Topic Article:
Loss-of-Function Mutations in the Filaggrin Gene and Alopecia Areata: Strong Risk Factor for a Severe Course of Disease in Patients Comorbid for Atopic Disease
Regina C Betz, Jana Pforr, Antonia Flaquer, Silke Redler, Sandra Hanneken, Sibylle Eigelshoven, Anne-Katrin Kortüm, Thomas Tüting, Julien Lambert, Jozef De Weert, Axel M Hillmer, Christine Schmael, Thomas F Wienker, Roland Kruse, Gerhard Lutz, Bettina Blaumeiser and Markus M Nöthen
Journal of Investigative Dermatology (2007)
127, 2539–2543; doi:10.1038/sj.jid.5700915

Filaggrin Gene Mutations Mediate Severity of Alopecia Areata When Associated with Atopic Dermatitis

Marianna L. Blyumin¹, Shasa Hu¹ and Robert S. Kirsner¹

_ Journal of Investigative Dermatology_ (2007) 127, 2494; doi:10.1038/sj.jid.5701137

Recently, several common loss-of-function mutations in the gene (FLG) coding for the epidermal protein filaggrin were found in several families with ichthyosis vulgaris (Smith et al., 2006). Furthermore, the association between ichthyosis vulgaris and atopic dermatitis (AD) has led to additional studies demonstrating that these same mutations are important risk factors for AD (Stemmler et al., 2007; Palmer et al., 2006). FLG mutations are related to other manifestations of atopy, such as asthma, but only in the presence of AD. Given the association of alopecia areata (AA) with AD, Betz et al. (2007) studied the association between AA and two common loss-of-function FLG gene mutations, R501X and 2282del4, which lead to premature termination of the first filaggrin repeat. In their study of a European cohort of 449 patients with AA, they did not find an association between AA and FLG gene mutations. However, 34% of the cohort had concomitant AD. In this subset of 145 patients, 37 patients had AD and asthma and 27 had AD, asthma, and allergic rhinitis. Within this subset, the investigators found highly increased FLG mutations in patients with AD; mutations were reported to an even greater extent in patients with AD and other manifestations of atopy, such as asthma or allergic rhinitis. Interestingly, in further investigations of the severity of AA in this subset, patients with AD and other signs of atopy who had FLG mutations were more likely to have a more severe form of AA, such as alopecia totalis and alopecia universalis. Through the following questions we will delve into this paper in greater detail.

QUESTIONS

1. What is the function of filaggrin protein in normal skin?

2. Mutations in filaggrin, associated with atopic dermatitis, have what implications in pathogenesis of atopic dermatitis and atopy?

3. What are the major findings of the present study?

4. What factors influence the subset analysis regarding atopic dermatitis and alopecia areata and the role of filaggrin mutations?

5. What are the clinical implications of the study?

ANSWERS

1. The article by Betz et al. evaluated the role of FLG in AA. FLG (OMIM ∗ 135940) is located on chromosome 1q21 and encodes profilaggrin, a 400-kDa polyprotein precursor containing numerous tandem filaggrin repeats. Profilaggrin is a main component of keratohyalin granules in human epidermis. After terminal differentiation of the granular cells in stratum corneum, profilaggrin is proteolytically cleaved into the 37-kDa filaggrin protein (Gan et al., 1990). Aggregation and compaction of keratin cytoskeleton by filaggrin and crosslinking by transglutaminases lead to the formation of the cornified envelope, which is the outermost barrier of the human body to water loss and infectious organisms and antigens (Hudson, 2006). Filaggrins bundle keratins in the corneocytes, allowing for flat and tight packaging, exemplified by the “bricks and mortar” compaction at the skin surface. Overall, filaggrin participates in epidermal differentiation and is the key protein in barrier function of the skin.

There are multiple known mutations of FLG (Barker et al., 2007). Smith et al. (2006) described two previously unreported loss-of-function mutations (R501Xand 2282del4) in FLG in a cluster of families with ichthyosis vulgaris. Furthermore, about 10% of the European population carries one of the FLG mutations (Barker et al., 2007). This article does not elucidate the history behind the two studied FLG mutations, but it does indicate the rationale for studying these mutations in AA. Recently, the pertinent FLG mutations have been identified as risk factors for AD (Palmer et al., 2006). Furthermore, AA has also been linked to atopic diseases, such as AD (Goh et al., 2006). Hence, Betz et al. studied whether mutations in FLG are likewise risk factors for AA.

2. How FLG mutations predispose individuals to AD has yet to be elucidated. One hypothesis is that a compromised cornified envelope from loss-of-function mutations in FLG predisposes affected individuals to exposure to potentially sensitizing environmental antigens (Hudson, 2006). As a major component of cornified envelope, filaggrin enhances cutaneous barrier function, shielding skin against various pathogens and allergens. When filaggrin’s aggregating utility is affected via FLG mutation (more severely affected if by loss-of-function mutations), the skin barrier is disrupted, allowing easier penetration. Consequently, these agents will more frequently interact with antigen presenting cells (APCs) in the skin. Depending on an individual’s genetic predisposition, the APCs can induce Th1 vs. Th2 immune responses. Finally, chronic activation of Th2 immune responses can invoke various atopic expressions, such as the allergic march (Marenholz et al., 2006).

