Frances J.D. Smith

Since the completion of the genome sequence of Saccharomyces cerevisiae in 1996 (refs 1, 2), there has been a large increase in complete genome sequences, accompanied by great advances in our understanding of genome evolution. Although little is known about the natural and life histories of yeasts in the wild, there are an increasing number of studies looking at ecological and geographic distributions, population structure and sexual versus asexual reproduction. Less well understood at the whole genome level are the evolutionary processes acting within populations and species that lead to adaptation to different environments, phenotypic differences and reproductive isolation. Here we present one- to fourfold or more coverage of the genome sequences of over seventy isolates of the baker's yeast S. cerevisiae and its closest relative, Saccharomyces paradoxus. We examine variation in gene content, single nucleotide polymorphisms, nucleotide insertions and deletions, copy numbers and transposable elements. We find that phenotypic variation broadly correlates with global genome-wide phylogenetic relationships. S. paradoxus populations are well delineated along geographic boundaries, whereas the variation among worldwide S. cerevisiae isolates shows less differentiation and is comparable to a single S. paradoxus population. Rather than one or two domestication events leading to the extant baker's yeasts, the population structure of S. cerevisiae consists of a few well-defined, geographically isolated lineages and many different mosaics of these lineages, supporting the idea that human influence provided the opportunity for cross-breeding and production of new combinations of pre-existing variations.

The Wellcome Trust Sanger Institute is one of the world's largest genome centers, and a substantial amount of our sequencing is performed with 'next-generation' massively parallel sequencing technologies: in June 2008 the quantity of purity-filtered sequence data generated by our Genome Analyzer (Illumina) platforms reached 1 terabase, and our average weekly Illumina production output is currently 64 gigabases. Here we describe a set of improvements we have made to the standard Illumina protocols to make the library preparation more reliable in a high-throughput environment, to reduce bias, tighten insert size distribution and reliably obtain high yields of data.

The Red Queen hypothesis proposes that coevolution of interacting species (such as hosts and parasites) should drive molecular evolution through continual natural selection for adaptation and counter-adaptation. Although the divergence observed at some host-resistance and parasite-infectivity genes is consistent with this, the long time periods typically required to study coevolution have so far prevented any direct empirical test. Here we show, using experimental populations of the bacterium Pseudomonas fluorescens SBW25 and its viral parasite, phage Phi2 (refs 10, 11), that the rate of molecular evolution in the phage was far higher when both bacterium and phage coevolved with each other than when phage evolved against a constant host genotype. Coevolution also resulted in far greater genetic divergence between replicate populations, which was correlated with the range of hosts that coevolved phage were able to infect. Consistent with this, the most rapidly evolving phage genes under coevolution were those involved in host infection. These results demonstrate, at both the genomic and phenotypic level, that antagonistic coevolution is a cause of rapid and divergent evolution, and is likely to be a major driver of evolutionary change within species.

Four distinct Plasmodium species are known to regularly infect humans: Plasmodium falciparum, P. vivax, P. malariae and P. ovale. The genome sequence of P. falciparum, the cause of the most severe type of human malaria, was completed in 2002 at the same time as the mosquito vector, Anopheles gambiae. In this week's Nature, which focuses on the malaria parasite, two further malaria genome sequences are described. First that of P. vivax, which contributes significant numbers to malaria incidence in humans, though in contrast to P. falciparum, the resulting disease is usually not fatal. The genome of this rather neglected species is presented together with a comparative analysis with the genomes of other Plasmodium species. Second, we publish the genome sequence of Plasmodium knowlesi. For long regarded as a monkey malaria parasite, it is increasingly becoming recognized as the fifth human-infecting Plasmodium species. In particular, it is prevalent in South East Asia where it is often misdiagnosed as another human malaria parasite P. malariae. As a model organism P. knowlesi stands out: not only is it a primate system, useful for work on vaccines, but it can be cultured in vitro and subjected to efficient transfection and gene knockouts. In a Review Article, Elizabeth Winzeler considers the progress made towards using the genome sequence to understand basic malaria parasite biology, and in particular the work on developing rational therapeutic approaches to combat P. falciparum infections. See also the Editorial. For a comprehensive collection of resources visit Nature's past malaria specials: Malaria killer blow ; Outlook on malaria ; Malaria web focus ; Malaria Insight ; Nature Medicine focus on malaria ; Focus on malaria Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the ‘kra’ monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia1,2. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated3, and it has a close phylogenetic relationship to Plasmodium vivax4, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or ‘hypnozoite’ in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone5) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome4 and other sequenced Plasmodium genomes6,7,8. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs9, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.

Eli Sprecher, Kathleen Green and colleagues show that biallelic mutations in DSG1 cause a syndrome featuring severe dermatitis, multiple allergies and metabolic wasting. The mutations abolish expression of desmoglein 1, resulting in loss of cell adhesion accompanied by increased expression of several allergy-related cytokines. The relative contribution of immunological dysregulation and impaired epithelial barrier function to allergic diseases is still a matter of debate. Here we describe a new syndrome featuring severe dermatitis, multiple allergies and metabolic wasting (SAM syndrome) caused by homozygous mutations in DSG1. DSG1 encodes desmoglein 1, a major constituent of desmosomes, which connect the cell surface to the keratin cytoskeleton and have a crucial role in maintaining epidermal integrity and barrier function. Mutations causing SAM syndrome resulted in lack of membrane expression of DSG1, leading to loss of cell-cell adhesion. In addition, DSG1 deficiency was associated with increased expression of a number of genes encoding allergy-related cytokines. Our deciphering of the pathogenesis of SAM syndrome substantiates the notion that allergy may result from a primary structural epidermal defect.

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 patient's siRNA-treated foot appears sufficiently promising to warrant additional studies of siRNA in this and other dominant-negative skin diseases.

Vici syndrome is a recessively inherited multisystem disorder characterized by callosal agenesis, cataracts, cardiomyopathy, combined immunodeficiency and hypopigmentation. To investigate the molecular basis of Vici syndrome, we carried out exome and Sanger sequence analysis in a cohort of 18 affected individuals. We identified recessive mutations in EPG5 (previously KIAA1632), indicating a causative role in Vici syndrome. EPG5 is the human homolog of the metazoan-specific autophagy gene epg-5, encoding a key autophagy regulator (ectopic P-granules autophagy protein 5) implicated in the formation of autolysosomes. Further studies showed a severe block in autophagosomal clearance in muscle and fibroblasts from individuals with mutant EPG5, resulting in the accumulation of autophagic cargo in autophagosomes. These findings position Vici syndrome as a paradigm of human multisystem disorders associated with defective autophagy and suggest a fundamental role of the autophagy pathway in the immune system and the anatomical and functional formation of organs such as the brain and heart.

Kindler syndrome is an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Linkage and homozygosity analysis in an isolated Panamanian cohort and in additional inbred families mapped the gene to 20p12.3. Loss-of-function mutations were identified in the FLJ20116 gene (renamed “KIND1” [encoding kindlin-1]). Kindlin-1 is a human homolog of the Caenorhabditis elegans protein UNC-112, a membrane-associated structural/signaling protein that has been implicated in linking the actin cytoskeleton to the extracellular matrix (ECM). Thus, Kindler syndrome is, to our knowledge, the first skin fragility disorder caused by a defect in actin-ECM linkage, rather than keratin-ECM linkage. Kindler syndrome is an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Linkage and homozygosity analysis in an isolated Panamanian cohort and in additional inbred families mapped the gene to 20p12.3. Loss-of-function mutations were identified in the FLJ20116 gene (renamed “KIND1” [encoding kindlin-1]). Kindlin-1 is a human homolog of the Caenorhabditis elegans protein UNC-112, a membrane-associated structural/signaling protein that has been implicated in linking the actin cytoskeleton to the extracellular matrix (ECM). Thus, Kindler syndrome is, to our knowledge, the first skin fragility disorder caused by a defect in actin-ECM linkage, rather than keratin-ECM linkage.

Filaggrin is a key protein involved in skin barrier function. Recently, mutations in the filaggrin gene, FLG, were identified in European families with ichthyosis vulgaris (IV) and shown to be an important predisposing factor for atopic dermatitis (AD).To study the role of FLG mutations in IV/AD in Japan.The known filaggrin mutations were studied by genotyping and new mutations identified by DNA sequencing.The European-specific mutations R501X and 2282del4 were absent from 253 Japanese individuals. We therefore sequenced the FLG gene in 4 Japanese families with IV and identified 2 novel mutations, 3321delA and S2554X. Immunohistologic and ultrastructural observations indicated that both truncation mutations lead to a striking reduction of keratohyalin granules in the epidermis. We screened 143 Japanese patients with AD for these FLG null mutations and identified them in 8 patients with AD (5.6%), including S2554X in 6 patients (4.2%) and 3321delA in 2 patients (1.4%). Both null variants were absent from 156 unrelated Japanese nonatopic and nonichthyotic controls, giving a significant statistical association between the FLG mutations and AD (chi(2)P value, .0015). This is the first report of FLG mutations in a non-European population.Our data indicate that FLG mutations in Japan are unique from those found in European-origin populations.Filaggrin null variants are also significant predisposing factors for AD in Japan and, on the basis of the recent European studies, may predict a more severe and persistent form of atopy.

Mutations in the filament aggregating protein (filaggrin) gene have recently been identified as the cause of the common genetic skin disorder ichthyosis vulgaris (IV), the most prevalent inherited disorder of keratinization. The main characteristics of IV are fine-scale on the arms and legs, palmar hyperlinearity, and keratosis pilaris. Here, we have studied six Irish families with IV for mutations in filaggrin. We have identified a new mutation, 3702delG, in addition to further instances of the reported mutations R501X and 2282del4, which are common in people of European origin. A case of a 2282del4 homozygote was also identified. Mutation 3702delG terminates protein translation in filaggrin repeat domain 3, whereas both recurrent mutations occur in repeat 1. These mutations are semidominant: heterozygotes have an intermediate phenotype most readily identified by palmar hyperlinearity and in some cases fine-scale and/or keratosis pilaris, whereas homozygotes or compound heterozygotes generally have more marked ichthyosis. Interestingly, the phenotypes of individuals homozygous for R501X, 2282del4, or compound heterozygous for R501X and 3702delG, were comparable, suggesting that mutations located centrally in the filaggrin repeats are also pathogenic.

BackgroundFilaggrin is a key protein involved in skin barrier function. Filaggrin (FLG) null mutations are important genetic predisposing factors for atopic disease.ObjectiveTo study the role of FLG null alleles in the clinical phenotype in children and young adults with asthma.MethodsFLG 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.ResultsThe filaggrin mutations were significantly associated with greater disease severity for asthma. Independent of eczema, mean FEV1/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 (χ2 = 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 β-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.ConclusionFLG mutations are associated not only with eczema-associated asthma susceptibility but also with asthma severity independent of eczema status.Clinical implicationsFLG status influences controller and reliever medication requirements in children and young adults with asthma. 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 FEV1/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 (χ2 = 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 β-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.

Pachyonychia congenita (PC) is a rare genodermatosis affecting the nails, skin, oral mucosae, larynx, hair, and teeth. Pathogenic mutations in keratins K6a or K16 are associated with the PC-1 phenotype whereas K6b and K17 mutations are associated with the PC-2 phenotype. Analysis of clinical, pathological, and genetic data from the literature and two research registries reveal that >97% of PC cases exhibit fingernail and toenail thickening, and painful plantar keratoderma. Prospective evaluation of 57 PC patients from 41 families revealed variable clinical findings: hyperhidrosis (79%), oral leukokeratosis (75%), follicular keratosis (65%), palmar keratoderma (60%), cutaneous cysts (35%), hoarseness or laryngeal involvement (16%), coarse or twisted hair (26%), early primary tooth loss (14%), and presence of natal or prenatal teeth (2%). Stratification of these data by keratin mutation confirmed the increased incidence of cyst formation and natal teeth among PC-2 patients, although cysts were more commonly seen in PC-1 than previously reported (25%-33%). Previously unreported clinical features of PC include development of painful oral and nipple lesions during breastfeeding, copious production of waxy material in ears, and inability to walk without an ambulatory aid (50%). Possible pathogenic mechanisms are discussed with respect to the clinicopathologic and genetic correlations observed.

Pachyonychia congenita type 2 (PC-2; Jackson-Lawler syndrome) is an autosomal dominant disorder characterized by hypertrophic nail dystrophy, mild focal keratoderma, multiple pilosebaceous cysts and other features of ectodermal dysplasia. Keratin 17 (K17) is a differentiation-specific keratin expressed in the nail bed, hair follicle, sebaceous gland and other epidermal appendages. Previously, we have demonstrated that PC-2 is caused by mutations in K17 and that similar mutations in this gene can present as steatocystoma multiplex with little or no nail dystrophy. Here, we describe three unrelated kindreds carrying K17 mutations. Two of these families have identical missense mutations (R94C) in the 1A domain of K17. However, while affected members of one kindred have the classical features of PC-2, affected persons in the other family have the steatocystoma multiplex phenotype. In a third family with PC-2, mutation N92S was detected, bringing the total number of distinct mutations reported in K17 thus far to 11. These results demonstrate that K17 mutations commonly underlie both PC-2 and steatocystoma multiplex and that the alternate phenotypes which arise from these genetic lesions in K17 are independent of the specific mutation involved.

Nuclear genetic disorders causing mitochondrial DNA (mtDNA) depletion are clinically and genetically heterogeneous, and the molecular etiology remains undiagnosed in the majority of cases. Through whole-exome sequencing, we identified recessive nonsense and splicing mutations in <i>FBXL4</i> segregating in three unrelated consanguineous kindreds in which affected children present with a fatal encephalopathy, lactic acidosis, and severe mtDNA depletion in muscle. We show that FBXL4 is an F-box protein that colocalizes with mitochondria and that loss-of-function and splice mutations in this protein result in a severe respiratory chain deficiency, loss of mitochondrial membrane potential, and a disturbance of the dynamic mitochondrial network and nucleoid distribution in fibroblasts from affected individuals. Expression of the wild-type <i>FBXL4</i> transcript in cell lines from two subjects fully rescued the levels of mtDNA copy number, leading to a correction of the mitochondrial biochemical deficit. Together our data demonstrate that mutations in <i>FBXL4</i> are disease causing and establish FBXL4 as a mitochondrial protein with a possible role in maintaining mtDNA integrity and stability.

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.

Severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome is a recently recognized syndrome caused by mutations in the desmoglein 1 gene (DSG1). To date, only 3 families have been reported.We studied a new case of SAM syndrome known to have no mutations in DSG1 to detail the clinical, histopathologic, immunofluorescent, and ultrastructural phenotype and to identify the underlying molecular mechanisms in this rare genodermatosis.Histopathologic, electron microscopy, and immunofluorescent studies were performed. Whole-exome sequencing data were interrogated for mutations in desmosomal and other skin structural genes, followed by Sanger sequencing of candidate genes in the patient and his parents.No mutations were identified in DSG1; however, a novel de novo heterozygous missense c.1757A>C mutation in the desmoplakin gene (DSP) was identified in the patient, predicting the amino acid substitution p.His586Pro in the desmoplakin polypeptide.SAM syndrome can be caused by mutations in both DSG1 and DSP. Knowledge of this genetic heterogeneity is important for both analysis of patients and genetic counseling of families. This condition and these observations reinforce the importance of heritable skin barrier defects, in this case desmosomal proteins, in the pathogenesis of atopic disease.

Scope Resistance of proteins to gastrointestinal digestion may play a role in determining immune‐mediated adverse reactions to foods. However, digestion studies have largely been restricted to purified proteins and the impact of food processing and food matrices on protein digestibility is poorly understood. Methods and results Digestibility of a total gliadin fraction (TGF), flour (cv Hereward), and bread was assessed using in vitro batch digestion with simulated oral, gastric, and duodenal phases. Protein digestion was monitored by SDS‐PAGE and immunoblotting using monoclonal antibodies specific for celiac‐toxic sequences (QQSF, QPFP) and starch digestion by measuring undigested starch. Whereas the TGF was rapidly digested during the gastric phase the gluten proteins in bread were virtually undigested and digested rapidly during the duodenal phase only if amylase was included. Duodenal starch digestion was also slower in the absence of duodenal proteases. Conclusion The baking process reduces the digestibility of wheat gluten proteins, including those containing sequences active in celiac disease. Starch digestion affects the extent of protein digestion, probably because of gluten‐starch complex formation during baking. Digestion studies using purified protein fractions alone are therefore not predictive of digestion in complex food matrices.

Type I and type II keratins form the heteropolymeric intermediate filament cytoskeleton, which is the main stress-bearing structure within epithelial cells. Pachyonychia congenita (PC) is a group of autosomal dominant disorders whose most prominent phenotype is hypertrophic nail dystrophy accompanied by other features of ectodermal dysplasia. It has been shown previously that mutations in either K16 or K6a, which form a keratin expression pair, produce the PC-1 variant (MIM 184510). Mutations in K17 alone, an unpaired accessory keratin, result in the PC-2 phenotype (MIM 184500). Here, we describe a family with PC-2 in which the K17 locus on 17q was excluded and linkage to the type II keratin locus on 12q was obtained (Z max 3.31 at straight theta = 0). Mutation analysis of candidate keratins revealed the first reported missense mutation in K6b, implying that this keratin is the previously unknown expression partner of K17, analogous to the K6a/K16 pair. Co-expression of these genes was confirmed by in situ hybridization and immunohistochemical staining. These results reveal the hitherto unknown role of the K6b isoform in epithelial biology, as well as genetic heterogeneity in PC-2.

Prevalent mutations in the FLG gene underlie the common skin disorder ichthyosis vulgaris and are significant risk factors for atopic dermatitis (eczema). The recent publication of a strategy to sequence this difficult gene identifies a spectrum of both prevalent and rare mutations that collectively have a significant impact on susceptibility to atopic disease.

The field of science and medicine has experienced a flood of data and technology associated with the human genome project. Over 10,000 human diseases have been genetically defined, but little progress has been made with respect to the clinical application of this knowledge. A notable exception to this exists for pachyonychia congenita (PC), a rare, dominant-negative keratin disorder. The establishment of a non-profit organization, PC Project, has led to an unprecedented coalescence of patients, scientists, and physicians with a unified vision of developing novel therapeutics for PC. Utilizing the technological by-products of the human genome project, such as RNA interference (RNAi) and quantitative RT-PCR (qRT-PCR), physicians and scientists have collaborated to create a candidate siRNA therapeutic that selectively inhibits a mutant allele of KRT6A, the most commonly affected PC keratin. In vitro investigation of this siRNA demonstrates potent inhibition of the mutant allele and reversal of the cellular aggregation phenotype. In parallel, an allele-specific quantitative real-time RT-PCR assay has been developed and validated on patient callus samples in preparation for clinical trials. If clinical efficacy is ultimately demonstrated, this “first-in-skin” siRNA may herald a paradigm shift in the treatment of dominant-negative genetic disorders.

RNA interference offers a novel approach for developing therapeutics for dominant-negative genetic disorders. The ability to inhibit expression of the mutant allele without affecting wild-type gene expression could be a powerful new treatment option. Targeting the single-nucleotide keratin 6a (K6a) N171K mutation responsible for the rare monogenic skin disorder pachyonychia congenita (PC), we demonstrate that small interfering RNAs (siRNAs) can potently and selectively block expression of mutant K6a. To test whether lead siRNAs could discriminate mutant mRNA in the presence of both wild-type and mutant forms, a dominant-negative PC cell culture model was developed. As predicted for a dominant-negative disease, simultaneous expression of both wild-type and mutant K6a resulted in defective keratin filament formation. Addition of mutant-specific siRNAs allowed normal filament formation, suggesting selective inhibition of mutant K6a. The effectiveness of our siRNA in skin was tested by co-delivering a firefly luciferase/mutant K6a bicistronic reporter construct and mutant-specific siRNAs to mouse footpads. Potent inhibition of the fluorescent reporter was demonstrated using the Xenogen IVIS200 <i>in vivo</i> imaging system. Additionally, wild type-specific siRNAs knocked down the expression of pre-existing endogenous K6a in human keratinocytes. These results suggest that efficient delivery of these "designer siRNAs" may allow effective treatment of numerous genetic disorders including PC.

The Gram-negative bacterium Photorhabdus asymbiotica (Pa) has been recovered from human infections in both North America and Australia. Recently, Pa has been shown to have a nematode vector that can also infect insects, like its sister species the insect pathogen P. luminescens (Pl). To understand the relationship between pathogenicity to insects and humans in Photorhabdus we have sequenced the complete genome of Pa strain ATCC43949 from North America. This strain (formerly referred to as Xenorhabdus luminescens strain 2) was isolated in 1977 from the blood of an 80 year old female patient with endocarditis, in Maryland, USA. Here we compare the complete genome of Pa ATCC43949 with that of the previously sequenced insect pathogen P. luminescens strain TT01 which was isolated from its entomopathogenic nematode vector collected from soil in Trinidad and Tobago.We found that the human pathogen Pa had a smaller genome (5,064,808 bp) than that of the insect pathogen Pl (5,688,987 bp) but that each pathogen carries approximately one megabase of DNA that is unique to each strain. The reduced size of the Pa genome is associated with a smaller diversity in insecticidal genes such as those encoding the Toxin complexes (Tc's), Makes caterpillars floppy (Mcf) toxins and the Photorhabdus Virulence Cassettes (PVCs). The Pa genome, however, also shows the addition of a plasmid related to pMT1 from Yersinia pestis and several novel pathogenicity islands including a novel Type Three Secretion System (TTSS) encoding island. Together these data suggest that Pa may show virulence against man via the acquisition of the pMT1-like plasmid and specific effectors, such as SopB, that promote its persistence inside human macrophages. Interestingly the loss of insecticidal genes in Pa is not reflected by a loss of pathogenicity towards insects.Our results suggest that North American isolates of Pa have acquired virulence against man via the acquisition of a plasmid and specific virulence factors with similarity to those shown to play roles in pathogenicity against humans in other bacteria.

Palmoplantar keratodermas (PPKs) are a group of disorders that are diagnostically and therapeutically problematic in dermatogenetics. Punctate PPKs are characterized by circumscribed hyperkeratotic lesions on the palms and soles with considerable heterogeneity. In 18 families with autosomal dominant punctate PPK, we report heterozygous loss-of-function mutations in AAGAB, encoding α- and γ-adaptin-binding protein p34, located at a previously linked locus at 15q22. α- and γ-adaptin-binding protein p34, a cytosolic protein with a Rab-like GTPase domain, was shown to bind both clathrin adaptor protein complexes, indicating a role in membrane trafficking. Ultrastructurally, lesional epidermis showed abnormalities in intracellular vesicle biology. Immunohistochemistry showed hyperproliferation within the punctate lesions. Knockdown of AAGAB in keratinocytes led to increased cell division, which was linked to greatly elevated epidermal growth factor receptor (EGFR) protein expression and tyrosine phosphorylation. We hypothesize that p34 deficiency may impair endocytic recycling of growth factor receptors such as EGFR, leading to increased signaling and cellular proliferation.

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.

Keratin 9 (K9) is a type I intermediate filament protein whose expression is confined to the suprabasal layers of the palmoplantar epidermis. Although mutations in the K9 gene are known to cause epidermolytic palmoplantar keratoderma, a rare dominant-negative skin disorder, its functional significance is poorly understood. To gain insight into the physical requirement and importance of K9, we generated K9-deficient (Krt9−/−) mice. Here, we report that adult Krt9−/−mice develop calluses marked by hyperpigmentation that are exclusively localized to the stress-bearing footpads. Histological, immunohistochemical, and immunoblot analyses of these regions revealed hyperproliferation, impaired terminal differentiation, and abnormal expression of keratins K5, K14, and K2. Furthermore, the absence of K9 induces the stress-activated keratins K6 and K16. Importantly, mice heterozygous for the K9-null allele (Krt9+/−) show neither an overt nor histological phenotype, demonstrating that one Krt9 allele is sufficient for the developing normal palmoplantar epidermis. Together, our data demonstrate that complete ablation of K9 is not tolerable in vivo and that K9 is required for terminal differentiation and maintaining the mechanical integrity of palmoplantar epidermis. Keratin 9 (K9) is a type I intermediate filament protein whose expression is confined to the suprabasal layers of the palmoplantar epidermis. Although mutations in the K9 gene are known to cause epidermolytic palmoplantar keratoderma, a rare dominant-negative skin disorder, its functional significance is poorly understood. To gain insight into the physical requirement and importance of K9, we generated K9-deficient (Krt9−/−) mice. Here, we report that adult Krt9−/−mice develop calluses marked by hyperpigmentation that are exclusively localized to the stress-bearing footpads. Histological, immunohistochemical, and immunoblot analyses of these regions revealed hyperproliferation, impaired terminal differentiation, and abnormal expression of keratins K5, K14, and K2. Furthermore, the absence of K9 induces the stress-activated keratins K6 and K16. Importantly, mice heterozygous for the K9-null allele (Krt9+/−) show neither an overt nor histological phenotype, demonstrating that one Krt9 allele is sufficient for the developing normal palmoplantar epidermis. Together, our data demonstrate that complete ablation of K9 is not tolerable in vivo and that K9 is required for terminal differentiation and maintaining the mechanical integrity of palmoplantar epidermis. epidermolytic palmoplantar keratoderma keratin 9 K9-deficient small interfering RNA

We studied a kindred with recessive epidermolysis bullosa simplex in which the affected members lacked expression of the basal cell keratin 14. The patients had severe generalized skin blistering that improved slightly with age. The basal cells of the patients did not express keratin 14 and contained no keratin intermediate filaments. The expression of keratin 5, the obligate copolymer of keratin 14, was slightly reduced. The expression of keratin 15, the alternative basal cell keratin, was increased, suggesting upregulation or stabilization to compensate for the lack of keratin 14. The expression of keratin 16, keratin 17, and keratin 19 in the patient's skin was not different from controls. Immunoelectron microscopy showed a loose network of keratin 5/keratin 15 protofilaments in the basal cells. Keratin 15 filaments did not aggregate into higher order bundles. Sequence analysis of genomic DNA revealed a homozygous mutation in the 3'-acceptor splice site of intron 1 (1840 A-->C) in the affected individuals. This mutation led to the skipping of exon 2 in 24% of the KRT14 transcripts and to the use of a cryptic splice site in 76% of the transcripts. Premature termination codons were generated in all transcripts (codons 175+1 or 175+29), leading to a truncated keratin 14 protein within the helical 1B rod domain. The disorder was associated with circumscribed hyperkeratotic lesions with the histology of epidermolytic hyperkeratosis. The prognosis of keratin 14 ablation is much better in the human than in the mouse.

Recent progress in understanding the molecular organization of the cutaneous basement membrane zone (BMZ) has revealed an intricate network of structural proteins necessary for stable association of the epidermis to the underlying dermis. Molecular genetics of the cutaneous BMZ has also revealed that defects in as many as nine distinct genes within the dermal-epidermal junction which result in different forms of epidermolysis bullosa (EB), a group of heritable mechano-bullous disorders. We have recently demonstrated that a variant of EB associated with late-onset development of muscular dystrophy (EB-MD, MIM no. 226670) results from mutations in the gene encoding plectin (PLEC1), a cytoskeleton associated attachment protein present in the hemidesmosomal inner plaque and the sarcolemma of the muscle. Consequently, mutations in this multi-functional gene/protein system can result in phenotypic manifestations of EB-MD both in the skin and the muscle. In this overview, we will summarize the domain organization of plectin and the structure of the corresponding gene (PLEC1), as well as the genetic basis of EB-MD in families studied thus far. Elucidation of the molecular basis of this subtype of EB adds to our understanding of the structural and functional complexity of the cutaneous BMZ.

The striate form of palmoplantar keratoderma is a rare autosomal dominant disorder affecting palm and sole skin. Genetic heterogeneity of striate palmoplantar keratoderma has been demonstrated with pathogenic mutations in the desmosomal proteins desmoplakin and desmoglein 1. We have studied a four-generation family of British descent with striate palmoplantar keratoderma. Ultrastructural studies show that intermediate filaments of suprabasal keratinocytes are finer than those of the basal layer. In addition, desmosome numbers are normal, but their inner plaques and midline structures are attenuated. Microsatellite markers were used to screen candidate loci including the epidermal differentiation complex on 1q, the desmoplakin locus on 6p, the type I and II keratin gene clusters on chromosomes 12q and 17q, and the desmosomal cadherin gene cluster on chromosome 18q. Significant genetic linkage to chromosome 12q was observed using marker D12S368, with a maximum two-point lod score of 3.496 at a recombination fraction of 0. Direct sequencing of the keratin 1 gene revealed a frameshift mutation in exon 9 that leads to the partial loss of the glycine loop motif in the V2 domain and the gain of a novel 70 amino acid peptide. Using expression studies we show that the V2 domain is essential for normal function of keratin intermediate filaments.

In 1994, the molecular basis of pachyonychia congenita (PC) was elucidated. Four keratin genes are associated with the major subtypes of PC: K6a or K16 defects cause PC-1; and mutations in K6b or K17 cause PC-2. Mutations in keratins, the epithelial-specific intermediate filament proteins, result in aberrant cytoskeletal networks which present clinically as a variety of epithelial fragility phenotypes. To date, mutations in 20 keratin genes are associated with human disorders. Here, we review the genetic basis of PC and report 30 new PC mutations. Of these, 25 mutations were found in PC-1 families and five mutations were identified in PC-2 kindreds. All mutations identified were heterozygous amino acid substitutions or small in-frame deletion mutations with the exception of an unusual mutation in a sporadic case of PC-1. The latter carried a 117 bp duplication resulting in a 39 amino acid insertion in the 2B domain of K6a. Also of note was mutation L388P in K17, which is the first genetic defect identified in the helix termination motif of this protein. Understanding the genetic basis of these disorders allows better counseling for patients and paves the way for therapy development.

Clouston syndrome (hidrotic ectodermal dysplasia) is an autosomal dominant ectodermal dysplasia characterized by alopecia, palmoplantar hyperkeratosis, and nail dystrophy. Recently, mutations in the <i>GJB6</i> gene encoding the gap junction protein connexin 30 have been shown to cause this disorder. To date, all mutations have involved two codons: G11R and A88V. Here, we report a novel mutation V37E within the first transmembrane domain of connexin 30 in a spontaneous case of Clouston syndrome. The mutation was detected in genomic DNA, confirmed in reverse transcription polymerase chain reaction products, and was excluded from 100 ethnically matched control individuals by restriction enzyme analysis.

