1. Sources of variation and co-variation in the population Jaakko Kaprio University of Helsinki

2. Place Epidemiology examines determinants of disease in relation to place, time and person characteristics such as: - genes - behavior - environment - developmental stage Time Person

3. Development of life expectancy (U.S females in 1990, 1995 and projected ) Olshansky et al., Science 2001; 291:1491

4. Changes in cardiovascular risk factors explain for men 66% of the changes in mortality from stroke Vartiainen et al. BMJ 1995;310:901-4

5. In complex disease a person's susceptibility genotype and environmental history combine to establish present health status, and the genotype's norm of reaction determines future health trajectory Genes, developmental history and environment as determinants of health

6. The post-genomic era  Now that the full human genome sequence has been published, we have access to genetic information in an unprecedented manner: –3 billion base pairs in the human genome –c 20 000 to 30 000 genes  Thus, developments in molecular genetic analysis render it now possible to attempt identification of liability genes in complex, multifactorial traits, and to dissect out with new precision the role of genetic predisposition and environment/life style factors in these disorders.  New technologies and statistical tools are continuously introduced  Nonetheless, quantitative genetic methods provide an overall picture of the role of familial and genetic factors

7. Monogenic & Complex disorders The majority of human diseases are complex, i.e. multiple genetic and non-genetic causes Figure: Peltonen & McKusick Science 2001

8. Segregation and linkage  Do diseased family members share alleles at a locus more often than expected?  Are these alleles the same in many families?  Sibpairs or large pedigrees can be studied, depending on the disease or trait in question

9. Types of genes  Rare inborn errors of metabolism and other Mendelian gene variants (e.g. familial hypercholesterolemia) have major impact on individuals and families, but little effect on population level; – FH accounts for 1% of serum cholesterol variability in the population  see e.g. OMIM: http://www.ncbi.nlm.nih.gov/Omim/  However, they continue to account for only a small fraction of all cases

10. Characteristics of complex traits  Trait values are determined by complex interactions among numerous metabolic and physiological systems, as well as demographic and lifestyle factors  Variation in a large number of genes can potentially influence interindividual variation of trait values  The impact of any one gene is likely to be small to moderate in size  For diseases : Monogenic diseases that mimic complex diseases typically account for a small fraction of disease cases (examples in obesity, hypertension, dyslipidemias).

11. Susceptibility genes  Susceptibility genes increase disease risk only moderately and are context dependent. –total heritability of cholesterol levels is typically c 50% –Apo E account for 5-10% of variability in serum cholesterol in many populations, but effect of Apo E4 allele is small in individuals –presence of apo E4 moderately increases CHD and AD risk in many populations  For example frequency of apo E4 allele (associated with CHD and Alzheimer’s) is highest in nomadic populations [ e.g. Pygmies (0.407) and Khoi San (0.370), Papuans (0.368), some Native Americans (0.280), and Lapps (0.310) ] compared to.10 to.15 in populations of Mediterranean descent.

12. Genetic epidemiology and behavior genetics Strategies for family studies:  Does disease or behavior aggregate in families?  What are the causes of familial aggregation?  What is the model of genetic inheritance and which genes are responsible?  How do genes interact with the environment?

13. How to detect genetic effects and genes? Family studies: – provide estimates of heritability – information on mode of inheritance – adoption and twin studies as special cases Molecular genetic studies: – genome-wide association studies & snp- heritability – linkage in families – animal studies (e.g.’knockouts’) – known functional variants

14. What is heritability Heritability is the estimate of the proportion in total variance of a trait or liability to a disease that is accounted for by genetic variance - interindividual genetic differences. Genetic variance may arise from additive effects, due to different alleles at a locus, or may be due to dominance, the interactions of alleles Heritability is a characteristic of populations, not individuals or families, which is affected by both genetic and environmental effects

15. FAMILY STUDY  Provides estimates of the degree of family aggregation  Risks to siblings, parents, offspring as well as to other relatives can be estimated  Similarity of different types of relatives can permit modelling of genetic versus non-genetic familial influences

16. Obesity in families (Quebec Family study, 1996)

17. Genetic epidemiology  To disentangle genes and experience, we study special family groups:  Either family members sharing experiences but differing in shared genes, e.g. twin studies or  family members sharing genes, but differing in their shared experience, e.g. adoption studies

18. ADOPTION DESIGN Test for association between trait in adoptees and trait in biological parents (genetic correlation) & Test for association between trait in adoptees and trait in adoptive parents. STRENGTHS:relatively powerful WEAKNESSES: (1) poor generalizability (2) adoptive parents likely to provide ‘good homes’ (3) biological parents of adoptive children may have had multiple forms of psychopathology - selection (4) poor characterization of phenotypes of biological parents

