Keratins and epidermolysis bullosa simplex

Keratin intermediate filaments play an important role in maintaining the integrity of the skin structure. Understanding the importance of this subject is possible with the investigation of keratin defects in epidermolysis bullosa simplex (EBS). Nowadays, in addition to clinical criteria, new molecular diagnostic methods, such as next generation sequencing, can help to distinguish the subgroups of EBS more precisely. Because the most important and most commonly occurring molecular defects in these patients are the defects of keratins 5 and14 (KRT5 and KRT14), comprehending the nature structure of these proteins and their involved processes can be very effective in understanding the pathophysiology of this disease and providing new and effective therapeutic platforms to treat it. Here, we summarized the various aspects of the presence of KRT5 and KRT14 in the epidermis, their relation to the incidence and severity of EBS phenotypes, and the processes with which these proteins can affect them.     

A novel COL7A1 gene mutation causing pretibial epidermolysis bullosa: Report of a Chinese family with intra-familial phenotypical diversity

Pretibial epidermolysis bullosa (PEB) is an extremely rare subtype of dominant dystrophic epidermolysis bullosa (DDEB) caused by mutation of the COL7A1 gene. More than 730 mutations have been identified in patients with DDEB, but only five mutations have been found to be related to PEB. In this study, a novel heterozygous nucleotide G > T transition at position 6101 in exon 73 of COL7A1 was detected, which resulted in a glycine to valine substitution (G2034V) in the triple-helical domain of type-VII collagen. This is the first report to show that one mutation caused a broad range of severity of disease in one family with PEB. These data suggest that c.6101G > T may influence the phenotype of PEB. They also contribute to the expanding database on COL7A1 mutations.     

An inducible mouse model for epidermolysis bullosa simplex: implications for gene therapy

The Dowling-Meara variant of epidermolysis bullosa simplex (EBS-DM) is a severe blistering disease inherited in an autosomal-dominant fashion. Here we report the generation of a mouse model that allows focal activation of a mutant keratin 14 allele in epidermal stem cells upon topical administration of an inducer, resulting in EBS phenotypes in treated areas. Using laser capture microdissection, we show that induced blisters healed by migration of surrounding nonphenotypic stem cells into the wound bed. This observation provides an explanation for the lack of mosaic forms of EBS-DM. In addition, we show that decreased mutant keratin 14 expression resulted in normal morphology and functions of the skin. Our results have important implications for gene therapy of EBS and other dominantly inherited diseases.     

Modeling effects of mutations in coiled-coil structures: case study using epidermolysis bullosa simplex mutations in segment 1a of K5/K14 intermediate filaments

The sequence of a protein chain determines both its conformation and its function in vivo. An attempt is made to gain an understanding of the classes of deformations that can arise in an important structural motif, the alpha-helical coiled coil, as a consequence of mutations occurring in its underlying heptad substructure. In order to do so we consider the model structure of segment 1A in intermediate filaments and then investigate the structures arising from each of the 22 mutations observed in cytokeratin K5/K14 molecules that lead to variants of epidermolysis bullosa simplex. These are refined separately using a molecular dynamics protocol. The mutations often result in a significant distortion of the backbone over a turn or so of the alpha helix in either the chain itself or its constituent partner, leading to the likelihood of impaired chain aggregation and hence molecular assembly. One mutant (K14-L143P; 1A-28) gave rise to structural distortion along almost the entire length of segment 1A. The remaining structures showed less deformation, and normal-looking intermediate filaments are likely in vivo. In addition, an identical mutation in the same position in each of the chains in the heterodimer did not necessarily give equivalent structural distortions. Although proline mutations frequently lead to the most severe structural deformations, a non-proline substitution (K14-R125S; 1A-10) gave rise to the largest local structural disruption that was observed. Unexpectedly, mutations in positions a and d were not always of the greatest structural significance, although three in position a were shown by AGADIR to result in a significant increase in alpha-helix stability.     

Partial deletion of the LAMA3 gene is responsible for hereditary junctional epidermolysis bullosa in the American Saddlebred Horse

Laminin 5 is a heterotrimeric basement membrane protein integral to the structure and function of the dermal–epidermal junction. It consists of three glycoprotein subunits: the α3, β3 and γ2 chains, which are encoded by the LAMA3 , LAMB3 and LAMC2 genes respectively. A mutation in any of these genes results in the condition known as hereditary junctional epidermolysis bullosa (JEB). A 6589-bp deletion spanning exons 24–27 was found in the LAMA3 gene in American Saddlebred foals born with the skin-blistering condition epitheliogenesis imperfecta. The deletion confirms that this autosomal recessive condition in the American Saddlebred Horse can indeed be classified as JEB and corresponds to Herlitz JEB in humans. A diagnostic test was developed and nine of 175 randomly selected American Saddlebred foals from the 2007 foal crop were found to be carriers of the mutation (frequency of 0.026).     

