Whole Genome Analysis and the

Human Condition
Thane Kreiner, PhD
Senior Vice President, Corporate Affairs
 The Genome is Complex

ƒ 3 B base pairs

ƒ Only ~1.5% of genome

encodes ~30,000 genes

ƒ Millions of differences
between any two people

ƒ Much unknown about “dark

matter” of genome
 Packaging Genetic Information

* *
* * *
*
5”

11µm

11µm

5” Millions of identical
probes / feature

1.28cm
Up to ~1,400,000
features / chip

1.28cm
Hybridization

C G C: G
T A T : A
A T A: T
A T A: T
G C G: C
A T A: T
G C G: C
C G C: G
Combinatorics and the Human Genome

Combinatorial theory:
YN compounds can be synthesized in Y x N
steps
where Y= number of components, N= length

(A+C+G+T)25 = All possible 25-mers (~1015)

in 4 x 25 = 100 chemical steps

Fodor, S. P. et al. Science 251(4995), 767-73, 1997

Combinatorics and the Human Genome

Since we can produce the entire set of 25-mers

in 100 chemical steps (or fewer), we can design
any array, including the entire human genome
in no more than 100 chemical steps.
 Content—features per chip

Feature Size
11 um 8 um 5 um

49 1,400,000 2,600,000 6,500,000

Format

400 110,000 200,000 500,000

2500 8,000 16,000 40,000

 Biology Teacher Developed Curriculum

ƒ IISME
ƒ High school biology teacher a summer intern
at Affymetrix
ƒ Developed curriculum for teaching about
arrays and their uses
ƒ Available on-line at:

www.affymetrix.com
 Character Based Literacy Program

ƒ Developed at Santa Clara University’s Markkula Center

for Applied Ethics by Steve Johnson
ƒ Being implemented on a county-wide basis
ƒ Currently focused on integrating ethics into language
arts programs; plan to expand to biology

www.scu.edu/ethics/practicing/focusareas/education/
 DNA, RNA and Proteins

Eukaryotic Cell ƒ DNA

ƒ Cell’s information library;
Nucleus
organized in segments (genes)

Proteins Transcription

ƒ RNA
ƒ Short-lived copies of active
DNA genes; amount of RNA is
RNA proportional to gene activity

Translation

ƒ Proteins
ƒ Active gene products: cell
components, enzymes,
hormones, receptors
Cell membrane
 Genetic Information Analysis

G G A G G C
A A
C C
G G
T T

GGA GGC
DNA Analysis
DNA

RNA

Eukaryotic Cell
RNA Analysis
“The Ultimate Gene Gizmo: Humanity on a Chip”*

ƒ Single U133 Plus 2.0 array contains

>1.3M features
─ High resolution: ~22 probes/transcript
─ ~50,000 transcripts and variants (all
well-annotated genes)
ƒ Utilizes recent draft of human genome
sequence (public domain) for best
information
ƒ “100 Coolest Inventions”
– Time Magazine

* Science Magazine, 10 Oct 2003

 Understanding Cancer

Therapeutic
Diagnosis Classification Prognosis
Choice

Is it benign? Which class of What are my Which treatment?

cancer? chances?
 Classifying Leukemia

ALL

AML

Which class of
leukemia? MLL
Golub, T.R., et al. Science 286: 531-537, 1999;
Armstrong, S.A., et al. Nature Genetics 30: 41- 47, 2002
Classifying AML, ALL and MLL

> 95%
correct
diagnoses

Armstrong, S.A., et al. Nature Genetics 30: 41- 47, 2002

 Childhood Leukemia

ƒ Childhood acute lymphoblastic leukemia

curable in 80% of patients
ƒ Cause of treatment failure in 20% unknown
– Test leukemic cells from 173 children for drug
sensitivity
– Differential gene expression patterns predict
sensitivity or resistance to four commonly used
drugs:
ƒ Prednisolone (33 genes)
ƒ Vincristine (40 genes)
ƒ Asparaginase (35 genes)
ƒ Daunorubicin (20 genes)

Holleman, et al. New England Journal of Medicine 351: 533-542, 2004

Leukemia: Predicting Response to Therapy

Holleman, et al. New England Journal of Medicine 351: 533-542, 2004

 Whole Genome Expression Analysis

ƒ How do gene patents affect commercialization of

diagnostics based on gene expression patterns?
ƒ What regulatory paradigms best balance patient
benefit and potential harm for this type of test?
ƒ How will the ability to “personalize” cancer treatment
affect standardization of care through DRGs?

www.nuffieldbioethics.org
www.geneticage.org
Teaching Idea: Gene Expression Patterns

ƒ Provide background on arrays and gene

expression analysis
ƒ Ask students to make treatment
recommendations for children with leukemia
who have different gene expression patterns
ƒ Hold a class discussion on how gene patents
affect the ability to bring new diagnostics and
drugs to the benefit of these children
RESOURCES
ƒ www.affymetrix.com
ƒ www.genome.gov/Education/
 Genetic Information Analysis

