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Notes: Plectin is a 500kDa 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 (PLECI) 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 form abnormalities in plectin should be considered in the differential diagnosis of cutaneous blistering, hoarseness and stridor in infancy.

Notes: The Human Genome Mapping Project and allied rapid advances in genetic technology over the past decade have facilitated accurate association of allelic variations in several genes with specific skin phenotypes. Currently the genetic bases of the majority of the more common genodermatoses have been elucidated. In scientific terms this work has been extraordinarily successful and has yielded many new biological insights. These advances, although exciting, have yet to be translated into direct benefit for patients with these diseases. Genetic counselling has been greatly aided by gene identification, by the better understanding of genotype–phenotype correlation and by the disclosure of unexpected genetic mechanisms in some families. Knowledge of the molecular basis of these disorders has also been vital in enabling DNA-based prenatal diagnosis in several conditions and DNA-based preimplantation diagnosis has been used in a selected few. While this successful period of gene mapping is now nearing completion, progress towards the next goal, that of developing therapeutic strategies based on the knowledge of these underlying genetic mechanisms, has proven frustratingly slow. Despite the ready access to the skin compared with solid internal organs, the challenges of cutaneous gene therapy are legion and many technical issues need to be surmounted to enable gene replacement or modification of gene expression to have a useful role in these disorders. In this article we make a comprehensive review of progress to date in gene identification, genotype–phenotype correlation, prenatal diagnosis and cutaneous gene therapy, and we examine future directions for research in this field.

Notes: Background 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. Objectives To characterize a novel type II keratin in mice and humans. Methods 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. Results 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. Conclusions 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.

Notes: Background  Erythrokeratodermia variabilis (EKV) is an autosomal dominant or recessive genodermatosis characterized by the coexistence of randomly occurring, transient, erythematous patches and hyperkeratosis of the skin. The disorder has been mapped to chromosome 1p35.1 but is genetically heterogeneous. EKV may be caused by pathogenic mutations in one of two neighbouring connexin genes, GJB3 and GJB4, encoding the gap junction proteins Cx31 and Cx30.3, respectively. Twelve distinct mutations identified to date cluster either at the cytoplasmic amino-terminus or in the four transmembrane domains.Objectives  To report a large family with EKV and an unrelated sporadic case.Methods  DNA amplification and mutation analysis, followed by denaturing high-performance liquid chromatography to confirm the segregation of the mutations in the two families with EKV.Results  A novel, recurrent GJB3 mutation (625C→T; L209F) was identified in the family with EKV and in the unrelated sporadic case.Conclusions  This mutation is the first to affect a conserved residue in the cytoplasmic carboxy-terminus of any connexin gene with a cutaneous phenotype, emphasizing its structural and/or functional importance.

Notes: Background  Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis characterized by epidermolytic hyperkeratosis strictly confined to the palms and soles, and usually associated with mutations in the keratin K9 gene (KRT9). Mutations in the keratin K1 gene (KRT1) have been shown to underlie a variety of phenotypes typically involving generalized epidermolytic hyperkeratosis, but in some cases the phenotype can be more regionally restricted.Objectives  To identify the genetic defect in two unrelated families initially presenting with EPPK but where careful examination revealed hyperkeratosis extending on to the proximal wrist flexure.Methods  Linkage analysis and DNA sequencing.Results  We found that this phenotype is caused by a heterozygous missense mutation in the K1 gene, designated I479T. This mutation lies in the highly conserved helix termination motif of K1, previously shown to be important for keratin assembly and filament formation. In general, mutations in this region of keratins are associated with more severe disease phenotypes. However, K1 mutations in this region and the I479T mutation in particular have previously been associated with both severe and mild bullous congenital ichthyosiform erythroderma phenotypes. When further clinical enquiries were made, several affected individuals in the families studied here were found to have had transient flexural peeling and hyperkeratosis in the neonatal period.Conclusions  K1 mutations may underlie a phenotype closely resembling EPPK. A history of transient flexural peeling and hyperkeratosis in childhood and palmoplantar keratoderma which extends beyond the boundary of the palmoplantar margins may indicate a K1 mutation rather than a K9 defect. As K1 mutations are also associated with severe widespread phenotypes, with important implications for prognostic and genetic counselling, whole body examination is recommended for patients presenting with EPPK.

Notes: 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.

Notes: Background  Lipoid proteinosis (LiP) is a rare autosomal recessive disorder characterized by a hoarse voice, warty skin infiltration and scarring. Mutations within the extracellular matrix protein 1 (ECM1) gene cause LiP. Since the early 1970s it has been recognized that South Africa has one of the largest groups of LiP patients worldwide, suggesting a probable founder effect. As LiP patients present with considerable clinical variability, this group of patients offers a unique opportunity for genotype–phenotype correlation.Objectives  To assess the clinical features and the molecular basis of LiP in patients from the Namaqualand area of the Northern Cape province of South Africa and to examine molecular evidence for a founder effect.Subjects and methods  The LiP patient cohort consisted of 29 Coloured patients from Namaqualand and a further seven Caucasoid patients from other areas of South Africa. The control group included 100 healthy geographically and ethnically matched individuals from Namaqualand. Samples were collected after informed consent and with ethics committee approval from the University of the Witwatersrand. LiP patients were examined clinically and a structured recording sheet was completed. A brief neurological evaluation was also performed. The LiP founder effect was investigated at the molecular level by ECM1 mutation detection and haplotype analysis.Results  The most consistent clinical signs for a diagnosis of LiP in this group were a hoarse voice and thickened sublingual frenulum leading to restricted tongue movement. Homozygosity for a nonsense mutation in exon 7 of the ECM1 gene, Q276X, was identified in all patients (Coloured and Caucasoid). Despite this genetic homogeneity, considerable clinical variability in skin presentation and psychiatric involvement was observed. Haplotype analysis using markers from a 9·98-Mb region around the ECM1 locus confirmed the founder effect with a founder core haplotype, 19-Q276X-12 (ND1-ECM1-D1S2343), in all but four LiP-associated alleles (n = 58). A LiP carrier rate of 1 in 9 was observed among the 100 Namaqualand controls, predicting a LiP incidence of 1 in 324 in this community.Conclusions  Although several consistent clinical features in LiP patients homozygous for the Q276X mutation in the ECM1 gene were observed, there remains considerable clinical variability. This suggests the action of genetic and environmental modifiers of disease severity. Strong molecular evidence supports a single founder effect for the high prevalence of LiP in South Africans, both Coloured and Caucasoid.