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The Allergy Gene

By W. H. Irwin McLean The Allergy Gene How a mutation in a skin protein revealed a link between eczema and asthma. Greg Betza It was a tense Friday afternoon in October 2005. Four of us in the lab had been working furiously that week in the fear that our results would be scooped at any moment. (It was an unfounded worry, but we had no way of knowing that at the time.) We had recently found the first mutation in a gene associated with a relatively common skin dise

By | December 1, 2010

The Allergy Gene

How a mutation in a skin protein revealed a link between eczema and asthma.

Greg Betza

It was a tense Friday afternoon in October 2005. Four of us in the lab had been working furiously that week in the fear that our results would be scooped at any moment. (It was an unfounded worry, but we had no way of knowing that at the time.) We had recently found the first mutation in a gene associated with a relatively common skin disease, but our results didn’t fully make sense, and we suspected there must be a second mutation hidden in this gene. A second mutation might explain the strange genetic patterns we had seen in affected families, and it also seemed to hint at a much bigger story.

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1

That may have been enough to start celebrating, but there was more riding on the finding than just this one rare disease. When Alan went back over his patient records, he noticed that ichthyosis vulgaris sufferers also exhibited another, very common skin disease—eczema—much more frequently than the general population. I pulled together everyone in the lab on Monday, holding a sort of emergency meeting of the war cabinet, to discuss the possibility that we had not just one but two really huge stories. “Whatever you’re doing,” I told them, “put it to bed—everyone works on this for the next couple of months.” It was a once-in-a-lifetime opportunity.

There was a chance that this enormous, bizarre skin protein not only was responsible for flaky skin, but could offer a new genetic and cellular explanation for eczema,2 as well as for other common allergies.

The hunt

I had thought about filaggrin for years. As a postdoc in Birgit Lane’s lab in Dundee back in 1995, I had worked on a similarly large and clunky skin protein called plectin. The protein contained six repeated regions, each of which had additional small internal repeats. This fractal-like gene, with a large 7,000-base-pair exon, was unlike anything I’d ever tried to clone. It was the great white whale of my postdoctoral work. We’d come into the lab every few days with a new idea for how to get at it, building a sizable toolbox of techniques and tricks that we used to finally elucidate the plectin gene’s sequence.

My interest in plectin started during my early postdoctoral work, which involved genetic disorders of keratin genes. Keratins form the filamentous cytoskeleton within epithelial cells of the epidermis. The stacked sheets of cells forming the skin’s outermost layer function as a protective barrier between the organism and its environment. Epithelial cells gain their mechanical strength from this tough keratin cytoskeleton in their cytoplasm, and defects in keratin genes make the skin more fragile. In the case of the epidermal keratins, this manifests as skin blistering in response to trauma as mild as rubbing, often accompanied by overgrowth and thickening of the skin, as the epidermis tries in vain to compensate for the fragility of the cells. Some 23 of the 54 keratin genes are known to be linked to various human genetic diseases.

FLLAGRIN’S FUNCTIONS
Normal skin (above left): In the granular layer of normal skin, the large profilaggrin protein is dephosphorylated and enzymatically cut into 10-12 smaller filaggrin molecules (1). As these cells move up to form the stratum corneum, they flatten their shape by collapsing their keratin structure—a process aided by interaction with filaggrin proteins (2). In the stratum corneum, filaggrin degrades into amino acids essential for maintaining moisture in the outer layers of skin (3). The intact skin barrier of healthy skin keeps allergens, pathogens (bacteria and viruses) and chemical irritants out of the body (4).
Eczema (above right): Mutations in the filaggrin gene greatly reduce the amount of filaggrin protein in the skin or lead to its complete absence (1), resulting in cracks in the skin barrier (2) that expose the lower layers to allergens that are usually kept out, thus causing eczema (3). Once foreign material, such as an allergen, passes through the defective skin barrier, it is spotted by cells of the immune system, leading to inflammation of the skin and other allergic responses (4). If a child is exposed to allergens through the skin, that exposure is more likely to prime the immune system to react aggressively to that allergen later in life, explaining the coincidence of asthma and eczema seen in patients throughout the world.

