foreachprotein,thelongestisoformisshown.Thenovelandthepreviouslyreportedcausalmutationsin
AlgerianUSHpatientsareindicatedinredandinblack,respectively.Abbreviations:IQ,isoleucine-glutaminemotifs;MyTH4,myosintailhomology
4domain;FERM,band4.1-ezrin-radixin-moesindomain;SH3,srchomology3domain;Nter,N-terminaldomain;PDZ,PSD95-discslarge-ZO1
domain;CC,coiledcoildomain;PST,proline-serine-threonine-richregion;EC,extracellularcadherindomain;TM,transmembranedomain;Ank,
ankyrindomain;SAM,sterilealphamotifdomain;LamG/TspN/PTX,N-terminalthrombospondin/pentaxin/lamininG-likedomain;LamNter,laminin
N-terminaldomain;LamEGF-like,laminin-typeEGF-likedomain;LamG-like,lamininG-likedomain;FNIII,fibronectintypeIIIdomain.
doi:10.1371/journal.pone.0161893.g001
protein.Theothermissensemutationofcadherin-23,Asp1501Asn,occursintheloopbetween
EC14andEC15.Sequencealignmentofthe27ECrepeatsoftheproteinshowsthepresenceof
asparticacidresiduesatpositionsequivalenttoAsp1501(S2Fig),andthesolved3D-structure
ofEC1-EC2indicatesthattheequivalentasparticacidresidueintheEC1-2linkercontributes
totheCa2+-bindingsite(Fig2B).ThesubstitutionofthenegativelychargedAsp1501byAsnis
predictedtodisruptthecorrespondingCa2+-bindingsite(Fig2B).
Thenovelin-framedeletionofthreenucleotidesidentifiedinUSH1G(p.Ala397del)
removesanalanineresiduefromthefirstα-helixofthesterilealphamotif(SAM)domain.
Thishelix(αA)ispartoftheSAMdomainhydrophobiccoreformedbyabundleoffourα-
helices.ModelingofthemutatedSAMdomainlackingtheAla397residuepredictsadisruption
ofthehydrophobiccore(Fig3A).Inaddition,Ala397contributestotheinterfaceofinteraction
betweensans(USH1G)andharmonin,thePSD95-discslarge-ZO1(PDZ)domain-containing
Table2. PredictedpathogenicityofthesixmissensemutationsfoundinAlgerianUSHpatients.
Gene CDH23 MYO7A USH2A
Mutation Tyr39His Gly291Arg Asp1501Asn Gly163Arg Arg212His Thr5006Met
SIFT Deleterious(0) Deleterious(0) Deleterious(0) Deleterious(0) Deleterious(0) Deleterious(0)
Mutation Diseasecausing Diseasecausing Diseasecausing Diseasecausing(1) Diseasecausing(1) Diseasecausing
Taster (0.94) (0.94) (0.7) (0.99)
PolyPhen-2 Probablydamaging Probablydamaging Probablydamaging Probablydamaging Probablydamaging Probablydamaging
(1) (1) (1) (1) (1) (1)
doi:10.1371/journal.pone.0161893.t002
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UsherSyndromeinAlgeria
Fig2.Predictedeffectofthep.Tyr39Hisandp.Asp1501AsnCDH23mutations.(A)Ontheleftside,ribbondiagramofprotocadherin-15
EC1-EC2(pink)boundtocadherin-23EC1-EC2(blue).Ca2+ionsaredepictedasgreenspheres.TheTyr39residueofcadherin-23isshown
inyellow.Ontherightside,detailedcartoonofthecadherin23-protocadherin15interactioninterface.TheTyr39residueengagesinvander
WaalsinteractionswithIle22andVal114ofprotocadherin-15,andthesubstitutionofthisresiduebyHis(orange)isnotpredictedtochange
theseinteractions.(B)Ontheleftside,ribbondiagramshowingthebackboneofcadherin-23EC1-EC2andthesidechainsoftheamino-acid
residuesinvolvedattheCa2+-bindinginterfaceoftheEC1-2linker.Ontherightside,detailedcartoonofthecorrespondingCa2+-bindingsite
intheEC14-15linker.TheAsp1501residueisshowninyellow.Lowerpanel:thesubstitutionofthisresiduebyAsn(orange)causessteric
clashesorchargeincompatibilityinallthreemodelingrotamers,andisthereforepredictedtoimpairCa2+-binding.
doi:10.1371/journal.pone.0161893.g002
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Fig3.Predictedeffectofthep.Ala397delUSH1Gmutation.(A)Ontheleftside,ribbondiagramshowingthehydrophobiccoreoftheSAMdomain
ofUSH1G/sans.ThesidechainsofthehydrophobicresiduesLeu392,Phe395,Leu396,andLeu399andofthepolarresidueHis400oftheαAhelix
arerepresentedingreenandinblue,respectively.Ontherightside,theribbondiagramshowsthedisruptionofthehydrophobiccoreoftheSAM
domainbytheHis400polarresidueinthepresenceofthep.Ala397delmutation.(B)Ontheleftside,ribbondiagramshowingtheoverallstructureof
theNter-PDZ1domainofUSH1C/harmonin(cyan)interactingwiththeSAMdomainofUSH1G/sans(orange).TheαAhelixoftheSAMdomain
interactswiththesurfacegrooveoftheharmoninPDZ1domain.NotethemajorinvolvementoftheSAMdomainresiduesThr394,Ala397,andHis400
inthisinteraction.Ontherightside,theribbondiagramshowstheabnormalSAM-PDZ1interactioninterfaceinthepresenceofthep.Ala397del
mutation:theLeu151andThr156residuesofthePDZ1domaincannotinteractwiththemissingAla397residueandtheHis400residueoftheSAM
domain,respectively.
