Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disease

Question

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disease in which patients exhibit numerous phenotypes that are characteristic of premature aging. The disease is associated with mutations in the gene coding for the protein Lamin A. The mutation that causes HGPS is a dominant mutation. The lamin A gene is depicted below before mRNA processing events occur. The exons are numbered in the boxed regions. The introns are the sequences between each exon 4. 5' 4 5 H6 10 H 11 12 3' In people that don't suffer from HGPS, the lamin A gene gets transcribed then translated into a Pre-lamin A protein that gets post-translationally processed into Lamin A. In HGPS patients, there are issues with this post-translational processing leading to aberrations in Lamin A protein levels. Shown below is a RT-PCR analysis of the lamin A gene in wild-type controls (lanes 1 and 2) and two HGPS patients (lane 3 and 4). These two patients each have a point mutation in the coding region of exon 11 of lamin A GGC>GGT (a C is changed to a T). This point mutation DOES NOT change the amino acid that is encoded at this codon. In both cases a glycine is coded for. -639 bp 489 bp Shown below is a western blot in which the same patients as described above were used to analyze Lamin A, pre- Lamin A, and Lamin C protein levels. (One antibody that can bind to all three of these proteins was used. Lamin C is a related protein that is encoded by a separate gene.) prelamin A amin C a) b) Describe the results that are shown in the RT-PCR and western blot above. (4 points) Based on these results explain what is happening as a result of the C to T mutation in the HGPS patients. (8 points)

Explanation / Answer

From the RT PCR and Western blot, it is clear that there is a single product in the WT sample. However, there are two products in the patient's sample.

The patient's sample shows two bands in both RT-PCR and Western blot. This suggests that there are two RNA products in the patient which give rise two different proteins.

It is given that there is a mutation in the last exon of the lamin A gene. The mutation is a substitution which replaces GGC with GGT. Both the codons encode the same amino acid, Glycine. It does not produce any stop codon. So, the effect of the mutation can not be at the protein level. It must be at the transcript level.

In eukaryotic splicing mechanism, AG-GT is the splice site acceptor-donor sites.

There is a generation of additional GT site in the last exon due to the mutation. This GT site is located internal to the exon (not at the end) and could act as an alternate splice site.

If this splice site is utilized, a short transcript/protein would be produced.

However, the efficiency of alternative splicing is less. Hence, in mutants, we still observe a WT band.

The smaller band in patient's sample corresponds to the alternatively spliced short transcript which in turn produces a truncated protein.

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