Presentation on theme: "Molecular Genetic Methods in Psychology www. well. ox. ac"— Presentation transcript:
1Molecular Genetic Methods in Psychology www. well. ox. ac Molecular Genetic Methods in PsychologyTom Price
2Recap: 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 inmolecular biology”
5DNA 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 complementThe sequence of bases along a strand is called the ‘DNA sequence’
6DNA ReplicationDuring 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.
7Transcription & 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.
8Genes A gene is a region of DNA whose sequence encodes a protein. Start of transcriptionexonsintronsA 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.
9ChromosomesHumans have ‘diploid’ chromosomes: each contains 2 DNA molecules, one from each parentHumans 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.
10Genetic VariationGenetic 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
11Polymorphisms 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…
12Meiosis and Recombination FatherSpermMotherDuring meiosis, the chromosomes duplicate, then cross over (‘recombine’) to produce a haploid gamete (sperm/egg)The gamete derives genetic variants from both parentsMeiosis is the basis for heredityMeiosisEggFertilisationChild
13Alleles and GenotypeAlleles = the genetic variants that exist at a particular genetic location (locus)Genotype = the alleles present at a locuscp. Phenotype = trait(s) of organismHomozygous = 2 of same alleleHeterozygous = different allelesAllele frequency = % of allele in a population
14How 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 ADHDFunctional 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
15Positional CloningThe identification of a gene based solely on its position in the genomeMost widespread strategy in human genetics in the last 15 yearsStrengths:No knowledge of gene product requiredVery strong track record in single-gene disordersWeaknesses:Understanding of function not a certain outcomePoor track record with multifactorial traits
16Sequencing of Human Genome Facilitates Positional Cloning Collins, F.S. Positional cloning moves from perditional to traditional, Nat Genet, 9: , 1995
18Mendel’s Laws: I. Segregation There are two elements of heredity governing a trait in each individual, and these segregate (separate) during reproduction.-+AllelesDominantRecessive
19Mendelian Disorders Measured phenotype caused by a single gene May have multiple mutations in geneMay be additional (environmental) causesFollow clear segregation in familiesTypically rare in populationExamplesDuchenne Muscular DystrophyCystic Fibrosis (1989)Huntingdon’s Disease (1993)~1200 have been mapped
20Pedigree AnalysisGenetic disorders, e.g. PKU caused by a recessive allele, have characteristic patterns of inheritance within families.above: autosomal dominantbelow: autosomal recessive
21Mendel’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.
22Linkage 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 linkageDegree of linkage is a measure of genetic distanceLinkage is measured by the recombination fraction, θ = proportion of recombinantsθ = 0: no linkageθ = 0.5: complete linkage
23Recombinants & 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 = 0.28We cannot classify any of the individuals in generations I and II as recombinant or nonrecombinant, or identify recombinants arising from oogenesis in individual II2.
24MarkersA polymorphic ‘marker’ locus can be informative about a disease locus over 106 base pairs awayOriginally, phenotypic markers used in place of genotype e.g. blood groups and APOe4 in Alzheimer’s DiseaseSequencing of genome ￫ many markersThe vast majority of markers have no effect on phenotype.
25Trait co-segregates with marker allele within families Genetic LinkageTrait co-segregates with marker allele within familiesRequirements:Many families with trait of interestInformative markers
26Linkage AnalysisPaternal 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 )
28Nonparametric 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.
29Netherton Syndrome Linkage Chavanas et al., Am J Hum Genet, 66: , 2000
31Netherton Syndrome Gene Chavanas et al. 2000, Nature Genetics
32Linkage: 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: ProblemsFor complex traits, there have been many unreplicated findings“True linkage is hard to find”
34Multifactorial (‘Complex’) Traits No clear segregation pattern in familiesCaused by > 1 genePossibly triggered / moderated by environmentEach gene (environment) may have small effectEpistasis or intragenic interactions likelyPleiotropy, environmental influences, gene x environment interactions likelyEpigenetic influences possibleMeasurement of phenotype not highly reliableHeterogeneity
35Why such limited success with Complex Trait Linkage studies? PowerPower calculations have always indicated need for many 100’s, probably thousands of families to detect genes of even moderate effectN ~ 200 for most studies conducted to dateFor QTL, this is about enough to detect a locus explaining 25% of the total variance in the traitHope for ‘low-hanging’ fruitIf there are one or a few monogenic-like loci within oligogenic spectrum, could lead to pathway informationNot supported by data.Practical problems: errors in data
36A ‘Link’ in the ChainLinkage analysis can do no more than point to broad regions – ‘linkage hotspots’ – at best ~20cM, ~200 genesMore powerful methods must be used to ‘home in’ on the crucial gene.
