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



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
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
* Correspondingauthor:JorgeGalindo-García.DepartamentodeProducciónAnimal,CentroUniversitariodeCienciasBiológicasyAgropecuarias,Universidad
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G/Gwild-type,0.14fortheG/Aheterozygote,and0.7fortheA/Ahomozygote.Conclusion: The genic
Keywords: genetic disease, molecular marker, PCR-RFLP, SOD1 gene.
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
Rev Colomb Cienc Pecu 2018; 31(2):150-154
Ayala-Valdovinos MA et al. Canine degenerative myelopathy mutation
sustitucióndebasesenelgenSOD1recientementeidenticadocomoc.118G>A.Objetivo: Determinar las
muestras deperrosde 22razasmediantela técnicadereacción encadenade lapolimerasay polimorsmosde
razasdiferentes.Lasfrecuenciasgénicasencontradasfueronde0,78paraelalelo Gy de0,14paraelalelo
para el homocigoto A/A. Conclusión:Lafrecuenciaencontradaparalamutaciónesaltaenlaspoblaciones
causante de MDC en perros reproductores resultaría útil para reducir su frecuencia.
Palabras clave: enfermedad genética, gen SOD1, marcador molecular, PCR-RFLPs.
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
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
A/A. Conclusão:Afrequênciaencontradaparaamutaçãoéaltanaspopulaçõesestudadas.Aimplementação
Palavras-chave: doença genética, gen SOD1, marcador molecular, PCR-RFLPs.
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
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
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).
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
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
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).
Rev Colomb Cienc Pecu 2018; 31(2):150-154
Ayala-Valdovinos MA et al. Canine degenerative myelopathy mutation
The identication of the DM causing mutation
could be useful to dierentiate this disease from
illnesses, such as intervertebral disk disease and
diskospondylitis(Joneset al.,2005).Theobjectiveof
genicfrequencies for DM in Guadalajara, Mexico,
using the polymerase chain reaction and restriction
fragment length polymorphisms (PCR-RFLP)
Materials and methods
Ethical considerations
Regulation Committee of the Academic Centre of
Study samples
22 dog breeds and associated crossbreeds. Breeds
were selected taking in account previous reports
which had identified them as DM-susceptible
Australian Shepherd, Belgian Malinois, Border
Collie, Boxer, Cocker Spaniel, Collie, Dalmatian,
and crossbreeds,GoldenRetriever,KerryBlueTerrier,
Welsh Corgi, Poodle, Pug, Rhodesian Ridgeback,
Rottweiler, and crossbreeds, Shetland Sheepdog,
andSiberian Husky.Blood samples were stored in
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
Sample genotyping
We extracted DNA from blood samples using
the Quick DNA™ Universal Kit (Zymo Research,
Orange, CA, USA). For genotyping through PCR-
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
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:
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
electrophoresiswith GelRed™(Biotium,Hayward,
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
sample;47animalsfrom 11breedswereidentiedas
analyzed. Exactly half of these breeds carried the
studied mutation. Zeng et al. (2014) published a
Rev Colomb Cienc Pecu 2018; 31(2):150-154
Ayala-Valdovinos MA et al. Canine degenerative myelopathy mutation
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
the assertion that the SOD1:c.118G>A mutation
is widespread across different dog populations.
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
that,intheaforementionedbreed,theSOD1 mutation
is present in various dog populations, regardless of
Capucchio et al. (2014) found a total A allele
Welsh Corgiand Boxer breedsdisplay an elevated
Aallele frequency,while also showing the highest
byAwanoet al. (2009),sevenDM-aecteddogswere
particularly high frequency. A molecular marker
thatcouldbeusedtoassistwithbreeding selection
would allow breeders to reduce this genetic factor
its presentation in future dog generations.
This study was funded by the University of
Clinic of the Veterinary Medicine and Animal Science
Conicts of interest
The authors declare that they have no conicts
Rev Colomb Cienc Pecu 2018; 31(2):150-154
Ayala-Valdovinos MA et al. Canine degenerative myelopathy mutation
Awano T, Johnson GS, Wade CM, Katz ML, Johnson GC,
a SOD1 mutation in canine degenerative myelopathy that
Capucchio MT,Spalenza V, Biasibetti E, Bottero MT, Rasero
R, Dalmasso A, Sacchi P. Degenerative myelopathy in German
Shepherd Dog: Comparison of two molecular assays for the
identication of the SOD1: c. 118G>A mutation. Mol Biol
NakamaeS,Kobatake Y, Suzuki R, TsukuiT,Kato S,Yamato
O, Kamishina H.Accumulation and aggregate formation
of mutant superoxide dismutase 1 in canine degenerative
A, Guo J. Breed distribution of SOD1 alleles previously
associated with canine degenerative myelopathy.J VetIntern
... In the studied group of animals, the frequency of the mutant allele (A) was 0.17, which is slightly higher than that previously reported for Belgian Malinois (0-0.14) [9,[18][19][20] but similar to the frequency reported in other breeds such as the Airedale Terrier, Australian Terrier, Chinese Crested, Chow Chow, French Bulldog, Irish Setter, and Puli [9,18] (Table-2). ...
