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BCR/ABL transcripts VS mutations

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Hi all,

So, I have been wondering about the difference between the various bcr/abl transcripts and the various gene mutations.

To my understanding these are two different things. Please read and let me know if I get them right:

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The transcripts have to do with the breakpoints in the BCR and ABL genes, which result in different combinations of the fusion gene, and different molecular weight (?). Therefore, there are various transcripts and sub-categories:

Based on the major BCR break point (M-BCR), you get the transcript p210, and two different variants,  b2a2 (e13a2) and  b3a2 (e14a2).

Based on the minor BCR break point (m-BCR), you get the transcript p190, variant e2a2.

Based on the micro BCR break point (μ-BCR), you get the transcript p230, variant e19a2.

Plus, I think there are more combinations based on different ABL break points, other than a2.

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1867492/

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Mutations. Where to begin. I have no clue what's going on here, apart from the infamous T315I, which I understand is a single base pair substitution. I have also read about P-loop mutations, and how they can affect imatinib binding, but nothing more than that.

So I have some questions towards you, guys:

1) How common are they? Not T315I in particular, but in general. If it is a not-so-important mutation, I guess it is possible that a lot of us have one, and we just don't know, right?

2) Upon DX, I was told that I have the p210 transcript, variant b3a2. I was also told that I don't have T315I. Looking back, and checking my lab results now that I know more, I don't understand where the second claim comes from. Is T315I detectable in a usual PCR test (if they know they have to look for it)? Or is mutation testing a different test?

3) Is it ok if you don't have a mutation testing upon DX? I'm 4 months into treatment after DX, and I would say I am doing pretty well on Nilotinib. Is there any reason to ask for mutation testing, if the trend of my q-PCR doesn't alarm my doctor?

4) When you get a mutation, what's the symptom? I guess, since you are tested on the transcripts that you have, and you can get the mutations on those transcripts, you will see your q-PCR rising? Is it possible that your blood test counts are affected first?

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So, what's your take on the above? 

Regards,

Koralia

Adding one more question to the pile:

Can T315I appear on all various transcripts?

Hi Koralia,
Regarding your questions 2 & 3 as far as I know the above mentioned mutation is not detectable in a usual PCR test I think it's a different test to check whether you have it or not.I also believe that this test is requested only if your PCR results start to rise even though you are under treatment.This could be a sign that there may be a mutation.Talking about my case I havent been checked for this mutation.
About your last question I don't know what are the symptoms but I know for sure(I have asked my doctor) that first the molecular results are affected and then your blood tests.
I hope I've been of some help.
Best
Vicky

Hi Vicky,

Thanks for your reply.

That's what I suspected as well. 

I also have another question, to anyone in the forum that can answer:

In order to find the transcript (p210) during my DX, my doctor had ordered a qualitative PCR, which showed p210. Then, I get my 3-monthly q-PCRs, which actually test p210 expression. If another transcript appears in the meantime, will the q-PCR be able to detect that as well, or is it focused on a single transcript?

Can anyone with more than one transcript answer this question?

 

BRs,

Koralia

PCR is designed to amplify transcripts from a specific DNA target region.

https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-s...

What makes PCR so powerful is that it can be designed and built to transcribe DNA in less than a day - but as you suspected, the DNA target has to be known ahead of time.

In CML, it took many years and much research to "discover" the gene and DNA segments responsible for bcr-abl transcripts. Subsequent research revealed several transcript mutations which give rise to different bcr-abl clones (molecular weight) essentially producing the same disease whether P190, 210 or T315i.

... but one has to know what the target is in order to "build" a PCR test for it. Because we know much about CML, patients suspected of having CML (i.e. high white blood cell count), first get a FISH test to identify the gene under the microscope. That is the first clue that one has CML - then blood samples are sent for PCR analysis where they already know to look for p190, p210. When they find that - they can prescribe appropriate drugs.

The problem in CML (and cancer in general) is that often more than one clone is present - so even if P210 is "dominant", a patient might have T315i lurking around. Only after treatment begins do we learn whether it works or not. The good news is for the vast majority of patients, the dominant clones are what is present only and the drug works (over 95% !!). For a minority of patients, T315i is a problem, but for which we now have drugs that can attack.

One final comment - It's all about proteins. What these transcripts do is generate proteins of slightly different shape and weight. Protein is amazing stuff. It literally is the stuff of life. Everything living is made of protein. There are Millions and millions of proteins - it is what makes each of us unique. But the drugs to target CML proteins only work well with a few particular shapes (protein is a three dimensional molecule with funny shapes) - lock and key sort of thing. So your CML gene can produce a slightly different CML protein for which the drug doesn't 'work'. And for someone else, it does. Ongoing research is focused on discovering all protein variations so that drugs can be designed to shut them down. Unfortunately, as fewer and fewer patients are left with untreatable CML (very few indeed), the financial incentive to build these last remaining drugs is not cost effective. For those patients, transplant is the remaining option.

Hi Scuba,

Thanks for the reply :)

I still have some unanswered questions, I hope I'm not tiring you all!

1) About transcripts: My transcript was not identified by FISH, but by qualitative PCR. Indeed, I was searched for three variants, p190, p210, p230. I came back positive for p210. So the transcript can also be identified by qualitative PCR, correct?

2) About q-PCR: My lab tests against ABL1 control gene. Does this mean that this is the DNA target region? If I understand correctly how the various known transcripts are created, all three most common ones (p190,p210,p230) contain the same "part" of ABL, breakpoint a2. Does it make sense to assume that, if you get another transcript on the way, it will also appear on this test, since the control gene is ABL and the breakpoint in the fusion gene is the same?

Or - and this is actually my main question - is it possible that I am only tested e.g. for p210, and I think I have reached a certain level (e.g. MMR), but another transcript has gone wild and I know nothing about?

3) To my understanding, T351I doesn't cound as a transcript, but as a mutation. Therefore, you can have e.g. transcript p210, with mutation T315I (I've definitely seen a paper with this combination). You might not know it, if you don't get tested for mutations, but as long as your q-PCR is decreasing, then you don't have to worry about mutations. Does that sound right?

Best,

Koralia

Hi Koralia,

Here are some answers to your follow up questions.

1. You are correct the transcript type is verified by qualitative PCR. Each type (p190, P210 or other) requires different PCR probes. But once you have been confirmed for a specific transcript, like p210, labs typically don't check of other breakpoints at subsequent PCR tests.

2. Abl is the normal gene and the PCR probes specifically for that measurement are the same for everyone regardless of BCR-ABL transcript type. It is very uncommon for another transcript type (as you say to go wild) to appear.  That is if you have p210 it is very unlikely p190 will show up.  However, some people do have multiple breakpoints within p210 (i.e. a2b2 and a2b3) but those will both be detected and quantitated individually using the same PCR probes. Hope this answers this question.

3.You are correct. T315I is a specific point mutation within the bcr-abl construct.  That is a Threonine amino acid has been substituted with an Isoleucine amino acid at position 315 of the protein chain.   The transcript type remains p210. In general yes, you don't need to worry about point mutations if your qPCR results remain stable and low.

Excellent summary.

Well, thank you both! Now it makes sense!

😊

Regards,
Koralia