The VHL tumor suppressing gene and a Mutation of it

The von Hippel-Lindau tumor supressor gene is located on human chromosome 3p25, a group A chromosome. VHL disease is caused by germ line mutations of this gene, that means that the mutation is passed on to offspring. Inheritance of the gene results in an increased likelyhood for having certain types of tumours. The type of tumours that develop depend upon the type of mutation. Truncation mutations, wherein the long sequence of genes are shortened, put the indiviual at increased risk for renal cell carcinomas and hemangioblastomas. There is also a slight increase in risk of pheochromocytoma; adrenal tumours. If the mutation is a Missense type, there is an increased likelyhood for developing these tumors also, however some more so than others, depending upon the location of the missense mutation. Missense mutations for what will become the Alpha-domain of the protein product of the gene result in increased likelyhoods across the board.
The protein product of the VHL gene, pVHL, enables the degradation of another chemical called "HIF-alpha".

This degradation ability (actually it helps marks HIF-alpha for degradation) is thought to be connected to its ability to bind to complexes containing elongin B and C, an cullin 2 (Cul2). Elongin C and Cul2 are homologous to Skp1 and Cdc53 (respectively), and because of this are suspected to target certain proteins for covalent modification with Ubiquitin, and thus target them for degradation.
So the loss of this marking (with Ubiquitin) function results HIF-alpha not being degraded in the cells lysosomes. Marking a normally produced protein with another molecule to signal that it should be degraded and destroyed by an entirely different set of chemicals (in an entirely different cellular organelle, the lysosome) is an off-handed and perhaps seemingly needlessly complex way to control things. But thats just so typical of evolutionary processes anyway, working in subtle and diverse manners to have dramatic affects.




a schematic action of ubiquitin, the mutation interupts this process

Misssense mutation RC 161/2 QW effects the Alpha-domain. I will be making this mutation in a genetic sample and analysing it. The numbers in the name 161/2 indicate amino-acid positions along the primary structure chain of the protein. RC is what is normally there, Argenine and Cysteine. QW is what they are replaced by in the mutation, Glutamine and Tryptophan. This results in the wrong amino acids being in that position, as the coding is off.



The universal genetic code




Specifically the Elongin C binding region will be mutated, as it occurs over positions 157-172.
Proteins affect other proteins and materials (in which cases they are called substrates) by having binding regions that have very specific shapes, sort of like a lock and a key. What also comes into play are the charges and chemical properties of the amino acids that make up these keyed regions. This can have an effect on the protein's ability to bind, or can also effect changes in the proteins tertiary structure.

The interesting thing about pVHl is that one domain, the alpha-domain, starts by binding with Elongin C, and from that a series of other proteins. The Beta-domain of the protein, meanwhile, has bound to HIF-alpha.

HIF-alpha is a transcription factor, its responsbile for the expression of some growth factors, some of which cause new blood vessel growth, aka angiogenesis. This is why VHL disease tumors are so highly vascularized, because the HIF-alpha builds up and this results in the genes that bring about angiogenesis being amplified.

HIF-alpha will be bound to ubitquitin by the complex attached to the alpha-domain, this 'marks' it for digestion/destruction by proteosomes, which apparently is triggered by an increase in the oxygen concentration of the local environment.

pVHL and the specific action underconsideration in schematic

With the RC 161/2 QW missense mutation, [b]the ability to bind Elongin C is severly comprimised[/b], and thus the chain of events leading to ubiquitinization and ultimate degradation is broken. The mutation [b]does not necessarily affect the ability of pVHL to bind to HIF-alpha[/b] itself. Because its a missense mutation, one can expect [b]the Disease Phenotype that results from it to be of the type 2B[/b]. This is a disease phenotype that results in very great risk of developing any of the three types of tumors.

Manufacturing the mutation for analysis

The specific mutation will be created via PCR. PCR will also clone the mutated genetic segement so as to permit further analysis.

In normal PCR, a primer is mixed with DNA and DNA polymerase enzymes cause the replication of the desired segement. Two primers will be used, a forward and a reverse, so as to yeild opposing strands of DNA. Three stages are required to create the missense mutation via PCR.

a general example of PCR with primers

In Stage A, a forward primer with the "START" codon, ATG, is run up to the point where the mutation will be. A reverse primer with the mutation sequence is permited to run in the oppositte direction. This results in everything downstream (or upstream, its relative) from the mutation being copied.

In Stage B, the same is done, but for everything in the opposite direction along the genetic sequence. A forward primer with the desired mutation is used, and the reverse primer has a "STOP" codon (TAA, TAG, or TGA)This yeilds two halves, with the mutation at opposing ends.

In Stage C, the strands of DNA are denatured/unzipped. Opposite strands are then re-annealed, resulting in one strand of what would be a double helix. PCR is then repeated, and this fills in the opposing strand to make up a full gene sequence.

Following is the sequence I'll use for stages A & B

1. First 41.5 micro-liters of cold water are pipetted into a 0.5 micro-liter PCR.
2. Then 1 micro-liter of the first primer, the forward start primer is pipetted.
3. Then 1 micro-liter of the second primer, the reverse mutation primer, is pipetted.
4. Then 5 microliters of a buffer, 10x PCR Buffer, is pipetted.
5. 0.5 micro-liters of Vent Polymerase is then added.

Now another 0.5 ml PCR tube is prepared for Stage B. The above process is repeated, with the exception that different primers are used; again the forward primer has the mutation, whereas the reverse primer now has the "STOP" codon. While preparing this Stage B tube, the Stage A tube is closed and placed on ice. The seemingly insiginificant heat of the room temperature and handling of the tube is enough to start the process prematurely.

When both tubes are ready, only now is the final step taken.

1 micro-liter of Template DNA is added to tube A, and 1 micro-liter is added to tube B.

The tubes are now sealed, labeled, and placed into the PCR device, set at 95 degrees Celsius. The mixture will be heated and denature over the course of 5 minutes. After which, there will be 30 cycles consisting of:

• Denaturation for 1 minute at 95 degrees Celsius
• Annealing for 1 minute at 50 degrees Celsius
• Extension for 1 minute at 72 degrees Celsius

After that, the process is completed with a 10 minute extension at 72 degrees celsius. The products of the PCR will now be ready for gel electrophoresis and puryfication.