Dog Genomics

1. Dog Genomics Chris Chu, Neva Watson and Melissa Steinmetz

2. History of dog genomics • Dogs were domesticated around 15000 BC • From domestication, different dogs were bred for their different • skills and traits • This leads to breeding for certain traits, thus specific breeds • have different traits that can be used for “genetic dissections” • July 2004, the first sequence was made available to the public

3. History of dog genomics • This first public dog genome was used to explore more than • 400 breeds of dogs

4. Why dog genomics? • There are many genetic diseases that humans and dogs • have in common • This is because they have syntenic parts of the genome • A few shared diseases are (so far, 223) Hepatic fibrosis Hodgkin's Disease Scoliosis Narcolepsy video 1 video 2 Dog models for human diseases

5. Olfactory Receptors • First discovered in 1991 in the Rattus norvegicus • a.k.a. the brown rat, discovered by Buck and Axel • The Olfactory Receptors are the first molecule that • detects a physical interaction of the odorant and then • triggers an olfactory response • In vertebrates, it is the largest gene family, comprising of 3-5% of total gene content

6. Olfactory Receptors • The Olfactory Receptors are a member of the G-protein • coupled receptors super family

7. Olfactory Receptors • 900 genes in humans and 1500 genes in mice were discovered to • be related to the olfactory receptors, but only 21 genes so far • have been discovered in canines. • The sense of smell in canines is many times more sensitive than • in humans. This might be due to the olfactory epithelium surface area, • Or, this heightened smell maybe a result of OR genes

8. Comparison of the canine and human olfactory receptor gene repertoiresPascale Quignon,1 Ewen Kirkness,2 Edouard Cadieu,1 Nizar Touleimat,1 Richard Guyon,1 Corinne Renier,1,3 Christophe Hitte,1 Catherine André,1 Claire Fraser,2 and Francis Galibert1 http://pets.webshots.com/photo/2391627310056833250yqkSCy

9. Identification of OR sequences 1. PCR amplification of canine DNA • Used 5 primer pairs to amplify regions between TM3 and TM7 • Sequenced 774 clones • 190 unique sequences • 10 previously identified OR genes

10. Identification of OR sequences 2. Compare canine DNA with human OR genes • Sequenced 3Gb canine genome at Celera Genomics • Sequenced 2kb and 1kb DNA inserts from standard poodle with 1.2X sequence coverage • Assembled 1.09 million contigs and 0.85 million singletons • Screened for sequences able to encode for a consensus AA sequence of all known human OR genes • MAYDRYVAICXPLHY • 737 unique sequences identified • 100 match unique PCR sequences

11. Where are they located? • Radiation Hybrid panel used to determine physical locations of OR sequences on canine chromosomes • What is a Radiation Hybrid panel?

12. Making a Radiation Hybrid Panel • Measured doses of radiation used to “break” DNA in target cells • Fuse irradiated cells with cultured rodent cells • Use selective marker to select for hybrids • Overlap hybrids to span the genome • Use PCR to identify markers http://opbs.okstate.edu/~melcher/mg/MGW1/MG1228.html

13. RH Mapping of OR Sequences • RHDF5000 panel • Canine fibroblasts irradiated at 5000 rads, then fused with thymidine kinase-deficient hamster cells • culture fused cells in HAT media • Yielded 126 hybrids • Used neighboring RH markers to map 562 canine OR sequences • Computed lod-scores with neighboring markers • OR regions spanned over 24 chromosomes • 37 chromosomal regions = 33 clusters of 2-124 genes + 4 solo genes • Chromosomes 18 and 21 contain 41% mapped OR sequences

14. RH Mapping of OR Sequences http://www-recomgen.univ-rennes1.fr/Dogs/OR_pnas.dir/ORcaryotype.html

15. Characterization of OR Pseudogenes • Translated canine OR nucleotide sequences • 417 fully characterized OR sequences have uninterrupted open reading frames • 244 exhibit frameshift mutations and/or in-frame stop codons • 102 sequences contained mutations near sequence ends • 142 putative pseudogenes were amplified and sequenced • 67 reclassified as genes • 75 confirmed as pseudogenes

16. Pseudogenes • 75 confirmed pseudogenes • 30% sequences with one mutation • 72% sequences with two mutations • 97% sequences with more than two mutations • 102 unconfirmed pseudogenes • 81 sequences with one mutation • 12 sequences with two mutations • 9 sequences with more than two mutations • (81 * 30%) + (12 * 72%) + (9 * 97%) = 42 estimated pseudogenes • Total estimated pseudogenes 75 + 42 = 117 pseudogenes • 117 pseudogenes out of 661 OR sequences = 18% pseudogenes

17. Canine OR Classification • Aligned regions between TM2 and EC2 for 403 OR sequences to construct a phylogram • 51 families and 202 subfamilies identified • Families contain between 1 and 35 genes • Class I contains 10 families • Class II contains 41 families

