Filaggrin Mutations Associated with Skin and Allergic Diseases

Alan D. Irvine, M.D., W.H. Irwin McLean, Ph.D., D.Sc., and Donald Y.M. Leung, M.D., Ph.D.

N Engl J Med 2011; 365:1315-1327October 6, 2011

Article

References

Mutations in the filaggrin gene (FLG) are among the most common and profound single-gene defects identified to date in the causation and modification of disease. FLG encodes an important epidermal protein abundantly expressed in the outer layers of the epidermis.1 Approximately 10% of persons of European ancestry are heterozygous carriers of a loss-of-function mutation in FLG, resulting in a 50% reduction in expressed protein.2 The critical role of filaggrin in epidermal function underlies the pathogenic importance of this gene in common dermatologic and allergic diseases. The spectrum of such diseases encompasses monogenic disorders of keratinization through complex abnormalities of epidermal transport of lipids and allergens. FLG mutation carriers have a greatly increased risk of common complex traits, including atopic dermatitis (which affects 42% of all mutation carriers), contact allergy, asthma, hay fever, and peanut allergy. These genetic variants also influence the severity of asthma and alopecia areata and susceptibility to herpetic infection.

Filaggrin Identification and Genomics

In 1977, Beverly Dale identified a highly insoluble, histidine-rich protein that co-purified with keratin intermediate filament proteins in epidermal extracts.3 The purified protein condensed and aligned keratin intermediate filaments in vitro and, accordingly, was named filaggrin (for filament aggregating protein).4 Antibodies against this protein were used to identify both a 37-kD filaggrin protein and a high-molecular-weight proprotein, profilaggrin,5 now known to have a mass of more than 400 kD.6 Subsequent attempts to clone filaggrin messenger RNA failed to isolate the entire coding sequence, but fragments of the sequence suggested that the majority of the approximately 15-kb message encoded multiple copies of the filaggrin protein flanked by short, unique sequences.7 The latter include an S100-type calcium-binding domain, A and B domains of uncertain function at the N-Figure 1

Genomic and Protein Organization of Filaggrin.

terminal, and a unique tail sequence at the C terminal (Figure 1Figure 1

Genomic and Protein Organization of Filaggrin.).

FLG comprises three exons, the first of which is noncoding.1 Exon 2 encodes part of the S100 domain, and exon 3, one of the largest exons in the genome at more than 12.7 kb, encodes almost the entire profilaggrin protein (Figure 1).1 The human genome reference sequence comprises 10 near-identical filaggrin repeats within exon 3, but Southern blot analysis of population samples,8 confirmed by cloning and sequencing,9 has shown that there are common size-variant alleles in the general population, with 10, 11, or 12 repeats. Until recently, the highly repetitive nature of exon 3 of FLG hampered routine diagnostic analysis of this gene in human disease.6,9

FLG is located in the epidermal differentiation complex, a cluster of approximately 60 genes involved in epithelial differentiation, on chromosome 1q21.10 FLG is one of a subcluster of genes encoding seven fused S100 proteins. These proteins — filaggrin, filaggrin-2, hornerin, trichohyalin, trichohyalin-like 1, cornulin, and repetin — share a common protein-domain organization, having an S100 calcium-binding motif at the N-terminal with an extended, highly repetitive tail.1 The epidermal differentiation complex contains several other families of genes, including those encoding classical S100 proteins, loricrin, involucrin, small proline-rich proteins, and late cornified envelope (LCE) proteins. Copy-number variation within a cluster of LCE genes has recently been implicated as a potential causative variant underlying susceptibility to psoriasis.11 An unusual gain-of-function mutation in loricrin is the cause of a monogenic genodermatosis called loricrin keratoderma.12

Cellular Features of Filaggrin

The epidermis is a complex, highly dynamic, self-renewing barrier tissue covering a very large fraction of the body. The epidermal differentiation program is illustrated in Figure 1 in the Supplementary Appendix, available with the full text of this article at NEJM.org. Cell proliferation in the epidermis is limited to epidermal stem cells in the innermost cell layer, the basal layer (Fig. 1 in the Supplementary Appendix). After cell division, daughter cells exit the cell cycle and migrate upward to form the spinous cell layers, where cell junctions are strengthened and additional keratin proteins are expressed. Closer to the skin surface, the cells of the granular layer contain dense cytoplasmic granules primarily composed of profilaggrin, with other protein components required for the formation of squames, the flattened, dead cells of the outermost stratum corneum that are responsible for the barrier function of the skin (Fig. 1 in the Supplementary Appendix). Two important barrier functions of this stratified, cornified squamous epithelium are to prevent water loss through the huge surface area of the human body and to block the entry of foreign substances (pathogens, antigens, allergens, and chemical irritants) from the external environment.1,13

