Molecular Genetic Methods in Psychology www.well.ox.ac.uk/~tprice/presentations.xml

1. Molecular Genetic Methodsin Psychologywww.well.ox.ac.uk/~tprice/presentations.xml Tom Price

2. Recap: Heredity • ‘Heritable’ characteristics are influenced by genetic variation (Mendel’s pea plants) • Traits are correlated within families (Galton) • Twin and adoption studies provide evidence of heritability

3. How? • Crick and Watson (1952) provided the mechanism. “the single biggest advance in molecular biology”

4. Central Dogma

5. DNA • DNA exists in the nucleus in twin strands • Each strand consists of A, C, G, T ‘bases’ on a sugar-phosphate ‘backbone’ • Each base binds only to its complement • The sequence of bases along a strand is called the ‘DNA sequence’

6. DNA Replication • During replication the DNA molecule unwinds, with each single strand becoming a template for synthesis of a new, complementary strand. Each daughter molecule, consisting of one old and one new DNA strand, is an exact copy of the parent molecule.

7. Transcription & Translation • DNA is first transcribed (copied) to a molecule of messenger RNA in a process similar to DNA replication. • The mRNA molecules then leave the cell nucleus and enter the cytoplasm to be translated into protein in the ribosomes. Triplets of bases (codons) in the mRNA form the genetic code that specify the particular amino acids that make up an individual protein.

8. Start of transcription exons introns Genes • A gene is a region of DNA whose sequence encodes a protein. • The human genome contains ~30,000 genes. • Only about 10% of the genome is known to include the protein- coding sequences (exons) of genes.

9. Chromosomes • Humans have ‘diploid’ chromosomes: each contains 2 DNA molecules, one from each parent • Humans have 23 ‘autosomal’ chromosomes and 1 sex chromosome (XX for females, XY for males) The extra copy of chromosome 21 identifies this individual as having Down syndrome.

10. Genetic Variation • Genetic variants (polymorphisms) arise by mutation, either spontaneously or from radiation, viruses, cancer, toxins… • Mutations in coding regions can change the gene product (‘coding variations’) – or not (‘silent mutations’) • Variations in non-coding regions can affect transcription (‘gene expression’) • Most variation occurs in ‘junk’ DNA

11. Polymorphisms • Deletion (e.g. Williams Syndrome) • Polysomy (e.g. Down Syndrome) • Variable-number repeat (e.g. Fragile X) • Single-Nucleotide Polymorphism (e.g. FOXP2 mutation in KE family with severe speech disorder) • Insertions, inversions, translocations…

12. Father Sperm Meiosis and Recombination Mother • During meiosis, the chromosomes duplicate, then cross over (‘recombine’) to produce a haploid gamete (sperm/egg) • The gamete derives genetic variants from both parents • Meiosis is the basis for heredity Meiosis Egg Fertilisation Child

13. Alleles and Genotype • Alleles = the genetic variants that exist at a particular genetic location (locus) • Genotype = the alleles present at a locus • cp. Phenotype = trait(s) of organism • Homozygous = 2 of same allele • Heterozygous = different alleles • Allele frequency = % of allele in a population

14. How to Find A Gene • Candidate genes: • You already have good reason to believe it is implicated. e.g. pharmacological evidence: dopamine transporter & receptor genes in ADHD • Functional genes: • Candidate based on what it is known to do. e.g. expression patterns in relevant tissue. BUT ~15,000 genes expressed in the brain

15. Positional Cloning • The identification of a gene based solely on its position in the genome • Most widespread strategy in human genetics in the last 15 years • Strengths: • No knowledge of gene product required • Very strong track record in single-gene disorders • Weaknesses: • Understanding of function not a certain outcome • Poor track record with multifactorial traits

16. Sequencing of Human Genome Facilitates Positional Cloning Collins, F.S. Positional cloning moves from perditional to traditional, Nat Genet, 9:347-350, 1995

17. Positional Cloning

18. Alleles + - Alleles + - + + - - Mendel’s Laws: I.Segregation There are two elements of heredity governing a trait in each individual, and these segregate (separate) during reproduction. Dominant Recessive

19. Mendelian Disorders • Measured phenotype caused by a single gene • May have multiple mutations in gene • May be additional (environmental) causes • Follow clear segregation in families • Typically rare in population • Examples • Duchenne Muscular Dystrophy • Cystic Fibrosis (1989) • Huntingdon’s Disease (1993) • ~1200 have been mapped

20. Pedigree Analysis • Genetic disorders, e.g. PKU caused by a recessive allele, have characteristic patterns of inheritance within families. • above: autosomal dominant • below: autosomal recessive

21. Mendel’s Laws: II.Independent Assortment • Traits are inherited independently of each other. NB. This is law is violated for traits governed by genes close by on the same chromosome. Alleles of these ‘linked’ loci will tend to co-segregate during recombination.

