I'm posting in response to the discussion a couple of days ago about mutation status where it was suggested that a borderline mutated/unmutated status suggested the presence of two clones. I don't think that is so, I think that it simply means that the % of nucleotides that differ from the germ line sequence is too close to the cut-off value to make a clear call that would be meaningful in terms of prognosis.
In the example shown in the photo, my own kappa variable light chain is aligned with a kappa variable light chain from the GenBank database. My sequence varies by 12 nucleotides from the database sequence, representing a 3.97% difference. If a "cut off" value of 4% represented something, my result would probably be given a borderline mutated/unmutated status. There are several different ways that the IGVH gene sequence can be determined, and they all are subject to error. My own IGVH sequence varies by a 7.1% deviation from germ line but I couldn't find my sequence this morning so I am using my kappa light chain as an example.
gardening-girl
Awoke to freezing temps this morning..no more tomatoes or peppers to harvest.
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Thanks Gardengirl. I harvested all of the green tomatoes and made a lovely chutney to can. Now to work on preparing the garden for winter. I'm still getting used to the late tomato season out here in the PNW and I didn't plant any winter vegetables.
I appreciate your detailed explanation. You Chris and Neil seem to have a handle on the science and I've decided to keep reading but avoid jumping to any conclusions. I just don't understand it well enough. (Science, not understood looks like magic, right?)
I just read your profile and you have an impressive journey and grasp of what you have experienced. Thank you for responding to this conversation topic and for laying out your understanding in a clear way. I really appreciate it.
Which is/are the germ line sequence? What is a germ line sequence. And what Been trying to interpret IGVH info; Chris tried to help but I do get lost. Is the 12 divided by 302 to equal .0397, the nucleotide difference. If so what's the 302 represent? # of total nucleotides tested for matching?
Oh wait... I see 302 proteins. And the Subject is you? And the Query is the database sequence? Oh I see.. it's not 6 separate queries labeled 2, 62, 122, 182, 242, & 302 but rather one one long peptide chain with 302 separate proteins of yours being compared to the way a normal kappa variable light chain looks like... and there's a 12 nucleotide difference or 3.97%.
Does anything I'm writing make sense? Sorry for thinking out loud without the proper credentials. Please correct my work.
Gene, it looks like you pretty much figured the sequence alignment out, but I'll go over it explaining it a little better, maybe.
In the alignment above, two DNA sequences are being compared. My sequence is numbered 2 through 302 (query) and the reference sequence from the GenBank database is numbered 89114966 through 89115266. There are 12 nucleotide changes out of 301 nucleotides being compared. Which is actually 3.98 I miscalculated yesterday, there are 301 nucleotides being compared, not 302. For some reason the 1st nucleotide did not show up in the alignment and I didn't notice that until today.
Now to answer your question about the germ line sequences for IGVH:
In B-cells the variable (V) region of the immunoglobulin(IG) Heavy Chain (H) gene ie IGVH undergoes nucleotide base changes as the B-cell matures. The DNA sequence of the IGHV before this mutational process occurs is called the "germ line" sequence of the gene. It represents a very early stage in the natural development a B-cell. If a B-cell becomes leukemic very early in this developmental process, all of the CLL cells arising from it will have an IGVH sequence similar to the germ line sequence. If however a B-cell becomes a leukemic cell later in the developmental process it will have an IGVH with significant changes in the DNA sequence and will be designated as being mutated.
Ideally when the mutation status of a CLL cell is evaluated in a particular patient it would be compared to the patient's own germ line sequence which could be obtained from any cells in the body other than B-cells (well not RBCs since they don't have any DNA). However this is hardly ever done. Instead the patient's DNA sequence is compared to a collection of germ line sequences in large DNA databases. The article tagged by Chris a couple of days ago discusses how the database used in the analysis can affect the results of the comparison.
First off, I must say that you are very smart in making the incomprehensible somewhat more comprehensible, but the more I get into stuff like this - magnifying some micro-processes of life - I get dizzy from what seems truly incomprehensible - the stupefying awe-inspiring, overpowering genius of the creator-in-chief (as when I look up on a starry starry night). To my puny brain even the "screw-ups" like leukemia are amazingly breathtaking.
