Filaggrin gene mutations and new SNPs in asthmatic patients: a cross-sectional study in a Spanish population

BackgroundSeveral null-mutations in the FLG gene that produce a decrease or absence of filaggrin in the skin and predispose to atopic dermatitis and ichthyosis vulgaris have been described. The relationship with asthma is less clear and may be due to the influence of atopy in patients with associated asthma.MethodsFour hundred individuals were included, 300 patients diagnosed with asthma divided into two groups according to their phenotype (allergic and non-allergic asthma) and 100 strictly characterized controls. The coding region and flanking regions of the FLG gene were amplified by PCR. We proceeded to the characterization of potential gene variants in that region by RFLP and sequencing and analysed their association with lung function parameters, asthma control and severity, and quality of life.ResultsWe identified two null-mutations (R501X and 2282del4), seven SNPs previously described in databases and three SNPs that had not been previously described. One of the SNP identified in this study (1741A > T) was more frequently detected in patients with non-allergic asthma, worse FVC, FEV1 and PEF values and a higher treatment step. In addition, lowered spirometric values were observed in the non-allergic group carrying any of the nonsynonymous SNPs.ConclusionsIn the association study of genetic variants of the FLG gene in our population the 1741A > T polymorphism seems to be associated with non-allergic asthma.

Related papers:

title: The Atopic March: Progression from Atopic Dermatitis to Allergic Rhinitis and Asthma
abstract: The development of atopic dermatitis (AD) in infancy and subsequent allergic rhinitis and asthma in later childhood is known as the atopic march. This progressive atopy is dependent on various underlying factors such as the presence of filaggrin mutations as well as the time of onset and severity of AD. Clinical manifestations vary among individuals. Previously it was thought that atopic disorders may be unrelated with sequential development. Recent studies support the idea of a causal link between AD and later onset atopic disorders. These studies suggest that a dysfunctional skin barrier serves as a site for allergic sensitization to antigens and colonization of bacterial super antigens. This induces systemic Th2 immunity that predisposes patients to allergic nasal responses and promotes airway hyper reactivity. While AD often starts early in life and is a chronic condition, new research signifies that there may be an optimal window of time in which targeting the skin barrier with therapeutic interventions may prevent subsequent atopic disorders. In this review we highlight recent studies describing factors important in the development of atopic disorders and new insights in our understanding of the pathogenesis of the atopic march.

title: The cornified envelope: a model of cell death in the skin
abstract: The epidermis functions as a barrier against the environment by means of several layers of terminally differentiated, dead keratinocytes — the cornified layer, which forms the endpoint of epidermal differentiation and death. The cornified envelope replaces the plasma membrane of differentiating keratinocytes and consists of keratins that are enclosed within an insoluble amalgam of proteins, which are crosslinked by transglutaminases and surrounded by a lipid envelope. New insights into the molecular mechanisms and the physiological endpoints of cornification are increasing our understanding of the pathological defects of this unique form of programmed cell death, which is associated with barrier malfunctions and ichthyosis.

title: Epithelial structural proteins of the skin and oral cavity: function in health and disease.
abstract: Epithelial tissues function to protect the organism from physical, chemical, and microbial damage and are essential for survival. To perform this role, epithelial keratinocytes undergo a well-defined differentiation program that results in the expression of structural proteins which maintain the integrity of epithelial tissues and function as a protective barrier. This review focuses on structural proteins of the epidermis and oral mucosa. Keratin proteins comprise the predominant cytoskeletal component of these epithelia. Keratin filaments are attached to the plasma membrane via desmosomes, and together these structural components form a three-dimensional array within the cytoplasm of epithelial cells and tissues. Desmosomes contain two types of transmembrane proteins, the desmogleins and desmocollins, that are members of the cadherin family. The desmosomal cadherins are linked to the keratin cytoskeleton via several cytoplasmic plaque proteins, including desmoplakin and plakoglobin (gamma-catenin). Epidermal and oral keratinocytes express additional differentiation markers, including filaggrin and trichohyalin, that associate with the keratin cytoskeleton during terminal differentiation, and proteins such as loricrin, small proline-rich proteins, and involucrin, that are cross-linked into the cornified envelope by transglutaminase enzymes. The importance of these cellular structures is highlighted by the large numbers of genetic and acquired (autoimmune) human disorders that involve mutations in, or autoantibodies to, keratins and desmosomal and cornified envelope proteins. While much progress has been made in the identification of the structural proteins and enzymes involved in epithelial differentiation, regulation of this process is less clear. Both calcium and retinoids influence epithelial differentiation by altering the transcription of target genes and by regulating activity of enzymes critical in epithelial differentiation, such as transglutaminases, proteinases, and protein kinases. These studies have furthered our understanding of how epithelial tissue and cell integrity is maintained and provide a basis for the future treatment of skin and oral disorders by gene therapy and other novel therapeutics.

title: Filaggrin mutations, atopic eczema, hay fever, and asthma in children.
abstract: Mutations in the filaggrin gene (FLG) have been shown to play a significant role in ichthyosis vulgaris and eczema, 2 common chronic skin diseases. However, their role in the development of other atopic diseases such as asthma and rhinitis has not yet been clarified in large population-based studies.



To study the effect of FLG mutations at the population level and their effect on other atopic phenotypes.



