linkage analysis

Question 1

Define genetic variations.

Answer 1

• differences in the DNA sequence between individuals in a population.
• variation can be inherited or due to environmental factors.

Question 2

What 4 different effects of genetic variation can have?

Answer 2

1. alteration of the amino acid sequence.
2. changes in gene regulation (gene expression)
3. physical appearance of an individual (eg. eye colour, genetic disease risk)
4. silent or no apparent effect.

Question 3

Why is genetic variation important?

Answer 3

1. genetic variation underlies phenotypic differences among different individuals.
2. genetic variation determines our predisposition to complex diseases and responses to drugs and environmental factors.
3. genetic variation reveals clues of ancesteral human migration history.

Question 4

What are mechanisms of genetic variation?

Answer 4

1. mutation/polymorphism
   - > germ line mutations : passed on to descents
   - > somatic mutations : not transmitted to descendants
   - > de novo mutations : new mutations not inherited by either parent.
2. homologous recombination: shuffling of chromosomal segments between partner (homologous) chromosomes of a pair.
3. gene flow: movement of genes from one population to another (eg. migration)

Question 5

compare and contrast rare variant (mutation) and common variant (polymorphism).

Answer 5

• mutation is a rare change in DNA sequence that is different to the ref sequence (normal).
• polymorphism is a DNA sequence variant that is common in the population. In this case no single allele is regarded as ‘normal’ allele, there are 2 or more equally acceptable alternatives.

Question 6

How do you determine if variant is mutation or polymorphism?

Answer 6

• the arbitrary cut -off point between a mutation and a polymorphism is a minor allele frequency (MAF) of 1%
• for a variant to be considered polymorphism the least common (minor) allele must be present in >/= 1% of the population.

Question 7

what is meiosis and recombination?

Answer 7

meiosis = creation of haploid gamete from sperm and egg.
=> Genetic recombination (stage I)
- homologous (maternal and paternal) chromosomes line up at the centre of nucleus and crossing over of arms of the chromosomes occurs, exchange in genetic material = introducing variation.
=> RANDOM recombination.

Question 8

what is crossing over?

Answer 8

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

Question 9

Define a genotype.

Answer 9

genetic make up of an individual.

Question 10

Define phenotype.

Answer 10

physical expression of the genetic makeup.

Question 11

Define alleles.

Answer 11

• alternative versions called alleles.

- an organism inherits 2 alleles one from each parents the alleles can be same ( homozygous) or different (heterozygous)

Question 12

Define haplotype.

Answer 12

a group of alleles that are inherited together from a single parent (haplo = single)

Question 13

what is homozygosity and heterozygosity?

Answer 13

homo = chromosomes with the same allele of gene on maternal and paternal chromosome at the same loci 
hetro= different allele of gene on maternal and paternal chromosome.

Question 14

When can linkage analysis be applied?

Answer 14

• mendelian/monogenic : disease caused by a single gene , with little or no impact from the environment (eg. PKD)

Question 15

When can linkage analysis not be applied?

Answer 15

• non-mendelian/polygenic disease or traits caused by the impact of many different genes, each having only a small individual impact on the final condition (e.g. psoriasis)
• multifactorial : diseases resulting from interaction between multiple genes and often multiple environmental factors (eg. heart disease)

Question 16

What is linkage analysis?

Answer 16

method used to map the location of a disease gene in the genome.
‘linkage’ refers to the assumption of two things being physically linked to each other.

Question 17

What do we use genetic markers for?

Answer 17

• to identify genetic ‘markers’ to identify the location of a disease gene based on physical proximity.

Question 18

What is the importance of genetic mapping?

Answer 18

like we know which way is north and south on a map we can work out what genes are proximal and distal in a chromosome by using genetic mapping to establish the locations of genes on the chromosomes.

Question 19

What is the importance of genetic mapping?

Answer 19

like we know which way is north and south on a map we can work out what genes are proximal and distal in a chromosome by using genetic mapping to establish the locations of genes on the chromosomes.

=> uses an observed locus (genetic marker) to draw inferences about an unobserved locus (disease gene)

Question 20

What are 2 types of maps?

Answer 20

1. genetic maps = look at information in blocks like zones in a tube map
   => pre - 2001 , =>centimorgans = 1% chance of recombination.
   => because we had no genome sequence to refer to these types of maps used recombinant events to determine distance along the chromosome : further away = more likely crossing over.
2. physical maps = look at information on the physical distances between landmarks (e.g. stations on a tube map)
   => post 2001
   => 1 megabase = 1 million base-pairs

Question 21

What are principles of genetic linkage?

Answer 21

• genetic linkage is the tendency for alleles at neighbouring loci to segregate together at meiosis
• cross -overs are more likely to occur between loci separated by some distance rather than between loci close together on the chromosome
• therefore to be linked, two loci must lie very closely together.