... and 0.04 vs. 0-0.06 for heterozygosity and homozygosity, respectively) [9,[18][19][20] and other breeds (Airedale Terrier, American Hairless Terrier, Beagle, Belgian Sheepdog, Borzoi, Boxer, Boykin, Chow Chow, Spaniel, and Kuvasz) ( Table-2) [6][7][8][9][18][19][20][21][22][23][24]. ...
... and 0.04 vs. 0-0.06 for heterozygosity and homozygosity, respectively) [9,[18][19][20] and other breeds (Airedale Terrier, American Hairless Terrier, Beagle, Belgian Sheepdog, Borzoi, Boxer, Boykin, Chow Chow, Spaniel, and Kuvasz) ( Table-2) [6][7][8][9][18][19][20][21][22][23][24]. ...
Full-text available
Background and aim: Canine degenerative myelopathy (CDM) is an adult-onset fatal disorder associated with a point mutation of the superoxide dismutase 1 (SOD1) gene (SOD1:c.118G>A). This study aimed to determine the allele and genotype frequencies of this mutation in a group of Belgian Malinois dogs in Greece. Materials and methods: Samples (n=72) of whole blood were collected from 72 purebred dogs of the Hellenic Armed Forces; these samples were processed for DNA isolation, polymerase chain reaction, and digestion with the restriction endonuclease AcuI. Sample testing was conducted in compliance with ISO17025 accreditation requirements. Results: The observed relative genotype frequencies were 71% for the homozygous (GG), 25% for the heterozygous (AG), and 4% for the homozygous mutant (AA) alleles. These frequencies were close to those expected, indicating no significant departure from Hardy-Weinberg equilibrium (HWE, p=0.395). The frequency of heterozygous animals indicates that a high risk of developing CDM in forthcoming generations exists in the tested population because mating among carriers would result in 25% AA progeny. The medical record of the group of study animals indicated selection against leishmaniosis, as applied throughout generations by owners and breeders. The potential association of this selection with the HWE status of the study population was discussed. Conclusion: The SOD1:c.118G>A mutation was common in the tested group of dogs; thus, they are suitable for a follow-up investigation on the development and progression of CDM. A case-control study on animals with evidence of sensitivity to infectious myelopathy could provide new insights into disease pathogenesis.
Full-text available
Previous reports associated 2 mutant SOD1 alleles (SOD1:c.118A and SOD1:c.52T) with degenerative myelopathy in 6 canine breeds. The distribution of these alleles in other breeds has not been reported. To describe the distribution of SOD1:c.118A and SOD1:c.52T in 222 breeds. DNA from 33,747 dogs was genotyped at SOD1:c.118, SOD1:c.52, or both. Spinal cord sections from 249 of these dogs were examined. Retrospective analysis of 35,359 previously determined genotypes at SOD1:c.118G>A or SOD1:c.52A>T and prospective survey to update the clinical status of a subset of dogs from which samples were obtained with a relatively low ascertainment bias. The SOD1:c.118A allele was found in cross-bred dogs and in 124 different canine breeds whereas the SOD1:c.52T allele was only found in Bernese Mountain Dogs. Most of the dogs with histopathologically confirmed degenerative myelopathy were SOD1:c.118A homozygotes, but 8 dogs with histopathologically confirmed degenerative myelopathy were SOD1:c.118A/G heterozygotes and had no other sequence variants in their SOD1 amino acid coding regions. The updated clinical conditions of dogs from which samples were obtained with a relatively low ascertainment bias suggest that SOD1:c.118A homozygotes are at a much higher risk of developing degenerative myelopathy than are SOD1:c.118A/G heterozygotes. We conclude that the SOD1:c.118A allele is widespread and common among privately owned dogs whereas the SOD1:c.52T allele is rare and appears to be limited to Bernese Mountain Dogs. We also conclude that breeding to avoid the production of SOD1:c.118A homozygotes is a rational strategy.
Full-text available
Canine degenerative myelopathy (DM) is a fatal neurodegenerative disease prevalent in several dog breeds. Typically, the initial progressive upper motor neuron spastic and general proprioceptive ataxia in the pelvic limbs occurs at 8 years of age or older. If euthanasia is delayed, the clinical signs will ascend, causing flaccid tetraparesis and other lower motor neuron signs. DNA samples from 38 DM-affected Pembroke Welsh corgi cases and 17 related clinically normal controls were used for genome-wide association mapping, which produced the strongest associations with markers on CFA31 in a region containing the canine SOD1 gene. SOD1 was considered a regional candidate gene because mutations in human SOD1 can cause amyotrophic lateral sclerosis (ALS), an adult-onset fatal paralytic neurodegenerative disease with both upper and lower motor neuron involvement. The resequencing of SOD1 in normal and affected dogs revealed a G to A transition, resulting in an E40K missense mutation. Homozygosity for the A allele was associated with DM in 5 dog breeds: Pembroke Welsh corgi, Boxer, Rhodesian ridgeback, German Shepherd dog, and Chesapeake Bay retriever. Microscopic examination of spinal cords from affected dogs revealed myelin and axon loss affecting the lateral white matter and neuronal cytoplasmic inclusions that bind anti-superoxide dismutase 1 antibodies. These inclusions are similar to those seen in spinal cord sections from ALS patients with SOD1 mutations. Our findings identify canine DM to be the first recognized spontaneously occurring animal model for ALS.
