Si mon P t e ersen J ones

Transcription

1 Simon Petersen Jones

2 What is a DNA based Test Uses sample of DNA Blood Cheek Swab Identifies the genotype (not phenotype) of the animal

3 There are Two Categories of Test: Mutation Detection Direct detection of presence/absence of disease causing mutation Many potential ways of doing this Linked marker tests Genotypes animal for polymorphic marker, one version of which is known to be linked to the disease allele

4 How to create a DNA based test 1. First identify the genetic mutation that causes disease OR very closely map the disease on the genome 2. Be 100% sure that the mutation causes the disease or the mapping information is accurate 3. If the suspect mutation is proven to cause the disease then it is very easy to create a rapid and accurate test for the identification of the mutation BUT using a linked marker before the mutation is identified is more problematic.

5 How to prove a DNA change is the cause of the disease Genetic information Statistically significant association between the DNA change and the disease status BUT can be some problems: Region surrounding the mutation will be associated with the disease Is disease fully penetrant? What is mutation predicted to do? Null mutation Missense mutation Any physiological supporting evidence? Functional? Gene expression?

6 Mutation detection tests Directly identify if the presence or absence of the causal mutation Assuming no sample mix up and appropriate controls these are Extremely accurate Extremely specific Only detects that gene mutation

7 Mutation Detection Tests Size of PCR product for insertion/deletions Sequencing of PCR product Allele specific PCR PCR restriction enzyme digestion test Mismatch PCR restriction enzyme digestion test TaqMan Assay

8 DNA based test for RPE65 Mutation PCR a 135bp fragment spanning mutation site Resolve on acrylamide or high percentage agarose gel see 4bp difference in size N N N A C C

9 Allele-specific eespec c PCR test TEST DNA Amplify in PCR that is designed to normal affected carrier amplify only the normal gene Amplify in PCR that is designed to amplify only the mutant gene

10 PCR Diagnostic Test for PRA in Cardigan Welsh Corgis 1a 1b 2a 2b 3a 3b 4a 4b 5a 5b 6a 6b M a = PCR for normal b = PCR for mutant 7a 7b 8a 8b Na Nb Ca Cb Aa Ab -ve a -ve b M

11 PCR Restriction Enzyme Tests A B Restriction enzyme digestion U A/A B/B A/B A

12 rcd1 PRA in Irish IihSetters Mutation in cgmp phosphodiesterase beta subunit Nucleotide 2420 G A Normal TGG = tryptophan Mutant TAG = stop codon Exon 21

13 Mismatch PCR Test for rcd1 PRA in Irish Setters Mutant allele Normal allele PCR Bfa1 Bfa1 Restriction enzyme digestbsr1 Bsr1

14 Mismatch PCR Test for rcd1 in Irish Setters M U A C N Clements et al (1993) Curr Eye Res. 12: Petersen-Jones et al (1995) J Small Anim Pract. 36:

15 Codon 616 1bp Deletion in PDE6A in Cardigan Welsh Corgis Normal 1921 TACCAGATGAAGTCCCAGAACCCACTGGCCAAGCTCCATGGGTCCTCCATCTTGGAAAGA 610 Y Q M K S Q N P L A K L H G S S I L E R CACCACTTGGAGTTCGGCAAAACGTTGCTGCGAGATGAGAGCCTGAATATCTTT H H L E F G K T L L R D E S L N I F Mutant 1921 TACCAGATGAAGTCCCAGACCCACTGGCCAAGCTCCATGGGTCCTCCATCTTGGAAAGAC 610 Y Q M K S Q T H W P S S M G P P S W K D ACCACTTGGAGTTCGGCAAAACGTTGCTGCGAGATGAGAGCCTGA T T W S S A K R C C E M R A * 28 abnormal amino acids starting at position 616 before a premature stop codon. Normal protein is 861 amino acids.

16 Normal Allele.GTCCCAGAACCCACTGGCCAAGCTCCATG.. TAGGTGACCGGTTCGAGGTAC HinfI PCR.GTCCCAGAATCCACTGGCCAAGCTCCATG.CAGGGTCTTAGGTGACCGGTTCGAGGTAC.GTCCCAG.CAGGGTCTTA

17 Mutant Allele.GTCCCAGACCCACTGGCCAAGCTCCATG.. TAGGTGACCGGTTCGAGGTAC HinfI PCR.GTCCCAGATCCACTGGCCAAGCTCCATG.CAGGGTCTAGGTGACCGGTTCGAGGTAC

18 a) Normal allele HinfI PCR HinfI 53bp 75bp 20bp b) Mutant allele HinfI PCR 53bp 94bp

19 Results - Mismatch PCR- Restriction Enzyme Digest Test M A N C U

20 Real Time PCR

21 TaqMan Assay

22 TaqMan Assay

23 TaqMan Assay

24 TaqMan Assay For Diagnostic Test Allele Discrimination

25 Unaffected Carrier Affected

26 Linked marker test Identifies presence or absence of a DNA marker known to be closely linked to the disease locus An indication of animals likely to be affected or unaffected

27 PRCD Mutation prcd 1? * Different breeds of dog * ** * *

28 Marker/Linkage Test for PRCD 2 2 Pattern A = normal 1 2 * 1 2 Pattern B most = carriers * ** * 1 1 Pattern C most = affected

29 Linked Marker Test for prcd Uses DNA markers very close to the defective gene Divides dogs into 3 groups Group A all dogs in this group are homozygous normal Group B many dogs in this group are carriers but some may be normal Group C all affected dogs are in this group, BUT also contains some carriers and some normals

30 Laboratories for PRA Testing Commercial labs University i labs Is lab reputable?

31 Advantages of DNA based Tests Mutation detection tests allow genotype of animal to be ascertained for recessive affected carrier homozygous normal can use carriers for breeding and prevent PRA affected dogs from being born Can be performed at any age Mutation detection tests are theoretically 100% accurate & 100% specific (barring human error)

32 Collection of Samples for DNAbased Genetic Tests Follow testing Lab s Labs instructions Identification of animal Careful labeling bli of samples Require a source of cells with nuclei Blood sample Cheek swab

33 Blood Samples for DNA Extraction Follow testing Lab s Labs instructions Citrate or EDTA tubs DO not allow to clot Label clearly, accurately and immediately Keep cool send with ih ice pack

34 Cheek Swabs for DNA Extraction Follow testing Lab s instructions Not within ihi a few hours of feeding dog Cytology brush to brush gently inside cheek Aiming to collect cells Label sample clearly, accurately & immediately

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37 Limitations of DNA based Tests Only applicable for a specific gene mutation Only a few tests are applicable for multiple li l breeds Some conditions are very genetically heterogeneous e.g. need many different tests for PRA

38 Advantages Offered by DNA based Tests Detect affected animals prior to onset of clinical signs Mutation detection tests for recessive disease identify: Homozygous normal Heterozygous Homozygous affected Enable retention of heterozygotes in breeding pool

39 Ui Using Carriers of Recessive Disease for Breeding If have a mutation detection DNA based test Aim is to avoid producing affected animals If carrier animal has other outstanding features it can be used for breeding Cross carrier with genetically normal animal Test offspring Use carriers if disease has high incidence Avoiding carriers will restrict gene pool Risk of incidence of other hereditary disorder Risk of losing desirable features

40 Future for Eye Screening in the DNA Era Can we hang up our ophthalmoscopes? Tests very specific ophthalmoscopy hh not specific Possibility of new forms of hereditary eye disease Previous low incidence emerge via popular sire effect Occurrence of new hereditary eye disease causing i gene mutations We must continue frequent eye screenings