Albert Pinhasov Department of Molecular Biology Ariel University Center

1. Genome-based Diagnostics:Hype, Hope or Reality? Albert Pinhasov Department of Molecular Biology Ariel University Center Actual Issues of laboratory medicine of the XXI century Kazakhstan May 22, 2012

2. We now know how God wrote the book of life BillClinton But….. Do we know how to read the book !?

3. Genetic code • Human Genome contains about 3-billion nucleotides • Approximately 99.9% of our DNA sequence is identical • the 0.1% difference between any two individuals But………… • At the DNA level, this 0.1% difference translates into 3 million sites of genomic variation

4. DNA Sequencing • “Sequencing” DNA is simply the elucidation of the order of the bases in an organism’s DNA strand • Early techniques were developed in the 1970’s • A variety of approaches now exist • The biggest limitation to sequencing is that the genome is big

5. DNA Sequencing • First generation sequencing • Next generation sequencing • Third generation sequencing

6. Sanger sequencing’ has been the only DNA sequencing method for 30 years

7. Next Generation Sequencing • Miniaturization enables massively parallel analysis • Carrying out millions of sequencing reactions simultaneously in each of 10 million tiny wells • Computer analysis of huge amounts of information allows “assembly" of a given sequence

8. Cost of Human Genome Next Generation Sequencing

9. Efforts are made REVEAL

10. Information-based medicine Pharmacoproteomics Systems medicine Individualized therapy Customized drug therapy Personalized Medicine Genotype-based therapy Integrated healthcare Predictive medicine Pharmacoge-nomics Translational medicine

11. Personalized Medicine • What is it? • What does it mean to us? • How it impacts our life? • How it changes our future?

12. Advances in science and technology Convergence of molecular biology, genetics, biotechnology, bioinformatics Transformational changes in medicine Shift towards prevention Reclassification of disease Integration and coordination Health as a national asset Symptom based care Fragmented, lack of coordination Inefficient use of information Huge gaps provider knowledge re genomics Powerful investors in current system resist change Costs growing and unsustainable Personalized Medicine Vision vs. Reality

13. Molecular Diagnostics are Transforming Medicine Molecular diagnostics is >a many billion market growing at >20% annually Recurrence monitoring Drug selection Disease detection Disease predisposition Pre-natal testing Key questions What diseases is this patient at risk for? Is the baby healthy? “ Has this patient a disease? What drugs should I prescribe? How has the disease returned?

14. Current challenges in genomic diagnostics • Personal training • Equipment • Data analysis • Standardization

15. Role of Central laboratories in development of genomic resources • Developing algorithms • Writing standard operating procedure (SOP) • Quality Control Scheme organization • Training courses at peripheral level

16. Peripheral laboratories need • Well trained people • SOP and quality assurance culture • Appropriate laboratory equipment • Sample collection and transportation experience • Data collection and registration experience

17. Clinical and Molecular Diagnostics of neurodegenerative and psychiatric conditions

18. Neurodegenerative disorders • Alzheimer’s Disease (AD) • Parkinson Disease (PD) • Epilepsy • Migraine • Multiple sclerosis (MS)

19. Alzheimer Disease • Signs & Symptoms: • Memory loss for recent events • Progresses into dementia and almost total memory loss • Inability to converse, loss of language ability • Affective/personality disturbance • Death from opportunistic infections, etc.

20. Confirmation of Diagnosis Neuronal (b-amyloid) plaques Neurofibrillary tangles From http://www.rnw.nl/health/html/brain.html From http://www.rnw.nl/health/html/brain.html Brain Atrophy

21. AD: Lab diagnostic’s view • There is currently no biomarker of AD for early detection. • To date there is no definitive blood test available that can discriminate dementia patients from healthy individuals. • A combination of characteristic plaque markers tau and amyloid b (Ab) may constitute a specific and sensitive cerebrospinal fluid marker for AD.