So here's my rudimentary take: as a B-cell matures its IGVH gene region changes or mutates - mutation in this case not having its pejorative meaning but rather meaning a natural process needed for maturation (similar to how the term 'germ' is not how we use it in the vernacular). So the more a B-cell mutated the more mature the B-cell has become and this matters for a B-cell that becomes leukemic because the later the stage of maturation and the higher % of IGVH mutation, the less aggressive the descendant cells will be when cells replicate.
This so reminds me of my Biology classes when I tried to answer an essay question explaining such complex processes with such minimal knowledge of the lowest course level (like being in Biology Kindergarten, but yet still in the first few weeks of Kindergarten given building blocks to organize using only the most simplest of blocks while knowing to construct anything of value I'd need to use the more complex shaped ones available only to first graders; in other words I think I need a few introductory genetic courses, before I can actually show any real understanding and proficiency). So what grade would a K-Biology student be given for the explanation above?
What I'm left wondering about with CLL is the difference among different percentages of mutation on the IGHV test. If less than 2% is considered unmutated what does it mean if someone is 1.9% & someone is 2.9%? Is 2.9% squarely in the mutated bucket? Is 1.9% truly unmuated? And is 7% or 8% mutated much better for one's prognosis than being 4% or 5%? Also can someone be 80% mutated or 90% or 99% mutated? I've only ever seen percentages under 10%.
Thanks Teach (feel free to say class is over & leave me wondering, if you'd rather not use up any more of your free time to give me free lessons).
Gene, first of all I love it that you are working to figure this stuff out! Second of all, I agree that it is indeed awe-inspiring that any cells (or living organisms for that matter) actually work at all!
Your statement "because the later the stage of maturation and the higher % of IGVH mutation, the less aggressive the descendant cells will be when cells replicate." is a logical conclusion from what I wrote above. However I over simplified my description. I was equating a more mature B-cell with one having undergone "affinity maturation". However, not all B-cells undergo mutations as they mature, only those that have interacted with a T-cell will undergo significant mutation, and that process is called affinity maturation or antigen driven selection. B-cells that don't interact with T-cells will not have as many mutations. I don't think researchers know why the prognosis differs between mutated and unmutated B-cells, or in other words between cells that have undergone affinity maturation and those that haven't.
Jeff Sharman has an excellent little blog on mutated vs unmutated CLL.
Your question about a higher % sequence difference from the germ line being better? There are some reports that more mutations are better (>5% better than 2%). To quote: "In particular, we found that patients affected by B-CLL with evidence of antigen-driven selection (called 'significantly mutated', sM >5%) had longer survivals even within the good prognosis subgroup with more than 2% mutations of IgVH genes,…" They conclude that the higher mutation rate (>5%) provides stronger evidence that the cells have undergone affinity maturation.
Hope the >5% figure works for me as I'm 5.1% IGVH mutated. After > 5 years I must say, so far so good.
Thanks for taking the extra time outside of your office hours for the remediation. I must confess that unfortunately when it comes the full maturation of my other B-cells - brain cells - like the CLL cells their development seems to have been stunted at a certain point especially when it comes to math and science without the high level functioning needed for high level thought processes. Tho' probably closer to the truth is that I have not provided my brain cells with the necessary fundamental building blocks of information they need for such complex topics (my brain cells are a bit ego-sensitive, so for their sake let me put most the blame on my lack of motivation and discipline where it belongs).
I've read through the nature.com article previously but will read the Sharman blog & see what I can glean. Thanks. May I ask what your background is that allows you to glean at such an incredibly high level? Must say I find that ability almost as awe-provoking as I find the complexity of life occurring at the electron-microscopic level (there's another reason I couldn't have become a scientist - my head would be constantly exploding from thoughts of all the metaphysical possibilities).
Thanks again. I appreciate your time, help.... and most of all the grade inflation.
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