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 n alternative 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
3
1
2
4
5
6
1
1
2
1
4
3
3
3
5
3
5
1
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 5050 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 gt 3

39
Sample Linkage Curve
LOD