ACVO Short Symposium: Genetics of Inherited Eye Diseases
GENETIC TESTING:
when is it indicated and what do the results tell me?
Gregory M. Acland
Professor of Medical Genetics
Baker Institute for Animal Health
Department of Clinical Sciences
Cornell University, Ithaca NY
607 256 5684
[email protected]
Wednesday, November 4, 2009, 1-5pm
Genetic Tests: Definition
(for present purposes)
The genetic tests to be discussed include only DNA
based tests, designed to evaluate an individual’s genotype,
at relatively few loci.
This definition (artificially) excludes
i) tests using, e.g., enzymes or other non-DNA biomarkers;
ii) tests designed to yield genotypes for populations;
iii) genome-wide genotyping systems.
Categories of Genetic Tests
1. Identity Testing
• Forensics
• Parentage testing
• Individual “Barcoding”
• Genealogical/Ancestry Studies
• Species Determination
Categories of Genetic Tests
2. Genotyping for Hereditary Traits
• marker-based vs mutation-detection-based
• veterinary medical diagnostics
• selection tool for animal breeders
• disease diagnosis & carrier detection
• genotyping for desirable or undesirable traits
Identity Testing: DNA “Fingerprinting”
Several technics can distinguish individuals by DNA analysis.
1.
“Jeffrey’s Method”: DNA Fingerprinting using Minisatellites
2.
DNA Fingerprinting using Microsatellites
3.
Mitochondrial DNA Genotyping
4.
Y-chromosome Genotyping
5.
SNP Genotyping
“Jeffrey’s Method”: DNA “fingerprinting” using Minisatellite Probes
This was the first, original method of identity testing by
DNA analysis. Relies on core sequence motifs that are
repeated in tandem arrays as minisatellites. Jeffrey’s original
core sequence = (GGAGGTGGGCAGGAAG)n. A
hybridization probe based on a tandem-repeat of such a core
sequence can detect many highly variable loci
simultaneously and provide an individual-specific DNA
'fingerprint’. Originally developed for human identity testing,
since adapted and extended to multiple species.
Jeffreys AJ, Wilson V and Thein SL Hypervariable 'minisatellite' regions in human DNA. Nature 1985 314: 67-73.
Jeffreys AJ, Wilson V, Thein SL. Individual-specific 'fingerprints' of human DNA. Nature 1985 316(6023):76-9.
DNA Fingerprinting using microsatellites
AKC DNA Profile
17 Microsatellites
3-9 alleles per marker
 heterozygosity = 0.1 to 0.8
 2 multiplex reactions
 99% power of exclusion for
parentage
 3.2 x 10-8 chance of duplicate
genotype

DeNise S, Johnston E, Halverson J, Marshall K, Rosenfeld D, McKenna S, Sharp T, Edwards J. Power
of exclusion for parentage verification and probability of match for identity in American Kennel Club
breeds using 17 canine microsatellite markers. Anim Genet. 2004 Feb;35(1):14-7.
AKC DNA Certification
Mandatory Identity Testing



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Frequently used sires
Sires collected for AI using stored semen
Compliance audit - kennel inspection
Customer complaint evaluation
Voluntary testing

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Individual certificate
Parentage certificate
DNA Fingerprinting:
Mitochondrial DNA
Y-chromosome Specific Genotyping
Mitochondrial DNA (mtDNA) is also used for both forensic and genealogical DNA
testing. Because there are many copies of mtDNA per cell, mtDNA may be available from
samples too degraded to yield sufficient gDNA for analysis.
Typically, e.g. for human forensics, the HV1 and HV2 regions of the mtDNA are
amplified and sequenced to identify single nucleotide differences compared to the
“reference” human mitochondrial sequence. MtDNA polymorphisms are inherited
maternally, and can thus match individuals of common maternal descent, but can serve
effectively to exclude a given candidate, when other methods fail. Heteroplasmy (multiple
mtDNA genotypes in a single individual) and unstable poly-C mtDNA polymorphisms can
complicate analysis of mtDNA data.
