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