Ecogenetics, Evolutionary Biology,
Genomics, and Medicine
Gilbert S. Omenn, M.D., Ph.D.
University of Michigan
Conference “Darwin y Medicina”
Mexico City, UNAM, 25 April, 2008
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15-16 February, 2001
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It’s a New World
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New Biology---New Technology
Genome Expression Microarrays
Comparative Genomics
Proteomics
Bioinformatics, Computational Medicine,
and Evolutionary Biology
Path to predictive, personalized,
preventive (P3) healthcare
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Breakthrough
of the
Year, 2005
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“Medicine Needs Evolution”
Nesse, Stearns, Omenn: Science 24 Feb 2006
• Evolution: a vibrant foundation for all biology
• Evolutionary thinking can help researchers
and clinicians answer common questions:
o Persistence of genes predisposing to
depression, high blood pressure, diabetes, and
other diseases
o Usefulness of cough, fever, diarrhea
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Multi- and Interdisciplinary Research will be
Required to Solve the “Puzzle” of Complex Diseases
and Conditions
Genes
Behavior
Diet/Nutrition
Infectious agents
Environment
Society
??? [evolution]
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Eco-Genetics
Known inherited predispositions to:
Foods
Food additives
Infectious agents
Drug chemicals
Pesticides
Inhaled pollutants
Sensory stimuli
Allergic & sensitizing agents
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Eco-Genetics of Food, Nutrition,
and Cultural Sensitivity
• Anthropology and biology of lactase
persistence
• “Everybody needs milk”
• “Got Milk?”
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Digestion of Milk from the Diet
• Nearly all mammals digest the sugars in
Mother’s milk until weaning.
• The enzyme lactase in the intestine does it.
• Lactase production usually drops 90%
during the first four years of life in humans,
though the rate varies widely.
• Certain human populations have a mutation
(on chromosome 2) which results in
persistence of lactase activity (gain of
function).
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Lactose Intolerance by Group
Human Groups
Percent Intolerant
Allele Frequency
Swedish
2%
0.14
Europeans in Australia
4%
0.20
Swiss
10%
0.32
American Caucasians
12%
0.35
Finns
18%
0.42
African Tutsi
20%
0.45
African Fulani
23%
0.48
American Blacks
75%
0.87
Australian Aborigines
85%
0.92
African Bantu
89%
0.94
Chinese
93%
0.96
Thais
98%
0.99
American Indians
100%
1.00
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Consequences
• Persistence of lactase in the intestine permits the
“unnatural” continuation of milk ingestion
throughout life, enhancing growth and
development. One benefit is higher calcium
intake, especially in northern climes where sun is
insufficient to activate vitamin D.
• People without this mutation shut off lactase
production and become “lactose malabsorbers”;
they may become “lactose intolerant” with
symptoms of excess gas (hydrogen) and diarrhea
upon eating lactose-containing dairy products.
• The Navajo used powdered milk supplies to paint
their adobe homes.
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Why Does Loss of Lactase Lead to
Symptoms?
• The lactose in milk remains undigested and
unabsorbed through the intestinal wall into the
bloodstream.
• Gut bacteria adapt to the abundance of lactose
(compared with glucose, etc) and metabolize the
lactose, generating lots of gas by fermentation.
• Gas can cause stomach cramps, bloating, diarrhea;
the osmotic pressure rises, reducing resorption of
water from the colon: a laxative effect.
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Are Lactose Maldigestion and Lactose
Intolerance Important in Mexico?
Rosado et al, J Nutrition 124:1052, 1994:
Children and adults (N=926) from Sonora,
Mexico, and Yucatan
Milk consumption (in 1982) was 219+/-22,
128+/-25, and 19+/-1 gm/day
Whole milk vs lactose-hydrolyzed milk: major
intolerance symptoms uncommon (0-11% of
children, 7-17% of 13-60 y/o); maldigestion by
hydrogen breath test, 2-43%, highest in Yucatan.
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Lactose Malabsorption in MexicanAmericans
Wotecki et al, Amer J Clin Nutr 30:470,1977
Prevalence 53% of Mexican-Americans and 15% of
Anglo-Americans after 50 gm lactose load
(glucose increase <25 mg/dl).
