PharmacoGenomics – personalized medicine.

1. DNA chip Usage: PharmacoGenomics –personalized medicine. Alina Starovolsky

2. SNP: “snip” Single Nucleotide Polymorphisms One-letter variations in the DNA sequence. SNPs contribute to differences among individuals. The majority have no effect, others cause subtle differences in countless characteristics, including risk for certain diseases.

3. Human genome diversity • 28% of the human genome are coding genes. (all the rest is “junk DNA”). • 1.4% are the exons. • 30,000 genes. • 40% of then have alterative splicing and thus there are more genes.

4. A multi-country effort (Japan, the United Kingdom, Canada, China, Nigeria, and the United States) to identify and catalog genetic similarities and differences in human beings. • Analyzing DNA from populations with African, Asian, and European ancestry. Together, these DNA samples should enable HapMap researchers to identify most of the common haplotypes that exist in populations worldwide

5. Polymorphism vs. mutation • Polymorphism is defined as a variation in more than 1% of the population. • Mutations Rare differences which occur in less than 1% of the population (usually much less than 1%). • Typically, mutations have been discovered in coding sequences of genes causing rare inherited diseases. • In Barley (שעורה): 1 out of 131 nucleotides is different between individuals (was calculated on 75 different genes). • In 4 types of chickens in comparison to their ancestor it was found that every 200 nucleotides there is an SNP.

6. Polymorphism in humans • Two random humans are expected to differ at approximately 1 in 1000 nucleotide pairs, whereas two random chimpanzees differ at 1 in 500 nucleotide pairs. • This is interpreted to mean that the human species is relatively young, perhaps too young to evolve subspecies. • However, with a geonome of approximate 3 billion nucleotides, on average two humans differ at approximately 3 million nucleotides. • Most of these SNPs are neutral, but some are functional and influence the phenotypic differences between humans. It is estimated that about 10 million SNPs exist in human populations. • Amino acid-altering non-synonymous coding-region SNPs would be rare and harder to be found because of expected selection against them in human evolution.

7. Pharmacogenomics“Medicine tailored to the individual” • The Study of how genetic differences influence variability in patients’ responses to drugs. • Personalized drugs.

8. SNPs rool • Genetic polymorphisms in drug-metabolizing enzymes, transporters, receptors, and other drug targets have been linked to inter-individual differences in the efficacy and toxicity of many medications. • Pharmacogenomic studies explainthe inherited nature of these differences in drug disposition and effects.

9. The DNA Chip:

10. SNP Genotyping • Using DNA chips, it is possible to measure many thousands of SNPs simultaneously in a small sample from a patient. • Can compare “genotypes” for SNP markers linked to virtually any trait.

11. Examples traits – complex and non complex diseases. • There are a number of classic “genetic diseases” caused by mutations of a single gene. • There are also many diseases that are the result of the interactions of many genes: • Athsma, heart disease, cancer. • Each of these genes may be considered to be a risk factor for the disease. • Groups of SNP markers may be associated with a disease without determining mechanism. • Pharmacogenomics – personalized drugs.

12. The Future Soon it will be able to profile variations between individuals’ DNA to predict responses to a particular medicine. It will provide information on the likelihood of efficacy and safety of a drug for an individual patient It Will change the practice and economics of medicine (Faster clinical trials. Less drug side effects.)

13. The ‘roots’ of pharmacogenetics Clinical observations of inherited differences in drug effects first documented in the 1950s. e.g. In African American population it was found that in response to the anti-malarial drug primaquine, they developed hemolyitic anemia due to polymorphic alleles of Glucose-6-phosphate dehydrogenase. D-glucose 6-phosphate + NADP+ = D-glucono-1,5-lactone 6-phosphate + NADPH (energy). Without enough normal G6PD to help red blood cells get rid of harmful oxidative substances, they can be damaged or destroyed, leading to a condition known as hemolytic anemia.

14. Cytochrome P450 The molecular genetic basis for the inherited traits began to be revealed in the late 1980s, with the initial cloning and characterization of a polymorphic human gene encoding the drug-metabolizing enzyme debrisoquin hydroxylase (CYP2D6). • Homozygousity for alleles of the Cytochrome P450 gene CYP2D6 (in ~10% of the Caucasian population) lead to dangerous vacular hypotension when receiving the hypertension drug debrisoquine.

