1、 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Chapter 15 Genomics and Medicine,The impact of genomics on the practice of medicine, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Contents,Medical promise of
2、genomics High-throughput methods for genotyping Cancer genomics Microbial genomics and medicine Finding new drug targets Developing vaccines DNA vaccines Gene therapy, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,What we hope to gain from genomics,Drug
3、, diagnostics, and prognostics development Genotyping to predict patient susceptibility to disease Personalized healthcare based on an individuals genomic features, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Long term returns,Personalized genotype da
4、tabases Used to assess health risks throughout life Adjustments to lifestyle and medical treatment Simulated cells Reduce the need for time consuming experiments Allow experiments that would otherwise be impossible New frameworks for clinical trials Pharmacogenomics, 2005 Prentice Hall Inc. / A Pear
5、son Education Company / Upper Saddle River, New Jersey 07458,Short term returns,Faster characterization of disease genes Better disease diagnosis / prognosis with microarrays Better methods for genotyping More efficient drug / vaccine development, 2005 Prentice Hall Inc. / A Pearson Education Compan
6、y / Upper Saddle River, New Jersey 07458,Advances in disease genetics,Detection of disease genes is most direct medical use of genomics information Over 1,000 disease genes were characterized by 2000 How to exploit this information?,250,0,1981,2000,Year of discovery,Disease genes, 2005 Prentice Hall
7、 Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Preimplantation diagnosis,Couples with at least one child suffering from cystic fibrosis underwent preimplantation diagnosis Biopsied cells from in vitro 3-day old embryos were genotyped Implanted embryos (NN and ND) in one c
8、ouple resulted in a healthy baby girl,DD,NN,DD,NN,BAD,ND,diagnosis,homoduplex,heteroduplex,biopsied cell,DNA added,1,2,3,4,5,6, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Single nucleotide polymorphisms (SNPs),Benefits of characterizing SNPs High den
9、sity SNP map will greatly facilitate finding disease genes Detection of SNPs can serve as a diagnostic for genetic diseases Millions of SNPs presently in public and private databases Fast, cheap, and accurate genotyping of SNPs still a challenge Smallest linkage disequilibrium studies still out of r
10、each Genotyping 30,000 SNPs in 1,000 individuals required 10-fold increase in technological capacity at end of 2001, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Biochemical basis for SNP genotyping,Hybridization with allele-specific oligonucleotides (
11、ASOs) Allele-specific primer extension Minisequencing Oligonucleotide ligation Restriction site cleavage Invasive cleavage, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Hybridization of allele-specific oligonucleotides,Uses hybridization to detect SNP
12、Heat or electric field used to denature hybrids Caveat Each SNP hybrid will denature with different parameters Microarrays overcome this to some degree,Allelle-specific probes,Stable,Unstable, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Allele-specifi
13、c primer extension,Use allele specific primers that include the SNP PCR will extend primers that match SNP, but not mismatches Detection Fluorescence assay that detects incorporation of primer into PCR product,Allele-specific primers,Extension,No extension,PCR, 2005 Prentice Hall Inc. / A Pearson Ed
14、ucation Company / Upper Saddle River, New Jersey 07458,Minisequencing,Use DNA polymerase to extend primer sequence by a single nucleotide Detection Colorimetric assay using antibodies to chemical group attached to nucleotide Luminometric detection of chemical released upon nucleotide addition Mass s
15、pectroscopy,One-nucleotide extension,Minisequencing primer,No extension,PCR, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Oligonucleotide ligation,Three probes used Increases cost Ligase ties together matched probes Detection methods Changes in mobilit
16、y Microarrays,Left probes,Match, ligation,Mismatch, no ligation,Right probe,Ligase, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Restriction site cleavage,Presence of SNP creates a site for cleavage by restriction enzyme Pattern of restriction fragment
17、s reveals presence (or absence) of SNP Method not sufficient for genome-wide SNP scan,Restriction enzyme,Cleavage,No cleavage, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Invasive cleavage,Highly specific probe binds to target sequence Probe causes ch
18、ange in conformation of double stranded oligonucleotide New conformation provides target for cleavage by FLAP endonuclease Cleaved signal sequence indicates SNP Requires a lot of DNA,Invader probe,Mismatch, cleavage,Match, no cleavage,Flap Endonuclease, 2005 Prentice Hall Inc. / A Pearson Education
