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Frequency of canine degenerative myelopathy SOD1:C.118G>A mutation in 22 dog breeds in guadalajara, Mexico

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Abstract

Background: Canine degenerative myelopathy (DM) is a late-onset disease that primarily affects large-breed dogs. The disease involves the spinal cord and produces progressive paresia and, eventually, complete loss of mobility. DM has been related to missense mutation c.118G>A in the SOD1 gene. Objective: To determine the genotypic and genic frequencies of DM in Mexico. Methods: In total, 330 samples from 22 different dog breeds were genotyped using the polymerase chain reaction and restriction fragment length polymorphisms (PCR-RFLP) technique. Results: The mutation was identified in 71 animals from 11 different breeds. Observed genic frequencies were 0.78 for the G allele and 0.14 for the A allele. Genotypic frequencies were 0.79 for the G/G wild-type, 0.14 for the G/A heterozygote, and 0.7 for the A/A homozygote. Conclusion: The genic frequency of this allele is high among the studied populations. A molecular marker program that identifies the DM mutation in breeding dogs should be implemented in order to reduce this frequency.
Rev Colomb Cienc Pecu 2018; 31(2):150-154
150
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
¤ Tocitethisarticle:Ayala-ValdovinosMA,Gomez-FernandezAA,Duifhuis-RiveraT,Aparicio-CidEA,Sánchez-ChiprésDR,Galindo-GarcíaJ.Frequency
ofcaninedegenerativemyelopathySOD1:c.118G>Amutationin22dogbreedsinGuadalajara,Mexico.RevColombCiencPecu2018;31(2):150-154.
* Correspondingauthor:JorgeGalindo-García.DepartamentodeProducciónAnimal,CentroUniversitariodeCienciasBiológicasyAgropecuarias,Universidad
deGuadalajara.CaminoRamónPadillaSámchez#2100,Nextipac,Zappopan,Jalico(México).E-mail:Jorge_galindo99@outlook.com
Abstract
Background:Caninedegenerativemyelopathy(DM)isalate-onsetdiseasethatprimarilyaectslarge-breed
dogs. The disease involves the spinal cord and produces progressive paresia and, eventually, complete loss of
mobility.DMhasbeenrelatedtomissensemutationc.118G>AintheSOD1 gene. Objective: To determine
thegenotypicandgenicfrequenciesofDMinMexico.Methods:Intotal,330samplesfrom22dierentdog
breedsweregenotypedusingthepolymerasechainreactionandrestrictionfragmentlengthpolymorphisms
(PCR-RFLP)technique.Results:Themutationwasidentiedin71animalsfrom11dierentbreeds.Observed
genicfrequencieswere0.78fortheGalleleand0.14fortheAallele.Genotypicfrequencieswere0.79for
theG/Gwild-type,0.14fortheG/Aheterozygote,and0.7fortheA/Ahomozygote.Conclusion: The genic
frequencyofthisalleleishighamongthestudiedpopulations.Amolecularmarkerprogramthatidentiesthe
DMmutationinbreedingdogsshouldbeimplementedinordertoreducethisfrequency.
Keywords: genetic disease, molecular marker, PCR-RFLP, SOD1 gene.
Resumen
Antecedentes: Lamielopatíadegenerativacanina(MD) esuna enfermedadprogresivadepresentación
tardía que afecta a la médula espinal, generalmente en caninos de razas grandes, y que produce paresis
progresivayeventual pérdida completa de la movilidad. Se harelacionadoconunamutaciónpuntualpor
151
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sustitucióndebasesenelgenSOD1recientementeidenticadocomoc.118G>A.Objetivo: Determinar las
frecuenciasgenotípicasygénicasparalapresentacióndeDMenMéxico.Métodos:Segenotipicaron330
muestras deperrosde 22razasmediantela técnicadereacción encadenade lapolimerasay polimorsmosde
longituddefragmentosderestricción(PCR-RFLPs).Resultados:Seidenticólamutaciónen71animalesde11
razasdiferentes.Lasfrecuenciasgénicasencontradasfueronde0,78paraelalelo Gy de0,14paraelalelo
A.Lasfrecuenciasgenotípicasfueronde0,79paraeltiposilvestreG/G,0,14paraelheterocigotoG/Ay0,7
para el homocigoto A/A. Conclusión:Lafrecuenciaencontradaparalamutaciónesaltaenlaspoblaciones
estudiadas.Laaplicacióndeunprogramadeselecciónasistidapormarcadoresmolecularescontralamutación
causante de MDC en perros reproductores resultaría útil para reducir su frecuencia.
Palabras clave: enfermedad genética, gen SOD1, marcador molecular, PCR-RFLPs.
