Disease and Gene Associations

Elizabeth R. HauserDuke University

Center for Human Genetics

North Carolina Genomics Symposium

March 18, 2005

Breaking News- March 14, 20053 Research Teams independently identify a gene for macular

degeneration

Significance of AMD result

• Affects 1 in 5 people over age 65• Complex disease

– Clearly a genetic component– Important environmental risk (e.g. Smoking)

• Multiple groups identified the same polymorphism– accounts for 20-50% of the overall risk in these studies

• Each group used a slightly different approach• Genetic analysis was an important step

– Started with linkage and proceeded to association

• Human Genome Project provided key information

The CFH gene for Age-related Macular Degeneration is the most recent example of a gene for a common disease, whose identification was greatly enhanced by the Human Genome Project.

These are exciting times in which to be doing research on genetic determinants of disease!

Outline

• Types of genetic disease• Evidence for the involvement of genes• Study designs and analysis methods• Taking complexity into account• Coronary artery disease example

Simple vs complex disease

• Disease definition=phenotype– Simple traits=> Easily defined– Complex traits=> Difficult to determine– How is the diagnosis made?

• Measurements• Instruments• May be expensive to collect

– There may be ambiguity in the definition of disease

• Affected well defined, Unaffected ?• Coronary artery disease requires specialized procedures

– Coronary catheterization– Stress tests– Clinical event, such as heart attack or bypass

operation

Monogenic Diseases Complex Diseases Environmental Diseases

Huntington Disease

Spinocerebellar Ataxia

Spastic Paraplegia

Tuberous Sclerosis

Alzheimer disease

Cardiovascular Disease

Autism

Parkinson Disease

Influenza

Hepatitis

Measles

Genes and Disease

- Genes- Environment

Causative or Mendelian

Gene• Gene directly leads to

disorder

• Recognizable inheritance patterns

• One gene per family

• Less common diseases– Cystic fibrosis,

muscular dystrophies

Complex or Susceptibility

Gene• Gene confers an

increased risk, but does not directly cause disorder

• No clear inheritance pattern

• Involves many genes or genes and environment

• Common in population– cancer, heart disease,

dementia

Defining what to study• As in any biomedical study, need to precisely define

the disease under study • Define primary phenotype and secondary phenotypes• Validity and Reliability• Understanding risk factors

– Genetic or Environmental?• Ethnic differences• Age/gender distribution

• Use epidemiologic information to refine the phenotype definition

• Consider comparability to other studies

Refining the phenotype-genes

• Idea: Make the effect of certain genes in the sample more easily detectable

• Genetic effects may be stronger for extremes of the risk factor distribution– restrict sample to people with onset at a very

young or very old age

• Genetic effects may be stronger for unusual presentations– restrict sample to individuals with coronary

artery disease (CAD) without lipid abnormalities– restrict sample to diabetics with nephropathy

Refining the phenotype-environment

• Minimize effect of known environmental confounders– restrict sample to nonsmokers– restrict sample to unmedicated people,

e.g. in hypertension studies

• Collect data in a genetically homogenous population such as a particular ethnic group or genetically isolated population– Reduce the number of genes contributing

to the phenotype

But, How do You Know Your Trait is

Genetic?

Familial aggregationFamilial aggregationFamilial aggregation is the clustering of affected individuals within families. Documenting the familial aggregation is often the first step in characterizing the genetic basis for a trait.

Major questions to ask yourself: Is there heterogeneity? Is it possible that there is a Mendelian

subset of families?

Oftentimes, Mendelian subsets of complex disease are characterized by early age of onset or increased severity.

Familial aggregation is the clustering of affected individuals within families. Documenting the familial aggregation is often the first step in characterizing the genetic basis for a trait.

Major questions to ask yourself: Is there heterogeneity? Is it possible that there is a Mendelian

subset of families?

Oftentimes, Mendelian subsets of complex disease are characterized by early age of onset or increased severity.

