Genetic Testing Prasit Phowthongkum MD, FRCP (T) Department of Medicine Faculty of Medicine...
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Transcript of Genetic Testing Prasit Phowthongkum MD, FRCP (T) Department of Medicine Faculty of Medicine...
Genetic TestingPrasit Phowthongkum
MD, FRCP (T)Department of Medicine
Faculty of MedicineChulalongkorn University
Topics
• 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
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:
• 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:– 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– 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– 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
• Confirmatory diagnosis
• Presymptomatic diagnosis
• Predictive diagnosis
• Susceptibility testing
• Prenatal diagnosis
• Preimplantation diagnosis
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
• 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
• Blood group
• Protein electrophoresis
• RFLPs
• VNTR-microsatellite
• VNTR-minisatellite
Specimen collection informationTesting 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 paraffin-embedded 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)
Cost-effectiveness
• 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