Y-STRs (STRs on the Y chromosome) are often used in genealogical DNA testing,
and sometimes in forensics. Y-STRs are inherited paternally, are only present in males, can
match individuals of common paternal descent, but can serve effectively to exclude a given
candidate.
DNA Fingerprinting using SNPs
 Single Nucleotide Polymorphisms (SNPs) offer increasingly
attractive and powerful ways to genotype individuals for a wide
range of applications.
 SNPs are very densely distributed across the genome of all
species examined.
 SNPS are relatively highly stable (i.e. compared to microsatellites
or minisatellites).
 Several high throughput SNP genotyping systems are available
that can be very cost effective for large scale studies.
Genetic testing using multiple SNPs
 For sufficiently high throughput, this is currently the most cost
efficient method for testing, for both identity testing and
diagnostic testing.
 Direct Cost per SNP genotype ranges from $$$ for 1 SNP locus
for one sample down to a few cents per locus per sample, for
systems that simultaneously evaluate hundreds of loci in hundreds
of samples.
 In many ways this is the future of genetic testing in veterinary
medicine.
Genetic testing using multiple SNPs
examples
1. Ancestry Determination in dogs.
Several companies currently offer “fingerprinting” analysis that purports
to identify the mix of “purebred” ancestry in a mixed breed dog.
e.g.
The Wisdom Panel™ MX Mixed www.whatsmydog.com
Breed Analysis Test
Canine Heritage™ Breed Test
www.metamorphixinc.com
DoggieDNAPrint™
www.doggiednaprint.com;
www.vetdnacenter.com
Genetic testing using multiple SNPs
examples
2. Prediction of Breeding Value for Dairy Bulls.
The Australian Dairy Cooperative Research Centre, in collaboration with
Affymetrix, has under development a system for testing DNA from
performance-tested Dairy Sires, correlating particular performance traits
with genotype at
multiple marker
sets, and thus allow
SNP-genotype
based identification
of elite cattle.
Simulated data based on
the work of Professor
Herman Raadsma,
University of Sydney, and
Dr Bruce Tier, Animal
Genetics and Breeding
Unit, Armidale, NSW.
Canine Parentage Testing Laboratories
American Kennel Club
Animal Health Trust, UK
DNA Diagnostics
GeneMark
Geneseek
HealthGene
IGENITY (Merial)
Midwest Microsystems
MMI Genomics
PawsitiveID
Therion International
Veterinary Diagnostics
Center
Veterinary Genetics
Laboratory, UC Davis
Vetgen
Vita-tech
www.akc.org/dna/index.cfm
www.aht.org.uk/
www.dnadiagnostics.com
www.lic.co.nz/
www.geneseek.com/
www.healthgene.com
www.igenity.com/
www.midwestmicro.com/
www.metamorphixinc.com/
www.pawsitiveid.net/
www.theriondna.com/
www.vetdnacenter.com/
www.vgl.ucdavis.edu/
www.vetgen.com/
www.vita-tech.com/home.cfm
Canine Genetic Disease Testing Laboratories
Alfort School of Veterinary
Medicine
Animal Health Diagnostic
Center, Cornell University
Animal Health Trust
DNA Diagnostics
Dogenes
HealthGene
Optigen
PennGen Laboratories
University of Missouri
Veterinary Diagnostics Center
Veterinary Genetics Laboratory
(UC Davis)
Vetgen
Vita-tech
www.labradorcnm.com
www.diaglab.vet.cornell.edu/
www.aht.org.uk
www.dnadiagnosticsinc.com/
www.dogenes.com/
www.healthgene.com
www.optigen.com
www.vet.upenn.edu
www.CanineGeneticDiseases.net
www.vetdnacenter.com/
www.vgl.ucdavis.edu/
www.vetgen.com/
www.vita-tech.com/
Genotyping for Hereditary Traits
1. Monogenic disorders, mutation based testing.
 Direct detection of causative mutation(s)
 Establish genotype of patient
Diagnosis
Prognosis
 Genotype relatives
Carrier detection
Selective breeding
Genotyping for Hereditary Traits
2. Monogenic disorders - Indirect DNA Tests based on
Linkage or Linkage Disequilibrium.
