4 QTL mappingLOCALIZE and then IDENTIFY a locus that regulates a trait (QTL)Nucleotide or sequence of nucleotides with variation in the population, with different variants associated with different trait levels.
5 For a heritable trait...Linkage:localize region of the genome where a QTL that regulates the trait is likely to be harbouredFamily-specific phenomenon:Affected individuals in a family share the sameancestral predisposing DNA segment at a given QTLAssociation:identify a QTL that regulates the traitPopulation-specific phenomenon:Affected individuals in a population share the sameancestral predisposing DNA segment at a given QTL
7 DNA structureA DNA molecule is a linear backbone of alternating sugar residues and phosphate groupsAttached to carbon atom 1’ of each sugar is a nitrogenous base: A, C, G or TTwo DNA molecules are held together in anti-parallel fashion by hydrogen bonds between bases [Watson-Crick rules]Antiparallel double helixA gene is a segment of DNA which is transcribed to give a protein or RNA productOnly one strand is read during gene transcriptionNucleotide: 1 phosphate group + 1 sugar + 1 base
8 DNA polymorphisms Microsatellites SNPs >100,000 Many alleles, (CA)n, veryinformative, even, easily automatedSNPs10,054,521 (25 Jan ‘05)10,430,753 (11 Mar ‘06)Most with 2 alleles (up to 4), not veryinformative, even, easily automatedAB
9 Diploid zygote >1 cell DNA organization22 + 12 (22 + 1)2 (22 + 1)2 (22 + 1)♂♂♁A -A -A -♁B -♂♁♂♁MitosisB -B -chr1A -A -A -- AA -- A♁♂♁B -B -B -- BB -- BA -- A- AB -chr1- B- BG1 phaseS phaseM phaseHaploid gametesDiploid zygote 1 cellDiploid zygote >1 cell
10 DNA recombination ♁ NR R R Diploid gamete precursor cell NR 22 + 122 + 1A -NR(♂)B -A -- Achr12 (22 + 1)2 (22 + 1)B -- B♁Meiosis- ARchr1♂(♂)(♁)♁- BA -- AA -- Achr1B -- BB -- BA -Rchr1chr1chr1chr1(♁)A -- AB -chr1Diploid gamete precursor cellB -- B- Achr1NR- BHaploid gamete precursorschr1Hap. gametes
11 DNA recombination between linked loci 22 + 1A -B -NR(♂)A -- AB -- B2 (22 + 1)♁Meiosis- ANR- B♂(♂)(♁)♁A -- AA -- AB -- BB -- BA -B -NR(♁)A -- AB -- BDiploid gamete precursor- A- BNRHaploid gamete precursorsHap. gametes
12 Human Genome - summaryDNA is a linear sequence of nucleotides partitioned into 23 chromosomesTwo copies of each chromosome (2x22 autosomes + XY), frompaternal and maternal origins. During meiosis in gamete precursors,recombination can occur between maternal and paternal homologsRecombination fraction between loci A and B (θ)Proportion of gametes produced that are recombinant for A and BIf A and B are very far apart: 50%R:50%NR - θ = 0.5If A and B are very close together: <50%R ≤ θ < 0.5Recombination fraction (θ) can be converted to genetic distance (cM)Haldane: eg. θ=0.17, cM=20.8Kosambi: eg. θ=0.17, cM=17.7
19 Parametric LOD score calculation Overall LOD score for a given θ is the sum of all family LOD scores at θeg. LOD=3 for θ=0.28
20 Parametric Linkage Analysis - summary QM1M2Mnθ0.50.5.4.18.104.22.168.1For each marker, estimate the θ that yields highest LOD score across all familiesThis θ (and the LOD) will depend upon the mode of inheritance assumedMOI determines the genotype at the trait locus Q and thus determines thenumber of meiosis which are recombinant or nonrecombinant. Limited toMendelian diseases.Markers with a significant parametric LOD score (>3) are said to be linkedto the trait locus with recombination fraction θ
21 Outline 1. Aim 2. The Human Genome 3. Principles of Linkage Analysis 4. Parametric Linkage Analysis5. Nonparametric Linkage Analysis
23 Approach Parametric: genotype marker locus & genotype trait locus (latter inferred from phenotype according to a specific disease model)Parameter of interest: θ between marker and trait lociNonparametric: genotype marker locus & phenotypeIf a trait locus truly regulates the expression of a phenotype, then tworelatives with similar phenotypes should have similar genotypes at amarker in the vicinity of the trait locus, and vice-versa.Interest: correlation between phenotypic similarity and marker genotypicsimilarityNo need to specify mode of inheritance, allele frequencies, etc...
25 Inheritance vector (M) Genotypic similarity between relativesIBS Alleles shared Identical By State “look the same”, may have thesame DNA sequence but they are not necessarily derived from aknown common ancestorM1M2M3M3IBD Alleles sharedIdentical By Descentare a copy of thesameancestoralleleQ1Q2Q3Q4M1M2M3M3Q1Q2Q3Q4IBSIBDM1M3M1M321Q1Q3Q1Q41Inheritance vector (M)1
26 Genotypic similarity between relatives - Inheritance vector (M)Number of alleles shared IBDProportion of alleles shared IBD -M1M3M2M311Q1Q3Q2Q4M1M3M1M3110.5Q1Q3Q1Q4M1M3M1M321Q1Q3Q1Q3
28 H:\manuel - Copy folder “Linkage” to C:\ PracticalAim(1) Estimate IBD with MERLIN; (2) IBD estimation can be influenced by genotyped individuals and allele frequencies; (3) computeH:\manuel - Copy folder “Linkage” to C:\1. Open with Notepad: pr1.ped pr1.dat pr1.map pr1.freq2. Start>Run>C:/Linkage/pfe32.exe3. Run Command Prompt4. Keep a File Explorer window openExercice1(1) Estimate IBD for pedigrees A, B and C in the previous slide(2) Change allele frequencies (pr1.freq) from to(i) and(ii)
29 Practical E F G A1A2 A3A4 A1A3 A1A3 A2A4 Exercice 2 A1A2 A1A3 A1A3 (1) Modify pr1.ped and estimate IBD probabilities and between twin 1 and twin 2 for pedigrees E, F and G:EFGA1A2A1A3A1A3A1A3A1A3A1A3A1A3A2A4P(IBD=0)0.080.000.00P(IBD=1)0.310.200.00P(IBD=2)0.610.801.000.770.901.00Allele frequencies on pr1.freq:
30 IBD at a marker Singlepoint IBD IBD at a ‘grid’ Multipoint IBD 5 cM M1 MnIBD at a markerSinglepoint IBD5 cMM1M2MnIBD at a ‘grid’Multipoint IBD
31 Statistics that incorporate both phenotypic and genotypic similarities Phenotypic similarity0.51Genotypic similarity ( )
33 VC ML – Quantitative & Categorical traits method0.51H1:H0:e.g. LOD=3
34 Genome-wide linkage analysis (e.g. VC) Individual LOD scores can be expressed as P values (Pointwise)LOD Chi-sq (n-df) P value(x4.6)True positivekTheoretical (Lander & Kruglyak 1995)LODLOD = 3.6, Chi-sq = 16.7, P =Type I error
35 Nonparametric Linkage Analysis - summary No need to specify mode of inheritanceModels phenotypic and genotypic similarity of relativesExpression of phenotypic similarity, calculation of IBDHE and VC are the most popular statistics used for linkage ofquantitative traitsOther statistics available, specially for affection traits
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