Canine degenerative myelopathy (DM) is an adult-onset progressive neurodegenerative disorder that has recently been linked to mutations in the superoxide dismutase 1 (SOD1) gene. We generated a polyclonal antibody against canine SOD1 to further characterize the mutant SOD1 protein and its involvement in DM pathogenesis. This antibody (SYN3554) was highly specific to canine SOD1 and had the ability to reveal distinct cytoplasmic aggregates in cultured cells expressing canine mutant SOD1 and also in the spinal neurons of symptomatic homozygotes. A similar staining pattern was observed in asymptomatic homozygotes. SOD1 aggregates were not detected in the spinal neurons of heterozygotes; the accumulation of SOD1 was also detected in the reactive astrocytes of homozygotes and heterozygotes to a similar extent. Our results support the hypothesis that the cytoplasmic accumulation and aggregate formation of the mutant SOD1 protein, especially in astrocytes, are closely associated with the pathogenesis of DM. Therefore, this disease is regarded as a spontaneous large-animal model of SOD1-mediated amyotrophic lateral sclerosis in humans. Copyright © 2015. Published by Elsevier Ltd.
Degenerative myelopathy (DM) is a late-onset, slowly progressive degeneration of spinal cord white matter which is reported primarily in large breed dogs. The missense mutation SOD1:c.118G>A is associated with this pathology in several dog breeds, including the German Shepherd Dog (GSD). The aims of the present study were to develop a tool for the rapid screening of the SOD1 mutation site in dogs and to evaluate the association of the polymorphism with DM in the German Shepherd breed. Two different techniques were compared: a minisequencing test and a real-time pcr allelic discrimination assay. Both approaches resulted effective and efficient. A sample of 47 dogs were examined. Ten subjects presented the symptoms of the illness; for one of them the diagnosis was confirmed by postmortem investigations and it resulted to be an A/A homozygote. In another clinically suspected dog, heterozygote A/G, the histopathological examination of the medulla showed moderate axon and myelin degenerative changes. GSD shows a frequency of the mutant allele equal to 0.17, quite high being a high-risk allele. Because canine DM has a late onset in adulthood and homozygous mutant dogs are likely as fertile as other genotypes, the natural selection is mild and the mutant allele may reach high frequencies. A diagnostic test, easy to implement, may contribute to control the gene diffusion in populations. The SOD1:c.118G>A mutation could be a useful marker for breeding strategies intending to reduce the incidence of DM.
Degenerative myelopathy (DM) is a common, slowly progressive, debilitating disease reported in several dog breeds, including the German Shepherd Dog and Pembroke Welsh Corgi. Boxer dogs present occasionally for a thoracolumbar myelopathy for which no cause is identified on MRI or cerebrospinal fluid analysis. Despite a lack of a histologic description of DM in the Boxer in the veterinary literature, such dogs are presumed to have DM. Here we report 2 histologically confirmed cases of DM in the Boxer breed in which histologic studies disclosed marked degenerative changes in the spinal cord that were most prominent in the thoracic and cranial lumbar segments. Lesions consisted of myelin vacuolation and degeneration, myelophagocytosis, reactive astrocytosis, and ellipsoid formation most prominent in the lateral and ventral funiculi. We present a detailed histologic description of DM in the Boxer dog and compare it to DM in other purebred dogs.
Post mortem examination was performed on 18 Pembroke Welsh Corgi dogs (mean age 12.7 years) with clinical signs and antemortem diagnostic tests compatible with a diagnosis of degenerative myelopathy. Tissue sections from specific spinal cord and brain regions were systematically evaluated in all dogs. Axonal degeneration and loss were graded according to severity and subsequently compared across different spinal cord segments and funiculi. White matter lesions were identified in defined regions of the dorsal, lateral, and ventral funiculi. The dorsolateral portion of the lateral funiculus was the most severely affected region in all cord segments. Spinal cord segment T12 exhibited the most severe axonal loss. Spinal nerve roots, peripheral nerves, and brain sections were within normal limits with the exception of areas of mild astrogliosis in gray matter of the caudal medulla. Dogs with more severe lesions showed significant progression of axonal degeneration and loss at T12 and at cord segments cranial and caudal to T12. Severity of axonal loss in individual dogs positively correlated with the duration of clinical signs. The distribution of axonal degeneration resembled that reported in German Shepherd dog degenerative myelopathy but differed with respect to the transverse and longitudinal extent of the lesions within more clearly defined funicular areas. Although these lesion differences may reflect disease longevity, they may also indicate a form of degenerative myelopathy unique to the Pembroke Welsh Corgi dog.