Identifying The Genes Involved In Mendelian Disorders

Question 1

What is independent assortment?

Answer 1

Independent assortment involves the independent arrangement of bivalents at the equator at metaphase I so that you end up with daughter cells that may randomly contain the same or different combinations of chromosomes to the parent cell.

Question 2

At what stages of meiosis do you get independent assortment?

Answer 2

Metaphase I (independent assortment of bivalents)

Question 3

Alleles at loci on the same chromosome will usually travel together. True or false?

Answer 3

True.

Question 4

What situation may result in alleles on the same chromosome not travelling together?

Answer 4

A crossover event between paired homologous chromosomes may separate alleles at loci on the same chromosome.

The loci will only be separated if a crossover occurs between the two loci.

Question 5

Explain why the chance of recombination between loci is a measure of the distance between them.

Answer 5

The loci will only separated if a crossover occurs between the two loci. Therefore the further apart the two loci the more likely it is that a crossover event will occur and homologous recombination will separate the two loci.

Question 6

If crossover occurs but it is not between the two loci we are looking at then the chromosomes will still be non recombinant with respect to our two loci. True or false?

Answer 6

True.

Question 7

What event that occurs during meiosis makes it possible to calculate the distances between genes (when given large populations or pedigrees)?

Answer 7

Homologous recombination due to crossing over.

Question 8

How can distances between genes be quantified?

Answer 8

1) . Their physical distance from each other in Mb.

2) . Their genetic distance as measured by frequency of recombination between the two genes per generation (cM).

Question 9

In what units is the genetic distance between genes measured?

Answer 9

The genetic distance between two loci is measured in centimorgans (cM).

1cM is equal to a recombination frequency of 1% and on average covers 1Mb of DNA.

However, the relationship between linear are genetic distance is not absolute. The frequency of recombination and thus the genetic distance between genes in different regions of the genome may differ depending on the sequence itself and the ancillary proteins that cover the DNA.

Question 10

Describe genetic mapping.

Answer 10

Genetic mapping depends on studying a minimum of 2 loci in a set of independent meiosis and then estimating the proportion of those meiosis where there is recombination between your loci.

If the recombination fraction for the two loci is significantly below 50% then the two loci are said to be linked and will be on the same chromosome.

Unlinked loci may be on different chromosomes or may be on the same chromosome by distant from each other.

Studying a series of linked loci and estimating the recombination fraction between the pairs of markers allows the loci to be arranged in order along a genetic map.

Question 11

What are the requirements for markers in genetic mapping experiments?

Answer 11

1) . Mapping looks at pairs of loci. Although any meiosis may involve a crossover between two loci we can only tell whether or not that has occurred if the person in which the meiosis takes place is heterozygous for both loci. The markers that we use therefore must be significantly polymorphic so that they are likely to be heterozygous.
2) . Markers must be easy to type using material that the family is willing to provide.
3) . Markers for positional cloning have to be available across the genome.
4) . Realistic pedigree collections require markers to be no more than 10 cM apart (approximately 300-500 markers spread across the genome).

Question 12

What two types of DNA marker are generally used for genetic mapping?

Answer 12

1) . Microsatellites - short tandem repeats. The number of repeats varies between individuals and they are highly polymorphic.
2) . SNPs - only 2 alleles therefore less likely to be informative. Are abundant and are amenable to high throughput genotyping as they are generally binary.

Question 13

How can we recognise recombinant in pedigrees using genetic mapping markers?

Answer 13

If we know which marker allele a disease segregates with then we can recognise recombinant offspring.

Non-affected individuals that have the disease marker allele and affected individuals that don’t have the disease marker allele are both likely to be recombinants.

Question 14

In many cases we may not know which marker allele is segregating with the disease just from the second generation individuals. What would we have to do in these cases in order to identify which marker is segregating with a disease?

Answer 14

In these cases we would have to use computer analysis to generate likelihoods.

These computer analysis will calculate the overall likelihood of observing a particular pedigree under two alternative hypothesis:

1) . Assuming the 2 loci under examination are linked.
2) . Assuming the 2 loci are unlinked.

The ratio of these two likelihoods provides evidence for or against linkage of the marker with the disease.

If the likelihood ratio supports lineage then the recombination fraction is the one that gives the most favourable ratio.

The output statistic from this analysis is the log of the likelihood ratio (LOD score).

Question 15

What LOD score is understood to give evidence of linkage between loci?

Answer 15

A LOD score of 3 or higher.

A LOD score of -2 or lower is significant evidence excluding linkage.

Question 16

How many and what kind of individual are usually required in linkage analysis?

Answer 16

A minimum of 10 meioses are required to provide statistically significant evidence of linkage.

We need heterozygous informative individuals.

Several families may need to be combined. We must be sure of the phenotype if families have been combined. We must be confident it is the same disease caused by a mutation at the same locus.

Question 17

Once genetic mapping has been used to identify the general region of the genome in which the disease gene lies has been identified what are the next steps in gene identification?

Answer 17

We must first narrow down the candidate region using additional markers. This will reduce the number of candidate genes to be tested.

Once the area has been reduced it can be screened for candidate genes. If information is available regarding the function of the candidate genes then this can be used to prioritise genes for further study. It would also be useful to look at the pattern of expression of the gene (is the gene expressed on tissues affected by the disease).

We then go on to screen these candidate genes in individuals affected by the disease to identify potential mutations.

Having identified candidate mutations it is important to assign likely functions to these changes.

A panel of unaffected individuals should be screened to ensure the change in the gene is not a polymorphism. The effects on the protein sequence and structure as well as possible effect on RNA structure should also be considered and backed up by experiments where possible.

Question 18

What are recombinant offspring?

Answer 18

Recombination offspring are those offspring whose genes are a non-parental allele combination.

Question 19

Give a summary of how you would go about identifying a disease causing mutation?

Answer 19

1) . Determine the area of the genome likely to contain the disease gene. This is done by analysing polymorphic markers across the genome and assessing recombination.
2) . Search the area for likely candidate genes.
3) . Search candidate genes for mutations.
4) . Assess the likely function of these mutations.

This will hopefully allow us to identify the genes causing the disease.

Question 20

What is the aim of linkage analysis or positional cloning?

Answer 20

To identify a genetic marker which is close to a disease gene.

Question 21

What does it mean if the recombination factor is significantly below 50%?

Answer 21

The loci are linked. For example if the recombination fraction between a marker locus and a disease locus is significantly below 50% the. That locus is linked to the disease locus.

Question 22

If we see a lot of recombinant individual when looking at a marker and a disease locus what does it mean?

Answer 22

Because recombination frequency is a function of distance if we see a lot of recombinant individuals it means that our marker and our disease locus are not located near to each other.

If we see lots of non recombinant parental combination it means it is more likely that the marker and the disease locus are close together which means we are narrowing down the position of the disease locus in the genome.

Question 23

If the sequence of the protein product of a gene is known how might you go about identifying the gene that encodes that protein?

Answer 23

1) . You can use the protein to work out a likely cDNA sequence using a table of the genetic code.
2) . An oligonucleotide probe corresponding to the cDNA sequence can then be designed and synthesised and used to screen the cDNA library to isolate a clone containing the cDNA.

This method was used to identify the alpha and beta globin genes.