Association analysis of the 2 common FLG-null mutations R501X and 2282del4 and 3 recently identified rare FLG variants (R2447X, S3247X, 3702delG) was performed on our cross-sectional population of German children (n = 3099) recruited as part of the International Study of Asthma and Allergies in Childhood II in Munich (n = 1159) and Dresden (n = 1940).



FLG variants increased the risk for eczema more than 3-fold (odds ratio [OR], 3.12; 95% CI, 2.33-4.173; P = 2.5 x 10(-14); population-attributable risk, 13.5%). Independent of eczema, FLG mutations conferred a substantial risk for allergic rhinitis (OR, 2.64; 95% CI, 1.76-4.00; P = 2.5 x 10(-6); population-attributable risk, 10.8%). Nasal biopsies demonstrated strong filaggrin expression in the cornified epithelium of the nasal vestibular lining, but not the transitional and respiratory nasal epithelia. In contrast, the association with asthma (OR, 1.79; 95% CI, 1.19-2.68; P = .0048) was restricted to asthma occurring in the context of eczema, and there was a strong association with the complex phenotype eczema plus asthma (OR, 3.49; 95% CI, 2.00-6.08; P = 1.0 x 10(-5)).



Our results suggest that FLG mutations are key organ specific factors predominantly affecting the development of eczema and confer significant risks of allergic sensitization and allergic rhinitis as well as asthma in the context of eczema.

title: Filaggrin exists in human nose.
abstract: Allergic diseases are manifested in various forms, such as atopic dermatitis (AD), bronchial asthma (BA) and allergic rhinitis (AR). Dermatitis research, such as that on Netherton syndrome, has progressed to the stage where it is now thought that lesions in the epithelial barrier predispose people to eczema, and that atopy might be a secondary disease.1 Filaggrin (FLG) has been known as a natural moisturizing factor and barrier-related protein. Mutation in the human FLG gene has been reported to be the most significant risk factor for AD. It has also been demonstrated that FLG gene mutations increased the risk of bronchial asthma in patients with atopic dermatitis. These findings have brought a breakthrough, where AD may be now considered as a barrier disorder disease.2 In contrast, FLG in the nose has not been well studied directly, although there are abundant mentions of FLG in relation to allergic rhinitis.3 In this study, for the first time, we attempted to evaluate FLG expression in human nasal mucosa through real time RT-PCR and demonstrated those alterations after various stimulations. Moreover, we also attempted to confirm the immunohistochemical localization of FLG in nasal mucosa, as presented previously in a preliminary study.4 Primary human nasal epithelial cells culture was performed in an air-liquid interface, as described by Gruenert et al.5 Human nasal epithelial cells were purchased from PromoCell GmbH (Heidelberg, Germany). The cell suspension of nasal epithelial cells was placed on a collagen-coated (Cellmatrix Type I-P, Nitta Gelatin, Japan) membrane filter (Millicel-CM, Millipore, MA, USA). Quantitative PCR analysis was performed, as described by Kinoshita et al.,6 after stimulation of the lipopolysaccharides with Escherichia coli (LPS, 1 mg/ml), Polyinosinicepolycytidylic acid potassium salt (Poly I: C 1 mg/ml), TNFa (20 ng/ml), and TNFa (20 ng/ml) þ Poly I: C 1 mg/ml for 24 h, respectively. The immunohistochemistry studywas performed using biopsies from three Japanese nonatopic patients. The anti-filaggrin primary antibody employed was an affinity-purified rabbit polyclonal IgG antibody (1/500 dilution; ab24584; Abcam PLC, Cambridge, UK). Stainingwas achievedwith aSimple Stain kit (Nichirei, Tokyo, Japan) and developed with 3, 3-diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide (Nichirei). The sectionswere counterstainedwith hematoxylin. The ethics committee of DokkyoMedical University Koshigaya Hospital approved the study.

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

title: Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema
abstract: 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; Fisher's 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.

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

title: Analysis of SPINK 5, KLK 7 and FLG genotypes in a French atopic dermatitis cohort.
abstract: The role of a genetically impaired epidermal barrier as a major predisposing factor in the pathogenesis of atopic disorders is currently under closer investigation. Variants on three candidate genes (SPINK5, KLK7 and FLG) have been associated with atopic dermatitis. A functional relevance has already been established for filaggrin variants, but not for SPINK5 and KLK7 polymorphisms. The objectives of this study were to confirm the association between SPINK5, KLK7, FLG variants and atopic dermatitis and to assess how variants influence selected phenotypic traits. This cross-sectional study was carried out over 20 months in 99 children and adults with atopic dermatitis (median age 7 years). The following items were analysed: SCORAD, TEWL, ichthyosis vulgaris, presence of asthma, total IgE serum levels. The SPINK5 E420K SNP, the KLK7 4bp insertion polymorphism and the filaggrin mutants (R510X and 2282del4) were analysed as described previously. The control group for genetic analysis was recruited in an ethnically matched, phenotypically anonymous cohort (n=102). The allelic frequencies were 0.525 for SPINK5, 0.26 for KLK7 polymorphisms, 0.101 and 0.075 for 2282del4 and R501X FLG mutants, respectively. The association of atopic dermatitis with filaggrin variants was confirmed, but not that of SPINK5 or KLK7 polymorphisms. SCORAD and TEWL measurements were not influenced by any of the variants. The SPINK5 polymorphism was associated with high IgE serum levels (p=0.011). Abnormal barrier genes do not influence the severity of atopic dermatitis. The SPINK5 gene polymorphism may modulate systemic immune effects favouring the IgE response to atopens. TEWL does not allow the characterization of subsets of patients with or without abnormal barrier genes.