After working on keratin genes for a number of years, we went on to characterize and sequence genes encoding proteins such as plectin that either bind to keratins and anchor the cytoskeleton to membranes, or that modify the keratins, giving epithelial cells a plethora of properties, from elasticity to hardness and water resistance. One of these proteins, filaggrin, was similar in complexity to plectin. Given the strong hints that it might be involved in ichthyosis vulgaris, by the mid-1990s I was just itching to try to sequence its gene and look for disease-causing mutations. But the gene was off limits. I considered Beverly Dale and colleagues at Seattle (who had initially identified the protein) friendly rivals, and I knew that her lab was working out FLG’s sequence. We liked those guys a lot. It was their gene, so we decided not to pursue it.

Then, in 2004, at a meeting in Utah, Frances Smith and I met one of Beverly’s colleagues, dermatologist Phil Fleckman. On the bus ride back from a social gathering near Park City, we started chatting about filaggrin. In the intervening years, a number of labs had had a go at sequencing filaggrin, but all had given up. Maybe it was finally our turn to try it. Phil sent us their DNA samples, and a few months later we had the first mutation, with the second mutation close on its tail.

When looking at the patient records for different varieties of ichthyosis vulgaris, Alan noticed that the patients had frequently also been diagnosed with atopic eczema, the most common skin disease. Atopic (or allergic) eczema affects about one in five children in industrialized nations. It is an inflammatory skin condition, usually starting in infancy or early childhood, with symptoms of red, inflamed, and intensely itchy skin. Eczema is very often accompanied by a plethora of allergies, including food and pet allergies, asthma, and hay fever.

A second mutation might explain the strange genetic patterns we had seen in affected families, and it also seemed to hint at a much bigger story.

Immediately, we went to the literature and found a 2001 study by Bill Cookson and his colleagues, now at Imperial College London. They had shown that there was an eczema susceptibility locus on chromosome 1. We scanned that locus in the human genome database—it was a very large area—and the filaggrin gene was bang-smack in the middle of it!

We went back to our DNA sample collection from about 50 Irish ichthyosis vulgaris patients and 200 members of the general population. It was a really tiny sample size for a case-control study. Most such genetic epidemiological studies for complex traits would have cases and controls that numbered in the thousands, so we weren’t too hopeful about seeing a statistically significant link to eczema. We counted the number of people with an filaggrin mutation in the eczema patient group and in the general population, and compared the results using a statistical test. If the mutations were significantly more common in patients with eczema compared to the control group, then we had found an eczema gene. A significant difference between the cases and controls is measured by a p value of 0.05 or lower. In complex-trait genetics at that time, you considered yourself lucky if you had something like 0.001. So we were totally blown away when the statistical result from our relatively small study came back as 10-17! This meant we had unbelievably strong evidence that the filaggrin gene was involved in eczema. From our more recent studies, we have shown that up to 50 percent of children with moderate to severe eczema carry one or two mutations in the filaggrin gene.3

A neighboring lab in Dundee, headed by Colin Palmer, had an ongoing study of the genetics of asthma. We analyzed their samples of 600 school kids from Dundee, Scotland, all of whom had asthma, as well as 1000 random population controls, for the two filaggrin mutations. When we sorted the kids that had both eczema and asthma from those with just asthma, the population divided into two groups of almost exactly 300 children each. Kids with asthma alone showed no association with the filaggrin mutations, but children with both diseases again showed a powerful statistical association.

That one simple experiment showed that at the molecular level there are two types of asthma: an asthma driven by a skin-barrier deficiency or eczema mechanism, and an asthma driven by a different mechanism. We published our initial asthma findings along with the eczema work. It was an unreal month-long rollercoaster ride. Six months later we had figured out how to fully sequence the filaggrin gene, and discovered that there are a number of common mutations in the gene that arose thousands of years ago in early human populations, in addition to many rare mutations that have arisen more recently. Not only was there an association between the a mutation in the gene and disease in people of European descent, we also found that there are specific combinations of rare and common mutations in other, nonwhite population groups—in Asia, for example—making filaggrin an important eczema/allergy gene worldwide.4

The genetic basis of allergies

What at first appeared to be a surprising association began to make sense on a cellular and molecular level as we continued to study the protein. Filaggrin is crucial for the formation of the stratum corneum, the layer of dead cells at the surface of the skin, and also for the hydration of this crucial barrier layer. People who have mutations in one or both copies of the filaggrin gene produce dry and flaky skin that is permeable to allergens or chemical irritants. When the barrier is broken, foreign material is able to pass through these skin layers. We think that childhood eczema—which usually first occurs within the first few months of life—is an indication that foreign pathogens and irritants have passed through an abnormally porous skin layer, activating a strong allergic immune response, and thus priming the body to react to antigens that it would not normally encounter by this route. Later in life, when the child’s immune system comes into contact with those same allergens, perhaps through the lungs, it reacts aggressively, causing the inflammation in the lungs that results in shortness of breath. In fact, many children with eczema have multiple allergies to house dust, pet hair, and other substances.

