Introduction to Linkage Analysis

1. Introduction to Linkage Analysis March 2002

2. 3 Stages of Genetic Mapping • Are there genes influencing this trait? • Epidemiological studies • Where are those genes? • Linkage analysis • What are those genes? • Association analysis

3. Where are those genes?

4. Outline • How is genetic information organized? • Chromosomes • Sequence • Examples of genetic variation • Changes that have observable effects • Genetic markers • Linkage analysis • Strategy for surveying variation in families

5. Genetic Information • Human Genome • 22 autosomes • X and Y • Sequence of 3 x 109 base-pairs • ~17-20 bp can identify unique sequence in the genome • Variation • Most sequence is conserved across individuals • 1 in 103 base-pairs differs between chromosomes

6. DNA • Polymer of 4 bases • Purines • (A)– Adenine • (G)– Guanine • Pyrimidines • (C) – Cytosine • (T)– Thymine • Double Helix • Complementary Strands • Hydrogen Bonds

7. Some Types of DNA Sequence • Genes • ~30,000 in humans • Exons, translated into protein • Introns, transcribed into RNA, but not protein • Promoters • Enhancers • Repeat DNA • Pseudogenes

8. Genetic Code • DNA  RNA  Protein • DNA: 4 bases (A,T,C,G) • RNA: 4 bases (A,U,C,G) • Proteins: 20 amino-acids • Universal Genetic Code • Translation between DNA/RNA and protein • Three bases code for one amino-acid

9. Genetic Code

10. Example of CFTR Variants

11. Phenotype vs. Genotype • Genotype • Underlying genetic constitution • Phenotype • Observed manifestation of a genotype • Different changes within CFTR all lead to cystic fibrosis phenotype

12. Common types of DNA variants • Tandem repeats • Microsatellites • Single nucleotide polymorphisms • Insertions • Deletions

13. Repeat Length Polymorphisms • Variable Number Tandem Repeats • VNTRs • Typical repeat units of 10 – 100s bp • E.g.: ~110 bp repeat in IL1RN gene • Microsatellites • Simple repeat sequences • Most popular are 2, 3 or 4 bp • E.g.: ACACACAC … • D naming scheme (e.g., D2S160)

14. Microsatellites • Most popular markers for linkage analysis • Large number of alleles (10 is common) • Can distinguish and track individual chromosomes in families • Relatively abundant • ~15,000 mapped loci

15. SNPs • Single Nucleotide Polymorphisms • Change one nucleotide • Insert • Delete • Replace it with a different nucleotide • Many have no phenotypic effect • Some can disrupt or affect gene function

16. A little more on SNPs • Most SNPs have only two alleles • Easy to automate their scoring • Becoming extremely popular • Typing Methods • Sequencing • Restriction Site • Hybridization

17. Classifying Genotypes • Each individual carries two alleles • If there are nalternative alleles … • … there will be n (n + 1) / 2 possible genotypes • 3 possible genotypes for SNPs, typically more for microsatellites and VNTRs • Homozygotes • The two alleles are the same • Heterozygotes • The two alleles are different

18. Genes in an individual • Sexual reproduction • One copy inherited from father • One copy inherited from mother • Each individual has • 2 copies of each chromosome • 2 copies of each gene • These copies may be similar or different

19. Meiosis • Leads to formation of haploid gametes from diploid cells • Assortment of genetic loci • Recombination or crossover

20. What happens in meiosis…

21. Recombination 1- 

22. Recombination • Actual • No. of recombinants between two locations • An average of one per Morgan • Observed • Usually, only odd / even number of crossovers between two locations can be established

23. Recombination and Map Distance

24. Intuition for Linkage Analysis • Millions of variations that could be responsible for disease • Impractical to investigate individually • Within families, they organized into limited number of haplotypes • Sample modest number of markers to determine whether each stretch of chromosome is shared

25. Tracing Chromosomes

26. Tracing Chromosomes 1 2 3 4 5 6 1 1 4 2 1 3 3 3 5 3 1 5

27. IBD • At each location, try to establish whether siblings (or twins) share 0, 1 or 2 chromosomes • Inference may be probabilistic

28. Example of Scoring IBD • Parental genotypes are available • Siblings are IBD = 2 • Share maternal and paternal chromosomes

29. Example of Scoring IBD II • Parental genotypes unavailable • IBD between siblings may be 0, 1 or 2 • Likelihood of each outcome depends on frequency of allele A

30. Example of IBD scoring III • Looking at multiple consecutive markers helps infer IBD • Especially without parental genotypes • IBD = 2 may be quite likely

31. Notation •  - IBD sharing (0, ½ and 1) • Z0 - probability  = 0 • Z1 - probability  = ½ • Z2 - probability  = 1

32. Typical IBD information

33. Model

34. No Linkage

35. Linkage

36. Hypothesis • Test evidence for linked genetic effect • Fit two models • Full model (Q,A,C,E) • Restricted model (A,C,E) • Maximum likelihood test • Compare likelihoods using ²

37. Analysis • Estimate  along chromosome • For example, using Genehunter or Merlin • Test hypothesis at each location • Summarize results in linkage curve • Chi-squared is 50:50 mixture of 1 df and point mass zero

38. Lod scores • Often, report results as lod scores • Genome is large, many locations tested • Threshold for significance is usually LOD > ~3

39. Sample Linkage Curve LOD