Recent Advances in Canine Hereditary Diseases

Transcription

Recent Advances in Canine Hereditary Diseases
2/6/14 Recent Advances in Canine
Hereditary Diseases
Urs Giger
Disclosures
§  Scientific advisor to various companies including
IDEXX, Purina, Royal Canin, and Waltham.
§  Research support from various organizations including
§  National Institutes of Health
§  Canine Health Foundation
§  Winn Feline Foundation
PD. Dr. med. vet. MS
Dipl. ACVIM & ECVIM-CA (Internal Medicine)
Dipl. ECVCP (Clinical Pathology)
§  Director of the Genetic Disease Testing Laboratory
Acknowledgements
Section of Medical Genetics
School of Veterinary Medicine
University of Pennsylvania
Philadelphia
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Large Breed
variations:
~400 dog breeds
§  Selected on basis of morphological
& behavioral characteristics.
§  Breeds are genetic isolates.
§  Some are geographically localized.
§  170 AKC registered breeds
§  All breeds are closely related.
§  Many breeds have narrow gene pools;
minimal genetic diversity.
§  Thus, many diseases are breed specific.
A – Adenine
DNA Polymorphisms
C – Cytosine
Ø  Single base changes are
called SNPs
Dog 1
Ø  Some are variable between
breeds and individuals of a
GG
breed.
Ø  Currently used for GenomeDog 2
Wide Association Studies
(GWAS) to discover genetic
GT
traits.
Ø  Also approach for complex
traits
Dog 3
Ø  Previously used
TT
microsatellites (repeats)
Canine Genome Sequence
achieved during past decade
§  First dogs sequenced (US$100 millions)
§  “Shadow” Poodle Celera TIGR Institute 2x
§  “Tasha” Boxer at MIT in Boston
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~3 billion bases
~20,000 genes
CanFam 3.1 Genome Sequence updated
Many more dogs sequenced ($5,000)
SNP discovery in many breeds
§  Commercial microarrays (SNP chips)
G – Guanine
T – Thymine
SNP
Ø  Single Nucleotide
Polymorphisms (SNPs)
My co-investigators at Penn Vet
Many collaborators worldwide
Veterinarians in many different clinics
Numerous pet owners and breeders
CATCTGCATCG
CATCTTCATCG
Approach to Genetic Traits
•  Candidate Gene(s)
–  Based upon trait
–  Based upon comparison
–  Still many unknown genes and functions
•  Genomics
–  Genome-wide association studies (GWAS)
–  Fine mapping
•  Whole genome sequencing
–  Exomic sequencing
•  Biochemical, metabolic, hematological and
clinic studies will be again needed.
1 2/6/14 Population Structure of Dog Breeds
Hereditary Diseases in Dogs & Cats
Number of Diseases
900
K=2
K=2
K=3
K=3
Shiba Inu
Chow Chow
Akita
Alaskan Malamute
Basenji
Chinese Shar-Pei
Siberian Husky
Afghan Hound
Saluki
Tibetan Terrier
Lhasa Apso
Samoyed
Pekingese
Shih Tzu
Irish Wolfhound
Saint Bernard
Greyhound
Belgain Sheepdog
Belgian Tervuren
Borzoi
Collie
Shetland Sheepdog
Pug
Komondor
Whippet
Standard Poodle
Bichon Frise
Keeshond
Manchester Terrier
Norwegian Elkhound
Kuvasz
Great Dane
Welsh Springer Spaniel
Doberman Pinscher
Standard Schnauzer
Italian Greyhound
Old English Sheepdog
American Water Spaniel
Miniature Schnauzer
Australian Terrier
English Cocker Spaniel
Irish Setter
West Highland White Terrier
Pointer
Basset Hound
Cavalier King Charles Spaniel
Giant Schnauzer
Pharaoh Hound
Golden Retriever
Beagle
Bloodhound
Airedale Terrier
American Cocker Spaniel
American Hairless Terrier
Chesapeake Bay Retriever
Cairn Terrier
Portuguese Water Dog
German Shorthaired Pointer
Border Collie
Bedlington Terrier
Clumber Spaniel
Ibizan Hound
Rhodesian Ridgeback
Dachshund
Australian Shepherd
Chihuahua
