1. 1 MOLECULAR DIAGNOSITICS Prof. Fang Zheng, Tianjin Medical University

2. 2 Introduction to Molecular Diagnostics Molecular Diagnostics

3. 3 Outline Concept of Molecular Diagnostics History of Molecular Diagnostics Impact on Human Diseases Basis for Molecular Assay Management of the course

4. 4 Concept of Molecular Diagnostics History of Molecular Diagnostics Impact on Human Diseases Basis for Molecular Assay Management of the course

5. 5 1. Molecular Diagnosis Molecular diagnosis of human disorders is referred to as the detection of the various pathogenic mutations in DNA and /or RNA samples in order to facilitate detection, diagnosis, sub- classification, prognosis, and monitoring response to therapy.

6. 6 1. Molecular Diagnostics The use of molecular biology techniques to expand scientific knowledge of the natural history of diseases, identify people who are at risk for acquiring specific diseases, and diagnose human diseases at the nucleic acid level.

7. 7 1. Molecular Diagnostics Molecular diagnostics combines laboratory medicine with the knowledge and technology of molecular genetics and has been enormously revolutionized over the last decades, benefiting from the discoveries in the field of molecular biology.

8. 8 The information revolution in molecular biology is permeating every aspect of medical practice The rate of disease gene discovery is increasing exponentially, which facilitates the understanding diseases at molecular level Molecular understanding of disease is translated into diagnostic testing, therapeutics, and eventually preventive therapies 1. Molecular Diagnostics: Emergence

9. 9 1. Molecular Diagnostics: Significance To face the new century, the medical practitioner not only understand molecular biology, but must also embrace the use of this rapidly expanding body of information in his medical practice, whether practicing family medicine, oncology, obstetrics and gynecology, pathology, or any other medical specialty.

10. 10 To introduce essential concepts in molecular diagnostics that impact on the identification of novel markers of human diseases To develop and apply useful molecular assays to monitor disease, determine appropriate treatment strategies, and predict disease outcomes. 1. Molecular Diagnostics: Goal

11. 11 Concept of Molecular Diagnostics History of Molecular Diagnostics Impact on Human Diseases Basis for Molecular Assay Management of the course

12. 12 2. History of Molecular Diagnostics 1865Gregor Mendel, Law of Heredity 1866 Johann Miescher, Purification of DNA 1953 1970 Recombinant DNA Technology 1977 DNA sequencing 1985 In Vitro Amplification of DNA (PCR) 2001The Human Genome Project Watson and Crick, Structure of DNA The Molecular Biology Timeline Sickle Cell Anemia Mutation 1949

13. 13 2. History of Molecular Diagnostics  Pauling introduced the term molecular disease in the medical vocabulary, based on their discovery that a single amino acid change leads to a sickle cell anemia.  In principle, their findings have set the foundations of molecular diagnostics.  Sickle cell anemia is a genetic disease which is caused by a single nucleotide change in the 6th aa of the  -chain of hemoglobin. Sickle cell anemia

14. 14 Sickle Cell Anemia Figure A. Normal red blood cells flowing freely in a blood vessel. The inset image shows a cross- section of a normal red blood cell with normal hemoglobin. Figure B. Abnormal, sickled red blood cells clumping and blocking blood flow in a blood vessel. The inset image shows a cross- section of a sickle cell with abnormal hemoglobin.

15. 15 J.D. Watson and F.H.C. Crick (1953) A structure for deoxyribose nucleic acid. Nature 171:737 “We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.). This structure has novel features which are of considerable biological interest.” Discovery of DNA Structure One of the most important biological discovery in the 20th century

16. 16 J.D. Watson and F.H.C. Crick (1953) Discovery of DNA Structure

17. 17 Rosalind E. Franklin 1920–1958 The structure of DNA was determined using X-ray diffraction techniques. Much of the original X-ray diffraction data was generated by Rosalind E. Franklin.

18. 18

19. 19 1962 J. Watson & F. Crick: DNA structure Max Perutz & John Kendrew: Protein sequence 1958 Frederick Sanger: Insulin sequence 1980 Frederick Sanger: DNA sequencing 1984 Cesar Milstein & Georges Kohler: Monoclonal Ab …… Discovery of DNA Structure Laboratory of Molecular Biology ， (LMB) (Cavendish Laboratory ) 1955- 12 scientists received Noble Prize

20. 20 2. History of Molecular Diagnostics  The first seeds of molecular diagnostics were provided in the early days of recombinant DNA technology.  cDNA cloning and sequencing were invaluable tools for providing the basic knowledge on the primary sequence of various genes.  DNA sequencing provided a number of DNA probes, allowing the analysis via southern blotting of genomic regions, leading to the concept and application of restriction fragment length polymorphism ( RELP ) track a mutant allele from heterozygous parents to a high-risk pregnancy.