18. About schizophrenia • Does not mean split personality! • Afflicts approximately 1% of the world’s population. • US spends 40 billion $ per year. M=F for rate, onset: male(15-25), female(25-35). • 10% of the people with the disorder commit suicide. • Wide spectrum of illness Characterized by two categories of symptoms: - positive symptoms - negative symptoms

19. Negative symptoms: Flattened emotional response. Lack of initiative and persistence. Anhedonia (inability to experience pleasure). Social withdrawal. Positive symptoms: (more responsive to drug treatment) Thought disorders. Delusions. Hallucinations. disorganized speech. (e.g. frequent incoherence) grossly disorganized or catatonic behavior.

20. What causes schizophrenia? • The Genetic Risk – known to “run in the family” Each of the genetically identical girls was to become schizophrenic before the age of 28…

21. What causes schizophrenia? • Viral infection in the 2nd trimester of pregnancy • Brain abnormality (enlarged lateral ventricles, low metabolic rate of the prefrontal cortex, abnormal cell arrange in the hippocampus). Usually correlated to negative symptoms • Social influence– highest in poor socioeconomic groups, stressful live events.

22. What causes schizophrenia? • The Gray matter is the cortex of the brain which contains nerve cells body. parietallobelogic hearing

23. What causes schizophrenia? • Biochemistry - “dopamine hypothesis” - dopamine levels increase in the brain. (Dopamine is a neurotransmitter that transports signals between nerve endings in the brain). • (antipsychotic drugs = dopamine antagonists, L-dopa, cocaine, amphetamine) – only effective only for the positive symptoms.

24. Dopamine D2 receptor • Found on chromosome 11q22-23 • Binding site of many psychoactive drugs • Chlorpromazine

25. ANTIPSYCHOTIC DRUGS TYPICAL ATYPICAL D2 Receptor Other dopamine receptors and 5HT2 receptor Treat mainly positive symptom Efficacy – 60% Treat negative symptoms too, Efficacy – 85%(less relapses)

26. THE PHARMACOGENOMIC HYPOTHESIS: DRUG EFFICACY RELEATE TO GENETIC REASONS • Drug mechanism- identify how drug ‘works’block dopamine receptors • Target – identify those gene products implicated in the mechanism of the drugDopamine receptor • Candidate gene – identify the gene that have been found to be associated with the diseaseDRD2 receptor(dopamine receptor D2). • Gene variants141 C Del/Ins, TaqI A

27. 141C Del/Ins polymorphism • deletion of cytosine 141 in the promoter region upstream from the transcription start site • Associated with schizophrenia in Japanese, Swedish and Portuguese population • In vitro – del allele is directly related to DRD2 expression • Individuals with no del allele had lower striatal density of dopamine receptor

28. TaqI polymorphism • localized 9.5 kb downstream from the DRD2 gene • restriction fragment length polymorphism creating A1 and A2 allels • A1 allele -lower density of DRD2 in the caudate nuclei and striaum • A2 allele - decrease in the binding potential of the D2 receptor • Controversy about the linkage to schizophrenia

29. Wu S,. Xing Q,. Gao R,. Li X, Gu N,. Feng G,.& He L. (2005).Response to chlorpromazine treatment may be associated with polymorphisms of the DRD2 gene in Chinese schizophrenic patients. Neurosci Lett. 376(1):1-4.

30. Purpose of the study: examine whether the DRD2 gene contribute to the therapeutic effect of chlorpromazine in schizophrenia by investigating the potential genetic role of the 141C Ins/Del and TaqIA polymorphism in the DRD2 gene Patients : - Chinese population - mean age – 27.3 - 2 or more characteristic symptoms according to the DSM –3R (Diagnostic and Statistical manual of Mental Disorder ). - first time to be treated with chlorpromazine - 8 weeks of treatment Assessment: clinical symptoms were evaluated by BPRS (brief psychiatric rating scale) by two psychiatrics (given no information about the patient’s genotype).

31. 141C Ins/Del Genotype frequency Ins/Ins Ins/Del Del/Del Responders 61 53 (86.9) 6 (9.8) 2 (3.3) Non responders 74 53 (71.6) 21 (28.4) 0(0) Results 1 : the frequency of Dell allele is higher in non responders than in responders P=0.01 

32. TaqI A Genotype frequency A2/A2 A1/A2 A1/A1 Responders 61 18 (29.5) 27 (44.3) 16 (26.2) Non responders 74 22 (29.8) 32 (43.2) 20(27.) Results 2 : no association between A1 allele and the drug response NO SIGNIFICANT RESULTS!