For the preceding 20 years or so, the study of eczema and asthma had been dominated by immunological theories. The predominant hypothesis dictated that these diseases were caused by some malfunction of the immune system. Indeed, patients who suffer from both eczema and asthma have an overactive Th2 immune system, the arm of the adaptive immune response that spurs IgE antibody production against allergens. While Th2 cytokines are indeed present, we have shown that it’s not a primary malfunction in the immune system, but a genetic malfunction in the skin that initiates eczema and, in some cases, asthma. Skin-barrier function is now center stage in eczema research. It should be noted, however, that filaggrin is not the only predisposing gene for eczema, and it is likely that others will emerge, some of which are likely to involve immune system malfunction.

Excited by these findings, we quizzed our colleagues for information about other diseases that might possibly be affected by filaggrin-gene mutations. The next natural target was psoriasis, another disease characterized by itchy, flaky, red patches of skin. We found no association with any filaggrin mutation, although it appears that a nearby gene is involved in psoriasis susceptibility.

Curing a genetic allergy?

The question now is how to prevent eczema, asthma, and allergies from occurring in patients with the susceptibility mutations in their filaggrin gene. If it were possible to find a cure for eczema in adults, could we also cure asthma, or would it be too late, with the immune system permanently primed against allergens? I would argue that it might at least help. If the skin barrier is not repaired, then the immune system will constantly produce more antibodies in response to allergens that continue to get past the barrier. Stopping that assault may have some beneficial effects. The alternate possibility is that we may need to prevent eczema in young children before they become sensitized. If the latter proves true, would it be sufficient to curb eczema early in life, as the immune system develops? These are all questions we are gearing up to answer.

We’ve recently published work on a mouse model with a mutation in the murine filaggrin gene that is highly analogous to the human mutations. These mice have many of the hallmarks of eczema, including elevated IgE and Th2 cytokine responses, thus providing compelling experimental evidence for the skin-barrier hypothesis. The plan is to create a mouse model in which we can switch the filaggrin gene on or off at will as the mouse develops, to define the most efficacious period for intervention to prevent eczema.

We’ve also started to look for ways to help the body replace filaggrin at the cellular level. We are currently searching small-molecule chemical libraries for new classes of compounds that might induce skin cells to produce more filaggrin protein. Results look promising, and we are hopeful that in a few years’ time, new drugs or creams that enhance skin-barrier function will be available to treat these common diseases.

Faculty Member References:

1. F.J. Smith et al., “Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris,” Nat Genet, 38:337-42, 2006.
2. C.N. Palmer et al., “Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis,” Nat Genet, 38:441-46, 2006. F1000 ID 1011547, Free F1000 Evaluation
3. A. Sandilands et al., “Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema,” Nat Genet, 39:650-54, 2007. F1000 ID 1085751, Free F1000 Evaluation
4. P.G. Fallon, “A homozygous frameshift mutation in the mouse FLG gene facilitates enhanced percutaneous allergen priming,” Nat Genet, 41:602-08, 2009. F1000 ID 1927974, Free F1000 Evaluation
5. A. Sandilands et al., “Filaggrin in the frontline: role in skin barrier function and disease,” J Cell Sci, 122:1285-94, 2009.

