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


• 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


• 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


• Both groups published results simultaneously

• Celera – Science

February 2001

• IHGSC – Nature

February 2001

http://www.nature.com/cgi-taf/DynaPage.taf?file=/nature/journal/v409/n6822/index.html


Nature 409, 818 - 820 (15 February 2001)


• 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

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– Methods for finding genes– Easy in prokaryotes

Human Genome

• Gene discovery– Difficult in eukaryotes

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”

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

13-1

13_06.jpg

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


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

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


• 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


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

• More polymorphisms were found


• 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


• Exon trapping was key

• Compare cloned exons between normal and Huntington disease patients


• 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


Gene

Gene

Gene

Gene

Environment

Disease A Disease B Disease C

Multifactorial disorders


• 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

Genetic Component in Complex Disorders

• Relative risk

r= frequency in relative of affected person

Population frequency


• Family Studies

Class of relative Proportion of genes shared

Examples

First degree 50% Parent/child, siblings

Second degree 25% Grandparent/grand-child, aunt/niece

Third degree 12.5% Cousins


• Problem of environmental impact

Congenital Malformations

Cleft lip Pyloric stenosis

General population 0.001 0.001

First degree relatives X40 (0.04) X10 (0.01)

Second degree relatives X7 X5

Third degree relatives X3 X1.5


Disorder Monozygotic Dizygotic

Breast cancer 6.5 5.5

Type I diabetes 30 5

Type II diabetes 50 30

Multiple sclerosis 20 6

Peptic ulcer 64 44

Rheumatoid arthritis 50 8

Tuberculosis 51 22


Disorder Monozygotic Dizygotic

Alcoholism 40 20

Autism 60 7

Schizophrenia 44 16

Alzheimer’s 58 26

Dyslexia 64 40

• In polygenic diseases, risk (susceptibility) alleles increase the phenotypic value

• Traits may appear continuously variable

• Traits may appear discontinuous



• How to find susceptibility gene?– Four main approaches

1. Candidate gene

2. Parametric linkage analysis

3. Non-parametric linkage analysis

4. Population association studies

Candidate gene

• Before searching the whole genome, think about what genes may be involved– Eg., Type I diabetes – Some genes involved in cell-mediated

immunity are located on chromosome 6 (Human leukocyte antigen region)

– Linkage between Type I diabetes and HLA was closely examined

• After a small genomic region is isolated, determine best candidate gene

Parametric Linkage Analysis

• Standard LOD score analysis, as used for single-gene disorders


• Eg., breast cancer susceptibility genes• Collect family history of >1500 breast cancer

patients

– Some family histories showed multiple cases occurring at early ages – could be a Mendelian allele segregating

– Best model suggested a dominant single-gene allele with a population frequency of 0.0006 – this suggested about 5% of total breast cancers



patients

– Now, look for families with multiple breast cancer cases with early onset

– Genotype family members and look for linkage

– Linkage (significant LOD score) to breast cancer was found to a marker on 17q21



patients

– The gene involved was cloned, like other single-gene disorders

– Breast cancer (BRCA) 1 gene– tumor suppressor gene involved in genomic stability

– LOH leads to high penetrance of breast cancer, as well as ovarian cancer



patients

– However, examination of BRCA1 mutations outside of affected families suggests lower penetrance


• Other successes in finding Mendelian risk factors in polygenic diseases

– HNPCC – non-polyposis colon cancer• MSH1, MLH1, PMS1, PMS2

– FAP – familial polyposis colon cancer• APC

– Premature heart disease - hypercholesterolemia• Mutation of the LDL receptor


• Familial hypercholesterolemia– Autosomal dominant


• Familial hypercholesterolemia

• 200 mg/dl - 350 mg/dl - dietary, common

• 400 mg/dl - 600 mg/dl - heterozygous, uncommon

• >600 mg/dl - homozygous, rare


• Familial hypercholesterolemia

• Autosomal dominant; allele frequency about 1:150


• Spectacular misfires as well:– Bi-polar disease (manic depression)– Initial linkage to HRAS and INS on

chromosome 11– LOD scores of 4.08 and 2.63– Two individuals in extended family

misdiagnosed– Lowered LOD score to 1.03 and 1.75

Non-parametric Linkage Analysis

• Genomic regions surrounding risk alleles will be inherited from a common ancestor in affected individuals to a greater frequency than by chance – also called autozygosity mapping

• Search for commonly inherited regions by polymorphic microsatellites, SNP’s, etc.