Kerry Blue Terrier
Schipperke
Irish Terrier
Flat Coated Retriever
Soft Coated Wheaten Terrier
Pomeranian
Labrador Retriever
Presa Canario
Rottweiler
Bullmastiff
Newfoundland
German Shepherd Dog
French Bulldog
Miniature Bull Terrier
Bulldog
Boxer
Mastiff
Bernese Mountain Dog
Greater Swiss Mountain Dog
K=4
K=4
Ancient/Asian
Modern/Hunting
Herding
Mastiff
Ostrander, 2007 & 2012
800
1990 – 3 mutations
2013 – 170 mutations
700
600
500
400
300
200
100
0
1950 1960 1970 1980 1990 2000 2005 2010
~230 Hereditary
Diseases in Cats
2013 – 24 mutations
“Inherited Diseases in Dogs” [IDID], http://www.vet.cam.ac.uk/idid
Dogs
“Mendelian Inheritance in Animals”, http://www.angis.org.au/databases/BRIX/omia
Autosomal
Recessive (AR)
Inheritance
Modes of Disease
Inheritance
v  most common
v  asymptomatic carriers
v  “skips generations”
•  Most diseases are inherited in an autosomal recessive manner
PC
P
XD
AR
Canine (IDID)
AD
Inherited Diseases in Dogs (http://
www.upei.ca/~cidd/intro.htm)
XR
XD
XR
PC
AR
AD
Human (OMIM)
Males - squares
Online Mendelian Inheritance in Man
Females - circles
AR = autosomal recessive; AD = autosomal dominant;
XR = X-linked recessive; XD = X-chromosomal dominant;
PC = polygenic, complex, autosomal recessive.
Affected – filled white
~2013
The Dog and Its Genome. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2006:249-289.
Autosomal Recessive
Inheritance
Carrier Female
D/d
Normal Male
Carrier Female
D/D
D/d
Soon complex traits
more common
Normal Affected
Hereditary Diseases
Carrier Male
Simple – single gene defects
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D/d
Autosomal recessive – dominant
X-chromosomal recessive – dominant
Still some variation between affecteds
Complex – polygenic – genetic association
50%
Heterozygous
Carriers
D/d
D/d
50%
Normal
(“Clear”)
D/D
25%
Affected
D/D
d/d
50%
Heterozygous
Carriers
D/d
D/d
25%
Normal
(“Clear”)
D/D
The majority of the mutant alleles underlying a
recessively inherited disorder are spread in the
population by heterozygous, clinically asymptomatic/
unaffected dogs.
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Major and modifying genes, predisposing
Environmental influences
Large variation in time of onset and severity
Mitochondrial (rare)
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Maternal transmission (mitochondrial DNA)
Excertional myopathies, sensory ataxic neuropathy
2 2/6/14 Hereditary Diseases
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Congenital malformations
–  Developmental anomalies
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Inborn errors of metabolism
–  Enzyme, receptor, transporter defects
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Genetic predispositions
–  Infections
–  Immunodeficiencies
–  Inflammations
–  Behavioral disorders
–  Immune diseases
–  Pharmacogenetics
–  Degenerative processes –  Cancer
•  All 3 can overlap and likely have a metabolic basis
Clinical
Signs
•  Puppy & kitten mortality
complex
•  Fading puppy or kitten
syndrome
•  Failure to thrive
•  Poor weight gain
•  Growth retardation
Top 10 Canine Health Concerns
(AKC Canine Health Foundation)
#1 Hip Dysplasia
#6 Lymphoma
#2 Allergies
#7 Patella Luxation
#3 Epilepsy
#8 Cataracts
#4 Hemangiosarcoma
#9 Bloat
#5 Hypothyroidism
#10 Atopic Dermatitis
Others: Osteosarcoma, Autoimmune Disease, Renal
Dysplasia, Portosystemic/hepatic Shunt, Elbow
Dysplasia, Deafness, Progressive Retinal Atrophy
Still need a lot of research to define these complex traits.
