Transcript Comparison of the canine and human olfactory receptor gene

Dog Genomics
Chris Chu, Neva Watson
and Melissa Steinmetz
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
History of dog genomics
•This first public dog genome was used to explore more than
400 breeds of dogs
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
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 35% of total gene content
Olfactory Receptors
•The Olfactory Receptors are a member of the G-protein
coupled receptors super family
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
Comparison of the canine and human
olfactory receptor gene repertoires
Pascale 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
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
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
Where are they located?
• Radiation Hybrid panel used to determine
physical locations of OR sequences on canine
chromosomes
– What is a Radiation Hybrid panel?
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
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
RH Mapping of OR Sequences
http://www-recomgen.univ-rennes1.fr/Dogs/OR_pnas.dir/ORcaryotype.html
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
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
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
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
So what does it all mean?
• 794 of 906 unique human OR genes identified
• Complete canine OR gene repertoire: about
1,300 genes
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
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)
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
Canine
Olfactory
Receptor Genes
Classification
indicates OR family
indicates OR sub-family
•
http://www-recomgen.univrennes1.fr/Dogs/OR_pnas.dir/ORcanineClassif.html
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
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
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
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 successive
duplications.
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
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
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
Duchenne Muscular Dystrophy
• One of nine muscular dystrophies
–
–
–
–
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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
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
Questions?