 Linkage based tests useful in humans for genetic
counselling in families. Less so for dogs.
 Linkage Disequilibrium based tests more useful -applicable on individual basis.
Useful when a mutation is mapped but not identified.
Requires:
polymorphic markers very close to disease locus
identification of least-closely-related affecteds-IBD
Genotype affecteds and nonaffected relatives
Establish marker haplotype associated with disease
Predict genetic status
Significant risk of false positives
Gene Tests for Canine Hereditary Traits
Ophthalmologic Disorders 1. PRA related disorders - Dogs
Trait
Cord1/PRA
Gene
RPGRIP1
crd
LCA-CSB
Dominant PRA
prcd-PRA
rcd1-PRA
rcd2-PRA
rcd3-PRA
Type A -PRA
XLPRA
NPHP4
RPE65
Opsin
PRCD
PDE6B
rd3
PDE6A
RPGR
* = new improved test coming soon (?)
Breeds
MLHDax,
Springer Spaniel *
SWHDax
Briard
Mastiff
multiple
Irish Setter, Sloughi
Collie
Cardigan Corgi
Miniature Schnauzer
Husky, Samoyed
Gene Tests for Canine Hereditary Traits
Ophthalmologic Disorders 2. PRA related disorders - Cats
Trait
Adult onset
PRA
Gene
CEP290
Breeds
Abyssinian, Somali and
Ocicat
Gene Tests for Canine Hereditary Traits
Ophthalmologic Disorders 3. Other traits - Dog
Trait
Cataract
CEA
cmr1, cmr2,
cmr3
Lens Luxation
Merle
Oculoskeletal
Dysplasia 1
Oculoskeletal
Dysplasia 2
* = not yet published
Gene
HSF4
Breeds
Boston Terrier,
Staffordshire Bull Terrier
NHEJ1
multiple
bestrophin
Massive breeds, Coton de
Tulear, Llaponian Herder
*
multiple (mainly terriers?)
SILV/Pmel17 multiple
*
Labrador Retriever
*
Samoyed
Gene Tests for Canine Hereditary Traits
Coat Color etc.
Trait
Brown vs Black
Yellow vs Black
Brindle vs
“Dominant
Black”
Merle
Gene
TYRP1
MCR1
K
Breeds
multiple
multiple
multiple
SILV
multiple
Gene Tests for Canine Hereditary Traits
Coat Color etc
Gene Tests for Canine Hereditary SystemicTraits
i. Hematologic/Immune disorders
Trait
CLAD
Cyclic
Neutropenia
Factor IX def.
(Hemophilia B)
Factor VII def.