Genetic Disease Testing
Methods
•  Signalment (breed)
•  Physical examinations
•  Imaging (Rads, U/S, CT)
•  Eye examination
•  Routine laboratory tests
•  Failing biological system
analysis
–  Metabolites
•  Protein assays
–  Quantity
–  Activity
•  DNA analysis
–  Mutation tests
–  Linkage tests
Phenotype
–  The observed clinical
findings as determined by
the genotype and the
environment
•  Carriers/heterozygotes of
recessive disorders are
asymptomatic
Genotype
–  The genetic constitution
or more specifically the
alleles present at one
gene locus
•  Homozygous (affected)
•  Heterozygous (carrier)
•  Complex traits
Genetic Disease Testing:
Affected Animals
•  Identify diseased animals
•  Discover animals at risks
•  Prior to developing signs
•  Prior to selling into homes
•  Prior to breeding
•  Prior to training dogs
Physical examinations
Imaging (x-rays, U/S, CT)
Eye examination
Pathology
Laboratory tests
Failing biological systems
Protein assays
DNA tests
3 2/6/14 Inborn Errors of Metabolism
•  Currently refers to single gene defects
•  With the better characterization of hereditary
disorders, practically all genetic defects could be
considered to be an inborn error of metabolism
including malformations and susceptibility to disease.
Metabolic Genetic
Screening Tests
Sir Archibald
Garrod 1910
Metabolic consequences in a pathway
WBC granulations
A
Spillover into
alternative
pathway
Accumulation
of substrates
E
B
Enzyme deficiency
or metabolic block
C
Lack of
products
D
•  Enzyme deficiencies
•  Structural proteins
•  Receptors, adhesion
molecules, ion channels
•  Plasma proteins
Severe lipemia
Metabolic Genetic Screening Tests
“Vivian” Giant
Schnauzer puppy
Urine
Paper Chromatography
Spot Tests
MPS spot
+
Clinitest
Ketostix
Nitroprusside
test
+
Amino Acid
+
+
MPS
Carbohydrate
Amino acid analyzer
electrophoresis chromotography
Chondroitin sulfate
Dermatan sulfate
Heparin sulfate
Keratan sulfate
Glucose
Lactose
Fructose
Galactose
Organic Acid
Cystine
Citrulline
Ornithine
Glutamine
Taurine
Alanine
Tyrosine
Leucine
Sarcosine
Valine
Glycine
Lysine
Arginine
+
MS/Gas
chromatography
Lactate
MMA
Isovaleric acid
Oxalate
Ketones
Cystinuria
Cystine
Ornithine
Lysine
Arginine
•  Renal tubular and intestinal transport defect of
cystine and dibasic amino acids (COLA)
•  Cystine precipitates in slightly acidic urine.
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Cystine can lead to crystalluria &
calculi formation
& obstruction.
normal >99% resorption
Border Collie puppies
-Giant Schnauzers
-Beagles
-Border Collies
-Australian Shepherds
-Kommodor
-recently a cat
Canine Cystinuria
1823
1935
1936 
1995
2000 
2013
Lassaigne: First cystine calculi found
Morris et al: Metabolic defect identified
Green et al: Genetic basis in Irish Terriers (X-chrom.?)
Autosomal-recessive trait in Newfoundlands
Type I cystinuria caused by mutation SLC3A1 in Newfis
Various mutations and androgen-dependent cystinuria
rBAT protein
SLC3A1
rBA
T
Heavy chain
b0,+AT protein
SLC7A9
Light chain
b0,+AT
Palacin, M. et al. Physiology 2005
4 2/6/14 >70 Breeds with Cystinuria
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Newfoundland
Irish Terrier
Mastiff
English Bulldog
Labrador Retriever
Australian Cattle Dog
Miniature Pinscher
Basset Hound
Dachshund
Diagnosis Cystinuria
100%
cystine
3+
•  Hexagonal crystals in acidic urine (highly variable)
•  Yellow-brown calculi: Crystallography, chemical analysis
•  Nitroprusside (cystine) test positive:
–  Always positive in type I & II cystinuria
… and many others
Cystine
Ornithine
Lysine
Arginine
COLA
≤178
≤100
≤200
≤100
≤500
Normal
Also common in humans: 1:7000
(µmol/g creatinine)
Molecular Basis of Cystinuria
Cystinuria in Newfoundlands/Landseers
(Samples NOT representative of population)
Dogs
%
%
%
Mutant
Allele
1996-99
998
2
26
72
15%
2000-03
1795
0.5
18
82
9%
2004-08
1425
0.3
4
94
2%
2009-13
844
0.1
0.3
99
0.2%
Year
Amino acid transporter system bo,+ in the
proximal tubule of the kidney encoded by two genes:
tubule
SLC3A1 = transporter subunit rBAT
4+
POSITIVE
NEGATIVE
(heavy chain)
- 784 amino acids
Tested
Affecteds
Carriers
Normal
Canadian
Champion
Terry
PennGen screening test results; biased
SLC7A9 = transporter subunit bo,+AT
(light chain)
- 490 amino acids
bo,+ is responsible for the reabsorption of COLA from the tubules
Molecular basis incompletely defined in dogs and humans
C* mutant allele
C normal allele
Cystinuria
Type I
New Classification
of Canine Cystinuria
Type I A
Type IIA
Type IIB
Inheritance
Autosomal
recessive
Autosomal dominant
Autosomal dominant
Sex limited
Gene
SLC3A1
SLC3A1
SLC7A9
Undetermined
Males and Females
Males and Females
Males and Females
Intact Adult Males
No
No
No
Yes
≥ 8000
≥ 8000
unknown
Phenotype - Genotype
Cystinuric Newfi puppy from
normal x carrier mating
How could this ever happen?
