Rev Colomb Cienc Pecu 2018; 31(2):150-154

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Revista Colombiana de Ciencias Pecuarias

Original articles

Frequency of canine degenerative myelopathy SOD1:c.118G>A mutation in 22 dog breeds in Guadalajara, Mexico¤

Frecuencia de la mutación SOD1:c.118G>A de mielopatía degenerativa canina en 22 razas de perros en Guadalajara, México

Frequência da mutação SOD1: c.118G>A de mielopatía degenerativa canina em 22 raças de cães no Guadalajara, México

Miguel A Ayala-Valdovinos, Zoot, MV, MSc, PhD; Ana A Gomez-Fernandez, IS; Theodor Duifhuis-Rivera, Zoot, MV, MSc; Eva A Aparicio-Cid, Zoot, MV; David R Sánchez-Chiprés, Zoot, MV, MSc, PhD; Jorge Galindo-García*, Zoot, MV, MSc, PhD.

Departamento de Producción Animal, Centro Universitario de Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, México.

(Received: November 06, 2016; accepted: September 22, 2017)

doi: 10.17533/udea.rccp.v31n2a08

¤ Tocitethisarticle:Ayala-ValdovinosMA,Gomez-FernandezAA,Duifhuis-RiveraT,Aparicio-CidEA,Sánchez-ChiprésDR,Galindo-GarcíaJ.FrequencyofcaninedegenerativemyelopathySOD1:c.118G>Amutationin22dogbreedsinGuadalajara,Mexico.RevColombCiencPecu2018;31(2):150-154.

* Correspondingauthor:JorgeGalindo-García.DepartamentodeProducciónAnimal,CentroUniversitariodeCienciasBiológicasyAgropecuarias,UniversidaddeGuadalajara.CaminoRamónPadillaSámchez#2100,Nextipac,Zappopan,Jalico(México).E-mail:Jorge_galindo99@outlook.com

Abstract

Background:Caninedegenerativemyelopathy(DM)isalate-onsetdiseasethatprimarilyaffectslarge-breeddogs. The disease involves the spinal cord and produces progressive paresia and, eventually, complete loss of mobility.DMhasbeenrelatedtomissensemutationc.118G>AintheSOD1 gene. Objective: To determine thegenotypicandgenicfrequenciesofDMinMexico.Methods:Intotal,330samplesfrom22differentdogbreedsweregenotypedusingthepolymerasechainreactionandrestrictionfragmentlengthpolymorphisms(PCR-RFLP)technique.Results:Themutationwasidentifiedin71animalsfrom11differentbreeds.Observedgenicfrequencieswere0.78fortheGalleleand0.14fortheAallele.Genotypicfrequencieswere0.79fortheG/Gwild-type,0.14fortheG/Aheterozygote,and0.7fortheA/Ahomozygote.Conclusion: The genic frequencyofthisalleleishighamongthestudiedpopulations.AmolecularmarkerprogramthatidentifiestheDMmutationinbreedingdogsshouldbeimplementedinordertoreducethisfrequency.

Keywords: genetic disease, molecular marker, PCR-RFLP, SOD1 gene.

Resumen

Antecedentes: Lamielopatíadegenerativacanina(MD)esunaenfermedadprogresivadepresentacióntardía que afecta a lamédula espinal, generalmente en caninos de razas grandes, y queproduceparesisprogresivayeventualpérdidacompletadelamovilidad.Seharelacionadoconunamutaciónpuntualpor

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sustitucióndebasesenelgenSOD1recientementeidentificadocomoc.118G>A.Objetivo: Determinar las frecuenciasgenotípicasygénicasparalapresentacióndeDMenMéxico.Métodos:Segenotipificaron330muestrasdeperrosde22razasmediantelatécnicadereacciónencadenadelapolimerasaypolimorfismosdelongituddefragmentosderestricción(PCR-RFLPs).Resultados:Seidentificólamutaciónen71animalesde11razasdiferentes.Lasfrecuenciasgénicasencontradasfueronde0,78paraelaleloGyde0,14paraelaleloA.Lasfrecuenciasgenotípicasfueronde0,79paraeltiposilvestreG/G,0,14paraelheterocigotoG/Ay0,7para el homocigoto A/A. Conclusión:Lafrecuenciaencontradaparalamutaciónesaltaenlaspoblacionesestudiadas.Laaplicacióndeunprogramadeselecciónasistidapormarcadoresmolecularescontralamutacióncausante de MDC en perros reproductores resultaría útil para reducir su frecuencia.

