Single Gene Disorders

Question 1

Define single gene disorders.

Answer 1

Single gene disorders are defined as they are caused by variation in the DNA sequence of a specific gene. (E.g. CF)

Question 2

How can single gene disorders be isolated?

Answer 2

Positional cloning.

Question 3

Define positional cloning.

Answer 3

Positional cloning is an approach to isolate genes of on the basis of their position on a gene map.

Question 4

Define linkage analysis and how it was used.

Answer 4

Linkage analysis of pedigrees first used to identify general location of a gene on a chromosome. (Look for co-segregation of a phenotype healthy/disease)

Question 5

Define RFLPs. How did RFLPs change the genetics scene? How did it lead to the isolation of certain genes?

Answer 5

DNA digestion with different REs and hybridsation with different probes generate several RFLPs which provide coverage across the genome to allow linkage with unknown genes of interest. - Enabled testing (carrier detection, antenatal testing) of many genetic conditions by pedigree and linkage analysis.
- Led onto the isolation of many genes such as the CF gene.

Question 6

Define chromosome walking and jumping and its relevance to positional cloning .

Answer 6

Walking; technique used to clone a gene (e.g., disease gene) from its known closest markers (e.g., known gene).
Jumping; similar to walking but is able to overcome the breakpoints of walking

Chromosome walking and jumping is used to identify clones from human genomic libraries that cover the region of interest.

Question 7

Define ortholog and paralog.

Answer 7

Ortholog; is a functionally equivalent gene or protein in another species.
Paralog; is a member of a gene or protein family within the same species.

Question 8

Draw out a genetics tree for CF.

Answer 8

Look at video

Question 9

Why might predicting phenotypes be difficult even with the knowledge of genotypes?

Answer 9

• Effect of some underlying mutations can be difficult to predict.
• Environmental factors may influence the phenotype.

Question 10

Describe the timeline of CF treatments.

Answer 10

• 20 years after discovery of the gene, Pearson (2009) discussed the lack of therapies developed since 1989.
• 2012 – Ivacaftor (Kalydeco, Vertex Pharmaceuticals)) approved by FDA. First drug to target cause of CF rather than symptoms - Targets patients with G551D mutation which causes chloride channel to fail to open.

Question 11

Can generic drugs, not focused on a specific gene product, be developed which would have merit of reducing costs(Typically price for treatment of CF is 300k.)?

Answer 11

• One class of generic drugs suppress stop codons so that translation continues (translational readthrough).
• Huge potential since nonsense mutations responsible for 5% - 70% of individual cases for various inherited diseases.

Question 12

Define compound heterozygosity.

Answer 12

When both alleles of a gene harbor mutations, but the mutations are DIFFERENT, it’s compound heterozygosity.

Question 13

Describe the ‘then and now’.

Answer 13

• Cloning of CF gene was a milestone in genetics and allowed for innovation for different methodologies used today.
  
  • For example, more polymorphisms available now and no need to chromosome walk to obtain DNA spanning the region - Instead, can download sequence from a database.
  • Expression analysis is now done by microarrays.

Question 14

After clones covering the entire region have been obtained, next step was to identify all genes within the region. What is the arduous process?

Answer 14

Homology to other mammalian genes
Expression of the gene in affected tissues
CpG islands in most mammalian genes