Gender
Androgen dependence
COLA
homozygous
µmol/g creat.
(normal ≤
500)
Breeds
Type III
≤ 4000
heterozygous
≤ 500
Newfoundland
Landseer
Labrador
≥ 3000
Australian Cattle Dog
≥ 700
Miniature Pinscher
Mastiff & Related
Breeds
Scottish Deerhound
Irish Terrier
5 2/6/14 Therapy for Cystinuria
Cystinuria Type III
•  Oft asymptomatic
–  Preventative measures
•  Obstruction (emergency)
Cysinuric Maned Wolves
Also cats, ferrets and servals
–  Surgery
–  Endoscopy
–  Lithotripsy (soft calculi)
–  Preventative measures
•  >70 canine breeds, frequently in Mastiffs, English
Bulldogs, Bassets, Dachshunds, Irish Wolfhounds
•  Medical
•  Mature male animals (sex-limited)
•  Castration
Allyson Berent
•  Marker test for Mastiff/Bulldogs now available
Medical Management of
Cystinuria
•  Urine alkalinization
•  Diuresis
–  Plenty of water
–  Frequent urination
–  No dehydration
–  Bicarbonate
–  Potassium citrate
–  pH >7.5
•  Diet
•  Chelating Substances
–  Special low protein diets
–  +/- alkalinization
–  No protein supplements
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–  No amino acid suppl.
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WSAVA Hereditary
Disease Committee
World Small Animal Veterinary Association
Assisting clinicians with diagnosis, treatment and
control of hereditary diseases and genetic
predispositions in dogs and cats.
–  2-MPG (Thiola)
–  D-Penicillamin
Infection prevention/
treatment
–  Antibiotics postoperative
–  Optimal surgerical tech.
DNA Testing Laboratories
Management of Hereditary Disorders
•  Prevention of the production of affected
animals is most important
•  Control of further spread of mutant alleles
Ø 46 labs identified
42 still offer tests
Ø 19 research; 23 commercial labs
27 dogs only; 5 cats only; 10 both
Ø 151 mutations: 137 in dogs; 27 in cats
Ø 135 tests offered
94 offered by multiple labs
Ø  http://research.vet.upenn.edu/
DNAGeneticsTestingLaboratorySearch/tabid/7620/Default.aspx
Slutsky et al. Veterinary J 2013.
–  maintain desirable traits and genetic diversity
•  Therapy is limited; there are ethical concerns
•  Surgical interventions
•  Supplementations
–  Vitamin B, Coagulation factors
•  Symptomatic therapy
•  Gene transfer experiments
–  Transplantations
–  Gene therapy
6 2/6/14 Treatment Options for
Coagulopathies
Development of novel therapies and
assessment of their efficacy and safety
Disease Specific Treatments:
• Correct the underlying disease
• Vitamin K supplementation
• Withdrawal of drugs/toxins
• Heparin reversal
?
AA
VF.
IX
Factor Replacement Therapy:
AA
VF.
IX
• Fresh Frozen Plasma (FFP; all coagulopathies)
• Cryoprecipitate (vWF, FVIII)
• Factor Products
• Purified concentrates (Human, Porcine)
• Recombinant products
25 g!
?
Experimental: Gene Therapy
70 kg!
20 kg!
