Colin S. Munro

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.

Mutations in ATP2A2, encoding a Ca2+ pump, cause Darier disease

Darier disease (DD) is an autosomal-dominant skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization. Recently we constructed a 2.4-Mb, P1-derived artificial chromosome contig spanning the DD candidate region on chromosome 12q23-24.1. After screening several genes that mapped to this region, we identified mutations in the ATP2A2 gene, which encodes the sarco/endoplasmic reticulum Ca2+ -ATPase type 2 isoform (SERCA2) and is highly expressed in keratinocytes. Thirteen mutations were identified, including frameshift deletions, in-frame deletions or insertions, splice-site mutations and non-conservative missense mutations in functional domains. Our results demonstrate that mutations in ATP2A2 cause DD and disclose a role for this pump in a Ca2+-signalling pathway regulating cell-to-cell adhesion and differentiation of the epidermis.

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 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.

Genes, growth factors and acanthosis nigricans

Summary Acanthosis nigricans (AN) occurs most commonly in association with hyperinsulinaemia and more rarely as a paraneoplastic syndrome. It is also a feature of several genetic disorders. Indirect evidence suggests a role for tyrosine kinase growth factor receptor signalling in the pathogenesis of AN. Defects in the insulin receptor gene causing insulin resistance and AN are well recognized, but recent data in several other syndromes of this association, including lipodystrophic disorders, have identified causative defects in other pathways. The mechanism of AN due to insulin resistance is most probably direct or indirect activation of the insulin‐like growth factor 1 receptor by high levels of circulating insulin. However, more direct evidence for abnormal tyrosine kinase receptor signalling in AN has been provided by studies of craniosynostosis and skeletal dysplasia syndromes with AN, which have identified activating mutations in fibroblast growth factor receptors.

Treatment of psoriasis with intermittent short course cyclosporin (Neoral®). A multicentre study

A 1 year, prospective multicentre study was performed to investigate the efficacy and safety of intermittent treatment with cyclosporin in psoriasis vulgaris. Subjects received cyclosporin (Neoral(r)) 5mg/kg per day until achieving 90% reduction in area affected, or for a maximum of 12 weeks. Those failing to demonstrate a satisfactory response were withdrawn. When further treatment was required, cyclosporin was recommenced. This cycle was repeated up to three times. Psoriasis activity was recorded using the area affected and sign scores for erythema, scaling and infiltration. Overall assessments of response and tolerability were recorded.

Heterozygous Null Alleles in Filaggrin Contribute to Clinical Dry Skin in Young Adults and the Elderly

