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Genetic Testing
Prasit Phowthongkum
MD, FRCP (T)
Department of Medicine
Faculty of Medicine
Chulalongkorn University
Topics
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Definition
Classification
Availability
Indication
Technical information
Turn around time
Interpretation
Cost-effectiveness
Adverse effects
Genetic testing
• Definition: the analysis of human DNA,
RNA, chromosomes, proteins, and certain
metabolites in order to detect heritable
disease-related genotype, mutations,
phenotypes, or karyotypes for clinical
puroses
•
Holtzman NA, Watson MS, eds. Promoting safe and effective genetic testing
in the United States: final report of the Task Force on Genetic Testing.
Baltimore: Johns Hopkins University Press, 1999
Type of Genetic Tests
• Cytogenetic tests
• Biochemical genetic tests
• Molecular genetic tests
Availability of Genetic Testing in
Thailand
• Cytogenetic testing
– Routine karyotypes: all university hospitals and few
private company in Bangkok
– High resolution karyotypes: Rajanukul hospital
– FISH:
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Duchenne Muscular Dystrophy
Prader Willi Syndrome
Angel Man Sydrome
CATCH (22q11 syndrome)
Subtelomeric FISH
– CGH
Molecular testing
• For patients with
inherited or at risk of
inherited disorders
and inherited
predisposition
conditions
• For non inherited
conditions
– Clinical purposes
• Diagnostic infectious
diseases, oncology
• Pharmacogenetics,
Toxicogenomics
– Non clinical purposes
• Forensic: paternity
testing, personal
identification
• Nutrigenomics
Availability of Genetic Testing in
Thailand
• Hematology:
– Thalassemia/hemoglobinopathies
– G 6 PD deficiency
– Hemophilia
– Factor V Leiden
– Protein S Deficiency
– Methemoglobinemia
• Neurology:
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Duchenne Muscular Dystrophy/ Becker Muscular Dystrophy
Spinal Muscular Atrophy
Spinocerebellar Ataxia type 1, 2, 3, 6, 7
Fragile X syndrome
Prader Willi Syndrome
Angel Man Syndrome
CADASIL
MERFF, MELAS
Wilson’s disease
Huntington’s disease
Parkinson disease
APOE
• oncology:
– APC for familial adenomatous polyposis (FAP)
– MSH1, MSH2, PMS2 for Hereditary Nonpolyposis
Coli Syndrome (HNPCC)
– BRCA1, BRCA2 for breast ovarian cancer syndrome
– MENIN, RET for MEN1, MEN2
– PTEN for cowden syndrome, Bannayan Ruvalcalba
Reiley Syndrome
– TP53 for Li Fraumeni Syndrome
• Connective tissue disorders:
– Marfan syndrome
• Dysmorpholgy:
– Achondroplasia
– Apert’s syndrome
– Etc.
• Metabolic diseases
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Congenital adrenal hyperplasia
Congenital adrenal hypoplasia
X-linked adrenoleucodystrophy
Metachromatic Leucodystrophy
Cystinosis
• EENT
– Leber’s Hereditary Optic Neuropathy
– Connexin (non syndromic deafness)
• Nephrology:
– Adult Polycystic Kidney Diseases
– Familial hypokalemic/hyperkalemic periodic paralysis
• Endocrinology
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MODY
VHL
Prop1, Pit1
LDL receptors
• Cardiology
– Brugada syndrome
Availability of genetic testing
• www.genetests.com
• A public funded medical genetics
information resource developed for
physicians, other health care providers,
and researchers, available at no cost to all
interested persons
• 621 laboratories testing for 1,420 diseases
• 1,130 clinical
• 290 research only
Indications
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Confirmatory diagnosis
Presymptomatic diagnosis
Predictive diagnosis
Susceptibility testing
Prenatal diagnosis
Preimplantation diagnosis
G banding
The high resolution G banding
The FISH image
Karyotypes
M-banding
Bioinformatics
• A patient who diagnosed Acute intermittent
porphyria biochemically
• Need: perform molecular diagnosis
• Knowledges: porphobilinogen deaminase
• Search gene information:
www.ensembl.org
PCR
• Amplify million folds of tiny amounts of DNA
• TemplatesDNA, primersDNA, dNTP, heat stable
DNA polymerase
• Initial heat for denature
• Template annealing at lower temperature
• Heat again for extending strand
• And repeats for douling products
• Too sensitive!
Modified PCR and non-PCR
amplifications techniques
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inverse PCR
quantitative PCR
real time PCR
nested PCR
RT-PCR
multiplex PCR
ARMS PCR
PCR mutagenesis
long template PCR
FRET assay, RCA, ERCA
Linkage analysis
• If two genes occur close together, they
said to be linked
• Crossing over during meiosis –
recombination of two loci
• Frequency of recombination- a relative
measure of distance
• Statistical measure of linkage- log of the
odds (LOD score) +3 = 95% that two
genes are linked
• Neurofibromatosis type I and II –
chromosome 17, 22
• CMT –chromosome 1q, 17p, and others
• Early onset and late onset-HD –
chromosome 4
• Duchene and becker muscular dystrophy
–chromosome X
• Single point linkage analysis
• Multi point linkage analysis
Polymorphic markers
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Blood group
Protein electrophoresis
RFLPs
VNTR-microsatellite
VNTR-minisatellite
• Gel electrophoresis
Automated sequencing
Specimen collection information
Testing
Specimen type
Collection tube
Shiping temperature
Delivery requirements
Congenital chromosome
studies
Whole blood
Sodium heparin
ambient
48 h
Hematologic chromosome
studies
Whole blood
Sodium heparin
ambient
48 h
Prenatal molecular or
cytogenetic studies
Amniotic fluid, chorionic villi
For villi: sterile tissue media
culture
refrigerated
overnight
Chromosome studies on
preduct of conception
Chorionic villi or fetal tissue
biopsy
Hanks or other sterile tissue
culture media
refrigerated
overnight
FISH or DNA testing for
lymphoma or solid tumors
Paraffin-embedded tissue
N/A
N/A
N/A
DNA testing
Whole blood
EDTA or ACD-B
ambient
48 h
RNA testing for solid tumors
Fresh frozen or paraffinembedded tissue
N/A
N/A
N/A
RNA testing for hematologic
disorders
Whole bolood or bone
marrow
EDTA
refrigerated
overnight
Biochemical genetic studies
Varies i.e. urine, plasma,
serum, skin biopsy
varies
varies
varies
Turn around time
• Low through put
• Medium through put
• High through put
Interpretation
• Diagnostic, predictive, susceptibility test
• Negative mutation detection:
– Wrong diagnosis (genetic heterogeneity,
phenocopies)
– Specimen labeling errors or laboratory errors
– Inadequate specimen quality or quantity, tissue
contamination
– False negative test (large gene rearrangements
,description of gene(s) or specific (exons) tested)
– Wrong person was test (false paternity)
• Something detected
– Pathogenic mutations
– Polymorphisms
– Mutations of unknown significance
Cost-effectiveness
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What is effective?
Who is the payer?
Prevalence esp. population screening
Severity of disorders
Availability of therapeutic or preventive
measures
Adverse effects
• Cost and coverage
• Emotional and psychological effects
• Genetic discrimination: jobs, insurance,
social
• Confidential
• Misinterpretations: wrong negative
confident, overemphasized