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Association studies of complex diseases

Association studies of complex diseases. COMPLEX DISEASES 1. 1. Incomplete penetrance (avaldumine) and phenocopy (fenokoopiad) 2. Genetic heterogeneity 3. Oligo/polygenic inheritance l i = l 1 l 2 l 3... l n (multiplicative effect) l i = l 1+ l 2+ l 3+...+ l n (addititive effekt).

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Association studies of complex diseases

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  1. Association studies of complex diseases

  2. COMPLEX DISEASES 1 • 1. Incomplete penetrance (avaldumine) and phenocopy (fenokoopiad) • 2. Genetic heterogeneity • 3. Oligo/polygenic inheritance li = l1l2l3...ln (multiplicative effect) li = l1+l2+l3+...+ln (addititive effekt). • BUT the contribution of every single ln is too small for enough statistical power power • 4. High frequency of disease-susceptibility alleles • 5. Influenced by environmental factors

  3. Quantitative 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: Susceptibility (vastuvõtlikkus) threshold(lävi) liability(kalduvus) liability > threshold = disease develops lR= the risk of the relatives of a patient compared to the general population risk lR > 1 genetic component of the disease exists COMPLEX DISEASES 2

  4. LINKAGE AND LD MAPPING Genetic Factor 1 Marker Genotypes MODE of INHERITANCE Genetic Factor 2 ??? CORRELATION To be TESTED Phenotype Genetic Factor n Cultural factors Polygenic background Individual Environment Common Environment

  5. Models for the interraction of loci in complex diseases (by Gabriel et al., 2002) GENOTYPE PHENOTYPE GENOTYPE PHENOTYPE EFFECT 1. Additive 3. Epistatic EFFECT + + individual no individual effect 4. Mixed Multipli- cative 2. Multipli- cative x x OR x x In combination only either combination only

  6. Genetic 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

  7. Parametric and non-parametric linkage analysis The method of choice in case: 1) a few major-susceptibility loci are expected 2) 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 variability Major drawbacks: 1)needs defining of the genetic model 2) needs information about allele frequencies and penetrance 3) needs exact diagnosis 4) needs large pedigrees and exensive sample size 5) does not work for disease heterogeneity 6) resolution limit 7) Dilemma: non-stringent analysis =>false-positive results stringent analüüs => low power

  8. Strategies used for successful complex disease 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

  9. Association studies 1 1) Candidate gene based: * metabolically and physiologically relevant * previously mapped by linkage to a chromosomal region * expression analysis *chromosomal rearrangements involved 2) Whole-genome scan: *single loci ->too expensive, laborious and lacks statistical power * needs existance of LD based HAPMAP

  10. CANDIDATE GENE/ SYSTEM BIOLOGY APPROACH EXACT DIAGNOSIS PHENOTYPIC MEASUREMENTS WELL-DEFINED CANDIDATE GENES OR BIOLOGICAL SYSTEMS WELL-DEFINED PHENOTYPE FOLLOW-UP STUDIES: CLINICAL AND GENETIC LIFESTYLE RECORDS POLYMORPHISM DISCOVERY ASSOCIATION STUDIES: 1) CASE-CONTROL 2) SIB-PAIR(TDT, HRR) STATISTICAL METHODS AND MODELS POPULATION STUDIES: VARIATION AND GENETIC STRUCTURE STUDIES OF POSITIVE ASSOCIATION RESULTS IN BIOLOGICAL SYSTEMS AND MODELS (mRNA, protein, knock-out)

  11. Association studies 2 Direct Indirect Based on polymorhisms potentially in allelic association (linkage disequilibrium with disease susceptibility variant) Both, candidate gene or region and whole genome scan approaches Based on candidate Pathological polymorhism e.g. changing aminoacid in a peptide ONLY candidate gene/ Candidate polymorhism approach

  12. Knowledge of linkage disequilibrium and haplotype structure of a target gene

  13. Familiy material: 1. ASP affected-sib-pair , APM (affected pedigree member) IBS- identity by descent, IBS- identity by state 2.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 Association studies 3

  14. 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

  15. Hirschhorn et. al.,2002: • summerized association studies conducted 1986-2000: 166 putative associations studies 3 or more times • ONLY 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?

  16. 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

  17. ARCAGE study: Number of case-control pairs required to provide an 80% power to detect a main effect at 1 % significance level.