title: Filaggrin mutations in children with severe atopic dermatitis.
abstract: Atopic dermatitis (AD) results from strong genetic and environmental interactions. AD shows genetic linkage to Chromosome 1q21. This region contains the epidermal differentiation complex (EDC), which consists of genes that form essential components of epidermal surfaces. Filaggrin (FLG) is one of these. Mutations in FLG/(R501X and 2282del4) are reported to be strongly associated with AD and to influence asthma accompanying AD. We investigated these effects in families recruited through a child with severe AD. We genotyped two panels of families, totalling 426, containing 990 affected and unaffected children. We found significant associations with AD (P=0.0001), asthma (P=0.006), and atopy (P=0.002). The FLG mutations were present in 26.7% of patients with AD, but were also present in 14.4% of children without AD. They were weakly associated with disease severity. The variants were not independently associated with asthma. The overall LOD score for genetic linkage of markers to the region was 3.57. This fell to 2.03 after accounting for the FLG mutations, indicating the presence of other genetic variants influencing AD at this locus. Our results provide further confirmation of the importance of mutations in FLG and the skin barrier in AD pathogenesis. The results indicate that investigations of other genes within the EDC should be undertaken.

title: Filaggrin null mutations and childhood atopic eczema: a population-based case-control study.
abstract: 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.



We sought to investigate the significance of 5 common FLG null mutations in childhood atopic eczema in an unselected population cohort.



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.



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).



FLG null mutations are significantly associated with mild-to-moderate atopic eczema in childhood, with a recessive pattern of inheritance.

title: The burden of disease associated with filaggrin mutations: a population-based, longitudinal birth cohort study.
abstract: Atopic disease is a major health problem. Mutations in the filaggrin gene (FLG) confer major susceptibility to eczema and related asthma.



We sought to determine the natural history and burden of atopic disease conferred by the 2 most common FLG mutations in a large, population-based birth cohort study.



We analyzed the effect of the most common null alleles (R501X and 2282del4) on several atopic phenotypes in a cohort of approximately 7000 English children born in 1990-1991.



FLG null alleles associated strongly with eczema; eczema associated with these mutations presents in early life and is more persistent (hazard ratio for eczema resolution for those with FLG mutations to FLG wild type, 0.67; 95% CI, 0.58-0.77; P = 5 x 10(-8)). FLG mutations conferred a population asthma risk of 1.80 (95% CI, 1.34-2.41; P = .00019); asthma risk was especially high in the context of eczema (odds ratio, 3.16; 95% CI, 2.25-4.43; P = 1.4 x 10(-11)). Strong associations were identified with sensitization to grass, house dust mite, and cat dander and sensitization to multiple allergens (odds ratio, 2.12; 95% CI, 1.03-4.37; P = 5.42 x 10(-27)).



FLG mutations are strong genetic determinants of eczema, early wheeze, asthma in the context of eczema, and atopic sensitization. They confer risk of a particular trajectory for eczema, with increased duration of disease and greater risk of asthma and multiple allergic sensitizations. FLG alleles help define the risk profile of children with eczema and help define the "eczema plus early wheeze" and "eczema plus asthma" phenotypes.

title: Filaggrin gene mutations are associated with asthma and eczema in later life.
abstract: Atopic conditions are common in middle and later life. Although a genetic component in these conditions has long been suspected, only recently have mutations in the filaggrin gene (FLG) emerged as a major cause in children and young adults. Filaggrin is an epidermal protein involved in aggregating the keratin cytoskeleton1 and is critical in forming the skin barrier. There are several mutations in the FLG locus associated with a loss of functional protein,2 but 2 (R501X and 2282del4) are common in populations of European  In children and younger adults FLG mutations are strongly associated with eczema, and in those with eczema, FLG is also associated with  It is hypothesized that the FLG mutation–compromised skin barrier allows percutaneous induction of TH2 cytokines in epithelia and the development of a systemic atopic tendency.5 
 
The effects of FLG mutations in middle-aged or older groups are not known. We aimed to estimate the effect of the 2 common FLG mutations on the prevalence of asthma and atopic conditions in persons aged 50 years and older in England using data from the English Longitudinal Study of Ageing (ELSA). 
 
ELSA is a follow-up study of respondents to the Health Survey for England (HSE; http://www.dh.gov.uk/), an annual cross-sectional survey of the community-living population. All HSE respondents in 2001 were asked about allergic conditions, and most respondents donated a blood specimen for IgE measurement. 
 
In 2002 and 2004, ELSA respondents were asked about diagnosed asthma, chronic bronchitis, or emphysema. In 2004, respondents were also asked to participate in a nurse-led physical examination and to provide blood samples. The identification of asthma in elderly subjects is complex, with overlaps in presentation with chronic obstructive pulmonary disease and even cardiac failure. We therefore used several case definitions in our analysis: “any obstructive airways disease” (diagnosed asthma, emphysema, or chronic bronchitis), “diagnosed asthma,” “self-reported wheezing in the last 12 months,” and “symptomatic asthma.” We defined patients with symptomatic asthma as those with doctor-diagnosed asthma (in 2002 and 2004) and wheezing in the previous 12 months. 
 
