Linkage Analysis

Question 1

What is genetic variation?

Answer 1

Refers to a difference in the DNA sequence between individuals in a population

It can be inherited or due to environmental factors (e.g. drugs, exposure to radiation, etc.)

Question 2

What are some effects of genetic variation?

Answer 2

1) Alteration of the amino acid sequence that is encoded by a gene
2) Changes in gene regulation (when and where a gene is expressed)
3) Physical appearance of an individual (eye colour, genetic disease risk)
4) Silent/no apparent effect

Question 3

Why is genetic variation important?

Answer 3

1) It underlies the phenotypic differences amongst different individuals
2) Genetic variations determine our predisposition to complex diseases and responses to drugs and environmental factors
3) Genetic variation reveals clues of ancestral human migration history

Question 4

List and describe the mechanisms of genetic variation

Answer 4

Mutation/polymorphism: errors in DNA replication. This may affect single nucleotides or larger portions of DNA

Germ-line mutation: passed on to descendants

Somatic mutations: not transmitted to descendants

De novo mutations: new mutation not inherited from either parent

Gene flow: the movement of genes from one population to another (e.g. migration)

Genetic recombination: shuffling of chromosomal segments between homologous chromosomes of a pair

Question 5

What is the difference between a mutation and a polymorphism?

Answer 5

A mutation is a rare change in the DNA sequence that is different to the normal sequence. The normal allele is prevalent in the population and the mutation changes this to a rare ‘abnormal’ variant

By contrast, a polymorphism is a DNA sequence variant that is common in the population. In this case, no single allele is regarded as the ‘normal’ allele. Instead, there are two or more equally acceptable alternatives

The arbitrary cut-off point between a mutation and a polymorphism is a minor allele frequency of 1%

Question 6

How does genetic recombination occur in meiosis?

Answer 6

Crossing over is the reciprocal breaking and re-joining of the homologous chromosomes during meiosis. This results in the exchange of chromosome segments and new allele combinations

The homologous chromosomes line up together and this is when crossing over can occur between the sister chromatids. After this, we get an exchange of genetic information between maternal and paternal chromosomes

Question 7

What is the difference between recombinant and non-recombinant alleles?

Answer 7

Recombinant alleles: a mixture of paternal and maternal material

Non-recombinant alleles: original to the chromosome

Question 8

Define genotype

Answer 8

The genetic makeup of an individual - details the two alleles an individual carries for a specific gene or marker

Question 9

Define phenotype

Answer 9

The physical expression of the genetic makeuo

Question 10

What are alleles?

Answer 10

Genes are found in alternative versions, called alleles. For each characteristic, an organism inherits two alleles, one from each parent

Question 11

What is the difference between a homozygous and heterozygous genotype?

Answer 11

Homozygous genotype: two identical alleles

Heterozygous genotype: two different alleles

Question 12

Define haplotype

Answer 12

A group of alleles that are inherited together from a single parent

Question 13

What are the different classifications of genetic disease?

Answer 13

MENDELIAN/MONOGENIC: disease that is caused by a single gene, with little or no impact from the environment (e.g. PKD).

NON-MENDELIAN/POLYGENIC: diseases of traits caused by the impact of many different genes, each having a small individual impact on the final condition (e.g. psoriasis).

MULTIFACTORIAL: diseases or traits resulting from an interactions between multiple genes and often multiple environmental factors (e.g. heart disease).

Question 14

What is linkage analysis?

Answer 14

The method used to map the location of a disease gene in the genome

Question 15

What is the importance of maps in linkage analysis?

Answer 15

Maps provide a context to orientate yourself and calculate distance between landmarks

Question 16

What are the two types of maps used in linkage analysis?

Answer 16

Genetic maps look at the information in blocks or regions

Physical maps provide information in the physical distances between landmarks based on their exact location

Question 17

How did the physical map for genetic mapping come about?

Answer 17

After 2001, we can now use physical mapping thanks to the human genome project.

We measure the distance on a genome in centimorgans. We use these centimorgan blocks to identify where we are on the chromosome

Question 18

List some principles of genetic linkage

Answer 18

• to be linked, the two loci must lie very close together
• haplotypes mark chromosomal segments which can be tracked through pedigrees and populations
• cross-overs are more likely to occur between loci separated by some distance than close together

Question 19

Describe linkage mapping using genetic markers

Answer 19

It used an observed locus to draw inferences about an unobserved locus

If a marker is linked to a disease locus, the same marker alleles will be inherited by two affected relatives more often than expected by chance

If the marker and the disease locus are unlinked, the affected relatives in a family are less likely to inherit the same marker alleles

Question 20

Describe the two genetic markers used in linkage analysis

Answer 20

1) Microsatellite markers: it is used less commonly now. They are highly polymorphic short tandem repeats of 2 to 6 basepairs. They may differ in length between chromosomes, they are relatively widely spaced apart
2) They are now the genetic marker of choice (since an SNP will be one of two possible bases). They are less heterozygous than micro-satellites, but they’re spaced much closer together. It is also much more informative

Question 21

List some difference between micro-satellite markers and SNPs

Answer 21

MICROSATELLITE MARKERS:

• 400 (or 300) microsatellite markers are used
• average spacing between them - 9 cM (or 20 cM)
• has a PCR-based system
• fluorescently-labelled primers
• manual assignment of genotypes
• labour intensive
• whole genome scan takes 2-3 months

SNPs:

• ~6000 SNPs are used
• they are spaced throughout the genome
• it has a micro-array-based system
• the genotype are assigned automatically
• it is highly automated
• the data is returned within 1-2 months

Question 22

What is microsatellite genotyping typically used for?

Answer 22

• DNA fingerprinting from small amounts of material
• standard test using 13 core loci
• paternity testing
• linkage analysis for disease gene identification

Question 23

How do SNP genotyping arrays work?

Answer 23

It provides a genome-wide coverage of SNP markers. The SNPs are proxy markers, not the casual disease variants. It can amplify thousands of markers in a single environment.

Alleles are identified by relative fluorescence:

• homozygous for allele 1 - green signal
• homozygous for allele 2 - red signal
• heterozygous for 1/2 - yellow signal

Question 24

What are SNP genotyping arrays typically used for?

Answer 24

• linkage analysis in families: we do the homozygosity mapping and mapping of Mendelian traits
• GWAS in populations: for non-Mendelian disorders and multifactorial traits

Question 25

How is statistical analysis of linkage performed?

Answer 25

The probability of linkage can be assessed using an LOD (logarithm of the odds) score.
It assesses the probability of obtaining test data if the two loci are linked, to the likelihood of observing the same data purely by chance. Hence, it calculates a likelihood of observed vs. unexpected (no linkage, θ=0.5)

You would apply this calculation with every marker that we genotype to generate an LOD score:
Z = log10((odds that the loci are linked, θ<0.5)/(odds that loci are unlinked, θ=0.5)

The recombination fraction (θ) is the proportion of recombinant births.

Question 26

What do LOD scores tell us?

Answer 26

The higher the LOD score, the higher the likelihood of linkage.

LOD scores can be calculated across the whole genome using genotype data for many genetic marker in multiple members of a family.

LOD scores are additive - different families linked to the same disease locus will increase the overall score.

A LOD score of ≥3 is considered evidence for linkage. It is the equivalent to odds of 1000:1 that the observed linkage occured by chance. This translates to a p value of approximately 0.05.
A LOD score of ≤-2 is considered evidence against linkage.