G G A G G C
A A
C C
G G
T T

GGA GGC
DNA Analysis
DNA

RNA

Eukaryotic Cell
RNA Analysis
Genetics: Preserving a way of life

ƒ Old order Amish have roots at

the time of the Reformation in
Europe
ƒ Isolated in Switzerland,
Southern Germany starting
around 1536
ƒ Small founder population
(~200) of Amish arrived in PA in
the 1720s: “religious tolerance”
ƒ ~ 12 generations since arrival
ƒ Custom not to marry “outside”
Autosomal Recessive Traits
Clinic for Special Children
Strasburg, PA

www.clinicforspecialchildren.org
 Two Generations, Nine Families,
21 Infant Deaths

Puffenberger, EG et al. Proc Natl Acad Sci U S A 101, 11689-94, 2004.

The DNA of any two people is 99.9% identical

A A
1
G G
C C
. .
. .
. .

T T
1000 G C
A A
. .
. .
. .

G G
2000
A C
Whole Genome Assay (WGA):
Scalable Complexity Reduction

Hybridization-
General complexity
based allele
reduction scheme AA BB AB
discrimination
Genome Coverage of 400
Microsatellite Marker Panel

Blue = microsatellites
Red = at least 1 SNP/100 kb
Black = Gaps
Median intermarker distance: 105 kb

Mean intermarker distance: 210 kb

Average Heterozygosity 0.37

 Identifying TSPYL

Puffenberger, EG et al. Proc Natl Acad Sci U S A 101, 11689-94, 2004.

Challenges in Newborn Screening

ƒ Genetic literacy
ƒ Public perception of genetic determinism
ƒ Ownership/control of information
ƒ Varying disease prevalence
ƒ Public health infrastructure
ƒ Regulatory issues
ƒ Off label use
– Pre-implantation genetic diagnosis
– Prenatal screening
– Predisposition testing?
 Genetic Screening to Identify
Inherited Disorders

ƒ Types of screening: ƒ Impact of disease

– Carrier – Death in childhood
– Preimplantation genetic – Moderate disability/variably
diagnosis shortened lifespan
– Prenatal – Mild or no disability/not
– Newborn necessarily shorten lifespan
– Predisposition – Adult onset
– Diagnostic

Leib, et al., in press

Genetic Tests

ƒ In the U.S. alone, over 500 laboratories offer

tests for more than 1000 genetic diseases
ƒ Over 15,000 entries in the Online Inheritance
in Man (OMIM) database
ƒ With the accelerated discovery of disease-
related loci, many more tests will emerge
ƒ Many social and policy issues, e.g.:
– What tests should be permitted and under what
conditions?
– How is off-label use handled?
– Who decides?
Teaching Idea: Genetic Testing

ƒ Provide background on Mendelian disorders

ƒ Have students work through several case
examples (autosomal recessive, autosomal
dominant, X-linked)
ƒ Form student committees that set policy for
different types of screening
RESOURCES
ƒ www.genetests.org
ƒ www.ncbi.nih.nlm.gov/omim/
Autism

ƒ Neurological disorder
ƒ Disrupts children’s ability to speak and relate to
others at 18 months
ƒ Complex disease, probably > 20 genes involved
ƒ Growing epidemic: 1 in 200 children, up from 1 in
10,000 only 10 years ago
 All Disease is a Result of Genes Interacting
with the Environment…

Exercise Diet Drugs

Genetics Environment
Diabetes
HIV / AIDS
Tay-Sachs Lead Toxicity
Cancer
Cystic Fibrosis Heart Disease

Infectious Disease Lifestyle Choices Exposures

NAAR Autism Genome Project

ƒ National Association for Autism Research

ƒ 170 scientists collected 6000 samples from 1500
families
ƒ Mapping 10K to be used in largest study of
genetics of autism
ƒ Project involves families, doctors, foundations,
NIH, and companies

Video
 Genes and Complex Conditions

ƒ Most common, complex conditions result from

the interaction of many genes with each other
and the environment
ƒ In many cases, the contribution of an
individual gene to the trait may be so small
that it is not possible to reliably ascertain its
contribution v. environmental influences
ƒ Possibility exists for “genetic determinism” and
inappropriate uses of genetic information
Genetic Discrimination Example:
Burlington Northern

ƒ 2000: Burlington Northern began secret genetic

testing of its workforce
– The railroad hoped to prove a pre-existing condition, thus
reducing their workers’ compensation costs.