Kindler syndrome (OMIM 173650) is a rare autosomal recessive disorder characterized by trauma-induced blister formation (especially in childhood) and photosensitivity. Other features include mucocutaneous scarring and progressive poikiloderma. There is also an increased risk of skin and mucous membrane malignancy. The disorder was recently mapped to 20p12.3 and pathogenic mutations were identified in a new gene, KIND1. This gene encodes a 677 amino acid protein, kindlin-1, a component of focal contacts in keratinocytes. In this study, we identified four new recurrent mutations in KIND1 in 16 individuals with Kindler syndrome from 13 families of Pakistani (676insC), UK Caucasian (E304X), Omani (W616X), or Italian (958–1G > A) origins. Haplotype analysis demonstrated common ancestral mutant alleles for each mutation, apart from one of the six Pakistani families in which the mutation 676insC (which occurs in a repeat of seven cytosines) was present on a different genetic background. All mutations were homozygous, apart from the three UK Caucasian cases that were all compound heterozygotes (second allele mutations: L302X, 1161delA, 1909delA). All mutations were associated with markedly reduced or absent skin immunostaining with an antikindlin-1 antibody. These loss-of-function KIND1 mutations demonstrate the importance of kindlin-1 in maintaining epithelial integrity, although the mechanism linking this mutant protein to photosensitivity and poikiloderma remains to be determined. Delineation of these recurrent mutations is also relevant to optimizing mutation detection strategies in Kindler syndrome patients from particular ethnic backgrounds. Kindler syndrome (OMIM 173650) is a rare autosomal recessive disorder characterized by trauma-induced blister formation (especially in childhood) and photosensitivity. Other features include mucocutaneous scarring and progressive poikiloderma. There is also an increased risk of skin and mucous membrane malignancy. The disorder was recently mapped to 20p12.3 and pathogenic mutations were identified in a new gene, KIND1. This gene encodes a 677 amino acid protein, kindlin-1, a component of focal contacts in keratinocytes. In this study, we identified four new recurrent mutations in KIND1 in 16 individuals with Kindler syndrome from 13 families of Pakistani (676insC), UK Caucasian (E304X), Omani (W616X), or Italian (958–1G > A) origins. Haplotype analysis demonstrated common ancestral mutant alleles for each mutation, apart from one of the six Pakistani families in which the mutation 676insC (which occurs in a repeat of seven cytosines) was present on a different genetic background. All mutations were homozygous, apart from the three UK Caucasian cases that were all compound heterozygotes (second allele mutations: L302X, 1161delA, 1909delA). All mutations were associated with markedly reduced or absent skin immunostaining with an antikindlin-1 antibody. These loss-of-function KIND1 mutations demonstrate the importance of kindlin-1 in maintaining epithelial integrity, although the mechanism linking this mutant protein to photosensitivity and poikiloderma remains to be determined. Delineation of these recurrent mutations is also relevant to optimizing mutation detection strategies in Kindler syndrome patients from particular ethnic backgrounds. Kindler syndrome Kindler syndrome (KS) was reported first in 1954 as an unusual inherited skin condition in a 14-y-old girl with trauma-induced blister formation, nail dystrophy, progressive poikiloderma, hyperkeratosis of the palms and soles, webbing between toes and fingers, and sensitivity to sunlight (Kindler, 1954Kindler T. Congenital poikiloderma with traumatic bulla formation and progressive cutaneous atrophy.Br J Dermatol. 1954; 66: 104-111Crossref PubMed Scopus (157) Google Scholar). A number of similar patients have subsequently been identified who have helped define KS as a distinct autosomal recessive disorder (OMIM 173650). Many of the clinical features of blistering and scarring in children with KS resemble those seen in the inherited skin disorder dystrophic epidermolysis bullosa, and the underlying skin pathology often involves morphologically and structurally similar disruption of skin adhesion at the dermal–epidermal junction (Fine et al., 2000Fine J.D. Eady R.A. Bauer E.A. et al.Revised classification system for inherited epidermolysis bullosa. Report of the Second International Consensus Meeting on diagnosis and classification of epidermolysis bullosa.J Am Acad Dermatol. 2000; 42: 1051-1066Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). The type VII collagen gene, COL7A1, however, which is mutated in dystrophic epidermolysis bullosa, has been excluded as a candidate gene for KS (Shimizu et al., 1997Shimizu H. Sato M. Ban M. et al.Immunohistochemical, ultrastructural, and molecular features of Kindler syndrome distinguish it from dystrophic epidermolysis bullosa.Arch Dermatol. 1997; 133: 1111-1117Crossref PubMed Google Scholar;Yasukawa et al., 2002Yasukawa K. Sato-Matsumura K.C. McMillan J. Tsuchiya K. Shimizu H. Exclusion of COL7A1 mutation in Kindler syndrome.J Am Acad Dermatol. 2002; 46: 447-450Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Moreover, transmission electron microscopy in KS may reveal additional pathologic findings including blister formation within basal keratinocytes, somewhat similar to cases of epidermolysis bullosa simplex, a disorder known to result from mutations in the genes encoding the intermediate filament proteins, keratins 5 or 14 (Irvine and McLean, 1999Irvine A.D. McLean W.H.I. Human keratin diseases: The increasing spectrum of disease and subtlety of the phenotype–genotype correlation.Br J Dermatol. 1999; 140: 815-828Crossref PubMed Scopus (322) Google Scholar). Alternatively, there may be features of reduplication of the lamina densa, or variable and inconsistent changes, including multiple planes of cleavage as well as alterations in elastic fibers (Hovnanian et al., 1989Hovnanian A. Blanchet-Bardon C. de Prost Y. Poikiloderma of Theresa Kindler: Report of a case with ultrastructural study, and review of the literature.Pediatr Dermatol. 1989; 6: 82-90Crossref PubMed Scopus (55) Google Scholar). Similar to patients with severe forms of epidermolysis bullosa, there is an increased risk of mucocutaneous malignancy in KS (Couwenberg et al., 1998Couwenberg S.M. Vuzevski V.D. Heule F. Siersema P.D. Kindler syndrome.Ned Tijdschr Geneeskd. 1998; 142: 1579-1580PubMed Google Scholar;Lotem et al., 2001Lotem M. Raben M. Zeltser R. Landau M. Sela M. Wygoda M. Tochner Z.A. Kindler syndrome complicated by squamous cell carcinoma of the hard palate: Successful treatment with high-dose radiation therapy and granulocyte-macrophage colony-stimulating factor.Br J Dermatol. 2001; 144: 1284-1286Crossref PubMed Scopus (49) Google Scholar;Mallipeddi, 2002Mallipeddi R. Epidermolysis bullosa and skin cancer.Clin Exp Dermatol. 2002; 27: 616-623Crossref PubMed Scopus (102) Google Scholar), but there are also further clinical differences, notably in the patients' response to ultraviolet irradiation. In KS, patients have an abnormal early erythema response to sun exposure as well as signs of progressive poikiloderma. Thus, for several years KS has been thought to represent an unusual, but distinct, variant of epidermolysis bullosa or an inherited poikiloderma syndrome. Using genome-wide linkage analysis on DNA obtained from consanguineous families, two groups have recently mapped the KS gene to 20p12.3 and identified 10 different pathogenic homozygous loss-of-function mutations in a novel gene (Jobard et al., 2003Jobard F. Bouadjar B. Caux F. et al.Identification of mutations in a new gene encoding a FERM family protein with a pleckstrin homology domain in Kindler syndrome.Hum Mol Genet. 2003; 12: 925-935Crossref PubMed Scopus (185) Google Scholar;Siegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin–extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). This gene, KIND1, encodes a 677 amino acid protein, kindlin-1. It is well conserved throughout evolution, with closely related homologs in Drosophila and Onchorhynchus, as well as Caenorhabditis elegans, suggesting that it has an important function across species (Rogalski et al., 2000Rogalski T.M. Mullen G.P. Gilbert M.M. Williams B.D. Moerman D.G. The UNC-112 gene in Caenorhabditis elegans encodes a novel component of cell–matrix adhesion structures required for integrin localization in the muscle cell membrane.J Cell Biol. 2000; 150: 253-264Crossref PubMed Scopus (159) Google Scholar;Schaller, 2000Schaller M.D. UNC112: A new regulator of cell–extracellular matrix adhesions?.J Cell Biol. 2000; 150: F9-F11Crossref PubMed Google Scholar;Siegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin–extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Humans possess three differentially expressed kindlin genes (encoding kindlin-1, kindlin-2, and kindlin-3), of which kindlin-1 is the major kindlin expressed by basal epidermal keratinocytes (Siegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin–extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). In this study, we searched for KIND1 mutations in 16 individuals with Kindler syndrome from 13 families to extend the mutation database and to identify any recurrent mutations in this genodermatosis that might have relevance to optimizing mutation detection strategies. Affected individuals 1–8 were from six Pakistani families residing in the UK. Cases 2 and 3 were siblings, as were cases 4 and 5, but none of the six families was known to be related to the others. Consanguinity was present within the individual families, however. Case 1 lived in southern England but the other families were spread across middle and northern England. The subjects' ages ranged from 3 mo to 30 y, there were six males and two females, and all had similar features of trauma-induced blistering, especially on the hands and feet, since infancy. Photosensitivity also began in infancy and consisted of erythema within minutes of sun exposure. Poikiloderma was often not apparent until 8–10 y and several cases were incorrectly diagnosed as dystrophic epidermolysis bullosa in early childhood. Most blistering ceased during teenage years. Thereafter, poikiloderma with prominent hyperpigmentation and hypopigmentation was the main clinical feature. Other features in adulthood included ectropion (cases 4 and 5) and dysphagia (case 5). In all children under the age of 7 y, the key investigation in establishing the diagnosis was transmission electron microscopy. Ultrastructurally, there was reduplication of the lamina densa, as well as variable focal areas of collagen lysis in the superficial dermis and occasional microclefts within the lamina lucida (data not shown). Kindler syndrome case 9 (female; 7 y) was from the Al Mudhabi region of Oman, whereas Omani case 10 (female; 19 y) was from the isolated island of Museira. Geographically and socially, their families were not known to be related, although both contained consanguineous marriages. The clinical features were similar to the British Pakistani KS cases. In addition, case 10 also had dysphagia with esophageal stenosis. There was also a history of a sibling of case 10 dying in early infancy because of severe skin fragility and secondary infection. Cases 11 (male; 23 y), 12 (female; 10 y), and 13 (female; 44 y) were unrelated white Caucasians from different parts of England. Although the clinical history of initial trauma-induced blistering and sun sensitivity was similar to the KS individuals above, the degree of poikiloderma was more subtle, reflecting the paler skin type (I–II; cf. types IV–V in subjects 1–10). The clinical features in case 11 closely resembled a mild form of dystrophic epidermolysis bullosa or possible non-Herlitz junctional epidermolysis bullosa, but again, reduplication of the lamina densa seen on transmission electron microscopy provided the clue to the correct diagnosis of KS. Affected Italian individuals 14 and 15 were brothers (34 and 36 y) from the rural south of the country, whereas case 16 (male; 46 y) was a seemingly unrelated individual from urban central Italy. All had trauma-induced blistering from birth until adolescence. Features of poikiloderma were subtle and more closely resembled postinflammatory pigmentation. In case 16, immunohistochemical analysis of epidermal basement membrane disclosed reduced immunostaining for type XVII collagen, but no pathogenic mutation has been detected in subsequent sequencing of the COL17A1 gene (GZ, unpublished data). At the age of 43 y, case 16 developed two squamous cell carcinomas on the upper lip and dorsum of the hand, both of which were excised and there have been no signs of any recurrence in 3 y follow-up. Sequencing of genomic DNA from affected individuals in the 13 families with KS disclosed pathogenic mutations in each case, as shown in Figure 2. In all affected individuals from the six Pakistani families (n=8), genomic DNA sequencing revealed a homozygous insertion of cytosine at position 676 in exon 5 of KIND1, denoted 676insC Figure 2a (GenBank no. AY137240; nucleotide numbers refer to cDNA with methionine initiation codon ATG as 1–2–3, etc.). In the two Omani families, sequencing of affected individuals' DNA disclosed a homozygous guanine to adenine change at nucleotide 1848 in exon 14 resulting in replacement of a tryptophan residue (TGG) by a stop codon (TGA), denoted W616X Figure 2b. In the three UK Caucasian patients, sequencing showed a heterozygous guanine to thymine change at nucleotide 910 in exon 7 that changes a glutamic acid residue (GAA) to a stop codon (TAA), denoted E304X Figure 2c. Compound heterozygosity for a further nonsense or frameshift mutation was shown in each case. These mutations comprised a thymine to adenine substitution at nucleotide 905 in exon 7, which changes leucine (TTA) to a stop codon (TAA), L302X Figure 2c; a single nucleotide deletion in exon 10, 1161delA; and a single nucleotide deletion in exon 15, 1909delA. In the two Italian families, three affected individuals were homozygous for an acceptor splice site mutation 958–1G > A in intron 7 Figure 2d. These new mutations, and the previously documented mutations in other patients with KS, are illustrated in Figure 3.Figure 3Schematic diagram showing the positions of all pathogenic mutations found in KIND1 and their positions within the gene. Mutations shown in bold are recurrent mutations, of which those identified in this study are underlined. Mutations in italics are also potential hotspot mutations. Exons (numbered 1–15) are to scale; introns in between are not to scale. Hatched areas denote start of 5′ and 3′ untranslated regions.View Large Image Figure ViewerDownload (PPT) Sequencing of genomic DNA in control and affected individuals disclosed that the KIND1 gene contains a number of highly polymorphic single nucleotide polymorphisms. Nine intragenic single nucleotide polymorphisms were identified. These comprised 1–29T/G, 114T/C, 151+20C/T, 152–4G/A, 479T/C, 532+8T/C, 532+34C/T, 533–17 A/C, and 722T/C, using the standard numbering system described above. Haplotype analysis of these polymorphisms in the affected individuals showed that the mutations W616X, E304X, and 958–1G > A occurred on similar genetic backgrounds for each mutation, consistent with propagation of common ancestral alleles in the Omani, UK Caucasian, and Italian gene pools, respectively Table I. Likewise, in five of the six Pakistani families, the mutation 676insC was shown on the same allelic background. In one case (patient 1), however, the mutated allele displayed an alternative haplotype with six out of nine intragenic polymorphisms being different.Table IAncestral KIND1 alleles associated with recurrent pathogenic mutations underlying Kindler syndromeKIND1 polymorphism676insC (Pakistani)E304X (UK Caucasian)W616X (Omani)958–1G>A (Italian)1–29T/CGTGG114T/CCTCC151+20C/TCCTT152–4G/AGGAA479T/CCTTT532+8T/CTTCC532+34T/CCCTT533–17A/CCAAA722T/CTTTT Open table in a new tab In control skin, immunofluorescence labeling with antikindlin-1 antibody showed bright, pan-epidermal staining, especially within the basal keratinocyte cytoplasm and at the dermal-epidermal junction Figure 4a. No specific staining was seen in the dermis. In contrast, all labeling in patient skin was markedly reduced and in some cases absent Figure 4b, c, d, e. In this study, we have identified seven new pathogenic mutations in KIND1 in patients with KS, bringing the total found thus far to 17, as summarized in Figure 3. Previously, two recurrent mutations, R271X and R288X, were identified (Siegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin–extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). The KIND1 mutation R271X was demonstrated in Panamanian, Caucasian American, and Omani subjects, whereas the mutation R288X was found in UK Caucasian and Turkish individuals with KS. Both of these mutations were shown on different respective genetic backgrounds consistent with hypermutable CpG dinucleotides (Cooper and Krawczak, 1993Cooper D.N. Krawczak M. Human Gene Mutation. BIOS Scientific Publishers Ltd, Oxford1993Google Scholar) producing potential hotspots for loss-of-function mutations in kindlin-1. By contrast, our study has delineated four new KIND1 mutations, 676insC, W616X, E304X, and 958–1G>A, occurring on the same genetic background in the Pakistani, Omani, UK Caucasian, and Italian gene pools, respectively. These mutations indicate propagation of an ancestral allele through different generations of these populations and are important starting points in the investigation into the molecular pathology of further cases of KS from the same ethnic background. Separate from this study, we have also identified the presence of the homozygous frameshift mutation 1714delA in an Italian individual with KS of North African Arabic origin (unpublished data). Interestingly, this mutation has already been reported in another subject of similar genetic background (Jobard et al., 2003Jobard F. Bouadjar B. Caux F. et al.Identification of mutations in a new gene encoding a FERM family protein with a pleckstrin homology domain in Kindler syndrome.Hum Mol Genet. 2003; 12: 925-935Crossref PubMed Scopus (185) Google Scholar), raising the possibility of a further common mutated ancestral allele in this population. Also noteworthy from our study is the Pakistani mutation 676insC, which appears to be both a hotspot and a founder mutation. This interpretation is supported by one individual (patient 1) having a different genetic background in KIND1 to the other seven affected subjects. The mutation 676insC occurs in a sequence of seven consecutive cytosines and may therefore have arisen in patient 1 through slipped mispairing (Cooper and Krawczak, 1993Cooper D.N. Krawczak M. Human Gene Mutation. BIOS Scientific Publishers Ltd, Oxford1993Google Scholar), compared to the propagation of a mutant allele in patients 2–8 inclusive. The immunofluorescence microscopy studies in patients with any of these four new recurrent mutations in KIND1 (as well as those reported bySiegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin–extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar) demonstrate the usefulness of the carboxy-terminal antikindlin-1 antibody as a potential diagnostic probe in KS. The marked reduction (or complete absence) of immunostaining in KS skin with this antibody appears to be a consistent and reliable finding. From a clinical perspective, it is clear that in nearly all the patients we studied there was a considerable delay in establishing a correct diagnosis of KS and refuting differential diagnoses of dystrophic epidermolysis bullosa or a poikilodermatous genodermatosis. Thus, the new antibody now allows for a rapid means of diagnosing KS. Nevertheless, it should be noted that immunostaining was not completely absent in all KS subjects (see Figure 4). Notably, some weak labeling was noted in patients with the homozygous mutations 676insC and 958–1G > A, as well as the individual who was a compound heterozygote for the mutations E304X/L302X. These mutations are sited in exon 5, intron 7, and exon 7, respectively. By contrast, antikindlin-1 antibody labeling in skin homozygous for exon 14 mutation W616X was completely absent. The relative differences may relate to putative alternative splicing of KIND1 (Siegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin–extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Indeed, preliminary assessment of potential KIND1 splice variants suggests that truncated isoforms spanning exons 2–8 and 9–15 may be expressed in normal keratinocytes (GHSA, unpublished data). The latter transcript would not be affected by mutations in exons 2–8 but could still react with the antibody made to the carboxy-terminal peptide in exon 15. This might explain the faint positive immunostaining in skin from cases with mutations upstream from exon 8, although other factors such as the consequences of the different mutations on RNA processing and transcript stability may influence the actual immunolabeling observed. From a clinicopathologic perspective, no major phenotypic differences were noted in patients with faint kindlin-1 immunostaining compared to those with a complete absence of antibody labeling. Despite the need for comprehensive characterization of normal splice variants in KIND1, the antibody remains a useful tool in establishing a diagnosis of KS. That said, one of the observations in the initial study characterizing the KIND1 gene was that some families with purported KS did not map to the KS locus on 20p12.3 (Siegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin–extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). This suggests that there may be a disorder that is clinically similar to but genetically different from KS. We have not yet had the opportunity to examine skin from such patients, however, to test the specificity of the new antikindlin-1 antibody. Similarly, one of the families reported by another research group showed evidence of genetic linkage to the KIND1 locus but the authors were unable to identify a mutation in this case (Jobard et al., 2003Jobard F. Bouadjar B. Caux F. et al.Identification of mutations in a new gene encoding a FERM family protein with a pleckstrin homology domain in Kindler syndrome.Hum Mol Genet. 2003; 12: 925-935Crossref PubMed Scopus (185) Google Scholar). In cases such as these, immunostaining for kindlin-1 in patient skin would be very useful to establish a diagnosis of KS in the absence of mutation data. The predicted protein structure of kindlin-1 reveals several domains of interest, in particular a bipartite FERM (band 4.1, ezrin, radixin, and moesin) domain separated by a pleckstrin homology domain, indicative of roles in plasma membrane adhesion structures and a possible link to signal transduction (Chishti et al., 1998Chishti A.H. Kim A.C. Marfatia S.M. et al.The FERM domain: A unique module involved in the linkage of cytoplasmic proteins to the membrane.Trends Biochem Sci. 1998; 23: 281-282Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar;Maffucci and Falasca, 2001Maffucci T. Falasca M. Specificity in pleckstrin homology (PH) domain membrane targeting: A role for a phosphoinositide-protein cooperation mechanism.FEBS. 2001; 506: 173-179Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar;Lemmon et al., 2002Lemmon M.A. Ferguson K.M. Abrams C.S. Plekstrin homology domains and the cytoskeleton.FEBS. 2002; 513: 71-76Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar;Siegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin–extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Kindlin-1 is involved in linking the actin cytoskeleton to the extracellular matrix (ECM) and has been demonstrated in vivo to locate at the ends of filamentous actin fibers within the cell, at the point where they insert into focal contacts, and thus to the ECM (Siegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin–extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). This makes KS the first inherited skin fragility disorder to be associated with a defect in actin cytoskeleton ECM linkage, unlike subtypes of epidermolysis bullosa, which are associated with defects in keratin intermediate filament-ECM linkage. This observation may shed light on the unusual morphology seen on transmission electron microscopy in KS, which displays features different to other inherited skin fragility disorders, including extensive basement membrane reduplication (Shimizu et al., 1997Shimizu H. Sato M. Ban M. et al.Immunohistochemical, ultrastructural, and molecular features of Kindler syndrome distinguish it from dystrophic epidermolysis bullosa.Arch Dermatol. 1997; 133: 1111-1117Crossref PubMed Google Scholar;Siegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin–extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). The actual mechanisms leading to basement membrane dysregulation remain to be elucidated, however. Likewise, the actin cytoskeleton–focal contact link to the clinical features of photosensitivity, poikiloderma, and squamous cell carcinoma is still a mystery to be explored. Identification of the specific interactions of kindlin-1 with other molecules by the yeast two-hybrid system or similar strategies should shed light on these unresolved issues. In essence, our study expands the mutation database of KIND1 mutations in KS, reports the utility of a new antikindlin-1 antibody in the rapid diagnosis of KS, and provides useful data in optimizing mutation screening in this genodermatosis. Following approval by the ethics committee of St. Thomas Hospital, London, and informed consent, DNA was extracted from peripheral blood samples taken from affected individuals, their parents, and clinically normal siblings (where possible) using a standard cold water lysis method (Sambrook et al., 1989Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY1989Google Scholar). PCR amplification of the KIND1 gene was performed as described elsewhere (Siegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin–extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). For PCR amplification, 500 ng of genomic DNA was used as template in an amplification buffer containing 1 μM of each primer, 1.5 mM of MgCl2, 50 μM of each nucleotide, and 2.5 U Taq polymerase (PE Biosystems, Warrington, UK) in a total volume of 50 μL in a PE Biosystems 9700 thermal cycler. The amplification conditions were 95°C for 5 min, followed by 35 cycles of 95°C for 45 s, annealing temperature (seeSiegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin–extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar) for 45 s, and 72°C for 45 s. Aliquots (5 μL) of the PCR products were analyzed by 2% agarose gel electrophoresis. PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Crawley, UK) and sequenced directly using Big Dye labeling in an ABI 310 genetic analyzer (PE Biosystems). Potential mutations were confirmed by restriction endonuclease digestion or bidirectional sequencing and assessed in up to 200 control chromosomes, selected to match the patients' ethnic backgrounds. The control samples were selected from 3000 archival DNA samples kept within the Genetic Skin Disease Group at St John's Institute of Dermatology, London, UK. To search for common sequence variants in KIND1 that would be useful for haplotype analysis, genomic DNA from 40 control individuals of mixed ethnic backgrounds was amplified using the PCR primers and methods detailed above (and inSiegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin–extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar) and screened using heteroduplex analysis, as described elsewhere (Ganguly et al., 1993Ganguly A. Rock M.J. Prockop D.J. Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: Evidence for solvent-induced bends in DNA heteroduplexes.Proc Natl Acad Sci USA. 1993; 90: 10325-10329Crossref PubMed Scopus (604) Google Scholar). PCR products displaying bandshifts were then investigated further by direct nucleotide sequencing. Skin biopsies were available from four patients (patients 6, 10, 11, and 16). Skin was washed in phosphate-buffered saline (PBS) for 30 min before being embedded in OCT compound (TAAB, UK) and snap frozen in isopentane cooled by liquid nitrogen. Five micron cryosections were fixed in 1:1 acetone and methanol at -20°C for 20 min, then rehydrated in PBS for 2×15 min, and incubated in 0.1% Triton X-100 in PBS prior to blocking with 10% normal goat serum in PBS for 20 min. Sections were then incubated in the same well with primary kindlin-1 rabbit polyclonal antibody for 1 h at 37°C. Details of the kindlin-1 antibody (made against the carboxy-terminal 15 amino acids) have been published elsewhere (Siegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin–extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). After washing, sections were incubated with goat antirabbit Alexa Fluor 488 conjugate (Cambridge BioSciences, Cambridge, UK) for 1 h. A negative control was treated with the same method but replacing antikindlin-1 antibody with 10% normal goat serum. The samples were thoroughly washed in PBS and subsequently mounted on coverslips. Microscopy was performed using a Nikon Optiphot Microscope with Kodak Microscopy Document System 290. We are grateful to the patients for their participation in this study, and to Dr E. Epstein Jr and Dr D. Siegel for initial collaborative studies on the KIND1 gene. The current work was supported by grants from Action Research, the Dystrophic Epidermolysis Bullosa Research Association (DEBRA, UK), the Barbara Ward Children's Foundation, and the British Skin Foundation. WHIM and FJDS are funded by a Wellcome Trust Senior Fellowship (to WHIM).

Pachyonychia congenita (PC) is an autosomal-dominant keratin disorder where the most painful, debilitating aspect is plantar keratoderma. PC is caused by mutations in one of four keratin genes; however, most patients carry K6a mutations. Knockout mouse studies suggest that ablation of one of the several K6 genes can be tolerated owing to compensatory expression of the others. Here, we have developed potent RNA interference against K6a as a paradigm for treating a localized dominant skin disorder. Four small interfering RNAs (siRNAs) were designed against unique sequences in the K6a 3'-untranslated region. We demonstrated near-complete ablation of endogenous K6a protein expression in two keratinocyte cell lines, HaCaT and NEB-1, by transient transfection of each of the four K6a siRNAs. The siRNAs were effective at very low, picomolar concentrations. One potent lead K6a inhibitor, which was highly specific for K6a, was tested in a mouse model where reporter gene constructs were injected intradermally into mouse paw and luciferase activity was used as an in vivo readout. Imaging in live mice using the Xenogen IVIS system demonstrated that the K6a-specific siRNA strongly inhibited bicistronic K6a-luciferase gene expression in vivo. These data suggest that siRNAs can specifically and very potently target mutated genes in the skin and support development of these inhibitors as potential therapeutics.