Epidermolysis bullosa simplex: Expression of gelatinase activity in cultured human skin fibroblasts

We have measured the baseline level of gelatinase in fibroblast-conditioned medium from 41 Scandinavian individuals. They comprised 12 healthy persons, 11 individuals with the skin disorder dominant epidermolysis bullosa simplex (DEBS), 16 patients with other types of epidermolysis bullosa (EB) and 2 siblings with prolidase deficiency. These results divide the cell strains into those with low and those with high activity levels. Although this dual biochemical trait occurred in all the groups of individuals, the high-activity trait was more frequent among the DEBS patients. The localized DEBS forms showed an elevated activity level, in contrast to the previously reported generalized DEBS Köbner forms. Although a high level was found in some individuals with other EB forms, the high incidence in four families with localized DEBS Weber-Cockayne (eight of eight) and a single family with generalized DEBS—mottled pigmentation (two of two) may result from a close linkage between an EB gene and a gene responsible for the biochemical trait. In addition, in the single complete family tested, the level of gelatinase activity in cultured fibroblasts seemed to be regulated by codominant alleles or genes. A raised baseline level of gelatinase activity in cultured skin fibroblasts may be the result of either an altered expression of gelatinase or an allelic variant of this enzyme with increased specific activity. Further studies of gelatinase in cultured fibroblasts may provide insight into the regulatory mechanism and genetics behind this activity and allow formal linkage studies versus DEBS.This work was supported in part by the Norwegian Research Council for Science and the Humanities (NAVF) and the Concerted Action on Hereditary Connective Tissue Diseases of the European Community (1990–1992, project leader, M. Matton).Part of this work was performed at the Department of Genetics, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo.     

A mutation in the LAMC2 gene causes the Herlitz junctional epidermolysis bullosa (H-JEB) in two French draft horse breeds

Epidermolysis bullosa (EB) is a heterogeneous group of inherited diseases characterised by skin blistering and fragility. In humans, one of the most severe forms of EB known as Herlitz-junctional EB (H-JEB), is caused by mutations in the laminin 5 genes. EB has been described in several species, like cattle, sheep, dogs, cats and horses where the mutation, a cytosine insertion in exon 10 of the LAMC2 gene, was very recently identified in Belgian horses as the mutation responsible for JEB. In this study, the same mutation was found to be totally associated with the JEB phenotype in two French draft horse breeds, Trait Breton and Trait Comtois. This result provides breeders a molecular test to better manage their breeding strategies by genetic counselling.     

Denaturing HPLC-based approach for detection of COL7A1 gene mutations causing dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is a rare clinically heterogeneous genodermatosis due to genetic defects in type VII collagen gene (COL7A1). Identification of COL7A1 mutations is a challenge since this gene comprises 118 exons and more than 300 mutations scattered over the gene have been reported. Here, we describe for the first time the use of denaturing high performance liquid chromatography (DHPLC) for COL7A1 mutation detection. To validate the method, exon-specific DHPLC conditions were applied to screen DNA samples from patients carrying known COL7A1 mutations. Abnormal DHPLC profiles were obtained for all known mutations. Subsequent DHPLC analysis of 17 DEB families of unknown genotype allowed the identification of 21 distinct mutations, 9 of which were novel. The DHPLC mutation detection rate was significantly higher compared with our mutation scanning rate with conventional techniques (97% vs 86%), indicating DHPLC as the method of choice for COL7A1 molecular characterization in DEB patients.     

Sustained phenotypic reversion of junctional epidermolysis bullosa dog keratinocytes: Establishment of an immunocompetent animal model for cutaneous gene therapy

Gene transfer represents the unique therapeutic issue for a number of inherited skin disorders including junctional epidermolysis bullosa (JEB), an untreatable genodermatose caused by mutations in the adhesion ligand laminin 5 (alpha3beta3gamma2) that is secreted in the extracellular matrix by the epidermal basal keratinocytes. Because gene therapy protocols require validation in animal models, we have phenotypically reverted by oncoretroviral transfer of the curative gene the keratinocytes isolated from dogs with a spontaneous form of JEB associated with a genetic mutation in the alpha3 chain of laminin 5. We show that the transduced dog JEB keratinocytes: (1) display a sustained secretion of laminin 5 in the extracellular matrix; (2) recover the adhesion, proliferation, and clonogenic capacity of wild-type keratinocytes; (3) generate fully differentiated stratified epithelia that after grafting on immunocompromised mice produce phenotypically normal skin and sustain permanent expression of the transgene. We validate an animal model that appears particularly suitable to demonstrate feasibility, efficacy, and safety of genetic therapeutic strategies for cutaneous disorders before undertaking human clinical trials.     