19. Adoption studies of obesity (Sörensen et al.1998)

20. The Classical Twin Study  Monozygotic (MZ) pairs are genetically alike  Dizygotic (DZ) pairs, like siblings, share on average half of their segregating genes  DZ pairs can be same-sexed or opposite-sex (male-female)  Increased similarity of twin pairs compared to unrelated subjects suggests familial factors  Increased similarity of MZ pairs compared to DZ pairs provides evidence for genetic factors

21. BMI in 25 year olds female twin pairs (rMZ= 0.78, rDZ = 0.37) FinnTwin16 study

22. The classical twin study modelling  Model contribution of additive (A) and non-additive (D)genetic effects, environmental effects shared by family members (C ) and unshared effects (E) (i.e. unique to each family member)  Competing models, e.g. E, AE, ACE can be statistically compared and tested against actual data  Mx – statistical program created by Mike Neale most commonly used in genetic modelling: http://views.vcu.edu/mx/

23. Twin similarity for life span at very old age

25. Extensions of the classical twin study I  Effect modification by age, sex and environmental factors, e.g. smoking or obesity  Assess genetic covariance over time through longitudinal models  Assess sex effects by comparison of like-sexed and same-sexed DZ pairs  Assess social interaction effects

26. Age dependence of genetic effects: CHD in twin brothers

27. a 2 m:0.20 f:0.47 c 2 m:0.42 f:0.18 e 2 m:0.39 f:0.35 a 2 m:0.84 f:0.90 e 2 m:0.16 f:0.10 PI at birthBMI at 16 y r m: 0.11, f: 0.09 r e m: 0.16, f: 0.07 Bivariate analyses indicate the genetic and environmental contributions to the relationship of relative weight at birth and in adolescence (Pietiläinen et al, Obes Res 2002) r a m: 0.21, f: 0.13 FinnTwin16

28. Different phenotypes, different effects of genes: smoking Genetic effects Non-genetic family effects Experimentation (age 12)11%73% Initiation/ever smoker (adolescents) 20-36%18-59% Initiation/ever smoker (adults) 28-80%4-50% Persistence/ cessation58-71%None Nicotine dependence (Fagerström or DSM-IV) 60-72%None

29. Models of Gene-Environment Interaction Purcell, S., Variance components models for gene-environment interaction in twin analysis. Twin Research, 2002. 5: p. 554-571

30. Parental Monitoring and Smoking Quantity (Dick et al, J Abn. Psych, 2006) Parental Monitoring Low High Standardized Variance

31. TWIN DESIGN: Weaknesses (1)Generalizability-having a same-age sibling?? -having a genetically identical same-age sibling?? (2)Relative rarity of twin pairs. (3)Non-orthogonal design -- need large sample sizes. (4)If major environmental risk-factors are not assessed, interaction of genetic effects and shared environmental effects will be confounded with genetic effects. (5)Weak for detecting parent-to-offspring environmental influences.

32. Assumptions of the classical twin study  Equality of environmental variances in MZ and DZ pairs Differences may arise from: çplacentation and in utero effects çFetal programming hypothesis implications çdifferential parental treatment çzygosity determination errors  Random mating

33. Perinatal mortality among twins by zygosity and chorionicity

34. Birthweights of twins East Flanders Prospective Twin Survey (Loos 1998) DZMZDCMZMC % of pairs641026 Mean Birthweight 2476g2401g2314g

35. FAMILY STUDY Ultimately, sampling regular families must be a key part of any genetic epidemiologic approach. *Provides tests of generalizability of findings using more specialized twin-family and adoption designs. *Allows adequate representation of minority groups. Numbers of minority twin pairs, eg. Swedish speaking twin pairs in Finland, available for study are often small.

36. How to detect genetic effects and genes? Molecular genetic studies: – candidate genes, genome-wide scans – association studies & linkage – animal studies (e.g.’knockouts’) Family studies: – provide estimates of heritability – information on mode of inheritance – adoption and twin studies as special cases

37. Increasing the genetic signal in the data...  ascertain pedigree units that are likely to segregate genes of relevance –Ex: pedigrees with quasi-Mendelian disease transmission –affected sib pair approach of linkage analysis  ascertain families on the basis of individuals with extreme or remarkable phenotypes –Ex: extremely discordant sibpairs –ascertain young individuals with the disease  ascertain individuals from isolated populations: –more homogenous genetically and culturally as well  ascertain intermediate phenotypes –physiologic phenotype is “closer” to sequence variants