Follow Disease as it is Passed from Parents to

Children

Follow Disease as it is Passed from Parents to Children

Follow Disease as it is Passed from Parents to Children

Follow Disease as it is Passed from Parents to Children

Follow Disease as it is Passed from Parents to Children

Twin StudiesTwin StudiesPurpose: Estimate the genetic component of a disease or associated phenotype

Usually assume that twins share a common environment which lessens the impact of environmental influences (although this may not be true for studies of adult twins)

Usually compare twins of same sex (especially useful if there are known differences in disease frequency in males and females)

Twins are same age so age-dependency is not a problem

Purpose: Estimate the genetic component of a disease or associated phenotype

Usually assume that twins share a common environment which lessens the impact of environmental influences (although this may not be true for studies of adult twins)

Usually compare twins of same sex (especially useful if there are known differences in disease frequency in males and females)

Twins are same age so age-dependency is not a problem

MZMZ DZDZ Type of DiseaseType of Disease

90%90% 90%90% Probably EnvironmentalProbably Environmental

100%100% 25%25%

Mendelian recessive, deviation Mendelian recessive, deviation from the expected frequencies from the expected frequencies may be due to incomplete may be due to incomplete penetrance penetrance

80%80% 16%16% ? ? ?? ? ?

72%72% 35%35% ? ? ?? ? ?

7%7% 7%7% May be the same as population May be the same as population frequencyfrequency

Twin StudiesTwin StudiesOne twin is affected, how often is the other?One twin is affected, how often is the other?

Review article: Martin et al. “A twin-pronged attack on complex traits” Nature Genetics 17: 387-392 (1997).

Review article: Martin et al. “A twin-pronged attack on complex traits” Nature Genetics 17: 387-392 (1997).

Comparison of disease frequency in adoptees with their biological vs. their adopted parents (or siblings). Given the adoptee is affected, what percent of parents have the disease?

Biologic Adoptive Type of diseaseSuggests strong genetic

85% 5% component, frequency in adoptive parents may reflect risk in the general population

Suggests strong environmental 5% 85% component, frequency in biologic

parent may reflect risk in general population

Comparison of disease frequency in adoptees with their biological vs. their adopted parents (or siblings). Given the adoptee is affected, what percent of parents have the disease?

Biologic Adoptive Type of diseaseSuggests strong genetic

85% 5% component, frequency in adoptive parents may reflect risk in the general population

Suggests strong environmental 5% 85% component, frequency in biologic

parent may reflect risk in general population

Twin Studies: AdoptionTwin Studies: Adoption

Segregation Analysis

• Test the disease distribution in families for concordance with specific genetic transmission models

• Very difficult studies to perform– Families need to be collected in a very precise

way

• Works best for single gene disorders• Not terribly successful for common

diseases

A measure of how “genetic” a trait or disease is: What is the rate of affection for relative of proband with the disease vs. the frequency of the disease in the general population?

A measure of how “genetic” a trait or disease is: What is the rate of affection for relative of proband with the disease vs. the frequency of the disease in the general population?

Risch N. Am J Hum Genet (1990): 46 pp. 222 - 253.Risch N. Am J Hum Genet (1990): 46 pp. 222 - 253.

Recurrence Risk to Relatives: I Recurrence Risk to Relatives: I

I=I=recurrence rate in relative of probandrecurrence rate in relative of proband

rate in general populationrate in general population

where ‘I’ indicates the degree of relationshipwhere ‘I’ indicates the degree of relationship

Values > 1.0 are generally taken to indicate evidence in favor of a genetic component. In general, the higher the value, the stronger the genetic component.

Values can be used to estimate the number of genes under different genetic models.

Note that the magnitude of the estimate is very dependent on the frequency in the population. For example, a common disorder may have frequency estimates of 3-6% depending on how a given study was performed but this results in small .

Values > 1.0 are generally taken to indicate evidence in favor of a genetic component. In general, the higher the value, the stronger the genetic component.

Values can be used to estimate the number of genes under different genetic models.

Note that the magnitude of the estimate is very dependent on the frequency in the population. For example, a common disorder may have frequency estimates of 3-6% depending on how a given study was performed but this results in small .

Recurrence Risk to Relatives: s Recurrence Risk to Relatives: s

Disease:

Alzheimer 4-5

Neural tube defects 25-50

Obesity 1.8

Autism 100-150

Cystic fibrosis 1000

Disease:

Alzheimer 4-5

Neural tube defects 25-50

Obesity 1.8

Autism 100-150

Cystic fibrosis 1000

Recurrence Risk to Relatives: s Recurrence Risk to Relatives: s

s:s:

Best Proof of All?

Connect genetic variation to the disease!

But, How Do We Find the Gene?

Locating a VariationLocating a Variation

30,000 Genes on 46 chromosomes30,000 Genes on 46 chromosomes

Locating a VariationLocating a Variation

Region carrying the variation

Region carrying the variation

Gene 1

Gene 3Gene 4

Gene 5

Gene 2

Gene 6

Locating a VariationLocating a Variation

Variation found in gene

Variation found in gene

The process of recombination in meiosis

creates a relationship between two genes that is a

function of the distance between them.

