canine genetics research at the aht · canine genetics research at the aht broad objective is to...
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Canine Genetics Research at the AHT
Broad objective is to identify genetic variants underlying inherited
disorders in dogs to :
1. enable development of DNA tests that:
a) allow breeders to reduce disease prevalence in specific breeds
b) aide differential diagnosis
2. improve understanding of pathophysiology of disease, that could lead to
improved treatment / therapies in dogs and other species
The Give a Dog a Genome project will enable us to identify
disease mutations more quickly and cost-effectively
Give a Dog a Genome
• On 25th January the Kennel Club contacted the Breed Health Co-ordinators
of all 215 registered breeds, inviting them to participate in Give a Dog a
Genome.
• In 2015 the Kennel Club Charitable Trust awarded the KCGC £50k to spend
on Whole Genome Sequencing (WGS).
• This is sufficient funding to sequence ~25 whole genomes.
• We invited each breed community to donate £1000
• We promised to match each donation with £1000 from the KCCT funding to
fund the sequencing of the whole genome of a dog of that breed.
• If successful this matched funding approach would enable us to sequence
50 whole genomes, creating largest canine genome bank in the UK
Talk Layout
• DNA – the basics – don’t panic
• whole genome sequencing – using it to identify a disease mutation
• A success story
• More about Give a Dog a Genome
Structure of DNA
G C
T A
G C
G C
G C
T A
G C
G C
T A
T A
5’
3’ 5’
3’
sugar-phosphate
chain
H
C
N C
C C
N
H
O O
CH3
T
N
C C
N C
C N
N C
H
A
H
N
H
N C
C C
N
O
H
C
C
N H
H
H
N
C C
N C
C N
N C
H
G O
H
N
H
H
TCCGTAGCTAGGCATGCATGGACTG
AGGCATCGATCCGTACGTACCTGAC
TCCGTAGCTAGGCATGCATGGACTG
Coding DNA
A A U G G G G G A U U U C C C mRNA
A T V W T Protein
A C T G A A A C C C C T T G G
A A T G G G G G A T T T C C C DNA
Transcription
Translation
Genetic Code
AUG used to both initiate protein synthesis and direct the incorporation
of methionine at internal positions of the polypeptide chain
• three special ‘stop’ codons that do not code for an amino acid but signal
the termination of translation
GCA
GCC
GCG
GCU
Ala
A
AGA
AGG
CGA
CGC
CGG
CGU
Arg
R
GAC
GAU
Asp
D
AAC
AAU
Asn
N
UGC
UGU
Cys
C
GAA
GAG
Glu
E
CAA
CAG
Gln
Q
GGA
GGC
GGG
GGU
Gly
G
CAC
CAU
His
H
AUA
AUC
AUU
Ile
I
UUA
UUG
CUA
CUC
CUG
CUU
Leu
L
AAA
AAG
Lys
K
AUG
Met
M
UUC
UUU
Phe
F
CCA
CCC
CCG
CCU
Pro
P
AGC
AGU
UCA
UCC
UCG
UCU
Ser
S
ACA
ACC
ACG
ACU
Thr
T
UGG
Trp
W
UAC
UAU
Tyr
Y
GUA
GUC
GUG
GUU
Val
V
UAA
UAG
UGA
Stop
Mutations
A mutation is a permanent change to the nucleotide sequence of an organism’s DNA
Types of mutation:
Insertion – the addition of one or more extra nucleotides into the DNA
Deletion – the loss of one or more extra nucleotides from the DNA
Substitution – the exchange of a single nucleotide
Inversion –the ‘flipping over’ of a stretch of nucleotides
Mutations can be small-scale, involving single, or small numbers of nucleotides, or large,
involving hundreds, or thousands of nucleotides.
Normal
Insertion
Deletion
Substitution
Inversion
Frame Shift Mutations
Insertions and deletions often alter the frame of the DNA (a frame shift),
which can significantly alter the gene product.
GCT GAA TGC TCG ATC GGC AAG ATC GAT GAT
A E C S I G K I D D
GCT GAA TGC TCG ATC GGC CAA GAT CGA TGA T
A E C S I G Q D R *
Substitutions
• Silent Mutations
GCT GAA TGC TCG ATC GGC AAA/G ATC GAT GAT
A E C S I G K I D D
• Missense Mutations
GCT GAA TGC TCG ATC GGC AAG ATC GAT/A GAT
A E C S I G K I D/E D
• Nonsense Mutations
GCT GAA TGC/A TCG ATC GGC AAG ATC GAT GAT
A E C/* S I G K I D D
Nomenclature
• Dictionary definition of mutation:
– a permanent change to the nucleotide sequence of an organism’s DNA
• Conventional definition of mutation:
– change to the nucleotide sequence of an organism’s DNA that is
deleterious
• We refer to positions in the DNA that can be different between individuals but
are not necessarily deleterious as variants
Variation
• There is a lot of variation within the canine genome
• Most of that variation is neutral
• Some of the variation is positive, or advantageous
• Some of the variation is deleterious (disease-causing)
• The challenge is to identify the deleterious variants within all the
background, non-deleterious variation
How much DNA is there?
