A Nonsense Variant in CCDC65 Gene Causes Respiratory Failure
Jan 17 2022 Maxime Ben Braiek 1
A Nonsense Variant in CCDC65 Gene Causes Respiratory Failure
Jan 17 2022 Maxime Ben Braiek 1
A Nonsense Variant in CCDC65 Gene Causes Respiratory Failure
Dec 24 2021 Maxime Ben Braiek 1
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BENJAMIN ROMIEUX. LYDIA VON AUW. CHARLES BOURGEOIS. AGATHE SALINA. CLAUDE BERNEY. FRÉDÉRIC SAEGESSER. CHARLES FRÉDÉRIC FAUQUEX. PAUL CHAUDET.
A Nonsense Variant in CCDC65 Gene Causes Respiratory Failure
Dec 24 2021 Ben Braiek 1
Identification of homozygous haplotypes carrying putative recessive
Maxime Ben Braiek1 Stéphane Fabre1*†
BTT Mens and Womens Rankings for pdf template.xlsx
Dec 16 2019 RINGLSTETTER Benjamin (GER). 197. 79. BELLER Alexander (CHI) ... JOLLOIS Benjamin (FRA). 24. 482. ALVAREZ Danny (ECU) ... ROMIEU Loic (FRA).
A Nonsense Variant in CCDC65 Gene Causes Respiratory Failure
Dec 24 2021 Maxime Ben Braiek 1
The Gascon Énonciatif System: Past Present
https://escholarship.org/content/qt12v9d1gx/qt12v9d1gx_noSplash_5d475992ae6ca4e08f26bb8b9b798c21.pdf?t=mtfcqd
A Nonsense Variant in CCDC65 Gene Causes Respiratory Failure
Dec 24 2021 Maxime Ben Braiek 1
C.;Allain,C.;Bardou,P.; Bordes,A.;
Plisson-Petit,F.; Portes,D.;Sarry,J.;
etal.A NonsenseVariant inCCDC65GeneCausesRespiratory Failure
AssociatedwithIncr easedLamb
MortalityinFr enchLacauneDairy
Sheep.Genes2022,13,45.https://
doi.org/10.3390/genes13010045AcademicEditor:
BeatrizGutierrez-Gil
Received:17November 2021
Accepted:20December 2021
Published:24December 2021
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distributedunderthe termsand conditionsofthe CreativeCommonsAttribution(CCBY) license(https://
creativecommons.org/licenses/by/4.0/).genesG C A T
T A C G
G C A TArticle
ANonsenseV ariantinCCDC65GeneCausesRespiratory
FailureAssociatedwith IncreasedLambMortality inFrenchLacauneDairySheep
BenBraiek
1,CaroleMoreno-Romieux 1,CharlotteAllain 2,PhilippeBardou 3,ArnaudBordes 1,Fr²d²ricDebat1,
4,FlorencePlisson-Petit
1,DavidPortes 2,JulienSarry 1,N²muelTadi 1,FlorentW oloszyn1
andSt phaneFabre 1,*1GenPhySE,Universit
²deToulouse, InstitutNationaldeRecherchePour L"agriculture, L"alimentationetL"environnement(INRAE),EcoleNationaleV²t²rinairedeToulouse(ENVT), 31326Castanet-Tolosan, France;
maxime.ben-braiek@inrae.fr(B.B.);car ole.moreno-romieux@inrae.fr (C.M.-R.);arnaud.bordes@inrae.fr(A.B.);
frederic.debat@inrae.fr(F.D.);florence.plisson-petit@inrae.fr (F.P.-P.);julien.sarry@inrae.fr(J.S.); nemuel.tadi@inrae.fr(N.T.); florent.woloszyn@inrae.fr(F.W.)2UEDomainede LaFage,Institut NationaldeRecher chePourL "agriculture,L "alimentationet
L"environnement(INRAE),12250Saint-JeanetSaint-Paul,France; charlotte.allain@inrae.fr(C.A.); david.portes@inrae.fr(D.P.)3Sigenae,InstitutNational deRecherche PourL"agricultur e,L"alimentation etL"environnement(INRAE),
31326Castanet-Tolosan, France;philippe.bardou@inrae.fr
4InstituteofGenetics, VetsuisseFaculty ,Universityof Bern,3012Bern,Switzerland;
cord.droegemueller@vetsuisse.unibe.ch *Correspondence:stephane.fabre@inrae.frAbstract:
