Frances J.D. Smith

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.

First-in-human Mutation-targeted siRNA Phase Ib Trial of an Inherited Skin Disorder

The rare skin disorder pachyonychia congenita (PC) is an autosomal dominant syndrome that includes a disabling plantar keratoderma for which no satisfactory treatment is currently available. We have completed a phase Ib clinical trial for treatment of PC utilizing the first short-interfering RNA (siRNA)-based therapeutic for skin. This siRNA, called TD101, specifically and potently targets the keratin 6a (K6a) N171K mutant mRNA without affecting wild-type K6a mRNA. The safety and efficacy of TD101 was tested in a single-patient 17-week, prospective, double-blind, split-body, vehicle-controlled, dose-escalation trial. Randomly assigned solutions of TD101 or vehicle control were injected in symmetric plantar calluses on opposite feet. No adverse events occurred during the trial or in the 3-month washout period. Subjective patient assessment and physician clinical efficacy measures revealed regression of callus on the siRNA-treated, but not on the vehicle-treated foot. This trial represents the first time that siRNA has been used in a clinical setting to target a mutant gene or a genetic disorder, and the first use of siRNA in human skin. The callus regression seen on the patients siRNA-treated foot appears sufficiently promising to warrant additional studies of siRNA in this and other dominant-negative skin diseases.

The Molecular Genetics of Keratin Disorders

Keratins are the type I and II intermediate filament proteins which form a cytoskeletal network within all epithelial cells. They are expressed in pairs in a tissue- and differentiation-specific fashion. Epidermolysis bullosa simplex (EBS) was the first human disorder to be associated with keratin mutations. The abnormal keratin filament aggregates observed in basal cell keratinocytes of some EBS patients are composed of keratins K5 and K14. Dominant mutations in the genes encoding these proteins were shown to disrupt the keratin filament cytoskeleton resulting in cells that are less resilient and blister with mild physical trauma.Identification of mutations in other keratin genes soon followed with attention focussed on disorders showing abnormal clumping of keratin filaments in specific cells. For example, in bullous congenital ichthyosiform erythroderma, clumping of filaments in the suprabasal cells led to the identification of mutations in the suprabasal keratins, K1 and K10. Mutations have now been identified in 18 keratins, all of which produce a fragile cell phenotype. These include ichthyosis bullosa of Siemens (K2e), epidermolytic palmoplantar keratoderma (K1, K9), pachyonychia congenita (K6a, K6b, K16, K17), white sponge nevus (K4, K13), Meesmann’s corneal dystrophy (K3, K12), cryptogenic cirrhosis (K8, K18) and monilethrix (hHb6, hHb1).In general, these disorders are inherited as autosomal dominant traits and the mutations act in a dominant-negative manner. Therefore, treatment in the form of gene therapy is difficult, as the mutant gene needs to be inactivated. Ways of achieving this are actively being studied. Reliable mutation detection methods from genomic DNA are now available. This enables rapid screening of patients for keratin mutations. For some of the more severe phenotypes, prenatal diagnosis may be requested and this can now be performed from chorionic villus samples at an early stage of the pregnancy.This review article describes the discovery of, to date, mutations in 18 keratin genes associated with inherited human diseases.

The Phenotypic and Molecular Genetic Features of Pachyonychia Congenita

Pachyonychia congenita (PC) is an autosomal dominant genodermatosis caused by heterozygous mutations in any one of the genes encoding the differentiation-specific keratins K6a, K6b, K16, or K17. The main clinical features of the condition include painful and highly debilitating plantar keratoderma, hypertrophic nail dystrophy, oral leukokeratosis, and a variety of epidermal cysts. Although the condition has previously been subdivided into PC-1 and PC-2 subtypes, the phenotypic characterization of 1,000 mutation-verified PC patients enrolled in the International PC Research Registry, coordinated by the patient advocacy group PC Project, shows that there is considerable overlap between these subtypes. Thus, a new genotypic nomenclature is proposed, in which PC-6a represents a patient carrying a mutation in the K6a gene, etc. Although a rare disorder, PC represents a good model for therapy development, and international efforts are ongoing to develop and deliver siRNA, gene, correction, small molecule, and other strategies to treat this painful, disabling skin condition. The special relationship between PC Project and the PC research community has greatly accelerated the development pathway from gene identification to clinical trials in only a few years and represents a paradigm of hope for other orphan diseases.

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.

Development of Allele-Specific Therapeutic siRNA for Keratin 5 Mutations in Epidermolysis Bullosa Simplex

Epidermolysis bullosa simplex (EBS) is an incurable, inherited skin-blistering disorder predominantly caused by dominant-negative mutations in the genes encoding keratins K5 or K14. RNA interference, particularly in the form of small interfering RNA (siRNA), offers a potential therapy route for EBS and related keratin disorders by selectively silencing the mutant allele. Here, using a systemic screening system based on a luciferase reporter gene assay, we have developed mutant-specific siRNAs for two independent EBS-causing missense mutations in the K5 gene (p.Ser181Pro and p.Asn193Lys). The specificity of the allele-specific inhibitors identified in the screen was subsequently confirmed at the protein level, where the lead inhibitors were shown to strongly knock down the expression of mutant proteins with negligible effect on wild-type K5 expression. In a cell-based model system, the lead inhibitors were able to significantly reverse the cytoskeletal aggregation phenotype. Overall, this approach shows promise for the treatment of EBS and paves the way for future clinical trials.

A Large Mutational Study in Pachyonychia Congenita

Pachyonychia congenita (PC) is a rare autosomal dominant skin disorder characterized predominantly by nail dystrophy and painful palmoplantar keratoderma. Additional clinical features include oral leukokeratosis, follicular keratosis, and cysts (steatocysts and pilosebaceous cysts). PC is due to heterozygous mutations in one of four keratin genes, namely, KRT6A, KRT6B, KRT16, or KRT17. Here, we report genetic analysis of 90 new families with PC in which we identified mutations in KRT6A, KRT6B, KRT16, or KRT17, thereby confirming their clinical diagnosis. A total of 21 previously unreported and 22 known mutations were found. Approximately half of the kindreds had mutations in KRT6A (52%), 28% had mutations in KRT16, 17% in KRT17, and 3% of families had mutations in KRT6B. Most of the mutations were heterozygous missense or small in-frame insertion/deletion mutations occurring within one of the helix boundary motif regions of the keratin polypeptide. More unusual mutations included heterozygous splice site mutations, nonsense mutations, and a 1-bp insertion mutation, leading to a frameshift and premature termination codon. This study, together with previously reported mutations, identifies mutation hotspot codons that may be useful in the development of personalized medicine for PC.