1. Linkage and LOD score Egmond, 2006 Manuel AR Ferreira Massachusetts General Hospital Harvard Medical School Boston

2. Outline 1. Aim 2. The Human Genome 3. Principles of Linkage Analysis 4. Parametric Linkage Analysis 5. Nonparametric Linkage Analysis Practical

3. 1. Aim

4. QTL mapping LOCALIZE 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... localize region of the genome where a QTL that regulates the trait is likely to be harboured identify a QTL that regulates the trait Linkage: Association: Family-specific phenomenon: Affected individuals in a family share the same ancestral predisposing DNA segment at a given QTL Population-specific phenomenon: Affected individuals in a population share the same ancestral predisposing DNA segment at a given QTL

6. 2. Human Genome

7. A DNA molecule is a linear backbone of alternating sugar residues and phosphate groups Attached to carbon atom 1’ of each sugar is a nitrogenous base: A, C, G or T Two DNA molecules are held together in anti-parallel fashion by hydrogen bonds between bases [Watson-Crick rules] Antiparallel double helix Only one strand is read during gene transcription Nucleotide: 1 phosphate group + 1 sugar + 1 base DNA structure A gene is a segment of DNA which is transcribed to give a protein or RNA product

8. DNA polymorphisms A B Microsatellites >100,000 Many alleles, (CA) n, very informative, even, easily automated SNPs 11,961,761 (11 Sept ‘06) Most with 2 alleles (up to 4), not very informative, even, easily automated

9. Haploid gametes ♁ ♂ ♂ ♁ G 1 phase chr1 S phase Diploid zygote 1 cell M phase Diploid zygote >1 cell ♁ ♂ ♁ A - B - A - B - A - B - A - B - A - B - A - B - ♂ ♁ A - B - A - B - ♂ ♁ - A - B - A - B - A - B - A - B DNA organization Mitosis 22 + 12 (22 + 1)

10. Diploid gamete precursor cell (♂)(♁) (♂) (♁) Haploid gamete precursors Hap. gametes NR R R ♁ A - B - - A - B A - B - - A - B A - B - - A - B A - B - - A - B ♂ ♁ A - B - A - B - - A - B - A - B DNA recombination Meiosis 2 (22 + 1) 22 + 1 chr1

11. Diploid gamete precursor (♂)(♁) (♂) (♁) Haploid gamete precursors Hap. gametes NR ♁ A - B - - A - B A - B - - A - B A - B - - A - B A - B - - A - B ♂ ♁ A - B - A - B - - A - B - A - B DNA recombination between linked loci Meiosis 2 (22 + 1) 22 + 1

12. Human Genome - summary Recombination fraction between loci A and B (θ) Proportion of gametes produced that are recombinant for A and B If A and B are very far apart: 50%R:50%NR - θ = 0.5 If A and B are very close together: <50%R - 0 ≤ θ < 0.5 Recombination fraction (θ) can be converted to genetic distance (cM) Haldane: eg. θ =0.17, cM=20.8 Kosambi: eg. θ=0.17, cM=17.7 DNA is a linear sequence of nucleotides partitioned into 23 chromosomes Two copies of each chromosome (2x22 autosomes + XY), from paternal and maternal origins. During meiosis in gamete precursors, recombination can occur between maternal and paternal homologs

13. 3. Principles of Linkage Analysis

14. Linkage Analysis requires genetic markers M1 M2 MnMn M1 M2 MnMn M1 M2 MnMn θ 0.5.4.3.15.3.4 0.5 Q θ.4.3.1.26.35 0.5.35.22.3.4

15. Linkage Analysis: Parametric vs. Nonparametric QM Phe A D C E Genetic factors Environmental factors Mode of inheritance Recombination Correlation Chromosome Gene Adapted from Weiss & Terwilliger 2000

16. 4. Parametric Linkage Analysis

17. M1M2Q1Q2M1M2Q1Q2 Linkage with informative phase known meiosis M2M5Q2Q2M2M5Q2Q2 M1M6Q1Q?M1M6Q1Q? M 1 Q 1 /M 2 Q 2 M3M4Q2Q2M3M4Q2Q2 M 1 Q 1 /M 3 Q 2 M 2 Q 2 /M 3 Q 2 M 1 Q 1 /M 4 Q 2 M 2 Q 2 /M 4 Q 2 M 2 Q 1 /M 3 Q 2 Chromosome M 1..6 Q 1,2 Autosomal dominant, Q 1 predisposing allele Gene ♁ ♂ NR: M 1 Q 1 NR: M 2 Q 2 R: M 1 Q 2 R: M 2 Q 1 θ MQ = 1/6 = 0.17 Informative Phase known (~20.8 cM) M1M1 Q1Q1 M2M2 Q2Q2 Estimate θ between M and Q

18. M 1 Q 1 /M 2 Q 2 M 1 Q 2 /M 2 Q 1 M1M2Q1Q2M1M2Q1Q2 M3M4Q2Q2M3M4Q2Q2 NR: M 1 Q 1 NR: M 2 Q 2 R: M 1 Q 2 R: M 2 Q 1 Q2Q2Q2Q2 Q1Q?Q1Q? P 1-θ θ M 1 Q 1 /M 2 Q 2 R: M 1 Q 1 R: M 2 Q 2 NR: M 1 Q 2 NR: M 2 Q 1 P M 1 Q 2 /M 2 Q 1 N 3 2 0 1 N 3 2 0 1 + + Informative Phase unknown Linkage with informative phase unknown meiosis M 1 Q 1 /M 3 Q 2 M 2 Q 2 /M 3 Q 2 M 1 Q 1 /M 4 Q 2 M 2 Q 2 /M 4 Q 2 M 2 Q 1 /M 3 Q 2 M1M1 Q1Q1 M2M2 Q2Q2 M1M1 Q2Q2 M2M2 Q1Q1 θ 1-θ