Werecentlydemonstratedthat theLacaunedeficienthomozygoushaplotype6 (LDHH6) potentiallyhostsa recessiveperinatal lethalmutation inLacaunedairysheepmappedon OAR3.In thepresent study,wehaveanalyzedthe whole-genomesequencesoftwoLacauneram heterozygous carriersofLDHH6. Aftervariantcalling andfilteringagainst thevariantsof 86non-carrierrams, we haveidentifieda singlenucleotidevariant (SNV)inthe twoLDHH6carriers whosevariantallele inducedapr ematurestop codon(p.Glu111*)intheCoiled-CoilDomainContaining 65(CCDC65)gene. CCDC65isinvolved intheassembly ofthenexin-dynein regulatorycomplex fortheformation of microtubulesinciliatedcells.Inorder toidentifythe phenotypeinhomozygous sheep,wegenerated at-riskmatings( n=17)between ramsandewes heterozygousfor thecandidatevariant inCCDC65. Atotalof 16lambswer ebornalive withfivegenotyped ashomozygouscarriers. Thehomozygous lambssuffer edfromrespiratoryproblems,and fourofthemdiedwithinthe firstmonthoflife.At necropsy,weobservedabroadhepatization oflunglobes possiblyinducedby infectiouspneumonia. Themanagementof thislethalr ecessiveallele(fr equencyof0.06) throughr easonedmatinginthe Lacaunesheepselection schemescouldr educelambmortality by2%.Keywords:
homozygoushaplotype deficiency;LDHH6;CCDC65;juvenilelethalmutation ;genetic disorder;primaryciliadyskinesia(PCD); ciliopathy;development;sheep 1.Introduction Healthandanimal welfarear emajorconcerns inlivestockpopulationswithimportant economicreper cussions.Duringthelastfewdecades,mortalityinthe firstmonthof life remainshigharound15-20% insheepand mostofthismortalityoccurswithin thefirstthr ee daysofpostnatal life[1,2].Therisk factorsforlamb mortalityare mainlydueto parameters
dependingonthe mother(nutrition,mothering ability,health status,parturitionconditions), thelambitself (lowbirthweight, vigor,colostr umintake,congenital malformations)and/or theirenvironment (extremeweather,predation, infectiousdiseases)[ 1 3 Althoughadequatefarm environment andmanagementpractices couldfavorlamb viability(reviewed inDwyeretal.[ 1]),itwould bepossibleto actonthe geneticaspectof Genes2022,13,45.https://doi.or g/10.3390/genes13010045https://www.mdpi.com/journal/genes
Genes2022,13, 452 of14 lamb survival.However ,theQTLappr oachfor mappinggenetic variants/lociaffecting
this traitis diffi cultduetolowheritability(h2< 0.1)and multifactorialnatur eof lamb
survival orlamb birthweight(i.e., bestpr oxyfor lambsurvival) [1,4-6]. Nevertheless,
thanks tothe useof highthr oughputgenomi ctools (singlenucleotidepolymorphism arrays, whole-genomesequences), thestudy ofgenetic disorders andanalysis ofassociated data havesuccessfully ledthe identification ofmany causalvariantsaf fectingthe viability of younganimals [ 7 Nowadays, twomain approaches areusedto identifyandcharacterizecausal variants associated withgenetic disorders. Thefirstoneis thehomozygosity-mapping basedon a case-controlappr oachusingonlyfew biologicalsamples from affected andnon-af fected animals [8-10]. Inthis pioneeringuse ofhomozygosity-mapping usinglar genumber of SNP arrayin cattle,Charlier etal. haveidentified three causal variantsr esponsiblefor congenital musculardystony types 1[OMIA 001450-9913]and 2[OMIA 001451-9913]in Belgian Bluecattle andichthyosis fetalis[OMIA 002238-9913]in ItalianChianina cattle[ 8]. In sheep,this approach wasusedsuccessfullyfor thefirst timein the microphthalmia[OMIA000649-9940] inT exel[
11]. Sincethe lastfew decades,this methodis effi cientto mapgenetic