TO THE EDITOR Null mutations in the epidermal barrier protein filaggrin (FLG) cause ichthyosis vulgaris (IV, Smith et al., 2006) and are a major risk factor for eczema and other atopic diseases (Palmer et al., 2006; Baurecht et al., 2007; Brown et al., 2008a; Henderson et al., 2008). About 10% of European patients are heterozygous for such null mutations (Sandilands et al., 2006), but clinical details of their skin phenotype are sparse. A recent study of 811 English children reported that mild ichthyosis, palmar hyperlinearity, and keratosis pilaris were commoner in carriers of null mutations (Brown et al., 2008a). We report the effect of heterozygosity for FLG null alleles, and age, on the prevalence of simple subjective or objective clinical features of ‘‘dry skin’’ in adults and elderly patients not selected for inflammatory skin disease. Patients referred for diagnosis of a discrete skin lesion to dermatology clinics in Glasgow were recruited in two age cohorts: young adults 18–40 years (192 patients; 82 men, 110 women), and the elderly 60–75 years (99 patients; 55 men, 44 women). Patients were asked not to apply body moisturizers for 48 hours before their appointment; sunbed users or those who had been on a sunny holiday within 6 weeks were excluded. The protocol was approved by the South Glasgow Research Ethics Committee, conformed to the Declaration of Helsinki Principles, and written informed consent was obtained. The study took place in all months of the year, in a climate without extreme variation in humidity, and all assessments took place before genotyping. A questionnaire asked patients whether they considered they had dry skin (possible responses, coded 0–3, were not at all, a little, moderately, or a lot), sensitive skin (same responses) and how often they used a moisturizer for the body (never, now and again, more than once a week, or daily), and sought a history of childhood eczema or other diseases. Trained observers made objective assessments of visible fine scale and of roughness in each of three body sites (volar forearms, lower legs, and lower back), of keratosis pilaris (upper arms only), and of increased palmar markings and texture. A simple scoring scale for each parameter was used: absent (0), just perceptible (1), obvious (2), and marked (3). The total score of these parameters (maximum 27) was used as a summary measure of ‘‘dry skin’’. DNA, extracted from whole blood using standard procedures, was genotyped for four FLG null mutations common in the UK (R501X, 2282del4, R2447X, and S3247X) as described (Palmer et al., 2006; Sandilands et al., 2007) with minor refinements (Table S1). Statistical comparisons were made in 284 patients in whom all four mutations were successfully typed, between those wild-type sequence at all loci (wt) and patients heterozygous for only one of the mutations (null). Of 284, 252 (89%) patients were wild type for all four mutations; 29 (10.2%) were heterozygous for one null mutation, and 3 (1%) were compound heterozygotes, R501X/R2447X, 2282del4/ S3247X, and R2447X/S3247X. The frequency of these alleles was consistent with the previous UK data (Table S2; Sandilands et al., 2007; Brown et al., 2008b). Four patients in whom the assessors commented on marked scaling consistent with IV included all three compound heterozygotes; the fourth was heterozygous only for R501X but on biopsy showed a reduced granular layer, consistent with previous immunohistochemical analysis indicative of an undetected second null mutation (Sandilands et al., 2007); for financial reasons, this patient was not subjected to full sequencing. Questionnaire results are shown in Figure 1a and Table S3. Women were highly significantly more likely than men to consider they had dry skin (39 vs 11%) or use body moisturizers regularly (43 vs 7%). Heterozygous carriers of null mutations were more likely to think they had dry skin Abbreviation: FLG, filaggrin; IV, ichthyosis vulgaris

The Gene for Hypotrichosis of Marie Unna Maps between D8S258 and D8S298: Exclusion of the hr Gene by cDNA and Genomic Sequencing

Hypotrichosis of Marie Unna (MU) is an autosomal dominant hair-loss disorder with onset in childhood. A genomewide search for the gene was performed in a large Dutch family using 400 fluorescent microsatellite markers. Linkage was detected with marker D8S258, and analysis of this family and a further British kindred with additional markers in the region gave a combined maximum two-point LOD score of 13.42, with D8S560. Informative recombinants placed the MU gene in a 2.4-cM interval between markers D8S258 and D8S298. Recently, recessive mutations in the hr gene were reported in families with congenital atrichia, and this gene was previously mapped close to the MU interval. By radiation-hybrid mapping, we placed the hr gene close to D8S298 but were unable to exclude it from the MU interval. This, with the existence of the semidominant murine hr allele, prompted us to perform mutation analysis for this gene. Full-length sequencing of hr cDNA obtained from an affected individual showed no mutations. Similarly, screening of all exons of the hr gene amplified from the genomic DNA of an affected individual revealed no mutations. Analysis of expressed sequences and positional cloning of the MU locus is underway.

Mutations in AQP5, Encoding a Water-Channel Protein, Cause Autosomal-Dominant Diffuse Nonepidermolytic Palmoplantar Keratoderma

Autosomal-dominant diffuse nonepidermolytic palmoplantar keratoderma is characterized by the adoption of a white, spongy appearance of affected areas upon exposure to water. After exome sequencing, missense mutations were identified in AQP5, encoding water-channel protein aquaporin-5 (AQP5). Protein-structure analysis indicates that these AQP5 variants have the potential to elicit an effect on normal channel regulation. Immunofluorescence data reveal the presence of AQP5 at the plasma membrane in the stratum granulosum of both normal and affected palmar epidermis, indicating that the altered AQP5 proteins are trafficked in the normal manner. We demonstrate here a role for AQP5 in the palmoplantar epidermis and propose that the altered AQP5 proteins retain the ability to form open channels in the cell membrane and conduct water.