33. conclusion: Del allele of the 141C Ins/Del polymorphism might predict therapeutic response to chlorpromazine in schizophrenia probably due to alteration of the D2 receptor density but that the A1 allele of the TaqI A polymorphism have no such effect Higher density of the D2 receptor low therapeutic response to chlorpromazine Del allele

34. Other studies: • (Suzuki A, Kondo T, Mihara K, Yasui-Furukori N, Ishida M, Furukori H, Kaneko S, Inoue Y, Otani K.(2001).The -141C Ins/Del polymorphism in the dopamine D2 receptor gene promoter region is associated with anxiolytic and antidepressive effects during treatment with dopamine antagonists in schizophrenic patients. Pharmacogenetics. 11(6):545-50) • Arranz, M.J., Li, T., Liu, X., Murray, R. Collier, D.A. Kerwin, R.W.(1998). Lack of association between a polymorphism in the promoter region of the dopamine-2 receptor gene and clozapine response. Pharmacogenetics. 8(6):481-4.

35. Diagnosis-systematized investigators blinded to the patient genotype Prior medical treatment Don’t separatepositive from negative symptoms AdvantagesDisadvantages

36. Non small cell lung cancer - NSCLC Lung carcinoma is the Leading cause of cancer deths in the USA and worldwide for both men and women.

37. Multi-center trial of EGFR inhibitor to treat advanced lung cancer (NSCLC) • Rationale: • EGFR (epidermal growth factor receptor) over-expressed in lung cancers (and other). • EGFR inhibitors block signal transduction and cell proliferation • Gefitinib : A drug that targets the ATP cleft within the EGFR. • Design: • 210 patients from Europe, Australia, South Africa, Japan • Objective tumor response in 19% of patients - mean survival 8 months • Response better among Japanese vs non-Japanese pts • (27.5% vs. 10.4% response; P = 0.002) • Response also better among female pts, adenocarcinoma pts, pts with prior hormonal/immuno treatment, pts with less morbidity • What is molecular basis of the differential response?

38. Lung cancer - EGFR inhibitors – EGFR somatic mutation Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC, Settleman J, Haber DA. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129-2139, 2004 Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, Naoki K, Sasaki H, Fujii Y, Eck MJ, Sellers WR, Johnson BE, Meyerson M. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304:1497-500, 2004

39. Activating mutations in EGFR underlying responsiveness of lung cancer to gefitinib • EGFR sequenced in pre-treatment tumor tissue from: • 9 responders (tumors that were available), 7 non-responders, 25 untreated patients Example of improvement after 6weeks treatment

40. Most of them were women, had never smoked, and had bronchoalveolar tumors

41. (9 tumors available from 25 responders) (25 untreated tumors evaluated) Overlap AA 747-750 8 out of the 9 patients that were checked for mutations in the tumors and responded to gefitinib had deletions in the tumor cells. And in 7 patients with no response no mutations were observed. (p<0.001)

42. Overlap

43. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 304:1497, 2004 119 primary lung tumors (58 Japan, 61 US), none treated before, EGFR somatic mutations in 15/58 (26%) of Japanese pts vs 1/61 (2%) of US pts. Among adenocarcinomas only, mutations in 14/41 (32%) of Japanese pts vs. 1/29 (3%) of US pts

44. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 304:1497, 2004 Pre-treatment tumors from treated patients: 6 responders, 4 non-responders

45. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 304:1497, 2004 exon 21 exon 18 exon 19 Sequence and substitutions alterations at kinase active site.

46. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 304:1497, 2004

47. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 304:1497, 2004 Mutations may stabilize interaction of EGFR with both ATP (enhancing phosphorylation) and with competitive inhibitor geftinib -> both enhanced inhibition by drug.

48. In general : Collect Drug Response Data • These drug response phenotypes are associated with a set of specific gene alleles. • Identify populations of people who show specific responses to a drug. • In early clinical trials, it is possible to identify people who react well and react poorly.

49. Make Genetic Profiles • Scan these populations with a large number of SNP markers. • Find markers linked to drug response phenotypes. • It is interesting, but not necessary, to identify the exact genes involved.

50. Profiles