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.

filaggrin ichthyosis vulgaris TO THE EDITOR Null mutations in the epidermal barrier protein filaggrin (FLG) cause ichthyosis vulgaris (IV, Smith et al., 2006Smith F.J. Irvine A.D. Terron-Kwiatkowski A. Sandilands A. Campbell L.E. Zhao Y. et al.Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris.Nat Genet. 2006; 38: 337-342Crossref PubMed Scopus (753) Google Scholar) and are a major risk factor for eczema and other atopic diseases (Palmer et al., 2006Palmer C.N. Irvine A.D. Terron-Kwiatkowski A. Zhao Y. Liao H. Lee S.P. et al.Common loss-of-function variants of the epidermal barrier protein major predisposing factor for atopic dermatitis.Nat Genet. 2006; 38: 441-446Crossref PubMed Scopus (2074) Google Scholar; Baurecht et al., 2007Baurecht H. Irvine A.D. Novak N. Illig T. Buhler B. Ring J. et al.Toward a major risk factor for atopic eczema: meta-analysis of filaggrin polymorphism data.J Allergy Clin Immunol. 2007; 120: 1406-1412Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar; Brown et al., 2008aBrown S.J. Relton C.L. Liao H. Zhao Y. Sandilands A. Wilson I.J. et al.Filaggrin null mutations and childhood atopic eczema: a population-based case–control study.J Allergy Clin Immunol. 2008; 121: 940-946Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar; Henderson et al., 2008Henderson J. Northstone K. Lee S.P. Liao H. Zhao Y. Pembrey M. et al.The burden of disease associated with filaggrin mutations: a population-longitudinal birth cohort study.J Allergy Clin Immunol. 2008; 121: 872-877Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). About 10% of European patients are heterozygous for such null mutations (Sandilands et al., 2006Sandilands A. O’Regan G.M. Liao H. Zhao Y. Terron-Kwiatkowski A. Watson R.M. et al.Prevalent and rare mutations in the gene encoding filaggrin cause ichthyosis vulgaris and predispose individuals to atopic dermatitis.J Invest Dermatol. 2006; 126: 1770-1775Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), 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., 2008aBrown S.J. Relton C.L. Liao H. Zhao Y. Sandilands A. Wilson I.J. et al.Filaggrin null mutations and childhood atopic eczema: a population-based case–control study.J Allergy Clin Immunol. 2008; 121: 940-946Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). 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 48hours 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., 2006Palmer C.N. Irvine A.D. Terron-Kwiatkowski A. Zhao Y. Liao H. Lee S.P. et al.Common loss-of-function variants of the epidermal barrier protein major predisposing factor for atopic dermatitis.Nat Genet. 2006; 38: 441-446Crossref PubMed Scopus (2074) Google Scholar; Sandilands et al., 2007Sandilands A. Terron-Kwiatkowski A. Hull P.R. O’Regan G.M. Clayton T.H. Watson R.M. et al.Comprehensive analysis of the gene encoding filaggrin uncovers prevalent mutations in ichthyosis vulgaris and atopic eczema.Nat Genet. 2007; 39: 650-654Crossref PubMed Scopus (471) Google Scholar) 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). Download .doc (.03 MB) Help with doc files Supplementary Table S1Primers and probes for FLG mutation screening by Taqman allelic discrimination 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., 2007Sandilands A. Terron-Kwiatkowski A. Hull P.R. O’Regan G.M. Clayton T.H. Watson R.M. et al.Comprehensive analysis of the gene encoding filaggrin uncovers prevalent mutations in ichthyosis vulgaris and atopic eczema.Nat Genet. 2007; 39: 650-654Crossref PubMed Scopus (471) Google Scholar; Brown et al., 2008bBrown S.J. Sandilands A. Zhao Y. Liao H. Relton C.L. Meggitt S.J. et al.Prevalent and low-frequency null mutations in the filaggrin gene are with early-onset and persistent atopic eczema.J Invest Dermatol. 2008; 128: 1591-1594Crossref PubMed Scopus (79) Google Scholar). 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., 2007Sandilands A. Terron-Kwiatkowski A. Hull P.R. O’Regan G.M. Clayton T.H. Watson R.M. et al.Comprehensive analysis of the gene encoding filaggrin uncovers prevalent mutations in ichthyosis vulgaris and atopic eczema.Nat Genet. 2007; 39: 650-654Crossref PubMed Scopus (471) Google Scholar); for financial reasons, this patient was not subjected to full sequencing. Download .doc (.03 MB) Help with doc files Supplementary Table S2Allele frequencies 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 “moderately” or “a lot” (Fisher's exact test; P=0.024), to due principally men, and not significant in women alone. Use of body moisturizers twice a week or more was commoner among carriers, but again this finding was significant only in men (P=0.035). Self-perceived sensitive skin (“moderately” or “a lot”) was no more common among carriers. Carriers were more likely to give a history of childhood eczema (P=0.009), but asthma and hay fever were not significantly more common in this study. Trends to fewer cases of acne and psoriasis in carriers were not significant. Download .doc (.24 MB) Help with doc files Supplementary Table S3Subjective features, history, and clinical signs In the objective assessments, the prevalence of fine scaling and roughness of the skin, keratosis pilaris, and of palmar hyperlinearity and roughness (a score of 1 or more) were all increased in carriers (Figure 1b). For most parameters this was significant by Fisher's exact test (Table S3). Distribution of total skin signs in wild-type and carrier populations is shown in Figure 2; 94% of young non-carriers had a score of 4 or less and thus a total score >4 was chosen to define clinical “dry skin”. By this criterion, using binary logistic regression, heterozygous carriers were more likely than non-carriers to have dry skin (odds ratio 7.3, 95% confidence interval 2.9–18.6, P=0.00002; P=0.003 in each age cohort). Independently of genotype, the elderly were more likely to have dry skin than younger adults (odds ratio 2.9, 95% confidence interval 1.3–6.3, P=0.007). Seventy percent of young adult carriers exhibited some features of dry skin (a score of 1 or more in any parameter) but so did 47% of young non-carriers. All the older carriers had some evidence of dry skin, but so did 70% of non-carriers. In identifying young carriers clinically, fine scale on the legs was helpful (53% of carriers vs 22% of non-carriers) but most older patients had scaling on the legs irrespective of genotype. In the latter group, fine scaling on the arms, or palmar hyperlinearity (63 vs 27%, and 50 vs 11%, respectively) provided better opportunities to make a clinical distinction. In this adult population, ascertained independently of dry or inflamed skin, the prevalence of null mutations was identical to previous UK populations who had not been ascertained. We chose to study patient perception and simple clinically assessed surface changes, to reflect the usage of the term “dry skin” by patients and practitioners in everyday life. This is less objective than physiological correlates of altered skin surface such as transepidermal water loss and stratum corneum hydration. Despite this, there was a clear effect of FLG mutation on the clinical measures. The finding that heterozygous carriers have an increased prevalence of dry skin is also consistent with studies with potential recruitment bias (for example, Novak et al., 2008Novak N. Baurecht H. Schafer T. Rodriguez E. Wagenpfeil S. Klopp N. et al.Loss-of-function mutations in the filaggrin gene and allergic contact sensitization to nickel.J Invest Dermatol. 2008; 128: 1430-1435Crossref PubMed Scopus (218) Google Scholar, ascertained for contact eczema; Kezic et al., 2008Kezic S. Kemperman P.M. Koster E.S. de Jongh C.M. Thio H.B. Campbell L.E. et al.Loss-of-function mutations in the filaggrin gene lead to reduced level moisturizing factor in the stratum corneum.J Invest Dermatol. 2008; 128: 2117-2119Crossref PubMed Scopus (216) Google Scholar, recruited by advertisement). We also confirm the recent population survey in English schoolchildren showing increased dry skin, an increased incidence of childhood eczema, and a surprisingly high prevalence of clinical IV (Brown et al., 2008aBrown S.J. Relton C.L. Liao H. Zhao Y. Sandilands A. Wilson I.J. et al.Filaggrin null mutations and childhood atopic eczema: a population-based case–control study.J Allergy Clin Immunol. 2008; 121: 940-946Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). However, even with the loosest criteria for dry skin, penetrance is at most 70% in young patients and nearly 50% of non-carriers have some positive findings. Signs such as keratosis pilaris and palmar hyperlinearity are suggestive but nonspecific, and though summary skin scores were highly significantly different between groups they have poor predictive values at an individual level. Less than a third gave a history of eczema. Thus, although null mutations in FLG are a major contributing factor to clinical dry skin and eczema in the population, additional factors influence expression of these phenotypes. These may include the number of FLG repeats per allele, which varies from 10 to 12 (Ginger et al., 2005Ginger R.S. Blachford S. Rowland J. Rowson M. Harding C.R. Filaggrin repeat number polymorphism is associated with a dry skin.Arch Dermatol Res. 2005; 297: 235-241Crossref PubMed Scopus (59) Google Scholar), or variations in the expression of FLG or other protein or lipid components of the cornified layer. The latter include ceramides, whose decline may be responsible for increasingly dry skin with age (Rogers et al., 1996Rogers J. Harding C. Mayo A. Banks J. Rawlings A. Stratum corneum lipids: the effect of ageing and the seasons.Arch Dermatol Res. 1996; 288: 765-770Crossref PubMed Scopus (290) Google Scholar). Further study of protein and lipid expression in the skin is required to identify the molecular correlates of clinical dry skin, impaired barrier function, and predisposition to eczema in carriers of FLG null mutations. We are grateful to Sarah Brown, Department of Dermatology, University of Newcastle upon Tyne, for recalculating mutant allele frequency data in her population study (Table S2) to permit comparison of the four polymorphisms studied. Clinical costs were met by the South Glasgow Dermatology Fund. Filaggrin research in the McLean/Smith laboratory is supported by grants from the British Skin Foundation, the National Eczema Society, and the Medical Research Council (reference no. G0700314) and by donations from anonymous families affected by eczema in the Tayside region of Scotland. Table S1. Primers and probes for FLG mutation screening by TaqMan allelic discrimination. Table S2. Allele frequencies. Table S3. Subjective features, history, and clinical signs.

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.

Pachyonychia congenita (PC) is a rare autosomal dominant keratinizing disorder characterized by severe, painful, palmoplantar keratoderma and nail dystrophy, often accompanied by oral leucokeratosis, cysts and follicular keratosis. It is caused by mutations in one of five keratin genes: KRT6A, KRT6B, KRT6C, KRT16 or KRT17.To identify mutations in 84 new families with a clinical diagnosis of PC, recruited by the International Pachyonychia Congenita Research Registry during the last few years.Genomic DNA isolated from saliva or peripheral blood leucocytes was amplified using primers specific for the PC-associated keratin genes and polymerase chain reaction products were directly sequenced.Mutations were identified in 84 families in the PC-associated keratin genes, comprising 46 distinct keratin mutations. Fourteen were previously unreported mutations, bringing the total number of different keratin mutations associated with PC to 105.By identifying mutations in KRT6A, KRT6B, KRT6C, KRT16 or KRT17, this study has confirmed, at the molecular level, the clinical diagnosis of PC in these families.

Infantile-onset X-linked spinal muscular atrophy (SMAX2) is a rare lethal disorder linked to mutations in the UBA1 (previously UBE1) gene, encoding ubiquitin-activating enzyme 1 that has an important role in the ubiquitin–proteasome pathway. Published pathological reports are scarce. Here we report a male infant who presented from birth with predominantly truncal hypotonia following an antenatal history of reduced fetal movements. He had a myopathic face, profound weakness, multiple contractures and areflexia. Creatine kinase was moderately raised. Brain MRI showed non-specific symmetrical periventricular white matter changes. Neurophysiology revealed evidence of motor and sensory involvement and muscle biopsy showed marked inflammatory changes with subtle features suggestive of acute denervation. UBA1 sequencing revealed a novel hemizygous missense mutation (c.1670A>T; p.Glu557Val). He died from progressive respiratory failure at 4 months. On post mortem assessment, in addition to severe ventral motor neuron pathology, there was widespread involvement of the sensory system, as well as developmental and degenerative cerebellar abnormalities. In contrast to typical SMN1-associated SMA, the thalamus was unaffected. These findings indicate that SMAX2 is more accurately classified as a motor sensory neuronopathy rather than a pure anterior horn cell disorder. Ubiquitin–proteasome pathway defects may not only cause neurodegeneration but also affect normal neuronal development.

Hereditary Fibrosing Poikiloderma (HFP) with tendon contractures, myopathy and pulmonary fibrosis (POIKTMP [MIM 615704]) is a very recently described entity of syndromic inherited poikiloderma. Previously by using whole exome sequencing in five families, we identified the causative gene, FAM111B (NM_198947.3), the function of which is still unknown. Our objective in this study was to better define the specific features of POIKTMP through a larger series of patients.Clinical and molecular data of two families and eight independent sporadic cases, including six new cases, were collected.Key features consist of: (i) early-onset poikiloderma, hypotrichosis and hypohidrosis; (ii) multiple contractures, in particular triceps surae muscle contractures; (iii) diffuse progressive muscular weakness; (iv) pulmonary fibrosis in adulthood and (v) other features including exocrine pancreatic insufficiency, liver impairment and growth retardation. Muscle magnetic resonance imaging was informative and showed muscle atrophy and fatty infiltration. Histological examination of skeletal muscle revealed extensive fibroadipose tissue infiltration. Microscopy of the skin showed a scleroderma-like aspect with fibrosis and alterations of the elastic network. FAM111B gene analysis identified five different missense variants (two recurrent mutations were found respectively in three and four independent families). All the mutations were predicted to localize in the trypsin-like cysteine/serine peptidase domain of the protein. We suggest gain-of-function or dominant-negative mutations resulting in FAM111B enzymatic activity changes.HFP with tendon contractures, myopathy and pulmonary fibrosis, is a multisystemic disorder due to autosomal dominant FAM111B mutations. Future functional studies will help in understanding the specific pathological process of this fibrosing disorder.

The filaggrin gene (FLG) is essential for skin differentiation and epidermal barrier formation. FLG loss-of-function (LoF) variants are associated with ichthyosis vulgaris and the major genetic risk factor for developing atopic dermatitis (AD).1Palmer C.N.A. Irvine A.D. Terron-Kwiatkowski A. Zhao Y. Liao H. Lee S.P. et al.Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.Nat Genet. 2006; 38: 441-446Crossref PubMed Scopus (2274) Google Scholar, 2Smith F.J.D. Irvine A.D. Terron-Kwiatkowski A. Sandilands A. Campbell L.E. Zhao Y. et al.Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris.Nat Genet. 2006; 38: 337-342Crossref PubMed Scopus (808) Google Scholar, 3van den Oord R.A.H.M. Sheikh A. Filaggrin gene defects and risk of developing allergic sensitisation and allergic disorders: systematic review and meta-analysis.BMJ. 2009; 339: b2433Crossref PubMed Scopus (363) Google Scholar Genetic stratification of patients with AD according to FLG LoF risk is a common practice for both research and clinical studies; however, few studies comprehensively sequence the entire FLG coding region. Most studies that include FLG genotyping have screened for common predominant LoF variants to report allele frequencies after full Sanger sequencing of a smaller batch of test patient samples or previously published data. This strategy potentially results in underreporting of the genetic contribution especially in ethnicities where FLG LoF variants are highly diverse.4Clark A.G. Hubisz M.J. Bustamante C.D. Williamson S.H. Nielsen R. Ascertainment bias in studies of human genome-wide polymorphism.Genome Res. 2005; 15: 1496-1502Crossref PubMed Scopus (361) Google Scholar Distinct LoF variants have been reported for most ethnicities studied to date. For example, 2 predominant sequence variants (p.R501X and c.2282del4) make up approximately 80% of the mutation burden in northern Europeans,5Sandilands A. Terron-Kwiatkowski A. Hull P.R. O'Regan G.M. Clayton T.H. Watson R.M. et al.Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema.Nat Genet. 2007; 39: 650-654Crossref PubMed Scopus (515) Google Scholar whereas in East Asian ethnicities, a larger FLG LoF mutation spectrum is found with fewer predominating variants.6Irvine A.D. McLean W.H.I. Leung D.Y.M. Filaggrin mutations associated with skin and allergic diseases.N Engl J Med. 2011; 365: 1315-1327Crossref PubMed Scopus (834) Google Scholar, 7Chen H. Common J.E.A. Haines R.L. Balakrishnan A. Brown S.J. Goh C.S.M. et al.Wide spectrum of filaggrin-null mutations in atopic dermatitis highlights differences between Singaporean Chinese and European populations.Br J Dermatol. 2011; 165: 106-114Crossref PubMed Scopus (106) Google Scholar However, routinely Sanger sequencing the entire FLG coding region for large cohorts is not always feasible, although desirable as it is essential to correctly stratify patients. To address this, we developed a robust and cost-effective high-throughput PCR-based method for analyzing the entire coding region of FLG using Fluidigm microfluidics technology and next-generation sequencing (NGS). We have applied this method to fully resequence cohorts of Chinese, Malay, and Indian patients with AD from the Singaporean population. We designed and optimized overlapping FLG-specific primer assays (containing NGS adapters) to span the entire FLG coding region including known intragenic copy number variation (CNV) (see Fig 1, A; see Table E1 in this article's Online Repository at www.jacionline.org). A total of 48 overlapping primer assays, with amplicons of maximum 500 base pairs, provided redundancy for sequencing reads across primer-binding sites and 100% coverage of FLG exon bases (see Fig 1, B). The Fluidigm Access Array 48.48 integrated fluidic circuit (IFC) chip (Fluidigm, San Francisco, Calif) generates 48 amplicons for 48 different DNA samples in parallel, simultaneously thermocycling 2304 PCR reactions at nanoliter volumes (see Fig E1 and this article's Methods section in the Online Repository at www.jacionline.org). Initially 96 DNA samples were assayed in IFC chips before Illumina MiSeq 2x250 bp read mode sequencing (4 samples failed); 14 samples from this batch of 96 were previously Sanger sequenced for the entire FLG coding region.5Sandilands A. Terron-Kwiatkowski A. Hull P.R. O'Regan G.M. Clayton T.H. Watson R.M. et al.Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema.Nat Genet. 2007; 39: 650-654Crossref PubMed Scopus (515) Google Scholar, 7Chen H. Common J.E.A. Haines R.L. Balakrishnan A. Brown S.J. Goh C.S.M. et al.Wide spectrum of filaggrin-null mutations in atopic dermatitis highlights differences between Singaporean Chinese and European populations.Br J Dermatol. 2011; 165: 106-114Crossref PubMed Scopus (106) Google Scholar The known FLG LoF variant profile was then used to validate LoF variant detection with the IFC and NGS method. We identified all FLG LoF variants originally identified by Sanger in the 14 samples as well as additional variants in 2 samples (see Table I) and documented LoF variants in the remaining 78 samples that passed quality control testing (see Table E2 in this article's Online Repository at www.jacionline.org). LoF variants were all confirmed by visual inspection using Integrated Genome Viewer before Sanger sequencing. In addition, we determined the FLG CNV of repeats 8 and 10 (an important risk factor for AD8Brown S.J. Kroboth K. Sandilands A. Campbell L.E. Pohler E. Kezic S. et al.Intragenic copy number variation within filaggrin contributes to the risk of atopic dermatitis with a dose-dependent effect.J Invest Dermatol. 2012; 132: 98-104Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) in the 92 samples using relative coverage-based metrics (see Fig E2 in this article's Online Repository at www.jacionline.org).Table IConcordance of FLG LoF variant detection for 14 previously fully Sanger-sequenced AD samples using our MiSeq 2x250 bp protocolSample IDSanger sequencingIllumina MiSeq assayIA-P003p.S1515Xp.S1515XIA-P009No LoF detectedNo LoF detectedIA-P014No LoF detectedNo LoF detectedIA-P017p.E2422Xp.E2422XIA-P021p.S406X; c.6950_6957del8p.S406X; c.6950_6957del8IA-P024c.1640delGc.1640delGIA-P025p.Q368X; c.3321delAp.Q368X; c.3321delAIA-P028c.7945delAc.7945delAIA-P062p.Q2417Xp.Q2417XIA-P063c.2952delCc.2952delCIA-P083∗Our NGS protocol detected additional LoF variants in 2 samples.c.9040_9058dup19c.9040_9058dup19; p.Q1790XIA-P084∗Our NGS protocol detected additional LoF variants in 2 samples.p.S1302Xp.S1302X; p.S1515XIA-P090c.4004del2c.4004del2IA-P094c.2282del4; p.R2447Xc.2282del4; p.R2447X∗ Our NGS protocol detected additional LoF variants in 2 samples. Open table in a new tab The IFC and NGS sequencing method was then used to analyze a further 334 Singaporean ichthyosis vulgaris and/or AD patient samples to obtain estimates of disease-associated LoF allele frequency in the 3 major ethnicities of Singapore—Chinese, Malay, and Indian. In 279 Chinese Singaporean samples (see Table E3 in this article's Online Repository at www.jacionline.org), we identified a further 11 additional LoF variants, raising the total number identified in this population to 33 (an increase from 22 variants identified in our previous study7Chen H. Common J.E.A. Haines R.L. Balakrishnan A. Brown S.J. Goh C.S.M. et al.Wide spectrum of filaggrin-null mutations in atopic dermatitis highlights differences between Singaporean Chinese and European populations.Br J Dermatol. 2011; 165: 106-114Crossref PubMed Scopus (106) Google Scholar) with 5 not previously reported in the literature (see Fig 1, C and D); 85 of these samples had also been previously Sanger sequenced7Chen H. Common J.E.A. Haines R.L. Balakrishnan A. Brown S.J. Goh C.S.M. et al.Wide spectrum of filaggrin-null mutations in atopic dermatitis highlights differences between Singaporean Chinese and European populations.Br J Dermatol. 2011; 165: 106-114Crossref PubMed Scopus (106) Google Scholar and the concordance profile was near identical (see Table E4 in this article's Online Repository at www.jacionline.org). A total of 14 LoF variants reached significance using Fisher exact test (P < .05) compared with population control data derived from ExAC (version 0.3.1) exome database (see Table E5 and the Methods section in this article's Online Repository at www.jacionline.org). The combined FLG LoF mutation allele frequency for Chinese Singaporean patients with AD is now 32.3%, an increase from 20.2% in our previous survey of 425 patient samples further supporting the biological importance of FLG mutations in AD.7Chen H. Common J.E.A. Haines R.L. Balakrishnan A. Brown S.J. Goh C.S.M. et al.Wide spectrum of filaggrin-null mutations in atopic dermatitis highlights differences between Singaporean Chinese and European populations.Br J Dermatol. 2011; 165: 106-114Crossref PubMed Scopus (106) Google Scholar Smaller cohorts of 19 Indian and 36 Malay patients were analyzed and we identified FLG LoF variants in 9 Indian samples and 9 Malay samples (see Fig 1, D; see Tables E6 and E7 in this article's Online Repository at www.jacionline.org). The identification of unreported FLG LoF variants in Indian and Malay ethnicities from Singapore confirms the diversity of FLG variants in AD between different ethnic groups. In total we identified 18 variants with limited overlap with the Chinese samples (4 out of 18) and a number of these are not present in the ExAC database, highlighting the contribution of rare, family-specific mutations in AD (11 of 18). This small but well-characterized study of Indian and Malay Singaporeans highlights the variation in combined allele frequencies between ethnicities, with 47.4% of Indian patients with AD and 25% of Malay patients with AD harboring FLG LoF variants. The presence of FLG LoF variants was strongly associated with AD; however, it was not significantly associated with increasing severity in the 334 patients analyzed in this study, possibly due to the small number of mild cases analzyed (mild cases in this cohort 5.3%; see Table E8 in this article's Online Repository at www.jacionline.org). In conclusion, we describe a multiplexed targeted resequencing method to study the FLG coding region. We highlight that comprehensive sequencing improves accuracy estimates of genetic contribution from FLG deleterious alleles and CNVs in AD. This strategy to study genetic variation does not rely on previous mutation spectrum information from any given population or ethnicity and therefore can identify rare, family-specific, or de novo variants globally. This approach outperforms exome sequencing because of its throughput and ability to analyze known CNVs that are not currently reflected in NCBI RefSeq for FLG. Amplicon resequencing is robust, reliable, and unbiased and has the potential for scalable sample preparation for small and large research or clinical studies, increasing accurate genotyping for improved outcomes. We have developed a cost-efficient FLG genotyping method (∼10 times cheaper than exome sequencing) for researchers and clinicians studying patients from any ethnicity that is vital to advance a precision medicine approach to AD. This method can facilitate research studies immediately and be developed for clinical genetic diagnostics in the future. We thank all the patients who participated in this study, and the research coordinators at the National Skin Centre, especially Nancy Liew and Veron Lu, for diligently collecting samples. MiSeq quality control was checked with the kind assistance of Christopher Wong, Hui Mann Seah, and Zhengzhong Qu, Polaris, Genome Institute of Singapore (GIS), A*STAR. We thank the GIS Sequencing platform, A*STAR, for its assistance. We also thank Dr Andy South for early discussions regarding Fluidigm Access Array technology for mutation detection. Download .docx (.04 MB) Help with docx files Online Repository text Download .docx (.02 MB) Help with docx files Table E1 Download .docx (.02 MB) Help with docx files Table E2 Download .docx (.05 MB) Help with docx files Table E3 Download .docx (.02 MB) Help with docx files Table E4 Download .docx (.02 MB) Help with docx files Table E5 Download .docx (.03 MB) Help with docx files Table E6 Download .docx (.02 MB) Help with docx files Table E7 Download .docx (.02 MB) Help with docx files Table E8 Download .docx (.03 MB) Help with docx files Table E9 Download .pdf (1.01 MB) Help with pdf files Fig E1 Download .pdf (.21 MB) Help with pdf files Fig E2 Download .docx (.05 MB) Help with docx files Legends for Figs E1 and E2

BackgroundPachyonychia congenita (PC) is a group of autosomal dominant disorders caused by mutations in one of five keratin genes (KRT6A, KRT6B, KRT6C, KRT16, KRT17). The establishment of an international registry containing clinical and molecular data led to the development of a disease classification based on the mutant gene and associated features.

Mutations inactivating the STS gene cause X-linked ichthyosis (XLI), whereas null mutations in the FLG gene cause ichthyosis vulgaris. Two brothers presented with XLI. One had a typical fine scaling, and the other was much more severely affected. Both patients carried STS missense mutation T165I. Furthermore, the more severely affected patient also carried heterozygous FLG mutation R501X, which was absent from his mildly affected brother. These data suggest that disrupting epidermal differentiation via different pathways can increase phenotypic severity. Owing to the high population frequency of FLG mutations, filaggrin is a possible genetic modifier in other genodermatoses.

The palmoplantar keratodermas (PPKs) are a large group of clinically and genetically heterogeneous genodermatoses. The gene defects underlying many PPKs still need to be resolved to facilitate definitive molecular diagnosis and genetic counseling. Dominant-negative mutations in any of the four identified keratin genes, KRT6A, KRT6B, KRT16, or KRT17, cause pachyonychia congenita (PC), characterized by hypertrophic nail dystrophy and other ectodermal features. In PC, focal PPK (FPPK) is the most painful and debilitating phenotypic feature. Some families presenting with FPPK alone, or with minimal nail changes, carry mutations in KRT16; however, most FPPK families do not harbor mutations in any of these keratin genes. Here, we report three unrelated families who presented with familial FPPK with minor or absent nail changes. The four PC/FPPK-related keratin genes were excluded; however, mutational analysis of the recently identified KRT6C gene, encoding keratin K6c, showed heterozygous in-frame deletion mutations in all three kindreds. Affected members of Families 1 and 2 carried the same mutation, p.Asn172del. In Family 3, the mutation p.Ile462-Glu470del co-segregated with the disease. KRT6C was shown to be expressed in the plantar epidermis using reverse transcription-PCR, consistent with the phenotype observed in this tissue. These data expand the genetic testing repertoire for the PPKs.

