Genetics Maps

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Genetics Maps
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Genetics Maps

• Genotyping individuals with STRs

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Genetics Maps

• By 1994, there was a 1 cM map based largely on
  microsatellites (STRs)

A comprehensive human linkage map with
centimorgan density. Murray JC, Buetow KH, Weber
JL, Ludwigsen S, Scherpbier-Heddema T, Manion F,
Quillen J, Sheffield VC, Sunden S, Duyk GM, et
al.Science. 1994 Sep 30265(5181)2049-54.
5840 loci total 3617 polymerase chain
reaction-formatted short tandem repeat
polymorphisms 427 genes 0.7 centimorgan density
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Genetics Maps
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Genetics Maps
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Physical Maps
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Physical Maps
Ordering clones based on Hybridization
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Physical Maps
Ordering clones based STS content
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Genetic Maps and Physical Maps are Aligned by STS
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Sequencing the Human Genome
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Gel run
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Sequencing the Human Genome
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Sequencing the Human Genome
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Sequencing the Human Genome

• Sequence 500 bp each reaction
• To sequence the Human genome, sequencing method
  needs to be
• FAST
• CHEAP
• In 1990 reality was
• SLOW
• EXPENSIVE (gt1 per base!)

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Sequencing the Human Genome

• International Human Genome Sequencing Consortium
• Primarily six institutes with high-throughput
  sequencing capabilities
• Whitehead Institute
• The Sanger Center
• Washington University
• DOE Sequencing Center
• Bayer College of Medicine (31 Jan 2005 161,489
  kb 2,935,479 kb)
• In 1990, the IHGSC began a 15 year plan to
  sequence the entire Human genome

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Sequencing the Human Genome
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Sequencing the Human Genome

• IHGSC Strategy - Shotgun sequencing of ordered
  BAC contigs
• Define BAC contig order based on STS
• Sequence each cluster of BACs within contig
  align based on sequence
• Anchor to genome by STS.

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Sequencing the Human Genome
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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
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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

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Sequencing the Human Genome
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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

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Sequencing the Human Genome

• Both groups published results simultaneously
• Celera Science
• February 2001
• IHGSC Nature
• February 2001

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

• Controversy! Science published Celeras sequence
  without requiring deposition to GenBank
• Celera provides full access, with a catch
• Celera provided Science with a copy in escrow

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Sequencing Your Human Genome

• For 500,000 you can have your DNA sequenced
• Sequence 1000 individual human genomes
• Personalized medicine

J. Craig Venter
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• Next Gen Sequencing

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• The proliferation of genetic testing resources

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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

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Human Genome

• Celera gt6500 genes
• Human Genome Sciences gt7000
• Incyte gt50,000
• Only a fraction may be awarded by USPTO, and only
  a fraction of these may be useful in treating
  human disease

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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

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Human Genome

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

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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

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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 bei.e.
  claims too broad without a working example
• How will basic science performed by
  Universities be rewarded?

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Human Genome

• Vioxx and Celebrex in news again this year
  increased risk of cardiovascular event i.e.
  heart attacks

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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

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Human Genome

• Gene discovery
• One approach is to examine transcriptome
• Exome

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Human Genome

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

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Human Genome

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

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Human Genome

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

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Human Genome

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

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Genetic Mapping of Mendelian Characters
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Identifying Disease-Causing Gene Variations

• Linkage analysis and Positional Cloning
• Clone disease gene without knowing anything
  except the approximate chromosomal location

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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

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Recombination
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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

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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

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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

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Positional Cloning

• Example - Huntingtons disease
• CAG
• Autosomal dominant
• 100 penetrance
• Fatal
• Late onset means patients often have children

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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

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Finding the Huntington Gene

• Blood samples taken
• Check for disease symptoms
• Paternity verified

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Finding the Huntington Gene

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

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Finding the Huntington Gene
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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

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Finding the Huntington Gene

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

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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

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Finding the Huntington Gene

• Exon trapping was key
• Compare cloned exons between normal and
  Huntington disease patients

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Finding the Huntington Gene
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Finding the Huntington Gene

• One exon, called IT15, contained an expanded CAG
  repeat.
• Mapping to 4 cM 1983
• Cloning of Huntington gene 1993

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