Presented at Hudson Valley Science Cafe

1. Insights from The Human Genome
Project about Risk of Disease
Toby G. Rossman, Ph.D.
The Nelson Institute of Environmental Medicine
NYU-Langone School of Medicine

2. In 2003, scientists in the Human Genome Project
obtained the DNA sequence of the 3 billion base
pairs making up the human genome.
The significance of this work to our health is an
ongoing project.

3. THE HOPE:

4. Genes and The Genome
Genes are made of DNA and provide the directions for
building all of the proteins that make our bodies function.
Genes are passed down by parents to their offspring.
The Genome is the sum total of all genetic material (DNA)
in a cell.

5. Introduction to DNA
A double helix (2 strands) with a
sugar-phosphate backbone.
Attached to each sugar are one
of 4 “bases”: A, G, T, C
Base pair:
C always pairs with G
A always pairs with T
The sequence of the bases (e.g.:
AATGCCCTGAACGTT) contain the
information (genetic code).

6. What DNA does
DNA is the genetic material
within the nucleus. It is many
DNA is the genetic material
genes long.
within the nucleus.
Replication
DNA is replicated before a cell
The process of transcription
divides. Each daughter cell
creates an an identical copy.
receives RNA using DNA
DNA information.
Transcription
The DNA in a gene is transcribed
The RNA leaves thecopy, using
to create an RNA nucleus.
information from one DNA strand. RNA
The process of translation
takes RNA leaves the nucleus.
The place in the Nucleus
cytoplasm. Translation
In the cytoplasm, the RNA is
I creates a protein using
translated to create a protein
RNA information. Protein
using RNA information.
Cytoplasm

7. The “Central Dogma” of biology
DNA
stores all information
transcription
mRNA
carries the message
Codon = 3 bases translation
Protein A protein is a string of amino acids

8. The Genetic Code
The genetic code is universal.
All known organisms use the same genetic code.
(Unity of life on earth; we are all related).
The genetic code is degenerate.
Some codons encode the same amino acid.
e.g. GGU, GGC, GGA, and GGG all encode glycine.
Degeneracy is mostly at the third base of the codon.
Some codons have additional functions.
AUG encodes methionine, but is also a start codon.
UAA, UAG, and UGA do not encode an amino acid.
These codons signal termination of the protein (stop codon).

9. DNA is packed into chromosomes (highly condensed)

10. Genes are present both in the nucleus and in mitochondria
Chromosomes located in cell nucleus
1 chromosome in
Autosomes mitochondria (hundreds
of copies in cell
cytoplasm)
1 2 3 4 5 6 7 8 9 10 11 12
Mitochondrial
DNA
13 14 15 16 17 18 19 20 21 22 X Y
mtDNA
Sex-
16,569 bp
Nuclear DNA
3.2 billion bp chromosomes Inherited only from
mother

11. An important technique: Hybridization
Heating the cellular DNA will separate the 2 strands.
You can then identify a piece of DNA by hybridization, using
a “probe” of the complementary strand (at least 12 bases).
This also works with RNA.
HOT
COLD

12. Painting the human chromosomes
Chromosome painting

13. A pair of homologous chromosomes (one from father and one from mother)
Most genes are in the 23 pairs of
chromosomes in the cell nucleus.
p Chromosomes contain thousands of
genes.
SOME DEFINITIONS
Gene – unit of DNA information about a trait
Alleles – slightly different versions of a gene
Q
Homozygous: both alleles are the same
Heterozygous: the alleles are different

14. Some important lessons from the Human
Genome Project
• The human genome is nearly the same in all people.
We are >99.9% alike.
• Only about 1.5% of the human genome contains
genes that are translated into proteins.
• Most of our DNA is transcribed into RNA, but not
translated.

15. Only a small % gets translated into protein
NEXT
SLIDE

16. The DNA in a gene is interrupted. It is not all expressed.
Exons are expressed; Introns are removed
Alternative splicing of exons allows more than 1 protein from each transcript
exon intron exon intron exon intron exon
EXON =
expressed
INTRON = not
expressed

17. What is all that DNA for?
It turns out that 90% of the genome is actively transcribed into RNA.
Although initially dismissed as “junk DNA”, recent evidence suggests
that many of the RNAs transcribed from this DNA play major biological
roles in control of gene expression, cellular development and
metabolism.
For example:
The Human Genome encodes only ~20,000 protein-coding genes. But
we have about 100,000 different proteins! Alternative splicing yields
many more than 20,000 proteins. RNA molecules may help control
which splicing event occurs.
We are at the tip of a very large iceberg!

18. Genetic variation
Single nucleotide polymorphisms (SNPs)
• Our genes are >99.9% alike (unless we are identical
twins).
• Gene sequences can differ at a single base (called
single nucleotide polymorphism or SNP).
• The human genome has at least 10 million SNPs.

