Use and Value of Genetic Tests for Patient Care

1. Use and Value of Genetic Tests for Patient Care • Review of Technologies • Real life examples Sherri J Bale, PhD, FACMG

2. Definition of Genetic Testing: • The analysis of human DNA in any of its forms or related products (chromosomes, RNA, proteins) • To detect disease-related genotypes, mutations, phenotypes, or karyotypes for clinical purposes Uses of Genetic Testing:

3. Genetic Tests for Constitutional Mutations • Cytogenetic Tests • Molecular Tests

4. Cytogenetic Test • Standard karyotype, used to look for gross chromosomal anomalies in children with development delays, congenital anomalies, mental retardation • FISH, used to look at 1 or 2 specific chromosomal regions suspected by the physician • BAC arrays, used to look at many (100s) chromosomal regions at once, using FISH technology • CGH array, used to look at MANY (50K-200K) regions at once, and identify specifically which genes are involved in the chromosomal anomaly.

5. Child with Multiple Congenital Anomalies and/or Autism CGH array - “molecular Karyotype” Standard karyotype

6. Patient with Tetralogy of Fallot, suspected 22q11 deletion • FISH test, 2 probes, used in baby, found deletion, and confirmed dx. Provided prognostic info to family. • Parents tested by FISH, found to be negative. Provided information re: risk in future children

7. Child with multiple anomalies and autism; no specific syndrome suspected • Karyotype normal • CGH array followed Karyotype. • Identified deletion involving end of one arm of chromosome 3 • Parents tested by FISH and dad found to be balanced carrier of the deletion • Prenatal diagnosis by quantitative PCR is now possible for the family.

8. Molecular Test – PCR for well-characterized mutations • KRAS gene test on tumor tissue from patients with colorectal cancer • Obtain tumor from patient • Extract DNA; PCR, then treat with enzyme that allows visualization of the mutation

9. Molecular Test – PCR, followed by sequencing, for identification of mutation • Used to identify mutation in a patient with inherited disease • Number of times PCR is done and how much sequencing is required depends on SIZE of gene, MANY UNITS. • Once mutation is identified, testing of parents, sibs, other relatives for ONLY that mutation, is needed. ONE or ONLY a FEW UNITS.

10. Molecular diagnosis of Gorlin Syndrome • 13 yo child presented to dentist with a jaw cyst – surgery performed but tooth was lost. Referred to geneticist. • Geneticist suspected Gorlin Syndrome • Molecular diagnosis involved PCR and sequencing, 26 “units” (large gene). Mutation identified. • Prognosis now known: This individual would develop many skin cancers, more jaw cysts. • Regular surveillance by dermatologist and dentist allowed early identification, less expensive treatment, and good clinical outcome: • Teeth were saved; Minimal damage to nose, ears, eyes

11. Use and Value of Genetic Tests • Diagnosis • Enables physicians to properly care for patient • Prognosis • Appropriate surveillance leading to early care and intervention • Risk Information • Is it inherited? What is the recurrence risk in future pregnancies? • Prenatal/Pre-symptomatic diagnosis • Allows informed decision making, preventive care

12. Human GeneticsTrends in Testing V.M. Pratt, Ph.D., FACMG

13. Diagnostic Laboratory TestingDriver of Healthcare Decisions • Diagnostic testing is foundation of healthcare • 70% of healthcare decisions based on diagnostic data • Diagnostic data yields information serving public and individual • Information can help identify trends for public health • Data enables physicians to care for individual patients • Data facilitates new test development • Continuum of diagnostic lab testing • From diagnosis to predictive and personalized medicine • Diagnostic data increasingly is providing actionable insights physicians can use to improve patients’ healthcare outcomes

14. Genes in the News

15. The Basics of Genetic Testing

16. DNA->RNA->Protein Cell membrane DNA Nucleus Chain of amino acids DNA bases mRNA Gene Protein Ribosome

17. Gene Tests – Three Common Methods Mutation Absent Mutation Present Chromosome Cytogenetics DNA Molecular Genetics Protein Biochemical Genetics

18. Types of Mutations • Single nucleotide - traditional • Missense • Nonsense (creation of stop codon) • Splicing • Regulatory sequences (promoter, 3’ end) • Deletions/Insertions – copy number variants (CNVs) • In frame • frameshift • Expansions (triplet repeat disorders) • Epigenetic (methylation) • Translocations and inversions

19. Alleles Quiet! I’ll speak for both of us! I’ll have to be in charge now! Dominant Allele Normal Allele Recessive Allele Damaged Allele

20. Human Chromosomes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

21. Gene Mutations Mismatch Deletion Insertion Deletion

22. General Principles • Hereditary disorders can affect multiple organs • Penetrance can be influenced by modifiers: genes + environment • Complexity of mutational spectrum varies

23. Different Genes – Different Functions Bone Cell Pancreas Cell Brain Cell

24. Disease Inheritance Is Complex Gene Changes in Cystic Fibrosis Mucus Production Gene Normal Mutation 1 Mutation 2 Mutation 3 No Symptoms Severe Symptoms Mild Symptoms No Symptoms

25. Why is Genetic Testing ordered? For couples who are having difficulty conceiving For couples who have experienced two or more miscarriages To make a diagnosis in an affected individual To see if pregnancy is at an increased risk for a genetic disorder To test people with a family history of a specific inherited disease; patients may want to know if they might develop the disease or pass it on to their children

