Hi guys

I don't mean to be a pain but I have been looking at the trialserve predictor again. And with my nephews deletion of 4-18 it says that 'no stop codon was found either in or out of frame', can anyone tell me what that means??

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There are 3 mutation types. Deletion, Duplication and Stop Codon. He does not have a stop codon, his mutation is a deletion....It says that on every one. I wish it didn't as it does cause confusion.
Thanks Lori
Lori you are quite correct there are 3 types of mutation, however they are deletion, duplication and point mutation.

Deletion is of course where regions of introns and/or exons are missing from the dystrophin gene. Duplication is where regions of introns and/or exons are inserted into the gene. Point mutations can be either transitions or transversions depending on whether it is a homologous change (transition: purine to purine or pyramidine to pyramidine) or heterologous (transversion: purine to pyramidine or vice versa). Anyone of these mutations can then be further categorised into the predicted functional changes of the mutation:

Nonsense (resulting in a premature stop codon) missense (resulting in an open reading frame even though a protein is produced with structural variations) and silent (where the gene fault does not result in any structural changes to the functionality or length of the protein).

Depending on where a mutation occurs will also have ramifications on whether gene splicing or transcription factors are affected (including faults in introns, not just exons), which again has ramifications on whether the reading frame is affected.

The difference between inframe and out of frame mutations is determined by how many nucleotides are affected. If coding nucleotides in multiples of 3 are inserted or deleted an in frame mutation will result, any other number, or multiple of, will result in a change of reading frame as DNA is always read as triplets (codons). This is called out of frame.

In frame verses out of frame is significant, as any fault that results in a frame shift (change of reading frame) may result in not only the amino acids around the mutation being incorrect, but every amino acid there-after (which is why there is then a possiblity of premature stop codons, and changes to the secondary structure (folding) of the protein). In frame faults can result in the amino acids at the point of mutation being incorrect, but the subsequent amino acids being as per normal, which can result in a functional protein being produced albeit a different length.

As far as the prediction software goes it is an indication, not a given prognosis for any individual, much the same as the weather report can not predict with 100% certainty the exact weather conditions on any given day. There are so many variables involved in mutations that the only way to get a true indication of the protein produced is by sequencing the entire gene and associated coding regions. However even this is not definitive as to the prognosis for a patient as it only takes into account mutations in Xp21 (the dystrophin gene) and does not take into account any mutations in other proteins that may directly or indirectly affect the function of dystrophin in the muscle fibre.

Hope that helps.
Hi Julie

You obviously know your stuff, I haven't a clue about a quarter of it but I am trying to learn. My nephew's deletion is from exons 4-18, would you know if that is in frame or out of frame?? Is it true that resulting in which exons are missing that there are different levels of severity of DMD??

Many thanks, Nadine

A deletion of exons 4-18 does not disturb the reading frame, thus in theory a truncated dystrophin should be produced. To test for this a Western Blot test should be done on a muscle specimen to test for the quantity and weight of dystrophin produced. If one is done and there is little to no dystrophin expressed then I would presume the deletion disturbs far enough into the introns to cause a splicing error and/or there is another mutation somewhere else that they didn't catch or there is a problem with starting the translation/transcription or a problem with stability of the protein produced (causing proteolytic degradation).

To know 100% certainty of the type of mutation occurring with dystrophin one must have their mRNA tested via a cDNA analysis. Looking at the gene will only tell you potential issues that may occur during translation, post translational mRNA is what encodes a protein so it is the true blueprint of the dystrophin protein. However, even with knowing an mRNA sequence there could also be issues with starting the transcription if there are issues with other RNA that aids in the control of the dystrophin transcription (i.e. the builders ability to follow the blueprint). There are also issues with proteasomes and how they "clean up" incomplete dystrophin proteins (i.e. clean up crew doesn't know the difference between scrap and functional material - known as proteolytic degradation). All these could lead to a reduction in dystrophin levels and cause BMD/DMD even if a truncated dystrophin protein is produced.

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