Of the 9432 respondents in ELSA 2004, 7156 underwent a nurseled clinical visit, during which 6231 donated DNA specimens. Of these, genotyping was successfully completed on 5432 subjects for R501X and 5335 subjects for 2282del4 mutations. The combined FLG genotype was available for 5289 individuals, of whom 2051 had been asked about atopy in the HSE 2001. Genotyping was performed by The Geneservice Ltd (http://www.geneservice.co.uk) using Applied Biosystems TaqMan probes. The R501X and 2282del4 mutations were both in Hardy-Weinberg equilibrium, and there were no discrepancies between duplicate samples. The 2 variants were combined to produce the FLG genotype, according to the method of Palmer et al.6 Associations were tested in age- and sex-adjusted linear or logistic regression models, with IgE models additionally adjusted for smoking status and exposure by using Stata SE Version 9 (StataCorp, College Station, Tex). 
 
In our sample 8.8% (n = 464) had at least 1 FLG mutation, and 0.25% (n = 13) were homozygous for either mutation or had copies of both, yielding a prevalence of 9% for FLG mutation presence. Although 11.7% reported doctor-diagnosed asthma and 17.7% reported attacks of wheezing in the previous 12 months, only 6.6% reported both diagnosed asthma and wheezing in the previous 12 months (ie, symptomatic asthma; Table I). 
 
 
 
TABLE I 
 
Sample characteristics of those genotyped for FLG 
 
 
 
FLG mutations were not associated with “any obstructive lung disease” or “diagnosed asthma.” However, there was a borderline association with “wheezing in the previous 12 months” (odds ratio [OR], 1.26; P = .05) and a significant association with symptomatic asthma (OR, 1.45; P = .03; Table II). 
 
 
 
TABLE II 
 
Association of FLG mutations with asthma and atopy 
 
 
 
Diagnoses of eczema were more common in those with mutations (OR, 1.69; P = .009; Table II), and a higher proportion of FLG carriers reported early ages of onset: 61% (n = 11/18 with data) of carriers compared with 26% of others (n = 36/138) had their skin conditions before the age of 20 years (OR, 4.93; 95% CI, 1.70–14.26; P = .003). FLG mutations were not associated with IgE levels. A history of dry skin in the past 12 months was also somewhat more common in those with mutations (OR, 1.47; P = .09; Table II), which is in line with recent findings by Novak et al.7 
 
The R501X mutation was independently associated with both eczema (OR, 2.08; 95% CI, 1.29–3.36; P = .003) and symptomatic asthma (OR, 1.59; 95% CI, 1.05–2.40; P = .03). These associations for the 2282del4 mutation were not statistically significant, but trends were in the same direction. 
 
In this first study of the effects of mutations in the FLG gene in middle and later life, there were strong associations between mutation status and eczema and symptomatic asthma. These associations showed a similar pattern to that found in children and young adults. We were unable to derive robust estimates of the risk of mutations resulting in both conditions together because of small numbers: in our data 41 individuals with symptomatic asthma also reported having eczema at baseline, of whom 8 had an FLG mutation. We found no association between mutation prevalence and age, providing no evidence of a significant loss of this genotype in the population because of increased mortality in those with FLG mutations. 
 
In large population studies, such as ELSA, detailed clinical diagnosis of eczema and asthma is not possible for logistic reasons: carefully diagnosed comparisons of cases and control subjects are likely to provide even stronger estimates of the associations. The prevalence of asthma in the genotyped population was 5.6% (95% CI, 4.7% to 6.5%) in men and 7.5% (95% CI, 6.5% to 8.4%) in women, which is comparable with published prevalences in middle and later life.8 We also note that the prevalence of the studied mutations in our sample (9%) is very similar to that seen in children in England (8.8%). However, our observed prevalence of eczema (13.0%; 95% CI, 11.6% to 14.5%) is somewhat higher than that reported by Gupta et al9 (ie, 9% of ≥45-year-olds) for the United Kingdom: the reason for this is unclear. 
 
Interestingly, only a minority of mutation carriers in our study actually had eczema or asthma (>80% of FLG mutation carriers had neither condition, which is consistent with the survival analysis by Henderson et al10), and work is needed to establish the factors that lead to either resilience or susceptibility in later life. More work is also needed on whether specific treatment approaches are possible for those with FLG mutations.

