Sequencing the Human Genome

1. Sequencing the Human Genome • In 1998, Celera Genomics announced plans to sequence the human genome… • …175,000 sequence reads per day, operating 24 hours a day, 7 days a week J. Craig Venter

2. Sequencing the Human Genome • Whole genome shotgun approach vs. Clone by Clone approach • By-passes the initial work of ordering clones • Celera performed about 32 million sequence reads, each 500 – 1000 bp

3. Sequencing the Human Genome

4. Sequencing the Human Genome • IHGSC published sequence reads every 24 hours to prevent patenting of DNA • Celera had access to IHGSC data • Debate over whether Celera could have shotgun sequenced the genome without IHGSC data

5. Sequencing the Human Genome • Both groups published results simultaneously • Celera – Science February 2001 • IHGSC – Nature February 2001

6. Sequencing the Human Genome Nature 409, 818 - 820 (15 February 2001)

7. Sequencing the Human Genome • Controversy! Science published Celera’s sequence without requiring deposition to GenBank • Celera provides full access, with a catch… • Celera provided Science with a copy in escrow

9. Sequencing Your Human Genome • For $500,000 you can have your DNA sequenced • Sequence 1000 individual human genomes • “Personalized” medicine J. Craig Venter

10. Human Genome • Legal considerations • Should DNA, or genes, be patentable? • In the past, USPTO considered genes as man-made chemicals • Copy DNA region, splice it together, and propagate it in bacteria, etc

11. Human Genome • Celera >6500 genes • Human Genome Sciences >7000 • Incyte >50,000 • Only a fraction may be awarded by USPTO, and only a fraction of these may be useful in treating human disease

12. Human Genome • 1994 U. of Rochester scientists isolate mRNA for COX-2 and clone gene • Suggest that compounds which inhibit COX-2 might provide pain relief from arthritis • Submit patent application in 1995

13. Human Genome • 1998 – Celebrex – inhibitor of cyclooxygenase-2 (COX-2) introduced as arthritis medication • Developed by Pfizer/Searle • Development began in early-90’s i.e. around time of U. of Rochester discovery

14. Human Genome • April 2000, U. of Rochester awarded patent covering COX-2 gene and inhibition of the peptide product thereof • The same day, U. of Rochester files lawsuit against Pfizer/Searle to block Celebrex sales • Claims that Pfizer/Searle infringes on their patent • They want royalties from the sale of the invention

15. Human Genome • 2003 – U. of Rochester patent found invalid • 2004 – Invalidation upheld by higher Court • U. of Rochester patent did not provide sufficient example of what the inhibitor would be…i.e. claims too broad without a working example • How will “basic science” performed by Universities be rewarded?

16. Human Genome • Vioxx and Celebrex in news again this year: increased risk of “cardiovascular event” i.e. heart attacks

17. Human Genome • Gene discovery • Methods for finding genes • Easy in prokaryotes

18. Human Genome • Gene discovery • Difficult in eukaryotes

19. Human Genome • Gene discovery • Average gene extends over 27 kb • Average 8.8 introns • Average 145 bp • Extremes: • Dystrophin gene 2.4 Mb • Titin gene contains 178 introns, coding for a 80,780 bp mRNA

20. Human Genome • Gene discovery • One approach is to examine “transcriptome”

21. Human Genome • Conservation of chromosome/gene location between organisms • Synteny • Exons tend to be conserved between species

22. Human Genome • Human vs. Pufferfish genome • Pufferfish genome about 1/7th the size of the human genome with similar number of genes

23. Human Genome • Predictive computer programs, e.g. GENSCAN • GENSCAN predicts the location of genes based on splicing predictions, promoter regions and other criteria

24. Human Genome • Online databases have formed to curate Human genome data • Ensembl (www.ensemble.org)

25. Genetic Mapping of Mendelian Characters

26. Identifying Disease-Causing Gene Variations • Linkage analysis and Positional Cloning • Clone disease gene without knowing anything except the approximate chromosomal location

27. Recombination • Recombination during meiosis separates loci • More often when they are farther apart • Less often when they are close • Recall discussion of the Genetic Map • Loci on separate chromosomes segregate independently • Loci on the same chromosome segregate as a function of recombination

28. Recombination 13-1

29. 13_06.jpg

30. Linkage analysis • Linkage analysis locates the disease gene locus • Linkage analysis requires • Clear segregation patterns in families • Informative markers close to the locus • Utilize LOD analysis to verify linkage • Calculate cM distance between Loci

31. Positional Cloning • Widely used strategy in human genetics for cloning disease genes • No knowledge of the function of the gene product is necessary • Strong for finding single-gene disorders

32. Positional Cloning • Linkage analysis with polymorphic markers establishes location of disease gene • LOD score analysis, and other methods are employed • Once we know the approximate location… • The heavy molecular biology begins

33. Positional Cloning • Example - Huntington’s disease • CAG… • Autosomal dominant • 100% penetrance • Fatal • Late onset means patients often have children

34. Finding the Huntington Gene – 1981-1983 • Family with Huntington's disease found in Venezuela • Originated from a single founder - female • Provided: • Traceable family pedigree • Informative meiosis • Problem was… only a few polymorphic markers where known at the time

35. Finding the Huntington Gene • Blood samples taken • Check for disease symptoms • Paternity verified

36. Finding the Huntington Gene • By luck, one haplotype segregated very closely with Huntington disease • Marker was an RFLP called G8 (later called D4S10)

38. Finding the Huntington Gene

39. Finding the Huntington Gene • Locate the region to the tip of the short arm of chromosome 4 by linkage with G8 (D4S10) • Maximum LOD score occurred at about 4 cM distance, i.e. 4 in 100 meiosis

40. Finding the Huntington Gene • Together this started an international effort to generate YAC clones of the 4 Mb region • More polymorphisms were found

41. Finding the Huntington Gene • Next, find an unknown gene in an uncharacterized chromosome location • Locate CpG islands • Cross-species comparisons • Further haplotype analysis suggested a 500 Kb region 3’ to D4S10

42. Finding the Huntington Gene • Exon trapping was key • Compare cloned exons between normal and Huntington disease patients

43. Finding the Huntington Gene

44. Finding the Huntington Gene • One exon, called IT15, contained an expanded CAG repeat…. • Mapping to 4 cM – 1983 • Cloning of Huntington gene – 1993

45. Complex Disease and Susceptibility Single gene disorders Mendelian Inheritance High penetrance Low environmental influence (but sometimes significant) LOD-based linkage analysis works great Genetic heterogeneity Low population incidence Gene Gene Disease

46. Complex Disease and Susceptibility Gene Gene Gene Gene Environment Disease A Disease C Disease B Multifactorial disorders

47. Single gene disorders Huntington’s Fragile X SCA1 DMD Werner’s syndrome Cystic fibrosis Multifactorial Heart disease Cancer Stroke Asthma Diabetes Alzheimer’s Parkinson’s Complex Disease and Susceptibility

48. Genetic Component in Complex Disorders • Relative risk lr= frequency in relative of affected person Population frequency

49. Genetic Component in Complex Disorders • Family Studies

50. Genetic Component in Complex Disorders • Problem of environmental impact