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 exorn 4. 5' 4 7 10 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 level:s 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. 234 -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.) amin A relamin A lamin 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

1. Both lamin A and C are transcribed from the same gene. In the RT-PCR, there are two fragments observed in the HGPS patients (lane 3 and 4). The 639 bp fragment is the wild type lamin A gene (as indicted in lane 1 and 2). The second band is a 489 is caused by the C to A mutation. This band is 150 bp deleted band which is obtained from the wild type lamin A. The transversion creates a crytic splice site, which results in this 150 bp deletion.

Lamin C show lower band in lane 1 and 2, while the lamin A band is larger band. Levels of both Lamin A and lamin C are reduced in HGPS patients (upper and lower bands in 3 and 4). Prelamin A is not be seen in controls, as it is processed to lamin A. However, as there is a defect in prelamin A processing to lamin A in HGPS, the prelamin A band is seen in observed only in HGPS patients (lane 3 and 4; middle band).

2. Prelamin A to lamin A processing is initiated by a distinct isoprenylation of a CAAX box cysteine. Next, farnesylation occurs wherein a farnesyl group is added to the C terminus. The farnseylated protein undergoes a C terminal cleavage to remove AAX from CAAX. The C is then methylated and undergoes an upstream endoproteolytic cleavage to release the mature lamin A, which is 72 kDa.

The transversions of C to T in HGPS patients converts a serine to a threonine, both polar uncharged amino acid. However, this transversions creates a cryptic site that cleaves off a 150 bp mutant allele that codes from a mutant protein due to 50 amino acid truncation of amino acids. The truncated protein retains a CAAX motif that is required for farnesylation but lack the endoproteolytic cleavage site. The endoproteolytic cleavage site is required for processing of lamin A from prelamin A. The prelamin A seen in HGPS is farnesylated but cannot undergo endoproteolytic cleavage. This 67 kDa protein is known as progerin.

Since lamin A and lamin C are obtained from prelamin A, levels of both will be reduced in HGPS.

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