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IMPLICATIONS OF GENETIC TESTING

IMPLICATIONS OF GENETIC TESTING. Huong Le Senior Hospital Scientist Department of Molecular & Clinical Genetics Royal Prince Alfred Hospital Sydney , Australia. OUTLINE. Introduction Types of Genetic test Genetic testing of large genes Familial Hypercholesterolemia (FH)

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IMPLICATIONS OF GENETIC TESTING

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  1. IMPLICATIONS OF GENETIC TESTING Huong Le Senior Hospital Scientist Department of Molecular & Clinical Genetics Royal Prince Alfred Hospital Sydney , Australia

  2. OUTLINE • Introduction • Types of Genetic test • Genetic testing of large genes • Familial Hypercholesterolemia (FH) • Hereditary Breast Cancer • Conclusions

  3. INTRODUCTION • Gene Mutations • Disrupt protein function associated with disease • DNA technology • Impact of Human Genome Project

  4. GENE http://www.ornl.gov/sci/techresources/Human_Genome/project/info.shtml

  5. DNA TECHNOLOGY COMBIMATRIX

  6. TYPES OF GENETIC TEST • Diagnostic • Confirm clinical diagnosis • Prenatal • Fetal prenatal diagnosis • Screening • Reproductive screening • Newborn screening • Community screening • Predictive • Huntington disease and cancer

  7. TYPES OF GENETIC TEST • Current diagnosis focuses on single gene disorders eg. CF, thalassaemias • The challenge ahead is to expand DNA diagnosis in • Disease involve with large gene / polygene • Multifactorial disorders

  8. Genetic testing of FAMILIAL HYPERCHOLESTEROLAEMIA • FH is an autosomal dominant disorder associated with premature atherosclerosis increased risk for coronary artery disease (CAD) • FH diagnosis criteria for index case • Cholesterol >7.5mmol/ or LDL > 4.9mmol/l in adult • Cholesterol > 6.7mmol/l or LDL > 4.0mmol/l if < 16 yrs • Plus family history of high cholesterol or MI (<55yrsM) or plus tendon xanthoma • Definite FH & Probable FH

  9. LDL Removed from Blood LDL APOB LDL RECEPTOR Cholesterol carried in “LDL” particle in Blood Receptor Recycles PCSK9 LDL broken down. Chol Bile excreted Genetic testing in FH patients-What genes ? Adapted from S. Humphries UK Most FH due to LDLR mutations, 5% by APOB, 2% PCSK9 BLOOD LIVER CELL Diet and New-made

  10. FH GENES • LDLR (Low Density Lipoprotein Receptor) gene • Location: 19p13.2 ; Structure: 44,36 kb and 18 exons • Heterogeneity: 900 LDLR mutations report & 5% are large deletion mutations • APOB (ApoB-100) gene • Location: 2p24-p23 ; Structure: 42,65 kb and 29 exons • Arg3500Glu & Arg3500Trp have shown to cause ligand-defective apoB-100 and associated with FH (5%) • Others

  11. S1 S2 S4 S5 S6 S7 S8… M1 M2 M3 M4 Bl PCR +ve PCR +ve DIAGNOSTIC TECHNIQUES • ARMS (Amplification Refractory Mutation System) Commercial kit : allow for detection of LDLR, APOB and PCSK9 mutations

  12. DIAGNOSTIC TECHNIQUES • DHPLC / WAVE system • A PCR product is injected onto a column of WAVE system. DNA variant will be detected base on heteroduplex formation • Use for screen for any sequence change in all exons including promoter region • Positive profiles will be confirmed by sequencing

  13. DIAGNOSTIC TECHNIQUES

  14. DIAGNOSTIC TECHNIQUES • Sequencing • Direct sequencing technology • Fluorescent dye terminator • AB DNA Analyzer 3730 with 96 capillaries • Sequencing analysis using SeqScape software • High throughput • Cheaper cost • Effective mutation detection

  15. SEQUENCING analysis

  16. DIAGNOSTIC TECHNIQUES • MLPA: Multiplex Ligation-dependent Probe Amplification • Detection of aberrant copy number of 40 genomic DNA sequences in one PCR based reaction. (Deletions, Duplications and Copy Number Polymorphisms) • Applications: gene dosage and genotyping

  17. MLPA (Multiplex ligation dependent probe amplification) MLPA results Probe design Schouten et al Nucleic Acids Res 30 e57 (2002)

  18. MUTATION DETECTION RATE • The sensitivity of this molecular test depends strongly on the certainty of the clinical diagnosis: • Definite FH cases have mutations detected in the LDLR gene about 70% of the time. • Probable FH cases have mutations detected in the LDLR gene about 40% of the time.

