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Molecular Genetic Methods in Psychology www. well. ox. ac

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1 Molecular Genetic Methods in Psychology www. well. ox. ac
Molecular Genetic Methods in Psychology 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 “the single biggest advance in
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 Genes A gene is a region of DNA whose sequence encodes a protein.
Start of transcription exons introns 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 Meiosis and Recombination
Father Sperm 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: Functional 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: , 1995

17 Positional Cloning

18 Mendel’s Laws: I. Segregation
There are two elements of heredity governing a trait in each individual, and these segregate (separate) during reproduction. - + Alleles 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 Trait co-segregates with marker allele within families
Genetic Linkage Trait co-segregates with marker allele within families Requirements: Many families with trait of interest Informative markers

26 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[ ] P( θ = 0.5 ) P( θ = 0 )

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: , 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 “True linkage is hard to find”
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 Power calculations 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 Allele 6 is ‘associated’ with disease
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 Linkage Association Requires families
Families or unrelateds Matching/ethnicity generally unimportant Matching/ethnicity important Few markers for genome coverage ( STRs) Many markers for genome coverage (105 – 106 SNPs) Weak design (allele-sharing based on covariances) Powerful design (based on mean differences) Yields coarse location Yields fine-scale location Good for initial detection, poor for fine-mapping Good for fine-mapping, poor for initial detection Powerful for rare variants Powerful for common variants, rare variants generally impossible

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: , 1998

51 Why limited success with association studies?
Small sample sizes → results overinterpreted Phenotypes are complex. Candidate genes difficult to choose Allelic/genotypic contributions are complex. Even true associations difficult to see. Background patterns of LD are unknown. Difficult to appreciate signal when can’t assess noise. Spurious results due to population stratification

52 Heterogeneity

53 Effects of Linkage Disequilibrium
Roses, Nature 2000

54 Alzheimer’s Disease Common Disease of old age:
Main cause of dementia in the elderly 4th leading cause of death Prevalence increases with age; much earlier onset in rare cases Progressive loss of memory, cognitive deterioration, and emotional disturbance Loss of neurons with many amyloid-containing plaques, neurofibrillary tangles

55 Genetic Epidemiology Early-onset disease is sometimes Mendelian and autosomal dominant. Standard lod score analysis in dominant early-onset families allowed mapping and subsequently cloning of three genes. Multicase late-onset families showed evidence of linkage to chromosome 19 when analyzed by the affected pedigree member method.

56 Apolipoprotein E 3 alleles: E2 (8%), E3 (77%), E4 (15%).
Risk relative to E3/E3 at age 65+ E3/E4: ~3 E4/E4: ~14 Accounts for ~20% of susceptibility APOe risk associated with age of onset, clinical manifestations of AD, selective effect on episodic memory

57 Investigation of APOe Risk
Mechanism currently not known Possible ethnic differences Genetic risk interacts with head injury, education, possibly nutrition (anti-oxidants?) Clinical trials of folic acid, statins, NSAIDs as protective factors.

58 Poster child for behavioural genetics?
AD & APOe Poster child for behavioural genetics? Or cautionary tale?

59 Further Reading Plomin R, DeFries JC, McClearn GE & McGuffin P. (2001). Behavioral Genetics (4th ed.). Worth. Strachan T & Read AP (1999). Human Molecular Genetics. Bios. (look online) Lahiri DK, Sambamurti K & Bennett DA. Apolipoprotein gene and its interaction with the environmentally driven risk factors: molecular, genetic and epidemiological studies of Alzheimer’s disease. Neurobiology of Aging 25:651–660.

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