Disease Gene Mapping

Question 1

What has made gene mapping in humans so difficult?

Answer 1

Inability to perform desired crosses and small number of progeny. This means research is restricted to analysing pedigrees with limited information.

Small offspring numbers.

Question 2

What gene is found to be linked to nail-patella syndrome?

Answer 2

The ABO blood type gene

Question 3

How can DNA markers be used?

Answer 3

They can help locate disease or other gene by linkage and physical location.

Question 4

What are the advantages of DNA markers?

Answer 4

DNA markers are rapid (PCR and sequencing)

Microsatellite markers are multiallelic, SNVs are biallelic

No need to wait for phenotype expression.

Phenotypes are often hard to distinguish due to not being discrete charaters.

DNA markers allow distinction between homozygous and heterozygous.

DNA markers must be well spread out on all chromosomes to ensure that there are no gapsin chromosomes

Question 5

Why is it important to know about variations?

Answer 5

Underlying phenotypic differences can be more well understood

They cause inherited diseases.

They allow tracking of ancestral human history

Question 6

What is the goal of a genetic study?

Answer 6

Identification of genetic risk factors causing human complex disease (Type II diabetes, hypertension, asthma)

Common complex disease and rare single gene (Mendelian) disease

Question 7

What evidence suggests that a disease or trait is genetic?

Answer 7

Twin studies (compare monozygotic to dizygotic twins)

Families segregation (Increased risk for disease among family members of an affected individual so frequency in first degree relatives is compared to frequency of disease in general population)

Question 8

How are genes that are involved in complex disease phenotypes identified?

Answer 8

Functional cloning: Disease -> Function -> Gene -> Map

Positional cloning: Disease -> Map -> Gene -> Function

Question 9

How are genes contributing to disease approached?

Answer 9

Candidates gene approach: Candidate genes are located in chromosome region suspected of being involved in the expression of disease traits. Can be identified by association and linkage with phenotypes.

Whole genome screen approach: Scanning whole genome without prior information, can discover potential genes playing roles for diseases.

Question 10

What are the advantages and disadvantages of using positional cloning to identify a gene?

Answer 10

Strengths:

No need to know the gene products required

Very strong track record in single-gene disorders.

Weaknesses:

Understanding of function not a certain outcome

Poor track record with multifactorial effects.

Question 11

How can gene mapping be used to identify disease phenotypes?

Answer 11

Marker genes located near the disease genotype are identified allowing the identification of traits that are linked to the genotype of the disease.

Question 12

How are genes identified that contribute to disease?

Answer 12

Linkage mapping (Measures segregation of alleles and a phenotype within a family. Use crossover during meiosis II. (Genes that are physically close together are more likely to be inherited together)

Linkage disequilibrium (Evaluate evidence of correlation between marker allele and a disease risk allele)

Question 13

What important gene was discovered by positional cloning?

Answer 13

1st gene responsible for human genetic disease discovered by positional cloning was cystic fibrosis. Geneticists attempted to identify the gene for CF and clones were isolated after linkage analysis found a region narrow enough.

Question 14

How were clones from the cystic fibrosis region identified?

Answer 14

Via chromosome walking and jumping.

Analysis of DNA sequences within the clones revealed 4 candidate genes

Additional studies eliminated 3 of the candidate genes

DNA sequencing revealed presence of a 3-bp deletion in the gene of the CF patient

Question 15

What is the recombination fraction?

Answer 15

The recombination fraction is the probability that in any meiosis there will be a recombination between them.

If θ=0.5 there is mendelian segregation and thus no linkage so recombination

If θ-0.0 there is very tight linkage and thus no recombination

Question 16

What is required if there is a higher recombination fraction?

Answer 16

More meioses are needed to obtain evidence that 2 genes are linked. (eg. 0.40 requires 343 meioses minimum.

Question 17

How is linkage mapping conducted?

Answer 17

Collect families with affected individuals

Genome scan - test markers evenly spaces across the entire genome.

Lod score (log of the odds) - What are the odds of observing the family marker data if the marker is linked to the disease (less recombination than expected) compared to if the marker is not linked to the disease.

Question 18

What are the problems with mapping in humans? How is this solved?

Answer 18

Can’t do controlled crosses

Crosses equivalent to tet-cross are extremely rare

Humans produce a very small number of progeny.

Solution - Combine results of many identical matings.(Lod score)

Question 19

How is the Lod score calculated?

Answer 19

2 probabilities calculated:

• First one assumes independent assortment
• Second one assumes a specific degree of linkage

Ratio (odds) of probabilities is calculated

Log of ratio is the Lod value

Repeat calculation for range of different degrees of linkage.

Question 20

What Lod score indicates significant support for a specific RF value?

Answer 20

> 3

3 represents an RF value that is 10^3 times as likely as there being no linkage.

2

Question 21

What is multipoint linkage mapping?

Answer 21

Using 1000s of markers, genetic maps have been constructed across the whole genome.

Multipoint mapping uses several markers at once to localise a disease gene relative to the other markers in the map.

More efficient process than using 1 markers at a time.

Question 22

What have the established successes and failures of linkage mapping been thus far?

Answer 22

It has been widely used to identify chromosomal regions linked to diabetes, breast cancer, alzheimer’s disease, and bipolar disorder.

Not every finding that was deemed significantly linked has been replicated convincingly.

Question 23

What are the important study design categories for gene linkage mapping?

