PART 2 - BEYOND THE GWAS – FUNCTIONAL SIGNIFICANCE
FOR EoE?
This is the exciting conclusion to parsing the findings in Kottyan,
et al., 2014 (1). Just in case you missed it or need a
refresher, here is a link to PART
1 – BACKGROUND TO UNDERSTAND EoE AND THE RESEARCH FINDINGS.
Brief summary of Part
1
After the researchers compared over 1.5 million regions of
the genome between EoE and control subjects, they identified 4 different
regions that were strongly associated with EoE.
Going back to the analogy used in Part 1 – the researchers identified
the flutes among the cacophony of the warming up orchestra. Now, they needed to analyze the melodies
those flutes were playing – stated biologically, they needed to figure out if
any of those flutes (i.e. – regions of DNA identified in the GWAS) played
faulty melodies (i.e. – errors in genes getting expressed that may lead to
EoE). Just because there is a difference in DNA between EoE patients and those
without EoE (controls) doesn’t necessarily mean that it is important
biologically – a flute could play the wrong note, but it may harmonize with the
intended note (e.g. make no difference to EoE). They were looking for a clear,
dissonant “wrong note.” Lead author and researcher on the paper, Dr. Leah
Kottyan relayed to me that in this line of work, identifying the differences in
DNA is the “easy” part. Now came the “hard” part.
An unlikely candidate
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| Image: CC license - Flickr photoisraelavila |
Out of the 4 candidate regions of DNA that passed the
“noise” test between EoE and controls, they honed in on a region of DNA sitting
next to a gene called CAPN14 (pronounced by saying all the letters C-A-P-N 14).
Abstract name, right (welcome to biology)?! In my attempt to make this more
relatable to you (and me) - I can’t help but call it Cap’n 14. Oh, childhood
parked in front of the TV on Saturday morning devouring a bowl of Cap’n Crunch
(with crunch berries, of course!). And if
you stay with me, my play on words may help solidify that ultimately genes serve
a function in the body. Could CAPN14 play a role in developing EoE?
Function of CAPN14?
While the authors identify a region of DNA near the CAPN14
gene, ultimately what matters is that genes (the instructions) get expressed as
a protein, and it is the protein that carries out a function in the body.
Proteins have diverse functions – anywhere from forming the structure of your
skin, to chemical signals allowing cells to communicate with each other, to the
enzymes that catalyze the chemical reactions breaking down food for nourishment.
Interestingly, very little is known of CAPN14, the protein!
The best a scientist can hope for in this type of study is
that candidate genes are researched by others in a different context or part of
the body so there are functional clues and you don’t have to start from scratch.
While little is known of CAPN14, we know it belongs to a family of proteins called
calpains. The famous cousins in this family are enzymes that break down other
proteins, but only when the appropriate signal is present. In other words,
cap’n will “crunch” proteins, but only in the presence of a crunch berry (in
cells, CAPN, the protein enzyme, works only when a specific signal – calcium -
is present). And the protein’s ability to crunch other proteins has been shown
by many other researchers to play many different roles inside of cells (2). What CAPN14 may be
“crunching” in the esophagus is an outstanding question to be addressed by
future work.
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| The pathway turning DNA instructions into proteins. Photo: "Gene2-plain" by Forluvoft - Own work. Licensed under Public domain via Wikimedia Commons |
The evidence for a damning role of CAPN14 in EoE
If some stretch of DNA is actually involved in EoE and not
just an innocent bystander, the researchers expected certain criteria to be
met:
1. Is the expression of the CAPN14 gene altered in the
esophagus of those with EoE vs. controls (i.e. is the pathway converting DNA
instructions into protein altered)?
Yes! Even more interesting is that the
greatest expression of CAPN14 occurs in those with active EoE. In those whose
EoE is inactive, expression of CAPN14 is dramatically decreased (still elevated
compared to those without EoE, though). So, not only is expression altered, but
expression appears to scale depending on how active the disease is.
2.
Can treating esophageal cells with a cell
signaling molecule (IL-13) that is known to induce EoE increase both CAPN14 gene
expression and protein function?
Yes! In fact, when IL-13 is applied
to esophageal cells, CAPN14 is the only member of the calpain family with
significantly altered expression levels. Furthermore, the authors took it a
step further, they looked to see if the calpain enzyme was active in esophageal
cells. The answer of course was - yes! It was significantly more active in
those treated with IL-13. Furthermore, the activity was stopped when a highly
specific chemical inhibitor of calpain was applied.