title: Analysis of SPINK 5, KLK 7 and FLG genotypes in a French atopic dermatitis cohort.
abstract: The role of a genetically impaired epidermal barrier as a major predisposing factor in the pathogenesis of atopic disorders is currently under closer investigation. Variants on three candidate genes (SPINK5, KLK7 and FLG) have been associated with atopic dermatitis. A functional relevance has already been established for filaggrin variants, but not for SPINK5 and KLK7 polymorphisms. The objectives of this study were to confirm the association between SPINK5, KLK7, FLG variants and atopic dermatitis and to assess how variants influence selected phenotypic traits. This cross-sectional study was carried out over 20 months in 99 children and adults with atopic dermatitis (median age 7 years). The following items were analysed: SCORAD, TEWL, ichthyosis vulgaris, presence of asthma, total IgE serum levels. The SPINK5 E420K SNP, the KLK7 4bp insertion polymorphism and the filaggrin mutants (R510X and 2282del4) were analysed as described previously. The control group for genetic analysis was recruited in an ethnically matched, phenotypically anonymous cohort (n=102). The allelic frequencies were 0.525 for SPINK5, 0.26 for KLK7 polymorphisms, 0.101 and 0.075 for 2282del4 and R501X FLG mutants, respectively. The association of atopic dermatitis with filaggrin variants was confirmed, but not that of SPINK5 or KLK7 polymorphisms. SCORAD and TEWL measurements were not influenced by any of the variants. The SPINK5 polymorphism was associated with high IgE serum levels (p=0.011). Abnormal barrier genes do not influence the severity of atopic dermatitis. The SPINK5 gene polymorphism may modulate systemic immune effects favouring the IgE response to atopens. TEWL does not allow the characterization of subsets of patients with or without abnormal barrier genes.

title: Loss-of-function variants of the filaggrin gene are associated with atopic eczema and associated phenotypes in Swedish families.
abstract: Recent studies have identified 2 loss-of-function variants, R501X and 2282del4, in the filaggrin gene as predisposing factors in the development of eczema. In this study, representing the first analysis of the variants in a Swedish population, we analysed transmission in 406 multiplex eczema families with mainly adult patients. In accordance with previous studies we found association between the filaggrin gene variants and atopic eczema (p=9.5 x 10(-8)). The highest odds ratio for the combined allele, 4.73 (1.98-11.29), p=3.6 x 10(-8), was found for the subgroup with a severe eczema phenotype, and association was also found with raised allergen-specific IgE, allergic asthma and allergic rhinoconjunctivitis occurring in the context of eczema. Our results support an important role for the filaggrin gene variants R501X and 2282del4 in the development and severity of atopic eczema and indicate a possible role for the subsequent progression into eczema-associated phenotypes.

title: Filaggrin loss-of-function mutations predispose to phenotypes involved in the atopic march.
abstract: Childhood eczema often precedes the development of asthma and allergic rhinitis in the so-called atopic march. Recently, 2 loss-of-function mutations in the gene encoding the epidermal barrier protein filaggrin were reported to be predisposing factors for eczema and concomitant asthma, suggesting a possible role in disease transition.



We aimed to assess the importance of filaggrin loss-of-function mutations in the susceptibility to eczema and associated clinical phenotypes.



The filaggrin mutations were genotyped and tested for association with allergic disorders in 2 large European populations including 1092 children with eczema.



Highly significant association of the filaggrin null mutations with eczema and concomitant asthma was replicated. Moreover, we found that these mutations predispose to asthma, allergic rhinitis, and allergic sensitization only in the presence of eczema. We show that the presence of 2 filaggrin null alleles is an independent risk factor for asthma in children with eczema, and that the 2 investigated mutations account for about 11% of eczema cases in the German population.



These results lend strong support to the role of filaggrin in the pathogenesis of eczema and in the subsequent progression along the atopic march. The fact that previous expression of eczema is a prerequisite for the manifestation of allergic airways disease and specific sensitization highlights the importance of the epidermal barrier in the pathogenesis of these disorders.



Our results suggest that the maintenance and repair of the epidermal barrier in infants with eczema may prevent the subsequent development of allergic airways disease.

title: Filaggrin mutations confer susceptibility to atopic dermatitis but not to asthma.
abstract: Loss-of-function mutations in the filaggrin gene (FLG) have been strongly associated with atopic dermatitis and allergic phenotypes in multiple populations. The role of these mutations in the development of asthma is less clear, particularly in patients who do not have coincident atopic dermatitis.



To determine whether FLG mutations are associated with asthma or asthma-related intermediate phenotypes.



We genotyped 2 loss-of-function FLG mutations (R501X and 2282del4) in white children (age 5-12 years) with mild to moderate asthma in the Childhood Asthma Management Program. We assessed the relationship of these mutations to asthma and allergy-related phenotypes in children with and without atopic dermatitis using both population-based and family-based tests of association.



Nearly 1/3 (185/646) of the participating children had atopic dermatitis. Although strong associations were observed between FLG mutations and atopic dermatitis (odds ratio, 2.4; P = 7.6 x 10(-5)) and between the mutations and total serum IgE level (P = .009 in the atopic dermatitis cohort), no association was noted with either asthma or asthma-related phenotypes, including FEV(1), FEV(1)/forced vital capacity, and methacholine PC(20) (P > .1 for all tests).



Although FLG loss-of-function mutations are consistently associated with atopic dermatitis and other allergic phenotypes, these mutations do not appear to influence either susceptibility to asthma or asthma severity phenotypes.



Filaggrin mutations that predispose to atopic dermatitis do not modulate the asthma phenotype.

title: Filaggrin null mutations are associated with increased asthma severity in children and young adults.
abstract: Filaggrin is a key protein involved in skin barrier function. Filaggrin (FLG) null mutations are important genetic predisposing factors for atopic disease.



To study the role of FLG null alleles in the clinical phenotype in children and young adults with asthma.



FLG mutations R501X and 2282del4 were assayed in 874 subjects 3 to 22 years old with asthma from Tayside. Lung function and disease severity were also studied.