Palabras clave: enfermedad genética, gen SOD1, marcador molecular, PCR-RFLPs.

Resumo

Antecedentes:Amielopatíadegenerativacanina(MD)éumadoençaprogressivadeapresentaçãotardiaqueafetaamedulaespinalgeralmentedecaninosderaçasgrandesequeproduzparesiaprogressivaeeventualmenteaperdacompletadamobilidade.Temsidorelacionadacomumamutaçãopontualporsubstituiçãodebasesno gen SOD1,recentementeidentificadocomoc.118G>A.Objetivo:DeterminarasfrequênciasgenotípicasegenéticasparaaapresentaçãodeDMnoMéxico.Métodos:Genotipagemde330amostrasdecãesde22raçaspormeiodatécnicadereaçãoemcadeiadapolimeraseepolimorfismosnocomprimentodefragmentosderestrição(PCR-RFLPs).Resultados:Amutaçãofoiidentificadaem71animaisde11raçasdiferentes.Asfrequênciasgênicasencontradasforamde0,78paraoaleloGede0,14paraoaleloA.Asfrequênciasgenotípicasforamde0,79paraotiposilvestreG/G,0,14paraoheterozigotoG/Ae0,7paraohomozigotoA/A. Conclusão:Afrequênciaencontradaparaamutaçãoéaltanaspopulaçõesestudadas.AimplementaçãodeumprogramadeseleçãoassistidapormarcadoresmolecularescontraamutaçãoquecausaMDCseriaútilparareduzirasuafrequência.

Palavras-chave: doença genética, gen SOD1, marcador molecular, PCR-RFLPs.

Introduction

Caninedegenerativemyelopathy(DM)isalate-onset neurodegenerative disease generally reported in large-breeddogsthataffects thewhitematterofthe spinal cord (March et al., 2009).DM slowlydegenerates the upper motor neurons of the pelvic limbs,causingprogressiveparesisandproprioceptiveataxia(Milleret al.,2009).Completelossofmobilityis observed 6 to 12months after the first signsappeared (Cappucchioet al., 2014).Thoracic-limbfunctionalityaswellasfecalandurinarycontinenceusuallyremainunaffecteduntiltheterminalstageofthedisease.Althoughgenetic,metabolic,nutritional,vascular,andimmune-mediatedetiologieshavebeenproposed,aspecificpathogenicprocessremainstobeidentified(Marchet al.,2009).

DMwas originally considered to affect onlytheGermanShepherd breed (Awanoet al., 2009).However, the pathology has since been identifiedin approximately 125 different dog breeds (Zenget al., 2014).A definitive diagnosis forDM can

only be confirmed by a postmortem spinal cordhistopathologic study (Cappucchio et al., 2014).DM-associateddegenerativedamageincludesaxonalloss and demyelization of the spinal cord, mainly inthecaudalthoracicarea(Nakamaeet al.,2015).Variations in the nervous lesionsdescribed for thediseaseexistbetweendifferentbreedsandevenwithinasinglebreed.Nonetheless,thedegenerativechangestowhitematterfuniculiobservedinallbreedsinvolvedilationofthemyelinsheath,axonalswelling,aswellas fragmentation and phagocytosis of myelin and axonaldebris(Marchet al.,2009).

DM appears to be an autosomal recessive,incompletely penetrant disease related to a missense mutation in the superoxide dismutase 1 (SOD1)gene (Capucchio et al., 2014), which can beidentifiedbyPCR.A2009reportfoundthatdogswith a confirmed postmortem histopathologicalDMdiagnosiswerehomozygous for theAallelein missense mutation SOD1:c.118G>A, whichpredicts ap.E40Kaminoacid substitution in theSOD1 gene(Zeng et al.,2014).

