Author: Neff, M.W. et.al. Presented by Katheryn McDonald and Todd Mercier

1. Breed distribution and history of canine mdr1-1∆, a pharmacogenetic mutation that marks the emergence of breeds from the collie lineage Author: Neff, M.W. et.al. Presented by Katheryn McDonald and Todd Mercier

2. Introduction • Goal of study: To identify breeds at risk for multi-drug sensitivity and level of susceptibility for each breed • Ivermectin • 1980’s, parasiticide • Causes a charge in the ligand-gated chloride ion channels of the peripheral nervous system, resulting in an influx that paralyzes nematode and arthropod parasites • Generally safe in domestic animals due to the blood-brain barrier

3. Introduction • Ivermectin • Key component of the blood-brain barrier is P-glycoprotein • Large protein complex that pumps drugs out of the brain and into bloodstream • Encoded by the multiple drug resistance gene (MDR1) • Lack of P-glycoprotein may lead to neurotoxicosis

4. Introduction • MDR1 • mdr1-1∆ • Causes frameshift with multiple premature stop codons, drastically shortening the P-glycoprotein • Allele probably results in a complete loss of P-glycoprotein function • First discovered in the collie breed

6. Objective • Determination of genetic linkage of the mdr1-1∆ allele in non-herding breeds to find other at risk canine populations • Determination of ancestral lineage in other breeds not previously thought to be related to the collie

7. Methods • Four classes of dogs were tested • Breeds from the collie lineage based on breed history • European herding breeds not thought to be related to the collie • Sighthounds and miscellaneous breeds that exhibited drug sensitivities • Multibreed panel of over a thousand samples from more than 90 breeds

8. Methods • DNA samples taken from buccal cells collected by oral swab • Genotyping for MDR1 accomplished with PCR (rapid duplication of DNA) • Genome location mapped for MDR1 using a canine/hamster radiation hybrid panel

9. Methods • Haplotype analysis • Loci were selected based on proximity to MDR1 • Primers were produced for 4 markers and MDR1 for PCR • Haplotypes reconstructed with PHASE program (assumes all haplotypes from a pop. were created in that population)

10. Methods • Statistical analysis • Deviation from Hardy-Weinberg equilibrium in each test was assessed by a χ2 test in each breed • Allele age determined by linkage disequilibrium (LD) at mdr1-1∆ • Distance between mdr1-1∆ and marker loci was 1cM or greater, suggests mutation is a weaker force than recombination • However, mutation may result in rare haplotypes

11. Results • 4000 samples surveyed, the mdr1-1∆ allele was present in nine breeds

12. Results • mdr1-1∆ present in predicted collie breeds • mdr1-1∆ not found in herding breeds traced back to Europe • mdr1-1∆ also found in two non-herding breeds, the Longhaired Whippet and the Silken Windhound (sighthounds)

13. Results • High frequency of the mdr1-1∆ allele in the Collie and Longhaired Whippet breeds suggests a founder effect for those breeds • So, separate mutation or are they related?

14. Results • Palindrome located 9 base pairs upstream of mdr1-1∆ could have served as mutational hotspot~ suggest possible unrelated mutations • MDR1 tested with radiation hybrid analysis, revealed marker alleles strongly associated with mdr1-1∆ were identical at 3 of the 4 loci for herding breeds and sighthounds • Identical allele by descent~ all dogs carrying the mdr1-1∆ allele are descendants of a dog from Great Britain before the genetic isolation of breeds

15. Discussion • mdr1-1∆ was not present in the Border collie, Bearded collie or the Austr. Cattle dog, yet do exhibit ivermectin sensitivity • Possible reasons • mdr1-1∆ present at a lower frequency • Another mutation is responsible

16. Discussion • Within breeds, genotype frequency is consistent with Hardy-Weinberg equil. • No evidence linking mdr1-1∆ with selection or non-random mating • Allele differences most likely arose from genetic drift and expansion of formal breeds since the 1800’s • Ultimately genetic drift and artificial selection have played a major role in genetic composition of breeds

17. Questions?