3.
Is there a possible mechanism for how CAPN14
expression is altered in EoE?
Yes! Remember in Part 1, where I
mentioned epigenetic regulation (changing how a gene gets expressed without
actually changing a gene’s A’s, T’s, C’s, or G’s)? Well, what they found is the
difference lies in a region of DNA controlling how much CAPN14 gets expressed.
IL-13 treatment resulted in DNA chemical tags (I like to think of them as DNA “jewelry”)
known to turn the protein production factory on (acetylation of DNA for those
familiar with epigenetic regulation).
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| Epigenetics markers = "DNA jewelry." Acetyl group turns "on" gene expression without modifying DNA's A's, T's, C's, or G's. Photo: Thermo Scientific |
Even more evidence
this is esophagus-specific
A big question for those studying allergic disorders is why
do only certain individuals who are “allergic” get EoE?... or food allergies?
…or eczema? ….or asthma? Why do those with EoE also tend to have another
allergic disorder? Scientists KNOW that many of the same cells and chemical
signals are shared between the various allergic disorders, which is why you
would expect regions of “dissonant” DNA to be the same for any allergic
disorder. For example, IL-13 is implicated in the other allergic disorders, not
just EoE.
Enter CAPN14. When the researchers looked at publicly available databases of tissue expression, they found that by and large, CAPN14 is highly expressed in the esophagus and head/neck region compared to any other bodily tissue. Could this be a smoking gun for the tissue specificity of EoE in those who skew toward “allergy” more generally? This, too, remains an outstanding question.
Many questions remain
This paper scratches the surface of EoE, but it opened a new
line of research in a completely unexpected place. Without the GWAS, CAPN14
would not likely be considered a strong candidate mediator. While the authors
know that the CAPN14 protein is present in the esophagus, they still do not
exactly understand what function it serves (i.e. a lot of work remains). Until
there is a better understanding of its function, it would not make sense to treat
people who have EoE with calpain inhibitors. In fact, the authors hypothesize
that CAPN14 may serve a protective role to quell inflammation! If this is the
case, you would want to promote calpain, not inhibit it.
What do the findings
mean for those who have EoE?
While my family doesn’t deal with EoE, my son has multiple
life-threatening food allergies. Is my son at risk for developing EoE,
especially if he were to go through an oral immunotherapy treatment for food
allergies? Fact is, I don’t know, nor can the findings from this paper alone
give me the answer. For any exciting scientific finding, I always try to ask
the questions I would want to know as a parent or individual dealing with the
disorder or potentially at risk:
Would a genetic test for the DNA region
around CAPN14 help?
While it’s true that there are private
companies that gladly offer genetic screenings for a non-trivial fee, screening
for the CAPN14 genetic variant alone will offer very little. In fact, only
about 9% of individuals with EoE have the CAPN14 variant in question, and for
those who have one copy of that variant, the risk of getting EoE is doubled.
Having that risk variant by no means guarantees EoE since there are individuals
who don’t have EoE who also have this variant. CAPN14 is one potentially
important factor among many. The important thing to note is that even without
the risk variant, CAPN14 is highly likely involved in a meshwork of complicated
cellular pathways for ALL individuals dealing with EoE.
What could this mean for the future?
CAPN14 was not the only factor that turned
up from their screen. In fact, the authors looked at gene expression for
200-some candidate genes associated with EoE. What they found was a striking
pattern of gene expression between controls and EoE (CAPN14 was one gene among
about 20 where there was over a 2 fold change in expression between those with
EoE and controls). The exciting thing, in my mind, is that combining multiple
markers may provide a way to help diagnose EoE. Currently, diagnosing EoE
relies on scoping combined with a biopsy of the esophagus under anesthesia.
Wouldn’t it be great if there was a pre-screening blood test that could help
diagnose EoE without so many scopings/biopsies?
THE TAKE-HOME MESSAGE
Many factors contribute to the complex interplay of nature
(DNA) and nurture (environmental effects on DNA expression) in both health and
disease, and this is true for EoE and other allergic disorders. Just like an
engine has many working parts that can fail for any number of reasons, so too
does the onset of disease. Any one of many different “parts” could be altered
and lead to the same end result (EoE). CAPN14 is clearly one piece of the
complex machinery that scientists are trying to reverse engineer. Stay tuned…
this is a story to keep following.
References (Freely available sources used where possible):
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