  18. ETHNIC ADMIXTURE Ethnic group 1 Ethnic group 2 True positive association: The frequency of risk allele is greater in both ethnic groups CASES 67% 67% CONTROLS 33% 33% False positive association: The frequency of risk allele is identical in controls and cases in both populations BUT:the allele is 2x as frequent in cases of pooled population compared to controls CASES 20% 80% CONTROLS 80% 20%

  19. Population staratification: solutions • 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)

  20. Haplotype blocks: *loci in LD combine into 3-5 common haplotypes defined by 6-8 SNPs *loci characterized by similar nucleotide variation Block size <1-170 Kb, average block for Europeans 18 kb, Africans 9kb Block 2 Block 3 A chromosomal segment Recombination hotspots: 1-2 kb regions characterized by extensive crossing-over events Reich et al., 2001, Johnson et al., 2001 Daly et al., 2001; Jeffreys et al., 2001; Goldstein et al., 2001; Gabriel et al., 2002

  21. Cardon & Abecasis, 2003

  22. 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

  23. Distribution of recombination rateRates given in cm/Mb, data from 4,088 STRP (Yu et a., 2001 1 cM=1 recombination event per 100 meioses 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

  24. Distribution of recombination across chromosome 19 Puurand, 2004

  25. Correlations of recombinationrates with sequence parameters Yu et al., 2001

  26. How to select SNPs for association studies? * * * Currently considered of high importance due to location of regulatory regions

  27. Whole genome association scan:SNP requirements for HAPMAPconstruction HAPMAP - genome map of haplotype blocks and variants

  28. 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

  29. CROHN DISEASE and NOD2 (Ogura et al., 2001) RESULT METHOD 1.LINKAGE MAPPING Multiple studies #16 pericentromere 2.POSITIONAL CANDIDATE APPROACH NOD2 gene CANDIDATE GENE structure 3. cDNA versus genomic clones 4.Mutatation search in patients (12) vrs. controls (4) 3020insC mutation in 3 patients: frameshift and truncated protein 5. Allele-specifi PCR for typing 3020Cins for #16 linked Families and case-control material TDT test: p=0.0046 (39 transmissions vrs. 17 nontr) Case-control: p=0.0018 (8.2 % in cases vrs. 4% in control) 6.1.TDT test for heterozygous parents and one affected child of the family 6.2. Case-contorl test for the significance of allele frequencydifference GRR for 3020insC heterozygotes 1.5 and homozygotes 17.6

  30. CROHN DISEASE and NOD2 (Hugot et al., 2001) RESULT METHOD #16 pericentromere 1.LINKAGE MAPPING Multiple studies 2.Locus REFINEMENT by additional typing and Linkage analysis 16q, ~ 5 cM region ~ 2 Mb candidate region 3. Physical mapping of the region 4.TDT test with 108 families P<0.05 for one of the markers 5. Replication TDTwith independent set of 76 families P<0.001 for the same marker Stong LD among most markes, SNPs as well as microsatellites 6. Sequencing of the 164 kb BAC clone An identified gene with a risk haplotype of 3 SNPs 7. Typing 11 identified SNPs 8. 1.Characterization of Unigene clusters 8.2. PDT tests for SNPs in the gene One SNP: 1 bp insertion in exon10 of NOD2 gene -> frameshift and truncated protein

  31. CROHN DISEASE and cytokine gene cluster on 5Q31 (Rioux et al., 2001; Daly et al., 2001) RESULT METHOD 18 cM peak on 5q31 1.LINKAGE MAPPING Genotype 18 cM region with 1 SSLP/0.35 cMfor 256 trios: 2. LD MAPPING 1 2 markers in significant TDT (p<0.001) Increase density of SSLPs, use microsatellite haplotypes for analysis 3.LD MAPPING 2 MULTILOCUS ANALYSIS: 435 KB HAPLOTYPE (p<3x10-6) Resequence known genes 4.CANDIDATE GENES NO CANDIDATE RISK ALLELES FOUND Resequence Genomic region in 8 patients SNP DISCOVERY 651 common SNPs Genomic SNPs in Crohn disease patients SNPs UNIQUE to RISK HAPLOTYPE across 250 kb SIGNIFICANT SNPS

  32. 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….

  33. Prerequisites for successful association 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

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