Molecular Genetics of Complex Traits www.well.ox.ac.uk/~tprice/presentations.xml

1. Molecular Genetics of Complex Traitswww.well.ox.ac.uk/~tprice/presentations.xml Tom Price

2. Linkage • Linkage = proximity of loci on chromosome • Close-by loci tend to be inherited together (co-segregate) • Can map genes by analysing co-segregation of trait and marker in families

3. Linkage Disequilibrium • LD = when alleles from different loci occur together with greater frequency than expected based on allele frequencies • LD is specific to the population • More LD in genetic isolates e.g. Iceland

4. Allelic Association • Allele correlates with trait in population • Close-by markers remain in LD with founding mutation over many generations

5. Positional Cloning Linkage then Association is a typical strategy

6. Positional Cloning of Complex Traits: Lack of Success ...Not surprisingly, progress in analyzing complex genetic disorders has been more modest. What success there has been has basically come from one of two approaches: (i) Identification of a sub-phenotype in pedigrees... (akin to Mendelian disorder) (ii) Genetic studies in isolated human populations (reduced genetic variation) Collins et al., Science, 278:1580-81, 1997 … and the situation has not improved since then

7. Quantitative Trait Locus • QTL = influential gene • Assumed to be one of many influential genes in a continuum of risk • Probabilistic model, not deterministic / Mendelian • Possible to model dominance and epistasis (gene-gene interaction) as well as additive genetic effects ← Genetic Risk → Disorder ← Genetic Risk →

8. Construct Validity Retardation/Disability Giftedness ← Intelligence → Is intelligence the same thing as lack of learning disability?

9. Learning Disability • Severe (IQ < 50): • Down Syndrome, Fragile X, many rare genetic disorders esp. on X chromosome • Birth trauma, hypoxia • Low familiality • Mild (IQ < 70): • Social correlates • High familiality Like IQ

10. Genetic Risk and IQ Severe Retardation ← IQ → • Severe retardation does not have the same genetic (or environmental) aetiology as either milder learning disabilities or IQ. • Genetic variants causing severe retardation do not influence IQ in the normal range.

11. IQ QTLs • Candidate gene studies have had limited success • APOe and cognitive decline in old age • Genes with variants causing disability generally do not have variants that affect IQ in the normal range • Positional cloning efforts have not led to replicated results • Presumably genes of small effect

12. Language Ability / Disability • Some evidence that developmental language disorders more heritable than language ability • Severe speech disorder in KE family due to a rare variant that does not affect common language impairments Lai et al. (2001) Nature 413:519-523.

13. Dyslexia / Reading Ability • Early successes: linkage hotspots • Possible overlap with language impairment on chromosome 15 • Cloning attempts unsuccessful

14. Even if we found a gene,what would we know? • Cognitive neuroscience only recently concerned with individual differences • Molecular machinery of learning still poorly understood • NMDA receptors & synaptic plasticity • Difficult to incorporate knowledge of molecular action into a systemic account of cognitive abilities

15. Neuroanatomy of Anxiety Stimulus processing: Cortex Hippocampus Thalamus Hypothalamus Amygdala Motor readiness Danger Efferent Systems Anxiety Sensory Systems Hormonal stress response: Chatecholamines Cortisol Threat Neurotransmitter Systems

16. HPA axis Hypothalamus PVN ACTH Pituitary CRH Cortisol Adrenal glands

17. Clinical Anxiety • Includes • Generalised Anxiety Disorder • Panic Disorder • Phobias • Evidence of moderate heritability exists for most diagnostic categories

18. Neuroticism • Personality trait • Many questionnaire items overlap with anxiety symptoms • Moderately heritable (0.3 – 0.6), little evidence of common environment

19. Neuroticism & Anxiety • Extreme neuroticism may manifest as clinical anxiety • Anxiety tends to be chronic: Neuroticism may be a more stable measure • Many different diagnostic categories for anxiety may have same genetic basis

20. Anxiety ← Genetic Risk → Neuroticism as DimensionAnxiety as Disorder • Less sensitive to diagnostic boundary • Assumes genetic influences affect both high and low risk subjects • Increase in power from using quantitative trait ← Neuroticism →

21. Construct Validity ? Anxiety ← Neuroticism → • What do you call a lack of neuroticism? • Does it have the same genetic basis as anxiety?

22. Shared Genetic Influences rG = 0.8 Genetic Risk Genetic Risk Multivariate twin data show overlapping genetic influences on GAD and Neuroticism: Genetic correlation rG = 0.8 Hettema et al., Am J Psychiatry. 2004; 161(9):1581-7 GAD Neuroticism

23. Endophenotype Other genes Genetic Risk Environment Ideal Endophenotype Phenotype

24. Endophenotypes • Complex traits may not relate closely to biological processes • An endophenotype is a proxy measure designed to be a behavioural marker of genetic risk, e.g. • cortisol dysregulation in anxious / affective disorders • sensory gating in schizophrenia • Endophenotypes are particularly important in animal models • Endophenotypes can have conceptual and measurement problems of their own