The filaggrin mutations were significantly associated with greater disease severity for asthma. Independent of eczema, mean FEV(1)/forced vital capacity of FLG wild-type individuals differed from those carrying either FLG null allele (0.89 vs 0.86; P = .012). Individuals bearing FLG null alleles were more likely to be prescribed increased medication (chi(2) = 10.3; P = .001), with the homozygote null individuals having an odds ratio of 6.68 (95% CI, 1.7-27.0; P = .008) for being prescribed long-acting beta-agonists in addition to inhaled steroids. FLG null alleles were also associated with increased rescue medication use (P = .004). Individuals with asthma and with FLG null alleles were more likely to have eczema, and individuals with eczema tended to have more severe asthma; however, the association of FLG null alleles with all markers of asthma disease severity was similar in children with and without eczema.



FLG mutations are associated not only with eczema-associated asthma susceptibility but also with asthma severity independent of eczema status.



FLG status influences controller and reliever medication requirements in children and young adults with asthma.

title: Filaggrin null mutations are associated with increased asthma exacerbations in children and young adults
abstract: 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.

title: An interaction between filaggrin mutations and early food sensitization improves the prediction of childhood asthma.
abstract: Asthma prediction in early infancy is essential for the development of new preventive strategies. Loss-of-function mutations in the filaggrin gene (FLG) were identified as risk factors for eczema and associated asthma.



We evaluated the utility of the FLG mutations for the prediction of asthma.



Eight hundred seventy-one individuals of the prospective German Multicenter Allergy Study cohort were genotyped for 3 FLG mutations. Information on asthma, eczema, and food sensitization was available from birth to 13 years of age. Pulmonary function was measured from 7 to 13 years of age. The predictive value of the FLG mutations and of atopic phenotypes in infancy was assessed for asthma.



In infants with eczema and sensitization to food allergens, the FLG mutations predicted childhood asthma with a positive predictive value of 100% (95% CI, 65.5% to 100%). This subgroup was characterized by a significant decrease in pulmonary function until puberty and represented 8.1% of all asthmatic children and 19.1% of patients with asthma after infantile eczema. We found a strong synergistic interaction between the FLG-null alleles and early food sensitization in the disease transition from eczema to asthma (relative excess risk due to interaction, 2.64; 95% CI, 1.70-3.98; P = .00040).



FLG mutations and food sensitization represent 2 distinct mechanisms interacting in the pathogenesis of asthma. In infants with eczema and food sensitization, genotyping of the FLG mutations allows the prediction of asthma before the onset of symptoms. Our findings might facilitate the development of early subgroup-specific interventions to prevent the progression from eczema to asthma.

title: Association of Filaggrin loss‐of‐function‐mutations with atopic dermatitis and asthma in the Early Treatment of the Atopic Child (ETAC) population
abstract: Many candidate gene studies for atopic dermatitis (AD) and associated phenotypes have been conducted so far, but replication of significant results has been a major problem. Two loss of function polymorphisms FLG R501X‐ and 2282del4, in the Filaggrin (FLG) gene encoding for an epidermal barrier protein were recently identified. They were reported to be predisposing factors for AD and concomitant asthma. Several groups confirmed the initial results in independent populations. The aim of this study is to further investigate the importance of these FLG variants in the development of AD and subsequent asthma symptoms in pre‐school children, we investigated children and parents of the Early Treatment of the Atopic Child (ETAC)‐trial. We genotyped 496 children and 488 parents of the ETAC population for the two FLG variants, evaluating an association by family based analysis (transmission disequilibrium test). We found a highly significant association of the FLG null variants R501X‐ and 2282del4 with AD (combined genotype p < 0.0001) and asthma (combined genotype p < 0.0001). The replication and its statistical significance underlines the importance of the FLG polymorphisms and the importance of the skin barrier function in the development of AD and subsequent asthma.

title: Filaggrin Gene Defects Are Independent Risk Factors for Atopic Asthma in a Polish Population: A Study in ECAP Cohort
abstract: FLG null variants of which 2282del4 and R501X are the most frequent in Caucasians are established risk factors for atopic dermatitis (AD) with an effect probably mediated through impairment of epidermal barrier. Among subjects with AD FLG defects are also consistently associated with asthma and allergic rhinitis (AR) but it is less clear to what extent these associations are also present independently from skin disease. The aim of the present study was to evaluate the role of 2282del4 and R501X in predisposing to these allergic phenotypes in a Polish population.  2282del4 and R501X were typed among 3,802 participants of the Epidemiology of Allergic Diseases in Poland (ECAP) survey, a cross-sectional population-based study using ECRHS II and ISAAC questionnaires, and ambulatory examination.  The FLG null variants were associated with AD (OR = 2.01, CI: 1.20–3.36, P = 0.007), allergic rhinitis (in particular persistent form, OR = 1.69, CI:1.12–2.54, P = 0.011), and asthma (in particular atopic asthma, OR = 2.22, CI:1.24–3.96, P = 0.006). Association with atopic asthma (but not persistent allergic rhinitis) was also present in the absence of AD, (OR = 2.02, CI: 1.07–3.81, P = 0.027) as well as in the absence of AD and history of broadly defined inflammatory skin disease (OR = 2.30, CI: 1.07–4.93, P = 0.03). Association to atopic asthma would have not been found if diagnosis was made by questionnaire only (OR = 1.15, CI: 0.58–2.32, P = 0.8). We did not observe an association between FLG variants and allergic sensitizations (P = 0.8) or total IgE. (P = 0.6).  In a Polish population FLG 2282del4 and R501X carriage increases risk for development of AD and atopic asthma (also in the absence of AD or history thereof). This suggests that interventions aimed at restoring epidermal barrier may have a general role in asthma prophylaxis/treatment.