• High throughput analysis critical


• Common to use Affected Sib-Pairs (ASP)

• Collect genotypic data for 100’s of ASP

• 300+ microsatellite markers genotyped for 10cM coverage

• Look for significant IBD (>chance occurrence)


• IBD: if parental alleles differ at locus, then sibs that have both alleles in common are identical by decent

• IBS: if parental alleles are not know, then we can only say sibs are identical by state

Population association studies

• Association studies are carried out on populations

• Look for alleles that segregate with the disease in a whole population– Direct causation– Natural selection– Linkage disequilibrium


• Linkage disequilibrium

• Combination of alleles at two closely linked loci occur more often than expected by chance from population frequencies

• Recombination reduces linkage disequilibrium


• Linkage disequilibrium vs. Linkage Mapping– Mapping is performed on families with few

informative meiosis; LD is determined on populations after many generations

– Mapping will show linkage over large distances; LD is visible only over short distances


• How to find susceptibility genes?– Four main approaches

1. Candidate gene

2. Parametric linkage analysis

3. Non-parametric linkage analysis

4. Population association studies

Alzheimer’s Disease (AD)

• North America – 0.1% at 60, 10% at 80, 30% at 90

• Early onset: <60

• Neurofibrillary tangles in the cerebral cortex and amyloid plaques in the brain

• Neuronal apoptosis occurs in the hippocampus and cerebral cortex – memory and learning


• Neurofibrillary tangles – polymerized tau protein

• Amyloid plaques – deposition of the -amyloid protein


• Apoptosis of neuronal cells– Sometimes called “Programmed cell death”– Energy-utilizing program of orderly self-

destruction– Organized dismantling of the cell to avoid

autoimmune reaction

Apoptosis

Apoptosis

• Activation of proteases (cysteine-aspartic acid specific; called Caspases)

• Cascade of “irreversable” proteolysis

• Activation of endonuclease – chops up the cells DNA – no going back now!

Apoptosis

• Apoptosis occurs:– During development– Removal of immunological cells– In cells with DNA damage– Defeated in cancer cells

• Neuronal cells maintain survival by exposure to “neurotrophins”

Search for Susceptibility Alleles for Alzheimer’s Disease

• Some clues as to causative agents of AD

– Down syndrome individuals develop clinical features of AD when they live >30 years

– Suggested that chromosome 21 may be involved in AD

– Parametric linkage analysis located a locus on chromosome 21q in early-onset familial AD

Causative genes in AD

• Amyloid precursor protein (APP) over-abundant in Alzheimer’s and Down syndrome individuals

• Amyloid precursor protein gene mapped to chromosome 21

• Trisomy 21 causes a over-expression of genes from chromsome 21, including APP


• APP – a causative agent of AD and involved in pathology of Downs syndrome

• Large transmembrane protein processed by , or -secretase

• -secretase generates A40 protein – non-toxic and the main protein in normal brain


• and-secretase generates A42 protein – toxic and insoluble – which forms plaques

• After APP was found by parametric linkage, mutations were found

• In familial AD, mutations in APP increased the amount of A42 cleavage


• More parametric linkage analysis within families of early-onset AD

– Presenilin I and II were discovered on chromosome 14 and 2

– Presenilin I is a -secretase – leading to increased A42 secretion


• 1% of AD is familial, and shows strong Mendelian inheritance of altered A42 generation

• What about risk alleles in sporadic AD? – 99% of cases


• Non-parametric linkage analysis was performed on Affected Pedigree Member (APM)

• 32 families in which 87 of 293 members showed AD

• Linkage with locus on chromosome 19

Causative genes in AD• In this region was the gene for Apolipoprotein E.

• ApoE was found in plaques and tangles– Good candidate

• A population association study was performed

• Three alleles of ApoE were identified:– ApoE2 (6%), ApoE3 (78%) and ApoE4 (16%)

• Strong LD was found for allele ApoE4 and several nearby SNP’s


• ApoE4 is a risk factor Alzheimer’s disease

ApoE4 dose

% affected Relative Risk

Age of onset

0 20 1 84.3

1 46.6 2.84 75.5

2 91.3 8.07 68.4

Summary

• Family, adoption and twin studies provide evidence of genetic component to complex disease

• Risk of disease is the combined effect of polygenes influenced by environment, thus termed multifactorial

• Combined affect of many common alleles each providing a small effect, or of a few uncommon alleles with large effect

• Candidate gene, parametric and non-parametric linkage analysis, and population association analysis are used to find risk factors for multifactorial disease

sequencing the human genome

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