19. Understanding SNPs
• Most variations are meaningless and do not affect our
ability to survive or adapt.
• For example: silent mutations in DNA, which change the
DNA, but do not change the amino acid the DNA codes
for.
• Other mutations may change the amino acid sequence of
a protein, but not the overall function of that protein.
• Some variation leads to disease. Single-gene disorders
include sickel cell anemia, cystic fibrosis and Huntington
disease.

20. There are other types of genetic variation besides SNPs
Eleanor Rigby picks up the rice in the church. (Original sequence)
Eleanor Rigby picks up the lice in the church. (SNP)
Eleanor Rigby picks up the church. (deletion)
Eleanor Rigby picks up the rice and beans in the church. (insertion)
Eleanor Rigby picku pt her icei nt hec hurch. (-1 frameshift)
Eleanor Rigby picks hcruhc eht ni ecir eht pu. (inversion)
Eleanor Rigby picks up the rice in the church. Eleanor Rigby picks up
the rice in the church. (gene duplification)
Rearrangement: Gene gets transposed to another chromosome.

21. Gene duplication: A major event in evolution
Gene duplications produce copy number variations (CNV) in
humans

22. Copy number variation (CNV)
Copy number for amylase-1 gene is higher in populations with a starchy diet.
Amylase digests starch.
50%
Perry et al.,
Nature
Genetics
2007
Gene copy number/cell

23. How different are human beings from one another?
 Differences in SNPs: about 10 million (~3/1000 bases)
 Large deletions and insertions: about 15 million base
differences
 Copy number variations: can lead to very large numbers of
base changes

24. Risk Factors for Disease
A risk factor increases your risk of developing a disease
or health problem.
Genes
“Environment” Behaviors and lifestyle (including diet)
+ = Environment
How can you tell which is more important? Sometimes a single
gene mutation leads to disease. THE EASY CASES

25. SICKLE CELL ANEMIA
An example of a single gene disease
• Caused by a mutant allele h of a hemoglobin gene H.
• 1/500 black Americans have the disease.
(homozygous hh)
• 1/10 is heterozygous (Hh) for the sickle cell gene
• The mutant gene is even more common among West
African Blacks.
• In some parts of Africa, the fraction of individuals with
this disease is 1/25
• The mutant allele confers some resistance to malaria.

26. Sickel cell anemia is caused by a single base change (SNP)
Fig. 11.2

27. Some other genetic disorders caused by single genes
Disorder Incidence (US) Symptoms
Cystic fibrosis 1/2,000 Caucasians Obstructive lung disorder,
1/17,000 African Americans infections, heart failure
1/9,000 Hispanics
Tay-Sachs disease 1/300,000 in US Destroys nervous system at about
8 months. Rarely live past age 2.
1/3500 in Ashkenazi Jewish
Huntington’s disease 1/20,000 (Western countries), Gradual deteriation of nervous
less freq. in Africa & Asia system
Maple syrup urine disease 1/9,000 to 1/300,000 Vomiting, seizures, mental retard.,
1/176 in PA Mennonites coma. Death in 2 years
Alpha-1-antitrypsin 1/2000 Early lung disorder (by 40 years)
BRCA1 or BRCA2 breast Will discuss later along with Increased risk for breast cancer
other cancer risk genes
cancer
Phenylketonurea (PKU) Mental retardation, excretion of
phenylalanine
Hemophilia B X-linked recessive Lack of clotting factor IX

28. Complexities in single gene diseases: Cystic Fibrosis
Cystic fibrosis (CF) is caused by an autosomal recessive mutation in
the gene CFTR (cystic fibrosis trans-membrane conductance
regulator).
However, it is not possible to predict the exact phenotype of this
disease from analysis of CFTR mutations. Other genes modify the
disease.

29. Polygenic inheritance
The Harder cases
• These underlie some of the more clinically important
human diseases including
• Heart disease
• Stroke
• Diabetes
• Schiozphrenia

30. Body Mass Index (BMI)
• At least 17 genes interact to control body weight: Genes
that affect how much we eat, metabolic rate and fat
distribution.
• One of these genes encodes a protein hormone called
leptin. Eating stimulates fat cells to secrete leptin. Leptin
travels to the brain and signals it to supress appetite and
increase metabolism to digest the food.
• Low levels of leptin indicate starvation, which triggers
hunger and decreases the metabolic rate.
• However, body weight is not entirely genetically
determined.