22. Linkage • Only ~1 recombination per chromosome • Loci that are close together on the same chromosome tend to be inherited together (‘linked’ or ‘in LD’) • The closer the loci, the more linkage • Degree of linkage is a measure of genetic distance • Linkage is measured by the recombination fraction, θ = proportion of recombinants θ = 0: no linkage θ = 0.5: complete linkage

23. Recombinants & Nonrecombinants Paternal alleles (where it can be worked out) • Grandchildren in generation III who received either A1B1 or A2B2 from their father are the product of nonrecombinant sperm; persons who received A1B2 or A2B1 are recombinant. Estimated recombination fraction = 2 / 7 = 0.28 • We cannot classify any of the individuals in generations I and II as recombinant or nonrecombinant, or identify recombinants arising from oogenesis in individual II2.

24. Markers • A polymorphic ‘marker’ locus can be informative about a disease locus over 106 base pairs away • Originally, phenotypic markers used in place of genotype e.g. blood groups and APOe4 in Alzheimer’s Disease • Sequencing of genome ￫ many markers • The vast majority of markers have no effect on phenotype.

25. Genetic Linkage Trait co-segregates with marker allele within families Requirements: • Many families with trait of interest • Informative markers

26. P( θ = 0.5 ) P( θ = 0 ) Linkage Analysis Paternal alleles (where it can be worked out) • We do not usually have this much information to work out recombinants / nonrecombinants. • If inheritance (e.g. dominant / recessive) is known, the likelihood of linkage can be calculated: LOD = log10[ ]

27. Single Gene Linkage Analysis

28. Nonparametric Linkage Analysis • In practice, complex inheritance is the norm, and nonparametric linkage analysis, which does not require the genetic model to be specified, is most commonly used. • A design employing affected sib pairs allows model-free analysis in nuclear families using programs like MAPMAKER/SIBS or GENEHUNTER. • LOD > 3.3 generally accepted as threshold for genome-wide significance.

29. Netherton Syndrome Linkage Chavanas et al., Am J Hum Genet, 66:914-921, 2000

30. Netherton Syndrome Haplotypes

31. Netherton Syndrome Gene Chavanas et al. 2000, Nature Genetics

32. Linkage: Success Stories • Linkage analysis has been successfully used to map many single gene disorders, e.g. early-onset Alzheimer’s Disease, many forms of mental retardation

33. Linkage: Problems • For complex traits, there have been many unreplicated findings “True linkage is hard to find”

34. Multifactorial (‘Complex’) Traits • 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

35. Why such limited success with Complex Trait Linkage studies? • Power • Powercalculations have always indicated need for many 100’s, probably thousands of families to detect genes of even moderate effect • N ~ 200 for most studies conducted to date • For QTL, this is about enough to detect a locus explaining 25% of the total variance in the trait • Hope for ‘low-hanging’ fruit • If there are one or a few monogenic-like loci within oligogenic spectrum, could lead to pathway information • Not supported by data. • Practical problems: errors in data

36. A ‘Link’ in the Chain • Linkage analysis can do no more than point to broad regions – ‘linkage hotspots’ – at best ~20cM, ~200 genes • More powerful methods must be used to ‘home in’ on the crucial gene.

37. The Next Link

38. (Allelic) Association Trait correlates with marker allele in population • Why? Markers remain in LD with the ‘founding’ mutation over many generations

39. Association = same ancestral origin Generation 1: a disease-causing mutation occurs on a chromosome Generation 2: about 50% of the children receive the mutation and the surrounding chromosomal segment from the mutated founder Generation 3: segments originating from the mutated founder chromosome get shorter … Generation N: very short segments around the mutated locus are conserved

40. Linkage: Allelic association within families

41. Allelic Association:Extension of linkage to the population • For both families, the same marker is ‘linked’ with the trait, but a different allele is implicated

42. Allelic Association:Extension of linkage to the population Trait is ‘linked’ with the same marker in all families: Allele 6 is ‘associated’ with trait.

43. Allelic Association Allele 6 is ‘associated’ with disease

44. Allelic Association:Three Common Forms • Direct Association • Mutant or ‘susceptible’ polymorphism • Allele of interest is itself involved in phenotype • Indirect Association • Allele itself is not involved, but a nearby correlated gene changes phenotype • Spurious association • Apparent association not related to genetic aetiology • Including: Natural selection , statistical artifact, and population stratification (see later)

45. Indirect & Direct Allelic Association Direct Association Measure trait relevance (*) directly, ignoring correlated markers nearby Indirect Association & LD Assess trait effects on D via correlated markers (Mi) rather than susceptibility/etiologic variants. Linkage Disequilibrium: correlation between (any) markers in population Allelic Association: correlation between marker allele and trait

46. Population Stratification • Recent admixture of populations • Requirements: • Group differences in allele frequency • Group differences in outcome • Leads to spurious association • In epidemiology, this is a classic matching problem, with genetics as a confounding variable Most oft-cited reason for lack of association replication

47. Population Stratification Association induced by sample mixing

48. Population Stratification: Solutions • Because of fear of stratification, complex trait genetics turned away from case/control studies • Family-based controls (e.g. TDT) • ‘Genetic control’: extra genotyping • Look for evidence of background population substructure and account for it.

49. Linkage v. Association

50. Association Study Outcomes Reported p-values from association studies in Am J Med Genet or Psychiatric Genet, 1997 Terwilliger & Weiss, Curr Opin Biotech, 9:578-594, 1998