Comments

Avatar of: stephen hauer

stephen hauer

Posts: 1

December 1, 2010

\nWhile my training is in Business ... The following is submtted for your Consideration.\n\nFor "Future" Mothers who may be genetically predisposed to allergy / Asthma or their Spouse ...\n\nBegin MOTHER Inhalant ImmunoTherapy before Conception.\n\nContinue thru 9 month gestation.\n\nContinue after Birth thru Nursing.\n\nMonitor Infant for Allergy MARCH / Eczema.\n\nIf Eczema...\n\nIgE test for FOODs & Inhalants.\n\nFor FOODs modify DIET ( maybe add Probiotics )\n\nFor Inhlants > expanded IgE testing followed by ImmunoTherapy DROPs.\n\nTest / Identify / Know / Neutralize Allergy MARCH before it proceeds to Allergic Asthma.\nWhile there is no cure for allergic ASTHMA > It can be Prevented.\n\nLet us work toward a Generation Free of Allergy / Allergic ASTHMA.\n\nStephen\n
Avatar of: anonymous poster

anonymous poster

Posts: 1

December 2, 2010

Congratulations on an impressive achievement. As a lifetime eczema/allergy/asthma suffer and a researcher (not in this field) it is really exciting to read of the impressive work that is being done in the field and to begin to understand the molecular basis of these problems.\n\nSadly I am not completely up to speed with the research and just wondered whether there is an explanation/hypothesis for the apparent dramatic rise in asthma and allergy that appears to be occurring in young children. Or is this an artefact of better census/statistics gathering? \n\nI wish the investigators continued success
Avatar of: anonymous poster

anonymous poster

Posts: 1

December 4, 2010

I read this article with great interest. I experienced cold urticaria fleetingly as a youth: hives that erupted after a plunge into cold water or sudden exposure to cold air. It is apparently not a rare condition and has multiple causes. My intuitive sense, which of course could be very wrong!, was of a cold induced change of conformation of a protein that caused it to be recognized as foreign and thus elicted an immune reponse. It would be interesting if my hypothetical protein could be one of these skin scaffold proteins. (I have not had eczema or psoriasis but my skin has always been thin, dry and flaky and sensitive to environmental insults. I no longer get hives but ever since have eased into swimming pools!)
Avatar of: anonymous poster

anonymous poster

Posts: 1

December 5, 2010

I am a General non acadamic Pathologist . I do have opportunity to use cytokeratin immunostaining in my practice. I also have interest in genetics . So I am able to understand this type of high class articles. I enjoyed very much reading it.\nIt is nicely explained . Congratulations for your finding out the mutation in that large protein. \n
Avatar of: Krishna Prasad

Krishna Prasad

Posts: 1

December 6, 2010

What is your opinion about Matriptase-Deficient Mice Exhibiting Ichthyotic Skin.\n\nMice lacking the serine protease matriptase (St14?/?), also known as MT-SP1, TADG15, and\nepithin, die perinatally due to severe skin barrier impairment (List et al., 2003). Mutations in\nthe human ST14 locus underlie autosomal recessive ichthyosis with hypotrichosis (ARIH:\nOMIM 610765) (Basel-Vanagaite et al., 2007; Alef et al., 2008; Desilets et al., 2008). Mice\npossessing one null and one hypomorphic allele of St14 (St14hypo/?), express ~1% of St14 at\nbirth and survive to adulthood (List et al., 2007). These St14hypo/? mice recapitulate many of\nthe hallmark features of ARIH, including impaired desquamation, hypotrichosis, and tooth\ndefects.The skin barrier is formed in the exterior layers of the epidermis and is comprised of cornified\nenvelopes (enucleated keratinocytes), held together by a lipid matrix (Segre, 2006a). The skin\nbarrier defect in both mice and humans with mutations in ST14 have been associated with\nimpaired filaggrin (FLG) processing (List et al., 2003; Desilets et al., 2008). FLG is expressed\nin the granular layer of terminally differentiating epidermal cells and encodes a 400 kDa\nprecursor protein, profilaggrin that undergoes dephosphorylation and proteolysis to yield 37\nkDa repeat filaggrin peptides. FLG peptides are thought to contribute to skin barrier function\nthrough aggregation of keratin intermediate filaments to form the chemically cross-linked\ncornified envelope as well as through hydration and acidification of the stratum corneum (Elias\net al., 2008; Steinert et al., 1981). Here, we examine the integration of (1) alterations in skin\nbarrier proteins; (2) innate immune response; and (3) selective shift in the microbiota of\nSt14hypo/? mice as an animal model of human ichthyotic phenotypes.\nThanks and regards,\nKrishna Prasad\nAurigene Discovery technologies Ltd.\nBangalore, INDIA
Avatar of: Ken Hoober

