genetic diagnostics in a real life...
TRANSCRIPT
Genetic Diagnostics in a Real Life Setting
David Barton
National Centre for Medical Genetics
Dublin, Ireland
Chromosome > DNA
Effect of point mutations/polymorphisms
How do we test – which tools to use?
• Mutation-specific tests
- Restriction enzyme digestion
- Southern blot
- Dot blot / reverse dot blot
- PCR hybridization
- PCR applications
• PCR/fragment sizing
• MLPA: large deletion/duplications
• Mutation detection
- Mutation scanning
o SSCP
o DGGE
o DHPLC
o HRM-CA
- Sequencing
o Sanger sequencing
o Next Generation Sequencing
Tools are tailored to the
underlying mutation type
How do we test – which tools to use?
• Mutation-specific test
- Restriction enzyme digestion
- Southern blot
- Dot blot / reverse dot blot
- PCR hybridization
- PCR applications
• PCR/fragment sizing
• MLPA: large deletion/duplications
• Mutation detection
- Mutation scanning
o SSCP
o DGGE
o DHPLC
o HRM-CA
- Sequencing
o Sanger sequencing
o Next Generation Sequencing
Multiplex Mutations – Cystic Fibrosis
• recessive genetic disease
• 1/1600 – 1/2500
• carriers : approx. 1 in 20 to 1 in 25
• localised on chromosome 7q31 (1985)
• gene : CFTR (1989)
Kerem B et al. Identification of the cystic fibrosis gene: genetic analysis. (Science, Sept.1989)
• diagnostic testing carrier testing prenatal testingneonatal screening (?), population screening (?)
Recessive disorder: implications
Distribution of Cystic Fibrosis Mutations
(Data from the CF Genetic Analysis Consortium- July, 1995)
ATP-binding R-domainMembrane spanning Membrane spanning ATP-binding
1 2 3 4 5 6a 6b 7 8 9 10 1112 13 1514a 14b 16 17a 17b 18 19 20 21 22 23 24
5' 3'
Missense
Nonsense
AA deletion
Frameshift
Splice site
Variation
Total
Amino acid
Read Out of Fluorescence-based Genotyping Assay
Mutantsignal
Normal signal
Normal/Normal
Heterozygous
Homozygous
No call
How do we test – which tools to use?
• Mutation-specific test
- Restriction enzyme digestion
- Southern blot
- Dot blot / reverse dot blot
- PCR hybridization
- PCR applications
• PCR/fragment sizing
• MLPA: large deletion/duplications
• Non-mutation-specific tests
- Mutation scanning
o SSCP
o DGGE
o DHPLC
o HRM-CA
- Sequencing
o Sanger sequencing
o Next Generation Sequencing
Duchenne Muscular Dystrophy
DMD Deletion - MalePeak Ratio
Size (bp)
Normal
Range
Deleted
How do we test – which tools to use?
• Mutation-specific test
- Restriction enzyme digestion
- Southern blot
- Dot blot / reverse dot blot
- PCR hybridization
- PCR applications
• PCR/fragment sizing
• MLPA: large deletion/duplications
• Mutation detection
- Mutation scanning
o SSCP
o DGGE
o DHPLC
o HRM-CA
- Sequencing
o Sanger sequencing
o Next Generation Sequencing
CG C T G A A A
A
C
Asp/Ala90
G C T G A A AA C
Asp90
Controle Patiënt
What do we test? one gene or multiple genes
Molecular testing
Molecular cytogenetic
testing
Cytogenetics
1 disease
1 (single) mutation
1 disease
1 gene
different diseases
1 (single) gene
1 disease
few genes
different diseases
many genes
(un)known defect
karyotyping
1 disease
1 chromosomal
anomaly
different diseases
different anomalies
total
genometotal
exome
Molecular testing
Real-Life Issues for Genetic Testing
• Who will manage the newest technologies?
• Who will do the interpretation ?
• Who will warrant the quality of the results ?
• Who will want genetic testing ?
• Who will need the information ?
• Who will (be able to) counsel the patient ?
• Who will own the genetic information ?
• Who will pay for the testing ?
• Who will be left out ?
• Who will decide about… ?
Geneva, some time in the distant past…
Things Do Go Wrong!
HD testing
PCR machine 1 PCR machine 2
www.eurogentest.org
www.eurogentest.org
Quality Assurance
• Quality needs to be an all-pervasive ethos in
diagnostic laboratories
• “Quality is not an act, it is a habit” - Aristotle
• Not just internal quality control, but
– External Quality Assessment
–Method validation
–Monitoring of quality inndicators
–Responding to the needs of service users
–Continuous quality improvement
Ἀριστοτέλης384-322 BC
www.eurogentest.org
EUGT Quality Survey
• 910 HMGT Labs in Europe
• 291 responses (32%)
• 81% public sector
• 60% university hospital
• 23% Accredited
• 26% Certified
www.eurogentest.org
Size of Labs
Samples received/year
1-50 5% (15)
51-200 13% (37)
201-500 16% (47)
501-1000 14% (39)
1001-2000 23% (65)
2001-5000 14% (39)
5001-10000 10% (30)
>10000 5% (15)
www.eurogentest.org
Quality Measures: Management
Quality Measure Acc Cert Not
SOPs 100* 99 69
Document control 100* 93 62
Diagnostic log books 98 82 58
Quality manual 98 78 43
Maintenance/calibration log books 98 77 50
Training records 98 76 37
Following turnaround times 97 76 46
Recording of non-conformities 97 76 46
Recording complaints and compliments 97 71 40
Documented internal audits 97 66 18
Recording diagnostic errors 95 77 61
Recording complaint response times 78* 66 31
www.eurogentest.org
Quality Measures: Technical
Quality Measure Acc Cert Not
Participation in EQA 100 77 65*
Internal Quality Control 98* 91 55
Validation of instruments 97 84 54
Performing internal audits 97 68 21
Systematic corrective/preventive actions 97 73 46
Validation of methods 95* 86 66
www.eurogentest.org
Accreditation
• Accreditation is the key to ensuring quality
• 77% of molecular genetics labs are not yet
accredited
• Accreditation should be made mandatory for all
clinical laboratories
• Information on the accreditation and EQA status of
labs should be easily available
www.eurogentest.org
Cautionary Notes
• Novelty is a risk factor
• Effective quality control requires an in-depth
knowledge of the technology
• We need to balance rapid access to the latest
tests/technologies with proper concern for patient
safety
• Accreditation provides a safe environment for this
to happen
• When it is research, call it research!