ResearchArticlebcepilepsy.weebly.com/uploads/1/1/8/8/11881867/... · 670 DiseaseMarkers M NDNDN D...

Hindawi Publishing CorporationDisease MarkersVolume 35 (2013) Issue 6 Pages 669ndash672httpdxdoiorg1011552013695918

Research ArticleHigh Frequency of a Single Nucleotide Substitution(c-6-180TgtG) of the Canine MDR1ABCB1 Gene Associatedwith Phenobarbital-Resistant Idiopathic Epilepsyin Border Collie Dogs

Keijiro Mizukami Akira Yabuki Hye-Sook Chang Mohammad Mejbah UddinMohammad Mahbubur Rahman Kazuya Kushida Moeko Kohyama and Osamu Yamato

Laboratory of Clinical Pathology Department of Veterinary Medicine Joint Faculty of Veterinary Medicine Kagoshima University1-21-24 Korimoto Kagoshima 890-0065 Japan

Correspondence should be addressed to Osamu Yamato osamvetkagoshima-uacjp

Received 13 June 2013 Revised 11 October 2013 Accepted 24 October 2013

Academic Editor Silvia Persichilli

Copyright copy 2013 Keijiro Mizukami et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A single nucleotide substitution (c-6-180TgtG) associated with resistance to phenobarbital therapy has been found in thecanine MDR1ABCB1 gene in Border Collies with idiopathic epilepsy In the present study a PCR-restriction fragment lengthpolymorphism assay was developed for genotyping this mutation and a genotyping survey was carried out in a population of472 Border Collies in Japan to determine the current allele frequency The survey demonstrated the frequencies of the TT wildtype TG heterozygote and GG mutant homozygote to be 600 303 and 98 respectively indicating that the frequencyof the mutant G allele is extremely high (249) in Border Collies The results suggest that this high mutation frequency of themutation is likely to cause a high prevalence of phenobarbital-resistant epilepsy in Border Collies

1 Introduction

Recently a single nucleotide substitution associated withphenobarbital-resistant idiopathic epilepsy was found inBorder Collies [1] which frequently present with severeepileptic seizures that are poorly controlled with antiepilepticdrugs [2] This polymorphic mutation is a substitution ofthymine for guanine at intron 1 near the 51015840-end of thecanine MDR1ABCB1 gene (c-6-180TgtG) where the mostimportant promoter elements are located [3] The mutationis not directly associated with the pathogenesis of idiopathicepilepsy but it may create resistance to phenobarbital therapyin epileptic Border Collies [1]

However the c-6-180TgtGmutation has been shown notto be a fully penetrant polymorphic mutation for pheno-barbital nonresponsiveness because phenobarbital-resistantidiopathically epileptic Border Collies in a previous case

control study included a homozygous TT wild-type dog[1] On the basis of data from a relatively small cohortof Border Collies it was hypothesized that the mutationmight be related to an upregulation of the gene and anoverexpression of P-glycoprotein (P-gp) encoded by theMDR1ABCB1 gene Therefore further studies are requiredto understand the regulatory effect of the mutation and itspotential clinical relevance For these purposes an accuratemethod of genotyping for the mutation should be developedand the frequency of the mutation should be determined in alarge normal population of dogs

In the present study a PCR-restriction fragment lengthpolymorphism (RFLP) assay was developed in order todiscriminate the genotypes of themutation and a genotypingsurvey was conducted in Japan by using samples fromclinically healthy Border Collies in order to determine thecurrent frequency of the mutant allele

670 Disease Markers

N D N D N DMTT TG GG

(bp)

800

500400300

200150

100

416313

191122103

(bp)

Figure 1 Electrophoretogram of agarose gel using PCR-restrictionfragment length polymorphism assay Fragment patterns in the 3genotypes such as homozygous wild type (TT) heterozygote (TG)and homozygous mutant type (GG) are shown with molecular sizemarkers (M) The amplified DNA without digestion (N) and DNAdigested with restriction endonuclease MboI (D) were analyzedsimultaneously by electrophoresis bp base pairs

