Association studies of complex Association studies of complex diseasesdiseases

COMPLEX DISEASES 1COMPLEX DISEASES 1• 1. Incomplete penetrance (avaldumine) and

phenocopy (fenokoopiad)

• 2. Genetic heterogeneity

• 3. Oligo/polygenic inheritancei = 123...n (multiplicative effect) i = 1+2+3+...+n (addititive effekt).• BUT the contribution of every single n is

too small for enough statistical power power

• 4. High frequency of disease-susceptibility alleles

• 5. Influenced by environmental factors

COMPLEX DISEASES 2• Quantitative Quantitative

characters:characters:• no 1:1 relationship

between genotype and ja phenotype

• The more loci are involved, the more the character resemble Gaussian distrubution

• G + E = Ph• Heriditability

(pärilikkus)

• Dichotomic characters:Dichotomic characters:• Susceptibility

(vastuvõtlikkus)

• threshold (lävi)

• liability (kalduvus)

• liability > threshold = disease develops

R= the risk of the relatives of a patient compared to the general population risk

R > 1 genetic component of the disease exists

Phenotype

GeneticFactor 2

GeneticFactor 1

GeneticFactor n

PolygenicbackgroundCommon

EnvironmentIndividual

Environment

Cultural factors

MarkerGenotypes

??????

LINKAGE AND LD MAPPING

CORRELATIONTo be TESTED

MODE of INHERITANCE

GENOTYPE GENOTYPE

EFFECTEFFECT

PHENOTYPE PHENOTYPE

individual

In combination only

no individual effect

either combination only

1. Additive

2. Multipli-cative

3. Epistatic

4. MixedMultipli-cative

+

+

x

x

x

OR

x

Models for the interraction of loci in complex diseases (by Gabriel et al., 2002)

Genetic mapping strategiesGenetic mapping strategies:

• Linkage mapping: studies co-segregation of tested markers and a disease in families under Mendelian models of inheritance

• Association analysis (linkage diseuilibrium mapping): compared the frequencies of tested alleles/genotypes in populations of cases and controls

Parametric and non-parametric linkage analysis

The method of choice in case:1) a few major-susceptibility loci are expected2) grouping of patients according to narrower phenotypes

is relevant (e.g. early-onset ja late-onset)3) study is carried out in an isolate population with lower

genetic variabilityMajor drawbacks:1)needs defining of the genetic model2) needs information about allele frequencies and

penetrance3) needs exact diagnosis4) needs large pedigrees and exensive sample size5) does not work for disease heterogeneity6) resolution limit7) Dilemma: non-stringent analysis =>false-positive

results stringent analüüs => low power

Strategies used for successful complex Strategies used for successful complex disease studies by linkage analysisdisease studies by linkage analysis:

• I. Grouping of patients:

• A. family vrs. Sporiadic cases (breast, colon cancer)

• B. Detailed diagnosis (diabetes, Hirschprung disease)

• II. Linkage study using only the selected subgroup of patients followed by positional cloning or positional candidate approach

Association studies 1Association studies 11) Candidate gene based:* metabolically and physiologically relevant* previously mapped by linkage to a chromosomal

region* expression analysis*chromosomal rearrangements involved 2) 2) Whole-genome scan: *single loci ->too expensive, laborious and lacks

statistical power

* needs existance of LD based HAPMAP

WELL-DEFINED PHENOTYPE

WELL-DEFINEDCANDIDATE GENES

OR BIOLOGICAL SYSTEMS

PHENOTYPICMEASUREMENTS

EXACT DIAGNOSIS

POLYMORPHISMDISCOVERY

LIFESTYLE RECORDS

ASSOCIATION STUDIES:

1) CASE-CONTROL2) SIB-PAIR(TDT, HRR)

FOLLOW-UP STUDIES: CLINICAL

AND GENETIC

CANDIDATE GENE/ SYSTEM BIOLOGY APPROACHCANDIDATE GENE/ SYSTEM BIOLOGY APPROACH

POPULATION STUDIES:VARIATION AND

GENETIC STRUCTURE

STATISTICALMETHODS AND

MODELS

STUDIES OF POSITIVE ASSOCIATION RESULTS IN BIOLOGICAL SYSTEMS AND MODELS (mRNA, protein, knock-out)

Association studies 2Association studies 2

Direct Indirect

Based on candidatePathological polymorhisme.g. changing aminoacid in a peptide

ONLY candidate gene/Candidate polymorhism approach

Based on polymorhisms potentially in allelic association (linkage disequilibrium with disease susceptibility variant)

Both, candidate gene or region and whole genome scan approaches

Knowledge of linkage disequilibrium and haplotype structure of a target gene

Association studies 3Familiy material:1. ASP affected-sib-pair , APM

(affected pedigree member) IBS- identity by descent, IBS-

identity by state2.TDT (Transmission

disequilibrium test)Tests candidate gene allele

transmission from heterozygous parents to SINGLE affected offspring

3. HRR (haplotype-relative risk

Compares marker-allele frequencies between chromosomes transmitted to patients and "control" non-transmitted chromosomes of their parents