32. Breast cancer risk differs in different countries: Heredity or Environment?
US

33. Breast cancer risk cannot be entirely genetic.
For example, diet, obesity, radiation exposure, and alcohol
use influence risk.
e.g. Alcohol Use
• Adult use associated with risk (about 10% increase for each
drink per day)
• All studies have reported impact of early age alcohol use on
breast cancer risk
• About twice the risk of breast cancer for women below 35 years
• Alcohol use increases estrogen levels
• Adequate folic acid (B vitamin) may decrease risk in women who
have more than 1 drink per day

34. Another example: stomach cancer
High incidence in Japanese men in Japan, but lower in Hawaii

36. Example of multifactor causation: breast cancer
or

37. Other inherited cancer syndromes
Syndrome Primary tumors Other tumors/traits
Dominant
Familial retinoblastoma retinoblastoma osteosarcoma
Hered. non-polyposis colon can. colorectal many
Familial adenomatous polyposis colorectal other GI, jaw, brain
Nevoid basal cell carcinoma skin jaw cysts, ovary, fibomas
Familial melanoma skin pancreas
Multiple endocrine neoplasia (1/2) pancreas(1), thyroid(2) other endocrine
Li-Fraumeni syndrome sarcoma, breast brain, leukemias
Recessive
Ataxia telangiectasia lymphoma cerebellar ataxia
Bloom’s syndrome solid tumors immunodeficiency,
small stature
Xeroderma pigmentosum skin abnormal pigmentation
Fanconi’s anemia AML skeletal abnormalities

38. Chemical carcinogens often cause DNA damage which
leads to mutations if the damage is not repaired
N2
H
O N
N
OP O
-
A
O ON N Mutation
O
O
N
H
Exposure to Metabolic -
OP O T
O ON O
mutagenic/carcinogenic activation
compounds O G*
O
N N
H
-
Cancer
OP O
O ON N NH
H
N2 O
H
HO
OH
O N
-
OP O
N O
O O
C DNA repair
O
DNA adduct
No Cancer

39. DNA repair defect diseases

40. Involvement of BRCA1 in DNA repair
Proteins known to bind to BRCA1’s BRCT domains—
such as Abraxas, BACH1, and CtIP—are all involved in
the DNA repair process.
This meshes with the prevailing view that faulty DNA
repair by a mutated BRCA1 results in genomic
instability, which ultimately leads to tumorigenesis.

41. 2007: The Year of GWA Studies
Pennisi E, Science 2007; 318:1842-43.

42. THE NEWEST METHOD FOR GENETIC ANALYSIS:
Genome-Wide Association Study (GWAS)
GWAS is an approach that involves rapidly scanning genetic
markers across the complete sets of DNA (genomes), of many
people to find genetic variations associated with a particular
disease.
Such studies are being carried out to find genetic variations
that contribute to common, complex diseases, such as
asthma, cancer, diabetes, heart disease and mental illnesses.

43. The Gene Chip (microarray)

44. Microarray chips
Oligonucleotide probes representing many
genes are spotted in an array.

46. Types of microarrays
Comparative Genomic Hybridization (CGH): for genomic gains
and losses or for a change in the number of copies of a
particular gene.
Microarray expression analysis: to determine the level of
expression of a gene. Reflects subject’s mRNA levels.
SNP or mutation analysis: In this case, gene sequences placed on
any given spot within the array will differ from that of other
spots, by only one (SNP) or a few specific nucleotides.

47. Genome-Wide Scan for Type 2
Diabetes in a Scandinavian Cohort:
SNP results
ttp://www.broad.mit.edu/diabetes/scandinavs/type2.html

48. Type 2 Diabetes
GWAS have accelerated the identification of type 2
diabetes susceptibility genes. There are now at least 19 loci
containing genes that increase risk of type 2 diabetes.
Individually, most of these variants confer a modest risk
(odds ratio [OR] = 1.1–1.25) of developing type 2 diabetes.
To date, these approaches have only identified two genes:
PPARG (peroxisome proliferator-activated receptor-r ) and
KCNJ11(potassium inwardly-rectifying channel J11)
robustly implicated in type 2 diabetes susceptibility.

49. GWAS for SNP Associations with
Myocardial Infarction show hot region
on Ch 9
Samani N et al., N Engl J Med 2007; 357:443-53.

50. GWAS for Age-Related Macular
Degeneration
Klein et al, Science 2005; 308:385-389.

51. Wellcome Trust GWAS of Seven Diseases
WTCCC,
Nature 2007;
447:661-678.

52. BIG SURPRISE: Most SNPs associated with
disease susceptibility are in introns and intergenic positions!!

53. Unique Aspects and problems of GWAS
• GWAS permits examination of inherited genetic variability
at unprecedented level of resolution.
• GWAS permits "agnostic" genome-wide evaluation.
• Once a genome is measured, it can be related to any trait.
• Most robust associations in GWA studies have not been
with genes previously suspected of association with the
disease.
• Many associations are in regions that do not harbor
genes.
But with more than 500,000 comparisons per study, the
potential for false positive results is unprecedented (and
expensive!).
Most associations are NOT robust.

