Huijia Chen

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.

Raman profiles of the stratum corneum define 3 filaggrin genotype–determined atopic dermatitis endophenotypes

Background Filaggrin (FLG) has a central role in the pathogenesis of atopic dermatitis (AD). FLG is a complex repetitive gene; highly population-specific mutations and multiple rare mutations make routine genotyping complex. Furthermore, the mechanistic pathways through which mutations in FLG predispose to AD are unclear. Objectives We sought to determine whether specific Raman microspectroscopic natural moisturizing factor (NMF) signatures of the stratum corneum could be used as markers of FLG genotype in patients with moderate-to-severe AD. Methods The composition and function of the stratum corneum in 132 well-characterized patients with moderate-to-severe AD were assessed by means of confocal Raman microspectroscopy and measurement of transepidermal water loss (TEWL). These parameters were compared with FLG genotype and clinical assessment. Results Three subpopulations closely corresponding with FLG genotype were identified by using Raman spectroscopy. The Raman signature of NMF discriminated between FLG-associated AD and non–FLG-associated AD (area under the curve, 0.94; 95% CI, 0.91-0.99). In addition, within the subset of FLG-associated AD, NMF distinguished between patients with 1 versus 2 mutations. Five novel FLG mutations were found on rescreening outlying patients with Raman signatures suggestive of undetected mutations (R3418X, G1138X, S1040X, 10085delC, and L2933X). TEWL did not associate with FLG genotype subgroups. Conclusions Raman spectroscopy permits rapid and highly accurate stratification of FLG-associated AD. FLG mutations do not influence TEWL within established moderate-to-severe AD.

Wide spectrum of filaggrin-null mutations in atopic dermatitis highlights differences between Singaporean Chinese and European populations

Background Null mutations in the filaggrin gene (FLG) cause ichthyosis vulgaris (IV) and predispose to atopic dermatitis (AD). Cohort studies in Europe and Japan have reported an FLG mutation carrier frequency of between 14% and 56%, but the prevalent European FLG mutations are rare or absent in Chinese patients with IV and AD.

Missing C‐terminal filaggrin expression, NFkappaB activation and hyperproliferation identify the dog as a putative model to study epidermal dysfunction in atopic dermatitis

Please cite this paper as: Missing C‐terminal filaggrin expression, NFkappaB activation and hyperproliferation identify the dog as a putative model to study epidermal dysfunction in atopic dermatitis. Experimental Dermatology 2010; 19: e343–e346.

Filaggrin mutations are associated with recurrent skin infection in Singaporean Chinese patients with atopic dermatitis

Background  Loss‐of‐function (null) mutations within the filaggrin (FLG) gene are a strong risk factor for atopic dermatitis (AD). We hypothesized that the absence or reduction of the filaggrin protein could compromise skin barrier and increase patients’ susceptibility to recurrent skin infection.

Association of Skin Barrier Genes within the PSORS4 Locus Is Enriched in Singaporean Chinese with Early-Onset Psoriasis

Psoriasis (OMIM#177900) is a common polygenic skin disorder affecting approximately 2% of the northern European population and 0.1% of the Han Chinese. Psoriasis patients suffer from chronic skin inflammation, manifested by erythematous scaly lesions. PSORS1-PSORS9 have been confirmed as psoriasis susceptibility loci in independent genetic studies on predominantly Caucasian populations, with psoriasis susceptibility loci (PSORS1, PSORS9) and additional loci at 9q33-34 and 2p22.3-11.2 reported in Han Chinese patients. In this study, we show the association of PSORS4 with psoriasis in Singaporean Chinese. Dense genotyping of single-nucleotide polymorphism-tagging candidate genes within the epidermal differentiation complex revealed significant association in the proximity of the involucrin gene (IVL); the strongest association was seen in early-onset psoriasis patients (P=0.0014). A follow-up genome-wide association screen localized the psoriasis susceptibility region to approximately 360 kb along chromosome 1 in the vicinity of IVL, small proline-rich region (SPRR) and proline-rich region 9 (PRR9) genes. The study of interactions between the causative variant(s) in this locus will provide insights into a possible role for epidermal barrier formation in the pathogenesis of psoriasis.

