Genetic Testing

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Genetic Testing

• Prasit Phowthongkum
• MD, FRCP (T)
• Department of Medicine
• Faculty of Medicine
• Chulalongkorn University

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Topics

• Definition
• Classification
• Availability
• Indication
• Technical information
• Turn around time
• Interpretation
• Cost-effectiveness
• Adverse effects

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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

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Type of Genetic Tests

• Cytogenetic tests
• Biochemical genetic tests
• Molecular genetic tests

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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

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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

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Availability of Genetic Testing in Thailand

• Hematology
• Thalassemia/hemoglobinopathies
• G 6 PD deficiency
• Hemophilia
• Factor V Leiden
• Protein S Deficiency
• Methemoglobinemia

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• 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
• Wilsons disease
• Huntingtons disease
• Parkinson disease
• APOE

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• 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

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• Connective tissue disorders
• Marfan syndrome
• Dysmorpholgy
• Achondroplasia
• Aperts syndrome
• Etc.
• Metabolic diseases
• Congenital adrenal hyperplasia
• Congenital adrenal hypoplasia
• X-linked adrenoleucodystrophy
• Metachromatic Leucodystrophy
• Cystinosis

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• EENT
• Lebers 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

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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

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Indications

• Confirmatory diagnosis
• Presymptomatic diagnosis
• Predictive diagnosis
• Susceptibility testing
• Prenatal diagnosis
• Preimplantation diagnosis

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G banding
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The high resolution G banding
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The FISH image
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Karyotypes
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M-banding
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Bioinformatics

• A patient who diagnosed Acute intermittent
  porphyria biochemically
• Need perform molecular diagnosis
• Knowledges porphobilinogen deaminase
• Search gene information www.ensembl.org

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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!

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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

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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

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• 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

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Polymorphic markers

• Blood group
• Protein electrophoresis
• RFLPs
• VNTR-microsatellite
• VNTR-minisatellite

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• Gel electrophoresis

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Automated sequencing
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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 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
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Turn around time

• Low through put
• Medium through put
• High through put

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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)

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• Something detected
• Pathogenic mutations
• Polymorphisms
• Mutations of unknown significance

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Cost-effectiveness

• What is effective?
• Who is the payer?
• Prevalence esp. population screening
• Severity of disorders
• Availability of therapeutic or preventive
  measures

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Adverse effects

• Cost and coverage
• Emotional and psychological effects
• Genetic discrimination jobs, insurance, social
• Confidential
• Misinterpretations wrong negative confident,
  overemphasized

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