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The identification of theDMcausingmutationcould be useful to differentiate this disease fromotherprogressivethoraco-lumbarsyndromecausingillnesses, such as intervertebral disk disease anddiskospondylitis(Joneset al.,2005).Theobjectiveofthepresentstudywastodeterminethegenotypicandgenic frequencies forDMinGuadalajara,Mexico,using the polymerase chain reaction and restriction fragment length polymorphisms (PCR-RFLP)technique.

Materials and methods

Ethical considerations

ThisstudywasapprovedbytheBioethicsInternalRegulation Committee of the Academic Centre of BiologicalandAgriculturalSciences,UniversityofGuadalajara,Mexico(CC/NN11-12/00/2012).

Study samples

Atotalof330bloodsampleswereobtainedfrom22 dog breeds and associated crossbreeds.Breedswere selected taking in account previous reportswhich had identified them as DM-susceptiblebreeds.Sampledbreedswere:AustralianCattledog,Australian Shepherd, BelgianMalinois, BorderCollie,Boxer,Cocker Spaniel,Collie, Dalmatian, EnglishBulldog,FrenchBulldog,GermanShepherd,and crossbreeds,GoldenRetriever,KerryBlueTerrier,LabradorRetriever,OldEnglishSheepdog,PembrokeWelshCorgi, Poodle, Pug,RhodesianRidgeback,Rottweiler, and crossbreeds, Shetland Sheepdog, andSiberianHusky.Bloodsampleswerestored inEDTAtubesandrefrigeratedbeforeprocessing.Somesampleswere submitted through the Small Animal Clinic of the Veterinary Medicine and Animal Science SchooloftheUniversityofGuadalajara. Othersweredonated to theUniversity ofGuadalajara by dogownersortheirveterinarians.

Sample genotyping

We extractedDNA from blood samples usingtheQuickDNA™UniversalKit (ZymoResearch,Orange,CA,USA).Forgenotyping throughPCR-RFLP,weusedtheThermoScientific™DreamTaq™

PCRkit(TakaraBio,Inc.,Kusatsu,Shiga,JAP)with20μLofreactionmixcontaining~100ngofbloodlysateDNA, 2 μL of 1X PCR buffer containing20mMMgCl2,1μLof10mMdNTPmix,0.5μLofDreamTaqDNAPolymerase,1.25pMolofbothprimers,andtheremainingvolumeofdouble-distilledwater (ddH2O). Sampleswere then digestedwiththeAcuI restriction endonuclease (NewEnglandBiolabs,Inc.,Ipswich,MA,USA)inaTechne® TC-5000 (Techne Inc.,Burlington,NJ,USA).Primersused to identify the gene encoding SOD1werethosedescribed byAwano et al. (2009;GenBank ID:NM_001003035).

Amplificationwasconducted inaTechne® TC-5000 (Techne Inc.,Burlington,NJ,USA) thermalcyclerwith the following PCR program: Initialdenaturingfor5minat95°C,with35consecutivecycles consisting of denaturing at 94 °C for 20 s,alignmentat50°Cfor30s,extensionat72°Cfor30 s, andfinal extension at 72 °C for 5min.Weanalyzedamplificationproductsby4%agarosegelelectrophoresiswithGelRed™(Biotium,Hayward,CA).Resultswerevisualizedunderultravioletlight.

Results

A total of 330 dogs from 22 breeds and theircrossbreeds were analyzed and genotyped toidentify the SOD1 genemutation associatedwithDM.Out of all analyzed animals, 24 representingfourbreedswere identifiedashomozygous for themutation(AA),correspondingto7.27%oftheentiresample;47animalsfrom 11breedswereidentifiedasheterozygousforthemutation(AG),correspondingto14.24%.TheresultingfrequencyfortheAallelewas0.14.Table1showstheAalleledistribution,accordingtobreed.ThemostrepresentativebreedforthisstudywasthePembrokeWelshCorgi,sinceitexhibitsthehighestAallelefrequencydespiteoccupyingthefifthplaceinnumberofsampledanimalsperbreed.