defects andtheir associatedvariants asshown bythe growing numberof discovered causal variants referencedintheOMIA: OnlineMendelian Inheritancein Animaldatabase ( https: //www.omia.org/ ; accessed2 September2021). Asexample, withthis approach, several pathogenic variantswer eidentifiedcausingjunctional epidermolysisbullosa in cattle[ 12,13] and sheep[ 14-16] andthe affected animalsdiedshortlyafter birthdue toskin disorders (injuries onlimb extremities, mucousmembranes).However, whenmutations affect viability ofdeveloping embryosduring gestation,phenotype orbiological samplesar enot available. Thus,these mutationsar emor eefficientlydetectable byasecondappr oachusing reversegenetic screens. Thisapproachis basedon theavailabilityoflar gesets ofSNP array genotyped animalspr ovidedbygenomicselectionfor example[ 17,18]. Reversescr eens identified haplotypesfor whichhomozygous carrieranimals are absentor significantlyless observed whencompar edtotheexpected numberbased onhaplotype frequency .In cattle, the originalworks ofV anRadenet al.[17] andFritz etal. [18] usingreverse geneticscreen followed bywhole-genome sequenceanalyses havesuccessfully identifiedcandidate causal variants inlinkage disequilibriumwith haplotypeswith significantpartial ortotal deficitin homozygous animals.Going onwith thisappr oach,numer ouse mbryoniclethalmutations wereidentified inHolstein andJersey breeds alteringAPAF1[19],SMC2[20,21],GART[18],TFB1M[
22],SDE2[23],CENPU[24] andCWC15[25] genes.Additionally ,neonatal/juvenile
lethal mutationsassociated withhomozygous haplotype deficiencywer ealsoidentifiedin AyshireandBraunvieh breeds affecting UBE3B[20] andTUBD1[26] genes. Recently,with theimplementation ofgenomic selectionin smallr uminants[ 27] and the availabilityoflar gege notypingdatasets,wehave identifiedhomozygousdeficient haplotypes segregatinginthe Lacaunedairy sheeppopulation bya reverse geneticscr een method [28]. Wehaveidentified 11haplotypes withsignificant deficitin homozygous animals, rangingfr om79to100%. Someof themwer eassociated withfertility lossat artificial insemination,and/or withincr easedstillbirth rate.Wether efore assumedthat these haplotypesar elikelytohost recessive mutationscausing embryonic/fetalor perinatal lethality,r espectively.Amongthesehaplotypes,LDHH6 (Lacaunedeficient homozygous haplotype 6)[OMIA 002342-9940]was themost frequent (12.1%of heterozygous carriers, located onOAR3:146.2-147.9 Mbon Oar_rambouillet_v1.0)and presented ahomozygous deficit of96% (three homozygousanimalswere observed,wher eas72 animalswereex- pected;p= 3.51027). LDHH6showed anincr easedstillbirth rateinat-riskmatings
between carrierrams anddaughte rsof carrierramswhencompar edto safematings. We have previouslyreported severalcandidategenesin theLDHH6 region ( WNT1 ,CCDC65 andPFKMgenes), butthe underlyingcausative variantis stillunknown [28]. In thepr esentstudy,we havetakenadvantageof whole-genomesequence datato identify thecausal variantassociated withthe LDHH6haplotype, studiedthe segregationGenes2022,13, 453 of14 of thevariant inseveral sheepbr eedsand validatedthe lethaleffect ofthe proposed causal
variant bygenerating at-riskmatings.2. Materialsand Methods
2.1. SequencingData
For variantcalling, publiclyavailable dataof 88ovine shortr eadIllumina HiSeq whole genome sequences(WGS) from 14breedsgenerated invarious INRAEandTeagasc resear ch projectswer eused.Amongthem, 24WGS were obtainedfr omdairy Lacaunesheep, and also genotypedwith theOvineSNP50 Beadchipfr omIllumina inthe frameworkofLacaune dairy sheepgenomic selectionpr ogram[ 27]. Adescription ofthe differ entbr eedsandall the accessionnumbers ofsequencing rawdata are availablein SupplementaryT ableS1.2.2. WGSV ariantCallingandAnnotation