Profilaggrin is expressed late in the epidermal differentiation program, in the granular cell layers of stratified, cornified epithelia14 (Fig. 1 in the Supplementary Appendix). The cytoplasm of the epidermal granular cells is packed with dense-staining keratohyalin granules, the main constituent of which is profilaggrin — a fact confirmed by the complete absence of granules in persons carrying homozygous null mutations in FLG (Fig. 1 in the Supplementary Appendix).6 These granules act as a reservoir of inactive profilaggrin, along with other proteins that are important for squame formation and maturation, such as loricrin. The transitional zone is essentially the uppermost layer of viable granular cells that are in the final process of differentiation into the flattened, tightly packed, and chemically cross-linked anuclear keratinocytes (squames), which make up the stratum corneum. A “bricks and mortar” model of the stratum corneum has been proposed,15 in which the flattened residual cells (squames) act as the bricks and the cornified cell envelope acts as the mortar. Together, they form a matrix of highly ordered and specialized lipids.16 The end products of filaggrin processing are found within the residual cytoplasm of these squames.

In the transitional cells, profilaggrin processing is initiated. The precise triggering event is unclear, but it is likely to involve dephosphorylation of the proprotein in concert with derepression of a protease cascade that leads to rapid liberation of the functionally active filaggrin monomers.1 A number of proteases have been shown to be involved in this process, with definitive data coming from studies of knockout and transgenic mice.17-20 Very recently, skin-specific retroviral-like aspartic protease has been shown to be specifically expressed in the transitional layer and to be the key enzyme responsible for cleavage of the individual filaggrin repeat units.21 Absence of the serine protease inhibitor LEKTI, encoded by SPINK5, leads to premature processing of profilaggrin and a complex phenotype that includes ichthyosis, hair-shaft abnormalities, and atopy (the Netherton syndrome).22

In the squames, filaggrin undergoes further modification. Fragments of filaggrin have been identified in covalent cross-links mediated by transglutaminase23; therefore, the protein contributes to the formation of the cornified cell envelope — the highly impermeable, proteolipid mortar structure of the stratum corneum. Arginine residues in filaggrin are converted to citrulline24; this is thought to facilitate further proteolysis into short peptides and, ultimately, a pool of hygroscopic amino acids and derivatives thereof, known as natural moisturizing factor (NMF).25 This process involves caspase 14 and other proteases.26,27

Important derivatives of amino acids include trans-urocanic acid and pyrrolidone carboxylic acid. Urocanic acid provides protection against ultraviolet radiation28 and modulates immune function.29 Pyrrolidone carboxylic acid is a chemical derivative of glutamine.30 Profilaggrin is one of the most histidine-rich and glutamine-rich proteins in the human genome, and it acts as a pool of these amino acids, which in turn modulate the pH of the stratum corneum, exert intracytoplasmic humectant activity (i.e., promote the retention of moisture), and possibly exert antimicrobial activity against staphylococcus.31 Measurement of epidermal NMF by means of in vivo confocal Raman spectroscopy has been shown to strongly correlate with filaggrin null mutation genotype.32 Measurements of urocanic acid and pyrrolidone carboxylic acid in epidermal tape strips strongly correlate with filaggrin genotype.33

The multifunctionality of filaggrin is clearly illustrated by comparing flaky tail mice (which have a loss-of-expression mutation in FLG) with caspase 14–knockout mice. In flaky tail mice, loss of profilaggrin and all downstream events leads to aberrant biogenesis of the stratum corneum (ichthyosis) as well as abnormally dry skin (xerosis).34 In contrast, caspase 14–null animals, in which profilaggrin-to-filaggrin processing is normal but filaggrin-to-NMF processing is absent, have xerosis and sensitivity to ultraviolet light without ichthyosis.26

Filaggrin deficiency has been experimentally shown to lead to a failure in the barrier function of the skin. The epidermis of filaggrin-deficient mice allows passive transfer of protein allergens. In cultures of human organotypic keratinocytes, the knockdown of FLG expression by RNA interference facilitates the uptake of fluorescent dyes and heavy-metal tracers.35,36 It is unclear how filaggrin deficiency leads to this defect in barrier function in terms of the bricks-and-mortar model; however, because assembly of lipid and protein components of the stratum corneum must be coordinated, bidirectional signaling should exist between these systems. Knockout of 12-lipoxygenase, an enzyme essential for lipid biogenesis in the stratum corneum,37 or of ATP-binding cassette transporter A12 (ABCA12), a lipid transporter,38 leads to aberrant profilaggrin processing, suggestive of lipid-protein signaling. It has been shown that maintaining a low pH in the stratum corneum is essential for lipid secretion and assembly.39

Diseases Associated with Filaggrin Mutations

Monogenic Disease

Figure 2

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