Mean consumption of milk and dairy products was
same in lactose absorbers and malabsorbers in
both groups, though GI symptoms after milk was
60% vs 24%.
Some reduced milk-drinking due to symptoms;
others continued for laxative benefit!
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What is the Diagnosis? What is
“Normal”?
• In Europe and North America, lactose
intolerance is regarded as a clinical
problem, a medical diagnosis. Those who
have symptoms may not know the cause;
lactose intolerance actually accounts for
many cases of “irritable bowel syndrome”.
• In populations where most people are
lactose malabsorbers, cultural nutritional
practices will dominate.
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Prevention of Symptoms
• Avoid milk
• Drink lactose-free milk (enzyme-treated)
• Lactose-reduced (L. acidophilus) milk, as in
yogurt and in hard cheeses
• Lactase pills
• Gene therapy??
Note: Milk sold for pet cats [remarkably, European
cat breeds have a mutation similar to the human
European mutation, whereas Asian breeds are
particularly sensitive!]
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When Did This Mutation Arise?
Most likely:
1. Separate events in Sweden and in the Arabian
Peninsula about 4000 BC, converging as they
spread, or
2. Single event in the Middle East about 4500
BC, with radiation from there.
3. Plus a more recent mutation in the East African
Tutsi.
A small survival advantage over the 6000-8000 years
since goats, sheep, and cattle were domesticated
can account for the population differences now
observed.
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An Associated Polymorphism of
Apolipoprotein A-IV-2 (His360Gln)
• Apo A-IV is a 46 kD plasma glycoprotein
synthesized in the intestine.
• His/Gln polymorphism [in Caucasians] at codon
360 (chromosome 11) modifies milkfat absorption
and chylomicron clearance.
• Worldwide distribution resembles lactase
persistence; across 29 population groups,
r = 0.94 (p<0.000001). Highest prevalence in
Iceland; probably carried into Europe by Vikings.
[Weinberg, Genetic Epi 17:285, 1999]
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Eco-Genetics
Known inherited predispositions to:
Drug chemicals
Pesticides
Inhaled pollutants
Foods
Food additives
Sensory stimuli
Allergic & sensitizing agents
Infectious agents
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Variation in Susceptibility to Infections
• Malaria (P. falciparum type): children with
sickle hemoglobin trait have greater survival.
• Malaria (P. vivax type): people lacking the
Duffy (Fy) red blood cell antigen are resistant
to this parasite, which enters the cell through
this surface antigen.
• People lacking CCR5 receptors on
lymphocytes are resistant to infection by HIV.
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Distinguishing between Host Defenses
and Infectious Agent Offenses
• Symptoms of host defenses include pain, nausea,
vomiting, diarrhea, fatigue, anxiety
• Treatment of these symptoms may suppress
defenses, promoting pathogen transmission and
harming patients
• Shigella diarrhea: faster recovery and fewer
complications without anti-diarrheal meds.
• Cough suppressants raise risk of pneumonia
• Acute phase response to infection, trauma, or
cancers both behavioral (fever) and physiological
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(sequestration of Fe, Zn). Counterpart in insects.
Evolution and Persistence of Virulence
• Host-parasite relationships are greatly influenced
by the rapidity of transmission.
• When transmission is rapid, pathogens can
disregard many host variables.
• When transmission is slow, pathogens benefit by
establishing a long-term balanced relationship
with the host.
• Modeling shows vaccines which prevent infection
constrain prevalence and virulence; vaccines that
offer only anti-toxin immunity have opposite
effects.
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Emergence of “Meta-Genomics”
• “Microbes run the world”—converting key elements
of life (C,N,O,S) into accessible forms; performing
photosynthesis; living in ecological and human host
niches, making nutrients, metals, and vitamins
available to the hosts, digesting foods, breaking
down toxins, fighting off disease-causing microbes.
• Microbial communities have adapted through
billions of years of environmental changes. New
molecular methods permit analysis of whole
communities, not just individual species or isolates.
