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

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:

• 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

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

• 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

• Gel electrophoresis

Automated sequencing

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)

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

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