19. Parametric LOD score calculation Overall LOD score for a given θ is the sum of all family LOD scores at θ eg. LOD=3 for θ=0.28 R

20. M1 M2 MnMn θ 0.5.4.3.1.3.4 0.5 Q For each marker, estimate the θ that yields highest LOD score across all families Markers with a significant parametric LOD score (>3) are said to be linked to the trait locus with recombination fraction θ This θ (and the LOD) will depend upon the mode of inheritance assumed MOI determines the genotype at the trait locus Q and thus determines the number of meiosis which are recombinant or nonrecombinant. Limited to Mendelian diseases. Parametric Linkage Analysis - summary

21. M1M2Q1Q1M1M2Q1Q1 M3M4Q1Q2M3M4Q1Q2 M2M3Q1Q1M2M3Q1Q1 M1M4Q1Q2M1M4Q1Q2 M1M4Q1Q1M1M4Q1Q1 M2M4Q1Q2M2M4Q1Q2 Practical: what is the most likely θ between M and Q? 1. Identify informative individual with offspring in the pedigree 2. Reconstruct possible phases of that individual and of all offspring 3. Classify the gametes that individual produces as R or NR 4. Count the number of R and NR gametes effectively produced 5. Express 6. Express LOD score M2M4Q1Q2M2M4Q1Q2

22. Practical: answers 2. Reconstruct possible phases of that individual and all offspring M3M4Q1Q2M3M4Q1Q2 NR: M 3 Q 1 NR: M 4 Q 2 R: M 3 Q 2 R: M 4 Q 1 P M 3 Q 1 /M 4 Q 2 R: M 3 Q 1 R: M 4 Q 2 NR: M 3 Q 2 NR: M 4 Q 1 P M 3 Q 2 /M 4 Q 1 N 1 3 0 1 N 1 3 0 1 M 2 Q 1 /M 3 Q 1 M 1 Q 1 /M 4 Q 2 M 1 Q 1 /M 4 Q 1 M 2 Q 1 /M 4 Q 2 1-θ θ θ M1M2Q1Q1M1M2Q1Q1 M2M3Q1Q1M2M3Q1Q1 M1M4Q1Q2M1M4Q1Q2 M1M4Q1Q1M1M4Q1Q1 M2M4Q1Q2M2M4Q1Q2 M2M4Q1Q2M2M4Q1Q2 + + 1. Identify informative individual with offspring in the pedigree3. Classify the gametes that individual produces as R or NR4. Count the number of R and NR gametes effectively produced5. Express6. Express LOD score

23. Outline 1. Aim 2. The Human Genome 3. Principles of Linkage Analysis 4. Parametric Linkage Analysis 5. Nonparametric Linkage Analysis

25. Approach Parametric: genotypes marker locus & genotypes trait locus (latter inferred from phenotype according to a specific disease model) Parameter of interest: θ between marker and trait loci Nonparametric: genotypes marker locus & phenotype If a trait locus truly regulates the expression of a phenotype, then two relatives with similar phenotypes should have similar genotypes at a marker in the vicinity of the trait locus, and vice-versa. Interest: correlation between phenotypic similarity and marker genotypic similarity No need to specify mode of inheritance, allele frequencies, etc...

26. Phenotypic similarity between relatives Squared trait differences Squared trait sums Trait cross-product Trait variance-covariance matrix Affection concordance T2T2 T1T1

27. Genotypic similarity between relatives IBS Alleles shared Identical By State “look the same”, may have the same DNA sequence but they are not necessarily derived from a known common ancestor IBD Alleles shared Identical By Descent are a copy of the same ancestor allele M1M1 Q1Q1 M2M2 Q2Q2 M3M3 Q3Q3 M3M3 Q4Q4 M1M1 Q1Q1 M3M3 Q3Q3 M1M1 Q1Q1 M3M3 Q4Q4 M1M1 Q1Q1 M2M2 Q2Q2 M3M3 Q3Q3 M3M3 Q4Q4 IBS IBD 2 1 Inheritance vector (M) 0 001 1

28. Genotypic similarity between relatives - M1M1 Q1Q1 M3M3 Q3Q3 M2M2 Q2Q2 M3M3 Q4Q4 Number of alleles shared IBD 0 M1M1 Q1Q1 M3M3 Q3Q3 M1M1 Q1Q1 M3M3 Q4Q4 1 M1M1 Q1Q1 M3M3 Q3Q3 M1M1 Q1Q1 M3M3 Q3Q3 2 Proportion of alleles shared IBD - 0 0.5 1 Inheritance vector (M) 0 011 0 001 0 000

29. Genotypic similarity between relatives - 2 2n ABC D

30. Statistics that incorporate both phenotypic and genotypic similarities Genotypic similarity ( ) Phenotypic similarity 0 0.51

31. Haseman-Elston regression – Quantitative traits Phenotypic (dis)similarity Genotypic similarity b ×=+ c 0 0.51

32. VC ML – Quantitative & Categorical traits method 0 0.51 H1:H1: H0:H0: e.g. LOD=3

33. Individual LOD scores can be expressed as P values (Pointwise) LODChi-sq (n-df)P value 2.19.670.0009 Genome-wide linkage analysis (e.g. VC) (x4.6) Type I error True positive LOD k Theoretical (Lander & Kruglyak 1995) LOD = 3.6, Chi-sq = 16.7, P = 0.000022

34. No need to specify mode of inheritance Nonparametric Linkage Analysis - summary Models phenotypic and genotypic similarity of relatives Expression of phenotypic similarity, calculation of IBD HE and VC are the most popular statistics used for linkage of quantitative traits Other statistics available, specially for affection traits