26. Continuum of Diagnostic Lab Testing Diagnostic ►Confirm diagnosis -Cystic Fibrosis Predictive ►Determine higher chance for disease before symptoms appear -Huntington Disease Personalized ►Tailor drug treatment to genotype -HIV Therapy PhysicianPatient Pharma

27. Common genetic disorders • Inherited (predictive or diagnostic) • Cystic fibrosis • Thrombophilia • Hereditary hemochromatosis • Fragile X syndrome • Acquired (predictive or diagnostic) • Chronic myelogenous leukemia (CML) • Pharmacogenetics (personalized) • Cytochrome P450s • HLA

28. New Assay/Biomarker ProgressionEvidence-based medicine Retrospective clinical trials Prospective clinical trials Clinical Research Test Translation Clinical Validity Clinical Utility Lab test developed -Analyticalvalidation Biomarker associated with disease Test canpredict clinical outcomes Benefits patients

29. Emerging Technologies and Testing

30. The genome is complex • High throughput DNA sequencing • microRNAs • Copy Number Variants (CNVs) • Epigenetics • methylation • Proteomics • Up and down regulation • Disease-specific patterns

31. New high throughput DNA sequencing methods • 454 (Roche) • 20 megabases per 4.5-hour run • capable of detecting mutations in an amplicon pool at low sensitivity • Reads: ~100 base pairs • Can’t read highly repetitive or long polymers • Solexa (Illumina) • Can sequence through homopolymers and repetitive sequences • 10's of millions of short (24-36 bp) reads • Single-end vs. paired end reads

32. MicroRNAs (miRNA) • Single-stranded RNA molecules • 21-23 nt • Transcribed from non-coding DNA • Regulate gene expression • Cancer • May enable classification of cancers (CUP) • Determine therapy

33. Copy Number Variants (CNVs)(Variome) • Large deletions or duplications of DNA • Usually cannot be detected by DNA sequencing • Newer technologies • aCGH • Impacts • Autism • Alzheimer disease • Parkinson disease • susceptibility to HIV-1 • some forms of color blindness

34. Array CGH Cross-platform identification and validation of CNVs Jennifer L. Freeman et al. Genome Res. 2006; 16: 949-961

35. Epigenetics • Changes in chromatin structure (how DNA is packaged) or alters gene activity without changing the DNA • DNA methylation • Modification of histones • Position effects • Cancer and imprinting disorders

36. Genetic Tests Find Mutations, NOT Disease Chances of Developing Breast Cancer by Age 65 100 10 90 9 80 8 70 7 60 6 50 5 40 4 30 3 20 2 10 1 0 0 Altered BRCA1 Normal BRCA1

37. Benefits of Gene Testing • Relief • Fewer Checkups • Informed Decisions • Intervention

38. Pharmacogenetics V.M. Pratt, Ph.D., FACMG

39. Personalized Medicine/Pharmacogenetics • Getting the right dose to the right patient at the right time

40. Chronic Myelogenous Leukemia (CML) - example • Philadelphia chromosome t(9;22) • Detectable by standard cytogenetics (karyotype analysis) • FISH • Fusion of BCR/ABL genes • FISH • Quantitative PCR • Treatable with Gleevac • Some people become resistant to Gleevac • Mutations in tyrosine kinase domains (DNA sequencing)

41. Example: Tamoxifen • Antiestrogen • blocks the activity of estrogen which can stop the growth of some breast tumors. • Used to treat estrogen receptor (ER)+ breast cancer • Metabolized by cytochrome P450 2D6 • Endoxifen (active form of Tamoxifen) • MEDCAC reviewing if should pay for Medicare population • FDA reviewing whether to revise the drug label to recommend CYP450 2D6 (CYP2D6) testing

42. Tamoxifen use • treat breast cancer that has metastisized. • treat early breast cancer in women who have already been treated with surgery, radiation, and/or chemotherapy. • reduce the risk of developing a more serious type of breast cancer in women who have had ductal carcinoma in situ (DCIS) and who have been treated with surgery and radiation. • reduce the risk of breast cancer in women who are at high risk for the disease due to their age, personal medical history, and family medical history.

43. Tamoxifen Metabolism

44. Various Assays/Platforms for CYP2D6

45. Example: Clopidogrel (Plavix) • inhibit blood clots in coronary artery disease, peripheral artery disease, and cerebrovascular disease. • Metabolized by cytochrome P450 2C19 to active form • 2-{1-[1-(2-chlorophenyl)-2-methoxy-2-oxoethyl]-4-sulfanyl-3-piperidinylidene}acetic acid • FDA-recently announced that clopidogrel cannot be taken with Prilosec (omeprazole) and Nexium (esomeprazole) • Inhibitors of 2C19

46. Clopidogrel use • Prevention of vascular ischemic events in patients with symptomatic artherosclerosis • Acute coronary syndrome without ST-segment elevation (NSTEMI) • ST elevation MI (STEMI) • It is also used, along with aspirin, for the prevention of thrombosis after placement of intracoronary stent

47. Various Assays/Platforms for CYP2C19

48. Laboratory Procedures and CodingPresented byKaye Jones, MLS(ASCP), CPCJan. 13, 2010

49. Objective • To improve your understanding of molecular diagnostic CPT coding

50. Molecular Codes • CPT codes 83890-83914 represent steps performed during molecular diagnostic procedures. • CPT codes are assigned based on the different steps and the number of times each type of step is performed. • Example: 83898 Amplification x3 83896 Nucleic acid probe, each x25