PFK
Gene
CD18
AP3B1
Breeds
Irish Setter
Collies
Factor IX
multiple
Factor VII
multiple
Phosphofructokinase Springer Spaniel,
American Cocker Spaniel
PK
Pyruvate Kinase
Basenji, Beagle,
Chihuahua, Dachshund,
WHW & Cairn terrier
Thrombasthenia ITGA2B
Otterhound, Great
Pyrenees
VWDI
VWD
many breeds
VWDII
VWD
several (Pointer) breeds
VWDIII
VWD
several breeds
X-SCID
IL-2Ry
Basset Hound, Cardigan
Corgi
Gene Tests for Canine Hereditary SystemicTraits
ii. Neuromuscular Disorders
Trait
Centronuclear
Myopathy
Cystinuria
Double Muscling
Fucosidosis
MPS IIIb
MPS VI
MPS VII
Myotonia congenita
Narcolepsy
Gene
PTPLA
Breeds
Labrador Retriever
SLC3A1
Myostatin
alpha-l-fucosidase
NAGLU
ASB
GUSB
CIC-1
Hypocretin
Neuronal Ceroid
Lipofuscinosis
cathepsin D
Newfoundland
Whippet
Springer Spaniel
Schipperke
Miniature Pinscher
German Shepherd
Miniature Schnauzer
Dachshund,
Doberman, Labrador
American Bulldog
Renal CystadenoCA &
Nodular Dermatofibrosis
Folliculin (FLCN,
BHD)
German Shepherd
Online Resources: Comparative Medical Genetics
Online Mendelian Inheritance
in Animals (OMIA)
Cambridge Vet School:
Inherited diseases, dog
NCBI: Online Mendelian
Inheritance in Man (OMIM)
also: BLAST, PUBMED,
Homologene, Entrez
Genome Browsers
UCSC
ENSEMBL
VISTA
omia.angis.org.au/
server.vet.cam.ac.uk/index.html
www.ncbi.nlm.nih.gov/
genome.ucsc.edu/
www.ensembl.org/index.html
genome.lbl.gov/vista/index.shtml
Reliability of Genetic Tests
Sensitivity & Specificity
in the context of
Genetic Testing
Reliability of Genetic Tests
Sensitivity = the probability that (frequency
with which) a truly affected individual will be
correctly diagnosed as affected by a test.
= TrueP ÷ TotalA
= TrueP ÷ (TrueP + FalseN)
Thus False Negatives lower Sensitivity
Reliability of Genetic Tests
Specificity = the probability (frequency) that
a truly nonaffected individual will be
correctly diagnosed as non-affected by a
test.
= TrueN / TotalN
= TrueN / (TrueN + FalseP)
Thus False Positives lower Specificity
Reliability of Genetic Tests
When (if) affected animals test as nonaffected:
TotalA = TrueP + FalseN
Reliability of Genetic Tests
When (if) non-affected animals test as affected:
TotalN = TrueN + FalseP
Reliability of Genetic Tests
For linked-marker-based tests, or mutation based tests for traits with
incomplete penetrance or complex inheritance, there is a risk of both
False Positives (nonaffecteds diagnosed as affecteds) and False
Negatives (affecteds diagnosed as nonaffecteds), thus Sensitivity and
Specificity are less than 100%.
For mutation based tests, in true Mendelian traits with complete
penetrance, there is essentially no risk of either False Positives or
False Negatives (excluding technical errors), thus both Sensitivity and
Specificity = 100%. That does not mean that there is no risk of error
or misinterpretation.
Reliability of Genetic Tests
The risks of error for mutation-based tests, in true Mendelian traits with
complete penetrance, represent not false positives or false negatives in
the traditional sense but:
 Sample errors - DNA sample does not come from the correct individual
(may be accidental or fraudulent).
 Technical errors - errors in handling and processing of samples, or in
reporting results.
 Application errors - testing for incorrect or inappropriate mutation.
 Interpretation errors - tests are for specific mutations, but diseases may be
caused by different mutations.
Quality control procedures can and should be implemented by a testing
laboratory to detect, reduce and (ideally) eliminate these errors.
Reliability of Genetic Tests
Where the trait is Mendelian but with incomplete penetrance, or is complex,
then false positives and false negatives can be significant, even when based
on direct mutation detection.
Examples:
• RPGRIP1 mutation in cord/PRA in MLHDax and Springer Spaniel.
Apparently mendelian single locus trait, but test has a very high rate of false
positives and false negatives, and thus both low sensitivity and specificity.
• True complex traits such as Hip Dysplasia -- there are unlikely to be simple
causative mutations but rather “risk alleles”. Interpretation of the meaning
and value for potential tests will also be complex.
Where to Test?
3 Comments for your consideration:
1. Declaration of Conflict of Interest.
2. Caveat Emptor
3. “There is hardly anything in the world that some man cannot make a little
worse and sell a little cheaper, and the people who consider price only are
this man's lawful prey.”
John Ruskin (1819-1900)