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