Aus Dem Institut Für Tierzucht Und Vererbungsforschung
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Tierärztliche Hochschule Hannover Institut für Tierzucht und Vererbungsforschung Analysis of the canine MDR1 gene in the dog breed Elo INAUGURAL-DISSERTATION zur Erlangung des Grades einer Doktorin der Veterinärmedizin - Doctor medicinae veterinariae - (Dr. med. vet.) vorgelegt von Silvia Fecht geb. Döpke aus Stadthagen Hannover 2007 Scientific supervisor: Univ.-Prof. Dr. Dr. O. Distl Examiner: Univ.-Prof. Dr. Dr. O. Distl Co-examiner: Univ.-Prof. Dr. R. Mischke Oral examination: 21.11.2007 To my family Parts of this work have been published or have been accepted for publication in the following journals: 1. Journal of veterinary medicine. A, Physiology, pathology, clinical medicine 2. Deutsche Tierärztliche Wochenschrift Contents Contents 1 Introduction ........................................................................................................................ 9 2 Review of prevalence, genetic aspects and adverse effects of the mdr1-1Δ mutation in dogs ............................................................................................................... 12 Summary........................................................................................................................ 13 Zusammenfassung ......................................................................................................... 14 Introduction.................................................................................................................... 14 The canine mdr1-1Δ mutation is associated with drug sensitivity ................................ 15 Drug neurotoxicity in dogs with the mdr1-1Δ mutation................................................ 16 Effects of the mdr1-1Δ mutation in other organs and on hormone release ................... 17 Prevalence of the mdr1-1Δ mutation in different dog breeds........................................ 18 Effects on dog breeding ................................................................................................. 19 Conclusions.................................................................................................................... 21 References...................................................................................................................... 22 3 Analysis of the canine mdr1-1Δ mutation in the dog breed Elo .................................. 32 Summary........................................................................................................................ 33 Introduction.................................................................................................................... 33 Materials and Methods................................................................................................... 36 Results............................................................................................................................ 38 Discussion...................................................................................................................... 39 Acknowledgements........................................................................................................ 41 References...................................................................................................................... 41 4 Haplotype analysis of the MDR1 flanking region in the dog breed Elo ...................... 46 Summary........................................................................................................................ 47 Zusammenfassung ......................................................................................................... 47 Introduction.................................................................................................................... 48 Materials and Methods................................................................................................... 50 Results and Discussion .................................................................................................. 51 Contents Acknowledgements........................................................................................................ 53 References...................................................................................................................... 53 5 Searching for functional polymorphisms in the exons 12, 21 and 26 of the canine MDR1 gene............................................................................................................ 58 Abstract.......................................................................................................................... 59 Introduction.................................................................................................................... 60 Materials and Methods................................................................................................... 62 Results............................................................................................................................ 66 Discussion...................................................................................................................... 68 Acknowledgements........................................................................................................ 70 References...................................................................................................................... 70 6 General Discussion ........................................................................................................... 76 7 Summary........................................................................................................................... 85 8 Erweiterte Zusammenfassung......................................................................................... 88 9 Appendix ........................................................................................................................... 99 10 List of publications......................................................................................................... 110 11 Acknowledgements......................................................................................................... 112 Abbreviations Abbreviations A adenine ABC ATP-binding cassette ABCB ATP-binding cassette sub-family B ACTH adrenocorticotropin hormone Ala alanine APS ammonium persulphate ATP adenosine 5'-triphosphate BLAST basic local alignment search tool bp base pairs C cytosine cDNA complementary deoxyribonucleic acid CFA Canis familiaris autosome CHO Chow-Chow cM centiMorgan CNS central nervous system DAL Dalmatian DMSO dimethyl sulfoxid DNA deoxyribonucleic acid dNTPs deoxy nucleoside 5’triphosphates (N is A, C, G or T) EDTA ethylenediamine tetraacetic acid EMBL European Molecular Biology Laboratory EUR Eurasian Dog F forward G guanine GAPDH glyceraldehyde-3-phosphate dehydrogenase GLM general linear model GSP German Spitz het heterozygous hom homozygous Abbreviations HPA axis hypothalamic-pituitary-adrenal axis HSA Homo sapiens autosome IRD infrared dye JSP Japanese Spitz M molar Mb mega base MDR multidrug resistance MGB minor groove binding mRNA messenger ribonucleic acid NCBI National Center for Biotechnology information no. number OES Old English Sheepdog PCR polymerase chain reaction PEK Pekingese POM Pomeranian Dog PSIC Position-Specific Independent Counts qRT-PCR quantitative Reverse Transcriptase-PCR R reverse RFLP restriction fragment length polymorphism RNA ribonucleic acid S.D. standard deviation SAS statistical analysis system Ser serine SNP single nucleotide polymorphism T thymine Ta annealing temperature TBE tris-borate-ethylenediamine tetraacetic acid TEMED N,N,N’,N’-tetramethylenediamine Thr threonine Introduction Chapter 1 Introduction 9 Introduction Introduction The MDR1 (multidrug resistance) gene encodes the transmembrane protein pump P- glycoprotein which is found in several mammalian tissues including the brain, intestine, liver, kidney, placenta and testis. The function of this ATP-dependent drug transporter is limiting the uptake of compounds from the gastrointestinal tract and contributing to their excretion via the liver, kidneys, and intestine. Moreover, P-glycoprotein in the blood-brain-barrier and other blood-tissue barriers protects sensitive organs from exposure to toxic compounds that may have entered the bloodstream. A deletion mutation in the canine MDR1 gene, mdr1-1Δ mutation, was found to be the cause of multiple drug sensitivity in several dog breeds from the Collie lineage such as the Collie, Australian Shepherd or Old English Sheepdog. This problem is commonly known as ‘ivermectin-sensitivity’ in the Collie. Affected dogs show severe neurotoxic adverse effects when exposed to drugs which are substrates of P- glycoprotein because of an increased permeability of the blood-brain-barrier caused by a nonfunctional P-glycoprotein. Additionally, elevated levels of cortisol can pass through the damaged blood-brain-barrier provoking suppression of the hypothalamic-pituitary-adrenal- axis, thereby resulting in reduced production of hormones. The prevalence of the mdr1-1Δ mutation in affected dog breeds ranges between 0.6 and 64% depending on breed and its geographic subpopulation. Previous studies showed that the mdr1-1Δ allele is identical by descent among affected breeds as evidenced by a single ancestral haplotype. An autosomal recessive inheritance pattern was revealed. No further sequence variations in the canine MDR1 gene have been reported in the literature as yet, whereas in the orthologous human ABCB1 (MDR1) gene, numerous single