54. Further Reading (and some criticisms)
Taft et al., Non-coding RNAs: regulators of disease. J.
Pathology 220:126-139, 2010
Roberts et al., The predictive capacity of personal genome
sequencing. Science Translational Medicine 2010
10.1126/scitranslmed.3003380 stm.sciencemag.org
Ioannidis et al., A compendium of genome-wide
associations for cancer: Critical synopsis and reappraisal.
J. Natl. Cancer Inst. 102:846-858, 2010
Bell, Our changing view of the genomic landscape of
cancer. J. Pathology 220:231-243, 2010
Vineis and Pearce, Genome-wide association studies may
be misinterpreted: genes versus heritability.
Carcinogenesis 32:1295-1298, 2011

55. Websites
CDC, Office of Genomics and Disease Prevention
www.cdc.gov/genomics/public/famhist.htm
"DNA Interactive" Site from Cold Spring Harbor Labs: http://www.dnai.org/index.htm
Howard Hughes Medical Institute's "Biointeractive" Site
http://www.hhmi.org/biointeractive/genomics/microarray.html
Learn Genetics — Genetic Science Learning Center
http://learn.genetics.utah.edu/
National Center for Biotechnology
http://www.ncbi.nlm.nih.gov/About/primer/snps.html
http://hapmap.ncbi.nlm.nih.gov/
Animations On How DNA Microarrays Work
http://www.imagecyte.com/animations/array2.html
http://www.bio.davidson.edu/courses/genomics/chip/chip.html

56. Some other things besides genes are important

59. Founder Effects
Occur when a population is established by a small number of
people. A mutation in one of the founders becomes prevelent in the
resulting population.
Afrikaners (S Africa) Familial hypocholesterolemia, APC, BRCA1/2, Blooms
French Canadians HED (skin disorder), congenital adrenal hyperplasia
Finns hMLH1, diastrophic dysplasia
Icelanders BRCA2
Dutch BRCA1/2, melanoma
Norwegians BRCA1
North Africans Allgrove syndrome
Swedes BRCA1/2
African Americans BRCA1
Germans (Black Forest) Von Hippel-Lindau disease
France (Rhone Alps) Hemophilia B
Sicilians Glycogen storage disease type II
South Italians CDA-II (anemia)

60. Steps in Chemical Carcinogenesis (also radiation)

61. HAPMAP
Testing all of the 10 million common SNPs in a person's
chromosomes would be extremely expensive. The
development of the HapMap will enable geneticists to take
advantage of how SNPs and other genetic variants are
organized on chromosomes. Genetic variants that are near
each other tend to be inherited together. For example, all of
the people who have an A rather than a G at a particular
location in a chromosome can have identical genetic
variants at other SNPs in the chromosomal region
surrounding the A. These regions of linked variants are
known as haplotypes.

62. Diseases and Traits with Published GWA Studies (n = 76, 11/17/08)
• Macular Degeneration • Lipids and Lipoproteins • Syst. Lupus
• Exfoliation Glaucoma • Warfarin Dosing Erythematosus
• Ximelegatran Adv. Resp. • Sarcoidosis
• Lung Cancer • Pulmonary Fibrosis
• Prostate Cancer • Parkinson Disease • Psoriasis
• Breast Cancer • Amyotrophic Lat. • HIV Viral Setpoint
• Colorectal Cancer Sclerosis • Childhood Asthma
• Bladder Cancer • Multiple Sclerosis
• Neuroblastoma • MS Interferon-β Response • Type 1 Diabetes
• Melanoma • Type 2 Diabetes
• TP53 Cancer Predispos’n • Prog. Supranuclear Palsy • Diabetic Nephropathy
• Chr. Lymph. Leukemia • Alzheimer’s Disease in • End-St. Renal Disease
ε4+ • Obesity, BMI, Waist, IR
• Inflamm. Bowel Disease
• Cognitive Ability • Height
• Celiac Disease
• Memory • Osteoporosis
• Gallstones
• Hearing • Osteoarthritis
• Irritable Bowel Syndrome
• Restless Legs Syndrome • Male Pattern Baldness
• QT Prolongation • Nicotine Dependence
• Coronary Disease • Methamphetamine • F-Cell Distribution
• Coronary Spasm Depend. • Fetal Hgb Levels
• Atrial Fibrillation/Flutter • Neuroticism • C-Reactive Protein
• Stroke • Schizophrenia • ICAM-1
• Subarachnoid • Sz. Iloperidone Response • Total IgE Levels
Hemorrhage • Bipolar Disorder • Uric Acid Levels, Gout
• Intracranial Aneurysm • Family Chaos • Protein Levels
• Hypertension • Narcolepsy • Vitamin B12 Levels
• Hypt. Diuretic Response • Attention Deficit • Recombination Rate