Filaggrin Null Mutations Are Not a Protective Factor for Acne Vulgaris

TO THE EDITOR Acne vulgaris is a very common skin disorder, affecting to some degree 88–94% of Singaporean adolescents (Tan et al., 2007; Yosipovitch et al., 2007). Genetic predisposition is a significant risk factor, as illustrated by familial and twin studies (Bataille et al., 2002; Ghodsi et al., 2009). The clinical features of acne include sebor-rhea, comedone formation, inflammatory pustules, nodules, and cysts, with resultant scarring. Important pathogenic mechanisms in acne include increased sebum production, hyperkeratinization and occlusion of the follicular duct, proliferation of Propionibacterium acnes, and an inflammatory reaction (Purdy and de Berker, 2006). P. acnes produces lipases, which liberate proinflammatory fatty acids from sebum and also triggers a cytokine response. Filaggrin is expressed in terminally differentiating keratinocytes and has a key role in epithelial barrier formation. Immunostaining demonstrates increased filaggrin expression in the sebaceous duct and infundibulum of acne vulgaris skin (Kurokawa et al., 1988), and P. acnes strains increase the expression of filaggrin and other differentiation-specific markers in normal human epidermal keratinocytes in vitro and in the suprabasal layers of human skin explants (Jarrousse et al., 2007). Similarly, inflammatory cytokines resulted in increased filaggrin expression in sebaceous gland explants (Guy and Kealey, 1998). However, it is not known whether differences in filaggrin expression represent a primary or secondary effect in the pathogenesis of acne. Null mutations in the filaggrin gene (FLG) result in reduced filaggrin expression and cause ichthyosis vulgaris (Smith et al., 2006). Such mutations are common in the general population, being carried by ~10% of Europeans and 7.3% of Singaporean Chinese (Sandilands et al., 2007; Chen et al., personal communication). This high carrier rate in different populations suggests a heterozygote advantage, and it has been proposed that a more permeable skin barrier may have been beneficial in evolutionary history (Irvine and McLean, 2006). The co-existence of FLG null mutations with other gene mutations that disrupt epidermal differentiation may increase phenotype severity (Liao et al., 2007; Gruber et al., 2009). It is also possible that heterozygosity for null mutations has other effects on skin physiology. Studying a cohort of 284 European dermatology patients not selected for dry skin (Sergeant et al., 2009) raised the possibility that carriage of one FLG null mutation could provide a protective effect against acne vulgaris. In the Sergeant study, the odds ratio of acne in the carrier group was 0.3 (95% confidence interval 0.1–1.0), but the difference between these individuals and the group without FLG mutations did not reach statistical significance (P = 0.08). In addition, this study relied¼ on a recalled history of acne. We therefore aimed to test the hypothesis that FLG null mutations are protective against the development of acne vulgaris by studying a well-documented group of patients with acne, and comparing with a population control group. A total of 287 Singaporean Chinese patients presenting with acne vulgaris to the National Skin Centre, a major dermatology outpatient facility in Singapore, were recruited: mean age 22 o years (SD 4.8), range 14–50 (27% <20 years of age and 95% were under 30 years), 76.3% were male. Acne vulgaris symptoms were reported for a mean o of 5.6 years (SD 4.2), range from <1 to 32 years. Patients with polycystic ovarian syndrome were excluded from this study. Acne severity was assessed using the Global Severity Assessment Score (Lehmann et al., 2002): 100 patients (34.8%) had mild acne, 129 (44.9%) had moderate acne, and 58 (20.3%) had severe acne. DNA samples from 440 unselected Singaporean Chinese population controls with a mean age of 44.6 years (SD 14.0), range 1–80, 44.1% male, for whom acne vulgaris status was unknown, were obtained from Singapore Bio-Bank, Singapore. This study was approved by the local domain specific ethical review board in accordance with the Declaration of Helsinki and all participants gave written, informed consent. Cases and controls were screened for all 22 population-specific FLG null mutations as recently reported (Chen et al., personal communication). In this group a total of 12 known FLG null mutations were detected: p.S406X, c.1249insG, c.2284del4, c.3321delA, p.S1302X, p.S15 15X, c.6950del8, p.Q2417X, p.E2422X, c.7945delA, p.S2706X, and p.R4307X, plus two mutations: c.6834del5 and c.8157delC, which to our knowledge are previously unreported. Fishers exact test and logistic regression analyses were used to compare the prevalence of FLG null mutations between cases and controls, using the statistical analysis package Stata (Version 9, Stata for Linux, StataCorp LP, College Station, TX). Power calculations were performed using Quanto version 1.2.4 (University of Southern California, http://hydra.usc.edu/gxe/). In this study, 8.2% of Singaporean Chinese acne vulgaris cases carried one or more FLG null mutations compared with 7.3% of the control population, a non-significant difference (Fishers exact test P = 0.783, odds ratio 1.2, 95% confidence interval 0.7–2.1), shown in Table 1. It is unlikely that our failure to demonstrate an association has occurred because of lack of power, as analysis of 256 acne vulgaris patients and 434 controls, assuming a population prevalence of 88% for acne vulgaris of comparable severity (Tan et al., 2007) would give a power of 99% to detect an odds ratio of 0.3 (Sergeant et al., 2009) for the combined FLG null genotype, with two-sided P = 0.05. The equivalent calculation using a population prevalence of 23% (based on Singaporean teenagers reporting treatment for their acne (Yosipovitch et al., 2007)) gives an estimated power of 90% for this study. Furthermore, the comprehensive screening of all 22 reported FLG null mutations from this carefully characterized Singaporean Chinese population means that the lack of association is unlikely to have occurred because of incomplete ascertainment of the FLG genotype. Table 1 Genotyping results and Fishers exact test to investigate the association between FLG null mutations and acne vulgaris in a case–control study Our finding, that in this Singaporean Chinese population the frequency of FLG null mutations in acne vulgaris patients is not statistically different from the ethnically matched controls, indicates that filaggrin haploinsufficiency is unlikely to have a generic protective effect in acne. It is likely that the overexpression of filaggrin in the pilosebaceous units reported in the disorder is a bystander effect, reflecting other alterations in keratinocyte differentiation, but not itself critical for acne pathogenesis.

