Linkage Analysis

Question 1

What is genetic variation?

Answer 1

Genetic variation refers to difference in the DNA sequence between individuals in a population.

The variation 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

Genetic variation can have different effects:

• alteration of the amino acid sequence (protein) that is encoded by a gene
• changes in gene regulation (where and when a gene is expressed)
• physical appearance of an individual (e.g. eye colour, genetic disease risk)
• silent or no apparent effect

Question 3

Why is genetic variation important?

Answer 3

1. Genetic variation underlies the phenotypic differences among 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) is an important source of genetic variation.

GENETIC RECOMBINATION: shuffling of chromosomal segments between partner (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 (reference) 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 tow or more equally acceptable alternatives.

The arbitrary cut-off point between a mutation and a polymorphism is a minor allele frequency (MAF) of 1% (i.e. to be classed as polymorphism, the least common allele must be present in ≥1% of the population).

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 (maternal and paternal) 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.

• Non-recombinant alleles: original to the chromosome
• Recombinant alleles: a mixture of maternal and paternal material

Question 7

Define genotype.

Answer 7

The genotype is the genetic makeup of the individual.

Question 8

Define phenotype.

Answer 8

The phenotype is the physical expression of the genetic makeup.

Question 9

What are alleles?

Answer 9

Genes are found in alternative versions called alleles.
For each characteristics, an organism inherits two allele, one from each parents; the alleles can be the same or different.

Question 10

Define genotype.

Answer 10

A genotype details the two alleles an individual carries for a specific gene or marker.

Question 11

What is the difference between a homozygous and a heterozygous genotype?

Answer 11

A homozygous genotype has identical alleles.

A heterozygous genotype has two different alleles.

Question 12

Define haplotype.

Answer 12

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

We can track what has been inherited in the maternal and paternal haplotype.

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

Linkage analysis is the method used to map the location of a disease gene in the genome.

The term ‘linkage’ refers to the assumption of two things being physically linked to each other.

Question 15

What is the importance of maps, and what are the two types of maps used in linkage analysis?

Answer 15

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

The two types of maps used in linkage analysis are:

• Genetic maps look at the information in blocks or regions (similar to zones on a tube map).
• Physical maps provide information in the physical distances between landmarks (e.g. stations on a tube map) based on their exact location.

Question 16

How did the physical map for genetic mapping come about?

Answer 16

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 17

List some principles of genetic linkage.

Answer 17

Genetic linkage is the tendency for alleles at the neighbouring loci to segregate together at meiosis. Therefore, to be linked, the two loci must lie very close together.

A haplotype defines multiple alleles at linked loci. 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 those close together.

Question 18

Describe linkage mapping using genetic markers.

Answer 18

It uses an observed locus (genetic marker) to draw inferences about an unobserved locus (disease gene).
If a marker is linked to a disease locus (e.g. M3 and M4), 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 (e.g. M5- M8), the affected relatives in a family are less likely to inherit the same marker alleles.

Question 19

Describe the two genetic markers used in in linkage analysis.

Answer 19

There are two types of markers used, both of which are highly variable in individuals:
MICROSATELLITE MARKERS: It is used less commonly now. They are highly polymorphic short tandem repeats of 2 to 6 basepairs. Microsatellites may differ in length between chromosomes (heterozygous). They are relatively widely spaced apart.

SINGLE NUCLEOTIDE POLYMORPHISMS: They are now the genetic marker of choice (since an SNP will be one of two possible bases). They are less heterozygous than microsatellites, but they’re spaced much closer together. It is also much more informative.

Question 20

List some difference between microsatellite markers and SNPs.

Answer 20

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 21

What is microsatellite genotyping typically used for?

Answer 21

• DNA fingerprinting from very small amounts of material
• standard test using 13 core loci, making the likelihood of a chance match 1 in 3,000,000,000,000
• paternity testing
• linkage analysis for disease gene identification

Question 22

How do SNP genotyping arrays work?

Answer 22

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 fluoresence:

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

Question 23

What are SNP genotyping arrays typically used for?

Answer 23

• linkage analysis in families (affected vs unaffected relatives): we do the homozygosity mapping (autosomal recessive) and mapping of Mendelian traits
• GWAS in populations (unrelated cases vs matched controls): for non-Mendelian disorders and multifactorial traits

Question 24

How is statistical analysis of linkage performed?

Answer 24

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 25

What do the LOD scores tell us?

Answer 25

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.