25. Complex Traits: Hard to Study • No clear segregation pattern in families • Caused by > 1 gene • Possibly triggered / moderated by environment • Each gene (environment) may have small effect • Epistasis or intragenic interactions likely • Pleiotropy, environmental influences, gene x environment interactions likely • Epigenetic influences possible • Measurement of phenotype not highly reliable • Heterogeneity

26. Phenotypic Complexity Weiss & Terwillinger, Nat Genet, 2000

27. Anxiety: Multiple Perspectives • Clinical diagnoses • Neuroticism • Physiological measures of fearfulness / stress e.g. • Skin conductivity • Peripheral cortisol (in serum/saliva) • Animal models of fearfulness

28. Peripheral Cortisol • Some clinical studies show cortisol ‘dysregulation’ in affective disorders/PTSD • Very mixed results • Measurement problems: • Circadian rhythm (peaks in morning) • Poor reliability • Base level or amplitude of stress response? • Blunted or increased stress response?

29. Animal Models • Animal models provide more powerful research designs than are available (on ethical grounds) for humans • Manipulation of genes and/or environment • Non-genetic animal models of anxious traits have proven extremely useful • Motherless chimps (Harlow: “Love at Goon Park”) • Maternal licking/grooming of rat pups (Meaney) • Very similar genomes to humans • Homology ~95% for mice, ~98% for chimps

30. Rodent Models of Anxiety • Unconditioned behaviour: • Open field activity / thigmotaxis / defecation • Latency to start eating in novel arena • Conditioned behaviour: • Startle to fear-conditioned stimulus • Freezing to footshock-conditioned stimulus • Passive vs. active avoidance

31. Validation of Rodent Models • Factor analysis reveals several components to unconditioned behaviour • Factors genetically separable • Anxiolytic drugs abolish fearfulness • Complicated by effects on activity (unconditioned) active vs. passive avoidance (conditioned). • Extensive anatomical, cellular, and molecular investigation of fear-potentiated startle • Amygdala involvement in establishment of conditioned fear

32. Animal Breeding Studies • Selective mating can be used to generate animal strains with particular characteristics for use in genetic studies, e.g. mice with high / low open field activity • Results in stable differences between lines • Differences generalise to other measures (e.g. defecation)

33. Rodent QTL Analysis Methods • Recombinant inbred strains • Cross C57BL/6J × DBA/2J strains • Recombinations known; DNA available as a public resource • Useful for coarse mapping: inbreed F2 progeny for greater resolution • Congenic Strains • These are identical except at a specific locus. • They are produced by repeated backcrossing • Used to explore the effect of changing just one genetic factor on a constant background. • Outbred stocks • Use naturally-occurring variation • Many recombinations so useful for fine mapping

34. Rodent QTL Analyses • Replicated linkage regions on 13 chromosomes • Some fine-mapping on chromosome 1 (~1cM) • A recent local success…

35. QTL Analysis in Outbred Lines Yalcin et al., Nat Genet. 2004;36(11):1197-202

36. 5HTTPR • Serotonin (5HT) Transporter Promoter Region • Functional variable-repeat polymorphism alters expression levels • Many association studies: • Short (less active) allele weakly increases anxiety (0.2 SD) and risk of bipolar disorder

37. 5HTTPR • Weak association might hide larger effects in different environments • Animal model evidence: short allele predicts low CSF serotonin metabolite levels ih rhesus monkeys, but only in deprived (peer-reared) and not in maternally reared monkeys. A similar interaction is seen for HPA stress response Caspi et al. (2003) Science 301: 386-389

38. Gene Targeting and Transgenics • Knock Outs lacking the gene of interest. • Models loss of function mutations • Conditional KOs lack gene in specific tissues • Transgenics contain foreign DNA inserts • Can be used to model overexpression • Much used in physiology • Developmental issues (in biological sense)

39. Epigenetics environmental influences on gene activity • Maternal care of rat pups increases expression of glucocorticoid receptor through increased 5HT activity • Long term aggravation of HPA stress response • Mechanism: methylation of GR promoter Weaver et al. (2004) Nat Neurosci 7:847-854

40. Random Mutagenesis • Induced by chemicals / radioactivity • Large-scale programs for Drosophilia, Mouse have led to important findings (e.g. CLOCK mutant with long circadian rhythm) (Vitaterna et al.,Science 1994;264:719-725) • Powerful • Laborious (esp. phenotyping)

41. Functional Genetics • High-throughput measures of cellular activity: • Transcription (microarrays) • Protein expression (Mass spectrometry) • Protein-DNA binding (‘ChIP’) • Systems Biology approach • Bioinformatic tools necessary to combine and analyse multivariate data

42. Behaviour: It’s Not All Biology Neither molecular genetics nor other biological investigations of behaviour (e.g. fMRI) will be intelligible without an understanding of the intermediary psychological processes. Psychologists define the phenotype

43. Further Reading Plomin R, DeFries JC, Craig IW, McGuffin P. (2002). Behavioral Genetics in the Postgenomic Era. APA. Deary IJ. (2001). Chapter 7: Wetware. In: Looking down on intelligence.