2 Methods and Materials

Control samples of each genotype which were determined bydirect DNA sequencing were used to evaluate the accuracyof the genotyping assay These samples were collected from 4wild-type (TT) 9 heterozygous (TG) and 10 homozygousmutant-type (GG) dogs DNA templates were preparedusing saliva spotted onto Flinders Technology Associatesfilter paper (FTA card Whatman International Ltd Piscat-away NJ USA) For the PCR-RFLP assay a 12mm discpunched out of the FTA card was used as a template afterquick washing as reported previously [4] The PCR test wasperformed by targeting the sequence around the mutationwith forward (51015840-GCA GTG GGG TGA GAA CTA GA-31015840)and reverse (51015840-CGC AAG CCA TGT AAG GTA TG-31015840)primers in a 20120583L reaction mixture containing 10 120583L of a2times PCR master mix (GoTaq Hot Start Green Master MixPromega Corp Madison WI USA) 125 pmol of primers1 120583L of GC enhancer solution (360 GC Enhancer AppliedBiosystems Foster City CA USA) and the treated disc of theFTA card as a template After denaturation at 95∘C for 10min45 cycles of amplification were carried out at a denaturingtemperature of 95∘C for 30 sec an annealing temperature of60∘C for 30 sec and an extension temperature of 72∘C for30 sec Extension during the last cycle was performed at 72∘Cfor 7min and 30 sec The PCR product was digested with aMboI restriction endonuclease (New England Biolabs IncIpswich MA USA) at 37∘C for 90min in a 10 120583L reactionmixture containing 8120583L of the PCR product 5U of MboIand 1 120583L of 10times restriction enzyme buffer (10timesNEbuffer NewEngland Biolabs Inc) included by themanufacturer Both theunprocessed and digested PCR products were subjected toelectrophoresis in 3 agarose gel (Agarose 21 Nippon GeneCo Ltd Tokyo Japan) The PCR-RFLP assay was designedto digest the wild-type sequence [darrGATC] into 3 fragments

and to not digest the mutant sequence [GAGC] resulting in 2fragments

The genotyping survey was carried out by using DNAtemplates extracted from saliva samples of 472 Border Colliesaged 2 months to 14 years in Japan These samples werecollected between 2006 and 2013 by the Japan Border CollieHealth Network a volunteer breedersrsquo association with theownersrsquo informed consent The PCR-RFLP assay establishedin this study was used for genotyping

3 Results

As shown in Figure 1 a 416-base pair (bp) DNA band wasamplified in theory in all the genotypes In the GG homozy-gous mutant-type dog the amplified band was digested into2 fragments (ie 313 and 103 bp bands) because there wasonly 1 restriction site available to theMboI whichwas presentin all the genotypes and unrelated to the target sequence Inthe TT homozygous wild-type dog the amplified band wasdigested into 3 fragments (ie 191 122 and 103 bp bands)because there were 2 restriction sites available one at thetarget sequence [darrGATC] and another at the same sequencepresent in all the genotypesThe amplification product in theTG heterozygous dog was digested into 4 fragments (ie 313191 122 and 103 bp bands) Owing to the inseparability of the122 and 103 bp bands these 2 bands externally appeared tobe 1 band therefore the digested PCR products in the TTwild-type and TG heterozygous dogs externally appearedas 2 and 3 fragments respectively The genotypes of all thedogs examinedwere consistent with the results of direct DNAsequencing

In the survey on 472 Border Collies 283 (600) wereTTwild-type dogs 143 (303)were TG heterozygous dogsand 46 (98) were GG homozygous mutant-type dogs Theoverall frequency of the mutant G allele was 249

4 Discussion

Pharmacogenetics is a relatively new discipline that inves-tigates how genetic variations are related to drug responseand it is expected to be an important tool for developingpersonalized medicine [5] Pharmacogenetic biomarkers rel-evant to various diseases drugs and genes have been foundand clinically used to maximize therapeutic efficacy reduceadverse drug reactions and determine the most appropriatedrug dosage required for efficacious and safe treatment [6]Information about pharmacogenetics in domestic animals isstill not as extensive as in humans however in veterinarymedicine there is great potential for advances in the comingyears because whole-genome sequencing of many specieshas been completed [7] The genetic marker c-6-180TgtGassociatedwith phenobarbital-resistant idiopathic epilepsy indogs may be an important pharmacogenetic biomarker inveterinary medicine in the near future

In the PCR-RFLP assay developed in the present studyall the genotypic variations of c-6-180TgtG could be easilydiscriminated by confirming the presence andor absenceof 313 and 191 bp fragments via agarose gel electrophoresis