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Metagenomics, A New Science Revealing
the Secrets of our Microbial Planet
[National Research Council U.S., 2007]
• Combines the power of genomics, bioinformatics, and
systems biology
• Involves study of sequences and functions of genomes of
many organisms simultaneously, including a great many not
culturable—as presented by Dr. Francino today
• Libraries already prepared from oceans, coral reefs, soils,
whale carcasses, thermal vents, and hot springs
• Expect quite different microbial communities in oral cavity,
respiratory passages, gut, genito-urinary, and skin
environments of people with different genotypes and different
environmental circumstances, including diets, diseases,
infections, and therapies.
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Framework for Regulatory
Decision-Making for Chemicals
Hazard Identification
Epidemiology
Lifetime rodent bioassays
Short-term, in vitro/in vivo tests
Structure / activity
Potency (dose/response)
Risk Characterization
Exposure analysis
Variation in susceptibility
Information
Risk Reduction
Substitution
Regulation / Prohibition
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Impetus for Susceptibility Analysis
 Clinical Occupational Medicine - “Why me, Doc?”
 OSH Act requires that standards be set “so that no
worker…shall suffer adverse effects.”
 Clean Air Act requires standards set to protect “most
susceptible subgroups.”
 Food Quality & Protection Act requires protection of
children and other vulnerable subpopulations.
 To discover mechanisms or markers for diseases that
may appear only years later -- and could be prevented:
getting inside the “black box”
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Eco-Genetic Polymorphisms
Tissue susceptibility
G6PD deficiency
Alpha -anti-trypsin deficiency
1
Biotransformation enzymes
Acetylator phenotype
Debrisoquine hydroxylation
(Carbon oxidation - P450)
Paraoxonase activity
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Occupational Bladder Cancer
(Huddersfield, U.K. Data)
% Slow Acetylators
Controls
57
Bladder CA
67
Dye workers (naphthylamines) 96
Clerical workers
40
Machine oils workers
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Acetylator Phenotype: bimodal distribution
of plasma level of active drug (INH) @ 6
hours after standard dose
Genetics: Single gene
Enzyme: N-acetyl-transferase in liver
Other drugs that require acetylation
hydralazine
dapsone & other sulfas
phenalzine
cyclophosphamide
What about occupational or environmental chemicals
that are detoxified by acetylation?
Industrial anti-oxidants
Naphthylamines
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Benzidines, biphenyls
Susceptibility to Chronic Beryllium Disease
• An immune-mediated pulmonary granulomatous
disease among exposed workers
• HLA-DP gene contains a Glu lysine mutation
at position 69 (Glu-69) predisposes to CBD:
a genetic biomarker of susceptibility
• Can be combined with lymphocyte proliferation
test for evidence of sensitization to beryllium
• Value-of-information from mandatory and
voluntary screening programs has been
estimated for risk reduction possibilities:
social, legal, and ethical issues are crucial32
Molecular Signatures
• Numerous studies underway using gene
expression microarrays and proteomics to
identify and utilize chemical-specific
“molecular signatures” for exposures, early
effects, and evidence of variation in
susceptibility.
• Model chemical: acetaminophen-toxigenomics and toxicoproteomics of liver
damage.
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The “Thrifty Genotype” and
Maladaptation to Modern Environments
• Humans adapted to the Paleolithic environment of hunters
and gatherers are maladapted to modern lifestyles.
• Phenotypes are obesity, high BP, metabolic syndrome, and
adult-onset diabetes.
• Most humans consume much more fat, sugars, and salt, but
less fiber and phytochemicals. Not simply digestive and
cardiovascular systems, but how our brains perceive and
process stimuli from food sources.
• The evolution from quadripedal to bipedal gait puts
pressure on the lower spine, with low back pain,
complications of childbirth, and tendency to fainting and
circulating problems.
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Evolution and High Blood Pressure
[A. B. Weder, Hypertension 2007; 49:260]
Sodium Hypothesis to explain higher incidence of high
blood pressure in African-Americans– “thrifty
genotypes” to maintain sodium levels, prevent
excretion in kidneys, in times of poor diets, including
slave ships.