Resumo
Antecedentes:Amielopatíadegenerativacanina(MD)éumadoençaprogressivadeapresentaçãotardiaque
afetaamedulaespinalgeralmentedecaninosderaçasgrandesequeproduzparesiaprogressivaeeventualmente
aperdacompletadamobilidade.Temsidorelacionadacomumamutaçãopontualporsubstituiçãodebases
no gen SOD1,recentementeidenticadocomoc.118G>A.Objetivo:Determinarasfrequênciasgenotípicas
egenéticaspara aapresentaçãodeDMnoMéxico.Métodos:Genotipagemde 330amostrasdecãesde22
raçaspormeiodatécnicadereaçãoemcadeiadapolimeraseepolimorsmosnocomprimentodefragmentos
derestrição (PCR-RFLPs).Resultados:Amutaçãofoi identicadaem71animais de11raçasdiferentes.
Asfrequências gênicasencontradasforam de0,78para oaleloGe de0,14para oaleloA.Asfrequências
genotípicasforamde0,79paraotiposilvestreG/G,0,14paraoheterozigotoG/Ae0,7paraohomozigoto
A/A. Conclusão:Afrequênciaencontradaparaamutaçãoéaltanaspopulaçõesestudadas.Aimplementação
deumprogramadeseleçãoassistidapormarcadoresmolecularescontraamutaçãoquecausaMDCseriaútil
parareduzirasuafrequência.
Palavras-chave: doença genética, gen SOD1, marcador molecular, PCR-RFLPs.
Introduction
Caninedegenerativemyelopathy(DM)isalate-
onset neurodegenerative disease generally reported
inlarge-breeddogsthat aects the whitematterof
the spinal cord (March et al., 2009). DM slowly
degenerates the upper motor neurons of the pelvic
limbs,causingprogressiveparesisandproprioceptive
ataxia(Milleret al.,2009).Completelossofmobility
is observed 6 to 12 months after the first signs
appeared (Cappucchio et al., 2014). Thoracic-limb
functionalityaswellasfecalandurinarycontinence
usuallyremainunaecteduntiltheterminalstageof
thedisease.Althoughgenetic,metabolic,nutritional,
vascular,andimmune-mediatedetiologieshavebeen
proposed,aspecicpathogenicprocessremainstobe
identied(Marchet al.,2009).
DM was originally considered to affect only
the German Shepherd breed (Awano et al., 2009).
However, the pathology has since been identied
in approximately 125 dierent dog breeds (Zeng
et al., 2014). A definitive diagnosis for DM can
only be confirmed by a postmortem spinal cord
histopathologic study (Cappucchio et al., 2014).
DM-associateddegenerativedamageincludesaxonal
loss and demyelization of the spinal cord, mainly
inthecaudalthoracic area (Nakamae et al., 2015).
Variations in the nervous lesions described for the
diseaseexistbetweendierentbreedsandevenwithin
asinglebreed.Nonetheless,thedegenerativechanges
towhitematterfuniculiobservedinallbreedsinvolve
dilationofthemyelinsheath,axonalswelling,aswell
as fragmentation and phagocytosis of myelin and
axonaldebris(Marchet 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 be
identiedbyPCR.A2009reportfoundthatdogs
with a conrmed postmortem histopathological
DMdiagnosiswere homozygous for theAallele
in missense mutation SOD1:c.118G>A, which
predictsap.E40K amino acidsubstitutionin the
SOD1 gene(Zeng et al.,2014).
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The identication of the DM causing mutation
could be useful to dierentiate this disease from
otherprogressivethoraco-lumbarsyndromecausing
illnesses, such as intervertebral disk disease and
diskospondylitis(Joneset al.,2005).Theobjectiveof
thepresentstudywastodeterminethegenotypicand
genicfrequencies for DM in Guadalajara, Mexico,
using the polymerase chain reaction and restriction
fragment length polymorphisms (PCR-RFLP)
technique.
Materials and methods
Ethical considerations
ThisstudywasapprovedbytheBioethicsInternal
Regulation Committee of the Academic Centre of
BiologicalandAgriculturalSciences,Universityof
Guadalajara,Mexico(CC/NN11-12/00/2012).
Study samples
Atotalof330bloodsampleswereobtainedfrom
22 dog breeds and associated crossbreeds. Breeds
were selected taking in account previous reports
which had identified them as DM-susceptible
breeds.Sampledbreedswere:AustralianCattledog,
Australian Shepherd, Belgian Malinois, Border
Collie, Boxer, Cocker Spaniel, Collie, Dalmatian,
EnglishBulldog,FrenchBulldog,GermanShepherd,
and crossbreeds,GoldenRetriever,KerryBlueTerrier,
LabradorRetriever,OldEnglishSheepdog,Pembroke
Welsh Corgi, Poodle, Pug, Rhodesian Ridgeback,
Rottweiler, and crossbreeds, Shetland Sheepdog,
andSiberian Husky.Blood samples were stored in
EDTAtubesandrefrigeratedbeforeprocessing.Some
samples were submitted through the Small Animal
Clinic of the Veterinary Medicine and Animal Science
SchooloftheUniversityofGuadalajara. Otherswere
donated to the University of Guadalajara by dog
ownersortheirveterinarians.
