Linda E. Campbell

Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis

Atopic disease, including atopic dermatitis (eczema), allergy and asthma, has increased in frequency in recent decades and now affects ∼20% of the population in the developed world. Twin and family studies have shown that predisposition to atopic disease is highly heritable. Although most genetic studies have focused on immunological mechanisms, a primary epithelial barrier defect has been anticipated. Filaggrin is a key protein that facilitates terminal differentiation of the epidermis and formation of the skin barrier. Here we show that two independent loss-of-function genetic variants (R510X and 2282del4) in the gene encoding filaggrin (FLG) are very strong predisposing factors for atopic dermatitis. These variants are carried by ∼9% of people of European origin. These variants also show highly significant association with asthma occurring in the context of atopic dermatitis. This work establishes a key role for impaired skin barrier function in the development of atopic disease.

Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris.

Ichthyosis vulgaris (OMIM 146700) is the most common inherited disorder of keratinization and one of the most frequent single-gene disorders in humans. The most widely cited incidence figure is 1 in 250 based on a survey of 6,051 healthy English schoolchildren. We have identified homozygous or compound heterozygous mutations R501X and 2282del4 in the gene encoding filaggrin (FLG) as the cause of moderate or severe ichthyosis vulgaris in 15 kindreds. In addition, these mutations are semidominant; heterozygotes show a very mild phenotype with incomplete penetrance. The mutations show a combined allele frequency of ∼4% in populations of European ancestry, explaining the high incidence of ichthyosis vulgaris. Profilaggrin is the major protein of keratohyalin granules in the epidermis. During terminal differentiation, it is cleaved into multiple filaggrin peptides that aggregate keratin filaments. The resultant matrix is cross-linked to form a major component of the cornified cell envelope. We find that loss or reduction of this major structural protein leads to varying degrees of impaired keratinization.

Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema

We recently reported two common filaggrin (FLG) null mutations that cause ichthyosis vulgaris and predispose to eczema and secondary allergic diseases. We show here that these common European mutations are ancestral variants carried on conserved haplotypes. To facilitate comprehensive analysis of other populations, we report a strategy for full sequencing of this large, highly repetitive gene, and we describe 15 variants, including seven that are prevalent. All the variants are either nonsense or frameshift mutations that, in representative cases, resulted in loss of filaggrin production in the epidermis. In an Irish case-control study, the five most common European mutations showed a strong association with moderate-to-severe childhood eczema (χ2 test: P = 2.12 × 10−51; Fishers exact test: heterozygote odds ratio (OR) = 7.44 (95% confidence interval (c.i.) = 4.9–11.3), and homozygote OR = 151 (95% c.i. = 20–1,136)). We found three additional rare null mutations in this case series, suggesting that the genetic architecture of filaggrin-related atopic dermatitis consists of both prevalent and rare risk alleles.

A homozygous frameshift mutation in the mouse Flg gene facilitates enhanced percutaneous allergen priming

Loss-of-function mutations in the FLG (filaggrin) gene cause the semidominant keratinizing disorder ichthyosis vulgaris and convey major genetic risk for atopic dermatitis (eczema), eczema-associated asthma and other allergic phenotypes. Several low-frequency FLG null alleles occur in Europeans and Asians, with a cumulative frequency of ∼9% in Europe. Here we report a 1-bp deletion mutation, 5303delA, analogous to common human FLG mutations, within the murine Flg gene in the spontaneous mouse mutant flaky tail (ft). We demonstrate that topical application of allergen to mice homozygous for this mutation results in cutaneous inflammatory infiltrates and enhanced cutaneous allergen priming with development of allergen-specific antibody responses. These data validate flaky tail as a useful model of filaggrin deficiency and provide experimental evidence for the hypothesis that antigen transfer through a defective epidermal barrier is a key mechanism underlying elevated IgE sensitization and initiation of cutaneous inflammation in humans with filaggrin-related atopic disease.