Case-control material:Tests for allele, haplotype

or genotype frequency differences between cases and controls

Association between an allele A (haplotype, genotype) and a disease D can be due to:

1) A gives susceptibility to D

2) A is linked to gene for D

3) false-positive result due to population structure

Genetic studies and epidemiology

Epidemiology: based on observing and measuring disease patterns in populations, and using association and statistical correlation to identify factors (including genetic) that affect those patterns

Hirschhorn et. al.,2002:• summerized association studies conducted

1986-2000: 166 putative associations studies 3 or more times

• ONLY 6!!!6!!! Have been constantly replicated

• WHY?• 1) limited knowledge of candidate-genes? • 2) limited sample set?• 3) limited list of tested polymorphisms?• 4) limited genetic models and statistical power?

Errors is association studies:Errors is association studies:• Small sample size• Unmatched control-group• Unknown genetic structure (LD structure,

variability etc.) of a population • Unknonwn background-LD around tested

candidate region• Failure to attempt study replication

ARCAGE studyARCAGE study: Number of case-control : Number of case-control pairs required to provide an 80% power to pairs required to provide an 80% power to

detect a main effect at 1 % significance leveldetect a main effect at 1 % significance level ..

OR Allele frequency

1% 5% 10% 20% 40%

1.2 96408 20170 10746 6159 4261

1.5 17727 3760 2028 1191 863

2.0 5418 1166 640 389 300

2.5 2848 621 346 216 176

3.0 1851 408 231 148 125

CASES

CONTROLS

CASES

CONTROLS

True positive association:The frequency of risk allele is greater in both ethnic groups

Ethnic group 1 Ethnic group 2

False positive association:The frequency of risk allele is identical in controlsand cases in both populationsBUT:the allele is 2x as frequentin cases of pooled population compared to controls

67% 67%

33% 33%

80% 20%

20%80%

ETHNIC ADMIXTUREETHNIC ADMIXTURE

Population staratification: Population staratification: solutionssolutions

• 1. TDT (transmission disequilibrium test)

• Needs family material and extra genotyping

• 2. Parallel case-controls studies in (I) several populations; (II) random sub-groups of the cases and controls sample sets

• 3. Statistical tests for detecting and correcting for stratification (e.g.Pritchard, 1999)

Haplotype blocks:Haplotype blocks:

Recombination hotspots: 1-2 kb regions characterizedby extensive crossing-over events

*loci in LD combine into 3-5 common haplotypes defined by 6-8 SNPs*loci characterized by similar nucleotide variation

Block 2 Block 3

A chromosomal segment

Reich et al., 2001, Johnson et al., 2001 Daly et al., 2001; Jeffreys et al., 2001; Goldstein et al., 2001; Gabriel et al., 2002

Block size <1-170 Kb, average block for Europeans 18 kb, Africans 9kb

Cardon &Abecasis, 2003

How to predict a haploblock?How to predict a haploblock?• Haploblocks vary extremely in size• The borders of the haploblocks are similar across

populations, but the haplotypes differs among them• The major determinant for the local pattrns of

human sequence variation and LD is the extreme variability in recombination rate (Reich et al., 2002, Jeffreys et al., 2001)

• The extent of the LD depends on regional CG content: the higher the CG content, the higher the recombination frequency (Eisenbarth, 2000, 2001)

• Correlation between the patterns of LD an isochores

Distribution of recombination rateDistribution of recombination rateRates given in cm/Mb, data from 4,088 STRP (Yu et a., 2001

1 cM=1 recombination event per 100 meioses

Range Mean±s.dSex average 0.0-0.6 1.3±0.80Male 0.0-7.9 0.92±0.96Female 0.0-8.8 1.68±1.07

0-0.5 0.5-1.5 1.5-3 >3.0Sex average 12% 58% 26% 4%Male 38% 45% 12% 5%Female 10% 38% 42% 10%

Recomination rate depends linearly on the size of a chromosome as well as the location along the chromosomal axis: shorter chromosomes and telomeres have are characterized by higher recombination rate

Distribution of recombination across chromosome 19

Puurand, 2004

Correlations of recombinationCorrelations of recombinationrates with sequence parametersrates with sequence parameters

Parameter 1 Parameter2 Relationship (R2) PSINE density LINE density Negative (0.2) <0.001

(A)n density Positive (0.8) <0.001STRPheterozygosity

None (0.00) 0.49

GC content Positive (0.05) <0.001(AC)n density Positive (0.001)(A)n density Positive (0.02) <0.001SINE density Positive (0.01) <0.001

Sex-averagedRecombinationRate *

LINE density Negative(0.02) <0.001Male Recomb.Rate

Chromosomeposition

Positive towardstelomeres (0.18)

<0.001

Yu et al., 2001

How to select SNPs for association studies?

*

*

* Currently considered of high importance due to location of regulatory regions

Whole genome association scan:SNP requirements for HAPMAP

construction

POPULATION Chromo-somesanalyzed

Min. SNPSpacing/av.block size

Haplo-Types/Block

SNPs forHAPMAP(gene-based)

SNPs forHAPMAP(genome)

1.Cosmopolitan(Stephens et al.)