Discussion

Atotalof330samplesfrom22dogbreedswereanalyzed.Exactly half of these breeds carried thestudied mutation. Zeng et al. (2014) published a

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Table 1. A allele distribution in 11 the breeds with the c.118G>A mutation.

Breed N G/G (%) G/A (%) A/A (%) q

Border Collie 80 79 (99) 1 (1) 0 (0) 0.01

Boxer 62 34 (55) 21 (34) 7 (11) 0.28

Poodle 33 29 (88) 4 (12) 0 (0) 0.06

German Shepherd 31 25 (81) 4 (13) 2 (6) 0.13

Pembroke Welsh Corgi 21 0 (0) 7 (33) 14 (67) 0.83

Belgian Malinois 18 14 (78) 3 (17) 1 (6) 0.14

English Bulldog 12 10 (83) 2 (17) 0 (0) 0.08

Old English Sheepdog 9 8 (89) 1 (11) 0 (0) 0.06

Rottweiler 7 6 (86) 1 (14) 0 (0) 0.07

Pug 3 2 (67) 1 (33) 0 (0) 0.17

Australian Shepherd 2 0 (0) 2 (100) 0 (0) 0.50

n: Number of dogs tested; G/G: Two normal alleles; G/A: Heterozygous; A/A: two mutant alleles; q: Mutant allele frequency; %: Percent of dogs per category.

retrospective studyabout the allelicdistributionofthe SOD1geneinvariousdogbreeds,wherethetotalAallelefrequencywasestimatedtobe0.37.Inourstudy,thetotalfrequencywas0.14,whichreinforcesthe assertion that the SOD1:c.118G>Amutationis widespread across different dog populations.SimilaritiesexistbetweenthehighAallelefrequenciesreportedbyvariousauthorsforthePembrokeWelshCorgibreed.Forexample,Zenget al.(2014)reportedafrequencyof0.79,andAwanoet al. (2009)reportedafrequencyof0.70.Bothvaluesaresimilartothatobtainedinourstudy(0.83).Theseresultsdemonstratethat,intheaforementionedbreed,theSOD1 mutation is present in various dog populations, regardless of geographicdistancing.ThesefindingsalsostateDMisnotrestrictedtoaspecificgeographiczone.

Capucchio et al. (2014) found a totalA allelefrequencyof0.17intheGermanShepherd.Theauthorsreferredtothisfigureasahighandriskyfrequencyforamutantallele.Manyofthefrequenciesfoundinourstudyweresimilar(GermanShepherd0.13,BelgianMalinois0.14,Boxer0.28),demonstratingthatthesefrequenciesrepresentanimportantfactortoconsiderintheconstantmanifestationofDM.BothPembrokeWelshCorgiandBoxerbreedsdisplayanelevatedAallele frequency,whilealso showing thehighestnumberofhomocygoticanimals.InastudyconductedbyAwanoet al. (2009),sevenDM-affecteddogswere

identifiedthroughhistopathology,allofwhichhadanA/Agenotype.ThisfindingsuggeststhatthereisanextremelyhighriskfortheA/AhomocygoticanimalstoeventuallyexhibitDMclinicalsigns.

Inconclusion,wewereabletoidentifytheSOD1 mutationassociatedwithDMin11dogbreedsthroughPCR-RFLP.TheSOD1:c.118G>AmutationispresentinvariousdogbreedsinMexico,insomecaseswithparticularly high frequency.Amolecularmarkerthatcouldbeusedtoassistwithbreedingselectionwould allowbreeders to reduce this genetic factorinfluencingDMincidence,whilehelpingtodiminishits presentation in future dog generations.

Acknowledgments

This studywas funded by the University ofGuadalajarathroughprojectP3E229355.TheAnimalClinic of the Veterinary Medicine and Animal Science SchooloftheUniversityofGuadalajaraprovidedthecaninebloodsamplesusedinthestudy.

Conflicts of interest

The authors declare that theyhaveno conflictsofinterestwithregardtotheworkpresentedinthisreport.

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