When BP increases, kidney output of Na+ and water are
increased, and BP falls: feedback.
Hypertension is viewed as an “emergent property” of the
entire cardiovascular system with feedback loops.
Epi studies: 30-50% of HBP risk due to heritable factors.
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Genes/Alleles Predisposing to High BP
• Angiotensinogen, AGT -6A
• G-Protein, beta-3 subunit, GNB3, 825T
These two are in genomic regions of broad linkage
disequilibrium and low haplotype diversity—hence
arose recently, were exposed to positive selection
• Beta-2 adrenergic receptor, 47A and 79C
• Epithelial sodium channel alpha-subunit (--946G)
• Cytochrome P450, CYP3A5*1
All 5 sodium-conserving ancestral variants are much
more prevalent in African (equatorial) populations.
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Growth & Development and Evolution of
High Blood Pressure (HBP)
Fetus is programmed to survive nutritional scarcity
in the mother; if born into an environment of
excess sodium and calories, leads to metabolic
syndrome, obesity, and high BP.
Fetal growth retardation with decreased glomerular
filtration capacity: set for higher range of BP
Thus, good maternal nutrition could prevent or delay
onset of some HBP cases.
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Allometry and Kidney Growth
• Kidney weight is proportional to body weight
with an allometric constant of 0.8, while blood
volume scales with constant 1.0.
• Kidney growth ceases at sexual maturity;
smaller as menarche and andrarche move to
younger ages.
Adaptation sets blood pressure and sodium
excretion higher.
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Uricase and Uric Acid
• Serum uric acid levels are higher in humans
that most other mammals (except guinea pigs
and Dalmatian dogs).
• Humans lack hepatic uricase.
• Uric acid can help maintain BP via
vasoconstriction in low sodium environments
• In high-sodium societies, high uric acid may
cause subtle renal injury, induce chronic salt
sensitivity, and lead to high BP.
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Dopamine-4 Receptor Repeat
Polymorphism and High Blood Pressure
• Dopamine release in the kidney increases renal
sodium excretion. In essential hypertension, DA
effect on sodium excretion is blunted.
• DA-4 receptors are expressed in juxtaglomerular
and cortical collecting cells; associations between
long allele in exon 3 and slight increases in
systolic BP, diastolic BP, and history of alcohol
use.
• DA-1 receptor (A48G) and GPCR kinase-4 gene
(GRK3gA142V) polymorphisms also associated
with high BP.
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Metabolic Syndrome in Mexico and in
Mexican Americans
[Bastarrachea et al, Human Biology 79:121, 2007]
• GEMM Family Study; so far, 375 individuals in
21 extended families.
• All metabolic and clinical endpoints significantly
heritable.
• Previous U.S. study: Mexican-Americans have
highest prevalence (32%) of metabolic syndrome
(ATP-III definition); non-Hispanic whites 24%,
African Americans 22%.
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Population Genetics: From Evolutionary
History to Genetic Medicine
[Jorde et al, Human Mol Genetics 2001]
• The effective human population size is surprisingly small,
about 10,000, far less than the census size, based on
mitochondrial DNA, Y chromosome variation, and
autosomal polymorphisms.
• Human population probably expanded rapidly in late
Pleistocene—tell-tale rare variants
• Genetic diversity between the major continental populations
is only 10-15% vs 85-90% within each continent.
• Much evidence supports African origin of modern humans,
with subset colonizing Asia and Europe about 100,000 years
ago. Most persuasive: haplotypes found outside Africa are
usually subsets of those found within Africa.
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Genome-wide Detection and Characterization
of Positive Selection in Human Populations (1)
Dense maps of human genetic variation: 3 million
polymorphisms (HapMap2).
Long-range haplotype methods identify alleles
segregating in a population undergoing recent
selection.
Cross-population comparisons help discover alleles
swept to near-fixation in a population.
Found >300 strong candidate regions; focused on 22
strongest regions.
.
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Genome-wide Detection and Characterization
of Positive Selection in Human Populations (2)
Identified 26 non-synonymous, coding, SNPs
showing regional evidence of positive selection.