Calpastatin is an endogenous specific inhibitor of calpain, a calcium-dependent cysteine protease. Here we show that loss-of-function mutations in calpastatin (CAST) are the genetic causes of an autosomal-recessive condition characterized by generalized peeling skin, leukonychia, acral punctate keratoses, cheilitis, and knuckle pads, which we propose to be given the acronym PLACK syndrome. In affected individuals with PLACK syndrome from three families of different ethnicities, we identified homozygous mutations (c.607dup, c.424A>T, and c.1750delG) in CAST, all of which were predicted to encode truncated proteins (p.Ile203Asnfs∗8, p.Lys142∗, and p.Val584Trpfs∗37). Immunohistochemistry shows that staining of calpastatin is reduced in skin from affected individuals. Transmission electron microscopy revealed widening of intercellular spaces with chromatin condensation and margination in the upper stratum spinosum in lesional skin, suggesting impaired intercellular adhesion as well as keratinocyte apoptosis. A significant increase of apoptotic keratinocytes was also observed in TUNEL assays. In vitro studies utilizing siRNA-mediated CAST knockdown revealed a role for calpastatin in keratinocyte adhesion. In summary, we describe PLACK syndrome, as a clinical entity of defective epidermal adhesion, caused by loss-of-function mutations in CAST. Calpastatin is an endogenous specific inhibitor of calpain, a calcium-dependent cysteine protease. Here we show that loss-of-function mutations in calpastatin (CAST) are the genetic causes of an autosomal-recessive condition characterized by generalized peeling skin, leukonychia, acral punctate keratoses, cheilitis, and knuckle pads, which we propose to be given the acronym PLACK syndrome. In affected individuals with PLACK syndrome from three families of different ethnicities, we identified homozygous mutations (c.607dup, c.424A>T, and c.1750delG) in CAST, all of which were predicted to encode truncated proteins (p.Ile203Asnfs∗8, p.Lys142∗, and p.Val584Trpfs∗37). Immunohistochemistry shows that staining of calpastatin is reduced in skin from affected individuals. Transmission electron microscopy revealed widening of intercellular spaces with chromatin condensation and margination in the upper stratum spinosum in lesional skin, suggesting impaired intercellular adhesion as well as keratinocyte apoptosis. A significant increase of apoptotic keratinocytes was also observed in TUNEL assays. In vitro studies utilizing siRNA-mediated CAST knockdown revealed a role for calpastatin in keratinocyte adhesion. In summary, we describe PLACK syndrome, as a clinical entity of defective epidermal adhesion, caused by loss-of-function mutations in CAST. Peeling skin syndrome (PSS) is characterized by continuous shedding of the stratum corneum of the epidermis with onset from birth or infancy and lasting throughout life.1Hacham-Zadeh S. Holubar K. Skin peeling syndrome in a Kurdish family.Arch. Dermatol. 1985; 121: 545-546Crossref PubMed Scopus (21) Google Scholar Skin peeling can be accompanied by erythema, vesicular lesions, or other ectodermal features including fragile hair and nail abnormalities.2Cassidy A.J. van Steensel M.A. Steijlen P.M. van Geel M. van der Velden J. Morley S.M. Terrinoni A. Melino G. Candi E. McLean W.H. A homozygous missense mutation in TGM5 abolishes epidermal transglutaminase 5 activity and causes acral peeling skin syndrome.Am. J. Hum. Genet. 2005; 77: 909-917Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar PSS can be divided into two clinical forms: acral PSS (APSS [MIM 609796]) and generalized PSS (GPSS [MIM 270300]). APSS involves the palmar, plantar, and dorsal surfaces of hands and feet and can be associated with mutations in transglutaminase 5 (TGM5 [MIM 603805]).3Kharfi M. El Fekih N. Ammar D. Jaafoura H. Schwonbeck S. van Steensel M.A. Fazaa B. Kamoun M.R. Fischer J. A missense mutation in TGM5 causes acral peeling skin syndrome in a Tunisian family.J. Invest. Dermatol. 2009; 129: 2512-2515Crossref PubMed Scopus (28) Google Scholar In addition, mutations in the gene encoding for the cysteine protease inhibitor cystatin A (CSTA [MIM 184600]) have recently been associated with autosomal-recessive exfoliative ichthyosis (MIM 607936) and also APSS.4Blaydon D.C. Nitoiu D. Eckl K.M. Cabral R.M. Bland P. Hausser I. van Heel D.A. Rajpopat S. Fischer J. Oji V. et al.Mutations in CSTA, encoding Cystatin A, underlie exfoliative ichthyosis and reveal a role for this protease inhibitor in cell-cell adhesion.Am. J. Hum. Genet. 2011; 89: 564-571Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 5Krunic A.L. Stone K.L. Simpson M.A. McGrath J.A. Acral peeling skin syndrome resulting from a homozygous nonsense mutation in the CSTA gene encoding cystatin A.Pediatr. Dermatol. 2013; 30: e87-e88Crossref PubMed Scopus (34) Google Scholar Individuals with inflammatory GPSS associated with mutations in corneodesmosin (CDSN [MIM 602593]) can also present with severe pruritus, food allergies, and repeated episodes of angioedema, urticaria, and asthma.6Oji V. Eckl K.M. Aufenvenne K. Nätebus M. Tarinski T. Ackermann K. Seller N. Metze D. Nürnberg G. Fölster-Holst R. et al.Loss of corneodesmosin leads to severe skin barrier defect, pruritus, and atopy: unraveling the peeling skin disease.Am. J. Hum. Genet. 2010; 87: 274-281Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar A similar entity termed SAM syndrome (severe dermatitis, multiple allergies, and metabolic wasting [MIM 615508]), which is caused by desmoglein 1 (DSG1 [MIM 125670]) deficiency, shares clinical features with inflammatory GPSS and atopy.7Samuelov L. Sarig O. Harmon R.M. Rapaport D. Ishida-Yamamoto A. Isakov O. Koetsier J.L. Gat A. Goldberg I. Bergman R. et al.Desmoglein 1 deficiency results in severe dermatitis, multiple allergies and metabolic wasting.Nat. Genet. 2013; 45: 1244-1248Crossref PubMed Scopus (231) Google Scholar A homozygous missense mutation was identified within CHST8 (MIM 610190), encoding N-acetylgalactosamine-4-O-sulfotransferase, in a large consanguineous family with non-inflammatory GPSS.8Cabral R.M. Kurban M. Wajid M. Shimomura Y. Petukhova L. Christiano A.M. Whole-exome sequencing in a single proband reveals a mutation in the CHST8 gene in autosomal recessive peeling skin syndrome.Genomics. 2012; 99: 202-208Crossref PubMed Scopus (36) Google Scholar However, the genetic basis of a number of PSS cases is still unresolved.9Pavlovic S. Krunic A.L. Bulj T.K. Medenica M.M. Fong K. Arita K. McGrath J.A. Acral peeling skin syndrome: a clinically and genetically heterogeneous disorder.Pediatr. Dermatol. 2012; 29: 258-263Crossref PubMed Scopus (15) Google Scholar, 10Chang Y.Y. van der Velden J. van der Wier G. Kramer D. Diercks G.F. van Geel M. Coenraads P.J. Zeeuwen P.L. Jonkman M.F. Keratolysis exfoliativa (dyshidrosis lamellosa sicca): a distinct peeling entity.Br. J. Dermatol. 2012; 167: 1076-1084Crossref PubMed Scopus (8) Google Scholar Here we show that homozygous loss-of-function (LOF) mutations in CAST (MIM 114090) underlie autosomal-recessive generalized PSS with leukonychia, acral punctate keratoses, cheilitis, and knuckle pads. We propose this clinical entity to be given the acronym PLACK syndrome. Via exome sequencing and Sanger sequencing we demonstrate that distinct homozygous LOF CAST mutations segregate with the disorder in all three families with PLACK syndrome. CAST encodes calpastatin, an endogenous protease inhibitor. Our findings emphasize the important role of the protease-inhibitor balance in epidermal homeostasis. We ascertained a 28-year-old Chinese female (individual 1, V-3 in Figure 1A) affected with PLACK syndrome who was born to second-cousin parents (Figure 1A). She was found to have trauma-induced recurrent blistering prominently on the extremities since infancy, which was worse in summer. In winter, asymptomatic skin peeling was more prominent, either spontaneously or after the remission of blistering, leaving underlying erythema. The blistering improved and was confined to distal extremities after puberty, and skin peeling progressed to involve nearly the entire body. She had mild pruritus and no history of atopic eczema, food allergy, allergic rhinitis, or asthma. Physical examination revealed generalized dry, scaly skin with superficial exfoliation and underlying erythema (Figure 1B). Cheilitis with dry, shedding scales was observed (Figure 1B). Several blisters were noted on her wrists and soles. Punctate palmoplantar keratoderma was seen, which coalesced into focal keratoderma predominately on the weight-bearing areas (Figure 1B). Knuckle pads with multiple hyperkeratotic micropapules were also found involving all the interphalangeal joints. Leukonychia affected the proximal half of the nails with mild distal onycholysis (Figure 1B). No other systemic abnormalities were identified. Serum total IgE level and blood eosinophil count were within normal range. Histological examination of a biopsy from the scaly skin of her leg showed hyperkeratosis, acanthosis, and intraepidermal clefting with irregular acantholysis (Figure 1C). We also investigated an affected Nepalese female (individual 2) from non-consanguineous parents who presented to dermatologists at age 3 with a 2-year history of painful lesions on the palms and soles. Clinical examination revealed punctate keratoses on the palms, extending onto the flexor aspect of the wrists and soles, plaques with hyperkeratotic micropapules over the interphalangeal joints, cheilitis of the upper lip, angular cheilitis, subtle telangiectasia on the cheeks, follicular hyperkeratosis on the extensor surface of the knees, and leukonychia of 70%–100% of the nails (Figures 1D and S1). At review at age 4, peeling areas had developed on the distal limbs including the extensor surface of the knees and elbows (Figure 1D). No history of atopic eczema, hay fever, or asthma was recorded and serum total IgE and eosinophil count were normal. An additional family with known consanguinity, previously reported as having a recessive form of pachyonychia congenita, was also enrolled in this study.11Haber R.M. Rose T.H. Autosomal recessive pachyonychia congenita.Arch. Dermatol. 1986; 122: 919-923Crossref PubMed Scopus (55) Google Scholar In brief, both affected European male siblings (individuals 3 and 4, now ages 54 and 58 years old) give a history of blistering and peeling of skin from the age of about 3 months on the hands, feet, knuckles, elbows, and knees. Skin fragility is induced by minor trauma and heat and continues to be the greatest problem for these individuals. Both individuals also have leukonychia, leukokeratosis, angular cheilitis, papules on the extensor surface of the fingers and toes, and punctate palmar keratoses and a plantar keratoderma, described in more detail by Haber and Rose.11Haber R.M. Rose T.H. Autosomal recessive pachyonychia congenita.Arch. Dermatol. 1986; 122: 919-923Crossref PubMed Scopus (55) Google Scholar We collected blood and saliva samples from the three families after obtaining informed consent. This project was approved by the Clinical Research Ethics Committee of the Peking University First Hospital, East London and City Health Authority Research Ethics Committee, and the Western Institutional Review Board, which all comply with all principles of the Helsinki Accord. After exclusion of pathogenic mutations in TGM5, CSTA, CDSN, and CHST8 by Sanger sequencing, we performed exome sequencing in individual 1 using 3 μg of genomic DNA. Exome capture was performed with the NimbleGen SeqCap EZ Library (Roche) for enrichment and then sequenced on an Illumina HiSeq2000 according to the manufacturer’s protocols. Variants were filtered against dbSNP137, the 1000 Genomes Project, HapMap8, and BGI internal databases, as described in our previous study. Because her parents were consanguineous, we focused on homozygous variants to identify the underlying defective gene. Among all the 148 variants fulfilling these criteria, only 15 variants lay in coding exons or splicing boundaries and were predicted to be “not tolerated” by SIFT.12Kumar P. Henikoff S. Ng P.C. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.Nat. Protoc. 2009; 4: 1073-1081Crossref PubMed Scopus (5006) Google Scholar These included a one-nucleotide duplication in CAST that was predicted to lead to a frameshift and truncation of the protein. By Sanger sequencing (primers are listed in Table S1), we confirmed that this mutation, c.607dup (p.Ile203Asnfs∗8; RefSeq accession numbers NM_001042440.3, NP_001035905.1), was homozygous in individual 1 and heterozygous in her unaffected parents, siblings, and daughters (Figures 1A and 2A). This mutation was not found in 200 ethnically matched controls. Exome sequencing was performed on genomic DNA from individual 2 using the NimbleGen SeqCap EZ Library SR (Roche) and sequenced on an Illumina HiSeq2000 according to the manufacturer’s protocols. Further details of analysis have been described previously.12Kumar P. Henikoff S. Ng P.C. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.Nat. Protoc. 2009; 4: 1073-1081Crossref PubMed Scopus (5006) Google Scholar Analysis of novel variants revealed a homozygous nonsense mutation (c.424A>T [p.Lys142∗]) in CAST. Sanger sequencing showed co-segregation with the disorder in this family (Figures 2A and S1A). Subsequent exome sequencing and Sanger sequencing also showed a homozygous frameshift mutation, c.1750delG (p.Val584Trpfs∗37), in CAST in individuals 3 and 4, which co-segregated with the disorder in the family (primers are listed in Table S1). All three identified mutations (including c.607dup) in CAST are absent in dbSNP137, HapMap8, the 1000 Genomes Project database, the National Heart, Lung, and Blood Institute Exome Sequencing Project (ESP) Exome Variant Server, and the BGI internal database, highly suggestive of the pathogenicity of these mutations based on their extreme rarity. All three mutations identified in CAST are predicted to encode truncated proteins and lead to a loss of function. To investigate the consequences of homozygosity for the c.607dup mutation in vivo, we examined the mRNA expression of CAST in the skin from individual 1 by using quantitative real-time PCR (qRT-PCR) (primers are listed in Table S2). The level of CAST transcription was determined on the basis of the comparative cycle threshold (2−ΔΔCt) method using cDNA templates generated from a corresponding area of the skin from a gender- and age-matched healthy control as the calibrator. In contrast to the control, we could detect only trace expression of CAST mRNA in individual 1, probably due to mechanisms of nonsense-mediated mRNA decay (Figure 2B). Immunohistochemical staining was subsequently performed with a calpastatin antibody (catalog number: sc-20779, Santa Cruz) in paraffin-embedded sections from the pretibial skin lesion of individual 1. A similar analysis was performed on paraffin-embedded sections taken from xerotic left thigh skin of individual 2, but using immunofluorescence. Consistent with the predicted effect of the two mutations and the results of the qRT-PCR analysis of the c.607dup mutation, calpastatin staining was absent or reduced in the skin from both individuals, whereas in the normal control skin, calpastatin is localized throughout the epidermis and has a cytoplasmic localization (Figures 2C and S2). No skin biopsy material was available from individuals 3 or 4. Calpastatin is a specific endogenous protease inhibitor of calpains (μ-calpain and m-calpain). Calpains are intracellular cysteine proteases that require calcium or epidermal growth factor for their catalytic activity.13Goll D.E. Thompson V.F. Li H. Wei W. Cong J. The calpain system.Physiol. Rev. 2003; 83: 731-801PubMed Google Scholar In skin, calpains have been reported as being involved in a range of cellular processes, including keratinocyte growth, migration, and cell cycle regulation.14Carragher N.O. Frame M.C. Focal adhesion and actin dynamics: a place where kinases and proteases meet to promote invasion.Trends Cell Biol. 2004; 14: 241-249Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar These processes are particularly important in epidermal terminal differentiation, characterized by expression of specific structural proteins. Immunohistochemical staining of lesional skin sections from individual 1 revealed a slight upregulation of loricrin (antibody catalog number ab24722, Abcam), keratin 1 (antibody catalog number ab24643, Abcam), and keratin 10 (antibody catalog number ab76318, Abcam) compared to control (Figure S3). The increased labeling of these proteins might be a compensatory mechanism for epidermal barrier disruption.15Pigors M. Kiritsi D. Cobzaru C. Schwieger-Briel A. Suárez J. Faletra F. Aho H. Mäkelä L. Kern J.S. Bruckner-Tuderman L. Has C. TGM5 mutations impact epidermal differentiation in acral peeling skin syndrome.J. Invest. Dermatol. 2012; 132: 2422-2429Crossref PubMed Scopus (30) Google Scholar In contrast, the staining of filaggrin in the epidermis of individual 1 (antibody catalog number ab81468, Abcam) and in the epidermis of individual 2 (antibody catalog number NCL-Filaggrin, Leica Biosystems), as well as the staining of the tight junction component claudin-116Furuse M. Hata M. Furuse K. Yoshida Y. Haratake A. Sugitani Y. Noda T. Kubo A. Tsukita S. Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice.J. Cell Biol. 2002; 156: 1099-1111Crossref PubMed Scopus (1185) Google Scholar (antibody catalog number 13255, Cell Signaling Technology) in the epidermis of individual 1 was indistinguishable from control (data not shown). Because increased activity of calpains can induce apoptosis,17Smith M.A. Schnellmann R.G. Calpains, mitochondria, and apoptosis.Cardiovasc. Res. 2012; 96: 32-37Crossref PubMed Scopus (191) Google Scholar in situ apoptotic examination of keratinocytes in the lesional skin biopsy from individual 1 was performed by a TUNEL assay (In Situ Cell Death Detection Kit, Roche). Skin sections from an unrelated healthy subject were used as a control. Images above the stratum basale were taken randomly with a fluorescence microscope (IX71, Olympus), with the same settings for both control and individual skin sections. Total cells and apoptotic cells (with fluorescent nuclei) in five random high-power fields (400×) were counted. The number of apoptotic cells in the imaged fields was significantly increased in lesional skin compared to normal control (Figures 3A and 3B ). Ultrastructural analysis of lesional skin sections from individual 1 was performed by transmission electron microscopy (TEM), which showed expanded intercellular spaces (Figure 3D), apoptotic chromatin condensation, and margination (Figure 3E), supporting the results of the TUNEL assay. To determine the functional consequences of CAST LOF mutations in vitro, we performed siRNA-mediated knockdown (KD) of CAST by using a specific siRNA pool (ON-TARGETplus Human CAST siRNA-SMARTpool, GE Healthcare Dharmacon) in the immortalized keratinocyte cell line, HaCaT. Non-targeting pool siRNA (ON-TARGETplus Non-targeting Pool, GE Healthcare Dharmacon) was used as a control. Immunocytochemistry of HaCaT cell monolayers treated with CAST siRNA and analysis of total protein lysates by immunoblotting showed robust CAST knockdown (Figures 4A–4C). We then used an in vitro mechanical-induced stress assay to investigate the role of calpastatin in keratinocyte adhesion in CAST siRNA-treated cells (CAST KD cells) and NTP siRNA-treated cells (NTP cells). For this we have used the Flexcell FX-4000 Tension System (Flexcell), which uses vacuum pressure to apply cyclic or static strain to cells cultured on flexible-bottomed culture plates. CAST KD cells (mimicking the homozygous LOF mutations) and NTP cells were subjected to mechanical stretch at a frequency of 5 Hz (five cycles of stretch and relaxation per second) and an elongation of amplitude ranging from 10% to 14% (increase in diameter across the silicone deformable membrane from 10% to 14%). Cells were stretched for 0 hr (non-stretched) and 4 hr. Immunocytochemistry with an in-house LL001 monoclonal keratin 14 antibody18Purkis P.E. Steel J.B. Mackenzie I.C. Nathrath W.B. Leigh I.M. Lane E.B. Antibody markers of basal cells in complex epithelia.J. Cell Sci. 1990; 97: 39-50Crossref PubMed Google Scholar performed on siRNA-treated cells before and after mechanical stress revealed breakage of the intercellular connections in CAST KD cell monolayers, independent of whether they had been subjected to mechanical stress. In contrast, NTP cells presented with stretched keratin filaments after stretching but no disruption in intercellular adhesion prior to mechanical stress (Figures 4D–4G). A recent study by Nassar et al. looking at CAST overexpression in a mouse model reported significant changes in the wound-healing process compared to normal mice.19Nassar D. Letavernier E. Baud L. Aractingi S. Khosrotehrani K. Calpain activity is essential in skin wound healing and contributes to scar formation.PLoS ONE. 2012; 7: e37084Crossref PubMed Scopus (46) Google Scholar CAST-overexpression mice showed a striking delay in wound healing with reduced proliferation and re-epithelialization, particularly in the early steps of the wound-healing process.19Nassar D. Letavernier E. Baud L. Aractingi S. Khosrotehrani K. Calpain activity is essential in skin wound healing and contributes to scar formation.PLoS ONE. 2012; 7: e37084Crossref PubMed Scopus (46) Google Scholar The possible effects of CAST LOF mutations on keratinocyte migration were assessed by performing a scratch assay on CAST KD cell monolayers compared to NTP cells. Because CAST KD scratch wounds appeared to close at the same rate as NTP scratch wounds, we concluded that cell migration in monolayers was not altered by CAST knockdown in three independent experiments (Figure S4). After observations in CAST LOF skin and our in vitro studies indicating a key role for calpastatin in keratinocyte adhesion, we examined desmosomal protein abundance. Immunofluorescence with an antibody targeting desmoplakin (DSP; 11-5F mouse monoclonal, a gift from David Garrod),20Parrish E.P. Steart P.V. Garrod D.R. Weller R.O. Antidesmosomal monoclonal antibody in the diagnosis of intracranial tumours.J. Pathol. 1987; 153: 265-273Crossref PubMed Scopus (91) Google Scholar the major protein of the desmosome, was performed on frozen skin sections from individuals 1 and 2. This showed an apparent increase in DSP abundance, with both a plasma membrane and cytoplasmic localization pattern compared to a specific membranous localization pattern in control skin (Figures 4H, 4I, S3G, and S3H). Furthermore, our in vitro studies displayed a general trend of DSP upregulation in CAST KD cells independent of mechanical stress when compared to NTP cells (data not shown). Calpain and its endogenous specific inhibitor calpastatin constitute an intracellular non-lysosomal proteolytic system ubiquitously distributed in mammal cells and many other organisms. By catalyzing the controlled proteolysis of target proteins, calpains play an important role in various cell functions, including cell proliferation, differentiation, mobility, and cell cycle progression, as well as cell-type-specific functions like cell fusions in myoblasts.13Goll D.E. Thompson V.F. Li H. Wei W. Cong J. The calpain system.Physiol. Rev. 2003; 83: 731-801PubMed Google Scholar, 21Barnoy S. Maki M. Kosower N.S. Overexpression of calpastatin inhibits L8 myoblast fusion.Biochem. Biophys. Res. Commun. 2005; 332: 697-701Crossref PubMed Scopus (40) Google Scholar Also, activation of calpains has been suggested to trigger apoptosis by cleaving either pro-apoptotic or anti-apoptotic proteins.22Tan Y. Dourdin N. Wu C. De Veyra T. Elce J.S. Greer P.A. Ubiquitous calpains promote caspase-12 and JNK activation during endoplasmic reticulum stress-induced apoptosis.J. Biol. Chem. 2006; 281: 16016-16024Crossref PubMed Scopus (204) Google Scholar It has been demonstrated in vitro that increased activity of m-calpain results in apoptosis of HaCaT cells.23Inoue A. Yamazaki M. Ishidoh K. Ogawa H. Epidermal growth factor activates m-calpain, resulting in apoptosis of HaCaT keratinocytes.J. Dermatol. Sci. 2004; 36: 60-62Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar LOF mutations in CAST lead to disinhibition of calpains, thus enhancing apoptosis of keratinocytes, as showed in our TUNEL and TEM results. Elevated apoptotic levels of keratinocytes can result in skin hyperkeratosis,24Lin Z. Chen Q. Lee M. Cao X. Zhang J. Ma D. Chen L. Hu X. Wang H. Wang X. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am. J. Hum. Genet. 2012; 90: 558-564Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 25Wang H. Cao X. Lin Z. Lee M. Jia X. Ren Y. Dai L. Guan L. Zhang J. Lin X. et al.Exome sequencing reveals mutation in GJA1 as a cause of keratoderma-hypotrichosis-leukonychia totalis syndrome.Hum. Mol. Genet. 2015; 24: 243-250Crossref PubMed Scopus (39) Google Scholar leading to the clinical phenotypes of acral punctate keratoses, knuckle pads with hyperkeratotic micropapules, and leukonychia. Previous studies have shown that calpain could promote focal adhesion disassembly by proteolysis of talin and focal adhesion kinase.26Franco S.J. Rodgers M.A. Perrin B.J. Han J. Bennin D.A. Critchley D.R. Huttenlocher A. Calpain-mediated proteolysis of talin regulates adhesion dynamics.Nat. Cell Biol. 2004; 6: 977-983Crossref PubMed Scopus (421) Google Scholar, 27Chan K.T. Bennin D.A. Huttenlocher A. Regulation of adhesion dynamics by calpain-mediated proteolysis of focal adhesion kinase (FAK).J. Biol. Chem. 2010; 285: 11418-11426Crossref PubMed Scopus (158) Google Scholar Our CAST KD experiments indicate that the calpain/calpastatin system might also be critical for intercellular adhesion. Increased calpain activity due to LOF mutations in CAST might lead to excessive proteolysis of certain epidermal desmosomal components, which though not extensively described in the present study, result in impaired desmosome function characterized by decreased resistance of the epidermis to mechanical stretch (blistering, skin peeling, and acantholysis). This speculation was further strengthened by the remarkable phenotypic overlap between PLACK syndrome and other inherited disorders with desmosome deficiency, including inflammatory GPSS,6Oji V. Eckl K.M. Aufenvenne K. Nätebus M. Tarinski T. Ackermann K. Seller N. Metze D. Nürnberg G. Fölster-Holst R. et al.Loss of corneodesmosin leads to severe skin barrier defect, pruritus, and atopy: unraveling the peeling skin disease.Am. J. Hum. Genet. 2010; 87: 274-281Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar SAM syndrome,7Samuelov L. Sarig O. Harmon R.M. Rapaport D. Ishida-Yamamoto A. Isakov O. Koetsier J.L. Gat A. Goldberg I. Bergman R. et al.Desmoglein 1 deficiency results in severe dermatitis, multiple allergies and metabolic wasting.Nat. Genet. 2013; 45: 1244-1248Crossref PubMed Scopus (231) Google Scholar and Netherton syndrome (MIM 256500), the last of which was caused by LOF mutations in SPINK5 (MIM 605010) encoding protease inhibitor LEKTI,28Chavanas S. Bodemer C. Rochat A. Hamel-Teillac D. Ali M. Irvine A.D. Bonafé J.L. Wilkinson J. Taïeb A. Barrandon Y. et al.Mutations in SPINK5, encoding a serine protease inhibitor, cause Netherton syndrome.Nat. Genet. 2000; 25: 141-142Crossref PubMed Scopus (701) Google Scholar leading to over-degradation of CDSN and DSG1, two major components of the desmosome.29Samuelov L. Sprecher E. Peeling off the genetics of atopic dermatitis-like congenital disorders.J. Allergy Clin. Immunol. 2014; 134: 808-815Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar The increased abundance of DSP in keratinocytes observed in the two affected individuals might be a compensatory mechanism in response to desmosomal damage. Although calpastatin was suggested to play diverse physiological roles in neurological, musculoskeletal, and ocular systems,30Carragher N.O. Calpain inhibition: a therapeutic strategy targeting multiple disease states.Curr. Pharm. Des. 2006; 12: 615-638Crossref PubMed Scopus (146) Google Scholar no significant related symptoms were observed in our affected individuals. Further studies are required to elucidate whether there is redundancy of the calpain proteolytic system in these organs, explaining the lack of abnormalities. Notably, Cast-knockout mice, which showed increased activity of calpains, exhibit no defect under normal conditions31Takano J. Tomioka M. Tsubuki S. Higuchi M. Iwata N. Itohara S. Maki M. Saido T.C. Calpain mediates excitotoxic DNA fragmentation via mitochondrial pathways in adult brains: evidence from calpastatin mutant mice.J. Biol. Chem. 2005; 280: 16175-16184Crossref PubMed Scopus (176) Google Scholar and only slight behavioral changes in a stressful environment.32Nakajima R. Takao K. Huang S.M. Takano J. Iwata N. Miyakawa T. Saido T.C. Comprehensive behavioral phenotyping of calpastatin-knockout mice.Mol. Brain. 2008; 1: 7Crossref PubMed Scopus (40) Google Scholar These phenotypic differences indicate different physiological functions of the calpain/calpastatin system between humans and mice. In summary, we describe the clinical features of an autosomal-recessive entity termed PLACK syndrome with generalized skin peeling, leukonychia, acral punctate keratoses, cheilitis, and knuckle pads, distinct from epidermolysis bullosa, pachyonychia congenita, and Bart-Pumphrey syndrome (MIM 149200), a syndrome associated with sensorineural deafness, leukonychia, and knuckle pads. In three families with PLACK syndrome, homozygous LOF mutations in CAST were identified, leading to reduced abundance of calpastatin, the only known inhibitor of calpains. Mutations of protease inhibitors can disrupt the skin barrier, impair keratinocyte adhesion, affect cell signaling, and cause various genetic skin conditions,4Blaydon D.C. Nitoiu D. Eckl K.M. Cabral R.M. Bland P. Hausser I. van Heel D.A. Rajpopat S. Fischer J. Oji V. et al.Mutations in CSTA, encoding Cystatin A, underlie exfoliative ichthyosis and reveal a role for this protease inhibitor in cell-cell adhesion.Am. J. Hum. Genet. 2011; 89: 564-571Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 33de Veer S.J. Furio L. Harris J.M. Hovnanian A. Proteases: common culprits in human skin disorders.Trends Mol. Med. 2014; 20: 166-178Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar such as Netherton syndrome caused by mutations in SPINK528Chavanas S. Bodemer C. Rochat A. Hamel-Teillac D. Ali M. Irvine A.D. Bonafé J.L. Wilkinson J. Taïeb A. Barrandon Y. et al.Mutations in SPINK5, encoding a serine protease inhibitor, cause Netherton syndrome.Nat. Genet. 2000; 25: 141-142Crossref PubMed Scopus (701) Google Scholar and Nagashima-type palmoplantar keratosis (MIM 615598) caused by mutations in SERPINB7 (MIM 603357).34Kubo A. Shiohama A. Sasaki T. Nakabayashi K. Kawasaki H. Atsugi T. Sato S. Shimizu A. Mikami S. Tanizaki H. et al.Mutations in SERPINB7, encoding a member of the serine protease inhibitor superfamily, cause Nagashima-type palmoplantar keratosis.Am. J. Hum. Genet. 2013; 93: 945-956Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 35Yin J. Xu G. Wang H. Zhao J. Duo L. Cao X. Tang Z. Lin Z. Yang Y. New and recurrent SERPINB7 mutations in seven Chinese patients with Nagashima-type palmoplantar keratosis.J. Invest. Dermatol. 2014; 134: 2269-2272Crossref PubMed Scopus (33) Google Scholar Our findings expand the spectrum of these conditions and explore new avenues for proteolytic pathways in skin. We are grateful to the patients and their family members for participation in this study. We gratefully acknowledge Dr. Deirdre Buckley (Bath, UK) who referred individual 2 to us. This work was supported in part by China National Funds for Distinguished Young Scientists (81425020 to Y.Y.), National Natural Science Foundation of China (81271744 to Y.Y. and 81201220 to Z.L.), Beijing Higher Education Young Elite Teacher Project (YETP0069 for Z.L.), and Shenzhen Key Laboratory of Cognitive Genomics (CXB201108250094A). F.J.D.S. and N.J.W. are supported by grants from the Pachyonychia Congenita Project. The Centre for Dermatology and Genetic Medicine at the University of Dundee is supported by a Wellcome Trust Strategic Award (098439/Z/12/Z to W.H.I.M.). This study is also funded, in part, by Barts and the London Charity (D.P.K.). Download .pdf (.46 MB) Help with pdf files Document S1. Figures S1–S4 and Tables S1 and S2 The URLs for data presented herein are as follows:1000 Genomes, http://browser.1000genomes.orgdbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/International HapMap Project, http://hapmap.ncbi.nlm.nih.gov/NHLBI Exome Sequencing Project (ESP) Exome Variant Server, http://evs.gs.washington.edu/EVS/OMIM, http://www.omim.org/Pachyonychia Congenita Project, http://www.pachyonychia.org/RefSeq, http://www.ncbi.nlm.nih.gov/RefSeq

Thirteen patients with pachyonychia congenita types 1 and 2 were studied, two of which had a family history of pachyonychia and 11 of which were sporadic cases. Heterozygous mis-sense or small in-frame insertion/deletion mutations were detected in the genes encoding keratins K6a, K16, and K17 in all cases. Three novel mutations, F174V, E472K, and L469R were found in the K6a gene. Two novel mutations, M121T and L128Q were detected in K16. Similarly, three novel mutations, L95P, S97del, and L99P were found in K17. In addition, we identified recurrent mutations N171del (three instances) and F174S in K6a and R94H in K17. Analysis of both phenotype and genotype data led to the following conclusions: (i) K6a or K16 mutations produce the pachyonychia congenita type 1 phenotype, whereas K17 (or K6b) mutations cause pachyonychia congenita type 2; (ii) the presence of pilosebaceous cysts following puberty is the best indicator of pachyonychia congenita type 2; (iii) prepubescent patients are more difficult to classify due to the lack of cysts; and (iv) natal teeth are indicative of pachyonychia congenita type 2, although their absence does not preclude the pachyonychia congenita type 2 phenotype. This study establishes useful diagnostic criteria for pachyonychia congenita types 1 and 2, which will help limit unnecessary DNA analysis in the diagnosis and management of this genetically heterogeneous group of genodermatoses.

The severe Dowling-Meara form of epidermolysis bullosa simplex is caused by dominant-negative mutations in keratins 5 and 14, which are specifically expressed in the basal keratinocytes of the epidermis. The most common mutation in the Dowling-Meara form of epidermolysis bullosa simplex patients is the missense mutation R125C in exon 1 of the K14 gene. We made a primary keratinocyte cell line from a sporadic case known to carry the R125C mutation as part of an ongoing gene therapy initiative. The full-length K14 cDNA was sequenced using keratinocyte mRNA. Unexpectedly, a second mutation was identified in K14: a heterozygous 1 bp insertion mutation (242insG) upstream of the R125C mutation. This frameshift mutation creates a premature termination codon immediately downstream, thereby nullifying the dominant-negative allele. The second mutation was only present in DNA derived from keratinocytes and was absent from lymphocyte DNA. This case represents a novel mechanism of revertant mosaicism and is an example of "natural gene therapy".

