Linkage analysis

Question 1

What does genetic variation refer to?

Answer 1

Refers to any position in the genome which varies between individuals

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

Question 2

What are the different effects caused by genetic variation?

Answer 2

• Alteration of amino acid sequence that is encoded by a gene
• Changes in gene regulation (where and when the gene is expressed)
• Physical appearance (eye colour, genetic disease)
• Silent or no apparent effect

Question 3

Why is genetic variation important?

Answer 3

Important because: -

• Underlies phenotypic differences among different individuals
• Determine over predisposition to complex diseases and responses to drugs and environmental factors
• Reveals clues of ancestral human migration history

Question 4

What are the 3 mechanisms by which genetic variation arises?

Answer 4

-Mutations/Polymorphism - errors in DNA repication, may affect single nucleotides or larger portions of DNA

• Germline mutations - passed onto descendants
• Somatic mutations - not transmitted to descendants
• denovo mutations - new mutation not inherited from either parent
• Genetic Recombination - shuffling of chromosomal segments between homologous chromosomes
• Gene flow- the movement of genes from one population to another

Question 5

What is the difference between a mutation and a polymorphism?

Answer 5

• MUTATION = rare change in DNA sequence
• POLYMORPHISM = DNA sequence variant that is common (in this case there is no single allele that 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 the MAF (minor allele frequency)

Question 6

Describe homologous recombination

Comment on non allelic homologous recombination

Answer 6

• Crossing over: reciprocal breaking and re-joining of the homologous chromosomes during meiosis
• Results in exchange of chromosome segments and new allele combinations
  
  • Non-Recombinant alleles = original to the chromosome
  • Recombinant alleles = a mixture of maternal and paternal material

Non allelic homologous recombination = occurs between two DNA sequences on chromosomes which have a sequence similarity but are not identical (is a cause of copy number variants = large sections of the genome which are repeated)

Question 7

What is a genotype?

What is a phenotype?

Answer 7

Genetic makeup of an individual which gives rise to the phenotype

Phenotype is the physical expression of the genetic makeup

Question 8

What is an allele?

Answer 8

An allele is an alternative version of a gene

• For each characteristic, an organism inherits two alleles, one from each parent the alleles can be the same or different
• A genotype details the two alleles an individual carries for a specific gene/marker

Question 9

What does homozygous and heterozygous mean?

Answer 9

• Homozygous = genotype has identical alleles
• Heterozygous = genotype has two different alleles

Question 10

What is a haplotype?

Answer 10

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

We are able to track what has been inherited in the maternal and paternal haplotype

Question 11

What are the three ways in which genetic diseases can be classified?

Answer 11

1. MENDELIAN/MONOGENIC - disease caused by a single gene e.g PKD (polycystic kidney disease)
2. NON-MENDELIAN/ POLYGENIC - disease or traits caused by the impact of many different genes, each onlt has small individual impact on the final condition e.g psoriasis
3. MULTIFACTORAL - disease or traits resulting from an interaction between multiple genes and often multiple environmental factors e.g heart disease

Question 12

How does penetrance differ depending on whether the disease is mendelian/monogenic or polygenic?

Answer 12

• Mendelian diseases have a high penetrance (percentage of individuals who carry mutation and develop symptoms of the disorder)
• Polygenic will require ‘multiple hits’ of the genes in order for expression of the phenotype

Question 13

What method is linkage analysis?

Answer 13

Method used to map location of a disease gene in the genome

• The term linkage refers to the assumption of two things being physically linked to one another

Question 14

What is the importance of maps?

Answer 14

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

Question 15

What are the two maps used in linkage analysis?

Answer 15

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

Physical mapping came about thanks to the human genome project we measure distance in the genome using centimorgans. These identify where we are on the chromosome

Question 16

What are the principals of genetic linkage?

Answer 16

• Genetic linkage is the tendency for alleles at neighbouring loci to segregate together at meiosis
• Therefore to be linked, two loci must be very close together
• A haplotype is defines multiple alleles at linked loci
• Cross overs are more likely to occur between loci separated by some DISTANCE than those close together

Question 17

Describe linkage mapping using genetic markers

Answer 17

• It uses an observed locus (genetic marker) to draw interences about an unobserved locus (disease gene)
  
  • If a marker is linked to a disease locus (e.g M3 and M4), the same marker 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 18

What 2 markers are used as genetic markers?

Answer 18

1. Microsatellite markers = highly polymorphic short tandem repeats of 2-6 bps. Microsatellites may differ in length between chromosomes (heterozygous). They are relatively widely spaced apart
2. SNPs = less heterozygous than microsatellites, more commonly used now, spaced closer together and are more informative

Question 19

List differences between microsatellite markers and SNPs

Answer 19

Microsatellite markers

• 400 (200) microsatellite markers
• Average spacing 9 cM (20 cM)
• PCR-based system
• Fluorescently-labelled primers
• Manual assignment of genotypes
• Labour intensive
• Whole genome scan 2-3 months

SNPs

• ~6,000 SNPs
• Spaced throughout the genome
• Microarray-based system
• Genotypes assigned automatically
• Highly automated
• Data returned within 1-2 months

Question 20

What is microsatellite genotyping used for?

Answer 20

• DNA fingerprinting from very small amounts of material
• Standard test uses 13 core loci making the likelihood of a chance match 1 in three trillion
• Paternity testing
• Linkage analysis for disease gene identification

Question 21

How do SNP genotyping arrays work?

Answer 21

• Provides genome-wide coverage of SNP markers
• SNPs are proxy markers; NOT the causal disease variants
• Can amplify thousands of markers in a single experiment
  
  • Alleles are identified by relative fluorescence
    
    • homozygous for allele 1 = green signal
    • homozygous for allele 2 = red signal
    • heterozygous (1/2) = yellow signal

Question 22

What are SNP genotyping arrays typically used for?

Answer 22

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

Question 23

How is statistical analysis of linkage performed?

Answer 23

Probability of linkage can be assessed using a LOD (logarithm of odds) score

• Assesses the probability of obtaining the test data if the two loci are linked, to the likelihood of observing the same data purely by chance
• i.e. calculates a likelihood ratio of observed vs. expected (no linkage, θ=0.5)

You can apply this calculation with every marker that we genotype to generate a LOD score

Question 24

What does the LOD score assess?

Answer 24

Assesses the probability of obtaining test data if the 2 loci are linked, to the likelihood of observing the same data purely by chance

Question 25

When is there no linkage in reference to the theta score and why?

Answer 25

When theta=0.5 there's no linkage because its just independent assortment

Question 26

Relationship between LOD score and linkage

Answer 26

**_The higher the LOD score, the higher the likelihood of linkage_** LOD scores are additive - different families linked to the same disease locus will increase the overall score * LOD score greater than or equal to 3 is considered evidence for linkage * LOD score less than or equal to -2 is considered evidence against linkage