ƒ The EEOC sued for discrimination based upon

predictive genetic testing

ƒ 2002: Case settled with Burlington Northern paying

$2.2 million to the workers
Teaching Idea: Genes and the Environment

ƒ Have students research common, complex

disorders such as heart disease, cancer,
autism, diabetes, etc.
ƒ Students make recommendations on if, when
and how a genetic test should be deployed
ƒ Hold a class discussion on other public health
measures that might be more effective
RESOURCES
ƒ www.nlm.nih.gov
ƒ www.affymetrix.com/
 Whole Genome Analysis Technology

1,000,000
100,000+
SNPs Validated
SNPs
First in a family of products
for whole genome association

10,000
SNPs

Complete solution for linkage

Multiple products over 18 months
Next product 500K on 2 arrays
100K SNP Arrays (121,575 SNPs)

Median intermarker distance: 8.0 kb

Mean intermarker distance: 22.5 kb

Average Heterozygosity 0.21

Genotyping an Island: Kosrae
 Whole Genome Association Study - Kosrae
Rockefeller University, Jeff Friedman

ƒ Obesity was virtually unknown prior to World War II

ƒ Diet primarily consisted of fish and vegetables
ƒ Now 85% are clinically obese due to introduction of US aid and food relief

ƒ Goal: Define associations between specific genes and obesity

and obesity-related diseases
ƒ Whole genome association study: 200,000+ SNPs
ƒ 3,000+ individuals (entire adult population)
ƒ High resolution view of genome: 600 million genotypes
The DNA of any two people is 99.9% identical

A A
1
G G
C C
. .
. .
. .

T T
1000 G C
A A
. .
. .
. .

G G
2000
A C
 Human Diversity: each variant arose only once

A A A A A A A A A
1
T G G G G G T G T
C C C C C C C C C
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .

T T T T T T T T T
1000
G C G G C G G G G
A A A A A A A A A
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .

G G G G G G G G G
2000
C C A A C A C A C
 Grouping people based on common haplotypes

A A A A A A A A A
1
G G G G G G T T T
C C C C C C C C C
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .

T T T T T T T T T
1000
C C G G G G G G G
A A A A A A A A A
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .

G G G G G G G G G
2000
C C A A A A C C C
Genome Coverage of Mapping 10K

Blue = microsatellites
Red = at least 1 SNP/100 kb
Black = Gaps
Median intermarker distance: 105 kb

Mean intermarker distance: 210 kb

Average Heterozygosity 0.37

10K Northern European Specific (18 SNPs)
10K Asian Specific (24 SNPs)
10K African Specific (240 SNPs)
Population Genetics: Out of Africa
African Ancestry
 African Lineage Database

ƒ 10,386 paternal lineages & 11,170 maternal lineages

ƒ From 135 indigenous African populations
– The data is a compilation of published sources, research collaborations,
and primary research
– The populations sampled are based on direction from historians,
anthropologists, and linguists.
– There is a specific concentration on regions that are known to have
participated in the TransAtlantic Slave Trade.
 Genetic Ancestry Information

ƒ Lineage based tests

– Mitochondrial DNA: maternal lineage
– Y chromosome: paternal lineage (non
recombining)

ƒ Autosomal marker based tests

– Ancestry informative markers in the
autosomes
– Substantial differences in allele
frequency across population groups

Shriver MD and Kittles RA, Nature Reviews Genetics 5: 611-618, 2004.

 Lineage Based Tests: Informative Value

Example: Inferring the genetic ancestry of African-

Americans
ƒ From 1619 to 1850, tens of millions of indigenous
West and Central Africans kidnapped and transported
to the Americas
ƒ Approximately 14 generations in 350 years
ƒ Each person has at most 16,384 ancestors (214)
ƒ Lineage based tests= .0061% min

Shriver MD and Kittles RA, Nature Reviews Genetics 5: 611-618, 2004.

 Race & Genetics

ƒ Tremendous healthcare spending disparities

between culturally defined racial groups
ƒ More genetic differences within racial groups than
among them
ƒ Lack of consistent terminology in scientific literature
– “Confounding” of cultural groups and continent of origin
 Biogeographical Ancestry Estimation

Shriver MD and Kittles RA, Nature Reviews Genetics 5: 611-618, 2004.

 Teaching Idea: Personal Genetic History

ƒ Students conduct on-line research on companies

offering personalized genetic history services
ƒ Each student picks a number of generations since
one ancestor arrived in the Americas (or elsewhere)
ƒ Student calculates maximum number of ancestors
to that generation and informative value of lineage
based markers
ƒ Hold a class discussion on biological and social
characterizations of race
RESOURCES
ƒ www.google.com
 Genetics: Ensuring Beneficial Uses

ƒ Genetic literacy essential to individual choice

ƒ Federal legislation to protect against misuse
ƒ Lower barriers to bring benefits to patients
– Gene patents
– Translational medicine
– Standards
– Regulatory pathways
– Reimbursement paradigms
ƒ Open dialogue with all players engaged

www.affymetrix.com/corporate/outreach/