Pachyonychia congenita (PC) is a rare autosomal dominant keratin disorder, subdivided into two major variants, PC-1 and PC-2. Predominant characteristics include hypertrophic nail dystrophy, focal palmoplantar keratoderma and oral leukokeratosis. Multiple steatocystomas that develop during puberty are a useful feature distinguishing PC-2 from PC-1. At the molecular level it has been shown that mutations in keratin K6a or K16 cause PC-1 whereas those in K6b or K17 lead to PC-2.To identify mutations in 22 families presenting with clinical symptoms of either PC-1/focal non-epidermolytic palmoplantar keratoderma (FNEPPK) or PC-2.Mutation analysis was performed on genomic DNA from PC patients by direct sequencing.Here, we report four new missense and five known mutations in K6a; one new deletion and three previously identified missense mutations in K16; plus one known mutation in K17.With one exception, all these heterozygous mutations are within the highly conserved helix boundary motif regions at either end of the keratin rod domain. In one sporadic case, a unique mutation in K16 resulting in deletion of 24bp was found within the central rod domain, in a child with a phenotype predominantly consisting of focal plantar keratoderma. The identification of mutations in cases of PC is prerequisite for future development of gene-specific and/or mutation-specific therapies.

atopic dermatitis filaggrin ichthyosis vulgaris Mutations in the gene encoding filaggrin (FLG) were identified as the underlying cause of ichthyosis vulgaris (IV; OMIM #146700) and also shown to predispose to atopic dermatitis (AD; Palmer et al., 2006Palmer C.N. Irvine A.D. Terron-Kwiatkowski A. Zhao Y. Liao H. Lee S.P. et al.Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.Nat Genet. 2006; 38: 441-446Crossref PubMed Scopus (2088) Google Scholar; Smith et al., 2006Smith F.J.D. Irvine A.D. Terron-Kwiatkowski A. Sandilands A. Campbell L.E. Zhao Y. et al.Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris.Nat Genet. 2006; 38: 337-342Crossref PubMed Scopus (755) Google Scholar). Although FLG is considerably difficult to analyze because of its large size (>12kb) and highly repetitive nature, PCR strategy that permits routine and comprehensive sequencing of the entire FLG has been developed recently (Sandilands et al., 2007Sandilands A. Terron-Kwiatkowski A. Hull P.R. O'Regan G.M. Clayton T.H. Watson R.M. et al.Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema.Nat Genet. 2007; 39: 650-654Crossref PubMed Scopus (474) Google Scholar). Using this methodology, we have identified four prevalent FLG mutations in Japanese patients with IV (Nomura et al., 2008Nomura T. Akiyama M. Sandilands A. Nemoto-Hasebe I. Sakai K. Nagasaki A. et al.Specific filaggrin mutations cause ichthyosis vulgaris and are significantly associated with atopic dermatitis in Japan.J Invest Dermatol. 2008; 128: 1436-1441Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). We also demonstrated that FLG mutations were significantly associated with AD and the frequency of these FLG mutations observed in our Japanese AD cohort was about 20%. However, the frequency in our cohort was still lower than that seen in analogous European case series, where it is up to 48% (Barker et al., 2007Barker J.N. Palmer C.N. Zhao Y. Liao H. Hull P.R. Lee S.P. et al.Null mutations in the filaggrin gene (FLG) determine major susceptibility to early-onset atopic dermatitis persists into adulthood.J Invest Dermatol. 2007; 127: 564-567Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar; Sandilands et al., 2007Sandilands A. Terron-Kwiatkowski A. Hull P.R. O'Regan G.M. Clayton T.H. Watson R.M. et al.Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema.Nat Genet. 2007; 39: 650-654Crossref PubMed Scopus (474) Google Scholar). Furthermore, it was reported that up to 37% of Japanese patients with AD had concomitant IV (Uehara and Hayashi, 1981Uehara M. Hayashi S. Hyperlinear palms: association with ichthyosis and atopic dermatitis.Arch Dermatol. 1981; 117: 490-491Crossref PubMed Scopus (46) Google Scholar; Uehara and Miyauchi, 1984Uehara M. Miyauchi H. The morphologic characteristics of dry skin in atopic dermatitis.Arch Dermatol. 1984; 120: 1186-1190Crossref PubMed Scopus (61) Google Scholar). Taken together, there might be further prevalent FLG mutations to be discovered in the Japanese population. Here we have studied a further Japanese family with IV and identified two further FLG mutations. A newly recruited Japanese family with IV was studied. The proband, a one-year-old Japanese boy, showed marked scaly dry skin on the extensor limbs and trunk (Figure 1a). Marked palmoplantar hyperlinearity was also evident (Figure 1b and c). A diagnosis of IV was made from these clinical observations. His mother and sister also showed scaly dry skin and palmoplantar hyperlinearity, but the clinical severity was mild compared to the proband (Figure 1d). Therefore, the inheritance pattern seemed semidominant. The proband, his mother, and his brother had concomitant AD. The medical ethical committee at Hokkaido University Graduate School of Medicine approved all the studies. The study was conducted according to the Declaration of Helsinki Principles. Participants or their legal guardians gave their written informed consent. Following informed consent, genomic DNA from all family members was extracted from peripheral blood according to standard procedures. Initially, all family members were screened for five FLG mutations identified in Japanese population so far, R501X, 3321delA, S2554X, S2889X and S3296X, by restriction enzyme digestion, fluorescent PCR, and direct DNA sequencing as described previously (Nomura et al., 2007Nomura T. Sandilands A. Akiyama M. Liao H. Evans A.T. Sakai K. et al.Unique mutations in the filaggrin gene in Japanese patients with ichthyosis vulgaris and atopic dermatitis.J Allergy Clin Immunol. 2007; 119: 434-440Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, Nomura et al., 2008Nomura T. Akiyama M. Sandilands A. Nemoto-Hasebe I. Sakai K. Nagasaki A. et al.Specific filaggrin mutations cause ichthyosis vulgaris and are significantly associated with atopic dermatitis in Japan.J Invest Dermatol. 2008; 128: 1436-1441Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar; Hamada et al., 2008Hamada T. Sandilands A. Fukuda S. Sakaguchi S. Ohyama B. Yasumoto S. et al.De novo occurrence of the filaggrin mutation p.R501X with prevalent mutation c.3321delA in a Japanese family with ichthyosis vulgaris complicated by atopic dermatitis.J Invest Dermatol. 2008; 128: 1323-1325Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). However, all individuals were wild type for these variants. Thus, we carried out full sequencing of the FLG as described previously (Sandilands et al., 2007Sandilands A. Terron-Kwiatkowski A. Hull P.R. O'Regan G.M. Clayton T.H. Watson R.M. et al.Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema.Nat Genet. 2007; 39: 650-654Crossref PubMed Scopus (474) Google Scholar), which led to the identification of a previously unreported nonsense mutation Q1701X in repeat 4 in the present family (Figure 1e). The proband turned out to be homozygous for this truncation mutation and his non-consanguineous parents and his sister heterozygous, whereas his brother wild type (Figure 1d). It was also confirmed that they carry no pathogenic mutations in the other FLG repeats. Then, we screened 118 unrelated Japanese patients with AD and 134 unrelated Japanese control individuals for Q1701X by direct DNA sequencing. The diagnosis of AD in our case series was made by experienced dermatologists, according to the AD diagnostic criteria by Hannifin and Rajka, 1980Hannifin J.M. Rajka G. Diagnostic features of atopic dermatitis.Acta Derm Venereol. 1980; 92: 44-47Google Scholar. Notably, mutation Q1701X was also identified in two Japanese patients with AD (1.7%), which brings the total number of recurrent FLG mutations so far identified in Japanese population to five. During the screening for Q1701X, we identified another previously unreported FLG mutation, S1695X, which is located only six amino acids upstream from Q1701X, in the general Japanese control population (Figure 1f). We screened 33 Japanese patients with IV and 118 with AD for S1695X, but all patients were wild type for this mutation. Only one heterozygote was identified in the control population. Therefore, S1695X seems to be an extremely rare FLG mutation in Japanese individuals. The control individuals had not been examined in relation to AD or IV status, that is, they were population controls rather than “hypernormal” controls, so no clinical details about the individual carrying S1695X are available. In total, there are at least seven FLG variants in the Japanese population, including five that are prevalent and two that are quite rare. The FLG genotype data in the Japanese AD case series and ethnically matched population control series are summarized in Table 1. In this study, case–control association analyses were performed by using Pearson's r2 statistics, as previously described (Palmer et al., 2006Palmer C.N. Irvine A.D. Terron-Kwiatkowski A. Zhao Y. Liao H. Lee S.P. et al.Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.Nat Genet. 2006; 38: 441-446Crossref PubMed Scopus (2088) Google Scholar). All alleles were observed to be in normal Hardy–Weinberg equilibrium. Here we demonstrate that about 25% of patients in our Japanese AD case series carry one or more of these seven FLG mutations (combined minor allele frequency=0.127, n=236) and these variants are also carried by 4% of general Japanese control individuals (combined minor allele frequency=0.019, n=268). There is significant statistical association between the seven FLG mutations and AD (r2P=1.75 × 10-6). Moreover, AD was manifested in heterozygous carriers of these FLG mutations with a Fisher's exact test odds ratio for AD of 6.8 (95% CI 2.5–18.5, P=3.7 × 10-5), implying a causal relationship between FLG mutations and AD. Taken together, these data strongly suggest that skin barrier impairment because of reduced filaggrin expression is important in the pathogenesis in AD.Table 1Atopic dermatitis case–control association analysis for FLG null variants in JapanR501X3321delAS1695XQ1701XS2554XS2889XS3296XCombinedGenotypesControlsCasesControlsCasesControlsCasesControlsCasesControlsCasesControlsCasesControlsCasesControlsCasesAA13411813311313311813411613311213210513411412991Aa001510021621304524aa0000000000000003Total134118134118134118134118134118134118134118134118For combined genotype, r2P=1.75 × 10-6; Fisher's exact test odds ratio=6.8 (95% CI 2.5–18.5). Open table in a new tab For combined genotype, r2P=1.75 × 10-6; Fisher's exact test odds ratio=6.8 (95% CI 2.5–18.5). To date, 24 FLG mutations, including the two identified in this study, have been reported in the European, Japanese, and Singaporean populations (Sandilands et al., 2007Sandilands A. Terron-Kwiatkowski A. Hull P.R. O'Regan G.M. Clayton T.H. Watson R.M. et al.Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema.Nat Genet. 2007; 39: 650-654Crossref PubMed Scopus (474) Google Scholar; Chen et al., 2008Chen H. Ho J.C.C. Sandilands A. Chan Y.C. Giam Y.C. Evans A.T. et al.Unique and recurrent mutations in the filaggrin gene in Singaporean Chinese patients with ichthyosis vulgaris.J Invest Dermatol. 2008; 128: 1669-1675Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar; Nomura et al., 2008Nomura T. Akiyama M. Sandilands A. Nemoto-Hasebe I. Sakai K. Nagasaki A. et al.Specific filaggrin mutations cause ichthyosis vulgaris and are significantly associated with atopic dermatitis in Japan.J Invest Dermatol. 2008; 128: 1436-1441Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Interestingly, mutations found in Japanese are different from those found in Europeans and Singaporean (Figure 1g), except in one case of the common European R501X mutation occurring as a very rare mutation on a different haplotype in the Japanese population (Hamada et al., 2008Hamada T. Sandilands A. Fukuda S. Sakaguchi S. Ohyama B. Yasumoto S. et al.De novo occurrence of the filaggrin mutation p.R501X with prevalent mutation c.3321delA in a Japanese family with ichthyosis vulgaris complicated by atopic dermatitis.J Invest Dermatol. 2008; 128: 1323-1325Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). These observations imply that every population is highly likely to have a unique set of FLG mutations. In conclusion, we have identified two further FLG mutations in the Japanese population. We also showed that at least about 25% of Japanese patients with AD carried one or more of FLG mutations. As we have sequenced more than 30 Japanese patients with IV, there is now little possibility that further highly prevalent mutations underlie the Japanese population. Taking the high frequency (up to 37%) of concomitant IV in patients with AD into account, however, it is still possible that there might be further multiple low-frequency FLG mutations to be discovered in the Japanese population. Further FLG mutation analysis will be necessary to understand the more precise genetic architecture of filaggrin-related AD in Japan. Irwin McLean has filed patents relating to genetic testing and therapy development aimed at the filaggrin gene. We thank the patients and their families for their participation. We also thank Ai Hayakawa for the fine technical assistance. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan to M Akiyama (Kiban B 20390304). Filaggrin research in the McLean laboratory is supported by grants from The British Skin Foundation; The National Eczema Society; The Medical Research Council (reference number G0700314); A*STAR, Singapore; and donations from anonymous families affected by eczema in the Tayside region of Scotland.

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. Methods: FLG mutations R501X and 2282del4 were assayed in 1135 individuals ranging from 3 to 22 years old with asthma from Tayside and Dumfries, Scotland. Asthma exacerbations over the previous 6 months were also studied. Results: The FLG mutations were significantly associated with greater risk of exacerbations in children with asthma. Exacerbations were significant for the R501X but not the 2282del4 mutation and the combined genotype compared to the wild‐type with odds ratios of 1.97 (95% CI, 1.19–3.22; P = 0.009) and 1.61 (95% CI, 1.08–2.40; P = 0.021), respectively. Individuals with FLG null alleles were more likely to require oral steroids (31.4% vs 19.5%; OR = 1.89; P = 0.021) for their exacerbations. There was also a 1.71‐fold increased risk (42.6% vs 30%; P = 0.041) of school absence owing to asthma exacerbations in asthmatic individuals with FLG null mutation. On sub‐group analysis, the effect of FLG mutations on asthma exacerbations is significant ( P = 0.045) only for participants with relatively mild asthma controlled on inhaled steroids, with inhaled albuterol according to need. Conclusion: In addition to their effect on asthma medication requirements reported previously, there is an association between the presence of FLG null mutations and the risk of asthma exacerbations in asthmatic children and young adults.

Epidermolysis bullosa simplex (EBS) is an inherited skin disorder caused by mutations in keratins K5 (keratin 5) and K14 (keratin 14), with fragility of basal keratinocytes leading to epidermal cytolysis and blistering. Patients present with widely varying severity and are classified in three main subtypes: EBS Weber-Cockayne (EBS-WC), EBS Köbner (EBS-K), and EBS Dowling-Meara (EBS-DM), based on distribution and pattern of blisters. We could identify K5/K14 mutations in 20 out of the 43 families registered as affected by dominant EBS in Scotland; with previous studies this covers 70% of all Scottish EBS patients, making this the most comprehensively analyzed EBS population. Nine mutations are novel. All mutations lie within five previously identified rod domain hotspots and the severest blistering was associated with mutations in the helix boundary motifs. In some cases, the same mutation caused symptoms of EBS-WC and/or EBS-K, both within and between families, suggesting a contribution of additional factors to the phenotype. In some patients, no mutations were found in K5, K14, or K15, suggesting involvement of other genes. The results confirm that EBS is best considered as a single disorder with a spectrum of phenotypic variations, from severe EBS-DM at one extreme to mild EBS-WC at the other.

Meesmann epithelial corneal dystrophy (MECD) is an inherited eye disorder caused by dominant-negative mutations in either keratins K3 or K12, leading to mechanical fragility of the anterior corneal epithelium, the outermost covering of the eye. Typically, patients suffer from lifelong irritation of the eye and/or photophobia but rarely lose visual acuity; however, some individuals are severely affected, with corneal scarring requiring transplant surgery. At present no treatment exists which addresses the underlying pathology of corneal dystrophy. The aim of this study was to design and assess the efficacy and potency of an allele-specific siRNA approach as a future treatment for MECD.We studied a family with a consistently severe phenotype where all affected persons were shown to carry heterozygous missense mutation Leu132Pro in the KRT12 gene. Using a cell-culture assay of keratin filament formation, mutation Leu132Pro was shown to be significantly more disruptive than the most common mutation, Arg135Thr, which is associated with typical, mild MECD. A siRNA sequence walk identified a number of potent inhibitors for the mutant allele, which had no appreciable effect on wild-type K12. The most specific and potent inhibitors were shown to completely block mutant K12 protein expression with negligible effect on wild-type K12 or other closely related keratins. Cells transfected with wild-type K12-EGFP construct show a predominantly normal keratin filament formation with only 5% aggregate formation, while transfection with mutant K12-EGFP construct resulted in a significantly higher percentage of keratin aggregates (41.75%; p<0.001 with 95% confidence limits). The lead siRNA inhibitor significantly rescued the ability to form keratin filaments (74.75% of the cells contained normal keratin filaments; p<0.001 with 95% confidence limits).This study demonstrates that it is feasible to design highly potent siRNA against mutant alleles with single-nucleotide specificity for future treatment of MECD.

Pachyonychia congenita (PC), a rare autosomal-dominant keratin disorder caused by mutations in keratin genes KRT6A/B, KRT16, or KRT17, is characterized by painful plantar keratoderma and hypertrophic nail dystrophy. Available studies assessing oral retinoid treatment for PC are limited to a few case reports.We sought to assess overall effectiveness, adverse effects, and patient perspective in patients with PC receiving oral retinoids.In a questionnaire-based retrospective cross-sectional survey of 30 patient with PC assessing oral retinoids (10-50 mg/d for 1-240 months), we determined the clinical score, satisfaction score, visual analog pain scale, and adverse effects.In 50% of patients there was thinning of hyperkeratoses (average improvement 1.6 on a scale from -3 to +3) (95% confidence interval 1.2-1.9, P < .001). In all, 14% observed amelioration of their pachyonychia; 79% did not experience any nail change. The self-reported overall satisfaction score with oral retinoid treatment was 2 or greater in 50% of the patients (mean 4.5 on a scale of 1-10). Although 33% reported decreased and 27% increased plantar pain with treatment, 40% did not notice any pain change. All patients experienced adverse effects, and 83% reported to have discontinued medication. Risk/benefit analysis favored lower retinoid doses (≤25 mg/d) over a longer time period (>5 months), compared with higher doses (>25 mg/d) for a shorter time (≤5 months).The retrospective, cross-sectional study design is prone to a recall bias.Oral retinoids are effective in some patients with PC. However, many patients discontinued medication because adverse effects outweighed the benefits. Careful dose titration is warranted in patients informed about potential adverse effects.

Epidermolytic palmoplantar keratoderma (EPPK) is one of >30 autosomal-dominant human keratinizing disorders that could benefit from RNA interference (RNAi)-based therapy. EPPK is caused by mutations in the keratin 9 (KRT9) gene, which is exclusively expressed in thick palm and sole skin where there is considerable keratin redundancy. This, along with the fact that EPPK is predominantly caused by a few hotspot mutations, makes it an ideal proof-of-principle model skin disease to develop gene-specific, as well as mutation-specific, short interfering RNA (siRNA) therapies. We have developed a broad preclinical RNAi-based therapeutic package for EPPK containing generic KRT9 siRNAs and allele-specific siRNAs for four prevalent mutations. Inhibitors were systematically identified in vitro using a luciferase reporter gene assay and validated using an innovative dual-Flag/Strep-TagII quantitative immunoblot assay. siKRT9-1 and siKRT9-3 were the most potent generic K9 inhibitors, eliciting >85% simultaneous knockdown of wild-type and mutant K9 protein synthesis at picomolar concentrations. The allele-specific inhibitors displayed similar potencies and, importantly, exhibited strong specificities for their target dominant-negative alleles with little or no effect on wild-type K9. The most promising allele-specific siRNA, siR163Q-13, was tested in a mouse model and was confirmed to preferentially inhibit mutant allele expression in vivo.

Keratin 7 (K7) is a Type II member of the keratin superfamily and despite its widespread expression in different types of simple and transitional epithelia, its functional role in vivo remains elusive, in part due to the lack of any appropriate mouse models or any human diseases that are associated with KRT7 gene mutations. Using conventional gene targeting in mouse embryonic stem cells, we report here the generation and characterisation of the first K7 knockout mouse. Loss of K7 led to increased proliferation of the bladder urothelium although this was not associated with hyperplasia. K18, a presumptive type I assembly partner for K7, showed reduced expression in the bladder whereas K20, a marker of the terminally differentiated superficial urothelial cells was transcriptionally up-regulated. No other epithelia were seen to be adversely affected by the loss of K7 and western blot and immunofluorescence microscopy analysis revealed that the expression of K8, K18, K19 and K20 were not altered in the absence of K7, with the exception of the kidney where there was reduced K18 expression.

Therapeutics based on short interfering RNAs (siRNAs), which act by inhibiting the expression of target transcripts, represent a novel class of potent and highly specific next-generation treatments for human skin diseases. Unfortunately, the intrinsic barrier properties of the skin combined with the large size and negative charge of siRNAs make epidermal delivery of these macromolecules quite challenging. To help evaluate the in vivo activity of these therapeutics and refine delivery strategies we generated an innovative reporter mouse model that predominantly expresses firefly luciferase (luc2p) in the paw epidermis--the region of murine epidermis that most closely models the tissue architecture of human skin. Combining this animal model with state-of-the-art live animal imaging techniques, we have developed a real-time in vivo analysis work-flow that has allowed us to compare and contrast the efficacies of a wide range nucleic acid-based gene silencing reagents in the skin of live animals. While inhibition was achieved with all of the reagents tested, only the commercially available "self-delivery" modified Accell-siRNAs (Dharmacon) produced potent and sustained in vivo gene silencing. Together, these findings highlight just how informative reliable reporter mouse models can be when assessing novel therapeutics in vivo. Using this work-flow, we developed a novel clinically-relevant topical formulation that facilitates non-invasive epidermal delivery of unmodified and "self-delivery" siRNAs. Remarkably, a sustained >40% luc2p inhibition was observed after two 1-hour treatments with Accell-siRNAs in our topical formulation. Importantly, our ability to successfully deliver siRNA molecules topically brings these novel RNAi-based therapeutics one-step closer to clinical use.

epidermolysis bullosa simplex TO THE EDITOR Epidermolysis bullosa simplex (EBS) is a mechanobullous genodermatosis characterized by an intraepidermal split through the cytoplasm of basal keratinocytes, which is mainly caused by dominant-negative mutations in the genes encoding keratins 5 and 14 (Coulombe et al., 1991Coulombe P.A. Hutton M.E. Letai A. et al.Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses.Cell. 1991; 66: 1301-1311Abstract Full Text PDF PubMed Scopus (533) Google Scholar; Lane et al., 1992Lane E.B. Rugg E.L. Navsaria H. et al.A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering.Nature. 1992; 356: 244-246Crossref PubMed Scopus (341) Google Scholar). Mutation analysis of KRT5 and KRT14 in a large biopsy-confirmed EBS population in the Netherlands, however, revealed that in 25% of unrelated cases no mutations could be identified in these genes (Bolling et al., 2011Bolling M.C. Lemming H.H. Jansen G.H. et al.Mutations in KRT5 and KRT14 cause epidermolysis bullosa simplex in 75% of the patients.Br J Dermatol. 2011; 164: 637-644PubMed Google Scholar). A similar percentage of EBS cases with wild-type KRT5 and KRT14 genes was reported for the EBS population in the United Kingdom (Rugg et al., 2007Rugg E.L. Horn H.M. Smith F.J. et al.Epidermolysis bullosa simplex in Scotland caused by a spectrum of keratin mutations.J Invest Dermatol. 2007; 127: 574-580Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). A missense mutation, Arg2000Trp, in PLEC, encoding the hemidesmosomal protein plectin, which connects the basal keratins to the hemidesmosomal plaque, was associated in cases with dominant EBS of hands and feet (EBS-Ogna) (Koss-Harnes et al., 2002Koss-Harnes D. Hoyheim B. Anton-Lamprecht I. et al.A site-specific plectin mutation causes dominant epidermolysis bullosa simplex Ogna: two identical de novo mutations.J Invest Dermatol. 2002; 118: 87-93Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar; Kiritsi et al., 2013Kiritsi D. Pigors M. Tantcheva-Poor I. et al.Epidermolysis bullosa simplex Ogna revisited.J Invest Dermatol. 2013; 133: 270-273Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). In this study, we investigated the frequency of PLEC mutations in biopsy-proven EBS probands that lacked mutations in KRT5 and KRT14. The study was performed according to the Declaration of Helsinki principles, and informed consent was obtained from the patients. PLEC mutation analysis was performed in 16 Dutch probands with a biopsy-proven EBS in which mutations in KRT5 and KRT14 had been excluded. PCR amplification of all exons and adjacent intronic sequences of PLEC (GenBank NM_000445) was performed using primers located in the flanking introns. Primers resulting in overlapping PCR products were used for the large exons 31 and 32 (information about primers and amplification conditions is available on request). Mutations found were excluded in at least 150 matched healthy control DNA samples. In 6 of the 16 probands (38%), we found three autosomal-dominant PLEC missense mutations (Table 1, Supplementary Files S1 and S2 online). The mutations segregated with the phenotype were not known as single-nucleotide polymorphisms and were absent in control DNA samples, thus strongly indicating pathogenicity. The missense mutations affected well-conserved residues (Supplementary File S2 online). Four unrelated probands (EB184, proband 1; EB149, proband 2; EB229, proband 3; and EB241, proband 4) carried the original Ogna mutation PLEC:c.5998C>T, p.Arg2000Trp. In EB149, the mutation was de novo. In probands 1, 3 and 4, the mutation segregated with the phenotype in an autosomal-dominant fashion in the family. In proband 5, a refugee from Iran, a new heterogygous missense mutation c.8668A>T, p.Thr2890Ser was detected. An affected brother carried the mutation, whereas an unaffected son did not carry the mutation. In proband 6, another new heterozygous missense mutation was detected: c.10597C>T, p.Arg3527Cys. This mutation was found in his affected mother as well. The unaffected siblings and father did not carry the mutation. Both new PLEC mutations are therefore dominant. The clinical features (Figure 1a, Supplementary File S1 online) of seasonal blisters of hands, feet, and lower legs are consistent with previous reports on EBS-Ogna (Gedde-Dahl, 1971Gedde-Dahl Jr., T. Epidermolysis Bullosa: A Clinical, Genetic and Epidemiological Study. The Johns Hopkins University Press, Baltimore, MD1971Google Scholar; Kiritsi et al., 2013Kiritsi D. Pigors M. Tantcheva-Poor I. et al.Epidermolysis bullosa simplex Ogna revisited.J Invest Dermatol. 2013; 133: 270-273Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Cardiological and neurological examination excluded cardiomyopathy and muscular dystrophy in all probands.Table 1Characteristics of the PLEC mutations found in this study and associated immunofluorescence microscopy findings in patient skin biopsiesProbandMutation c.1Numbering according to GenBank NM_000445.3 with one being the adenine from the ATG start codon.Mutation p.2GenBank NP_000436.2 (isoform 1c).InheritanceDomainConservationImmunofluorescence1 (EB184)5998C>T (CGG>TGG)Arg2000TrpADRodM, d, zf, frHD121 ↓, 10F6 absent in basal cell layer, and reduced throughout epidermis2 (EB149)5998C>T (CGG>TGG)Arg2000TrpSpRodM, d, zf, frHD121 ↓, 10F6 absent in basal cell layer, reduced throughout epidermis3 (EB229)5998C>T (CGG>TGG)Arg2000TrpADRodM, d, zf, frHD121 ↓, 10F6 absent in basal cell layer, and reduced throughout epidermis4 (EB241)5998C>T (CGG>TGG)Arg2000TrpADRodM, d, zf, frHD121 ↓, 10F6 absent in basal cell layer, reduced throughout epidermis5 (EB146)8668A>T (ACG<TCG)Thr2890SerADPlakin-repeat domain 1M, d, zfHD121∼normal, 10F6 slightly↓panepidermal6 (EB203)10579C>T (CGC>TGC)Arg3527CysADPlakin-repeat domain 3M, dHD121∼normal, 10F6 slightly↓panepidermalAbbreviations: AD, autosomal dominant; d, dog; fr, frog; M, mouse; Sp, sporadic; zf, zebrafish.1 Numbering according to GenBank NM_000445.3 with one being the adenine from the ATG start codon.2 GenBank NP_000436.2 (isoform 1c). Open table in a new tab Download .pdf (4.8 MB) Help with pdf files Supplementary Information Abbreviations: AD, autosomal dominant; d, dog; fr, frog; M, mouse; Sp, sporadic; zf, zebrafish. Electron microscopic analysis revealed abnormal hemidesmosomes and an intraepidermal split just above the hemidesmosomal plaque (Supplementary File S3b online). Immunofluorescence plectin staining with antibody HD121 in skin samples of probands 1–4 (p.Arg2000Trp) was reduced along the epidermal basement membrane zone, and the faint pericellular epidermal staining was absent (Supplementary File S3 online). In contrast, skin of probands 5 (p.Thr2890Ser) and 6 (p.Arg3527Cys) showed normal HD121 staining. Staining of plectin with 10F6 was absent in the basel cell layer in probands 1–4 or reduced in probands 5 and 6 (Table 1, Figure 1b). Expression of hemidesmosomal components type XVII collagen and integrin β4 was normal in all probands (data not shown). In hemidesmosomes, plectin is an important anchoring protein for basal keratins to integrin β4 (for review see Sonnenberg and Liem, 2007Sonnenberg A. Liem R.K. Plakins in development and disease.Exp Cell Res. 2007; 313: 2189-2203Crossref PubMed Scopus (227) Google Scholar). Plectin dimerizes with its central α-helical coiled-coil rod domain that stabilizes hemidesmosomes (Koster et al., 2004Koster J. van Wilpe S. Kuikman I. et al.Role of binding of plectin to the integrin beta4 subunit in the assembly of hemidesmosomes.Mol Biol Cell. 2004; 15: 1211-1223Crossref PubMed Scopus (103) Google Scholar). Disruptive amino acid substitutions in the rod domain, like the Arg>Trp substitution at position 2,000 (large, basic, and positively charged arginine to hydrophobic and neutral tryptophan), interfere with dimer formation and make it more vulnerable to proteolysis (Walko et al., 2011Walko G. Vukasinovic N. Gross K. et al.Targeted proteolysis of plectin isoform 1a accounts for hemidesmosome dysfunction in mice mimicking the dominant skin blistering disease EBS-Ogna.PLoS Genet. 2011; 7: e1002396Crossref PubMed Scopus (50) Google Scholar; Winter and Wiche, 2012Winter L. Wiche G. The many faces of plectin and plectinopathies: pathology and mechanisms.Acta Neuropathol. 2012; 125: 77-93Crossref PubMed Scopus (99) Google Scholar). Mutations p.Thr2890Ser and p.Arg3527Cys are located in the plakin-repeat domains PRD1 and PRD3, respectively (Figure 1c). These substitutions predict considerable alterations to protein folding. Both mutations do not directly affect the known intermediate filament-binding site that resides in PRD5-PRD6 (Steinbock et al., 2000Steinbock F.A. Nikolic B. Coulombe P.A. et al.Dose-dependent linkage, assembly inhibition and disassembly of vimentin and cytokeratin 5/14 filaments through plectin’s intermediate filament-binding domain.J Cell Sci. 2000; 113: 483-491PubMed Google Scholar). However, all PRDs are believed to be tightly packed to provide structural rigidity (Janda et al., 2001Janda L. Damborsky J. Rezniczek G.A. et al.Plectin repeats and modules: strategic cysteines and their presumed impact on cytolinker functions.Bioessays. 2001; 23: 1064-1069Crossref PubMed Scopus (32) Google Scholar). In addition, a phosphorylation site and binding sites for several cell signaling proteins have been identified in the plectin C-terminus (Osmanagic-Myers and Wiche, 2004Osmanagic-Myers S. Wiche G. Plectin-RACK1 (receptor for activated C kinase 1) scaffolding: a novel mechanism to regulate protein kinase C activity.J Biol Chem. 2004; 279: 18701-18710Crossref PubMed Scopus (95) Google Scholar). Threonine residues are often involved in phosphorylation. Alignment of the six PRDs revealed that in some PRDs the residue corresponding to Thr2890 within the 38-amino acid sequence is sometimes a serine (Choi et al., 2002Choi H.J. Park-Snyder S. Pascoe L.T. et al.Structures of two intermediate filament-binding fragments of desmoplakin reveal a unique repeat motif structure.Nat Struc Biol. 2002; 9: 612-620PubMed Google Scholar). However, the full segregation of the phenotype with the mutation within the family and the exclusion of the mutation in over 300 healthy control alleles strongly suggest pathogenicity. Immunofluorescence staining with anti-plectin antibody 10F6 in skin with the Ogna mutation Arg2000Trp shows severe reduction in the basal cell layer staining (Koss-Harnes et al., 1997Koss-Harnes D. Jahnsen F.L. Wiche G. et al.Plectin abnormality in epidermolysis bullosa simplex Ogna: non-responsiveness of basal keratinocytes to some anti-rat plectin antibodies.Exp Dermatol. 1997; 6: 41-48Crossref PubMed Scopus (31) Google Scholar). The Ogna mutation renders plectin’s 190-nm-long coiled-coil rod domain more vulnerable to cleavage by calpains (Walko et al., 2011Walko G. Vukasinovic N. Gross K. et al.Targeted proteolysis of plectin isoform 1a accounts for hemidesmosome dysfunction in mice mimicking the dominant skin blistering disease EBS-Ogna.PLoS Genet. 2011; 7: e1002396Crossref PubMed Scopus (50) Google Scholar). In agreement, the new C-terminal mutations in probands 5 and 6 apparently do not affect plectin staining as much as the Ogna mutation. Therefore, normal HD121 antigen mapping does not exclude EBS-plectin. In conclusion, we showed that in six of the 16 (38%) EBS probands with wild-type KRT5 and KRT14 genes (8% of a total EBS population of 78 probands), dominant PLEC missense mutations underlie non-syndromic EBS, making PLEC the third most candidate gene to screen for mutations in EBS patients. Furthermore, we add two mutations to the known PLEC:p.Arg2000Trp mutation causing EBS. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

To identify an allele-specific short interfering RNA (siRNA), against the common KRT12 mutation Arg135Thr in Meesmann epithelial corneal dystrophy (MECD) as a personalized approach to treatment.siRNAs against the K12 Arg135Thr mutation were evaluated using a dual luciferase reporter gene assay and the most potent and specific siRNAs were further screened by Western blot. Off-target effects on related keratins were assessed and immunological stimulation of TLR3 was evaluated by RT-PCR. A modified 5' rapid amplification of cDNA ends method was used to confirm siRNA-mediated mutant knockdown. Allele discrimination was confirmed by quantitative infrared immunoblotting.The lead siRNA, with an IC(50) of thirty picomolar, showed no keratin off-target effects or activation of TLR3 in the concentration ranges tested. We confirmed siRNA-mediated knockdown by the presence of K12 mRNA fragments cleaved at the predicted site. A dual tag infrared immunoblot showed knockdown to be allele-specific, with 70% to 80% silencing of the mutant protein.A potent allele-specific siRNA against the K12 Arg135Thr mutation was identified. In combination with efficient eyedrop formulation delivery, this would represent a personalized medicine approach, aimed at preventing the pathology associated with MECD and other ocular surface pathologies with dominant-negative or gain-of-function pathomechanisms.