38. Two basic Analysis Strategies 1.candidate gene analysis motto: study a few good genes 2.whole-genome searches (genome scans) motto: cast out a net that catches all the big fish

39. Association studies: Case-control design What is the difference between genes of cases (e.g. with disease or trait) and controls? s Selection of controls is major challenge, as in all case-control studies s High rate of false-positive studies: s many genes are available for study s population admixture confounding factor s Publication bias

40. Candidate Gene Studies  statistically straightforward: test the association between genotypes and phenotype with contingency tables, chi- square test, regression  principle: if an allele is more frequent in affecteds than unaffecteds  gene may be close to a disease gene  candidacy of a gene can come from a number of different sources: –biological insights (e.g. gene expressed in a certain tissue) –homology to other genes –functional studies in model organisms –member of a relevant gene family  Challenge: greater biological understanding of the genes

41. POPULATION STRATIFICATION Hypothetical Example (by Andrew Heath) Falsely infer that A1 allele is risk-factor for Roman Catholicism. OR = 2.28, 95%CI 1.39 - 3.73 NO ASSOCIATION NORTHERN EUROPEAN ANCESTRY (N=200) SOUTHERN EUROPEAN ANCESTRY (N=200) NOT A1 allele A1 allele NOT ROMAN CATHOLIC ROMAN CATHOLIC NOT ROMAN CATHOLIC ROMAN CATHOLIC 162 18 90% 18 2 10% 35 15 25% 105 45 75% 70% 30% 90% 10% NOT ROMAN CATHOLIC ROMAN CATHOLIC 197 33 123 47 NOT A1 allele A1 allele MINGLED IN AUSTRALIAN POPULATION (N=400)

42. Genome-wide association studies  Large scale case-control series  For example MI patients and matched controls without MI  Use of very large numbers of SNPs to identify all possible genes associated with the disease  Typically 100,000 to 500,000 SNPs  Different technology platforms (Affymetrix, Illumina)

43. Gene x Environment Interactions Kendler & Eaves, 1986 ProtectivePredisposing Environment Liability to Illness AA Aa aa ProtectivePredisposing Environment Liability to Illness AA Aa aa ProtectivePredisposing Environment Liability to Illness AA Aa aa Genes and environment have additive, independent effects Genes control degree of sensitivity to environmental influence Genes control susceptibility to environmental pathogenesis

44. Gene-environment correlations refer to genetic effects on individual differences in liability to exposure to particular environmental circumstances. (Background is the extensive evidence that environmental risk exposure is far from randomly distributed) Gene-environment interactions concern genetically influenced individual differences in the sensitivity to specific environmental factors. (Background is the extensive evidence of huge individual differences in vulnerability to all manner of environmental hazards)

45. Examples of social x biological interactive effects  Biology controls sensitivity to environment effects –E.g., family stress x serotonin metabolism => depression and anxiety risk (Caspi, Science 2003)  Social context generates undifferentiated risk; biology constrains pathologic specificity –E.g., childhood neglect => alcoholism in men, eating disorders in women  Biological susceptibilities are amplified during rapid or intense contextual change –E.g., biological or gender-based vulnerabilities to depression and alcohol use as indexed by pubertal development  Biology controls liability to experiencing predisposing environments –E.g. genes for skin color

46. Integration of information at different levels Developments in molecular genetics render it now possible to attempt identification of liability genes in complex, multifactorial traits, and to dissect out with new precision the role of genetic predisposition and environment/life style factors in these disorders. But, an integrative framework is needed Complex picture Gottesmann I, Science 1997

47. Complexity of Complex Diseases  Classical polygenic or "threshold" inheritance: a certain number of mutations at different loci must be present before a system is sufficiently challenged to result in disease.  Locus heterogeneity, in which defects in any of a number of genes or loci confer disease susceptibility independently of each other.  Epistasis, or gene interaction: interactive effects of mutations, genotypes, and/or their biologic products  Environmental vulnerability: gene products are influenced by environmental stimuli.  Gene × environment interactions: gene has a deleterious effects only in the presence of a particular environmental stimulus.  Time-dependent expression of genes  General aging of the system

48. Testing of epidemiological causal hypotheses – use of twins  Differences between MZ cotwins in a pair are due to environmental causes (in the very broadest sense)  somatic mutations and other genetic changes during development  prenatal environmental and birth order effects  differential treatment in childhood  different exposures ( occupational, lifestyle)  Exposure/disease discordant DZ pairs are fully matched on early childhood effects, and partially on genetic factors  Studies of exposure discordant twin pairs have increased power compared to unmatched case-control series, depending on the degree of familiality of the exposure