doi:10.1371/journal.pone.0161893.g003
proteinencodedbyUSH1C[14],[37],andtheabsenceofthisresidueispredictedtoaffectthe
dockingoftheSAMdomainαAhelixtothesurfacegrooveoftheharmoninPDZ1domain
(Fig3B).Incidentally,amongthetwelvepresumablypathogenicmutationspreviouslyreported
inUSH1G,therewereonlytwo(monoallelic)missensemutationsaffectingresidueslocatedin
theSAMdomain,p.Leu420Valandp.Arg447Trp,noneofwhichispredictedtoaffectthebind-
ingtoharmonin[38,39].
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UsherSyndromeinAlgeria
Fig4.DNAsequenceelectropherogramshowingtheboundariesofthelargedeletionidentifiedinPCDH15.ThePCRintronicprimersusedto
definetheboundariesofthedeletionidentifiedintheUSH1FpatientwerePCDH15-intron9Forward(5'-GAAATCCCTTGACCCCTTGT-3')and
PCDH15-intron14Reverse(5'-GCAAAGACTTGGAACCAACC-3'),leadingtoanampliconofapproximately1.2Kb.
doi:10.1371/journal.pone.0161893.g004
ThethreenovelnonsensemutationsarelocatedinMYO7Aexon39(p.Lys1810(cid:1)),encoding
thesecondmyosintailhomology4(MyTH4)domainofmyosinVIIa,inUSH1Cexon10(p.
(cid:1)
Glu260 ),encodingthePDZ2domainofharmonin(commontoallharmoninclassesandsub-
classes),andinCDH23exon10(p.Gln362(cid:1)),encodingthe4thECdomainofcadherin-23.In
addition,thenovelc.5850T>A(p.Ser1950Ser)synonymousmutationinCDH23ispredicted
tocreateaspliceacceptorsiteinexon44(encodingthe18thECdomainoftheprotein),which
alsoresultsinaprematurestopcodoninthematuretranscript.Allthesemutationsare
expectedtoresulteitherinatruncatedproteinorinnoproteinatallowingtononsensemedi-
atedmRNAdecay[40].
Finally,abiallelicdeletionencompassingPCDH15exons10to14(p.Glu293_Gln530del)
wasidentifiedintheUSH1Fpatient,andconfirmedbyquantitativePCRanalysisofthese
exons(datanotshown).Thislargein-framedeletionisexpectedtoaffectprotocadherin-15
EC3-EC5.ThepreciseboundariesofthedeletionwereestablishedbyPCRamplificationonthe
patient’sgenomicDNAwithspecificprimersinintrons9and14,followedbySangersequenc-
ingoftheamplicon(Fig4).
TheseresultshighlightthegeneticheterogeneityinAlgerianUSH1patients,eventhough
MYO7AandCDH23arepredominantcausalgenes,eachbeinginvolvedin6outof16patients
inourseries.ItisnoteworthythattheproportionofUSH2patientsamonghearingimpaired
childrenismostprobablyunderestimatedbecauseclinicaldiagnosisisusuallynotmadebefore
theonsetofthevisualloss,owingtotheabsenceofvestibularsymptomsinthisclinicalsubtype.
USH2patientswereindeedunderrepresentedinourseriesofUSHpatients,mostofwhom
werebelow15yearsold(seeTable1).Anadditionalstudy,focusedonUSH2Algerianpatients,
isthereforerequiredtodeterminethespectrumofmutationsinthesepatients.
SupportingInformation
S1Fig.DNAsequencingelectrophoregramsshowingthepointmutationsandsmalldele-
tionsidentifiedin17ofthe18patients(seeFig4forthelargebiallelicdeletionofPCDH15
PLOSONE|DOI:10.1371/journal.pone.0161893 September1,2016 8/11
UsherSyndromeinAlgeria
identifiedintheotherpatient).Referenceelectrophoregramsandsequencesareshownon
topofthevariantelectrophoregrams.Asterisksindicatethepositionsofthepointmutations.
Thepositionsofthedeletionsareindicatedbyframesinthereferencesequences,andbyverti-
calbarsonthevariantelectrophoregrams.Notethefourelectrophoregramsshowingpoint
mutationsattheheterozygousstatethatcorrespondtothetwocompoundheterozygous
patients(seeTable1).
(TIF)
S2Fig.Sequencealignmentofthe27ECrepeatsofhumancadherin-23.
(DOCX)
S1Table.PrimersusedtoamplifytheexonsoftheUSHgenes.
(DOCX)
Acknowledgments
Wethankallthefamilymembersfortheirparticipationinthestudy.WealsothankLuce
Smagghefortechnicalassistance.ThisworkwassupportedbygrantsfromtheTassiliProject,
theAlgeriangovernment,theBNPParibasFoundation,theERCgrant“Hairbundle”(ERC-
2011-AdG294570),ANR-15-RHUS-001(LIGHT4DEAF),andtheFrenchLABEXLIFE-
SENSESgrant[referenceANR-10-LABX-65].
AuthorContributions
Conceptualization:AZCP.
Formalanalysis:SAA.BahloulA.BehlouliCB.
Resources:ML.
Supervision:AZCP.
Validation:KBACRBYRZMDSMH.
Writing–originaldraft:SACBJPHA.BahloulCP.
Writing–review&editing:AZMMA.BahloulJPHCP.
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