Friday, August 22, 2014

Eosinophilic Esophagitis - Scientific Excitement - Part 2 of 2

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
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.

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).

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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