Johann W. Bauer

The classification of inherited epidermolysis bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB

BACKGROUND Since publication in 2000 of the Second International Consensus Report on Diagnosis and Classification of Epidermolysis Bullosa, many advances have been made to our understanding of this group of diseases, both clinically and molecularly. At the same time, new epidermolysis bullosa (EB) subtypes have been described and similarities with some other diseases have been identified. OBJECTIVE We sought to arrive at a new consensus of the classification of EB subtypes. RESULTS We now present a revised classification system that takes into account the new advances, as well as encompassing other inherited diseases that should also be included within the EB spectrum, based on the presence of blistering and mechanical fragility. Current recommendations are made on the use of specific diagnostic tests, with updates on the findings known to occur within each of the major EB subtypes. Electronic links are also provided to informational and laboratory resources of particular benefit to clinicians and their patients. LIMITATIONS As more becomes known about this disease, future modifications may be needed. The classification system has been designed with sufficient flexibility for these modifications. CONCLUSION This revised classification system should assist clinicians in accurately diagnosing and subclassifying patients with EB.

miR-17, miR-19b, miR-20a, and miR-106a are down-regulated in human aging.

Aging is a multifactorial process where deterioration of body functions is driven by stochastic damage while counteracted by distinct genetically encoded repair systems. To better understand the genetic component of aging, many studies have addressed the gene and protein expression profiles of various aging model systems engaging different organisms from yeast to human. The recently identified small non‐coding miRNAs are potent post‐transcriptional regulators that can modify the expression of up to several hundred target genes per single miRNA, similar to transcription factors. Increasing evidence shows that miRNAs contribute to the regulation of most if not all important physiological processes, including aging. However, so far the contribution of miRNAs to age‐related and senescence‐related changes in gene expression remains elusive. To address this question, we have selected four replicative cell aging models including endothelial cells, replicated CD8+ T cells, renal proximal tubular epithelial cells, and skin fibroblasts. Further included were three organismal aging models including foreskin, mesenchymal stem cells, and CD8+ T cell populations from old and young donors. Using locked nucleic acid‐based miRNA microarrays, we identified four commonly regulated miRNAs, miR‐17 down‐regulated in all seven; miR‐19b and miR‐20a, down‐regulated in six models; and miR‐106a down‐regulated in five models. Decrease in these miRNAs correlated with increased transcript levels of some established target genes, especially the cdk inhibitor p21/CDKN1A. These results establish miRNAs as novel markers of cell aging in humans.

Expression profiling of aging in the human skin

During the last years it was shown that the aging process is controlled by specific genes in a large number of organisms (C. elegans, Drosophila, mouse or humans). To investigate genes involved in the natural aging process of the human skin we applied cDNA microarray analysis of naturally aged human foreskin samples. For the array experiments a non-redundant set of 2135 pre-selected EST clones was used. These arrays were used to probe the patterns of gene expression in naturally aged human skin of five young (3-4 years of age) and five old (68-72 years of age) healthy persons. We found that in total 105 genes change their expression over 1.7-fold during the aging process in the human skin. Of these 43 genes were shown to be down-regulated in contrast to 62 up-regulated genes. Expression of regulated genes was confirmed by real-time PCR. These results suggest that the aging process in the human skin is connected with the deregulation of various cellular processes, like cell cycle control, cytoskeletal changes, inflammatory response, signaling and metabolism.

Ribosomal proteins Rpl10 and Rps6 are potent regulators of yeast replicative life span.

The yeast ribosome is composed of two subunits, the large 60S subunit (LSU) and the small 40S subunit (SSU) and harbors 78 ribosomal proteins (RPs), 59 of which are encoded by duplicate genes. Recently, deletions of the LSU paralogs RPL31A and RPL6B were found to increase significantly yeast replicative life span (RLS). RPs Rpl10 and Rps6 are known translational regulators. Here, we report that heterozygosity for rpl10Delta but not for rpl25Delta, both LSU single copy RP genes, increased RLS by 24%. Deletion of the SSU RPS6B paralog, but not of the RPS6A paralog increased replicative life span robustly by 45%, while deletion of both the SSU RPS18A, and RPS18B paralogs increased RLS moderately, but significantly by 15%. Altering the gene dosage of RPL10 reduced the translating ribosome population, whereas deletion of the RPS6A, RPS6B, RPS18A, and RPS18B paralogs produced a large shift in free ribosomal subunit stoichiometry. We observed a reduction in growth rate in all deletion strains and reduced cell size in the SSU RPS6B, RPS6A, and RPS18B deletion strains. Thus, reduction of gene dosage of RP genes belonging to both the 60S and the 40S subunit affect lifespan, possibly altering the aging process by modulation of translation.

