An Example Of Positional Cloning Flashcards
How was the gene for DSH identified?
Individuals were typed for 343 micro satellite markers across the genome.
LOD scores for most markers analysed were negative (no linkage) but a series of markers in the middle of chromosome 1 were different.
They next used additional markers to map the region at a finer level.
By looking at the haplotypes of affected individuals with additional markers the location of the disease gene was narrowed down by identifying the regions in the previously identified area that those affected possess.
Looking at recombinant haplotypes helped to narrow down the disease gene region. Affected individuals with recombinant haplotypes must have inherited the region of the chromosome with the gene in - therefore if they only have a part of the region we have identified it as being in then it must be in that part and we can narrow down the region further. Doing this in multiple individuals can narrow down the region a lot.
7 candidate genes in a 500kb region were identified.
Affected individuals were screened using single stranded confirmation polymorphism (sscp) analysis.
Changes were identified on one gene in each sample analysed. This was the DSRAD gene now known as ADAR.
If we look within a family all the affected individuals aphave the same ADAR mutation. Different families had changes in different exons/different mutations.
A mutation in which gene was found to be responsible for DSH?
Changes in the DSRAD gene now known as ADAR (double stranded RNA-specific adenosine deaminase).
What is single stranded confirmation polymorphism (sscp) analysis?
This technique relies on PCR amplification followed by separation of PCR products which have been separated into single stranded structures on a hi res non denaturing gel.
The ssDNA will form secondary structures in order to stabilise themselves and these secondary structures also affect the mobility of the PCR products within the gel.
If mutations are present compared to the normal sequence, these changes may also affect the secondary structure and hence the mobility. Therefore you will see different variants on the gel.
This method provides a way of narrowing down the region which is likely to contain the mutation, which will then be characterised by Sanger sequencing.
Today the whole region would likely just be sequenced by NGS rather than by sscp and then Sanger.
Are the mutations that were identified in the ARAD gene in DSH definitely pathogenic? How do we know this?
Two are nonsense mutations and thus are unlikely to be common polymorphic variants.
Two are mis-sense mutations involving conserved amino acids and these regions are likely to be important for the functions of the protein.
Mutations were not found in 116 unrelated normally pigmented adults.
The next step would be to work out how the gene mutations cause disease and why a 50% reduction in gene activity causes pigmentation problems.
What is Dyschromatosis Symmetrica Hyreditaria (DSH)?
DSH is the presence of hyperpigmented and hypopigmented macules on the back of the hands and on the dorsal surface of the feet.
It is an autosomal dominant disorder.
Most cases are reported in Japanese populations.