Found 2 genes in a common biological process with
positive selection in the same population:
• LARGE and DMD in West Africa, related to
infection by Lassa virus
• SLC24A5 and SLC45A2 in Europe, related to
skin pigmentation
• EDAR and EDA2R in Asia, involved in hair
follicle development.
[Sabeti et al. Nature 2007;449:913-919]
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Genome-wide Detection and Characterization
of Positive Selection in Human Populations (3)
• The most differentiated genes among 3
million markers studied globally are those
determining skin pigmentation.
• May have been linked to risk of ricketts
(failure of bone production) if there is not
sufficient vitamin D and activation of
vitamin D by sunlight, as in northern
European populations.
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A Genome-Wide Association Study of
Global Gene Expression (1)
[Dixon et al. Nature Genetics 2007;39:1202-1207]
• Aim: build a database to systematically examine
potential effects of disease-associated variants on
transcript expression.
• Created a global map of effects of polymorphisms on
gene expression in 400 children from 206 British
families [in asthma study]. Looking for regulatory
effects (non-coding SNPs).
• Genotyped 408,000 SNPs; measured gene expression
of 54,000 transcripts from 20,599 genes in
lymphoblastoid cell lines.
• 28% of transcripts, representing 6600 genes, had
narrow heritability H2 >0.3.
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A Genome-Wide Association Study of
Global Gene Expression (2)
• Highly heritable expression levels are best
evidence that trait is genetically regulated.
• LCL cells provide general indicator of gene
expression.
• Use Affy chips for gene expression, Illumina
beads to genotype
• Identified 88 genes separately associated with 3 or
more SNPs (cis and trans).
• Only 3 SNPs ass’d with >5 transcripts—no
evidence for “master regulators”
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A Genome-Wide Association Study of
Global Gene Expression (3)
• Used Gene Ontology to identify genes enriched
among highly heritable traits
• Greatest enrichment: “response to unfolded
proteins”--chaperonins and heat shock proteins
• May represent an evolutionary response to cellular
stress; unfolded proteins key in prion disease,
Alzheimer disease, diabetic pancreatic beta cells
• Cell cycle, RNA processing, DNA repair, and
immune response genes also highly heritable.
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Genome-Wide Association Study (4)
Applied to Childhood Asthma
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•
•
•
[Moffatt et al, Nature 2007; 448: 470-47
New susceptibility locus: non-coding SNPs
residing within a 206-kb segment on chromosome
17q23.
Of 19 genes in this region, the expression database
pointed to ORMDL3 (p<10-22), correlated
(r=0.67) with asthma. Codes a transmembrane
protein anchored in endoplasmic reticulum.
Disease-associated alleles accounted for 30% of
variance of expression.
Can expose LCL cells to asthma triggers.
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Other Disease-Associated GenomeWide Studies (5)
• Crohn’s disease: PTGER4, chromosome 5
• Type 2 adult-onset diabetes: PHACS on chr 11
• Fetal hemoglobin expression in adults: markers
upstream of HBS1L on chr 6q23.
• LDL and HDL cholesterol levels: 11 genes
confirmed, 7 novel genes identified
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Genome-Wide Disease Association Studies
[Willer et al, Nature Genetics 2008;40:161-169]
Study of 20,000 people revealed 18 genes which
influence cholesterol levels, of which 7 are newly
identified.
Those increasing total and LDL-cholesterol match
those predisposing to coronary heart disease.
However, no protective variants were found for
action via HDL-C. Maybe this explains recent
failures of estrogen replacement in Women’s
Health Initiative trials and of Pfizer HDL-Cenhancing drug in coronary disease patients.
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Genomic Evidence for Evolution of the Lung
[Torday & Rehan, Amer J Resp Cell Mol Biol 31:8, 2004]
• Parathyroid hormone-related protein (PTHrP) is a highly
conserved, stretch-regulated gene necessary for embryonic
transition from branching morphogenesis to alveolization
of the lung, beginning with the fish swim bladder.
• Growth and stretching up-regulate, while microgravity and
overdistension down-regulate, PTHrP and PTHrP receptor
mRNA in alveolar type II cells.