Genetic Markers• In order to use recombination need to have

genetic markers throughout the genome• Know where the markers are in the genome

– Human Genome Project tells us precisely where the markers are

• Unchanged from generation to generation• Follow transmission from parents to offspring• Be able to distinguish alleles

– Polymorphic- having more than one state (alleles)– Can follow markers and alleles from one

generation to the next

Observe Disease and Markers of Genes Passed Together from Parents to

Children

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Observe Disease and Markers of Genes Passed Together from Parents to

Children

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Observe Disease and Markers of Genes Passed Together from Parents to

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Observe Disease and Markers of Genes Passed Together from Parents to

Children

Observe Disease and Markers of Genes Passed Together from Parents to

Children

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Observe Disease and Markers of Genes Passed Together from Parents to

Children

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Observe Disease and Markers of Genes Passed Together from Parents to

Children

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Observe Disease and Markers of Genes Passed Together from Parents to

Children

The phenomenon of the co-transmission of disease and marker alleles within a given

family is called LINKAGE.

No recombination is taking place between the disease

and marker suggesting that they are close together on

the same chromosome.

Suppose that we see linkage and we can follow

transmission of marker alleles from parents to

offspring.

Suppose that in comparing many families, all diseased

people in all families get the A allele.

Now we have ASSOCIATION too.

• Alleles A and B at two loci are associated if the event that a gamete carries A is not independent of the event that the gamete carries allele B.

• Alleles are not associated if they occur together in the same gamete randomly.

• Association is population-specific.

ABA

BAB

aba

bab

AB

Allelic Association

ABA

bAb

aba

Bab

AB

We can test for genetic association in families or in

unrelated people.

Many genetic association studies are performed as

case control studies.

Information is gained when we can combine evidence for

genetic linkage with evidence for genetic

association.

How do we apply these ideas?

Coronary Artery Disease• Major cause of death and disability

throughout the world• 12 million Americans have coronary

artery disease– 7 million with myocardial

infarctions– 6.2 million with angina pectoris

• Well-defined risk factors: smoking, high cholesterol, physical inactivity, overweight, family history

What is the evidence that CAD is genetic?

Family history is a strong risk factor.

Evidence for Genes in CAD

• Familial aggregation– The clustering of affected individuals in families

• Twin studies– If one twin is affected, the other twin is affected

more often than by chance– A monozygous (identical) twin has higher risk

than a dizygous twin (Marenberg et al. 1994)– Relative risk to co-twins is increased at young

age– In old twins, rates are similar in MZ and DZ

twins

Estimation of relative risk (for CAD

• Shea at al (1984): relative risk to sibs 2-3.9– Controlled for known risk factors– Risk to relatives higher in low risk factor group– Suggests risk due to family history may be

independent of other known risk factors, especially at young ages

• Risanen (1989) risk increased in first degree relatives– Risk to brothers <55 was 6.7, sisters <55 2.8

Characteristics of Familial CAD

• Many family members affected, especially female relatives

• Early onset <55 in men; <65 in women• Multi-vessel disease• Multiple risk factors• Refractory to conventional therapy• Family history of related conditions (i.e.,

stroke, diabetes, hypertension, cholesterol abnormalities)

Complex or Multifactorial Inheritance

DISEASE

Smoking

Exercise

Diet

Age

Family History

CAD is a multi-factorial condition

"good" genes "bad" genes

"good" environment

low risk of CAD

medium risk of CAD

"bad" environment

medium risk of CAD

high risk of CAD

Genetics and Age of Onset

"good" genes "bad" genes

"good" environment

CAD at very old age

CAD at young age

"bad" environment

CAD at average Age

CAD at very young age

The GENECARD Study

• Goal: Identify genes predisposing to early onset CAD

• Use genome screen approach with a very large sample size (950 families) to map genes for early-onset CAD

• Ascertain siblings with CAD verified by medical record review

• Age of onset is the key feature– Males < 50 at diagnosis– Females < 55 at diagnosis

GENECARD Criteria• Inclusion Criteria

– Men who have had coronary atherosclerotic heart disease diagnosed at or before age 50, and women with a diagnosis at or before age 55, using any of the following criteria

• Angina or myocardial ischemia

• Cardiac catheterization indicating a blockage in at least one vessel of 50% or greater