Abstract: Pachyonychia congenita type 1 (PC‐1) is an autosomal dominant ectodermal dysplasia characterized by nail dystrophy, focal non‐epidermolytic palmoplantar keratoderma (FNEPPK) and oral lesions. We have previously shown that mutations in keratin 16 (K16) cause fragility of specific epithelia resulting in phenotypes of PC‐1 or FNEPPK alone. Here, we report 2 novel mutations in K16 causing distinct phenotypes. A heterozygous missense mutation (L124R) was detected in a kindred with PC‐1. In a family where mild FNEPPK was the only phenotype, a 23 bp deletion and a separate 1 bp deletion downstream were found in exon 6: [1244–1266del; 1270delG]. At the protein level, these mutations remove 8 residues and substitute 2 residues in the helix termination motif (HTM) of the K16 polypeptide. The HTM sequence is conserved in all known intermediate filament proteins and for convenience, this complex mutation was designated ΔHTM. Transient expression of K16 cDNAs carrying either the L124R or the ΔHTM mutation in epithelial cell line PtK2 produced aggregation of the keratin cytoskeleton. However, the aggregates observed with the ΔHTM mutation were morphologically different and appeared to be less disruptive to the endogenous cytoskeleton. Therefore, loss of the HTM sequence may render this mutant K16 less capable of contributing to filament assembly and decrease its dominant‐negative effect, resulting in the milder FNEPPK phenotype.

We studied three families suffering from nail abnormalities who had previously been diagnosed as pachyonychia congenita. No keratin gene mutations were detected. Sequencing of connexin 30 (<i>GJB6</i> gene) in these patients identified heterozygous missense mutations G11R and A88V that are known to be associated with Clouston syndrome. This unexpected finding expands the Clouston syndrome phenotype and suggests that some patients diagnosed with pachyonychia may in fact be suffering from Clouston syndrome.

Journal Article Pachyonychia congenita patients with mutations in KRT6A have more extensive disease compared with patients who have mutations in KRT16 Get access K.M. Spaunhurst, K.M. Spaunhurst Department of Dermatology, Stanford University School of Medicine, Stanford, CA, U.S.AUniversity of Southern California Keck School of Medicine, Los Angeles, CA, U.S.A Search for other works by this author on: Oxford Academic Google Scholar A.M. Hogendorf, A.M. Hogendorf Department of Pediatrics, Oncology, Hematology and Diabetology, Medical University of Lodz, Lodz, PolandDepartment of Pediatrics and Immunology of Developmental Age, Medical University of Lodz, Lodz, Poland Search for other works by this author on: Oxford Academic Google Scholar F.J.D. Smith, F.J.D. Smith Division of Molecular Medicine, University of Dundee, Dundee, U.K Search for other works by this author on: Oxford Academic Google Scholar B. Lingala, B. Lingala Department of Dermatology, Stanford University School of Medicine, Stanford, CA, U.S.A Search for other works by this author on: Oxford Academic Google Scholar M.E. Schwartz, M.E. Schwartz Pachyonychia Congenita Project, East Heritage Way, Salt Lake City, UT, U.S.A Search for other works by this author on: Oxford Academic Google Scholar A. Cywinska‐Bernas, A. Cywinska‐Bernas Department of Pediatrics and Immunology of Developmental Age, Medical University of Lodz, Lodz, Poland Search for other works by this author on: Oxford Academic Google Scholar K.J. Zeman, K.J. Zeman Department of Pediatrics and Immunology of Developmental Age, Medical University of Lodz, Lodz, PolandPolish Mother’s Memorial Hospital Institute, Lodz, Poland Search for other works by this author on: Oxford Academic Google Scholar J.Y. Tang J.Y. Tang Department of Dermatology, Stanford University School of Medicine, Stanford, CA, U.S.A Jean Y. Tang. E‐mail: tangy@stanford.edu Search for other works by this author on: Oxford Academic Google Scholar British Journal of Dermatology, Volume 166, Issue 4, 1 April 2012, Pages 875–878, https://doi.org/10.1111/j.1365-2133.2011.10745.x Published: 01 April 2012 Article history Accepted: 13 November 2011 Published: 01 April 2012

BackgroundPachyonychia congenita (PC) is a rare autosomal dominant skin disease, with chronic pain being the most prominent complaint. Histological studies showing alterations in sensory innervation, along with reports on alterations in mechanical sensitivity, suggest that PC may be a form of neuropathy.

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.

Palmoplantar keratodermas are a group of heterogeneous diseases characterized by thickening, and marked hyperkeratosis, of the epidermis of the palms and soles. Palmoplantar keratodermas can be divided into four major classes: diffuse, focal, punctate, and palmoplantar ectodermal dysplasias. All forms are genetic diseases inherited as autosomal dominant disorders. We studied a patient exhibiting a localized thickening of the skin in parts of the right palm and the right sole, following Blaschko's lines, that does not fit into any classes already described. We sequenced the keratin 16 cDNA derived from skin biopsy material from affected and non affected palms. The keratin 16 cDNA sequence from lesional epidermis showed a 12 base pair deletion (309-320del), which deletes codons 104-107. The mutation is predicted to delete four amino acids, GGFA, from the V1 domain of the keratin 16 polypeptide, close to the 1A domain. Full-length keratin 16 cDNA sequence derived from the unaffected palm was completely normal, consistent with a postzygotic mutation as is suggested by the mosaicism observed. We defined this new clinical entity, "unilateral palmoplantar verrucous nevus", rather than localized or focal epidermolytic palmoplantar keratodermas, as the lesions are present only on one side of the body and follow Blaschko's lines. This study is a report of a mosaic mutation in keratin 16 and also the association of a mutation in the V1 domain of a type I keratin associated with a human disease.

Epidermolytic palmoplantar keratoderma (EPPK, MIM #144200) is an autosomal dominant disorder in which hyperkeratosis confined to the palms and soles is characterized histologically by cytolysis of suprabasal keratinocytes. Mutations in the keratin 9 gene (KRT9), a type 1 keratin expressed exclusively in the suprabasal keratinocytes of palmoplantar epidermis, have previously been demonstrated in this disorder. Here, we have studied four Northern Irish kindreds presenting with EPPK. By direct sequencing of polymerase chain reaction products, heterozygous missense mutations in exon 1 of KRT9 were detected in all the families. These included a novel mutation M156T; as well as M156V in two kindreds; and R162Q in the remaining family. All mutations were confirmed by reverse strand sequencing and restriction enzyme analysis. The point prevalence of EPPK in Northern Ireland was found to be 4.4 per 100,000. To date, all reported EPPK mutations occur in the helix initiation motif at the start of the central coiled-coil rod domain of K9.

Keratins are the intermediate filament proteins specifically expressed by epithelial cells. The Human Genome Project has uncovered a total of 54 functional keratin genes that are differentially expressed in specific epithelial structures of the body, many of which involve the epidermis and its appendages. Pachyonychia congenita (PC) is a group of autosomal dominant genodermatoses affecting the nails, thick skin and other ectodermal structures, according to specific sub-type. The major clinical variants of the disorder (PC-1 and PC-2) are known to be caused by dominant-negative mutations in one of four differentiation-specific keratins: K6a, K6b, K16, and K17. A total of 20 human keratin genes are currently linked to single-gene disorders or are predisposing factors in complex traits. In addition, a further six intermediate filament genes have been linked to other non-epithelial genetic disorders. We have established a comprehensive mutation database that catalogs all published independent occurrences of intermediate filament mutations (http://www.interfil.org), with details of phenotypes, published papers, patient support groups and other information. Here, we review the genotype-phenotype trends emerging from the spectrum of mutations in these genes and apply these correlations to make predictions about PC phenotypes based on the site of mutation and keratin pair involved.

Pachyonychia congenita (PC) is a rare, autosomal dominant keratin disorder caused by mutations in four genes (KRT6A, KRT6B, KRT16, or KRT17). The International PC Research Registry is a database with information on patients' symptoms as well as genotypes. We sought to describe the heterogeneity of clinical symptoms and to investigate possible genotype-phenotype correlations in patients with two types of K16 mutations, p.Asn125 and p.Arg127, causing the PC-16 subtype of PC. We found that clinical symptoms depended on the type of amino-acid substitution. Patients with p.Asn125Asp and p.Arg127Pro mutations exhibited more severe disease than patients carrying p.Asn125Ser and p.Arg127Cys mutations in terms of age of onset of symptoms, extent of nail involvement, and impact on daily quality of life. We speculate that amino-acid substitutions causing larger, more disruptive changes to the K16 protein structure, such as a change in amino-acid charge in the p.Asn125Asp mutation or a bulky proline substitution in the p.Arg127Pro mutation, may also lead to more severe disease phenotypes. The variation in phenotypes seen with different substitutions at the same mutation site suggests a genotype-phenotype correlation. Knowledge of the exact gene defect is likely to assist in predicting disease prognosis and clinical management.

TO THE EDITOR Palmoplantar keratodermas (PPKs) are a group of genetically heterogeneous genodermatoses. Recently mutations in TRPV3 were identified as a cause of the rare form of PPK, Olmsted syndrome (OS; OMIM 614594; Lai-Cheong et al., 2012Lai-Cheong J.E. Sethuraman G. Ramam M. et al.Recurrent heterozygous missense mutation, p.Gly573Ser, in the TRPV3 gene in an Indian boy with sporadic Olmsted syndrome.Br J Dermatol. 2012; 167: 440-442Crossref PubMed Scopus (61) Google Scholar; Lin et al., 2012Lin Z. Chen Q. Lee M. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am J Hum Genet. 2012; 90: 558-564Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar; Danso-Abeam et al., 2013Danso-Abeam D. Zhang J. Dooley J. et al.Olmsted syndrome: exploration of the immunological phenotype.Orphanet J Rare Dis. 2013; 8: 79Crossref PubMed Scopus (38) Google Scholar; Kariminejad et al., 2014Kariminejad A. Barzegar M. Abdollahimajd F. et al.Olmsted syndrome in an Iranian boy with a new de novo mutation in TRPV3.Clin Exp Dermatol. 2014; 39: 492-495Crossref PubMed Scopus (13) Google Scholar; Duchatelet et al., 2014Duchatelet S. Pruvost S. de Veer S. et al.A new TRPV3 missense mutation in a patient with Olmsted syndrome and erythromelalgia.JAMA Dermatol. 2014; 150: 303-306Crossref PubMed Scopus (37) Google Scholar). OS was first reported in 1927 in an Italian American boy with painful palmoplantar keratoderma, deep fissures, pseudoainhum, curved thickened nails, and periorificial hyperkeratosis with fissuring (Olmsted, 1927Olmsted H.C. Keratodermia palmaris et plantaris congenitalis: report of a case showing associated lesions of unusual location.Am J Dis Child. 1927; 33: 757-764Crossref Google Scholar). About 50 clinical cases of OS have been described, and all generally exhibit the features described by Olmsted as well as some additional features (Mevorah et al., 2005Mevorah B. Goldberg I. Sprecher E. et al.Olmsted syndrome: mutilating palmoplantar keratoderma with periorificial keratotic plaques.J Am Acad Dermatol. 2005; 53: S266-S272Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In this study, we report the case of six families, referred to the Pachyonychia Congenita Project for the evaluation of painful plantar keratoderma, but lacking pseudoainhum or significant periorificial keratoderma. In each case, after no mutations were identified in the PC-associated keratin genes, KRT6A, KRT6B, KRT6C, KRT16, or KRT17, and in some cases, after other candidate genes including GJB6, DSP, DSG1, KRT5, and KRT14 had been screened, we identified heterozygous missense mutations in TRPV3, thus greatly expanding the phenotypic spectrum of OS. Samples were obtained with written, informed patient consent and ethical approval by a Western Institutional Review Board that complies with principles of the Helsinki Accord. An 18-year-old girl of European ancestry from Family 1 initially noted focal callus formation on the soles at age 4. Severe, painful plantar keratoderma now necessitates periodic use of a wheelchair. She has mild keratoderma on the hands, thin nail plates with koilonychia, and fine slow-growing hair. She has no periorificial keratoderma (Figure 1a, b, c,Supplementary Figure S1 online and Supplementary Table S1 online). A likely diagnosis was PC, but no causative mutations were identified in the PC-related keratin genes nor in other candidate genes. Therefore, a whole-exome sequencing approach was performed (Supplementary Methods online), and data were analyzed for sequence variants in known keratoderma genes. A heterozygous missense mutation, p.Gly573Cys; c,1717G>T, was identified in TRPV3 and confirmed by Sanger sequencing (Supplementary Methods online) but was not present in her unaffected parents or brother. This mutation has been reported in a sporadic case of Olmsted syndrome (Lin et al., 2012Lin Z. Chen Q. Lee M. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am J Hum Genet. 2012; 90: 558-564Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). TRPV3 was considered a candidate gene for five additional families in which no mutations were identified in the PC-associated keratin genes or in other candidate genes. Exons and intron/exon boundaries of TRPV3 were amplified by PCR for Sanger sequencing (Supplementary Methods online). A 7-year-old Brazilian girl in Family 2 presented with easily peeling hyperkeratosis on her toes and feet at about 18 months of age, and these have evolved into painful, focal hyperkeratosis. She has erythema and hyperkeratosis of the distal fingers and subungual hyperkeratosis. She has had transient periorificial hyperkeratosis and her nail plates are normal (Figure 1d, e, f,Table 1, Supplementary Figure S1 online). A previously unreported heterozygous missense mutation, p.Gly568Val; c.1703G>T, was identified (Supplementary Figure S2 online); this mutation was not detected in either of her unaffected parents. Amino acid, p.Gly568 is highly conserved across several species. This mutation is not in the dbSNP database or the NHLBI Exome Variant Server (http://evs.gs.washington.edu/EVS/).Table 1Clinical findings in patients with mutations in TRPV3ReportInheritanceTRPV3 mutationPlantar keratodermaPalmar keratodermaPseudoainhumPeriorificial keratodermaHairOlmsted's patientDiffuse-SDiffuse-SPresentPresentDryLin et al., 2012Lin Z. Chen Q. Lee M. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am J Hum Genet. 2012; 90: 558-564Abstract Full Text Full Text PDF PubMed Scopus (238) Google ScholarADp.Gly573Ser (4)1Individual families.M(1)2Individuals.; Mod(1); S(2)M(1)2Individuals.; Mod(1); S(2)PresentM(1)2Individuals.; Mod(1); S(2)Alopecia - M(1); Mod (1); S (2)Lin et al., 2012Lin Z. Chen Q. Lee M. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am J Hum Genet. 2012; 90: 558-564Abstract Full Text Full Text PDF PubMed Scopus (238) Google ScholarADp.Gly573Cys (1)MMAbsentMAlopecia - MLin et al., 2012Lin Z. Chen Q. Lee M. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am J Hum Genet. 2012; 90: 558-564Abstract Full Text Full Text PDF PubMed Scopus (238) Google ScholarADp.Trp692Gly (1)ModModPresentModAlopecia - MLai-Cheong et al., 2012Lai-Cheong J.E. Sethuraman G. Ramam M. et al.Recurrent heterozygous missense mutation, p.Gly573Ser, in the TRPV3 gene in an Indian boy with sporadic Olmsted syndrome.Br J Dermatol. 2012; 167: 440-442Crossref PubMed Scopus (61) Google ScholarADp.Gly573Ser (1)Diffuse-SDiffuse-SPresentMFine-dryDanso-Abeam et al., 2013Danso-Abeam D. Zhang J. Dooley J. et al.Olmsted syndrome: exploration of the immunological phenotype.Orphanet J Rare Dis. 2013; 8: 79Crossref PubMed Scopus (38) Google ScholarADp.Gly573Ala (1)Diffuse-SDiffuse-SAbsentSAlopecia-SDuchatelet et al., 2014Duchatelet S. Pruvost S. de Veer S. et al.A new TRPV3 missense mutation in a patient with Olmsted syndrome and erythromelalgia.JAMA Dermatol. 2014; 150: 303-306Crossref PubMed Scopus (37) Google ScholarADp.Leu673Phe (1)Diffuse-SDiffuse-SAbsentAbsentFine, dryDuchatelet et al., 2014Duchatelet S. Guibbal L. de Veer S. et al.Olmsted syndrome with erythromelalgia caused by recessive TRPV3 mutations.Br J Dermatol. 2014; 171: 675-678Crossref PubMed Scopus (23) Google ScholarARp.Gly568Cys; p.Gln216_Gly262del (1)Diffuse-S (1); Focal-Mod(1)NRAbsentAbsentFine-dryEytan et al., 2014Eytan O. Fuchs-Telem D. Mevorach B. et al.Olmsted syndrome caused by a homozygous recessive mutation in TRPV3.J Invest Dermatol. 2014; 136: 1752-1754Abstract Full Text Full Text PDF Scopus (35) Google ScholarARp.Trp521Ser (1)Diffuse-SDiffuse-SAbsentPresentSparseKariminejad et al., 2014Kariminejad A. Barzegar M. Abdollahimajd F. et al.Olmsted syndrome in an Iranian boy with a new de novo mutation in TRPV3.Clin Exp Dermatol. 2014; 39: 492-495Crossref PubMed Scopus (13) Google ScholarADp.Trp692Cys (1)Diffuse-SDiffuse-SPresentPresent (Mod)Sparse; fragileHe et al., 2015He Y. Zeng K. Zhang X. et al.A Gain of Function Mutation in TRPV3 causes focal palmoplantar keratoderma in a Chinese family.J Invest Dermatol. 2015; 135: 907-909Abstract Full Text Full Text PDF PubMed Scopus (23) Google ScholarADp.Gln580Pro (1)2Individuals.Focal-ModFocal-ModAbsentAbsentNormalFamily 1ADp.Gly573Cys (1)Focal-SFocal-MAbsentAbsentFineFamily 2ADp.Gly568Val (1)Focal-ModFocal-MAbsentMNormalFamily 3ADp.Gly568Asp (2)2Individuals.Focal-ModMAbsentMFineFamily 4ADp.Gly568Asp (1)Focal-ModFocal-MAbsentNRNormalFamily 5ADp.Gly573Ser (1)Focal-ModM/transientAbsentM/transientFragile, sparseFamily 6ADp.Gly573Ser (1)Focal-ModMAbsentAbsentNormalReportFollicular keratosisErythemaHyperhidrosisNailsLesional itchLesional painLeukokeratosisOlmsted's patientNRLesion border; dorsal handsHandsThickenedNRPresentNRLin et al., 2012Lin Z. Chen Q. Lee M. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am J Hum Genet. 2012; 90: 558-564Abstract Full Text Full Text PDF PubMed Scopus (238) Google ScholarScalpLesion borderNRNRS (4)2Individuals.PresentNRLin et al., 2012Lin Z. Chen Q. Lee M. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am J Hum Genet. 2012; 90: 558-564Abstract Full Text Full Text PDF PubMed Scopus (238) Google ScholarScalpLesion borderNRNRSPresentNRLin et al., 2012Lin Z. Chen Q. Lee M. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am J Hum Genet. 2012; 90: 558-564Abstract Full Text Full Text PDF PubMed Scopus (238) Google ScholarNRLesion borderNRNRSPresentNRLai-Cheong et al., 2012Lai-Cheong J.E. Sethuraman G. Ramam M. et al.Recurrent heterozygous missense mutation, p.Gly573Ser, in the TRPV3 gene in an Indian boy with sporadic Olmsted syndrome.Br J Dermatol. 2012; 167: 440-442Crossref PubMed Scopus (61) Google ScholarNRNRNRDystrophyNo“Functional impairment”NRDanso-Abeam et al., 2013Danso-Abeam D. Zhang J. Dooley J. et al.Olmsted syndrome: exploration of the immunological phenotype.Orphanet J Rare Dis. 2013; 8: 79Crossref PubMed Scopus (38) Google ScholarNRLesion borderNRDystrophySSNRDuchatelet et al., 2014Duchatelet S. Pruvost S. de Veer S. et al.A new TRPV3 missense mutation in a patient with Olmsted syndrome and erythromelalgia.JAMA Dermatol. 2014; 150: 303-306Crossref PubMed Scopus (37) Google ScholarNRErythromelalgiaPresentThin, brittleSSNRDuchatelet et al., 2014Duchatelet S. Guibbal L. de Veer S. et al.Olmsted syndrome with erythromelalgia caused by recessive TRPV3 mutations.Br J Dermatol. 2014; 171: 675-678Crossref PubMed Scopus (23) Google ScholarMErythromelalgiaPresentNormalSSNREytan et al., 2014Eytan O. Fuchs-Telem D. Mevorach B. et al.Olmsted syndrome caused by a homozygous recessive mutation in TRPV3.J Invest Dermatol. 2014; 136: 1752-1754Abstract Full Text Full Text PDF Scopus (35) Google ScholarNRDiffuse, palms and solesNRNRPresentPresentPresentKariminejad et al., 2014Kariminejad A. Barzegar M. Abdollahimajd F. et al.Olmsted syndrome in an Iranian boy with a new de novo mutation in TRPV3.Clin Exp Dermatol. 2014; 39: 492-495Crossref PubMed Scopus (13) Google ScholarNRNRNRDystrophic/absentSSNRHe et al., 2015He Y. Zeng K. Zhang X. et al.A Gain of Function Mutation in TRPV3 causes focal palmoplantar keratoderma in a Chinese family.J Invest Dermatol. 2015; 135: 907-909Abstract Full Text Full Text PDF PubMed Scopus (23) Google ScholarNRNRNRNRNRNRNRFamily 1MLesion borderHands and feetKoilonychia; thin platesAbsentSAbsentFamily 2AbsentLesion border; distal digits (hands)FeetNormalAbsentSAbsentFamily 3MLesion borderFeetKoilonychia; thin plates; onychoschiziaSSPresentFamily 4NRLesion borderNROnychoschiziaNRSAbsentFamily 5MLesion border; dorsal feetFeetKoilonychia; thin plates; onychoschiziaAbsentSAbsentFamily 6NRLesion borderNoNormalNRSAbsentAbbreviations: M, mild; Mod, moderate; NR, not reported; S, severe.1 Individual families.2 Individuals. Open table in a new tab Abbreviations: M, mild; Mod, moderate; NR, not reported; S, severe. The proband from Family 3, a 38-year-old European female, developed calluses on her feet at the age of 8–9 years. She now has severe plantar pain and difficulty in walking (Figure 1g, h, i, Table 1,Supplementary Figure S1 online). She has thin nail plates with koilonychia. Her father, two paternal uncles and grandmother were similarly affected showing autosomal dominant inheritance of the disorder. A previously unreported heterozygous missense mutation, p.Gly568Asp;c.1703G>A (Supplementary Figure S2 online), was identified in the proband and in one affected paternal uncle; it was not present in an unaffected paternal uncle nor in her mother, (her father is deceased). This mutation is not listed in dbSNP or the NHLBI Exome Variant Server. Interestingly, we found the same mutation, p.Gly568Asp, in Family 4 from South America. The first sign of a skin abnormality in the 25-year-old proband was peeling skin on her feet at age 4 years. Painful, focal keratoses formed on her feet and to a lesser extent on her hands. She has mild periungual hyperkeratosis on her fingers and toes, onychoschizia and longitudinal overcurvature of several toenails. She has no periorificial hyperkeratosis, and her hair is normal (Table 1). No clinical information or DNA samples were available from her parents or from other family members. A 55-year-old man of European ancestry from Family 5 presented with painful calluses on the soles of his feet. Focal hyperkeratoses with thin surrounding rim of erythema started on his heels as a child when he started to walk and spread to the soles of his feet. He has thin nail plates with koilonychia and sparse, fragile hair. He develops severe hyperhidrosis accompanied by burning pain in the feet and bright erythema on the dorsal hands and feet in response to extremes of temperature. He has no palmar hyperkeratosis, but has had transient perioral and periauricular hyperkeratosis (Figure 1j, k, l, Table 1,Supplementary Figure S1 online). He believes that etretinate and acitretin have significantly improved his quality of life. His father was also affected. A heterozygous missense mutation, p.Gly573Ser; c.1717G>A, the most commonly reported mutation to date in TRPV3, was identified in this individual. Mutation p.Gly573Ser, was also found in a 7-year-old girl of European ancestry (Family 6), who developed thickening of the skin on her heels at about 4 years of age (Table 1). She has severe plantar pain and now uses crutches to aid her mobility. Her parents are unaffected. The genetic basis of autosomal dominant OS was recently elucidated (Lin et al., 2012Lin Z. Chen Q. Lee M. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am J Hum Genet. 2012; 90: 558-564Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) when heterozygous missense mutation, p.Gly573Ser, was identified in TRPV3 in a Chinese family. Mutations in TRPV3 were subsequently identified in five additional Chinese families. All developed symptoms before 1 year of age, had varying severity of palmoplantar hyperkeratosis, periorificial hyperkeratosis, alopecia, and severe lesional pain and itch. All but one had constricting digital bands. Several heterozygous mutations have been reported at codons 573; p.Gly573Ser (Lai-Cheong et al., 2012Lai-Cheong J.E. Sethuraman G. Ramam M. et al.Recurrent heterozygous missense mutation, p.Gly573Ser, in the TRPV3 gene in an Indian boy with sporadic Olmsted syndrome.Br J Dermatol. 2012; 167: 440-442Crossref PubMed Scopus (61) Google Scholar), p.Gly573Ala (Danso-Abeam et al., 2013Danso-Abeam D. Zhang J. Dooley J. et al.Olmsted syndrome: exploration of the immunological phenotype.Orphanet J Rare Dis. 2013; 8: 79Crossref PubMed Scopus (38) Google Scholar), and p.Gly573Cys (Lin et al., 2012Lin Z. Chen Q. Lee M. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am J Hum Genet. 2012; 90: 558-564Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) and two mutations at codons 692; p.Trp692Gly (Lin et al., 2012Lin Z. Chen Q. Lee M. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am J Hum Genet. 2012; 90: 558-564Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) and p.Trp692Cys (Kariminejad et al., 2014Kariminejad A. Barzegar M. Abdollahimajd F. et al.Olmsted syndrome in an Iranian boy with a new de novo mutation in TRPV3.Clin Exp Dermatol. 2014; 39: 492-495Crossref PubMed Scopus (13) Google Scholar). The heterozygous missense mutation p.Leu673Phe was found in a patient with OS and erythromelalgia (Duchatelet et al., 2014Duchatelet S. Pruvost S. de Veer S. et al.A new TRPV3 missense mutation in a patient with Olmsted syndrome and erythromelalgia.JAMA Dermatol. 2014; 150: 303-306Crossref PubMed Scopus (37) Google Scholar). Homozygous missense and compound heterozygous mutations in TRPV3 have been shown to result in recessive OS with (Duchatelet et al., 2014Duchatelet S. Guibbal L. de Veer S. et al.Olmsted syndrome with erythromelalgia caused by recessive TRPV3 mutations.Br J Dermatol. 2014; 171: 675-678Crossref PubMed Scopus (23) Google Scholar) or without erythromelalgia (Eytan et al., 2014Eytan O. Fuchs-Telem D. Mevorach B. et al.Olmsted syndrome caused by a homozygous recessive mutation in TRPV3.J Invest Dermatol. 2014; 136: 1752-1754Abstract Full Text Full Text PDF Scopus (35) Google Scholar). Recently, the heterozygous missense mutation p.Gln580Pro was identified in a family with focal palmoplantar keratoderma (He et al., 2015He Y. Zeng K. Zhang X. et al.A Gain of Function Mutation in TRPV3 causes focal palmoplantar keratoderma in a Chinese family.J Invest Dermatol. 2015; 135: 907-909Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), more reminiscent of the cases described here. TRPV3 belongs to the family of transient receptor potential (TRP) cation channels and is widely expressed in keratinocytes and hair follicles (Peier et al., 2002Peier A.M. Reeve A.J. Andersson D.A. et al.A heat-sensitive TRP channel expressed in keratinocytes.Science. 2002; 296: 2046-2049Crossref PubMed Scopus (726) Google ScholarNilius et al., 2013Nilius B. Biro T. Owsianik G. TRPV3: time to decipher a poorly understood family member!.J Physiol. 2013; 592: 295-304Crossref PubMed Scopus (93) Google Scholar) as well as in other tissues including the brain, spinal cord, sensory neurons, and the cornea. Mutations in TRPV3 causing autosomal dominant OS were shown to be gain-of-function mutations resulting in increased TRPV3 activity (Lin et al., 2012Lin Z. Chen Q. Lee M. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am J Hum Genet. 2012; 90: 558-564Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). In this study, two, to our knowledge previously unreported, mutations were identified at codon 568. Interestingly another amino acid substitution at this position, p.Gly568Cys, was recently reported in combination with a splice site mutation, exhibiting autosomal recessive inheritance in this case (Duchatelet et al., 2014Duchatelet S. Guibbal L. de Veer S. et al.Olmsted syndrome with erythromelalgia caused by recessive TRPV3 mutations.Br J Dermatol. 2014; 171: 675-678Crossref PubMed Scopus (23) Google Scholar). TRPV3 forms a tetrameric complex, each subunit consists of six transmembrane domains (S1–S6) and a cytoplasmic amino and carboxy termini (Supplementary Figure S2 online). p.Gly568 is within the linker region between S4 and S5, near the boundary of S4. It is predicted that substitution of this glycine is less damaging than substitutions further within the S4–S5 linker such as p.Gly573 (Duchatelet et al., 2014Duchatelet S. Guibbal L. de Veer S. et al.Olmsted syndrome with erythromelalgia caused by recessive TRPV3 mutations.Br J Dermatol. 2014; 171: 675-678Crossref PubMed Scopus (23) Google Scholar). In silico prediction tools (PolyPhen and Mutation Taster) predict all three variants at codon 568, p.Gly568Asp, p.Gly568Val (this study), and p.Gly568Cys (Duchatelet et al., 2014Duchatelet S. Guibbal L. de Veer S. et al.Olmsted syndrome with erythromelalgia caused by recessive TRPV3 mutations.Br J Dermatol. 2014; 171: 675-678Crossref PubMed Scopus (23) Google Scholar) to be damaging. In our families, no other mutations were identified in TRPV3, and in Family 2, the parents were wild-type for p.Gly568 indicating a de novo mutation, p.Gly568Val, in the proband. Mutation p.Gly568Asp was shown to be dominantly inherited in Family 3; the mutation was identified in the proband and an affected paternal uncle (affected father is deceased). However, in the family reported with p.Gly568Cys in combination with a splice site mutation (Duchatelet et al., 2014Duchatelet S. Guibbal L. de Veer S. et al.Olmsted syndrome with erythromelalgia caused by recessive TRPV3 mutations.Br J Dermatol. 2014; 171: 675-678Crossref PubMed Scopus (23) Google Scholar) the unaffected father was heterozygous for p.Gly568Cys and the clinical phenotype of the two affected brothers was significantly different. Overall, these findings suggest that environmental factors/modifier genes may also be involved in determining the phenotypic variability. TRPV3 is involved in many cellular and physiological processes. Recently, Cheng et al., 2010Cheng X. Jin J. Hu L. et al.TRP channel regulates EGFR signaling in hair morphogenesis and skin barrier formation.Cell. 2010; 141: 331-343Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) demonstrated the important role of TRPV3 in regulating EGFR signaling in hair and skin barrier function using a TRPV3 knockout mouse model that developed a wavy hair coat and curly whiskers in addition to a red, dry scaly skin at birth, reminiscent of mice with a defective skin barrier. Although reported as a thermosensitive cation channel, activated at 30–33 °C, this thermosensory role is unclear (Nilius and Biro, 2013Nilius B. Biro T. TRPV3: a 'more than skinny' channel.Exp Dermatol. 2013; 22: 447-452Crossref PubMed Scopus (58) Google Scholar). Interestingly, coexistence of erythromelalgia with OS has been reported (Duchatelet et al., 2014Duchatelet S. Guibbal L. de Veer S. et al.Olmsted syndrome with erythromelalgia caused by recessive TRPV3 mutations.Br J Dermatol. 2014; 171: 675-678Crossref PubMed Scopus (23) Google Scholar, Duchatelet et al., 2014Duchatelet S. Pruvost S. de Veer S. et al.A new TRPV3 missense mutation in a patient with Olmsted syndrome and erythromelalgia.JAMA Dermatol. 2014; 150: 303-306Crossref PubMed Scopus (37) Google Scholar), and one of our patients has findings compatible with erythromelalgia. Many OS patients report hyperhidrosis (including four of ours). In this study, heterozygous missense mutations were identified in TRPV3 in six families, (two previously unreported and two recurrent mutations) with painful, palmoplantar keratoderma. Clinically, none were as severe as typical OS (Table 1). The cases we have described expand the phenotypic spectrum of Olmsted syndrome caused by mutations in TRPV3. Mutations in TRPV3 should be considered as a cause of painful PPK even in the absence of periorificial hyperkeratosis and pseudoainhum as described by Olmsted. In contrast and to avoid confusion, painful PPKs caused by mutations in genes other than TRPV3 probably should not be referred to as Olmsted syndrome. The authors state no conflict of interest We thank all the patients and families involved in this study and Dr Antonella Tosti, Miami, FL, USA and Dr Sherri Bale, GeneDx, MD, USA for referring patients. We also thank Professor Maurice van Steensel and Dr Eli Sprecher for valuable comments and discussions and to Holly Evans of PC Project for all her help with data preparation. FJDS and NJW are supported by grants from the Pachyonychia Congenita Project (to FJDS, www.pachyonychia.org) and Tenovus Scotland (to FJDS). The Centre for Dermatology and Genetic Medicine at the University of Dundee is supported by a Wellcome Trust Strategic Award (098439/Z/12/Z to WHIM). Supplementary material is linked to the online version of the paper at at http://www.nature.com/jid Download .doc (.09 MB) Help with doc files Supplementary Information

Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal protein keratins K3 and K12, respectively. To investigate the pathomechanism of this disease, we generated and phenotypically characterized a novel knock-in humanized mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by western blot, RNA-seq and quantitative real-time polymerase chain reaction. Mass spectrometry (MS) and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, and apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared with the wild-type (P < 0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease and is a valuable resource in understanding the pathomechanism of the disease. Although the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations.

PURPOSE: Meesmann corneal dystrophy is an autosomal dominant disorder characterized by fragility of the anterior corneal epithelium. We have previously demonstrated that this disease can be caused by mutations in the genes encoding keratins K3 or K12, the major intermediate filament proteins expressed in corneal epithelial cells. Here, we have carried out mutation analysis in a United States kindred presenting with typical features of Meesmann corneal dystrophy. METHODS: Exons 1 and 6 of the K12 gene (KRT12) were polymerase chain reaction amplified from the proband’s and control DNA and subjected to direct automated sequencing. RESULTS: A heterozygous missense mutation 1300A→G was detected in exon 6 of KRT12, predicting amino acid substitution I426V in the helix termination motif of the K12 polypeptide. The mutation was confirmed in the proband and excluded from 50 normal individuals by restriction enzyme analysis of polymerase chain reaction products. CONCLUSION: We report a novel mutation in a critical molecular overlap region of K12 in a United States family with Meesmann corneal dystrophy. The results confirm that mutations in the corneal keratins (K3 or K12) can underlie Meesmann corneal dystrophy.

Pachyonychia congenita type 1 (PC-1) is an autosomal dominant ectodermal dysplasia characterized by hypertrophic nail dystrophy, focal non-epidermolytic palmoplantar keratoderma and variable features of oral leukokeratosis and follicular keratosis. Previously, we have shown that this disease can be caused by mutations in type I keratin K16 and one mutation has been reported in its type II keratin expression partner, K6a. Mutation analysis for K6a has been hampered by the presence of multiple copies of the K6 gene in the human genome, of which some are expressed and others are pseudogenes. Here, we describe a mutation detection strategy where the entire KRT6A gene, approximately 7 kb, is specifically amplified by long-range PCR. Using this technique, we have detected two novel mutations in the 1A domain of the K6a polypeptide, N171K and F174S. Mutations were confirmed in the affected individuals and were excluded from 50 unaffected unrelated individuals by restriction enzyme analysis of KRT6A PCR products. Additionally, mutation N171K was confirmed by RT-PCR in mRNA derived from lesional palmoplantar epidermis of an affected individual, confirming the specificity of the genomic PCR for the functional K6a gene. This, together with a similar strategy which we have developed for the K16 gene, provide a robust system for mutation detection and prenatal diagnosis for patients with PC-1.

A young girl with clinical features of pachyonychia congenita type 1 was unusual in that the typical skin and nail changes were not noted until the age of 6 years. Direct sequencing of the KRT16A gene, encoding keratin K16, revealed a novel mutation K354N in the central 2B domain of the K16 polypeptide. The mutation created a new BsmI restriction site and therefore, the mutation was confirmed in the patient and excluded from both parents and 50 normal, unrelated individuals by BsmI digestion of KRT16A polymerase chain reaction products. This is the first time a mutation has been described in this location in a keratin other than K14, where similar mutations cause the milder Weber-Cockayne and/or Köbner types of epidermolysis bullosa simplex.

RNA interference (RNAi) is an evolutionarily conserved mechanism that results in specific gene inhibition at the mRNA level. The discovery that short interfering RNAs (siRNAs) are selective, potent, and can largely avoid immune surveillance has resulted in keen interest to develop these inhibitors as therapeutics. A single nucleotide-specific siRNA (K6a_513a.12, also known as TD101) was recently evaluated in a phase 1b clinical trial for the rare skin disorder, pachyonychia congenita (PC). To develop a clinical trial molecular end point for this type of trial, methods were developed to: (1) isolate total RNA containing amplifiable mRNA from human skin and callus material; (2) quantitatively distinguish the single-nucleotide mutant mRNA from wild-type K6a mRNA in both patient-derived keratinocytes and patient callus; and (3) demonstrate that repeated siRNA treatment results in sustained inhibition of mutant K6a mRNA in patient-derived keratinocyte cultures. These methods allow noninvasive sampling and monitoring of gene expression from patient-collected shavings and may be useful in evaluating the effectiveness of RNAi-based therapeutics, including inhibitors that specifically target single-nucleotide mutations. RNA interference (RNAi) is an evolutionarily conserved mechanism that results in specific gene inhibition at the mRNA level. The discovery that short interfering RNAs (siRNAs) are selective, potent, and can largely avoid immune surveillance has resulted in keen interest to develop these inhibitors as therapeutics. A single nucleotide-specific siRNA (K6a_513a.12, also known as TD101) was recently evaluated in a phase 1b clinical trial for the rare skin disorder, pachyonychia congenita (PC). To develop a clinical trial molecular end point for this type of trial, methods were developed to: (1) isolate total RNA containing amplifiable mRNA from human skin and callus material; (2) quantitatively distinguish the single-nucleotide mutant mRNA from wild-type K6a mRNA in both patient-derived keratinocytes and patient callus; and (3) demonstrate that repeated siRNA treatment results in sustained inhibition of mutant K6a mRNA in patient-derived keratinocyte cultures. These methods allow noninvasive sampling and monitoring of gene expression from patient-collected shavings and may be useful in evaluating the effectiveness of RNAi-based therapeutics, including inhibitors that specifically target single-nucleotide mutations. complementary DNA PCR cycle threshold keratin protein keratin gene nucleotide pachyonychia congenita immortalized keratinocytes derived from a PC patient with the K6a p.Asn171Lys mutation immortalized keratinocytes derived from a PC patient with the K16 p.Asn125Asp mutation quantitative reverse transcription-PCR rapid amplification of cDNA ends RNA interference small interfering RNA

gene encoding alpha- and gamma-adaptin-binding protein p34 base pair Online Mendelian Inheritance in Man (punctate) palmoplantar keratoderma receptor tyrosine kinase TO THE EDITOR Punctate palmoplantar keratoderma (punctate PPK or PPKP) is a rare autosomal dominant disorder of keratinization. Three variants of this disease have been described; PPKP1 (OMIM ♯148600, Buschke–Fischer–Brauer type) is characterized by the progressive development of discrete areas of hyperkeratosis on the palms and soles, followed by more extensive diffuse hyperkeratosis on the pressure-bearing areas of plantar skin. Linkage analyses of families affected by PPKP1 have previously identified a locus within 15q22–q24 (Martinez-Mir et al., 2003Martinez-Mir A. Zlotogorski A. Londono D. et al.Identification of a locus for type 1 punctate palmoplantar keratoderma on chromosome 15q22-q24.J Med Genet. 2003; 40: 872-878Crossref PubMed Scopus (31) Google Scholar; Gao et al., 2005Gao M. Yang S. Li M. et al.Refined localization of a punctate palmoplantar keratoderma gene to a 5.06-cM region at 15q22.2-15q22.31.Br J Dermatol. 2005; 152: 874-878Crossref PubMed Scopus (27) Google Scholar), but two Chinese pedigrees with a PPKP1 phenotype demonstrated linkage on chromosome 8q24.13–q24.21 (Zhang et al., 2004Zhang X.J. Li M. Gao T.W. et al.Identification of a locus for punctate palmoplantar keratodermas at chromosome 8q24.13-8q22.21.J Invest Dermatol. 2004; 122: 1121-1125Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Recently, nonsense mutations in AAGAB, the gene encoding alpha- and gamma-adaptin-binding protein p34, were reported in three PPKP1 families (Giehl et al., 2012Giehl K.A. Eckstein G.N. Pasternack S.M. et al.Nonsense mutations in AAGAB cause punctate palmoplantar keratoderma type Buschke-Fischer-Brauer.Am J Hum Genet. 2012; 91: 754-759Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Simultaneously, we applied whole-exome sequencing and reported heterozygous loss-of-function mutations in AAGAB in 18 PPKP1 kindreds (Pohler et al., 2012Pohler E. Mamai O. Hirst J. et al.Haploinsufficieny for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma.Nat Genet. 2012; 44: 1272-1276Crossref PubMed Scopus (59) Google Scholar). AAGAB is located on chromosome 15q22, within one of the previously reported linkage regions (Martinez-Mir et al., 2003Martinez-Mir A. Zlotogorski A. Londono D. et al.Identification of a locus for type 1 punctate palmoplantar keratoderma on chromosome 15q22-q24.J Med Genet. 2003; 40: 872-878Crossref PubMed Scopus (31) Google Scholar; Pohler et al., 2012Pohler E. Mamai O. Hirst J. et al.Haploinsufficieny for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma.Nat Genet. 2012; 44: 1272-1276Crossref PubMed Scopus (59) Google Scholar). We now report the AAGAB genotype in a further 12 PPKP1 patients from 6 independent kindreds of Scottish, English, and Mexican ancestry. This study was carried out in accordance with the Declaration of Helsinki Principles, and all subjects gave written informed consent. A 56-year-old Scottish woman reported a 15-year history of the progressive development of tender callosities on her soles and palms. Examination revealed multiple hyperkeratotic foci on the palmar skin of hands and fingers (Figure 1a) and diffuse hyperkeratosis on weight-bearing areas of plantar skin (Figure 1b). The proband’s parents were unaffected, but one of her three offspring demonstrated a PPKP1 phenotype. Sequencing of AAGAB using reported methodology (Pohler et al., 2012Pohler E. Mamai O. Hirst J. et al.Haploinsufficieny for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma.Nat Genet. 2012; 44: 1272-1276Crossref PubMed Scopus (59) Google Scholar) identified a heterozygous 1-bp deletion, mutation c.344del resulting in a premature termination codon (p.Asp115Valfs*7; Supplementary Figure S1 online). This mutation has previously been observed in two apparently unrelated Scottish families (Pohler et al., 2012Pohler E. Mamai O. Hirst J. et al.Haploinsufficieny for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma.Nat Genet. 2012; 44: 1272-1276Crossref PubMed Scopus (59) Google Scholar). Download .pdf (28.67 MB) Help with pdf files Supplementary Information A 74-year-old Scottish woman presented with a 25-year history of progressive hyperkeratosis of her plantar skin (Figure 1d), with lesser involvement of the palmar skin (Figure 1c). The proband’s parents were unaffected, but one of her three adult children had keratoderma. DNA sequencing identified a previously unreported heterozygous nonsense mutation, c.390G&gt;A, predicting the protein change p.Trp130*0, within AAGAB (Supplementary Figures S1 and S2b online). This mutation was not detected in ∼11,000 European and African American individuals’ exome sequencing data (NHLBI Exome Sequencing Project http://evs.gs.washington.edu/EVS/ accessed 27 March 2013), nor is it reported in the 1000 Genomes Catalogue of Human Genetic Variation (http://www.1000genomes.org/ensembl-browser accessed 27 March 2013). A 79-year-old Scottish man presented with a 30-year history of hyperkeratosis of the soles and palms (Supplementary Figure S3a and b online). His medical history included a cutaneous squamous cell carcinoma on the hand and a basal cell carcinoma on the nose, but these were not at sites affected by keratoderma. The patient’s parents, eight siblings, and two children were unaffected. DNA sequencing revealed a heterozygous c.870+1G&gt;A splice-site mutation within AAGAB (Supplementary Figure S1 online), previously reported in an unrelated Scottish family (Pohler et al., 2012Pohler E. Mamai O. Hirst J. et al.Haploinsufficieny for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma.Nat Genet. 2012; 44: 1272-1276Crossref PubMed Scopus (59) Google Scholar). The protein consequences of this mutation are unknown but is likely to represent a frameshift mutation. A 55-year-old female patient of English ancestry presented at the age of 30 years with typical features of PPKP1. She had multiple keratotic papules on palmar and plantar surfaces, which had gradually increased in number, becoming confluent and tender (Supplementary Figure S4a and b online). The patient has one affected and four unaffected siblings; her two daughters are similarly affected and her one grandson shows early signs of punctate keratoderma. DNA sequencing revealed a heterozygous frameshift mutation c.472del, leading to a premature termination codon (p.Gly158Glufs*0; Supplementary Figure S1 online), previously reported in five apparently unrelated Scottish families (Pohler et al., 2012Pohler E. Mamai O. Hirst J. et al.Haploinsufficieny for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma.Nat Genet. 2012; 44: 1272-1276Crossref PubMed Scopus (59) Google Scholar). Thirty-one members of this Mexican family over four generations demonstrate a PPK1P phenotype with an autosomal dominant pattern of inheritance. The affected living relatives showed multiple hyperkeratotic papules on the palms and soles with no associated nail disease (representative clinical images are shown in Figure 1e and f). Linkage analysis in this family has identified the AAGAB locus at chromosome 15q22 (Martinez-Mir et al., 2003Martinez-Mir A. Zlotogorski A. Londono D. et al.Identification of a locus for type 1 punctate palmoplantar keratoderma on chromosome 15q22-q24.J Med Genet. 2003; 40: 872-878Crossref PubMed Scopus (31) Google Scholar). AAGAB sequencing of DNA from five affected family members has now identified a previously unreported heterozygous 1-bp deletion in exon 3, c.275del predicting the protein change p.Leu92Leufs*18, in each affected individual (Supplementary Figures S1 and S2d online). This mutation was not detected in ∼11,000 European and African American individuals’ exome sequencing data (NHLBI Exome Sequencing Project http://evs.gs.washington.edu/EVS/ accessed 27 March 2013), nor is it reported in the 1000 Genomes Catalogue of Human Genetic Variation (http://www.1000genomes.org/ensembl-browser accessed 27 March 2013). A 42-year-old Scottish woman presented with punctate callosities affecting her palms and soles, which were present since early adulthood (Figure 1g and h). The proband’s mother was affected to a milder degree, and her 15-year-old son showed early signs of punctate keratoderma on the plantar surfaces. Sequencing of all exons of AAGAB in these three cases failed to identify any mutations. However, microsatellite linkage analysis in the pedigree was consistent with involvement of the AAGAB locus (Figure 2). Hemizygous inheritance of a microsatellite marker within intron 1 of AAGAB indicated a genomic deletion. A reduction in the gene dosage for AAGAB exon 1 in the proband compared with her unaffected father was confirmed using semiquantitative fluorescent PCR (Supplementary Figure S5a and b online), whereas exons 2 and 3 were amplified in equal quantities in the proband and control (Supplementary Figure S5c and d online). The precise size of this genomic deletion involving exon 1/intron 1 of AAGAB has not been defined, but it is consistent with the resultant haploinsufficiency of AAGAB. It has previously been shown that knockdown of p34 in HaCaT cells in vitro leads to increased stabilization of the EGFR protein, a receptor tyrosine kinase (Pohler et al., 2012Pohler E. Mamai O. Hirst J. et al.Haploinsufficieny for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma.Nat Genet. 2012; 44: 1272-1276Crossref PubMed Scopus (59) Google Scholar). In addition to EGFR, keratinocytes express several other receptor tyrosine kinases (RTKs). To further investigate the functional effects of AAGAB mutations, we tested the effect of gene knockdown on the transmembrane protein Axl, an RTK that is highly expressed in skin and is involved in signaling pathways to cellular proliferation (Postel-Vinay and Ashworth, 2012Postel-Vinay S. Ashworth A. AXL and acquired resistance to EGFR inhibitors.Nat Genet. 2012; 44: 835-836Crossref PubMed Scopus (25) Google Scholar). We investigated the effect of p34 knockdown on Axl in HaCaT keratinocytes and found a significant increase in the amount of Axl protein in cells treated with AAGAB-specific small interfering RNA (Supplementary Figure S6 online; methods are fully described in online Supplementary Tables S1 and S2 online). These six kindreds demonstrate replication of a newly reported genetic cause for punctate PPK1, further confirming AAGAB as a causative gene. AAGAB mutations result in hereditary deficiency of p34, leading to hyperproliferative hyperkeratosis through increased growth factor signaling, which is thought to result from impairment in the endocytosis and recycling of EGFR proteins (Pohler et al., 2012Pohler E. Mamai O. Hirst J. et al.Haploinsufficieny for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma.Nat Genet. 2012; 44: 1272-1276Crossref PubMed Scopus (59) Google Scholar). A total of 13 predicted loss-of-function mutations have now been reported; these are listed in Supplementary Table S3 online. We have identified recurrent mutations resulting in frameshift and deleterious splice site changes, as well as previously unreported variants. There is no apparent genotype–phenotype correlation within this small group of patients, but our clinical observation is that environmental factors and personal skin care regimes affect the degree of plantar hyperkeratosis. The PPKP1 families reported here demonstrate some diversity in the clinical appearance of plantar hyperkeratosis, whereas the punctate palmar keratoses have a more similar appearance in each kindred (Figure 1). An association with the development of malignancy in PPKP1 patients has been reported, including breast and colonic adenocarcinoma (Bennion and Patterson, 1984Bennion S.D. Patterson J.W. Keratosis punctate palmaris et plantaris and adenocarcinoma of the colon.J Am Acad Dermatol. 1984; 10: 587-591Abstract Full Text PDF PubMed Scopus (46) Google Scholar; Stevens et al., 1996Stevens H.P. Kelsell D.P. Leigh I.M. et al.Punctate palmoplantar keratoderma and malignancy in a four generation family.Br J Dermatol. 1996; 134: 720-726Crossref PubMed Scopus (47) Google Scholar). It is not clear whether there is truly an over-representation of malignancy in PPKP1 cases, and malignant neoplasms were not prevalent in the six kindreds reported here. Hyperkeratosis of the plantar skin localized to the weight-bearing areas may be explained by mechanical trauma, but the extremely focal distribution of hyperkeratosis on the palmar skin in the disorder is striking. The mechanism(s) responsible for producing this very focal disease on the background of a mutation expressed throughout the palmar skin are likely to be of clinical relevance, but they remain to be defined. It is possible that a second mutation has occurred within each focal area of hyperkeratosis. This was not identified by microdissection and sequence analysis (Pohler et al., 2012Pohler E. Mamai O. Hirst J. et al.Haploinsufficieny for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma.Nat Genet. 2012; 44: 1272-1276Crossref PubMed Scopus (59) Google Scholar), but further investigation is warranted. A clearer understanding of the genetic and/or environmental mechanisms by which focal keratoses arise may give further insight into the role of p34 in controlling cell division. Because of the role of EGFR and Axl in various malignancies, Axl-specific inhibitors are under development (Postel-Vinay and Ashworth, 2012Postel-Vinay S. Ashworth A. AXL and acquired resistance to EGFR inhibitors.Nat Genet. 2012; 44: 835-836Crossref PubMed Scopus (25) Google Scholar) and are potentially of relevance to this rare but distressing keratoderma. The p34 protein encoded by AAGAB has been functionally implicated in the biology of intracellular transport of membrane-bound, clathrin-coated vesicles. We hypothesized that a possible link between an inherited defect in vesicle transport and epidermal hyperproliferation might involve RTKs, which are turned over by endocytosis via clathrin- and AP2-dependent processes (Ceresa, 2006Ceresa B.P. Regulation of EGFR endocytic trafficking by rab proteins.Histol Histopathol. 2006; 21: 987-993PubMed Google Scholar; Rappoport and Simon, 2009Rappoport J.Z. Simon S.M. Endocytic trafficking of activated EGFR is AP-2 dependent and occurs through preformed clathrin spots.J Cell Sci. 2009; 122: 1301-1305Crossref PubMed Scopus (86) Google Scholar). The upregulation of Axl protein expression after small interfering RNA knockdown of AAGAB demonstrates the involvement of p34 in RTK turnover in addition to EGFR. The identification of molecular mechanisms in palmoplantar keratoderma serves to improve our understanding of the pathogenesis of cutaneous hyperkeratosis. It may also offer insight into the control of keratinization in physiological conditions, as well as more prevalent inflammatory skin diseases characterized by hyperproliferation and hyperkeratosis. We are grateful to the PPKP1 patients and their families for sharing clinical information and providing samples. This work was supported by the Wellcome Trust (Clinical Intermediate Fellowship WT086398MA to SJB; Programme Grant 092530/Z/10/Z to WHIM; Strategic Award 098439/Z/12/Z to WHIM) and a project grant from the Pachyonychia Congenita Project to FJDS. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

epidermolysis bullosa simplex pachyonychia congenita TO THE EDITOR Homozygosity for dominant mutations in keratin genes is rare and has only been reported for epidermolysis bullosa simplex (EBS; Stephens et al., 1995Stephens K. Zlotogorski A. Smith L.T. et al.Epidermolysis bullosa simplex - a keratin 5 mutation is a fully dominant allele in epidermal cytoskeletal function.Am J Hum Genet. 1995; 56: 577-585PubMed Google Scholar; Hu et al., 1997Hu Z.L. Smith L. Martins S. et al.Partial dominance of a keratin 14 mutation in epidermolysis bullosa simplex--increased severity of disease in a homozygote.J Invest Dermatol. 1997; 109: 360-364Crossref PubMed Scopus (23) Google Scholar; Oldak et al., 2011Oldak M. Szczecinska W. Przybylska D. et al.Gene dosage effect of p.Glu170Lys mutation in the KRT5 gene in a Polish family with epidermolysis bullosa simplex.J Dermatol Sci. 2011; 61: 64-67Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The majority of pathogenic variants reported in 23 keratin genes are heterozygous missense or small in-frame insertion/deletion mutations inherited in an autosomal dominant manner (http://www.interfil.org; Szeverenyi et al., 2008Szeverenyi I. Cassidy A.J. Chung C.W. et al.The Human Intermediate Filament Database: comprehensive information on a gene family involved in many human diseases.Hum Mutat. 2008; 29: 351-360Crossref PubMed Scopus (267) Google Scholar). A small number of recessive cases have been reported, mostly due to nonsense mutations (Yiasemides et al., 2008Yiasemides E. Trisnowati N. Su J. et al.Clinical heterogeneity in recessive epidermolysis bullosa due to mutations in the keratin 14 gene, KRT14.Clin Exp Dermatol. 2008; 33: 689-697Crossref PubMed Scopus (29) Google Scholar). Here, we report homozygosity for dominant missense mutations in keratin 17 that modify the pachyonychia congenita (PC) phenotype. PC is an autosomal dominant skin disorder caused by heterozygous mutations in any one of the genes encoding keratins K6a, K6b, K16, or K17 (McLean et al., 2011McLean W.H. Hansen C.D. Eliason M.J. et al.The phenotypic and molecular genetic features of pachyonychia congenita.J Invest Dermatol. 2011; 131: 1015-1017Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The main characteristics are palmoplantar keratoderma, plantar pain, and nail dystrophy. Additionally, oral leukokeratosis, follicular keratoses, and epidermal cysts often occur. PC due to mutations in K17 (termed PC-17) is more frequently associated with neonatal teeth and widespread pilosebaceous cysts in adults. Family 1, of Hispanic ancestry, showed autosomal dominant inheritance (Figure 1a). A consanguineous marriage between affected individuals resulted in three affected (one homozygous (proband) and two heterozygous) and one unaffected offspring. Both parents had some characteristics of PC. The father had thickened nails, mild plantar hyperkeratosis and steatocysts; the mother had steatocysts but reported neither nail changes nor keratoderma. The 10-year-old proband had features typical of PC but was much more severely affected than other family members and had additional features (Figure 1b–g). Unusually, all nails were thickened at birth. He also had several neonatal teeth. At 4 months he developed leukokeratosis, and by 7 months had blistering on his hands and feet, and thickening of palmoplantar skin that is now localized to pressure points (Figure 1b, c, e, and f). He continues to have painful blisters and has follicular keratosis (Figure 1g). He has no steatocysts to date, although these generally develop at puberty. The most unusual feature, not previously associated with PC, was hair loss, first noted at 7 months and has continued during his lifetime (Figure 1d). On examination he had a circumscribed area in the occipital region where the hairs were much shorter and thinner. Eyebrows were normal. His affected brother (25 years old) and sister (13 years old) had thickened toenails, slight thickening of fingernails with splinter hemorrhages and widespread steatocysts. Neither sibling had keratoderma, blistering, neonatal teeth nor alopecia (Figure 1h–j). The brother's 1-year-old daughter had nail involvement and a neonatal tooth. Genomic DNA was obtained with informed consent and appropriate ethical approval that complies with the Declaration of Helsinki Principles (Western IRB study no. 20040468). DNA extraction and mutation detection were performed according to the published protocols that avoid pseudogene contamination (Wilson et al., 2011Wilson N.J. Leachman S.A. Hansen C.D. et al.A large mutational study in pachyonychia congenita.J Invest Dermatol. 2011; 131: 1018-1024Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Two primer sets were used for each mutation hotspot exon to ensure against polymorphism. By DNA sequencing of KRT17, a homozygous dominant missense mutation was identified, designated p.Asn92Ser (protein), c.275A>G (DNA), in the proband of Family 1. The mildly affected parents and siblings were heterozygous (Figure 1k–n). Mutation p.Asn92Ser is the most commonly reported mutation in KRT17, occurring in 36% of PC-17 families (Wilson et al., 2011Wilson N.J. Leachman S.A. Hansen C.D. et al.A large mutational study in pachyonychia congenita.J Invest Dermatol. 2011; 131: 1018-1024Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Family 2 was of Middle Eastern ancestry. The proband, a 32-year-old male, had thickened finger and toenails by 2 years of age (Figure 2a and b). Painful blisters, calluses, fissures, and ulcerations were present on hands and feet (Figure 2c and e), and he had follicular hyperkeratosis (Figure 2f and g) but no oral leukokeratosis. He developed generalized alopecia at the age of 3 years and now has almost total alopecia (Figure 2d, at age 8). At the age of 7, the patient received oral etretinate (Tigason; 1mgkg-1 per day) for 3 months, which reduced the follicular hyperkeratosis but did not improve the plantar blisters, alopecia, or pachyonychia. His parents were reportedly unrelated but unfortunately were not available for examination or DNA sampling. The father was reported to have steatocysts. The proband of Family 2 was homozygous for dominant missense mutation p.Arg94Cys, c.280C>T in KRT17 (Figure 2h and i). The proband was homozygous for microsatellite markers spanning type I keratin locus. Mutation p.Arg94Cys has been reported in several cases of dominant PC-17 (http://www.interfil.org). To our knowledge, homozygous dominant missense mutations have not been previously reported in PC. However, both these mutations are commonly seen as heterozygous mutations, with p.Asn92Ser being the most commonly reported mutation in KRT17. Interestingly, both mutations, p.Asn92Ser and p.Arg94Cys, have been reported to show phenotypic variation: most commonly giving rise to PC-17 (http://www.interfil.org) but occasionally steatocystoma multiplex (Covello et al., 1998Covello S.P. Smith F.J.D. Sillevis Smitt J.H. et al.Keratin 17 mutations cause either steatocystoma multiplex or pachyonychia congenita type 2.Br J Dermatol. 1998; 139: 475-480Crossref PubMed Scopus (143) Google Scholar; Wang et al., 2009Wang J.F. Lu W.S. Sun L.D. et al.Novel missense mutation of keratin in Chinese family with steatocystoma multiplex.J Eur Acad Dermatol Venereol. 2009; 23: 723-724Crossref PubMed Scopus (7) Google Scholar). Intra-familial phenotypic variation, as seen here between affected heterozygous members of Family 1, has previously been reported (Smith et al., 1997Smith F.J.D. Corden L.D. Rugg E.L. et al.Missense mutations in keratin 17 cause either pachyonychia congenita type 2 or a phenotype resembling steatocystoma multiplex.J Invest Dermatol. 1997; 108: 220-223Crossref PubMed Scopus (129) Google Scholar). Of the three reported cases of homozygosity for a dominant mutation in another keratin disorder, EBS, two mutations were described as partially dominant (or semidominant)—homozygous individuals were more severely affected than heterozygotes (Hu et al., 1997Hu Z.L. Smith L. Martins S. et al.Partial dominance of a keratin 14 mutation in epidermolysis bullosa simplex--increased severity of disease in a homozygote.J Invest Dermatol. 1997; 109: 360-364Crossref PubMed Scopus (23) Google Scholar; Oldak et al., 2011Oldak M. Szczecinska W. Przybylska D. et al.Gene dosage effect of p.Glu170Lys mutation in the KRT5 gene in a Polish family with epidermolysis bullosa simplex.J Dermatol Sci. 2011; 61: 64-67Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). However, in a third EBS family, homozygosity for a mutation in KRT5 did not alter clinical severity (Stephens et al., 1995Stephens K. Zlotogorski A. Smith L.T. et al.Epidermolysis bullosa simplex - a keratin 5 mutation is a fully dominant allele in epidermal cytoskeletal function.Am J Hum Genet. 1995; 56: 577-585PubMed Google Scholar). In the families studied here, the features typical of PC were more severe in the homozygous cases than heterozygotes, again indicating a gene dosage effect. The additional feature of hair loss, seen in both cases, is particularly interesting as alopecia has not been previously reported in the cases of mutation-confirmed PC. K17 is expressed in the hair follicle (Moll et al., 2008Moll R. Divo M. Langbein L. The human keratins: biology and pathology.Histochem Cell Biol. 2008; 129: 705-733Crossref PubMed Scopus (973) Google Scholar), and we hypothesize that the increased dosage of mutant K17 in the homozygotes crosses a threshold where hair abnormalities become evident. K17-null mice develop hair fragility during the first week after birth resulting in severe alopecia (McGowan et al., 2002McGowan K.M. Tong X. Colucci-Guyon E. et al.Keratin 17 null mice exhibit age- and strain-dependent alopecia.Genes Dev. 2002; 16: 1412-1422Crossref PubMed Scopus (100) Google Scholar), showing that this protein is critically important for hair follicle function. Individuals homozygous or compound heterozygous for dominant mutations have a much greater risk of having affected offspring (essentially 100%), compared with heterozygous carriers of a dominant mutation (50%). Therefore, in terms of genetic testing, homozygous or compound heterozygous mutations should be especially sought in PC patients who present with alopecia. As four keratin genes are involved in dominant PC, bigenic inheritance is also a possibility in unusually severe cases resembling those reported here. We thank the patients for participating in this study. FJDS and NJW are supported by the Pachyonychia Congenita Project (http://www.pachyonychia.org).