Exacerbation of X-linked ichthyosis phenotype in a female by inheritance of filaggrin and steroid sulfatase mutations

BACKGROUND X-linked ichthyosis (XLI) is a relatively common, recessive condition caused by mutations in the steroid sulfatase (STS) gene. Common loss-of-function mutations in the filaggrin gene (FLG) cause ichthyosis vulgaris and predispose individuals to atopic eczema. OBJECTIVE To test the hypothesis that co-inheritance of FLG mutations can act as a genetic modifier in XLI. METHODS An unusually severe XLI phenotype in addition to eczema and mild childhood asthma was investigated in a female Indian patient by fluorescent in situ hybridization (FISH) for the common STS gene deletion. Direct sequencing of the entire FLG gene was also performed. RESULTS FISH analysis revealed that the proband was homozygous for the common STS genomic deletion mutation. Further investigation revealed a frame-shift mutation 3672del4 in the gene encoding filaggrin (FLG), leading to premature termination of profilaggrin translation. Interestingly, her father, who had a very typical mild presentation of XLI, did not carry this FLG mutation in addition to his STS deletion. Her mother was a heterozygous carrier of the FLG mutation and consistent with this, had mild symptoms of ichthyosis vulgaris; she was also a heterozygous carrier of the STS deletion. CONCLUSION This is the second reported case of the modifying effects of FLG null alleles on XLI and strengthens the hypothesis that filaggrin defects can synergize with STS deficiency to exacerbate the ichthyosis phenotype.

A rare connexin 26 mutation in a patient with a forme fruste of keratitis–ichthyosis–deafness (KID) syndrome

Background  Keratitis–ichthyosis–deafness (KID) syndrome is a rare ectodermal dysplasia characterized by generalized erythrokeratotic plaques, sensorineural hearing loss, and vascularizing keratitis. Cutaneous changes and hearing loss typically present in early childhood, whereas ocular symptoms present later. Mutations in the connexin (Cx) 26 gene, GJB2, are now established to underlie many of the affected cases, with the majority of patients harboring the p.D50N mutation.