21. 21 2. History of Molecular Diagnostics The PCR Revolution Kary Mullis 1985 41y Invention of PCR 1993 49y Received the Noble Prize

22. 22 2. History of Molecular Diagnostics The PCR Revolution PCR has greatly facilitated and revolutionized molecular diagnostics. Its most powerful feature - large amount of copies of the target sequence generated by its exponential amplification, which allows the identification of a known mutation within a single day.

23. 23 2. History of Molecular Diagnostics The PCR Revolution PCR markedly decreased need for radioactivity, allowed molecular diagnostics to enter the clinical laboratory. PCR either is used for the generation of DNA fragments to be analyzed, or is part of the detection methods

24. 24 U.S. Government project coordinated by the Dept. of Energy and NIH U.S. Government project coordinated by the Dept. of Energy and NIH Goals of the Human Genome Project Goals of the Human Genome Project (1990 – 2006) – To identify all of the genes in human DNA; – To identify all of the genes in human DNA; – To determine the sequences of the 3 billion bases that make up human DNA; – To determine the sequences of the 3 billion bases that make up human DNA; – To create databases; – To create databases; – To develop tools for data analysis; and – To develop tools for data analysis; and – To address the ethical, legal, and social issues that arise from genome research – To address the ethical, legal, and social issues that arise from genome research 2. History of Molecular Diagnostics Human Genome Project

25. 25 U.S. Government project coordinated by the Dept. of Energy and NIH U.S. Government project coordinated by the Dept. of Energy and NIH Goals of the Human Genome Project Goals of the Human Genome Project (1990 – 2006) – To identify all of the genes in human DNA; – To identify all of the genes in human DNA; – To determine the sequences of the 3 billion bases that make up human DNA; – To determine the sequences of the 3 billion bases that make up human DNA; – To create databases; – To create databases; – To develop tools for data analysis; and – To develop tools for data analysis; and – To address the ethical, legal, and social issues that arise from genome research – To address the ethical, legal, and social issues that arise from genome research 2. History of Molecular Diagnostics Human Genome Project

26. 26 Concept of Molecular Diagnostics History of Molecular Diagnostics Impact on Human Diseases Basis for Molecular Assay Management of the course

27. 27 Discovery of potential novel molecular markers of human diseases Identification of novel molecular markers of human diseases Utility of molecular markers to develop useful molecular assays for detection, diagnosis, and prediction of disease outcomes 3. Impact on Human Diseases: Novelty

28. 28 3. Impact on Human Diseases: Advantage Monitor diseases more accurately Allows for early treatment and better patient care Determine most appropriate treatment Reduces or eliminates unnecessary treatment Reduces or eliminates inadequate treatment Yields greater cost effectiveness Reduce patient morbidity and mortality

29. 29 Diagnostic-Identity of a disease Diagnostic-Identity of a disease Prognostic-Outcome of a disease Prognostic-Outcome of a disease Predictive-Possibility of a disease Predictive-Possibility of a disease Therapeutic-Response of a disease to treatment Therapeutic-Response of a disease to treatment 3. Impact on Human Diseases: Practical application

30. 30 HEMATOLOGY INFECTIOUS DISEASE IDENTITY TESTING GENETIC DISEASE SOLID TUMORS Molecular Pathology 3. Impact on Human Diseases

31. 31  Molecular Genetics Single gene disorders 病种多，特定家系中发病率高，对群体影响小， 遗传性基因携带者的筛查 Polygenic disorders 病种少，特定家系中发病率高，对群体影响大 遗传易感性的检测 Chromosomal disorders 3. Impact on Human Diseases

32. 32  Molecular Oncology Diagnostic testing Disease prognosis Determination of predisposition 3. Impact on Human Diseases

33. 33  Hematopathology Diagnostic testing Determination of clonality  Identity Testing Parentage Clinical testing 3. Impact on Human Diseases

34. 34  Infectious Disease Qualitative and quantitative detection of infectious agents Microbial identity testing Genotyping/drug resistance testing 3. Impact on Human Diseases

35. 35 Concept of Molecular Diagnostics History of Molecular Diagnostics Impact on Human Diseases Basis for Molecular Assay Management of the course

36. 36 4. Basis for Technology: Fundamental (1) Advance in the understanding of the structure and chemistry of nucleic acids have facilitated the development of technologies that can be employed effectively in molecular diagnostics.