Disease Markers 671

(Figure 1) The restriction site that is present in both allelesand unrelated to the target sequence did not interfere withthe determination of all the genotypes but provided assuranceof the digestive function of the restriction endonucleaseFurthermore other genotyping assays (including a TaqManprobe-based real-time PCR method) could not be estab-lished because nucleic acid sequences around the mutationare rich in GC pairs and include a partially duplicatedrepeat (GenBank reference no NC 006596 and accessionno AAEX030092951) which might interfere with specifichybridization by designed probes Therefore the PCR-RFLPassay is a very useful tool for the genotyping of c-6-180TgtG

The genotyping survey showed that the mutant G allelefrequency in Border Collies is extremely high (approximately25) compared to that in other molecularly defined fatalinherited diseases in Border Collies such as neuronal ceroidlipofuscinosis (41) [8] and trapped neutrophil syndrome(56) [9] The high frequency of the G allele in JapaneseBorder Collies cannot be generalized to Border Collie pop-ulations in different countries however because populationstratification can easily occur in a geographic region unknow-ingly by using dogs with a single genotype for breeding inpure dog breeds (ie the founder effect) [10] The G allelefrequency in this study was consistent with that in a smallpopulation of European Border Collies (24) although thispopulation did not consist of randomly selected normalanimals [1]

The phenotype of the mutant G allele seems to act in adominant fashion [1] as a consequence approximately 40Border Collies (GG and TG genotypes) examined in thisstudy may show resistance to phenobarbital therapy if theybecome affected with epilepsy This might be the reason whydrug-resistant epilepsy frequently occurs in Border Colliesalthough there are other possible causes [2 11] further studiesare needed to clarify this issue

Previous studies hypothesized that phenobarbital resis-tance is due to an upregulation of the MDR1ABCB1 geneand the subsequent overexpression of this genersquos productP-gp in the brains of Border Collies with drug-resistantepilepsy [1] P-gp is expressed at the luminal membrane ofthe endothelial cells of brain capillaries and restricts the entryof drugs (including ivermectin and phenobarbital) into thecentral nervous system by an efflux-based transport mech-anism [4 12] P-gp is also expressed in various mammaliantissues such as the brush border membrane of epithelial cellsin the intestinal tract the luminal membrane of proximaltubules in the kidney and the canalicular membrane of liverhepatocytes [13] Overexpression of P-gp in these organsdiminishes oral drug bioavailability and promotes drugelimination into urine and bile [14] Therefore c-6-180TgtGinBorder Colliesmay not only affect phenobarbital resistancebut may also affect the pharmacokinetics of other P-gpsubstrate drugs Additionally in veterinary medicine thereis another well-known mutation in the canineMDR1ABCB1gene (c227 230del) that causes a P-gp defect and subse-quently substrates drug intoxication including ivermectintoxicosis [15] however the allele frequency of c227 230delis low (025) in Border Collies [4] It is necessary tofurther study the association between P-gp and c-6-180TgtG

and the interaction between these mutations in the canineMDR1ABCB1 gene before the pharmacogenetic informationrelated to c-6-180TgtG can be applied to clinical practice

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

The authors are grateful to Mr Kawamichi of the Japan Bor-derCollieHealthNetwork for collecting samples fromBorderCollies This study was supported financially by Grants(nos 25292181 OY and 25-4919 KM) from the Ministry ofEducation Culture Sports Science andTechnology of Japan

References

[1] L Alves V Hulsmeyer A Jaggy A Fischer T Leeb and MDrogemuller ldquoPolymorphisms in the ABCB1 gene in pheno-barbital responsive and resistant idiopathic epileptic BorderColliesrdquo Journal of Veterinary Internal Medicine vol 25 no 3pp 484ndash489 2011

[2] V Hulsmeyer R Zimmermann C Brauer C Sauter-Louis andA Fischer ldquoEpilepsy in Border Collies clinical manifestationoutcome and mode of inheritancerdquo Journal of VeterinaryInternal Medicine vol 24 no 1 pp 171ndash178 2010

[3] H Takane D Kobayashi T Hirota et al ldquoHaplotype-orientedgenetic analysis and functional assessment of promoter variantsin the MDR1 (ABCB1) generdquo Journal of Pharmacology andExperimental Therapeutics vol 311 no 3 pp 1179ndash1187 2004