MPS Disorders in Dogs & Cats
WBC
inclusions due
to MPS
storage
(New Zealand, Australia)
MPS
Enzyme
Breeds
MPS I
α-iduronidase
Plotthound, Rottweiler, DSH
MPS II
Iduronate sulfatase
Labrador Retriever
MPS IIIA NAGLU enzyme
Dachshund, Huntadog
MPS IIIB
N-acetylglucosaminidase
Schipperke
MPS VI
Arylsulfatase B
Miniature Pinscher, Schnauzer, &
Poodle; Chesapeake Bay, Siamese
and DSH cats
MPS VII
β-glucuronidase
Mixed breed dog, German Shepherd,
Rat & Brazilian Terriers, DSH cats
MPS IIIB
MPS I
MPS VII
Normal Patient 1 Patient 2 Normal
6 µl
MPS IIIA
MPS IIIB
9 µl
MPS Spot Test in Urine
Mucopolysaccharides (MPS)
Glycosaminoglycans (GAGs)
MPS VI in Miniature
Pinschers
MPS II
MPS VII
MPS
degradation
Improving the Genetic
Health of Your Puppies
7 2/6/14 Control of Genetic
Diseases
Considerations
Recommendations
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Severity of disease
Onset of clinical signs
Specific diagnosis
Detection of carriers
Accuracy of test
Frequency of disease
Breed gene pool
Breed health club
Registry
Laws
Genetic Disease
Screening
GOAL:
Control of genetic
diseases with
recessive traits in
future generations
Identify carriers:
Do not breed affecteds
Screen all breeders
Breed clear to clear
Breed clear to carriers
–  Test all offspring
–  Select clear in next
generation
•  Do not select only against
one disease
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simple autosomal recessive disease
Polycystic Kidney & Liver
Disease
Protein Assays
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First litter
Second litter
Normal Carrier
Affected
~90 Diseases
Specialized laboratory
Concurrent control (100%)
Specific tissue required
Special shipping required
Age dependent
•  Identifies affected animals
–  Low activity/quantity <20%
•  May identify carriers
–  Intermediate activity ~50%
•  Potential overlap between
–  Carriers and normals
–  Limits their usefulness
DNA
Sources
Screening Tests
•  Physical findings, routine lab tests determine phenotype
–  Only affecteds are recognized clinically
•  Metabolic screening and protein assays
•  Genotypic identification is precise: Affecteds, Carriers, Normals
–  Inexpensive, simple, once done per animal; DNA is stable
Genotype
Allele
Protein
Phenotype
Normal
2 normal/wild-type
100+25%
Healthy
Carrier
1 mutant + 1 normal
50+25%
Healthy
Affected
2 mutant
0-20%
Diseased
M
3 Carriers
3 Normals
Obligate carriers
Test matings
Protein assays
DNA tests
3 Affecteds
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stable
EDTA blood
Cheek swabs
Other tissues
Serum & urine not useful
M
Mutant allele
Normal allele
8 2/6/14 DNA Mutation
Analysis
•  Most accurate
•  Genotype is independent of age
–  Testing at birth possible
•  Identifies affecteds
–  Diseased animals
–  Animals at risk of becoming ill
•  Discovers carriers
(asymptomatic)
•  Stable samples
–  EDTA blood, swab, brush
•  Simple submission
–  Regular mail
•  Quick test
•  Relatively inexpensive
Control of Genetic
Diseases
normal
affected
carrier
Considerations
Recommendations
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Severity of disease
Onset of clinical signs
Specific diagnosis
Detection of carriers
Accuracy of test
Frequency of disease
Breed gene pool
Breed health club
Registry
Laws
Do not breed affecteds
Screen all breeders
Breed clear to clear
Breed clear to carriers
–  Test all offspring
–  Select clear in next
generation
•  Do not select only against
one disease
Recent Advances in Canine
Hereditary Diseases
Urs Giger
PD Dr. med. vet. MS
Dipl. ACVIM & ECVIM-CA (Internal Medicine)
Dipl. ECVCP (Clinical Pathology)
Section of Medical Genetics
School of Veterinary Medicine
University of Pennsylvania
Philadelphia
Section of Medical Genetics
Faculty, Fellows & Residents
Many Collaborators
Referring Clinicians & Dog Owners
Supported in part by National Institutes of
Health (RR02512) and Canine Health and
other Foundations
penngen@vet.upenn.edu
http://www.vet.upenn.edu/penngen
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