22. AD: genetic markers

23. Prevalence of APOE genotypes in Alzheimer’s disease (AD) and controls Jarvik G, Larson EB, Goddard K, Schellenberg GD, Wijsman EM (1996) Influence of apolipoprotein E genotype on the transmission of Alzheimer disease in a community-based sample. Am J Hum Genet 58:191-200

24. Odds of Alzheimer’s Disease by APOE and Age Christensen et al., Genetics in Medicine, 2008 Cupples et al., Genetics in Medicine, 2004 Farrer et al., JAMA, 1997

25. Clinical laboratory Diagnostics of Depression

26. Research Strategies Neuropeptides Neurotrophins PACAP BDNF Depression

27. N TM3 TM2 TM4 Asp - TM1 TM5 TM7 TM6 AC AC γ β ATP αs αs GDP GTP cAMP GDP cAMP cAMP cAMP PACAP Receptor N Extracellular space TM3 TM2 TM4 PACAP Asp - TM1 TM5 TM7 TM6 γ β αs C i3 loop GTP GDP Gs protein Cytoplasm

28. cAMP cAMP cAMP cAMP C C C C C C CREB CREB PO4 PO4 CBP CBP CBP CBP PO4 PO4 C R PKA R C Cytoplasm Nucleus RNA pol II Transcription of BDNF gene CREB TBP DNA CREB TATA

29. BDNF in MDD patients

30. Pre-symptomatic Genetic Testing: Shifting the Emphasis from Reaction to Prevention

31. Pre-symptomatic testing • Genetic and biochemical analysis of healthy individuals who are at increased risk for a specific disorder.

32. Aims of Pre-symptomatic testing • Early detection of disease manifestation • To improve clinical outcome in positive cases

33. Huntington Disease: A Paradigm for Presymptomatic Genetic Testing • Heritable neuropsychiatric disorder • First described by George Huntington in 1872 • Prevalence vary among different populations • Highest -5–10 per 100,000 • Onset –typically fourth decade of life. • death usually occurring in the sixth decade. • No known medical cure • No preventive measures available.

34. Huntington Disease: cause • Single-gene disorder • Autosomal dominant • One mutated copy of the gene is necessary for an individual to be affected • each child of an affected parent is at 50% risk to inherit the disease since only • Responsible Gene – HTT

35. Huntington Disease: symptoms • Movement abnormalities • (e.g., chorea, dystonia, bradykinesia • Psychiatric disturbances • (e.g., depression and anxiety) • Cognitive impairment • (e.g., inattention and executive dysfunction)

36. Huntington disease - a triplet repeat disease • The pathogenic mutation in the HTT gene is unstable, expanded CAG trinucleotide repeat in the first exon which encodes an abnormal polyglutamine tract • A run of > 34 glutamine residues causes the protein to aggregate in the brain cells and cause progressive cell death CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG …... CAG

37. HD alleles categorization • Normal: 26 or fewer CAG repeats. • Intermediate: 27–35 CAG repeats. • These individuals will not develop HD, but may be at risk of having a child with an HD-causing allele. • Reduced penetrance: 36–39 CAG repeats. • These individuals are at risk for, but may not develop, HD • more likely to have later onset disease. • Full penetrance: 40 or more CAG repeats. • These individuals will develop HD.

38. Advantages of predictive testing for Huntington disease • Uncertainty of gene status removed. • If negative: • concerns about self and offspring reduced • If positive: • make plans for the future • arrange surveillance/treatment if any • inform children/decide whether to have children

39. Disadvantages of predictive testing for Huntington disease • If positive: • removes hope • introduces uncertainty (if and when) • known risk to offspring • impact on self/partner/family/friends • potential problems with insurance/mortgage. • If negative: • expectations of a ‘good’ result • ‘survivor’ guilt.

40. Potential outcomes of pre-symptomatic HD genetic testing. • Severe Psychological Distress • International testing protocols were established to protect at-risk individuals and to provide guidance for clinical and laboratory personnel

41. Our Expectations • Greater effectiveness of health care delivery • Improved health • Improved quality of life