title: Meta-analysis of filaggrin polymorphisms in eczema and asthma: robust risk factors in atopic disease.
abstract: The discovery of filaggrin (FLG) null mutations as a major risk factor for eczema represents a milestone toward the understanding of an important mechanism in this complex disease. However, published studies demonstrate differences concerning design and effect size, and conflicting results for asthma have been reported.



We sought to provide a more accurate estimate of FLG effect sizes and to better refine FLG risk profiles within the broad and inclusive eczema diagnosis. We also sought to provide a more detailed and conclusive estimate of the risk for asthma associated with FLG null alleles.



We performed a meta-analysis of 24 studies on FLG mutations and eczema involving 5,791 cases, 26,454 control subjects, and 1,951 families as well as 17 studies on asthma involving 3,138 cases, 17,164 control subjects, and 1,511 offspring.



Both case-control and family studies showed strong associations with eczema. Case-control studies were heterogeneous, whereas family studies yielded more homogeneous results. Combined analysis showed that FLG haploinsufficiency strongly increases the eczema risk (odds ratio [OR], 3.12; 95% CI, 2.57-3.79) and is associated with more severe and dermatologist-diagnosed disease. FLG mutations are also significantly associated with asthma (OR, 1.48; 95% CI, 1.32-1.66). However, although strong effects for the compound phenotype asthma plus eczema (OR, 3.29; 95% CI, 2.84-3.82) were observed, there appears to be no association with asthma in the absence of eczema.



This meta-analysis summarizes the strong evidence for a high eczema risk conferred by FLG mutations and refines their risk profiles, suggesting an association with more severe and secondary care disease. FLG mutations are also a robust risk factor for asthma and might help define the asthma endophenotype linked with eczema.

title: Development and validation of the Mini Asthma Quality of Life Questionnaire.
abstract: The 32-item Asthma Quality of Life Questionnaire (AQLQ) has shown good responsiveness, reliability and construct validity; properties that are essential for use in clinical trials, clinical practice and surveys. However, to meet the needs of large clinical trials and long-term monitoring, where efficiency may take precedent over precision of measurement, the 15-item self-administered MiniAQLQ has been developed. The MiniAQLQ was tested in a 9-week observational study of 40 adults with symptomatic asthma. Patients completed the MiniAQLQ, the AQLQ, the Short Form (SF)-36, the Asthma Control Questionnaire and spirometry at baseline, 1, 5 and 9 weeks. In patients whose asthma was stable between clinic visits, reliability was very acceptable for the MiniAQLQ (intraclass correlation coefficient (ICC)=0.83), but not quite as good as for the AQLQ (ICC=0.95). Similarly, responsiveness in the MiniAQLQ (p=0.0007) was good but not quite so good as for the AQLQ (p<0.0001). Construct validity (correlation with other indices of health status) was strong for both the MiniAQLQ and the AQLQ. Criterion validity showed that there was no bias between the instruments (p=0.61) and the correlation between them was high (r=0.90). The Mini Asthma Quality of Life Questionnaire has good measurement properties but they are not quite as strong as those of the original Asthma Quality of Life Questionnaire. The choice of questionnaire should depend on the task at hand.

title: Adaptation of the Asthma Quality of Life Questionnaire to a second language preserves its critical properties: the Spanish version.
abstract: To test the metric proprieties of the Spanish version of the Juniper Asthma Quality of Life Questionnaire (AQLQ), we studied 116 adult asthmatic patients with a wide range of disease severity (53 patients were recruited from the respiratory outpatient department, 38 from a primary health care centre and 25 were patients admitted into hospital due to acute asthma). The patients were assessed twice, at recruitment and after 2 months. The AQLQ showed a high internal consistency (Cronbach's alpha = 0.78 to 0.96) and a high 2-week reproducibility (ICC = 0.82 to 0.92). Expected significant differences in AQLQ scores were observed according to disease severity as measured by symptoms, medication, use of services and recruitment setting. The cross-sectional and longitudinal correlations between AQLQ and the overall St. George's Respiratory Questionnaire were strong, moderate to strong between AQLQ and dyspnea and weak to moderate between AQLQ and FEV(1). The changes in AQLQ scores were significantly different in patients who either improved or deteriorated from those patients who remained stable (P <.0001 and P <.01, respectively, for the overall AQLQ). We conclude that the Spanish version of the AQLQ is reliable, valid and sensitive to changes.

title: Development of the asthma control test: a survey for assessing asthma control.
abstract: Asthma guidelines indicate that the goal of treatment should be optimum asthma control. In a busy clinic practice with limited time and resources, there is need for a simple method for assessing asthma control with or without lung function testing.



The objective of this article was to describe the development of the Asthma Control Test (ACT), a patient-based tool for identifying patients with poorly controlled asthma.