Reads mappingand variantscalling were performedusing Nextflowv20.10.0 and Sarekpipeline v2.6.1[ 29]. GenomeAnalysis Toolkit (GATK)bestpractices werefollowed as implementedin theSar ekpipeline. Inthisproject, thefollowing stepswer eper - formed: aligningthe reads withBWAv0.7.17-r1188 [30] againstthe ovinegenome assembly Oar_rambouillet_v1.0 (GCF_002742125.1),marking duplicater eads(MarkDuplicates), base quality recalibration(BQSR)and callinggermline smallvariants (HaplotypeCaller inGVCF mode) withGA TKv4.1.7.0[31], annotationof smallvariants withSnpEf fv4.3t [32] and quality controlwithMultiQC v1.8[ 332.3. Identificationof CandidateCausal Variants
Based onOvineSNP50 Beadchipgenotyping, twoLacaune animalswer edetected as LDHH6 heterozygouscarriers(others asnon-carri ers)among the24 sequencedLacaune genomes [28]. AllSNPs, smallinsertion anddeletion variantslocated withinthe LDHH6 regionextended by1 Mbfr omeach sidewer eextractedfrom OAR3(Oar_rambouillet_v1.0; NC_040254.1:145,243,481-148,946,399pb) usingSnpSift Filter[ 32]. Theselectionfilter of candidate polymorphismswas asfollows: (i)variant allelecompar edto theRambouillet referencegenome;(ii) inthe heterozygote statein the2 heterozygousLDHH6-carriers Lacaune sheep;and (iii)in ther eference homozygousstate inallLDHH6-noncarriers Lacaune sheepand inother non-related breeds. Thesevariantswere checkedmanually using theIntegrative GenomicsV iewer(IGV) [34] toconfirm thebioinformatics variant
calling priorto furtherinvestigation.2.4. BiologicalSamples andCCDC65-Specific GenotypingAssay
Blood samples(3 ml)wer ecollected byjugularveinpunctur ewith theV enojectsystem containing EDTA(Ter umo,Tokyo,Japan)anddir ectlystoredat 4C or20C depending on furtheruse. Earsamples were obtainedwith atissuesamplingunit (TSU,Allflex Europe, Vitr , France)taking anear punch(1 mm3) directlyplacedin theTSU storagebuf fer at4 C.
Ear biopsieswere placedtwiceconsecutivelyin 180L of50 mMNaOH, heated10 min at 95 C, neutralizedwith 20L of1 MHCl, andthen vortexedduring 10s. Partof the blood sampleswas usedfor extractionof genomicDNA asdescribed inBodin etal. [35]. All othersamples were usedfordirect genotypingwithout DNApurification onwhole blood oron neutralizedNaOH treatment solutionof earbiopsies[ 36causal variant(Oar_rambouillet_v1.0, NC_040254.1:g.147,207,999C> A;XM_004006389.4:c.
521G >T) wasgenotyped eitherby RFLPor PACE (PCRallele competitiveextension)
analysis. RFLPwas resolved on2%agarose gelusing BsaJIr estrictionenzyme(News England Biolabs,Ipswich, MA,USA) aftereither afirst stepof Terra PCRDir ectPoly- merase Mixamplification (Takara Bio,Kusatsu,Japan)using 1L oftotal blood(or 5L ear biopsysolution), ora PCRusing GoTaq FlexiDNA Polymerase(Pr omega,Madi- son, WI,USA) with50 ngof purifiedDNA. Thefollowing amplificationprimers (for- ward5 0-GAGCTGCGTGTGTAAGATGA-30and reverse50-CCTCCAGCTCCATGTTGTAA- Genes2022,13, 454 of14 30) weredesignedusing Primer3Plussoftwar e[ 37]. PCRfor RFLPwas performedon an ABI2720thermocycler (AppliedBiosyst ems,W altham,MA,USA)with thefollow- ing conditions:5 minat 94C, 35cycles of30 sat 94C, 30s at58 C and30 sat
72C, followedby 5min finalextension at72 C. FluorescentPACE analysiswasdone with 15ng ofpurified DNAusing theP ACE-IR2x GenotypingMaster mix(3CR Bio- science) inthe presence of12M ofa mixof extended allelespecific forwardprimers(5 0- GAAGGTGACCAAGTTCATGCTGGACCTGTCAGAAGCCGAGG-30and 50-GAAGGTGA CCAAGTTCATGCTGGACCTGTCAGAAGCCGAGT-30) and30 M ofcommon reverse primer (50-AGGGCGTGGGCGTGCTGCT-30) ina finalvolume of10 L. Thetouch-down PCR amplificationcondition was15 minat 94C forthe hot-startactivation, 10cycles of