Loss-of-function variants in the filaggrin gene are a significant risk factor for peanut allergy

Background IgE-mediated peanut allergy is a complex trait with strong heritability, but its genetic basis is currently unknown. Loss-of-function mutations within the filaggrin gene are associated with atopic dermatitis and other atopic diseases; therefore, filaggrin is a candidate gene in the etiology of peanut allergy. Objective To investigate the association between filaggrin loss-of-function mutations and peanut allergy. Methods Case-control study of 71 English, Dutch, and Irish oral food challenge–positive patients with peanut allergy and 1000 non peanut-sensitized English population controls. Replication was tested in 390 white Canadian patients with peanut allergy (defined by food challenge, or clinical history and skin prick test wheal to peanut ≥8 mm and/or peanut-specific IgE ≥15 kUL−1) and 891 white Canadian population controls. The most prevalent filaggrin loss-of-function mutations were assayed in each population: R501X and 2282del4 in the Europeans, and R501X, 2282del4, R2447X, and S3247X in the Canadians. The Fisher exact test and logistic regression were used to test for association; covariate analysis controlled for coexistent atopic dermatitis. Results Filaggrin loss-of-function mutations showed a strong and significant association with peanut allergy in the food challenge–positive patients (P = 3.0 × 10−6; odds ratio, 5.3; 95% CI, 2.8-10.2), and this association was replicated in the Canadian study (P = 5.4 × 10−5; odds ratio, 1.9; 95% CI, 1.4-2.6). The association of filaggrin mutations with peanut allergy remains significant (P = .0008) after controlling for coexistent atopic dermatitis. Conclusion Filaggrin mutations represent a significant risk factor for IgE-mediated peanut allergy, indicating a role for epithelial barrier dysfunction in the pathogenesis of this disease.

Loss-of-Function Mutations in the Filaggrin Gene Lead to Reduced Level of Natural Moisturizing Factor in the Stratum Corneum

TO THE EDITOR Filaggrin is a key protein required for the formation of the stratum corneum (SC) barrier. Filaggrin is also essential for SC hydration, as it acts as a source of hygroscopic amino acids and their derivatives, known as natural moisturizing factor (NMF). The human gene encoding filaggrin (FLG) is highly polymorphic and to date, 15 null mutations have been detected of which four (R501X, 2282del4, R2447X, and S3247X) are prevalent at varying frequencies in the white European population (Sandilands et al., 2007). Homozygous or compound heterozygous FLG mutations underlie the common skin-keratinizing disorder ichthyosis vulgaris, and have been shown to be a major genetic predisposing factor for atopic dermatitis (AD) (Sandilands et al., 2006). Diminished filaggrin expression has been demonstrated in both ichthyosis vulgaris and AD skin (Seguchi et al., 1996; Sugiura et al., 2005; Smith et al., 2006). As filaggrin is the precursor protein for the amino-acid-derived components of the NMF, we hypothesized that carriers of FLG-null mutations have reduced level of NMF in the SC. To measure NMF in the SC of the palm (thenar eminence) and forearm skin, we used confocal Raman microspectroscopy (3510 Skin Composition Analyzer; River Diagnostics, Rotterdam, The Netherlands). The principles of this method and the procedure have extensively been described elsewhere (Caspers et al., 2001, 2003). The reference spectrum of NMF was constructed from a superposition of the spectra of pyrrolidone-5-carboxylic acid, ornithine, serine, proline, glycine, histidine, and alanine. In addition to NMF, skin barrier function as measured by transepidermal water loss was assessed on the volar forearm (Tewameter 210; Courage and Khazaka Electronic GmbH, Cologne, Germany). One hundred and forty-nine volunteers recruited by public advertisement, as well as 10 AD patients, were screened for four FLG mutations (R501X, 2282del4 R2447X, and S3247X). All subjects filled in a questionnaire on the history of skin diseases and allergies, and the Erlangen atopy questionnaire that also included a question on skin dryness. Signs of active disease (erythema, crusting, weeping, and lichenification) were assessed by a dermatologist. Having visible skin changes on the forearm was the exclusion criterion. Written informed consent was obtained from all subjects. The experimental protocol followed the Declaration of Helsinki Principles and was approved by the Ethical Committee of the Academic Medical Centre. Genomic DNA was extracted from buccal swab samples (Puregene DNA isolation kit; Gentra Systems, Minneapolis, MN). Polymorphisms were genotyped as reported previously (Sandilands et al., 2007). To compare data from two groups, we used twotailed Student’s t-test for unpaired samples. Sixteen carriers (12 female) of an FLG mutation and 23 individuals (15 female) wild type with respect to these mutations were included in the study. Of the 16 carriers, five were heterozygous for R501X, eight were heterozygous for 2282del4, and one was heterozygous for R2447X. One individual was homozygous for Abbreviations: AD, atopic dermatitis; FLG, human filaggrin-encoding gene; NMF, natural moisturizing factor; SC, stratum corneum