19、Company / Upper Saddle River, New Jersey 07458,An ideal SNP genotyping method,PCR is rate limiting step of most SNP genotyping techniques The ideal genotyping method Single molecule genotyping (i.e. no PCR) Example: atomic force microscopy with nanotube probes,AFM,labels,DNA,probe,height,DNA molecul
20、e, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Cancer genome projects,Cancer Genome Anatomy Project (CGAP) Established 1997 by National Cancer Institute (USA) Specializes in EST sequencing Human Cancer Genome Project (HCGP) Established 1999 by Brazili
21、an research groups Specializes in SAGE analysis Cancer Genome Project (CGP) Established 2000 by Wellcome Trust and Sanger Institute (United Kingdom) Specializes in genomic mutations leading to cancer, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Cancer
22、 genomics using ESTs and SAGE,Use EST/SAGE tags from normal and tumorous tissue Tags stored in publicly accessible databases Bioinformatics tools used to reveal patterns of gene expression that define cancerous states,normal,tumor,SAGE/EST,sequencing,tags,database and analysis, 2005 Prentice Hall In
23、c. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Searching for cancer-causing mutations in genomic DNA,Use human genome sequence to make PCR primers for target genes Compare PCR products from normal tissue and tumors using automated heteroduplex analysis Sequence genes when he
24、teroduplex analysis suggests tumor/normal differences Find genomic mutants,PCR,heteroduplex analysis,sequence mutants,Make PCR primers of target genes from normal and tumor tissue,1,2,3, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Benefits of cancer g
25、enomics,EST/SAGE projects Annotations for human genome sequence Understanding cancerous / normal tissue differences in gene expression Identifying cancer-specific splice variants Genetic polymorphisms associated with cancer Investigation of genomic DNA Genetic polymorphisms associated with cancer Id
26、entification of cancer-causing mutations, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Microarrays and cancer,Histology not always effective tool for prognosis/diagnosis Microarrays distinguish cancerous tissues on the basis of a gene expression profil
27、e Use in diagnosis Example: characterizing acute lymphoblastic leukemia Use in prognosis Example: metastasis in medulloblastoma, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Microarrays in the prognosis of metastasis,Identified 85 genes with different
28、levels of expression in metastatic and non-metastatic tumors 72% accuracy in predicting metastasis Identified genes that induce metastasis Could serve as potential drug targets,M+,M,Down- regulated,Up-regulated, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 0
29、7458,Microbial genomics and medicine,Hundreds of microbial genomes have been sequenced Opportunities for better understanding disease Reveal new drug targets Suggest vaccine candidates Most microbial genomes sequenced are pathogenic, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Sadd
30、le River, New Jersey 07458,Malaria,Infects 500 million, kills 2 million every year Mosquito-borne illness Drug and pesticide resistance emerged in the 1960s Global warming may increase size of endemic areas,1994,1966,1946, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, N
31、ew Jersey 07458,The life cycle of P. falciparum, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Advances in genomics and malaria,2003: human genome sequence completed 2002: Complete genome of mosquito 2002: Complete genome of P. falciparum Genomic approa
32、ches to combating malaria Genetically modified mosquitoes Efficient development of drugs / vaccines,genetically modified mosquitos,P. falciparum, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Using transgenic mosquitoes to control malaria,12-amino acid
33、peptide (SM1) found to inhibit Plasmodium entry to salivary glands Transgene used to transfect germline of mosquitoes CP promoter for gene expression during blood feeding GFP to detect transfection 4 copies of 12-amino acid peptide gene,AgCP promoter,GFP,SM14,HA1,CP signal, 2005 Prentice Hall Inc. /
34、 A Pearson Education Company / Upper Saddle River, New Jersey 07458,Ability of transgenic mosquitoes to infect was impaired,69-95% decrease in oocyst formation Infection of mice greatly reduced or eliminated Caveats P. falciparum can evolve to overcome transgenic mosquitoes Need more transgenes to r
35、educe this possibility,GM midgut,Wild-type midgut, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Using functional genomics to find drug targets,Functional genomics reduces the need for complex biochemical analysis Genome sequence sufficient to reveal un
36、discovered pathways Functional genomics can identify previously characterized proteins in a new species In some cases providing targets for pre-existing drugs Example: P. falciparum, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Bacterial homologs in P.