20 sat 94C, 54-62C for60 s(dr opping0.8 C percycle), then36 cyclesof 20s at94 C
and 60s at54 C performedon anABI9700 thermocyclerfollowed bya finalpoint read of the fluorescenceonan ABIQuantStudi o6 real-time PCRsystemandusing theQuantStudio software1.3 (AppliedBiosystems). Theaccuracy ofthe genotypingwas validatedby Sanger sequencing onfew samples. The presenceofthe CCDC65variant waschecked ina DNAset ofthe 2021cohort of 2952Lacaune malelamb candidatesfor genomicselection. DNAwas extractedbyLabogena (
http://www.labogena.fr , accessedon 2September 2021)for low-densitySNP chip genotyping(SheepLD v.3, onbehalfthedairy Lacaunebr eedindustry) andLDHH6 status wasestablished aspr eviouslydescribed [28]. ADNA diversitypanel of872 animals from25 French sheepbreeds[ 38] and8 Swisssheep breeds [39] wasalso specifically genotyped forthe CCDC65variant.2.5. Generationof HomozygousLambs
Dairy Lacauneewes (
n= 245)fr omtwoINRAEexperimental flocks(Langlade andLa Fage, agreementnumbers:D3142901 andA312031, respectively) were genotyped forthe CCDC65variant. Heterozygousewes( n= 17)wer eartificiallyinseminated (AI)withfresh semen fromheterozygous carrierrams( n= 3)s electedamongthegenotyped animalsfor genomic selectionwith aknown statusat theLDHH6 haplotype[ 28] andfurther genotyped as heterozygousatthe CCDC65locus. Anultrasound diagnosisof gestationwas realized between 45and 60days afterAI. Lambswer eweighted atbirth and15 daysafterbirth.Ear biopsies fromnewbornlambs (TSUAllflex) were collectedfor CCDC65genotyping atthe same timeof themandatory electronic eartag identification.2.6. EthicsStatement
by theFr enchMinistryofT eachingand ScientificResear chandlocalethical committee C2EA-115 (Scienceand AnimalHealth) inaccor dancewith theEur opeanUnionDirective2010/63/EU onthe protection ofanimalsusedfor scientificpurposes.
3. Results
3.1. ScreeningofWGS DataIdentifies aNonsense Variant inCCDC65 GeneAssociated
with LDHH6 Toidentify theputative causalvariant hostedby theLDHH6 haplotype,we consid- eredbiallelic polymorphisms(SNPs andInDels) proven from 88ovine WGScontaining24 Lacaunesequences and,among them,two LDHH6heter ozygouscarriers. Variant
searchanalysis andannotations were deliberatelylimited totheLDHH6r egion(OAR3:146,243,481-147,946,399 pb)extended by1 Mbfr omeach side.In this3.7Mbr egion,we
detected 53,632polymorphisms witha qualityscor e> 30.After filtering,weidentified carriers (Table1).Genes2022,13, 455 of14
Table1. Candidate SNPs,locations andfunctional annotations.Position Ref/AltQuality ScoreLocation AnnotationFunctional Consequence
a145,928,967 C/G347.3 Intergenic, downstreamofFAM186AModifier146,173,708 G/A396.0 Intergenic, upstreamofASIC1Modifier
146,566,556 C/T133.1 Intronic, FAM186BModifier
146,718,479 G/A272.7 Intronic, SPATS2Modifier
146,809,812 T/C604.4 Intronic, DNAJC22Modifier
147,207,999 C/A506.7 Exonic,CCDC65(c.521G >T) High,stop-gain (p.Glu111*)
147,345,297 G/A192.3 Intergenic, upstreamofTEX49Modifier
148,189,184 A/G596.7 Intergenic Modifier
148,212,194 A/C181.7 IntergenicModifier
148,417,713 A/G545.8 Intergenic,downstream ofZNF641Modifier
148,904,267 G/A439.1 Intronic,HDAC7Modifiera
variant annotationand effect predictedbySnpEf f[ 32].Among thoseSNPs, onlyone waspr edictedto highlyalter genefunction.ThisSNV (NC_040254.1:g.147,207,999C >A; XM_004006389.4:c.521G> T)leads toa nonsensevariant located inexon 3of theCoiled-Coil DomainContaini ng65(CCDC65) gene(Figur e1a,b). The variant shouldr esultinapr emature stopcodon andatruncatedprotein inposition 111 (XP_004006438.1:p.Glu111*) whilethe entire translatedCCDC65protein iscompo sedof