Regeneration of the entire human epidermis using transgenic stem cells

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

Squamous cell carcinoma in junctional and dystrophic epidermolysis bullosa

We report here on three patients suffering from recessive dystrophic epidermolysis bullosa and one suffering from generalized atrophic benign epidermolysis bullosa, all of whom developed cutaneous squamous cell carcinoma. Our observations and a review of the literature suggest that squamous cell carcinoma in generalized atrophic benign epidermolysis bullosa is very infrequent and has a better outcome compared to skin cancer in recessive dystrophic epidermolysis bullosa. These differences could be explained by the distinct pathophysiology and clinical course of each of these variants of epidermolysis bullosa. In contrast to UV-induced skin cancer, the tumours in epidermolysis bullosa develop on distal extremities at sites of chronic wound healing. The cases reported here underline the exceptional importance of early histopathological assessment of suspicious skin lesions in patients with epidermolysis bullosa.

Revertant mosaicism: partial correction of a germ-line mutation in COL17A1 by a frame-restoring mutation

Generalized atrophic benign epidermolysis bullosa is an autosomal recessive subepidermal blistering disease typified by null mutations in COL17A1. In 1 large kindred, affected individuals were homozygous for a 2-bp deletion in COL17A1, 4003delTC, which resulted in a downstream premature termination codon, nonsense-mediated mRNA decay, and abrogation of type XVII collagen synthesis. Interestingly, 1 of these patients, although phenotypically identical to her affected siblings, showed focal expression of type XVII collagen in epidermal basement membrane in a pattern suggestive of revertant mosaicism. When studies of randomly obtained epidermal, oromucosal, and peripheral blood cells failed to identify the genetic basis of this apparent mosaicism, microscopic subpopulations of potentially revertant epidermal cells (i.e., those overlying basement membrane containing type XVII collagen) were selectively isolated using laser capture microdissection. Analysis of DNA and RNA from these cells revealed a second mutation, 4080insGG, on 1 allele of COL17A1. This 2-bp insertion corrected the reading frame just proximal to the premature termination codon, countered nonsense-mediated mRNA decay, and allowed protein production by patient keratinocytes in vivo and in vitro. These studies elucidate the molecular basis of a novel form of revertant mosaicism in humans.

Spliceosome-Mediated Trans-Splicing: The Therapeutic Cut and Paste

Spliceosome-mediated RNA trans-splicing (SMaRT) is an RNA-based technology to reprogram genes for diagnostic and therapeutic purposes. For the correction of genetic diseases, SMaRT offers several advantages over traditional gene-replacement strategies. SMaRT protocols have recently been used for in vitro phenotypic correction of a variety of genetic disorders, ranging from epidermolysis bullosa to neurodegenerative diseases. In vivo studies are currently bringing trans-splicing RNA therapy toward clinical application. In this review, we summarize the progress made toward the medical use of SMaRT and provide an outlook on its upcoming applications.

Matrix metalloproteinase-7 activates heparin-binding epidermal growth factor-like growth factor in cutaneous squamous cell carcinoma

Background  Tumour‐specific expression of matrix metalloproteinase (MMP)‐7 has been noted in cutaneous squamous cell carcinomas (SCCs) in patients with recessive dystrophic epidermolysis bullosa (RDEB).

Development of spliceosome-mediated RNA trans-splicing (SMaRT™) for the correction of inherited skin diseases

Abstract: Gene therapy of large genes (e.g. plectin and collagen genes) is hampered by size limitations for insertions of the currently used viral vectors. To reduce the size of these insertions spliceosome‐mediated RNA trans‐splicing (SMaRT™), which provides intron‐specific gene‐correction at the pre‐RNA level, can be an alternative approach. To test its applicability in skin gene therapy, SMaRT™ was used in the context of the 4003delTC mutation in the collagen XVII gene (COL17A1) causing generalized atrophic benign junctional epidermolysis bullosa. A β‐galactosidase (β‐gal) trans‐splicing assay system was established using intron 51 of COL17A1 as the target for trans‐splicing. In this system, intron 51 is flanked by the 5′exon and the 3′exon of the β‐gal gene, the latter containing two in‐frame stop codons. Cotransfection of a pre‐trans‐splicing molecule consisting of the binding domain of intron 51 and the 3′exon of β‐gal without the stop codons resulted in a 300‐fold increase of β‐gal activity compared to controls. A 2–3‐fold increase in efficiency was obtained through an elongation of the binding domains. Replacement of the complete 3′end of the COL17A1 gene was shown using a collagen XVII mini‐gene construct. The β‐gal assay was used in human keratinocytes to evaluate the influence of a keratinocyte‐specific spliceosome background. Reverse transcription polymerase chain reaction and β‐gal activity assay showed functional correction of the stop‐codons in cultured human keratinocytes and in an immortalized GABEB cell line harbouring the 4003delTC mutation. These results demonstrate that SMaRT™ is feasible in a keratinocyte‐specific context and therefore may be applied in skin gene therapy.