Ken Hoober

Posts: 1

December 8, 2010

It was gratifying to see the maturity of the work on filaggrin. The first evidence for the protein was obtained in Dr. Isadore Bernstein's lab at the University of Michigan during the 1960's. Dr. Bernstein was interested in finding a marker for differentiation and realized that the epidermis is an excellent, stratified differentiating organ. Dr. Fukuyama in Bernstein's lab observed by radioautography an unusual pattern of incorporation of amino acids into the granular layer. With these data as a basis, I purified a protein fraction with an unusual amino acid composition that explained the histological data (J.K. Hoober and I.A. Bernstein. 1966. PNAS 56:594-601). Further work by Dr. Bernstein's lab characterized the cDNA and localized the gene to chromosome region Iq21. I congratulate all those who have made remarkable progress on this protein.
Avatar of: VINCENT CONNORS

VINCENT CONNORS

Posts: 3

December 8, 2010

Wish I'd of had the time to read this last week prior to starting my lectures on hypersensitivities - what a nice concise tie in it would have been for those struggling seniors! Great work!
Avatar of: Jordan Hubert

Jordan Hubert

Posts: 1

December 10, 2010

Very interesting article. It is exciting to finally see the light at the end of the tunnel in the work on filaggrin. I was struck, though, by the amount of time that was required: ?To sequence it once was an effort that took years; to do it repeatedly, as we would have to for our mutational screens, was much more challenging." This emphasizes the current gap between sequencing vs. analysis. In the past year, there has been a very significant acceleration in analysis: what used to take genomics companies like Knome several months to complete is now being done in a matter of days. Thanks to a tremendous amount of effort and software automation, genome analysis is moving more rapidly than ever. The challenge in 2011 is to accelerate the actual sequencing period as well.

December 10, 2010

This is great news! something that has taken years to achieve. Perhaps changes in the diet has played a role in the increase incidence of this condition. Since you mentioned that you have discovered a precursor to the 'FLG' something along the lines of "competitive inhibition" may be changing the signaling. Something interesting to also consider is the geographic location, notice that when you tested the sample from the US the genetic variation was there and stronger than sample from Ireland. At least you will now know what can be done to prevent this and bring about comfort to those who suffer from this type of disease or should I say 'condition'.\n
Avatar of: kate tuikhang

kate tuikhang

Posts: 1

December 12, 2010

Its always intersting to hav a good things. And it is better to know something than not knowing anything. We must leave the old one and adopt new technology according to the need of the society in this present scenario , where human are facing a huge threat from micro-organism... in the like of this I am really thankful and may you continue serve to human in days to come too...\n\nBe prosperous... rewards awaits you........
Avatar of: Sten Dreborg

Sten Dreborg

Posts: 1

December 19, 2010

The influence on the skin structure by mutant filagrin influences the penetration of allergens through the skin layers. This is true for contact dermatitis and for s.c. allergic contact eczema, i.e. when allergens from e.g. meat or vegetables penetrate te skin and causes hand eczema.\nHowever, according to accepted nomenclature eczema means what has been called atopic eczema, e.g. cow's milk that is ingested. \nIn my opionon filagrin can influence the allergic contact eczema and concat eczema caused by e.g. Ni but not the atopic eczema.
Avatar of: alberto rivas

alberto rivas

Posts: 1

December 19, 2010

Congratulations for your excelent article.After 12 years of researching allergies,both my wife ( at the moment following helminthic therapy ) and me were aware of the conection between skin problems and allergies, but it is good to have the idea scientifically proved. Considering treatments, I wonder if you have found the nutrient asociated with this particular SNIP,so ,by providing big doses of it ,perhaps the FLG gene mutation could be treated. Thank you. Alberto

December 28, 2010

I am so greatful for the research being done on the filaggrin gene. As I sat here itching while I read, I finally know why my skin has acted the way it has all my life. I just could not understand why I seemed to "shed" on a regular basis. I felt that the topical creams I have used for so long were only bandaids and that the true root of the problem just had not been found yet. Now my 7 year-old grandson and possibly my 1 month-old granddaughter are suffering with the same condition. I have hope for them, that they will have relief in their future. Thank you.
Avatar of: anonymous poster

anonymous poster

Posts: 1

January 3, 2011

Two of my cousins (sisters) had severe debilitating eczema in childhood and early adulthood. One of them developed sceroderma in mid-life and died of it after a few years. I have always wondered if there was a connection between the 2 conditions. What do you think?

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