498 aminoacids (Figure 1c). Thevariant localizesin thefirst Coiled-Coil(CC) domain
in ahighly conservedr egionof theproteinbetween mammalspecies andeven inalgaeChlamydomonas reinhardtii
) wherether oleof CCDC65wasoriginallystudied [40-43]
(Supplementary FigureS1).The resulting truncated proteinhaskept themajorpartof the NYD-SP28 domainshar edwithCCDC164also partof theN-DRC [44] buthas losttwo CC domains knownto beimplicated inpr otein-protein interaction[ 4243
].Figure 1. Nonsense variantin CCDC65gene associatedwith theLDHH6 lethalhaplotype. (a) LDHH6 haplotype (NC_040254.1:OAR3:146,243,481pb-147,946,399pb)extended by1 Mbfr omeach side. The blackbar indicatesthe limitsof LDHH6haplotype withthe firstand lastmarkers (Illumina OvineSNP50 OAR3_145545612.1an dOAR3_147275963.1).Vertical linesindicate thepositionsof the candidatecausal SNVin CCDC65(red)and 10other SNVsfully associatedwith LDHH6(blue);
Genes2022,13, 456 of14 (b)CCDC65gene structureandlocalizationofthe g.4732G> T(GeneID:101104220)/c.521G >T
(XM_004006389.4) variantin thethir dexon (UTR:untranslatedregion, CDS:coding sequence); (c) schematicr epresentationofCCDC65protein (XP_004006438.1)with oneN-terminal 'NYD-SP28" (Pfam PF14772)and three coiled-coil(CC)domains(UniP rotKB-W5QCQ9), andposition ofthe prematureC-terminalr esidue,pGlu111.3.2. VariantAssociationwith LDHH6and PopulationEstimation ofAllele Frequency
In ordertopr ovideadditional evidenceinfavorof theCCDC65polymorphism as the causalvariant, wehave genotypedthe c.521G> TSNP ina cohortof 2952Lacaune lambs withknown status attheLDHH6locus (SupplementaryFigur eS2). Thecontingency table indicateda clearassociation betweenthe LDHH6status andthe nonsensevariant in CCDC65(Table2, Fischer"sexacttestp<0.0001,without thehomozygouscarrierindividual). Based onthis genotyping,the c.521Tvar iantallele frequency wascalculatedat7%, and, as expected,the distributionof genotypeswas notconsistent withthe Hardy-W einberg equilibrium (Chi-squaretest,p< 0.001).Table2. Contingency tablebetween LDHH6status andgenotype atc.521G >T inCCDC65.Genotype +/+LDHH6/+ LDHH6/LDHH6T otal
G/G 25403 02543
G/T93990408
T/T0011
Total254940212952+/+: non-carriers;LDHH6/+: heterozygous carriersandLDHH6/LDHH6:homozygous carriers. Since 2017,all Lacaunemale lambcandidates forgenomic selectionwer egenotyped on low-densitysheep (SheepLDchip) betweenone andfive monthsof age,with agood representationofthe geneticdiversity inthe selectionschemes. Inthe Lacaunedairy breed, the populationconsists oftwo subpopulationswith separateselection schemesconducted by twobr eedingcompanies.Overall,LDHH6 heterozygous carrierfr equencyobserved is stable inthe populationbetween 2017and 2021.Nevertheless, thefr equencyof carriers was two-foldhigher forbr eedingcompany 1comparedto breeding company2 (Figure2).Figure 2. Evolution ofLDHH6 heterozygous carrierfrequencybetween 2017and 2021indairy Lacaune malelambs. Thefr equencyis indicatedeitherfor allcandidates togenomic selection(Total) or dependingon thebr eedingcompany (BC_1or2).3.3. Generationof At-RiskMatings toObtain HomozygoteLambs
The genotypingof theCCDC65variant intwo experimentalflocks ofdairy Lacaunequotesdbs_dbs25.pdfusesText_31[PDF] benjamin sabatier - Galerie Bertrand Grimont - Art Et De Divertissement
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