37、 falciparum,The apicoplast: an essential organelle in P. falciparum Self-replicating Possesses its own 35 kb genome Related to algae Using sequences from the P. falciparum genome project, a bacterial enzyme was discovered DOXP reductoisomerase, part of synthetic pathway Homologs in E. coli, B. subti
38、lis, and Synechocystis sp. Enzyme believed to be associated with apicoplast, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,The mevalonate and DOXP pathways,Synthesis of isopentenyl diphosphate Essential for synthesis of steroids DOXP pathway used by bac
39、teria and plants (chloroplasts) Mevalonate pathway used by animals, fungi Drug targeting DOXP pathway should have few human side-effects,DOXP,Mevalonate,isopentenyl diphosphate, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Can drugs that target DOXP re
40、ductoisomerase cure malaria?,GFP-labeling showed isolation of DOXP redectoisomerase in apicoplasts Previously developed antibacterial drug FR-900098 effectively inhibits DOXP reductoisomerase FR-900098 cured rats with Malaria with minimal toxicity,enzyme activity (%),drug concentration (nM),0.1,1,10
41、0,10,1000, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Finding vaccine candidates,Genomics for vaccine development Functional genomics reveals microbial surface proteins Surface proteins constitute potential antigens for use as vaccines Recombinant an
42、tigen proteins used to test new vaccines Example: N. meningitidis 32% of all meningococcal disease in U.S. Genome fully sequenced in 2000, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Challenges in N. meningitidis vaccine development,1960s: Using purif
43、ied membrane-associated polysaccharides, a vaccine was developed Worked well on adults But ineffective in most vulnerable population Children and infants No vaccine for serogroup B N. meningitidis Serogroup B vaccines using polysaccharides too similar to human polysaccharides Serogroup B vaccines us
44、ing surface proteins are too strain-specific, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Using genomics to overcome vaccine challenges,Identified 570 putative membrane proteins in serogroup B Meningococcus (MenB) Express proteins in E. coli Look for
45、positive immune responses in mice Select vaccine candidates expressed on the surface of multiple virulent strains of MenB Narrow down vaccine candidates to 7 antigens that lack phase variability Look for surface proteins in other Neisseria species, 2005 Prentice Hall Inc. / A Pearson Education Compa
46、ny / Upper Saddle River, New Jersey 07458,DNA vaccines,Inject naked DNA containing microbial gene into patient Somatic (or preferably antigen-presenting) cells produce DNA product, which constitutes antigen Host generates immune response to antigen,A gene gun, 2005 Prentice Hall Inc. / A Pearson Edu
47、cation Company / Upper Saddle River, New Jersey 07458,Components of a DNA vaccine plasmid,Origin of replication for rapid replication in bacteria Antibiotic resistance gene to select transfected bacteria Mammalian promoter PolyA tail for mRNA stabilization CpG motif for strong immune response Antige
48、n gene,Antibiotic resistance,Origin,Promoter,Gene insert,Poly A tail, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,DNA vaccines under development,600 DNA vaccines currently under development Examples of diseases being tackled Tuberculosis Malaria AIDS
49、Presently no DNA vaccines on the market,Clinical Trials (2002),phase,Total number 636, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Advantages and disadvantages compared to traditional vaccines,Advantages of DNA vaccines Induce humoral and cellular immune responses Manufactured very easily Inexpensive No refrigeration necessary Disadvantages Concerns about autoimmune disease Possibility of introducing foreign DNA into the human germline,