[4] K Mizukami H-S Chang A Yabuki et al ldquoRapid genotypingassays for the 4-base pair deletion of canineMDR1ABCB1 geneand low frequency of the mutant allele in Border Collie dogsrdquoJournal of Veterinary Diagnostic Investigation vol 24 no 1 pp127ndash134 2012

[5] J A Johnson ldquoPharmacogenetics in clinical practice how farhavewe come andwhere arewe goingrdquoPharmacogenomics vol14 pp 835ndash843 2013

[6] H Bakhouche and O Slanar ldquoPharmacogenetics in clinicalpracticerdquo Prague Medical Report vol 113 no 4 pp 251ndash2612012

[7] C M Mosher and M H Court ldquoComparative and veterinarypharmacogenomicsrdquoHandbook of Experimental Pharmacologyvol 199 pp 49ndash77 2010

[8] K Mizukami H-S Chang A Yabuki et al ldquoNovel rapidgenotyping assays for neuronal ceroid lipofuscinosis in BorderCollie dogs and high frequency of the mutant allele in JapanrdquoJournal of Veterinary Diagnostic Investigation vol 23 no 6 pp1131ndash1139 2011

[9] K Mizukami A Yabuk T Kawamichi et al ldquoReal-time PCRgenotyping assay for canine trapped neutrophil syndrome andhigh frequency of the mutant allele in Border Colliesrdquo TheVeterinary Journal vol 195 pp 260ndash261 2013

[10] P Quignon L Herbin E Cadieu et al ldquoCanine populationstructure assessment and impact of intra-breed stratification onSNP-based association studiesrdquo PLoSONE vol 2 no 12 ArticleID e1324 2007

[11] H A Volk D Arabadzisz J-M Fritschy C Brandt K Beth-mann and W Loscher ldquoAntiepileptic drug-resistant rats differ

672 Disease Markers

from drug-responsive rats in hippocampal neurodegenerationand GABAA receptor ligand binding in a model of temporallobe epilepsyrdquo Neurobiology of Disease vol 21 no 3 pp 633ndash646 2006

[12] A H Schinkel ldquoThe physiological function of drug-trans-porting P-glycoproteinsrdquo Seminars in Cancer Biology vol 8 no3 pp 161ndash170 1997

[13] F Thiebaut T Tsuruo H Hamada M M Gottesman IPastan and M C Willingham ldquoCellular localization of themultidrug-resistance gene product P-glycoprotein in normalhuman tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 84 no 21 pp 7735ndash77381987

[14] M F Fromm ldquoP-glycoprotein a defense mechanism limitingoral bioavailability and CNS accumulation of drugsrdquo Interna-tional Journal of Clinical Pharmacology and Therapeutics vol38 no 2 pp 69ndash74 2000

[15] K L Mealey S A Bentjen J M Gay and G H CantorldquoIvermectin sensitivity in Collies is associated with a deletionmutation of themdr1 generdquo Pharmacogenetics vol 11 no 8 pp727ndash733 2001

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

670 Disease Markers

N D N D N DMTT TG GG

(bp)

800

500400300

200150

100

416313

191122103

(bp)

Figure 1 Electrophoretogram of agarose gel using PCR-restrictionfragment length polymorphism assay Fragment patterns in the 3genotypes such as homozygous wild type (TT) heterozygote (TG)and homozygous mutant type (GG) are shown with molecular sizemarkers (M) The amplified DNA without digestion (N) and DNAdigested with restriction endonuclease MboI (D) were analyzedsimultaneously by electrophoresis bp base pairs

