Genetics Maps

Genetics Maps

• Genotyping individuals with STR’s

Genetics Maps

• By 1994, there was a ~1 cM map based largely on microsatellites (STR’s)

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 30;265(5181):2049-54.

5840 loci total3617 polymerase chain reaction-formatted short tandem repeat polymorphisms427 genes0.7 centimorgan density

Genetics Maps

Genetics Maps

Physical Maps

Physical Maps

Ordering clones based on Hybridization

Physical Maps

Ordering clones based STS content

Genetic Maps and Physical Maps are Aligned by STS

Sequencing the Human Genome

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Sequencing the Human Genome

Sequencing the Human Genome

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 (>$1 per base!)

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

Sequencing the Human Genome

Sequencing the Human Genome

• IHGSC Strategy - Shotgun sequencing of ordered BAC contigs

• Define BAC contig order based on STS

• Sequence each cluster of BAC’s within contig – align based on sequence

• Anchor to genome by STS….

Sequencing the Human Genome

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

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

Sequencing the Human Genome

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

Sequencing the Human Genome

• Both groups published results simultaneously

• Celera – Science

February 2001

• IHGSC – Nature

February 2001

Sequencing the Human Genome

Nature 409, 818 - 820 (15 February 2001)

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

Sequencing Your Human Genome

• For $500,000 you can have your DNA sequenced

• Sequence 1000 individual human genomes

• “Personalized” medicine

J. Craig Venter

• Next Gen Sequencing

• The proliferation of genetic testing resources

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

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

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

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

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

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?

Human Genome

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

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

Human Genome

• Gene discovery– One approach is to examine “transcriptome”– Exome

Human Genome

• Conservation of chromosome/gene location between organisms

• Synteny

• Exons tend to be conserved between species

Human Genome

• Human vs. Pufferfish genome

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

Human Genome

• Predictive computer programs, e.g. GENSCAN

• GENSCAN predicts the location of genes based on splicing predictions, promoter regions and other criteria

Human Genome

• Online databases have formed to curate Human genome data

• Ensembl (www.ensemble.org)

Genetic Mapping of Mendelian Characters

Identifying Disease-Causing Gene Variations

• Linkage analysis and Positional Cloning

– Clone disease gene without knowing anything except the approximate chromosomal location

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

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

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

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

Positional Cloning

• Example - Huntington’s disease

– CAG…

– Autosomal dominant– 100% penetrance– Fatal– Late onset means patients often have children

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

Finding the Huntington Gene

• Blood samples taken

• Check for disease symptoms

• Paternity verified

Finding the Huntington Gene

• By luck, one haplotype segregated very closely with Huntington disease

• Marker was an RFLP called G8 (later called D4S10)

Finding the Huntington Gene

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

Finding the Huntington Gene

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

• More polymorphisms were found

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

Finding the Huntington Gene

• Exon trapping was key

• Compare cloned exons between normal and Huntington disease patients

Finding the Huntington Gene

Finding the Huntington Gene

• One exon, called IT15, contained an expanded CAG repeat….

• Mapping to 4 cM – 1983

• Cloning of Huntington gene – 1993

Complex Disease and Susceptibility

Gene

Gene

Disease

Single gene disorders

Mendelian Inheritance

High penetrance

Low environmental influence (but sometimes significant)

LOD-based linkage analysis works great

Genetic heterogeneity

Low population incidence

Complex Disease and Susceptibility

Gene

Gene

Gene

Gene

Environment

Disease A Disease B Disease C

Multifactorial disorders