164(313 genes)

5.0 kb/NA NA 138 K 600 K

2.Cosmopolitan(Patil et al.)

20(# 21)

7.1 kb/7.8 kb

2.7 245 K 423 K

3.European(Daly et al.)

516(5q31)

16.7 kb/34.6 kb

2-4 NA 180 K

4.Cosmopolitan(Gabriel et al.)

400(54 loci)

NA/11-22 kb 3-5 NA 300 -1000 K

HAPMAP - genome map of haplotype blocks and variants

Mapping Example : Crohn disease• Chronic inflammatory disorder of gastrointestinal tract• 1/1000 in young adults, incidence increased last century• The effect of environmental factors on genetically

predisposed host• Repeated linkage to # 16 pericentromeric, IBD1 region• Two independent studies (Ogura et al., Hugot et al.)

showed the association to the same polymorphism of NOD2 gene, encoding protein with homology to plant disease resistance gene products

• Third study (Rioux et al., Daly et al.) with only Canadian families identifed linkage peak at 5q31 and LD mapping showed association of risk haplotypes to Crohn disease

CROHN DISEASE and NOD2 CROHN DISEASE and NOD2 (Ogura et al., 2001)(Ogura et al., 2001)

1.LINKAGE MAPPINGMultiple studies

2.POSITIONAL CANDIDATE APPROACH

3. cDNA versus genomic clones

4.Mutatation search in patients (12) vrs. controls (4)

5. Allele-specifi PCR for typing 3020Cins for #16 linked

Families and case-control material

6.1.TDT test for heterozygous parents and one affected child of the family

6.2. Case-contorl test for the significanceof allele frequencydifference

#16 pericentromere

NOD2 gene

CANDIDATE GENE structure

3020insC mutation in 3 patients: frameshift and truncated protein

TDT test: p=0.0046 (39 transmissions vrs. 17 nontr)

Case-control: p=0.0018 (8.2 % in cases vrs. 4% in control)

METHOD RESULT

GRR for 3020insC heterozygotes 1.5 and homozygotes 17.6

CROHN DISEASE and NOD2 CROHN DISEASE and NOD2 (Hugot et al., 2001)(Hugot et al., 2001)

1.LINKAGE MAPPINGMultiple studies

2.Locus REFINEMENT by additional typing and Linkage analysis

3. Physical mapping of the region

4.TDT test with 108 families5. Replication TDTwith independent

set of 76 families

6. Sequencing of the 164 kb BAC clone

#16 pericentromere

16q, ~ 5 cM region

~ 2 Mb candidate region

P<0.05 for one of the markers

Stong LD among most markes,SNPs as well as microsatellites

METHOD RESULT

An identified gene with a risk haplotype of 3 SNPs

P<0.001 for the same marker

7. Typing 11 identified SNPs

8. 1.Characterization of Unigene clusters8.2. PDT tests for SNPs in the gene

One SNP: 1 bp insertion in exon10of NOD2 gene -> frameshift and

truncated protein

CROHN DISEASE and cytokine gene cluster CROHN DISEASE and cytokine gene cluster on 5Q31 on 5Q31 (Rioux et al., 2001; Daly et al., 2001)(Rioux et al., 2001; Daly et al., 2001)

1.LINKAGE MAPPING

2. LD MAPPING 1

3.LD MAPPING 2

4.CANDIDATE GENES

SNP DISCOVERY

SIGNIFICANT SNPS

18 cM peak on 5q31

2 markers in significant TDT (p<0.001)

MULTILOCUS ANALYSIS:435 KB HAPLOTYPE (p<3x10-6)

NO CANDIDATE RISKALLELES FOUND

651 common SNPs

SNPs UNIQUE to RISK HAPLOTYPE across 250 kb

Genotype 18 cM region with 1 SSLP/0.35 cMfor 256 trios:

Increase density of SSLPs, use microsatellite haplotypes for analysis

Resequenceknown genes

Resequence Genomic region in 8 patients

Genomic SNPs in Crohn disease patients

METHOD RESULT

CROHN DISEASE mapping and success• 1) multiple independent sets samples

• 2) availabilty of family material

• 3) combined methodology and haplotype analysis: the first application of LD mapping involving a systematic search for LD across a linkage peak and exhaustive ascertainment of SNPs in the critical region

• 4) for NOD2 - the same mutation identified in two independent studies!!

• 5) for cytokine cluster on 5q31 - the causal mutation has not yet been found. May be it is just the effect of haplotype or genotype effect of several combined loci, which counts….

Prerequisites for successful Prerequisites for successful association studiesassociation studies:

• 1. Detailed phenotype and diagnosis: maximized probability for the similar genetic factors for cases

• 2. Structure of control population : suitable controls• 3. Available family material• 4. Replicated results with independent sample sets• 5. Exhaustively characterized candidate regions for

genes, polymorphisms, population variation and LD• 5. Stringent criteria and testing of alternative genetic

models