2 Methods and Materials

Control samples of each genotype which were determined bydirect DNA sequencing were used to evaluate the accuracyof the genotyping assay These samples were collected from 4wild-type (TT) 9 heterozygous (TG) and 10 homozygousmutant-type (GG) dogs DNA templates were preparedusing saliva spotted onto Flinders Technology Associatesfilter paper (FTA card Whatman International Ltd Piscat-away NJ USA) For the PCR-RFLP assay a 12mm discpunched out of the FTA card was used as a template afterquick washing as reported previously [4] The PCR test wasperformed by targeting the sequence around the mutationwith forward (51015840-GCA GTG GGG TGA GAA CTA GA-31015840)and reverse (51015840-CGC AAG CCA TGT AAG GTA TG-31015840)primers in a 20120583L reaction mixture containing 10 120583L of a2times PCR master mix (GoTaq Hot Start Green Master MixPromega Corp Madison WI USA) 125 pmol of primers1 120583L of GC enhancer solution (360 GC Enhancer AppliedBiosystems Foster City CA USA) and the treated disc of theFTA card as a template After denaturation at 95∘C for 10min45 cycles of amplification were carried out at a denaturingtemperature of 95∘C for 30 sec an annealing temperature of60∘C for 30 sec and an extension temperature of 72∘C for30 sec Extension during the last cycle was performed at 72∘Cfor 7min and 30 sec The PCR product was digested with aMboI restriction endonuclease (New England Biolabs IncIpswich MA USA) at 37∘C for 90min in a 10 120583L reactionmixture containing 8120583L of the PCR product 5U of MboIand 1 120583L of 10times restriction enzyme buffer (10timesNEbuffer NewEngland Biolabs Inc) included by themanufacturer Both theunprocessed and digested PCR products were subjected toelectrophoresis in 3 agarose gel (Agarose 21 Nippon GeneCo Ltd Tokyo Japan) The PCR-RFLP assay was designedto digest the wild-type sequence [darrGATC] into 3 fragments

and to not digest the mutant sequence [GAGC] resulting in 2fragments

The genotyping survey was carried out by using DNAtemplates extracted from saliva samples of 472 Border Colliesaged 2 months to 14 years in Japan These samples werecollected between 2006 and 2013 by the Japan Border CollieHealth Network a volunteer breedersrsquo association with theownersrsquo informed consent The PCR-RFLP assay establishedin this study was used for genotyping

3 Results

As shown in Figure 1 a 416-base pair (bp) DNA band wasamplified in theory in all the genotypes In the GG homozy-gous mutant-type dog the amplified band was digested into2 fragments (ie 313 and 103 bp bands) because there wasonly 1 restriction site available to theMboI whichwas presentin all the genotypes and unrelated to the target sequence Inthe TT homozygous wild-type dog the amplified band wasdigested into 3 fragments (ie 191 122 and 103 bp bands)because there were 2 restriction sites available one at thetarget sequence [darrGATC] and another at the same sequencepresent in all the genotypesThe amplification product in theTG heterozygous dog was digested into 4 fragments (ie 313191 122 and 103 bp bands) Owing to the inseparability of the122 and 103 bp bands these 2 bands externally appeared tobe 1 band therefore the digested PCR products in the TTwild-type and TG heterozygous dogs externally appearedas 2 and 3 fragments respectively The genotypes of all thedogs examinedwere consistent with the results of direct DNAsequencing

In the survey on 472 Border Collies 283 (600) wereTTwild-type dogs 143 (303)were TG heterozygous dogsand 46 (98) were GG homozygous mutant-type dogs Theoverall frequency of the mutant G allele was 249

4 Discussion

Pharmacogenetics is a relatively new discipline that inves-tigates how genetic variations are related to drug responseand it is expected to be an important tool for developingpersonalized medicine [5] Pharmacogenetic biomarkers rel-evant to various diseases drugs and genes have been foundand clinically used to maximize therapeutic efficacy reduceadverse drug reactions and determine the most appropriatedrug dosage required for efficacious and safe treatment [6]Information about pharmacogenetics in domestic animals isstill not as extensive as in humans however in veterinarymedicine there is great potential for advances in the comingyears because whole-genome sequencing of many specieshas been completed [7] The genetic marker c-6-180TgtGassociatedwith phenobarbital-resistant idiopathic epilepsy indogs may be an important pharmacogenetic biomarker inveterinary medicine in the near future

In the PCR-RFLP assay developed in the present studyall the genotypic variations of c-6-180TgtG could be easilydiscriminated by confirming the presence andor absenceof 313 and 191 bp fragments via agarose gel electrophoresis

Disease Markers 671








Acknowledgments


References












672 Disease Markers











of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 671








Acknowledgments


References












672 Disease Markers











of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















672 Disease Markers











of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















ResearchArticlebcepilepsy.weebly.com/uploads/1/1/8/8/11881867/... · 670 DiseaseMarkers M NDNDN D...

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