Yiwei Zhao

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.

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.

Filaggrin null mutations are associated with increased asthma exacerbations in children and young adults

Background:  Filaggrin (FLG) null mutations are important genetic predisposing factors for atopic asthma and have recently been shown to influence controller and reliever medication needs in asthmatic children. Our objective was to study the role of FLG null alleles in asthma exacerbations.

Loss-of-function mutations of an inhibitory upstream ORF in the human hairless transcript cause Marie Unna hereditary hypotrichosis

Marie Unna hereditary hypotrichosis (MUHH) is an autosomal dominant form of genetic hair loss. In a large Chinese family carrying MUHH, we identified a pathogenic initiation codon mutation in U2HR, an inhibitory upstream ORF in the 5′ UTR of the gene encoding the human hairless homolog (HR). U2HR is predicted to encode a 34–amino acid peptide that is highly conserved among mammals. In 18 more families from different ancestral groups, we identified a range of defects in U2HR, including loss of initiation, delayed termination codon and nonsense and missense mutations. Functional analysis showed that these classes of mutations all resulted in increased translation of the main HR physiological ORF. Our results establish the link between MUHH and U2HR, show that fine-tuning of HR protein levels is important in control of hair growth, and identify a potential mechanism for preventing hair loss or promoting hair removal.

Filaggrin null mutations and childhood atopic eczema: A population-based case-control study

BACKGROUND Null mutations within the filaggrin gene (FLG) are associated with moderate-to-severe atopic eczema; their role in mild-to-moderate eczema in the general population is unknown. OBJECTIVE We sought to investigate the significance of 5 common FLG null mutations in childhood atopic eczema in an unselected population cohort. METHODS Eight hundred eleven English children aged 7 to 9 years were screened for FLG mutations. Eczema cases were defined by using United Kingdom diagnostic criteria and skin examination. Asthma and seasonal rhinitis cases were defined by parental questionnaire. Association between phenotype and genotype was investigated using Fisher exact test and logistic regression analysis. RESULTS The 12-month period prevalence of atopic eczema was 24.2% (95% CI, 21.2% to 27.2%), with 96% (115/120) of cases having mild-to-moderate disease. The combined null genotype (carriage of > or = 1 FLG mutations) was significantly associated with atopic eczema (P = 1.2 x 10(-4)). The odds ratio (OR) for individuals carrying 2 null mutations was 26.9 (95% CI, 3.3-217.1), but heterozygote carriers showed no significant increase in risk (OR, 1.2; 95% CI, 0.7-1.9). Eight of 190 eczema cases (4.2%) carried 2 FLG null mutations and thus might be attributed to filaggrin deficiency. Asthma in the context of eczema showed significant association with the FLG null mutations (P = 7.1 x 10(-4)). There was no association of FLG with asthma independent of eczema (P = .15) and no association with seasonal rhinitis (P = .66). CONCLUSION FLG null mutations are significantly associated with mild-to-moderate atopic eczema in childhood, with a recessive pattern of inheritance.

Filaggrin haploinsufficiency is highly penetrant and is associated with increased severity of eczema: further delineation of the skin phenotype in a prospective epidemiological study of 792 school children

Background  Null mutations within the filaggrin gene (FLG) cause ichthyosis vulgaris and are associated with atopic eczema. However, the dermatological features of filaggrin haploinsufficiency have not been clearly defined.

