1Introduction to Gene-Finding:Linkage and AssociationDanielle Dick, Sarah Medland, (BenNeale)Aim of QTL mapping…LOCALIZE and then IDENTIFY a locus thatregulates a trait (QTL)• Locus: Nucleotide or sequence of nucleotides with variationin the population, with different variants associated withdifferent trait levels.2Location and Identification• Linkage• localize region of the genome where a QTL thatregulates the trait is likely to be harboured• Family-specific phenomenon: Affected individualsin a family share the same ancestral predisposingDNA segment at a given QTLLocation and Identification• Association• identify a QTL that regulates the trait• Population-specific phenomenon: Affectedindividuals in a population share the sameancestral predisposing DNA segment at a givenQTL3LinkageOverviewProgress of the Human Genome ProjectHuman Chromosome 44Genetic markers(DNA polymorphisms)ATGCTTGCCACGCEATGCTTGCCATGCESingle Nucleotide PolymorphismATGCTTGCCACGCEATGCTTCTTGCCATGCEMicrosatellite Markerscan be di(2), tri(3), or tetra (4)nucleotide repeatsDNA polymorphisms Can occur in gene, but be silent Can change gene product (protein) Alter amino acid sequence (a lot or a little) Can regulate gene product Upregulate or downregulate protein production Turn off or on gene Can occur in noncoding region This happens most often!5MutationsHow do we map genes? Deviation from Mendel’s IndependentAssortment Law Aa & Bb = ¼ AB, ¼ Ab, ¼ aB, ¼ ab We’re looking for variation from this6RecombinationRecombination Another way of introducing genetic diversity Allows us to map genes! Crossovers more likely to occur between genesthat are further away; likelihood of arecombination event is proportional to thedistance Interference – tend not to see 2 crossovers in a smallarea Alleles that are very close together are morelikely to stay together, don’t assort independently7Linkage Mapping (is a marker “linked”to the disease gene) Collect families with affected individuals Genome Scan - Test markers evenly spacedacross the entire genome (~every 10cM, ~400markers) Lod score (“log of the odds”) – what are the oddsof observing the family marker data if the markeris linked to the disease (less recombination thanexpected) compared to if the marker is not linkedto the diseaseThomas Hunt Morgan – discoverer of linkage8Linkage = Co-segregationA2A4A3A4A1A3A1A2A2A3A1A2A1A4A3A4A3A2Marker allele A1cosegregates withdominant diseaseLod scores >3.0 evidence for linkage <-2.0 can rule out linkage In between – inconclusive, collectmore families9Linkage = Co-segregationA2A4A3A4A1A3A1A2A2A3A1A2A1A4A3A4A3A2•Parametric Linkage usedvery successfully to mapdisease genes for Mendeliandisorders•Problematic for complexdisorders: requires diseasemodel, penetrance, assumesgene of major effect,phenotypic precisionNonparametric Linkage Based on allele-sharing More appropriate for phenotypes withmultiple genes of small effect, environment,no disease model assumed Basic unit of data: affected relative (oftensibling) pairs10x1/4 1/4 1/4 1/4IDENTITY BY DESCENTSib 1Sib 24/16 = 1/4 sibs share BOTH parental alleles IBD = 28/16 = 1/2 sibs share ONE parental allele IBD = 14/16 = 1/4 sibs share NO parental alleles IBD = 02222111 1111 1000011Genotypic similarity between relativesIBS Alleles shared Identical By State “look the same”, may have thesame DNA sequence but they are not necessarily derived froma known common ancestor - focus for associationIBD Alleles sharedIdentical By Descentare a copy of thesame ancestor allele- focus for linkageM1Q1M2Q2M3Q3M3Q4M1Q1M3Q3M1Q1M3Q4M1Q1M2Q2M3Q3M3Q4IBS IBD21Genotypic similarity – basic principals Loci that are close together are more likely to beinherited together than loci that are further apart Loci are likely to be inherited in context – ie with theirsurrounding loci Because of this, knowing that a loci is transmitted from acommon ancestor is more informative than simplyobserving that it is the same allele Critical to have parental data when possible12Linkage Markers…For disease traits (affected/unaffected)Affected sib pairs selected IBD = 2IBD = 1IBD = 01000250750500Expected 1 2 3 127 310Markers13For continuous measuresUnselected sib pairs1.000.250.750.50IBD = 0 IBD = 1 IBD = 2Correlation between sibs0.00So how does all this fit into Mx?14IDENTITY BY DESCENTSib 1Sib 24/16 = 1/4 sibs share BOTH parental alleles IBD = 28/16 = 1/2 sibs share ONE parental allele IBD = 14/16 = 1/4 sibs share NO parental alleles IBD = 02222111 1111 10000T2AEA Ca ac eT11 111Ee1Cc11 or .51 In biometrical modeling A is correlated at 1for MZ twins and .5 for DZ twins .5 is the average genome-wide sharing of genesbetween full siblings (DZ twin relationship)15 In linkage analysis we will be estimating anadditional variance component Q For each locus under analysis the coefficient ofsharing for this parameter will vary for each pair ofsiblings The coefficient will be the probability that the pair ofsiblings have both inherited the same alleles from acommon ancestorˆ!Q A C EPTwin1E C A QPTwin2MZ=1.0 DZ=0.5MZ & DZ = 1.01 1 1 11 1 1 1q a c ee c a qˆ!16LinkageHow do we do this?1.Genotyping data.Break down of time spent during a linkage/association studyCleaning &preparinggenotype dataRuning linkageanalysesEstimatingsignificanceMicrosatellite data Ideally positioned at equal genetic distancesacross chromosome Mostly di/tri nucleotide repeatshttp://research.marshfieldclinic.org/genetics/GeneticResearch/screeningsets.asp17Microsatellite data Raw data consists of allele lengths/calls (bp) Different primers give different lengths So to compare data you MUST know whichprimers were usedhttp://research.marshfieldclinic.org/genetics/GeneticResearch/screeningsets.aspBinning Raw allele lengths are converted to allelenumbers or lengths Example:D1S1646 tri-nucleotide repeat sizerange130-150 Logically: Work with binned lengths Commonly: Assign allele 1 to 130 allele, 2 to 133 allele … Commercially: Allele numbers often assigned based onreference populations CEPH. So if the first CEPH allelewas 136 that would be assigned 1 and 130 & 133 wouldassigned the next free allele number Conclusions: whenever possible start from the RAW allelesize and work with allele length18Error checking After binning check for errors Family relationships (GRR,
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