Question 22

How does distance from gene marker and recombination link?

Answer 22

scenario 1: disease gene is a long distance away from a genetic marker => independent assortment => high likelihood of recombination

scenario 2: disease gene is close to gene marker => non-independent assortment => large proportion of non-recombinants expected (ie, greater likelihood of co-segregation of marker with the gene)

Question 23

What are some methods of genetic linkage?

Answer 23

• genotype multiple genetic markers across the genome
• genotype multiple family members from families with the genetic trait
• identify which genetic markers co-segregate with the disease (phenotype) (ie which haplotypes are the same in all affected family members)
• these genetic markers are therefore ‘linked’ to the disease gene is likely to be located.

Question 24

What micro-satellite markers?

Answer 24

• less common now. Highly polymorphic short tandem repeats of 2 to 6 bp
• micro-satellites may differ may differ in length between chromosome (heterozygous)
• are relatively widely spaced apart.

=> 400 (200) microsatellite markers
=> average spacing 9cM (20cM)
=>PCR - based system
=> fluorescently -labelled primers
=>manual assignment of genotypes 
=>labour intensive
=>whole genome scan > 2-3 months

Question 25

What single nucleotide polymorphism?

Answer 25

- now the genetic marker of choice. biallelic (a SNP will be one of two possible bases) - lower heterozygosity than micro satellites, but spaced much closer together - more informative ``` =>6,000 SNPs => spaced throughout the genome => micro-array based automatically => highly automated => data returned within <1-2 months ```

Question 26

What is microsatellite marker genotyping typically used for?

Answer 26

- DNA fingerprinting from very small amounts of material - standard test uses 13 core loci making the likelihood of chance match 1 in three trillion - paternity testing - linkage analysis for disease gene identification.

Question 27

What is the mechanism of fluorescent marker genotyping?

Answer 27

- fluorescently - tagged PCR primers - Allows for multiplexing of PCR products with different colours and fragment lengths - fragment sizes separated down to 1bp resolution

Question 28

What is SNP genotyping microarrays?

Answer 28

- provides genome - wide coverage of SNP markers - SNPs are proxy markers ; NOT the casual disease variants - Can amplify thousands of markers in a single experiment - alleles are identified by relative fluroescence => homozygous for allele 1 = green signal => homozygous for allele 2 = red signal => heterozygous (1/2) = yellow signa

Question 29

what is SNP genotyping microarrays typically used for?

Answer 29

- linkage analysis in families (affected vs unaffected relatives) => homozygosity mapping (autosomal recessive) and mapping of Mandelian traits - GWAS in populations (unrelated cases VS matched controls) => non- Mendelian disorders and multifactorial traits

Question 30

outline how linkage mapping using genetic markers is done?

Answer 30

- uses an observed locus(genetic marker) to draw inferences about an unobserved locus (disease gene) - 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 31

how do you build a haplotype trees?

Answer 31

- determine which allele is inherited from which parent - paternal haplotype drawn on the left - maternal haplotype drawn on the right

Question 32

how do we use statistical analysis of linkage to identify disease genes.

Answer 32

=> linkage software => probability of linkage can be asses using LOD score => assesses the probability of obtaining test data if the two loci are linked, to the likelihood of observing same data purely by chance => i.e calculates a likelihood ratio of observed vs expected => higher the LOD score, the higher the likelihood of linkage.

Question 33

What do the LOD tell?

Answer 33

- a LOD score >= 3is considered evidence for linkage => equivalent to odds of 1000: 1 observed linkage happened by chance =>translates to a p value of 0.05 - a LOD score of <= -2 is considered evidence against linkage.

Question 34

What is an example of an autosomal dominant disease gene and what are associated features?

Answer 34

Adams-Oliver syndrome => terminal transverse limb defects (TTLD) => scalp aplasia cutis congenita (ACC) associated features: - neurological anomalies - cardiac malformations - vascular defects (eg. cutis marmorata telangiectatica congenita, dilated veins)

Question 35

What are stages of variant validation by exome sequencing?

Answer 35

1. variant profile - affected individual (SNVs, CVNs, short indels) ``` 2.Filtering strategy zygosity exclude known/common variants - dbSNP -1000 genomes - control variant profiles ``` Linkage - prior linkage scans - variant profiles of relatives Predicted functionality nonsense/missense/splice/silent prior biological knowledge: - candidate genes and pathway 3. filtering parameters zygosity = heterozygous excludes known/ common variants = alleles frequency < 0.01 in control population linkage = chr 3q13 peak (115-128Mb) predicted functionality = nonsense > splice > missense biological knowledge = candidate genes and pathways (disease causing variants in ARHGAP31 were identified through a candidate gene analysis of genes in the minimal linkage interval 4. causative variant