3. The authors first compared the prevalence of R501X and 2282del4 alleles in patients with AA and controls (Betz et al., 2007; Table 1). Two of the 444 AA patients were homozygous for R501X alleles, and 12 were heterozygous for R501X alleles, compared to 14 of the 455 heterozygous carriers of R501X in the control group. With respect to the 2282del4 mutation, 32/435 AA patients and 22/458 controls were heterozygous carriers. Thus, no significant differences were detected. The lack of differences in allele frequency suggests that FLG alleles are not risk factors for AA. Interestingly, the presence of FLG mutations became more significant after the investigators combined the two mutations and performed subset analyses. First, the authors divided the patients with AA into four groups: (1) AD, (2) AD + asthma, (3) AD + asthma + allergic rhinitis, and (4) the remainder (i.e., patients with AA alone without any of the three major atopic diatheses). Supporting previous studies linking FLG with AD, groups 1, 2, and 3 had significantly higher frequencies of FLG mutations (either R501X or 2282del4) compared with the control population (P = 0.006, 0.0001, and 0.001, respectively) (Betz et al., 2007; Table 2). Overall, AA patients with AD had a 15.2% frequency of FLG mutations in this study. Interestingly, the investigators included odds ratio (OR) calculations in this subset analysis. As an example, an odds ratio of 4.79 (2.17–10.55) was found for group 3. This should be interpreted with caution, as this OR is only for FLG mutations in patients with AD + asthma + allergic rhinitis who also happen to have AA. In other words, the atopic diathesis is a confounder in the context of studying FLG and AA, and a direct association between AA and FLG cannot be concluded, especially given the investigators’ original null hypothesis (that there was no allelic frequency difference between AA patients and controls).

The investigators then divided the AA patients into two groups based on severity: mild and severe. Those with alopecia totalis, alopecia totalis/universalis, and/or alopecia universalis were designated severe. These patients (N = 238) had 26 heterozygous mutations in FLG and 4 homozygous or compound heterozygous mutations, which was significantly higher than in the control population. There were no differences between mild AA patients and the control group in allelic frequencies. A similar subset analysis based on family history and onset age of AA did not reveal significant differences in FLG between AA patients and controls.

In the final set of analyses, the investigators further divided AA patients based on atopic association and severity of AA to test the effect of comorbidity and FLG mutations on AA severity (Betz et al., 2007; Table 3). Severe AA was found to be significantly associated with AD + asthma or AD + asthma + allergic rhinitis but not with AD alone. However, the next set of analyses found that the presence of a FLG mutation was associated with severe AA in all comorbidity subgroups (groups 1, 2, and 3). Based on these findings, one may infer that, while AD alone is not associated with severe AA, the presence of an FLG mutation in AD patients is associated with more severe disease.

4. There are several notable limitations to this study. One, acknowledged by the investigators, is the potential for the study sample’s not being representative of all patients with AA, based on the recruitment methods and unclear diagnostic process. Furthermore, segregation of mild and severe AA was somewhat arbitrary. Although rational, this definition of mild disease, for example, would include someone with nearly total hair loss who was refractory to treatment. Many clinicians would include this patient as having severe AA.

An important limitation was that this article did not clearly define the diagnostic criteria used to define patients as having AD, asthma, allergic rhinitis, or atopy, because the investigators relied on patients’ reporting of these diseases. Moreover, the manner in which the self-defined “experienced medical doctors” in the study made the diagnosis of AD is not defined, nor is how the authors generated an appropriate sample subset of AA patients with comorbid atopic diseases. Thus, the 34% of AA patients in the study who reportedly had associated AD may be an overrepresentation.

The most important drawback of the study is the subgroup exploratory analysis. By not accounting for multiple testing and appropriately lowering the P value for multiple subgroup tests, the authors may have created a statistical anomaly. This may also explain the results of the tertiary linkage between FLG mutations, atopy, and AA severity.

5. It is difficult to draw conclusions that directly influence clinical practice. We do learn that patients who have concomitant AA and AD as well as other manifestations of atopy, should they possess a FLG mutation, are more likely to have a severe form of AA such as alopecia totalis or universalis. It is possible that patients with severe AA and AD, although not directly studied, are more likely to have FLG mutations than those with less severe AA. However, it would be advisable to interpret these implications with caution. Although the paper rejects the hypothesis that FLG mutations are risk factors for AA, at best, it does consider a potential and indirect role of filaggrin in AA and its comorbidity with atopic dermatitis.

Studies regarding disease associations and FLG mutations are emerging as an exciting frontier with ties to diverse reactive inflammatory medical and dermatologic diseases, including asthma, rheumatoid arthritis, and psoriasis. Therefore, although it remains early in our understanding of barrier function and systemic inflammatory disease, this study alerts us to stay tuned for further elucidations of these fascinating filaggrin discoveries.

REFERENCES

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Betz RC, Pforr J, Flaquer A, Redler S, Hanneken S, Eigelshoven S et al. (2007) Loss-of-function mutations in the Filaggrin gene and alopecia areata: strong risk factor for a severe course of disease in patients comorbid for atopic disease. J Invest Dermatol 127:2539–43

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^1^Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, Florida, USA