37. 37 4. Basis for Technology: Platform  Amplification Techniques PCR polymerase chain reaction 多聚酶链反应 LCR ligase chain reaction 连接酶链反应 NASBA nucleic-acid sequence-based amplification 核酸序列依赖的扩增 Molecular Technologies in the Clinical Laboratory  DNA Sequencing

38. 38 4. Basis for Technology: Platform  Electrophoretic Methods SSCP (single-strand conformation polymorphism) 单链构象多态性 DGGE ( denaturing gradient gel electrophoresis) 变性梯度凝胶电泳法 Molecular Technologies in the Clinical Laboratory  Hybridization Techniques Southern hybridization Blot Northern hybridization Blot

39. 39 4. Basis for Technology: Platform  Biochip Technology DNA micro-array Protein micro-array Molecular Technologies in the Clinical Laboratory  Recombinant DNA Technology

40. 40 4. Basis for Technology: Target specialty Genetically-based diseases can be diagnosed Specificity can be controlled Single base changes can be detected Expression of gene product is not required Targets can be amplified >10 5 Nucleic acids are targeted by molecular assays

41. 41 4. Basis for Molecular Assays: Diseases Cause (etiology) Mechanism (pathogenesis) Structural alterations (morphologic/molecular) Functional consequences (clinical significance)

42. 42 4. Basis for Molecular Assay: Pathogenesis (1) Diagnostic Distinguishing variants of human disease based on presence of specific molecular markers (chromosome translocations in Burkitt’s lymphoma: c-myc) Understanding molecular pathogenesis of human disease enables effective utilization of molecular assays

43. 43 4. Basis for Molecular Assay: Pathogenesis (1) Prognostic Prediction of likely patient outcomes based on presence of specific molecular markers (gene mutations predicting clinical course in cancer) Understanding molecular pathogenesis of human disease enables effective utilization of molecular assays

44. 44 4. Basis for Molecular Assay: Pathogenesis (2) Understanding molecular pathogenesis of human disease enables effective utilization of molecular assays Therapeutic Prediction of response to specific therapies based on presence of specific molecular markers (gene mutations predicting poor drug sensitivity in lung cancer: p53, k-ras)

45. 45 4. Basis for Molecular Assay: Molecular biology (1)  Genetic Lesions in Human Disease Identification of genetic markers Identification of disease-related genes Molecular targets for assay development

46. 46 4. Basis for Molecular Assay: Molecular biology (1)  Characterization of Gene Sequences Facilitates characterization of disease-causing mutations Molecular targets for assay development

47. 47 4. Basis for Molecular Assay: Molecular biology (2) Completion of the sequence of the human genome will enable identification of all human genes and establishment of disease-gene relationships, facilitating development of numerous new molecular assays.

48. 48 4. Basis for Molecular Assay: Molecular biology (4) Improvements in medicine Improvements in medicine Microbial genome research Microbial genome research DNA forensics/identity DNA forensics/identity Improved agriculture and livestock Improved agriculture and livestock Better understanding of evolution and human migration Better understanding of evolution and human migration More accurate risk assessment More accurate risk assessment Beneficial outcomes from human genome project

49. 49 4. Basis for Molecular Assay: Molecular biology (5) Use of genetic information Use of genetic information Privacy/confidentiality Privacy/confidentiality Psychological impact Psychological impact Genetic testing Genetic testing Reproductive options/issues Reproductive options/issues Education, standards, and quality control Education, standards, and quality control Commercialization Commercialization Conceptual and philosophical implications Conceptual and philosophical implications Human genome project: Ethical, Legal, and Social Implications

50. 50 What’s So Great About Molecular Diagnostics ? As many as 5,000 diseases have direct genetic causes High sensitivity and increased specificity for most tests adds diagnostic utility Potential for simple standardized procedures an automation rapid throughput Increased number of techniques for infectious diseases and tumor diagnostics A viable reflex for equivocal morphology Prices are falling 5. Conclusion

51. 51 The ultimate goal of the molecular diagnostics is to provide molecular information that will combine with and complement information related to patient history and symptomology, clinical laboratory results, histopathological findings, and other diagnostic information to provide a more sensitive, precise, and accurate determination of disease diagnosis and/or guidance toward appropriate and effective treatment options. 5. Conclusion

52. 52 E N D