18. Human OR Classification • Aligned 714 OR sequences between TM2 and EC2 regions and constructed phylogram • 61 families and 285 subfamilies identified • Families contain between 1 and 93 OR gene sequences • Class I contains 10 families • Class II contains 51 families • Combined canine and human phylograms http://www-recomgen.univ-rennes1.fr/Dogs/OR_pnas.dir/ORHuCa.html

19. So what does it all mean? • 794 of 906 unique human OR genes identified • Complete canine OR gene repertoire: about 1,300 genes

20. Canine OR Pseudogenes • 18% ORs are pseudogenes • 20% mice • 63% calculated for humans • In all chromosomes with OR sequences • Largest clusters (chromosomes 18 and 21) • 4% and 10% pseudogenes • Gene families also show uneven distribution

21. Human OR Pseudogenes • Even gene distribution • Small clusters and isolated genes have high pseudogene levels • Largest clusters have lower pseudogene levels • Even in Families too • Except family 41 (87% pseudogenes)

22. Classifications • Two classes • Class I: families not species specific • Class II • One dog family (#38) is dog specific • One human family (#41) contain more genes then dog

23. Canine Olfactory Receptor Genes Classification • http://www-recomgen.univ-rennes1.fr/Dogs/OR_pnas.dir/ORcanineClassif.html indicates OR family indicates OR sub-family

24. Orthologous Dog and Human Clusters • 33 OR clusters and 4 isolated canine genes • 80 clusters and 62 isolated human genes • Why the difference? • Human from genome sequencing • Dog from RH-mapping • Some human genes with no dog counterpart

25. Orthologous Dog and Human Clusters Continued • Nucleotide similarity • Dog and human clusters paired • 20 canine clusters to 29 human • No human ortholog clusters found for 13 canine clusters • Clusters dog specific • No OR clusters found in orthologous regions of human or mouse

26. Evolution of the OR Repertoire • Only half canine OR genes identified • Equal or higher # of genes in canine clusters • Larger canine repertoire then human • Table 1 • 2 Clusters on chromosome 20 correspond to 2 human clusters • Canine clusters have more genes than human • Orthologous human family is scattered over 13 chromosomes

27. Evolutionary Conclusions • Similar clustered genomic organization of OR genes in dogs, mice and humans • Strong genomic conservation • suggests OR genes evolved from a common mammalian ancestral repertoire by successiveduplications.

28. Conclusions • Dog olfactory epithelium can express up to 20 times more ORs than humans • Dog OR repertoire is about 30% larger then humans • Lower # of pseudogenes

29. What else? • Dogs are used to study a variety of Human illnesses • Epilepsy • DMD • Hemophilia B http://research.nhgri.nih.gov/dog_genome/breen2001/breenmaps_data/cfax.html

30. Lafora • Seizures • begin in the teenage years • increase in frequency until they cause death, usually within five years after the onset of the first symptoms. • Identified one gene in humans • knew there was at least a second gene, because some families couldn't be linked to the first gene. • Dogs: myoclonic seizures • characterized by brief short jerks of a muscle or a group of muscles. • signs develop between the ages of six and nine, • death follows within three years • Jan. 7, 2005 issue of Science investigators report that affected dogs carry two copies of a gene with an expansion mutation. • repetitions of base pairs have also been implicated in other neurodegenerative diseases, such as Huntington's disease. http://www.avma.org/onlnews/javma/mar05/050315h.asp

31. Duchenne Muscular Dystrophy • One of nine muscular dystrophies • Degerative diseases affecting voluntary muscles • Absence of dystrophin • Early childhood (2-6years) • Generalized muscle weakness • X-linked recessive • Golden Retriever Muscular Dystrophy • point mutation in the consensus splice acceptor in intron 6 of the canine dystrophin gene. • exon 7 is skipped during processing of the GRMD dystrophin messenger RNA. • Possible Treatments • Plasmid or virus gene therapy • Mutation correction using short DNA fragments • Blocking proteasome degradation pathways can stabilized any truncated dystrophin proteins http://www.mdausa.org/disease/dmd.html

32. Hemophilia B • Recessive bleeding disorder • mutations in the Factor IX (FIX) gene on the X chromosome • An essential part of the blood coagulation cascade • Clotting factor deficiencies result in bleeding into joints, soft tissue, and muscles • Occurs in 1 in 30,000 males • Identified the first known mutation to cause hemophilia B in the dog • a missense mutation resulting in the complete absence of detectable protein • multiple cases have been described in different breeds and distinct mutations for five of these have been reported (Table 1) • Treatment • standard: intravenous infusion of FIX concentrates to prevent or treat bleeding episodes. • While treatment is effective and generally safe, it is expensive and inconvenient • Molecular therapies are being investigated • provide patients with treatment options • evaluate the overall efficacy of such approaches

33. Questions?