38(Allelic) Association Trait correlates with marker allelein populationWhy?Markers remain in LD with the ‘founding’ mutation over many generations
39Association = same ancestral origin Generation 1: a disease-causing mutation occurs on a chromosomeGeneration 2: about 50% of the children receive the mutation and the surrounding chromosomal segment from the mutated founderGeneration 3: segments originating from the mutated founder chromosome get shorter…Generation N: very short segments around the mutated locus are conserved
41Allelic Association: Extension of linkage to the population For both families, the same marker is ‘linked’ with the trait, but a different allele is implicated
42Allelic Association: Extension of linkage to the population Trait is ‘linked’ with the same marker in all families:Allele 6 is ‘associated’ with trait.
43Allele 6 is ‘associated’ with disease Allelic AssociationAllele 6 is ‘associated’ with disease
44Allelic Association: Three Common Forms Direct AssociationMutant or ‘susceptible’ polymorphismAllele of interest is itself involved in phenotypeIndirect AssociationAllele itself is not involved, but a nearby correlated gene changes phenotypeSpurious associationApparent association not related to genetic aetiologyIncluding: Natural selection , statistical artifact, and population stratification (see later)
45Indirect & Direct Allelic Association Direct AssociationMeasure trait relevance (*) directly, ignoring correlated markers nearbyIndirect Association & LDAssess trait effects on D via correlated markers (Mi) rather than susceptibility/etiologic variants.Linkage Disequilibrium: correlation between (any) markers in populationAllelic Association: correlation between marker allele and trait
46Population Stratification Recent admixture of populationsRequirements:Group differences in allele frequencyGroup differences in outcomeLeads to spurious associationIn epidemiology, this is a classic matching problem, with genetics as a confounding variableMost oft-cited reason for lack of association replication
47Population Stratification Association induced by sample mixing
48Population Stratification: Solutions Because of fear of stratification, complex trait genetics turned away from case/control studiesFamily-based controls (e.g. TDT)‘Genetic control’: extra genotypingLook for evidence of background population substructure and account for it.
49Linkage v. Association Linkage Association Requires families Families or unrelatedsMatching/ethnicity generally unimportantMatching/ethnicity importantFew 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 locationYields fine-scale locationGood for initial detection,poor for fine-mappingGood for fine-mapping,poor for initial detectionPowerful for rare variantsPowerful for common variants,rare variants generally impossible
50Association Study Outcomes Reported p-values from association studies inAm J Med Genet or Psychiatric Genet, 1997Terwilliger & Weiss, Curr Opin Biotech, 9: , 1998
51Why limited success with association studies? Small sample sizes → results overinterpretedPhenotypes are complex. Candidate genes difficult to chooseAllelic/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
53Effects of Linkage Disequilibrium Roses, Nature 2000
54Alzheimer’s Disease Common Disease of old age: Main cause of dementia in the elderly4th leading cause of deathPrevalence increases with age; much earlier onset in rare casesProgressive loss of memory, cognitive deterioration, and emotional disturbanceLoss of neurons with many amyloid-containing plaques, neurofibrillary tangles
55Genetic EpidemiologyEarly-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.
56Apolipoprotein E 3 alleles: E2 (8%), E3 (77%), E4 (15%). Risk relative to E3/E3 at age 65+E3/E4: ~3E4/E4: ~14Accounts for ~20% of susceptibilityAPOe risk associated with age of onset, clinical manifestations of AD, selective effect on episodic memory
57Investigation of APOe Risk Mechanism currently not knownPossible ethnic differencesGenetic risk interacts with head injury, education, possibly nutrition (anti-oxidants?)Clinical trials of folic acid, statins, NSAIDs as protective factors.
58Poster child for behavioural genetics? AD & APOePoster child for behavioural genetics?Or cautionary tale?
59Further ReadingPlomin 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.