Statins Downregulate K6a Promoter Activity: A Possible Therapeutic Avenue for Pachyonychia Congenita

Pachyonychia congenita (PC) is a keratinizing disorder predominantly caused by mutations in keratin 6a (K6a) (∼50% of cases) or K6b, K16, or K17. One means of treating PC is identification of small-molecule inhibitors of PC-related keratins. Here, we cloned the human K6a promoter, and using a cell-based reporter gene assay, a chemical library was screened for K6a inhibitors. One compound, compactin, the precursor of all cholesterol-lowering statins, was of particular interest. We found that, surprisingly, simvastatin and other statins inhibit K6a promoter activity and K6a protein expression. Further investigation showed that this effect works through cholesterol/mevalonate pathway inhibition rather than an off-target effect. Inhibition of both basal and IFN-γ-inducible K6a expression by statins was demonstrated. Both these K6a inhibitory effects were found to be mediated by Stat1 transcription factor, but only the IFN-γ-inducible promoter activity was controlled via the Stat/JAK pathway. The repressive effect of statins was found to be mediated by the isoprenoid pathway downstream of mevalonate (the intermediate following 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase) but upstream of cholesterol, specifically the geranylgeranylation pathway. These data set the scene for further unraveling signaling pathways that control the K6a promoter, as well as facilitating clinical trials for statins in PC patients.

Filaggrin mutations are a major predisposing factor in childhood-onset adult-persistent atopic dermatitis