Sample genotyping
We extracted DNA from blood samples using
the Quick DNA™ Universal Kit (Zymo Research,
Orange, CA, USA). For genotyping through PCR-
RFLP,weusedtheThermoScientic™DreamTaq™
PCRkit(TakaraBio,Inc.,Kusatsu,Shiga,JAP)with
20μLofreaction mixcontaining~100ngofblood
lysate DNA, 2 μL of 1X PCR buer containing
20mMMgCl2, 1μLof10mMdNTPmix,0.5μL
ofDreamTaqDNAPolymerase,1.25pMolofboth
primers,andtheremainingvolumeofdouble-distilled
water (ddH2O). Samples were then digested with
theAcuI restriction endonuclease (New England
Biolabs,Inc.,Ipswich,MA,USA)inaTechne® TC-
5000 (Techne Inc., Burlington, NJ, USA). Primers
used to identify the gene encoding SOD1werethose
described by Awano et al. (2009; GenBank ID:
NM_001003035).
Amplicationwas conductedin a Techne® TC-
5000 (Techne Inc., Burlington, NJ, USA) thermal
cycler with the following PCR program: Initial
denaturingfor 5 minat95°C,with35 consecutive
cycles consisting of denaturing at 94 °C for 20 s,
alignmentat 50 °C for30s,extensionat72°C for
30 s, and nal extension at 72 °C for 5 min. We
analyzedamplicationproductsby4%agarosegel
electrophoresiswith GelRed™(Biotium,Hayward,
CA).Resultswerevisualizedunderultravioletlight.
Results
A totalof 330 dogs from 22 breeds and their
crossbreeds were analyzed and genotyped to
identify the SOD1 gene mutation associated with
DM. Out of all analyzed animals, 24 representing
four breeds were identied as homozygous for the
mutation(AA),correspondingto7.27%oftheentire
sample;47animalsfrom 11breedswereidentiedas
heterozygousforthemutation(AG),correspondingto
14.24%.TheresultingfrequencyfortheAallelewas
0.14.Table1showstheAalleledistribution,according
tobreed.Themostrepresentativebreedforthisstudy
wasthePembrokeWelshCorgi,sinceitexhibitsthe
highestAallelefrequencydespiteoccupyingthefth
placeinnumberofsampledanimalsperbreed.
Discussion
Atotalof330samplesfrom22dogbreedswere
analyzed. Exactly half of these breeds carried the
studied 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; %: Percen t of dogs per category.
retrospective study about the allelic distribution of
the SOD1geneinvariousdogbreeds,wherethetotal
Aallelefrequency wasestimatedtobe 0.37.Inour
study,thetotalfrequencywas0.14,whichreinforces
the assertion that the SOD1:c.118G>A mutation
is widespread across different dog populations.
SimilaritiesexistbetweenthehighAallelefrequencies
reportedbyvariousauthorsforthePembrokeWelsh
Corgibreed.Forexample,Zenget al.(2014)reported
afrequencyof0.79,andAwanoet al. (2009)reported
afrequency of 0.70.Bothvaluesaresimilar to that
obtainedinourstudy(0.83).Theseresultsdemonstrate
that,intheaforementionedbreed,theSOD1 mutation
is present in various dog populations, regardless of
geographicdistancing.ThesendingsalsostateDM
isnotrestrictedtoaspecicgeographiczone.
Capucchio et al. (2014) found a total A allele
frequencyof0.17intheGermanShepherd.Theauthors
referredtothisgureasahighandriskyfrequencyfor
amutantallele.Manyofthefrequenciesfoundinour
studyweresimilar(GermanShepherd0.13,Belgian
Malinois0.14,Boxer0.28),demonstratingthatthese
frequenciesrepresentanimportantfactortoconsider
intheconstantmanifestationofDM.BothPembroke
Welsh Corgiand Boxer breedsdisplay an elevated
Aallele frequency,while also showing the highest
numberofhomocygoticanimals.Inastudyconducted
byAwanoet al. (2009),sevenDM-aecteddogswere
identiedthroughhistopathology,allofwhichhadan
A/Agenotype.Thisndingsuggeststhatthereisan
extremelyhighriskfortheA/Ahomocygoticanimals
toeventuallyexhibitDMclinicalsigns.
Inconclusion,wewereabletoidentifytheSOD1
mutationassociatedwithDMin11dogbreedsthrough
PCR-RFLP.TheSOD1:c.118G>Amutationispresent
invariousdogbreedsinMexico,insomecaseswith
particularly high frequency. A molecular marker
thatcouldbeusedtoassistwithbreeding selection
would allow breeders to reduce this genetic factor
inuencingDMincidence,whilehelpingtodiminish
its presentation in future dog generations.
Acknowledgments
This study was funded by the University of
GuadalajarathroughprojectP3E229355.TheAnimal
Clinic of the Veterinary Medicine and Animal Science
SchooloftheUniversityofGuadalajaraprovidedthe
caninebloodsamplesusedinthestudy.
Conicts of interest
The authors declare that they have no conicts
ofinterestwithregardtotheworkpresentedinthis
report.
154
Rev Colomb Cienc Pecu 2018; 31(2):150-154
Ayala-Valdovinos MA et al. Canine degenerative myelopathy mutation
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