• The canine genome contains 2.4 x 109 nucleotides.
• This is nearly two and a half billion ‘letters’ of DNA.
• If each one measured 1 mm the DNA in the dog would stretch from
Land’s End to John O’Groats and back again.
• There are about 20,000 genes.
Canine Reference Genome
• In 2004 the Canine Genome Sequencing Project produced a high-
quality draft sequence of a female boxer named Tasha, known as
CanFam1.0
• By comparing Tasha with many other breeds, the project also
compiled an initial set of ~2.5 million single nucleotide
polymorphisms (SNPs)
• The current version of the canine reference genome is CanFam3.1
DOG 3 CGATTATGATCAACTAGACTACGCATGACACACCGATCGATCGATTCGATCGG
DOG 1 CGATTATGATCAACTAGACCAGGCATGGCATACCGATCGATCGAATCGCTCGT
DOG 2 CGATCATGATCAACTGGACTAGGCATGACATACCGATCGATCGAATCGATCGT
CASE CGATTATGATCAACTGGACTAGGCATGACACACCGATCGGTCGAATCGCTCGT
REF CGATCATGATCAACTAGACTAGGCATGACATACCGATCGATCGAATCGATCGT
Affected Dog = CASE
CASE CGATTATGATCAACTGGACTAGGCATGACACACCGATCGGTCGAATCGCTCGT
REF CGATCATGATCAACTAGACTAGGCATGACATACCGATCGATCGAATCGATCGT
DOG 4 CGATCATGATCAACTAGACTACGCATGACATACCGATCGATCGAATCGATCGT
DOG 5 CGATCATGATCAACTGGACTAGGCATGACATACCGATCGATCGAATCGATCGT
DOG 6 CGAACATGATCAACTGGACTAGGCATGACATACGGATCGATCGAATCGATCGT
DOG 7 CGATCATGATCAACTAGACTACGCATGACATACCGATCGATCGAATCGATCGT
DOG 8 CGATCATGATCAACTGGACTAGGCATGACATACGGATCGATCGAATAGATCGT
It’s a bit more complicated than that…
Number of homozygous variants with both alleles unique to sample
14809_MS 2,827,515
17289_BGVP 3,299,219
23005_V 3,418,818
24093_BC 3,763,033
24604_WSS_AB 2,952,464
25078_WSS_AB 3,062,121
25314_SHY 4,119,944
25852_SBT 3,469,799
26042_BOT 3,185,908
26102_LR 4,387,742
26133_G 3,012,348
26569_FBD 3,559,523
7897_FCR 3,095,933
CKCS_17377 2,597,150
Dennis5runs_merged 3,555,431
FCR_25382 3,022,227
FCR_25384 3,199,004
ISP_21897 4,084,161
ST_25135 1,986,209
SV_25951 3,048,115
V_21485.fastq 3,305,723
Filtering by effect of mutation
Sequence ontology Term Effect score
start_lost 5
stop_gained 5
splice_acceptor_variant 5
splice_donor_variant 5
inframe_deletion 5
inframe_insertion 5
missense_variant 5
3_prime_UTR_truncation 4
5_prime_UTR_truncation 4
regulatory_region_ablation 4
non_coding_exon_variant 4
splice_region_variant 4
conserved_intergenic_variant 3
conserved_intron_variant 3
nc_transcript_variant 3
coding_sequence_variant 3
feature_elongation 2
feature_truncation 2
intron_variant 2
non_coding_transcript_exon_variant 2
regulatory_region_variant 2
3_prime_UTR_variant 2
5_prime_UTR_variant 2
downstream_gene_variant 1
intergenic_region 1
intragenic_variant 1
intergenic_variant 1
Filter by segregation score
Cas
e 1
Cas
e 1
Cas
e 2
Cas
e 2
Cas
e 3
Cas
e 3
Co
ntr
ol 1
Co
ntr
ol 1
Co
ntr
ol 2
Co
ntr
ol 2
Co
ntr
ol 3
Co
ntr
ol 3
Seg score 1: AA/AB/BB
G G G G G G A A A A A A 6
G G G G G G A A A A G A 5
A G G G G G A A A A A A 5
G G G G G G G G A A A A 5
G G G G G G G G A G A A 4
We expect: Cases AA, Carriers AB, Controls BB
The Genome Bank
• The genomes of two different dogs will differ in ~2-3 million places.
• The more breeds that are represented in the bank of genomes the
more variation across the genome we will capture.
• The more variation we capture the more effectively we can filter out
benign (or neutral) variants.
• The more effectively we can filter out neutral variants, the more
quickly (& cheaply) we will be able to identify disease mutations.
• Sequencing the genomes of single representatives of 50 different
breeds will capture far more variants than multiple dogs of the same
breed.
The Genome Bank
• The genome bank is a permanent resource that can be used to
facilitate inherited disease investigations in ANY BREED
• Dogs affected with a specific inherited diseases can act as controls
for studies of a different disease. e.g. the genome from a dog
affected with PRA can act as a control for epilepsy.