The transfer of normal genes into somatic cells is one strategy to treat patients with genetic diseases. However, this strategy has still encounters technical problems including effi cacy of gene transfer rate and practical clinical safety. Thus, other strategies including pharmacological therapy or gene correction, are receiving increasing attention. Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the COL7A1 encoding type VII collagen. This study examined the feasibility of antisense oligoribonucleotide (AON) therapy for DEB. AON was designed to induce skipping of a targeted exon containing a premature termination codon mutation, resulting in restoration of the open reading frame. We targeted exon 70 of COL7A1 since a recurrent mutation 5818delC was localized to exon 70 in Japanese DEB patients. We initially designed and synthesized two AONs to modulate splicing of exon 70 and found that one AON induced effective skipping of normal exon 70 containing the 16 amino acids. Attachment and migration analyses showed that recombinant collagen without contribution of exon 70 had similar effect to normal type VII collagen. Next, we synthesized mutation-specifi c AON by deleting cytosine at 5818. Introduction of this AON into DEB keratinocytes harboring 5818delC without expression of type VII collagen showed that the AON induced skipping of exon 70 in the abnormal 5818delC allele. Furthermore, 6.2 % of the DEB keratinocytes started to express type VII collagen in vitro after application of the mutation-specifi c AON. Injection of the AON into rat model grafted the DEB keratinocytes and fi broblasts induced detectable of type VII collagen expression at the basement membrane zone. We conclude that skipping of targeted exons using mutation-specifi c AON may show potential for future gene therapy for DEB patients.We recently reported that ABCA12 works as an epidermal keratinocyte lipid transporter, and that defective ABCA12 results in a loss of the skin lipid barrier, leading to harlequin ichthyosis (HI), one of the most devastating genodermatoses. In the present study, precise expression pattern of ABCA12 was studied in human embryonic and fetal skin of 7-22 weeks estimated gestational age (EGA) and newborn skin samples. For controls, we also studied the expression of transglutaminase 1 (TGase1) that is known to cross-link several precursor proteins in the formation of the cornifi ed cell envelope during keratinocyte terminal differentiation. In twolayered epidermis (about 6-9 weeks EGA), both ABCA12 and TGase1 were only expressed in periderm cells. In three-layered epidermis (10-13 weeks EGA), ABCA12 staining was seen not only in periderm, but also throughout the entire epidermis, while TGase1 staining was restricted to the periderm. A similar pattern was observed during four- or more-layered epidermal development (14-22 weeks EGA). In newborn skin, ABCA12 and TGase1 were seen only in the upper epidermal layers, mainly the granular layer. These staining patterns were similar to those in normal adult skin. Next, we studied ABCA12 mRNA expression in extracts of the fetal skin (at 10, 14, 15 weeks EGA). In 15 weeks EGA, the expression level of ABCA12 mRNA was signifi cantly increased compared with that in the early development (10 and 14 weeks EGA). This increasing pattern of ABCA12 mRNA expression is consistent with ABCA12 immunofl uorescent staining during human epidermal development. The unique pattern of ABCA12 expression during human epidermal development might imply severe symptoms of HI patients with ABCA12 mutations around the birth. 2006 ESDR ABSTRACTS www.Mutations in ABCA12 lead to harlequin ichthyosis and lamellar ichthyosis. The keratinocyte lipid transporter ABCA12 is a member of the ATP-binding cassette transporter family, and members of the ABCA subfamily have closely related functions as lipid transporters. Previously, we reported that the pathomechanism of harlequin ichthyosis involves the defective function of the lipid transporter ABCA12.To further elucidate the precise distribution pattern and function of ABCA12, we performed double-labeling immunofl uorescence staining for ABCA12 and for Golgi-associated or lamellar granule-associated molecules both on normal human epidermis and cultured normal human keratinocytes. We studied the precise localization of ABCA12 and other molecules using confocal laser scanning microscope. In normal human epidermis, ABCA12 was observed mainly in the granular layers with glucosylceramide (one of the major lamellar granule contents) and transglutaminase 1 (a cornifi ed cell envelope-associated keratinization marker), but not always colocalized with GM130 and TGN46 (Golgi-related molecules) that were expressed from the lower epidermis. In normal human keratinocytes cultured in high Ca++ concentration medium, ABCA12 colocalized with GM130 (a cis-Golgi- associated molecule), TGN46 (a trans-Golgi-associated molecule) and glucosylceramide. Transglutaminase 1 was restricted to the cell membrane and ABCA12 localization was within the cytoplasm distinct from transglutaminase 1 localization. The present results suggest that ABCA12 is mainly expressed in differentiated, granular layer keratinocytes with glucosylceramide and transglutaminase 1, and, at the subcellular level, ABCA12 is distributed from the cisside of the Golgi apparatus to trans-Golgi network, lamellar granules at the cell periphery. Our results suggest that ABCA12 may play an important role in lipid transport from the cis-side of the Golgi apparatus through the trans-Golgi network, to the cell periphery via lamellar granules in human epidermal granular layer keratinocytesHarlequin ichthyosis (HI) is a devastating genodermatosis that is often fatal during the neonatal period. Until the identifi cation of ABCA12-encoding a keratinocyte lipid transporter, as the causative gene for HI, prenatal diagnosis (PNDx) had been performed for more than 20 years by electron microscopic examination of fetal skin biopsy samples. We report here the fi rst case of DNA-based PNDx for HI. The proband, the fi rst child of healthy non-consanguineous French parents, showed a typical HI phenotype and died soon after birth. ABCA12 immunostaining was markedly reduced in the proband’s skin. Direct sequence analysis of ABCA12 revealed that the proband was a compound heterozygote for two novel mutations: a maternal nonsense mutation p.Ser1249X in exon 26 and a paternal missense mutation p.Arg2479Lys occurring at the last codon of exon 50. p.Ser1249X leads to an approximate 52 % truncation of the ABCA12 protein losing both ATP-binding cassette active sites. p.Arg2479Lys involves a highly conserved codon among diverse species in the second ABCA12 ATP-binding cassette. For their third pregnancy, the parents requested PNDx. Direct sequence analysis using fetal genomic DNA from amniotic fl uid cells at 17 weeks of pregnancy revealed that the fetus was a compound heterozygote for both mutations. The fetus was predicted to be affected and the parents requested the pregnancy to be terminated. The aborted fetus showed typical signs of HI. Analysis of ABCA12 transcripts of cultured keratinocytes from the abortus showed the presence of six abnormally spliced products arising from the allele carrying the missense mutation. Four of them lead to premature termination codons while the two others produced deleted proteins missing 21 and 31 amino acids in the second ATP-binding cassette. These results indicated residual expression of ABCA12. The present report paves the way for molecular PNDx of HI in the earlier stages of pregnancy