Structural basis of the interaction between integrin α6β4 and plectin at the hemidesmosomes

The interaction between the integrin α6β4 and plectin is essential for the assembly and stability of hemidesmosomes, which are junctional adhesion complexes that anchor epithelial cells to the basement membrane. We describe the crystal structure at 2.75 Å resolution of the primary α6β4–plectin complex, formed by the first pair of fibronectin type III domains and the N‐terminal region of the connecting segment of β4 and the actin‐binding domain of plectin. Two missense mutations in β4 (R1225H and R1281W) linked to nonlethal forms of epidermolysis bullosa prevent essential intermolecular contacts. We also present two structures at 1.75 and 2.05 Å resolution of the β4 moiety in the absence of plectin, which reveal a major rearrangement of the connecting segment of β4 on binding to plectin. This conformational switch is correlated with the way α6β4 promotes stable adhesion or cell migration and suggests an allosteric control of the integrin.     

Cytological and immunocytochemical evaluation of thyroid and breast masses in patients with a previous neoplasm: case reports

The diagnosis of secondary tumours represents one of the most important fields in the application of fine needle aspiration cytology (FNAC). We studied two patients, one with a history of breast cancer and one with a previous tumour of the thyroid, who showed a second mass, in the thyroid and in the breast, respectively, during follow up. The aim of our study was to evaluate if cytology, performed on FNAC smears, may distinguish a metastatic lesion from a second primary tumour, or if further immunocytochemistry should be performed. Our data demonstrate that, while cytology may be indicative of a second primary tumour, the histotype should be confirmed by immunocytochemical staining.     

以肺部病变为主要表现的韦格纳肉芽肿1例并文献复习

目的:促进韦格纳肉芽肿的早期诊断和治疗,提高患者的生存率.方法:回顾性分析我院收治的1例韦格纳肉芽肿患者的发病诊治过程的临床资料并结合文献进行复习.结果:入院后行胸部CT、抗中性粒细胞胞浆抗体(ANCA)等检查,明确诊断后给予糖皮质激素、细胞毒类药物等治疗后短期缓解,5个月后死亡.结论:韦格纳肉芽肿的临床表现多种多样,诊断困难,实验室检查无特异性,临床上对于多系统受累的疾病应警惕此病,应结合患者临床表现及辅助检查做出早期诊断,及早治疗,从而提高患者的生存率.     

Review: Preimplantation genetic diagnosis (PGD) as a reproductive option in patients with neurodegenerative disorders

Preimplantation genetic diagnosis (PGD) was introduced in the late 1980s and represents an option for couples at risk of transmitting an inherited, debilitating or neurological disorder to their children. From a cleavage or blastocyst stage embryo, cell(s) are collected and then genetically analyzed for disease; enabling an unaffected embryo to be transferred into the uterus cavity. Nowadays, PGD has been carried out for several hundreds of heritable conditions including myotonic dystrophy, and for susceptibility genes involved in cancers of the nervous system. Currently, advanced molecular technologies with better resolution, such as array comparative genomic hybridisation, quantitative polymerase chain reaction, and next generation sequencing, are on the verge of becoming the gold standard in embryo preimplantation screening. Given this, it may be time for neurological societies to consider the published evidence to develop new guidelines for the integration of PGD into modern preventative neurology. Therefore, the main aim of this review is to illustrate the option of PGD to enable conception of an unaffected baby, and to assist clinicians and neurologists in the counseling of the patient at risk of transmitting an inherited disease, to explore the genetic journey throughout in vitro fertilization IVF with PGD.     

Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium.     

Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses.

Previously we demonstrated that transgenic mice expressing mutant basal epidermal keratin genes exhibited a phenotype resembling a group of autosomal dominant human skin disorders known as epidermolysis bullosa simplex (EBS). EBS diseases affect approximately 1: 50,000 and are of unknown etiology, although all subtypes exhibit blistering arising from basal cell cytolysis. We now demonstrate that two patients with spontaneous cases of Dowling-Meara EBS have point mutations in a critical region in one (K14) of two basal keratin genes. To demonstrate function, we engineered one of these point mutations in a cloned human K14 cDNA, and showed that a K14 with an Arg-125----Cys mutation disrupted keratin network formation in transfected keratinocytes and perturbed filament assembly in vitro. Since we had previously shown that keratin network perturbation is an essential component of EBS diseases, these data suggest that the basis for the phenotype in this patient resides in this point mutation.     

Medulloblastoma in the nevoid basal-cell carcinoma syndrome: case reports and review of the literature

The nevoid basal-cell carcinoma syndrome (NBCCS) is a rare autosomal-dominant inherited disorder. Its clinical manifestations are multiple basal-cell nevi and cysts of the jaw along with skeletal anomalies and various combinations of numerous other defects. NBCCS is characterized by a marked propensity for developing cancers. One of the most frequently reported tumour is brain medulloblastoma. We are reporting two cases of NBCCS and medulloblastoma. A review of the case reports demonstrates certain prominent features of medulloblastoma associated with NBCCS. The patients generally are males, presenting at an unusually young age, under 5 or 2 years and show a longer survival rate. Its lay down to search for NBCCS in early medulloblastoma's, especially under 2 years.