Introduction to Linkage Analysis

March 2002

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

Where are those genes?

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

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

DNA Polymer of 4 bases

Purines (A) – Adenine (G) – Guanine

Pyrimidines (C) – Cytosine (T) – Thymine

Double Helix Complementary Strands Hydrogen Bonds

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

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

Genetic Code

Example of CFTR Variants

Position Mutation Effect482 G->C Arg-117 -> His-177

1609 C->T Gln-493 -> STOP1654 Deletion of 3 nucleotides Deletion of Phe-5082566 AT insertion Frameshift3659 C deletion Frameshift

Phenotype vs. Genotype Genotype

Underlying genetic constitution Phenotype

Observed manifestation of a genotype

Different changes within CFTR all lead to cystic fibrosis phenotype

Common types of DNA variants

Tandem repeats Microsatellites Single nucleotide polymorphisms Insertions Deletions

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)

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

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

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

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

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

Meiosis Leads to

formation of haploid gametes from diploid cells

Assortment of genetic loci

Recombination or crossover

What happens in meiosis…

Recombination

Non-RecombinantGametes

RecombinantGametes

/

/

/

/

1-

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

Recombination and Map Distance

0.00

0.20

0.40

0.60

0.80

1.00

0.00 0.20 0.40 0.60 0.80 1.00

Distance

Ob

se

rve

d R

ec

om

bin

ati

on

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

Tracing Chromosomes

Tracing Chromosomes

1 1 2 1

31 2 4

43 3 3 5 6

5 53 1

IBD At each location, try to establish

whether siblings (or twins) share 0, 1 or 2 chromosomes

Inference may be probabilistic

Example of Scoring IBD Parental genotypes

are available

Siblings are IBD = 2 Share maternal and

paternal chromosomes

A/C A/C

A/A A/A

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 A/A A/A

Example of IBD scoring III Looking at multiple

consecutive markers helps infer IBD Especially without

parental genotypes

IBD = 2 may be quite likely

A/AC/GA/TG/G

A/AC/GA/TG/G

Notation - IBD sharing (0, ½ and 1)

Z0 - probability = 0 Z1 - probability = ½ Z2 - probability = 1

sharing IBD estimated ,ˆ 121

2 ZZ

Typical IBD information

Pair Chr. Pos (cM) z0 z1 z2 pi-hat5378-5479 3 10 0.00 0.01 0.99 0.9955378-5479 3 20 0.00 0.01 0.99 0.9955378-5479 3 30 0.00 0.50 0.50 0.7505378-5479 3 40 0.00 1.00 0.00 0.5005378-5479 3 50 0.01 0.98 0.01 0.500

Model

Twin1

Q A C E

Twin2

QACE

= 0.0, 0.5, 1.0

0.5 [DZ], 1.0 [MZ]

1.0

No Linkage

Linkage

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

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

Lod scores Often, report results as lod scores

Genome is large, many locations tested Threshold for significance is usually LOD >

~3

2

10

6.4

),,(

),,,(log

ECAL

ECAQLLOD

Sample Linkage Curve

LOD