• The sequence of a dog of any breed will help investigations in other
breeds.
DOG 3 CGATTATGATCAACTAGACTACGCATGACACACCGATCGATCGATTCGATCGG
DOG 1 CGATTATGATCAACTAGACCAGGCATGGCATACCGATCGATCGAATCGCTCGT
DOG 2 CGATCATGATCAACTGGACTAGGCATGACATACCGATCGATCGAATCGATCGT
CASE CGATTATGATCAACTGGACTAGGCATGACACACCGATCGGTCGAATCGCTCGT
REF CGATCATGATCAACTAGACTAGGCATGACATACCGATCGATCGAATCGATCGT
Affected Dog = CASE
DOG 4 CGATCATGATCAACTAGACTACGCATGACATACCGATCGATCGAATCGATCGT
DOG 5 CGATCATGATCAACTGGACTAGGCATGACATACCGATCGATCGAATCGATCGT
DOG 6 CGAACATGATCAACTGGACTAGGCATGACATACGGATCGATCGAATCGATCGT
DOG 7 CGATCATGATCAACTAGACTACGCATGACATACCGATCGATCGAATCGATCGT
DOG 8 CGATCATGATCAACTGGACTAGGCATGACATACGGATCGATCGAATAGATCGT
Cerebellar cortical degeneration
Video provided by Joe Fenn, Royal Veterinary
College
• Two four month old cases (littermates) presented separately at • Joe Fenn, Royal Veterinary College neurology department
– Signs of ataxia at 2-3 months of age – Marked head tremor at four months
• Most forms of cerebellar ataxia display an autosomal recessive mode of inheritance
• Genetically different across breeds
• Could we genome sequence one of the cases and identify causative mutation ?
• Genome of Vizsla case compared to genomes of 13 other dogs
• different breeds, non-ataxic
Cerebellar cortical degeneration
Genome sequencing variants amongst our 14 canine genomes:
15,223,374
Variants with a high effect score: 50,655
Variants HOMOZYGOUS in the Vizsla only 290
Variants in known candidate genes: 1
• exon 26 splice donor site mutation (GT>AT) in sorting nexin 14 gene (SNX14)
• an altered SNX14 splicing pattern for a CCD case was demonstrated by RNA analysis
• no SNX14 protein could be detected in CCD case cerebellum by western blotting.
• additional case also homozygous
• 107 additional Vizslas genotyped, one carrier identified
0
100
200
300
400
500
600
700
10 12 14 16 18 20 22 24 26 28 30
Nu
mb
er
of
va
ria
nts
aft
er
se
g s
co
re a
nd
eff
ec
t s
co
re f
ilte
r (m
ax
imu
m)
Number of controls
WGS comparisons: effect of the number of controls on filtered variants
Give a Dog a Genome
• 75 breeds have signed up to GDG since its official launch
• All have donated at least £1000
• The KCGC has recently donated a further £25k to support this project…
• …taking total raised >£150k
• Process of selecting dogs to sequence is now underway.
Choosing Dogs to Sequence
• Depending on the health information we receive from the BHCs we
will decide to sequence:
a) A dog affected with a disorder that is a demonstrable health concern in
the breed, or
b) An older (> 8yo), apparently healthy dog.
• We will invite owners of suitable dogs to send swabs directly to us.
• We will extract DNA and measure quantity and quality from all
suitable candidates, including archived DNA samples.
• We will select the most suitable dog, based on robust clinical
diagnosis and good quality DNA.
• The identify of the dog we select will be kept confidential
• First dogs will be sequenced during October
Healthy Dogs
• We are requesting that healthy, older dogs have a full eye
examination, including gonioscopy, so we can use them as controls
for all our eye studies.
• The gonioscopy is required for our glaucoma studies.
• Because gonioscopy requires a considerable degree of expertise we
request that the eye examination is done by an ophthalmologist that
is a DipECVO, i.e.a recognised specialist.
• To help meet these rigorous criteria the AHT will reimburse the
costs for up to 3 dogs to have a BVA certificate, including
gonioscopy.
Health Summary
• > 80 health concerns listed by 66 breeds
• About half concerns are listed by single breeds only
Health concern Number of concerned
breeds
Idiopathic Epilepsy 15
Cataract, Hereditary 7
PRA - early & late onset 6
Hip dysplasia 6
Hypothyroidism (autoimmune thyroiditis?) 6
Lymphoma 6
Auto-immune mediated conditions 5
Cancer/ tumours 5
Dilated Cardiomyopathy 5
Patella luxation 5
Glaucoma 4
Cardiac 4
Degenerative Myelopathy (CDRM) 4
Elbow Dysplasia 4
Osteosarcoma 4
Acknowledgements
• Kennel Club Charitable Trust
• Louise Burmeister
Ellen Schofield
Mike Boursnell
Oliver Forman
Bryan McLaughlin
Kat Lawson
• Breed Health Co-Ordinators