3D analysis of facial morphology

Dense surface models can be used to analyze 3D facial morphology by establishing a correspondence of thousands of points across each 3D face image. The models provide dramatic visualizations of 3D face‐shape variation with potential for training physicians to recognize the key components of particular syndromes. We demonstrate their use to visualize and recognize shape differences in a collection of 3D face images that includes 280 controls (2 weeks to 56 years of age), 90 individuals with Noonan syndrome (NS) (7 months to 56 years), and 60 individuals with velo‐cardio‐facial syndrome (VCFS; 3 to 17 years of age). Ten‐fold cross‐validation testing of discrimination between the three groups was carried out on unseen test examples using five pattern recognition algorithms (nearest mean, C5.0 decision trees, neural networks, logistic regression, and support vector machines). For discriminating between individuals with NS and controls, the best average sensitivity and specificity levels were 92 and 93% for children, 83 and 94% for adults, and 88 and 94% for the children and adults combined. For individuals with VCFS and controls, the best results were 83 and 92%. In a comparison of individuals with NS and individuals with VCFS, a correct identification rate of 95% was achieved for both syndromes. This article contains supplementary material, which may be viewed at the American Journal of Medical Genetics website at http://www.interscience.wiley.com/jpages/0148‐7299/suppmat/index.html.

Levels of filaggrin degradation products are influenced by both filaggrin genotype and atopic dermatitis severity

To cite this article: Kezic S, O’Regan GM, Yau N, Sandilands A, Chen H, Campbell LE, Kroboth K, Watson R, Rowland M, Irwin McLean WH, Irvine AD. Levels of filaggrin degradation products are influenced by both filaggrin genotype and atopic dermatitis severity. Allergy 2011; 66: 934–940.

Filaggrin loss-of-function mutations are associated with enhanced expression of IL-1 cytokines in the stratum corneum of patients with atopic dermatitis and in a murine model of filaggrin deficiency

Background Filaggrin (FLG) mutations result in reduced stratum corneum (SC) natural moisturizing factor (NMF) components and consequent increased SC pH. Because higher pH activates SC protease activity, we hypothesized an enhanced release of proinflammatory IL-1 cytokines from corneocytes in patients with atopic dermatitis (AD) with FLG mutations (ADFLG) compared with that seen in patients with AD without these mutations (ADNON-FLG). Objectives We sought to investigate SC IL-1 cytokine profiles in the uninvolved skin of controls and patients with ADFLG versus patients with ADNON-FLG. We also sought to examine the same profiles in a murine model of filaggrin deficiency (Flgft/Flgft [FlgdelAPfal] mice). Methods One hundred thirty-seven patients were studied. NMF levels were ascertained using confocal Raman spectroscopy; transepidermal water loss and skin surface pH were measured. IL-1α, IL-1β, IL-18, IL-1 receptor antagonist (IL-1RA), and IL-8 levels were determined in SC tape strips from 93 patients. All subjects were screened for 9 FLG mutations. Flgft/Flgft (FlgdelAPfal) mice, separated from maFlgft/maFlgft (flaky tail) mice, were used for the preparation and culture of primary murine keratinocytes and as a source of murine skin. RT-PCR was performed using primers specific for murine IL-1α, IL-1β, and IL-1RA. Results SC IL-1 levels were increased in patients with ADFLG; these levels were inversely correlated with NMF levels. NMF values were also inversely correlated with skin surface pH. Skin and keratinocytes from Flgft/Flgft mice had upregulated expression of IL-1β and IL-1RA mRNA. Conclusions ADFLG is associated with an increased SC IL-1 cytokine profile; this profile is also seen in a murine homologue of filaggrin deficiency. These findings might have importance in understanding the influence of FLG mutations on the inflammasome in the pathogenesis of AD and help individualize therapeutic approaches.

Filaggrin loss-of-function mutations are associated with early-onset eczema, eczema severity and transepidermal water loss at 3 months of age

Background  Filaggrin loss‐of‐function (FLG) mutations are associated with eczema and skin barrier impairment, but it is unclear whether skin barrier impairment precedes phenotypic eczema in FLG mutation carriers.