A 22-item survey was administered to 471 patients with asthma in the offices of asthma specialists. The specialist's rating of asthma control after spirometry was also collected. Stepwise regression methods were used to select a subset of items that showed the greatest discriminant validity in relation to the specialist's rating of asthma control. Internal consistency reliability was computed, and discriminant validity tests were conducted for ACT scale scores. The performance of ACT was investigated by using logistic regression methods and receiver operating characteristic analyses.



Five items were selected from regression analyses. The internal consistency reliability of the 5-item ACT scale was 0.84. ACT scale scores discriminated between groups of patients differing in the specialist's rating of asthma control (F = 34.5, P <.00001), the need for change in patient's therapy (F = 40.3, P <.00001), and percent predicted FEV(1) (F = 4.3, P =.0052). As a screening tool, the overall agreement between ACT and the specialist's rating ranged from 71% to 78% depending on the cut points used, and the area under the receiver operating characteristic curve was 0.77.



Results reinforce the usefulness of a brief, easy to administer, patient-based index of asthma control.

title: Validation of the Spanish Version of the Asthma Control Test (ACT)
abstract: Validation of the Spanish version of the Asthma Control Test (ACT).  A total of 607 asthmatic patients were assessed. The psychometric properties of ACT were evaluated. The ACT capacity to predict the physician's assessment of asthma control was assessed using the area under the receiving operating characteristics (ROC) curve (AUC), sensitivity, specificity, and positive-negative predictive values.  ACT's Cronbach α was 0.84. The intraclass correlation coefficient was 0.85. The AUC was 0.86, with a sensitivity of 71% and a specificity of 85% for a score of ≤19.  The Spanish version of ACT is shown to be a reliable and valid tool for evaluating and discriminating asthma control.

title: Assessing the Probability That a Positive Report is False: An Approach for Molecular Epidemiology Studies
abstract: Too many reports of associations between genetic variants and common cancer sites and other complex diseases are false positives. A major reason for this unfortunate situation is the strategy of declaring statistical significance based on a P value alone, particularly, any P value below.05. The false positive report probability (FPRP), the probability of no true association between a genetic variant and disease given a statistically significant finding, depends not only on the observed P value but also on both the prior probability that the association between the genetic variant and the disease is real and the statistical power of the test. In this commentary, we show how to assess the FPRP and how to use it to decide whether a finding is deserving of attention or "noteworthy." We show how this approach can lead to improvements in the design, analysis, and interpretation of molecular epidemiology studies. Our proposal can help investigators, editors, and readers of research articles to protect themselves from overinterpreting statistically significant findings that are not likely to signify a true association. An FPRP-based criterion for deciding whether to call a finding noteworthy formalizes the process already used informally by investigators--that is, tempering enthusiasm for remarkable study findings with considerations of plausibility.

title: Why do we not find polyps in the lungs? Bronchial mucosa as a model in the treatment of polyposis.
abstract: The link between lower and upper airways has been reported since the beginning of 1800 s. They share the same pseudostratified ciliated columnar epithelium lining and the concept of one airway, one disease is quite well widespread. Nasal polyposis and asthma share basically the same inflammatory process: predominant infiltration of eosinophils, mucus cell hyperplasia, edema, thickened basal membrane, polarization for Th2 cell immune response, similar pro-inflammatory mediators are increased, for example cysteinyl leukotrienes. If the lower and upper airways share a lot of common epithelial structural features so why is the edema in the nasal mucosa able to increase so much the size of the mucosa to the point of developing polyps? The article tries to underline some differences between the nasal and the bronchial mucosa that could be implicated in this aberrant change from normal mucosa to polyps. This paper creates the concept that there are no polyps with the features of nasal polyposis disease in the lower airway and through it is developed the hypothesis of the nasal polyps origin could partially lie on the difference between the upper and lower airway histology.

title: The immunology of asthma
abstract: Asthma is a common disease that affects 300 million people worldwide. Given the large number of eosinophils in the airways of people with mild asthma, and verified by data from murine models, asthma was long considered the hallmark T helper type 2 (TH2) disease of the airways. It is now known that some asthmatic inflammation is neutrophilic, controlled by the TH17 subset of helper T cells, and that some eosinophilic inflammation is controlled by type 2 innate lymphoid cells (ILC2 cells) acting together with basophils. Here we discuss results from in-depth molecular studies of mouse models in light of the results from the first clinical trials targeting key cytokines in humans and describe the extraordinary heterogeneity of asthma.

title: Causes of epidermal filaggrin reduction and their role in the pathogenesis of atopic dermatitis.
abstract: The epidermis protects human subjects from exogenous stressors and helps to maintain internal fluid and electrolyte homeostasis. Filaggrin is a crucial epidermal protein that is important for the formation of the corneocyte, as well as the generation of its intracellular metabolites, which contribute to stratum corneum hydration and pH. The levels of filaggrin and its degradation products are influenced not only by the filaggrin genotype but also by inflammation and exogenous stressors. Pertinently, filaggrin deficiency is observed in patients with atopic dermatitis regardless of filaggrin mutation status, suggesting that the absence of filaggrin is a key factor in the pathogenesis of this skin condition. In this article we review the various causes of low filaggrin levels, centralizing the functional and morphologic role of a deficiency in filaggrin, its metabolites, or both in the etiopathogenesis of atopic dermatitis.