W3 - Linkage Analysis

Question 1

What are germline mutations?

Answer 1

Passed onto descendants - present on all cells.

Question 2

What are somatic mutations?

Answer 2

They are not transmitted to descendants and present in some cells of the body. Occurs due to external insult like radiation, smoking etc - causes cancers.

Question 3

What are de novo mutations?

Answer 3

New mutations often in the embryonic stage of development. Not inherited from either parent. Can be passed onto descents.

Question 4

What is homologous recombination?

Answer 4

Shuffling of chromosomal segments between partner (homologous) chromosomes of a pair.

Crossing over: Breaking and rejoining of the homologous chromosomes during meiosis. Results in exchange of chromosome segments and new allele combinations.

Crossovers are more likely to occur between Loci separated by some distance than between loci close together on the chromosome.

Question 5

What is gene flow?

Answer 5

The movement of genes from one population to a number (eg. Migration) is an important source of genetic variation.

Question 6

What is the difference between mutations and polymorphism?

Answer 6

• Mutation = Rare change. Normal allele is prevalent in the population - the mutation changes this to a rare abnormal variant.
• polymorphism = DNA variant common in the population. No allele is the normal allele. Instead or two or more equal acceptable alternatives.

The arbitrary cut-off point between mutations on a polymorphism as a minor allele frequency of 1%. Ie. For variant to be classified as a polymorphism, the least common allele must be present in less than 1% of the population.

Question 7

What is a haplotype?

Answer 7

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

Multiple alleles at linked loci. These chromosomal segments can be tracked through pedigree and population.

Question 8

What are the three classification of genetic disease?

Answer 8

• Mendelian/ Monogenic
• Non-Mendelian/ Polygenic
• Multifactorial

Question 9

What is a Mendelian/Monogenic disease?

Answer 9

Disease that is caused by a single gene with little or no impact on the environment. Eg. Polycystic kidney disease. 

Question 10

What is non-Mendelian/ polygenic disease?

Answer 10

Disease or trait caused by the impact of many different genes each having only a small individual impact on the final condition. Eg. Psoriasis

Question 11

What is a multifactorial disease?

Answer 11

Disease or traits resulting from interaction between multiple genes and of multiple environmental factors eg heart disease.

Question 12

What is linkage analysis?

Answer 12

Linkage analysis is a method used to map the location of the disease gene into Genome.
The term linkage refers to the assumption of two things being physically linked to each other

Assumption made= can use genetic markers to identify the location of a disease gene based on the physical proximity.

Question 13

What is the difference between genetic maps and physical maps of Genome?

Answer 13

Maps allow orientation and calculation of distance.

Genetic: looks at information in blocks or regions. Used before 2001. Used CentiMorgans.

Physical: provide information on the physical distance between landmarks. Used after 2001 and uses Megabase.

Question 14

What are the two types of genetic markers?

Answer 14

• Microsatellite Markers
• Single nucleotide polymorphisms 

Question 15

What are Microsatellite markers?

Answer 15

Less common now – highly polymorphic short tandem repeats of 2 to 6bp.
Microsatellites my different length between chromosomes (heterozygous)
A relatively widely spaced apart 

 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

Question 16

What are single nucleotide polymorphism Markers?

Answer 16

No the genetic marker of choice- Biallelic (a SNP will be one of two possible bases).
Lower heterozygosity than Microsatellites, but space to much closer together.
More informative

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

Question 17

What is microsatellite genotyping used for?

Answer 17

Typically used for:

 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 18

How is microsatellite genotyping done?

Answer 18

There are lots of polymorphic repeat units.
You can amplify it across the region using PCR.
Can we run on a gel.

Smaller repeat units = shorter fragments.

Question 19

How is fluorescent genotyping done?

Answer 19

• Fluorescently tagged PCR primers
• allows for multiplexing of PCR products with different colours and fragment lengths.
• fragment sizes separated down to 1bp resolution. 

The peaks and numbers of the charts can be used to identify maternal and paternal inheritance.

Question 20

What are Single nucleotide polymorphism (SNP)?

Answer 20

• Single base change
• most common type of variation
  -Go to occur approximately one per 1000 bases
• Human genome is 3 billion base pairs

Question 21

What are SNP genotyping microarrays?

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 microarrays used for?

Answer 22

Typically used for:
 Linkage analysis in families (affected vs unaffected relatives)
 homozygosity mapping (autosomal recessive) and mapping of

Mendelian traits
 GWAS in populations (unrelated cases vs matched controls)
 non-Mendelian disorders and multifactorial traits

Question 23

What can you tell with linkage mapping using genetic markers?

Answer 23

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

If the market and the disease locus of unlinked, the infected relatives and family are less likely to inherit the same marker alleles.

Question 24

How do we build haplotypes?

Answer 24

Haplotypes are half the genetics.

We look at the markers of both parents and affected individual to figure out the haplotypes from the mother and father.

We look at the affected members eg, mother and you and work out which regions are the same.

If your brother is affected too, we can do the same for him and see the similarities and differences to narrow down the location of the affected gene.

Can build up pedigrees.

Question 25

What is the statistical analysis of linkage?

Answer 25

Creating the haplotypes genomes would be laborious individually. We use software instead.

The probability of linkage can be assessed using a LOD score.

LOD = logarithm of the 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

(Refer to Power point slides for the formula)

 Recombination fraction is the proportion of recombinant births (i.e. R / NR+R)
 Maximum recombination fraction = 0.5, which is equivalent to independent assortment
 The higher the LOD score, the higher the likelihood of linkage

Question 26

What is parametric analysis?

Answer 26

Specifies the pedigree structure and inheritance pattern (model)

Question 27

What is non-parametric analysis?

Answer 27

Detects allele sharing between affected individuals.

Question 28

How old is LOD scores additive?

Answer 28

Different families linked to the same disease Locus will increase the overall score.

Question 29

What are LOD scores indicative of?

Answer 29

 A LOD score ≥ 3 is considered evidence for linkage
 Equivalent to odds of 1000:1 that the observed linkage occurred by chance
 Translates to a p-value of approximately 0.05
 A LOD score ≤ -2 is considered evidence against linkage