• An acute myocardial infarction diagnosed by enzymes or electrocardiogram

• Unstable angina

• Coronary Artery Bypass Graft (CABG)

• Percutaneous Transluminal Coronary Angioplasty (PTCA)

•

GENECARD

• Exclusion Criteria– Substance abuse in the absence of diagnosed

coronary stenosis– Congenital heart disease– History of chest irradiation– End stage renal disease– Myocarditis as a primary etiology of chest pain

GENECARD: Study Requirements

• Family history-at least 2 siblings with early CAD

• Blood sample

• Medical history with medical record confirmation

• Measurement of hips and waist

• Risk factor interview

• Measurement of blood pressure

Linkage Analysis

• Assume the affected people in the same family have the disease because of the same gene.

• Idea: If the gene causing the disease in this family is close to a genetic marker (linked), then we should see less recombination than we would expect under the hypothesis of no linkage.

• Genotype markers across the genome.• Look for markers that are shared more

often by family members with CAD.

Several Intervals Are Linked to CAD

Chromosome 1 ~ 22

Hauser ER. Et al., AJHG, 2004 Sep;75(3):436-47 6

GENECARD: Chromosome 3

CAD can have different clinical characteristics in

different people. What if we divide our families into

subsets based on presence or absence of additional

conditions:Acute Coronary Syndrome,

Diabetes, Metabolic Syndrome.

GENECARD: Chromosome 1

Different facets of the disease may have different

genetic contributions.

It is often useful to consider disease subtypes or other

clinical covariates to develop more genetically similar sets

of families.

CATHGEN: CAD Association Study

• Identify cases and controls from the Duke Coronary Catheterization Lab– Cases have significant atherosclerosis– Controls have minimal atherosclerosis

• Genotype markers in regions of linkage• Look for alleles that appear more often in

cases and controls

Genotype and Allele Comparisons

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0 500 1000 1500 2000 2500 3000 3500

Map Position (cM)

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Young Affecteds vs. Old Normals - Genotype Young Affecteds vs. Old Normals - Allele

Young Affecteds vs. Old Affecteds - Genotype Young Affecteds vs. Old Affecteds - Allele

Old Affecteds vs. Old Normals - Genotype Old Affecteds vs. Old Normals - Allele

GC Affecteds vs. Old Normals - Genotype GC Affecteds vs. Old Normals - Allele

1.3

1 2 3 4 5 6 7 8 9 1011 12 13

14155 16 17 18 19 20 2122 X

Preliminary Candidate Gene Association Study in CATHGEN

Conclusions• Gene identification studies of complex

disease are fun, exciting and challenging.• These studies require input from individuals

with different expertise: Clinicians, Epidemiologists Molecular Biologists, Bioinformaticians, Statisticians, Geneticists.

• The Human Genome Project has accelerated our understanding of genetic architecture.

• Genes for complex disease will be discovered at a fast rate.

• Next steps are studies that identify the gene function as it relates to disease.

GENECARD CollaboratorsDUCCS NetworkWilliam KrausChristopher GrangerElaine DowdySusan EstabrooksLiling HuangStephanie DeckerTeresa PeaceJerome AndersonSherry JamesonAlan BartelCathy GarveyPaul CampbellJanet PattersonBrian CrenshawTeresa SchraderCharlie DennisKim DeRosaJames HeinsimerNancy Howald

International NetworkDavid CrossmanSheila FrancisKaren EgglestonJonathan HainesDouglas VaughnBrendan McAdamWilliam HillegasPaula ClevengerChris JonesKath RocheVincent MooserVincent JominiNicolas RedondiBernhard Winkelmann

Glaxo-Smith-KlineJulia PerrySanjay SharmaScott SundsethLefkos MiddletonAllen RosesVincent Mooser

Duke CHGElizabeth HauserMargaret Pericak-VanceJeffery VanceMichael HauserSilke SchmidtMargaret JamisonSandra WestDonny AsperKruti DesaiJason FlorJason GibsonAdam MacLaurinGeorge WardGeorge WillisCarol HaynesColette BlachRodney JonesLin Hu

William HerzogTania GeshoffMicheal HindmanJennifer KaneMike RotmanVirginia RemenyKent SalisburyDianne OskinsCharise PattenAlan WisemanMary DuquetteBrent MuhlesteinChloe MaycockSandra ReynaRichard GoulahGina KavanaughSebastian PalmeriCasey CasazzaFred McNeerSusan MarpleJeff MichelSteve RoyalBrian Hilbourn