• Both surfactant homeostasis and alveolar capillary
perfusion are under PTHrP control.
• Phylogeny across vertebrate species reveals evolutionary
amplification of PTHrP signaling mediating development,
modeling, and remodeling of the alveolar wall.
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PTHrP Signaling Between Germ Layers
By simultaneously stimulating activities of PTHrP and
PTHrP-receptor, alveolar wall distention increases
surfactant production and alveolar capillary blood flow,
with ventilation/perfusion (V/Q) matching.
V/Q matching is the evolutionary integration of
cell/molecular interactions by which the lung and
vasculature have adapted to the progressive increase in
metabolic demand for oxygen.
PTHrP activates cyclic AMP-dependent protein kinase A,
which drives adipose differentiation related protein
(ADRP) to promote trafficking of lipid substrate for
surfactant and leptin to stimulate differentiation of alveolar
type II cells, during thinning of the alveolar wall due to
inhibition of fibroblasts by PTHrP.
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Evidence from Ontology and Phylogeny
Three dramatically different types of lung:
• Single-chambered lung of fish, frogs, lizards:
100X diameter; interstitial muscle dominates
• Bronchoalveolar lung of mammals: conducting
and terminal airways are convoluted, facilitating
gas exchange; increased saturation and greater
turnover of surfactant protects the smaller alveoli
• Cross-current saccular lung of birds: the exception
to prove the rule; lack alveoli, lungs are stiff and
fixed to thorax, yet possess type II cells that
express PTHrP and surfactant. PTHrP’s role in
bone morphogenesis links hollow bones with air
sacs of the respiratory system of birds.
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Effects of Deletion of PTHrP;
Clinical Insights and Advances
• Models of what pathology might result in humans with
mutations of PTHrP have been produced as knock-outs in
mice.
• Deletion of PTHrP results in failed alveolization and death
due to pulmonary insufficiency within minutes to hours of
birth. The lung develops normally up to the stage of
canalization, coincident with the point in development
when PTHrP is produced in mice, rats, and humans.
Functionally, the lung fails to produce surfactant due to
immaturity of the embryonic lung tissue layers.
• These abnormalities could account for various types of
surfactant-deficient syndromes in human newborns.
• PPARgamma agonists can help premature babies with
bronchopulmonary dysplasia (Torday & Rehan, Peds Res
2007).
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Admixture Studies in Mexico
[Jimenez-Sanchez, Science 300:295, 2003]
• Mexico has a characteristic genetic population
structure from admixture of 65 native Indian
groups with those of Spanish ancestry.
• Genomic medicine was designated a priority in
health care and medical research during the tenure
of Dr. Julio Frenk as Secretary of Health.
• A 15-year plan for Genomic Medicine began with
the establishment of INMEGEN, Instituto de
Medicina Genomica in the M-NIH, a Consortium
for the INMEGEN, and public debate on ELSI.
58
Genome-wide Admixture Panel for
Hispanic/Latino Populations
[Mao et al, Amer J Human Genetics 80:1171, 2007]
• Admixture mapping is a powerful method for identifying
genetic risk factors for complex traits and diseases having
prevalence differences across populations.
• Here a panel of 2120 “ancestry-informative markers”
maximizes differences between Native American and
European populations while minimizing differences
between Mexican (Maya and Nahua) and South American
(Aymara and Quechua) populations.
• Can reduce by 100X the genotyping required for wholegenome association studies in these populations.
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Mao, 2007: PCO 3D Plot of Genetic Distances with Affy 500K Array
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Mao et al, 2007: Principal coordinate analysis and 3D plot
separating European from MesoAmerican, Maya from others.
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SUMMARY
• Genome-wide studies and databases are directly
useful for analyses of the risk factors and
pathways for specific diseases in different
populations around the world and in diverse
populations within individual countries such as
Mexico and the United States.
• Evolutionary biology provides helpful insights for
a wide array of developmental, metabolic, and
disease phenotypes in humans.
• As we celebrate 200th anniversary of Darwin’s
birth, evolution by natural selection is a
foundation for all of biology, including medicine.
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