Nail dystrophy is a hallmark of the autosomal dominant disorder pachyonychia congenita (PC), accompanied by keratoderma and other ectodermal defects [ [1] Eliason M.J. Leachman S.A. Feng B.J. Schwartz M.E. Hansen C.D. A review of the clinical phenotype of 254 patients with genetically confirmed pachyonychia congenita. J Am Acad Dermatol. 2012; 67: 680-686 Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar ]. We recently analysed three cases initially diagnosed as PC, but who presented with nail dysplasia only. None had mutations in any of the four keratin genes associated with PC (KRT6A, KRT6B, KRT16 & KRT17) [ [2] Wilson N.J. Leachman S.A. Hansen C.D. McMullan A.C. Milstone L.M. Schwartz M.E. et al. A large mutational study in pachyonychia congenita. J Invest Dermatol. 2011; 131: 1018-1024 Crossref PubMed Scopus (60) Google Scholar ] or in KRT6C (associated with keratoderma and trivial changes) nor in GJB6 (Clouston syndrome is in the differential diagnosis for PC). The frizzled 6 gene (FZD6) was analysed because mutations in this gene were shown to cause autosomal recessive nail dysplasia [ 3 Frojmark A.S. Schuster J. Sobol M. Entesarian M. Kilander M.B. Gabrikova D. et al. Mutations in Frizzled 6 cause isolated autosomal-recessive nail dysplasia. Am J Hum Genet. 2011; 88: 852-860 Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar , 4 Naz G. Pasternack S.M. Perrin C. Mattheisen M. Refke M. Khan S. et al. FZD6 encoding the Wnt receptor frizzled 6 is mutated in autosomal-recessive nail dysplasia. Br J Dermatol. 2012; 165: 1088-1094 Crossref Scopus (30) Google Scholar ]. FZD6 mutations were identified in these three unrelated families with clinically similar nail dystrophy, where all fingernails and toenails were discoloured and thickened from birth.

Mutations in keratin genes underlie a variety of epidermal and nonepidermal cell‐fragility disorders, and are the genetic basis of many inherited palmoplantar keratodermas (PPKs). Epidermolytic PPK (EPPK) is an autosomal dominant disorder that can be due to mutations in the keratin 1 gene, KRT1. Epidermolytic ichthyosis (EI), the major keratinopathic ichthyosis, is characterized by congenital erythroderma, blistering and erosions of the skin. Causative mutations in KRT1 and KRT10 have been described, with PPK being present primarily in association with the former. We report four unrelated cases (one with sporadic EI and three with autosomal dominant PPK), due to two novel and two recurrent KRT1 mutations. Mutations in KRT1 are not only scattered throughout the keratin 1 protein, as opposed to being clustered, but can result in a range of phenotypes as further confirmed by these mutations, giving a complex genotype/phenotype pattern.

Pachyonychia congenita type 1 (PC-1) is an autosomal dominant ectodermal dysplasia characterized by severe nail dystrophy, focal non-epidermolytic palmoplantar keratoderma (FNEPPK) and oral lesions. We have previously shown that mutations in keratin K16 cause fragility of specific epithelia resulting in phenotypes of PC-1 or FNEPPK alone. These earlier analyses employed an RT-PCR approach to avoid amplification of K16-like pseudogenes. Here, we have cloned the K16 gene (KRT16A) and two homologous pseudogenes (ψKRT16B and ψKRT16C), allowing development of a genomic mutation detection strategy based on a long-range PCR, which is specific for the functional K16 gene. We report a novel heterozygous 3 bp deletion mutation (388del3) in K16 in a sporadic case of PC-1. The mutation was detected in genomic DNA and confirmed at the mRNA level by RT-PCR, showing that our genomic PCR system is reliable for K16 mutation detection. Using this system, we carried out the first prenatal diagnosis for PC-1 using CVS material, correctly predicting a normal fetus. This work will greatly improve K16 mutation analysis and allow predictive testing for PC-1 and the related phenotype of FNEPPK. Copyright © 1999 John Wiley & Sons, Ltd.

We and others have previously shown that ichthyosis bullosa of Siemens, an autosomal dominant disorder characterized by epidermal thickening and blistering, is caused by mutations in the late-differentiation keratin K2e. Here, we have determined the genomic organization and complete sequence of the KRT2E gene, which consists of nine exons, spanning 7634 bp of DNA. The gene was mapped by high-resolution radiation-hybrid mapping to the interval between microsatellite markers D12S368 and CHLC.GATA11B02.1112. Several intragenic polymorphisms were detected, including an 18 bp duplication in exon 1, corresponding to the V1 domain of the K2e polypeptide. Genomic polymerase chain reaction conditions were optimized for all exons, and two novel mutations, N192Y in the 1A domain and E482K in the 2B domain of K2e, were found in ichthyosis bullosa of Siemens families. Mutations were excluded from 50 normal unrelated individuals by restriction analysis. These results emphasize that mutations in K2e underlie ichthyosis bullosa of Siemens and provide a comprehensive mutation detection strategy for ongoing studies of keratinizing disorders.

Keratins are a multigene family of intermediate filament proteins that are differentially expressed in specific epithelial tissues. To date, no type II keratins specific for the inner root sheath of the human hair follicle have been identified.To characterize a novel type II keratin in mice and humans.Gene sequences were aligned and compared by BLAST analysis. Genomic DNA and mRNA sequences were amplified by polymerase chain reaction (PCR) and confirmed by direct sequencing. Gene expression was analysed by reverse transcription (RT)-PCR in mouse and human tissues. A rabbit polyclonal antiserum was raised against a C-terminal peptide derived from the mouse K6irs protein. Protein expression in murine tissues was examined by immunoblotting and immunofluorescence.Analysis of human expressed sequence tag (EST) data generated by the Human Genome Project revealed a fragment of a novel cytokeratin mRNA with characteristic amino acid substitutions in the 2B domain. No further human ESTs were found in the database; however, the complete human gene was identified in the draft genome sequence and several mouse ESTs were identified, allowing assembly of the murine mRNA. Both species' mRNA sequences and the human gene were confirmed experimentally by PCR and direct sequencing. The human gene spans more than 16 kb of genomic DNA and is located in the type II keratin cluster on chromosome 12q. A comprehensive immunohistochemical survey of expression in the adult mouse by immunofluorescence revealed that this novel keratin is expressed only in the inner root sheath of the hair follicle. Immunoblotting of murine epidermal keratin extracts revealed that this protein is specific to the anagen phase of the hair cycle, as one would expect of an inner root sheath marker. In humans, expression of this keratin was confirmed by RT-PCR using mRNA derived from plucked anagen hairs and epidermal biopsy material. By this means, strong expression was detected in human hair follicles from scalp and eyebrow. Expression was also readily detected in human palmoplantar epidermis; however, no expression was detected in face skin despite the presence of fine hairs histologically.This new keratin, designated K6irs, is a valuable histological marker for the inner root sheath of hair follicles in mice and humans. In addition, this keratin represents a new candidate gene for inherited structural hair defects such as loose anagen syndrome.

The molecular basis of Meesmann's epithelial corneal dystrophy (MECD) has recently been attributed to mutations in the cornea specific keratin genes KRT3 and KRT12. The mechanisms by which these mutations cause the Meesmann's phenotype are not clear. This study presents new data, examines clinical, histological, ultrastructural, and molecular aspects of MECD, and compares the features seen in this condition with those observed in other well studied keratin diseases such as epidermolysis bullosa simplex.A two generation family with typical features of Meesmann's epithelial corneal dystrophy (MECD) was studied. All family members were examined under a slit lamp. Biopsy material from elective keratoplasty was studied by histopathological and ultrastructural analysis using standard techniques. Direct automated sequencing of genomic DNA was used for mutation detection, mutations were confirmed by restriction digest analysis.The abnormal corneal epithelium was acanthotic and contained numerous dyskeratotic cells and intraepithelial vesicles. By electron microscopy abnormally aggregated and clumped keratin filament bundles were detected in basal and suprabasal keratinocytes from the centre of the cornea. Direct sequencing of the patients' genomic DNA revealed a novel missense mutation (423T>G) in exon 1 of the cornea specific keratin 12 (KRT12) gene. This mutation predicts the amino acid change N133K within the helix initiation motif of the K12 polypeptide. Comparative studies with well established keratin disorders of other human epithelia underscore the pathogenic relevance of K3 and K12 gene mutations in Meesmann's epithelial corneal dystrophy. The morphological data presented here illustrate the disruptive effects of keratin gene mutations on the integrity of corneal keratinocytes.A clinical, histopathological, and ultrastructural study of a previously unreported family with MECD is presented. In this family the disease is ascribed to a novel mutation in KRT12. A molecular mechanism is proposed for MECD based on the comparison with other well characterised keratin diseases.

Keratins are cytoplasmic intermediate filament proteins expressed by epithelial cells. Keratin 7 (K7) is expressed in a wide range of epithelial structures in humans. We have cloned and fully sequenced the human and mouse K7 genes and mRNAs, and the K7 mRNA from the marsupial Potorous tridactylis, from which the widely used simple epithelial cell lines PtK1 and PtK2 are derived. Percentage identity plots comparing the mouse and human genomic sequences revealed a number of conserved CpG islands associated with the K7 gene. There was considerable conservation of introns between the two species, which may indicate the presence of intronic regulatory elements. Only the most proximal 500 bp of the promoter was conserved, although an additional conserved sequence island was found 2–3 kb upstream. Protein sequence comparisons between the three species allowed identification of conserved regions of the keratin variable domains that may be candidates for protein–protein interactions and/or regulatory modification. From the mouse sequence, we generated a polyclonal rabbit antibody specific for murine K7. This antibody was used to perform a survey of K7 expression in the mouse. The expression pattern was similar to the reported human distribution, with substantial expression observed in lung, bladder, mesothelium, hair follicle, and ductal structures. We also noted previously unreported expression of K7 in the gastrointestinal tract and filiform papillae of the tongue and specific K7 expression in a range of “hard” epithelial tissues. The distribution of K7 in mouse and availability of genomic sequence from the 129/Sv mouse strain will allow the generation and analysis of transgenic mice expressing mutant forms of K7 and to predict the phenotype of human genetic disorders caused by mutations in this keratin.

Pachyonychia congenita (PC), a rare autosomal‐dominant keratin disorder caused by mutations in keratin genes KRT6A/B, KRT16 or KRT17, is characterized by painful plantar keratoderma and hypertrophic nail dystrophy. Loss‐of‐function mutations in the filaggrin (FLG) gene underlie the most prevalent skin disorder of cornification, ichthyosis vulgaris (IV), which presents with generalized scaling and is also associated with atopic dermatitis. Recently, FLG mutations have been reported to increase phenotype severity of X‐linked ichthyosis and alopecia areata. We report a parent–child trio in which the mother and the son have PC and the father has IV. Both the mother and the son are carriers for the KRT16 mutation p.Leu132Pro. The son, who is much more severely affected than his mother, in addition carries the heterozygous FLG mutation p.R2447X, which was inherited from the father. This observation suggests that coinheritance of mutations in KRT16 and FLG may aggravate the PC phenotype and that FLG could serve as a genetic modifier in PC.

dear editor, Loricrin keratoderma (syn. Camisa syndrome, OMIM 604117) is a rare autosomal dominant genodermatosis characterized by palmoplantar keratoderma and ichthyosis.1, 2 It is caused by mutations in loricrin, a small basic protein synthesized in the upper granular layer, which becomes a major constituent of the cornified cell envelope.3 Seven distinct mutations in loricrin have been reported in 15 unrelated pedigrees to date.4, 5, 6 We report a multi‐generation family with prominent ichthyosis and palmoplantar involvement due to a novel mutation in loricrin. The proband was a 14‐year‐old boy who presented with generalized dryness and scaling affecting his trunk and all four limbs, previously thought to be ichthyosis vulgaris, which was reported from early childhood (Fig. 1a). Clinical examination revealed widespread, prominent ichthyosis and mild diffuse transgredient hyperkeratosis of palms and soles. There was no evidence of atopic dermatitis or keratosis pilaris. His mother and eight other family members were similarly affected (Fig. 1b). Subsequently, a further child was born with a collodion membrane followed by generalized ichthyosis. Figure 1 (a) Pedigree of family with loricrin keratoderma. Filled symbols represent affected individuals. Whole exome sequencing was performed on one affected family member. *marks individuals who were screened for loricrin mutation by Sanger sequencing. (b–d) ... Biopsies of affected skin were processed for light and electron microscopy by standard methods (upper back of the proband's mother) or for electron microscopy only (affected acral skin from the proband's grandfather).7 Following informed consent, genomic DNA was extracted from blood or saliva samples from 10 affected and unaffected family members (Fig. 1a). A whole exome sequencing approach was taken to analyse the proband's DNA (Methods S1; see Supporting Information). Light microscopy of skin from the proband's mother showed mild hyperkeratosis, a normal granular layer and no significant parakeratosis (Fig. 2a). Electron microscopy of (i) affected acral skin demonstrated mild intracellular oedema, abundant keratohyaline granules in upper layers, with desmosomes and keratin filaments appearing intact and of (ii) affected upper back skin, vacuolar changes and disruption of suprabasal keratinocytes (Fig. ​(Fig.3).3). Whole exome sequencing directed at relevant epidermal genes revealed a novel heterozygous duplication mutation in the loricrin gene in exon 2 (designated c.806dupG), with an insertion of a single base pair resulting in a frameshift leading to a delayed termination codon and elongation of the protein by 22 amino acids (Fig. 2d). The mutation was confirmed by Sanger sequencing (Methods S2; see Supporting Information) and was present in affected individuals but was not in unaffected family members (Figs 1a, ​a,2b,c).2b,c). This mutation is not on the dbSNP database or NHLBI Exome Variant Server (http://evs.gs.washington.edu/EVS/). Figure 2 (a) A biopsy from the proband's mother's skin showing mild hyperkeratosis, a normal granular layer and no significant parakeratosis (original magnification × 20). (b–d) Mutation analysis. (b) Normal loricrin sequence in exon 2, showing ... Figure 3 Semithin sections and ultrastructural images. In semithin sections (a) there was epidermal acanthosis and compact hyperkeratosis with a pattern of diffuse vacuolar change. These vacuoles (small arrows) were predominantly observed in the suprabasal keratinocyte ... Six different heterozygous insertion mutations in loricrin in 14 unrelated pedigrees have previously been reported and one heterozygous deletion in a further pedigree2, 4, 5, 6, 8, 9, 11, 12, 13 (see Supporting Information; Table S1). All six insertion mutations are single base‐pair insertions leading to delayed termination codons with the most frequent mutation 730insG being present in eight of the 14 published families.2, 4, 8, 9, 11, 12, 13 This region of the loricrin gene is thought to be a mutation hotspot because of the presence of six consecutive guanine nucleotides.12All single‐base‐pair insertion and deletion mutations lead to a frameshift and delayed termination, thus elongating the protein by 22 amino acids and changing the Gly‐Lys‐rich domain into an Arg‐Leu‐rich terminal domain,2 except for two pedigrees of Brazilian origin, where the new protein is 25 amino acids longer than wild‐type protein.5 The mechanism of action of these mutations is thought to relate to preferential localization of mutant loricrin in the nucleus due to the formation of nuclear localization sequences within the arginine‐rich mutant loricrin.14 It has been suggested that the abnormal nuclear protein may disrupt the apoptotic process in terminal differentiation of keratinocytes in mouse models, thus supporting the hypothesis that the phenotype of loricrin keratoderma is caused by the synthesis of mutant loricrin rather than by the lack of wild‐type loricrin.14, 15 No clear genotype‐phenotype associations for pedigrees with specific mutations have currently been identified.4, 5, 13 The phenotype of loricrin keratoderma is heterogeneous. The common clinical features in both the 15 previously reported pedigrees and our new pedigree are palmoplantar keratoderma, usually of a honeycomb pattern, and generalized ichthyosis. Other features, including knuckle pads, pseudoainhum/hyperconstricting bands with autoamputation of digits and collodion babies, are variably reported. Previously, Gedicke et al.13 have suggested that the term ‘mutilating keratoderma with ichthyosis’ is not entirely suitable, due to the variation in phenotypes previously reported. Similarly, given the prominence of generalized ichthyosis with lesser palmoplantar involvement in this pedigree, and the presence of generalized ichthyosis in all previously reported pedigrees, we suggest that the condition could be described as loricrin ichthyosis rather than loricrin keratoderma.

The group of reticulate pigmentary disorders includes the rare autosomal dominant Dowling-Degos disease (DDD) and Galli-Galli disease (GGD;.OMIM 179850, OMIM 615327, OMIM 615696). 1 In light of substantial clinical, histological and mutational overlap between GGD and DDD they are considered to belong to the same entity. 2-3 Mutations in KRT5 (encoding keratin 5) have been associated with GGD/DDD since 2006. 2-5 With the development of whole exome sequencing (WES), mutations in POFUT1 (encoding protein O-fucosyltransferase 1) 6-7 and POGLUT1 (encoding protein O-glucosyltransferase 1) 8 have been shown to underlie some cases of GGD/DDD. We report mutations in POGLUT1 in 3 families of European ancestry. This article is protected by copyright. All rights reserved.

Plectin is a 500 kDa protein involved in cytoskeleton-plasma membrane attachment with a wide tissue distribution including cutaneous and airway epithelia, muscle and neuronal tissue. Recently, mutations in the gene encoding plectin (PLEC1) have been implicated in the pathogenesis of an autosomal recessive variant of epidermolysis bullosa simplex in which cutaneous blistering starting in the neonatal period is associated with muscular dystrophy in later life. In this study, we report two unrelated patients, both of consanguineous parentage, who presented with cutaneous blistering and a hoarse cry from birth. Both experienced inspiratory stridor and respiratory distress, necessitating emergency tracheostomy in one case. Immunoreactivity to monoclonal antibodies against plectin was absent or markedly reduced in skin biopsies from both patients. Electron microscopy revealed a low intraepidermal plane of cleavage and hypoplastic hemidesmosomes with a reduced association with keratin intermediate filaments. Direct sequencing of PLEC1 in each case demonstrated two novel homozygous frameshift deletion mutations, 5069del19 and 5905del2, which both create downstream premature termination codons. Although currently neither patient has symptoms of muscle disease, the identification of mutations in PLEC1 may be predictive for the future development of muscular dystrophy. Recessive epidermolysis bullosa simplex resulting from abnormalities in plectin should be considered in the differential diagnosis blistering, hoarseness and stridor in infancy.

Journal Article Steatocystoma multiplex, oligodontia and partial persistent primary dentition associated with a novel keratin 17 mutation Get access J.K. Gass, J.K. Gass Department of Dermatology, Box 46, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, U.K. Search for other works by this author on: Oxford Academic Google Scholar N.J. Wilson, N.J. Wilson Cancer Research UK Cell Structure Research Group Search for other works by this author on: Oxford Academic Google Scholar F.J.D. Smith, F.J.D. Smith Epithelial Genetics Group, Division of Molecular Medicine, University of Dundee, Dundee, U.K. Search for other works by this author on: Oxford Academic Google Scholar E.B. Lane, E.B. Lane Cancer Research UK Cell Structure Research GroupA*STAR Institute for Medical Biology, Biopolis, Singapore Search for other works by this author on: Oxford Academic Google Scholar W.H.I. McLean, W.H.I. McLean Epithelial Genetics Group, Division of Molecular Medicine, University of Dundee, Dundee, U.K. Search for other works by this author on: Oxford Academic Google Scholar E. Rytina, E. Rytina Department of Histopathology, Addenbrooke’s Hospital, Cambridge, U.K. Search for other works by this author on: Oxford Academic Google Scholar I. Salvary, I. Salvary Department of Dermatology, James Paget University Hospitals NHS Trust, Great Yarmouth, U.K. E‐mail: julia.gass@addenbrookes.nhs.uk Search for other works by this author on: Oxford Academic Google Scholar N.P. Burrows N.P. Burrows Department of Dermatology, Box 46, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, U.K. Search for other works by this author on: Oxford Academic Google Scholar British Journal of Dermatology, Volume 161, Issue 6, 1 December 2009, Pages 1396–1398, https://doi.org/10.1111/j.1365-2133.2009.09383.x Published: 01 December 2009

Scope Factors such as food processing, the food matrix, and antacid medication may affect the bio‐accessibility of proteins in the gastrointestinal tract and hence their allergenic activity. However, at present they are poorly understood. Methods and Results Roasted peanut flour was incorporated into either a chocolate dessert or cookie matrix and bio‐accessibility were assessed using an in vitro digestion system comprising a model chew and simulated gastric and duodenal digestion. Protein digestion was monitored by SDS‐PAGE and immunoreactivity analyzed by immunoblotting and immunoassay. IgE reactivity was assessed by immunoassay using serum panels from peanut‐allergic subjects. Roasted peanut flour proteins proved highly digestible following gastro‐duodenal digestion even when incurred into a food matrix, with only low molecular weight polypeptides of M r &lt; 8 kDa remaining. When gastric digestion was performed at pH 6.5 (simulating the effect of antacid medication), peanut proteins are not digested; subsequent duodenal digestion is also limited. IgE reactivity of the major peanut allergens Ara h 1, Ara h 2, and Ara h 6, although reduced, was retained after oral‐gastro‐duodenal digestion irrespective of digestion conditions employed. Conclusion Peanut allergen bio‐accessibility is unaffected by the dessert or cookie matrices whilst high intra‐gastric pH conditions render allergens more resistant to digestion.

RNA interference offers a novel approach for treating genetic disorders including the rare monogenic skin disorder pachyonychia congenita (PC). PC is caused by mutations in keratin 6a (K6a), K6b, K16, and K17 genes, including small deletions and single nucleotide changes. Transfection experiments of a fusion gene consisting of K6a and a yellow fluorescent reporter (YFP) resulted in normal keratin filament formation in transfected cells as assayed by fluorescence microscopy. Similar constructs containing a single nucleotide change (N171K) or a three-nucleotide deletion (N171del) showed keratin aggregate formation. Mutant-specific small inhibitory RNAs (siRNAs) effectively targeted these sites. These studies suggest that siRNAs can discriminate single nucleotide mutations and further suggest that "designer siRNAs" may allow effective treatment of a host of genetic disorders including PC.

Keratin 7 is expressed in simple epithelia but is expressed at low or undetectable levels in gastrointestinal epithelial cells. In the pancreas, it is present in ductal but not in acinar cells. K7 mRNA is overexpressed in pancreatic cancers. Here we use luciferase reporter assays to analyze the tissue-specific regulatory elements of murine keratin 7 (Krt7) promoter in vitro and in vivo. All elements required for appropriate cell and tissue specificity in reporter assays are present within the Krt7 -234 bp sequence. This fragment appears more selective to pancreatic ductal cells than the Krt19 promoter. GC-rich sequences corresponding to putative Sp1, AP-2 binding sites are essential for in vitro activity. Krt7-LacZ transgenic mice were generated to analyze in vivo activity. Sequences located 1.5 or 0.25 kb upstream of the transcription initiation site drive reporter expression to ductal, but not acinar, cells in transgenic mice. LacZ mRNA was detected in the pancreas as well as in additional epithelial tissues--such as the intestine and the lung--using both promoter constructs. An AdK7Luc adenovirus was generated to assess targeting selectivity in vivo by intravenous injection to immunocompetent mice and in a xenograft model of pancreatic cancer. The -0.25 kb region showed pancreatic selectivity, high activity in pancreatic cancers, and sustained transgene expression in xenografts. In conclusion, the krt7 promoter is useful to target pancreatic ductal adenocarcinoma cells in vitro and in vivo.

Reticulate pigmentary disorders include the rare autosomal dominant Galli-Galli disease (GGD) and Dowling-Degos disease (DDD). Clinical diagnosis between some of the subtypes can be difficult due to a degree of overlap between clinical features, therefore analysis at the molecular level may be necessary to confirm the diagnosis.To identify the underlying genetic defect in a 48-year-old Asian-American woman with a clinical diagnosis of GGD.Histological analysis was performed on a skin biopsy using haematoxylin-eosin staining. KRT5 (the gene encoding keratin 5) was amplified from genomic DNA and directly sequenced.The patient had a history of pruritus and hyperpigmented erythematous macules and thin papules along the flexor surfaces of her arms, her upper back and neck, axillae and inframammary areas. Hypopigmented macules were seen among the hyperpigmentation. A heterozygous 1-bp insertion mutation in KRT5 (c.38dupG; p.Ser14GlnfsTer3) was identified in the proband. This mutation occurs within the head domain of the keratin 5 protein leading to a frameshift and premature stop codon.From the histological findings and mutation analysis the individual was identified as having GGD due to haploinsufficiency of keratin 5.

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of PQS-dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat substrate(s) responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the primary signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. This defect was also phenocopied by deletion of cytB or cytC1. Thus, either lack of the Rieske sub-unit or mutation of cytochrome bc1 genes results in the perturbation of PQS-dependent autoinduction resulting in eDNA deficient biofilms, reduced antibiotic tolerance and compromised virulence factor production.

Pachyonychia congenita (PC) is a group of inherited ectodermal dysplasias, the characteristic phenotype being hypertrophic nail dystrophy. Two main clinical subtypes, PC-1 and PC-2, are inherited as autosomal dominant disorders, but other less well characterized clinical forms also exist. The PC-1 phenotype may be distinguished by the absence of the epidermal cysts found in PC-2, and it has been shown to be caused by mutations in either keratin K16 or its expression partner, the K6a isoform of K6. Mutations in K16 have also been shown to cause a milder related phenotype, focal non-epidermolytic palmoplantar keratoderma. Recently, we have developed a long-range polymerase chain reaction (PCR) strategy which allows specific amplification of the entire functional K16 gene (KRT16A), without amplification of the two K16 pseudogenes (ψKRT16B and ψKRT16C), enabling mutation analysis based on genomic DNA. Here, using this methodology, we describe novel mutations R127P and Q122P in the helix 1A domain of K16 in two families presenting with PC-1. Both mutations were excluded from 50 normal unrelated individuals by restriction enzyme analysis of K16 PCR fragments. In one family, ultrastructural analysis was performed, revealing distinctive tonofilament abnormalities. Specifically, keratin filament bundles were greatly condensed, but did not form the dense amorphous aggregates seen in a number of other keratin disorders. In the second kindred, autosomal dominant cataract was present in some but not all members affected by PC. As the cataract phenotype did not fully cosegregate with the K16 mutation, and given that K16 is not expressed in the lens, these two phenotypes may be coincidental.