Answer 23

Linkage analysis analysis looks through families for co-segregation of disease and gene variants

Association studies detect association between genetic variants and disease across families: exploits linkage disequilibrium. (more appropriate for complex disease)

Question 24

What kind of studies are association studies?

Answer 24

Case-control studies

Cohort designs

Parents - affected child trios (TDT= Transmission disequilibrium testing)

Question 25

What do case-control studies do to identify markers for disease?

Answer 25

Collect affected and unaffected subjects. Then they compare frequency of genetic components between the 2 groups.

Question 26

What do TDTs do to identify disease?

Answer 26

Collect affected children and their parents.

Compare the distribution of the transmitted allele to the distribution of the non-transmitted allele from parents.

Question 27

What are the advantages and disadvantages to using population based case-control studies?

Answer 27

Advantages: Quite powerful to detect small genotypic effects, even in modest samples of case and controls.

Easy to collect the cases and controls for general population samples

Disadvantage: Population stratification. If there are underlying differences in the cases and controls that are unrelated to disease risk false positive results are more likely.

Question 28

What are the advantages and disadvantages of family-based association studies?

Answer 28

Advantage: Resistant to potential bias from population stratification

Disadvantage: Requires at least one parent to be heterozygous at the marker
being tested, therefore power of this approach is significantly lower

Question 29

What assumption is there when using association studies to identify disease causing mutations?

Answer 29

That the mutation has the same ancestral origin and over time it has gotten smaller until very short segments are conserved around the mutated locus.

Question 30

What is linkage disequilibrium?

Answer 30

Nonrandom association of alleles within a population.

Alleles at neighbouring loci tend to cosegregate.

Question 31

What are haplotypes?

Answer 31

polymorphisms within a single gene that form various combinations. (If there are 5 polymorphisms and alleles are equally frequent and independent then 2^5 kinds of haplotypes are produced

Question 32

Why are there less combinations of haplotypes produced than expected?

Answer 32

Because alleles are not independently assorted.

Question 33

What is linkage disequilibrium?

Answer 33

The occurrence in members of a population of combinations of linked genes in non-random proportions.

Question 34

How can SNP haplotypes be used for mapping?

Answer 34

A haplotype is a chromosomal region defined by a specific array of marker alleles it carries

Haplotypes are inherited through generations as blocks which eventually break up due to recombination.

Question 35

What are the issue with using association studies?

Answer 35

Spurious association is caused by 2 factors in population stratification:

A difference in proportion of individuals from 2 or more subpopulations in case and controls (confounding effect of differences between people of different subpopulations)

Subpopulations have different allele frequencies at the locus.

Question 36

How has the problem of population stratification been solved?

Answer 36

Using family based controls (eg. TDT - a type of family association study looking at proportion of children getting the disease from heterozygous parents)

Genetic control: Extra genotyping looking for evidence of background population substructure and account for it

Question 37

How are analyses different between linkage and association analyses?

Answer 37

Linkage analyses look for relationship between a marker and disease within a family

Association analyses look for relationship between a marker and disease between families

Question 38

How is localisation resolution different between linkage and association analysis?

Answer 38

Due to low number of generations involved in linkage analysis we can’t get to very specific variants. However, we can work with relatively few markers due to being in the same family.

Association analysis gives a more fine-scaled resolution due to marker genes being present in a huge number of people who are distantly related. However, due to how numerous the related markers are, they can be tedious (and expensive).

Question 39

What are genome wide association studies?

Answer 39

GWASs typically focus on associations between single-nucleotide polymorphisms (SNPs) and traits like major human diseases.

Question 40

What happens to shared segments with increasing number of generations between them and their common ancestor?

Answer 40

The more distant a common ancestor the smaller each shared segment is but number of people sharing the segment will be greater.

Question 41

How are genomewide association studies carried out?

Answer 41

Extract DNA - Genotype

Calculate which of 300 - 500k SNPs and/or haplotypes are more frequent in cases than controls.

Use whole genome chips

Question 42

What is a SNP chip?

Answer 42

Extension of microarray technology allowed automated genotyping of huge numbers of SNPs across the genome

Question 43

What assumption is made in Genome Wide Association studies?

Answer 43

Common complex diseases are caused by common variants (Now genomes can just be sequenced for this information)

Question 44

What are the pros and cons of using microsatellites as genetic markers?

Answer 44

Pros:

Highly multi-allelic/High hereozygosity/informative

Cons:

Much fewer in numbers and have complex genotyping assays

Question 45

What are the pros and cons of using SNPs as genetic markers?

Answer 45

Pros:

Very frequent

Simplified genotyping platforms

Cons:

Biallelic and not very informative.

Question 46

What do quantile-quantile plots tell us?

Answer 46

2 types of observed test statistics generated in GWAS

In case control studies a chi-squared comparison of absolute genotype counts is calculated for each variant

Question 47

Why haven’t GWS been as successful as had been hoped?

Answer 47

Proposed hypothesis:

Different combinations of variants at multiple loci aggregate in specific individuals to increase disease risk.

Common variants are expected to be of ancient origin, they are more susceptibility factors and so have typically weak deleterious effects (i.e mild missense mutations or changes in gene expression)

Even cumulative contributions of identified variants are small

Question 48

What is missing heritability and what is the explanation?

Answer 48

Small proportion of genetic variance in complex diseases is explained by available GWAS data this is missing heritability. This can be explained by:

Large numbers of common variants having weak effects (People chosen for studies having the condition are the people with the common variants)

Rare variants have large effect

Gene-gene and gene-environmental interactions.