TRK Mutations

What is a mutation? Mutations today generally refer to alterations in the basic genetic code as it translates into proteins. “Alterations” may refer to changes in the way genes are positioned relative to one another. Before discussing simple changes in the coding of TRK protein kinases, an introduction to the genetic code might be helpful. This may lead to an understanding of why genetic alterations that result in “fusion proteins” are sometimes more dangerous than simple genetic code Trk mutations that generally result in small or no changes at all, as we shall see in this metaphorical example.

What is a protein?

A protein is a polymer of amino acids that hook together in an amino to carboxy terminal fashion, much like the children’s toy pop beads.  The “R” group can be anything from a simple hydrogen in the case of glycine, to a hydrophobic, aromatic group in the cases of tryptophan, phenylalanine, and tyrosine.

Proteins as Pop Beads A. Pop beads assemble into chains. The shape of the chain is determined by the shape of the bead. B. In proteins, amino acids are assembled into polymers. The shape of the protein is determined by amino acid side chain called “R”.

Changing the shape of a pop bead in a chain may impact the shape of the chain; substituting one amino acid for another may change the shape and function of a protein.

Mutations start with DNA

DNA is a double stranded molecule whose nucleotides code for proteins. For now we will ignore the structures of guanine, cytosine, adenine, and thymine.

  • Triplet combinations of these four bases code for all 20 amino acids.
  • Amino acid monomers are joined together to form proteins.
  • The sequence of nucelotides in the “sense strand” is transcribed into messenger RNA.  In mRNA,  thymine is replaced with uracil.
  • After  mRNA is transcribed from the chromosomal DNA in the nucleus, it is exported to the ribosomes in the cytosol.

  • Ribosomes translate the mRNA into proteins using transfer RNA (tRNA) that binds both mRNA codons and single amino acids.
  • The triplet codon directs tRNA addition of amino acids.
  • Methionine is always the first amino acid in a newly synthesized protein because the codon telling the ribosome to start translating the mRNA is also the codon for methionine.

The single letter code for methionine is M. The code for alanine is A, the single letter code for lysine is K.

 

Some types of simple mutations

A mutation is any permanent alteration in the nucleotide sequence of a gene.   Biochemists use both three letter and one letter names for amino acids.  For example, lysine may be written as “Lys” or as “K.” If single letter amino acid codes mean absolutely nothing to you, we can use that single letter amino acid code to write simple sentences like “MAKINGTHINGSISHARD,”  or “making things is hard.”

If you wish to follow along, a web based program to  translate mRNA, or DNA, sequence into the amino acid sequence of a protein is provided by Expasy.

“AUG” spells “start” and methionine

Translation starts at the first “AUG” codon of mRNA, which also happens to code for the amino acid methionine. The reading frame is considered to start here.  Other codons tell the ribosome to stop translating the mRNA into protein.

A.  Translation must be started in the correct place

When one submits a nucleotide sequence to Expasy for translation, the program translates reading from the first, second and third nucleotide.  When a nucleotide is deleted, a frame shift mutation has occurred. Arrows in Panel A indicate the start of the reading frame. Frame 1 starts at the A of the codon AUG and translates the message into our “Making things is hard” protein. Frame 2 starts at the U and considers UGG to be the first codon. It takes a bit of reading before the ribosome comes across the requisite start codon of AUG. When a protein is produced, it is the gibberish “Mg pist gvf hirg!” protein that resembles a drunken tweet.   The third reading frame starts with GGC. This codes for a G, or glycine, followed by a “Stop!” codon UAA. We have a meaningless AAU codon for asparagine because the ribosome needs to read “AUG” to start translation. Then we get another UAA stop codon. In this second frame shift we have also created that codes for a premature stop codon.

Simple mutations

B.  Some point mutations stop translation prematurely

In Panel B we have an example of how a T→ A point mutation in the first nucleotide of the lysine (K) codon leads to a  nonsense mutation that halts translation after only two amino acids. This is only a big deal if the cell requires a large amount of the   “making things is hard” protein.   The cell may get confused by the “Ma” protein.   There could be trouble if the enzyme end of a protein is the N-terminus (first to be translated) and the regulatory domain is in the C-terminus.  This is not the case for the Trk proteins.  Trk regulatory domains are in the N-terminus and enzyme, or catalytic, domains are in the C-terminus.

C.  Some point mutations result in livable changes, others do not

In Panel C we have an example of a T → G point mutation in the fist nucleotide in the lysine (K) codon. Instead of a lysine (K) we get an asparagine (Q). This is a missense mutation, non synonymous mutation. One may argue that this is a conservative substitution. The hypothetical  “Maqing things is hard” protein may still get the point across.

D.  Many point mutations do not change a thing

In panel D we have a T → G substitution in the third nucleotide in the lysine (K) codon. What we get is another lysine due to the degeneracy of the genetic code. This is called a silent as well as a synonymous mutation. Not all point mutations are even remotely bad. Some do not change any aspect of the business of the cell.

TRK genetic alterations, how do they compare with mutations?

We have illustrated the impact of point mutations using simple English sentences spelled out with single letter codes for amino acids. If we can continue on this path just a while longer, we can illustrate the impact of fusing the promoter of a house keeping gene to a gene that codes for a very useful protein that only gets expressed in low amounts or in defined stages of development. Promoters and chimeric TRK fusion proteins are beyond the scope of this introduction.

An individual might engage in some activities only at certain times. Other activities like drinking water are more housekeeping.

The promoters of house keeping genes signal the cell to make the protein on an ongoing, or “house keeping” basis.  These proteins control functions that need to happen on an ongoing basis.   Drinking water after exercising might be an example of a house keeping function.  Many other functions need to occur less frequently.  Having a glass of wine at a party on weekends could be one example for an individual.  In Trk alterations a production of a rare event protein is controlled by factors that maintain house keeping proteins.  An analogy at the individual level would be to drink wine instead of water after a a daily workout.

Important Information

Often innocuous mutations may occur in TRK genes in cancer patients. These simple mutations may be passenger mutations rather than driver mutations. TRK gene rearrangements appear to drive cancers. A Drilon 2017 review has summarized the results in two clinical trials testing a specific Trk inhibitor. The tumors bearing only simple mutations in the TRK gene did not respond to the inhibitor suggesting that some other event was causing the tumor to grow out of control. The tumors in which the TRK gene was fused with a house keeping gene decreased in size in response to the Trk inhibitor. A Phase II clinical trial testing the same specific Trk inhibitor. To be eligible a patient’s tumor must carry a TRK fusion.