  19. FH GENETIC TEST AT RPAH, SYDNEY

  20. FH GENETIC TEST AT RPAH FLOW CHART

  21. GENETIC TESTING OF BREAST CANCER • Multifactorial disorder • BRCA1 and BRCA2 are two genes associated with hereditary breast cancer and ovarian cancer • DNA testing for BRCA cancer predisposition mutations perform for • Proband • At risk relatives • Risk of developing cancer which is associated with BRCA mutations appears to be variable

  22. GENETIC TESTING OF BREAST CANCER • BRCA1 (hereditary breast /ovarian cancer) • Location: 17q12-21 • Structure: 81,16 kb and 23 exons & encoded a protein of 1863 amino acids • ~ 500 mutations have been reported • BRCA2 (hereditary breast /ovarian cancer) • Location: 13q12-3 • Structure: 84,19 kb and 28 exons & encoded a protein of 3418 amino acids • ~ 300 mutations have been reported

  23. BRCA-Associated Cancers: Lifetime Risk BRCA1 BRCA2

  24. GENETIC TESTING OF BREAST CANCER • Features that indicate increased likelihood of having BRCA mutations • Multiple cases of early onset breast cancer within one family • Ovarian cancer (with family history of breast or ovarian cancer) • Breast and ovarian cancer in the same woman • Bilateral breast cancer • Ashkenazi Jewish heritage • Male breast cancer

  25. GENETIC TESTING OF BREAST CANCER • In familial breast cancer, germ line mutations in the BRCA1 or BRCA2 genes are followed by somatic inactivation of the wild-type allele • BRCA1 and BRCA2 are thus thought to be tumour suppressors since loss of heterozygosity at either locus can lead to retention of the mutant allele in tumours from heterozygous carriers.

  26. BRCA mutation detection strategy at Prince of Wales Hospital, Sydney • Ashkenasi Jewish heritage ARMS: c.185delAG,c. 5382insC, c. 617delT • Sequencing of BRCA1 followed by BRCA2 using M13 labelled primers in a 96 well 20μl format with stepped annealing temperatures. • SeqScape analysis software • MLPA analysis

  27. GENETIC TESTING OF BREAST CANCER Adapt from Jennifer E. Axilbund, Cancer Risk Assessment Program The Johns Hopkins Hospital

  28. RESULT INTERPRETATION • Proband: • Mutation present: conferring high risk of cancer for the family members • Mutation absent: • Limited / low sensitive techniques • Mutation in other regions / genes • Other factors • Inconclusive result: • Important in clinical problem when • A variant with unknown clinical significant

  29. VARIANT ANALYSIS • Classification variants as deleterious or neutral based on: • Frequency of variant in cases and controls • Segregation of sequence variant with disease in family • Co-occurrence of variant with a known deleterious mutation in one or more tested individuals • Nature position of amino acid substitution plus degree of conservation among different species, protein modeling • Result of functional assay

  30. RESULT INTERPRETATION • At risk relative • Mutation present: increase risk of cancer • Mutation absent: similar risk as in general population having cancer predisposing BRCA1/2 mutations

  31. MUTATION DETECTION RATE • Only 1/3 cases have mutations in BRCA1/2 genes • Low detection rate of BRCA1/2 mutations • Limited / low sensitive techniques • Mutation in other regions / other genes • Other factors

  32. GENETIC COUNSELING • Genetic counseling is required to address issues that arising from the results of advance in genetic testing in order to assure appropriate genetic testing can be translated into clinical care • Information from genetic test can affect the lifestyles of individual and their family members • The challenge focuses on issues including privacy, informed consent, risk assessment, decision making, disease intervention, insurance and employment.

  33. FUTURE DIRECTION OF GENETIC TESTING • Microarray analysis • Facilitate analysis of all known mutations in a gene at same time or all mutations in many genes can be assayed in a single chip. • Applications have expanded in disease diagnosis, drug discovery and toxicological research (R. Uma & T. Rajkumar 2007) • Genome-wide approaches capable of analysing thousands of genes and gene products and provide a better biological understanding of breast cancer and other cancers

  34. FUTURE DIRECTION OF GENETIC TESTING • Identify breast cancer profile (e.g. known subtype) has potential to improve prognosis and predict the best individual therapeutic scheme • Whole genome sequencing • Aim at US $ 1000 personal genome project within 10 years • 454 from Roche company can do 20 MB in 4 hours

  35. MOLECULAR BIOMARKER USEFUL FOR TREATMENT RESPONSE(James et al Oncologist 2007;12;142-150)

  36. CONCLUSIONS • Identify family specific mutation  recommendation for predictive testing can be used to screen for at risk relatives • provide opportunities for earlier disease management thus reduce morbidity rate and enhance effectiveness of prevention • However the complexities of all possible genetic test results are still the challenge ahead in term of result interpretation / translation into clinical care • Issues arise from genetic testing also need to be addressed

  37. The future model of genetic services(Trent et al Encyclopedia of Diagnostic Genomics and Proteomics 2004; p676-681).

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