The cytoplasmic plaque protein desmoplakin (DP), which is located in desmosomes, plays a major role in epithelial and muscle cell adhesion by linking the transmembrane cadherins to the cytoplasmic intermediate filament network. Mutations of DP may cause striate palmoplantar keratoderma, arrhythmogenic right ventricular dysplasia, skin fragility/woolly hair syndrome, Naxos-like disease, and Carvajal syndrome. DP must be indispensable, because DP-/- mice are early abortive. Here, we report a patient with severe fragility of skin and mucous membranes caused by genetic truncation of the DP tail. The new phenotype is lethal in the neonatal period because of immense transcutaneous fluid loss. The phenotype also comprised universal alopecia, neonatal teeth, and nail loss. Histology showed suprabasal clefting and acantholysis throughout the spinous layer, mimicking pemphigus. Electron microscopy revealed disconnection of keratin intermediate filaments from desmosomes. Immunofluorescence staining of DP showed a distinct punctate intercellular pattern in the patient's skin. Protein analysis revealed expression of truncated DP polypeptides. Mutational analysis of the patient demonstrated compound heterozygosity for two DP mutations, 6079C-->T (R1934X) and 6370delTT, respectively. Aberrant mRNA transcripts that predict premature termination of translation with loss of the three intermediate filament-binding subdomains in the DP tail were detected by RT-PCR. The new dramatic phenotype, which we named "lethal acantholytic epidermolysis bullosa," underscores the paramount role of DP in epidermal integrity.

Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine; however, their potential clinical application is hampered by the low efficiency of somatic cell reprogramming. Here, we show that the synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNA-367/302s delivered as mature miRNA mimics greatly enhances the reprogramming of human primary fibroblasts into iPSCs. This synergistic activity is dependent upon an optimal RNA transfection regimen and culturing conditions tailored specifically to human primary fibroblasts. As a result, we can now generate up to 4,019 iPSC colonies from only 500 starting human primary neonatal fibroblasts and reprogram up to 90.7% of individually plated cells, producing multiple sister colonies. This methodology consistently generates clinically relevant, integration-free iPSCs from a variety of human patient’s fibroblasts under feeder-free conditions and can be applicable for the clinical translation of iPSCs and studying the biology of reprogramming.

Revertant mosaicism due to in vivo reversion of an inherited mutation has been described in the genetic skin disease epidermolysis bullosa (EB) for the genes KRT14 and COL17A1. Here we demonstrate the presence of multiple second-site mutations, all correcting the germline mutation LAMB3:c.628G→A;p.E210K, in 2 unrelated non-Herlitz junctional EB patients with revertant mosaicism. Both probands had a severe reduction in laminin-332 expression in their affected skin. Remarkably, the skin on the lower leg of patient 078-01 (c.628G→A/c.1903C→T) became progressively clinically healthy, with normal expression of laminin-332 on previously affected skin. In the other proband, 029-01 (c.628G→A/c.628G→A), the revertant patches were located at his arms, shoulder, and chest. DNA analysis showed different second-site mutations in revertant keratinocytes of distinct biopsy specimens (c.565-3T→C, c.596G→C;p.G199A, c.619A→C;p.K207Q, c.628+42G→A, and c.629-1G→A), implying that there is not a single preferred mechanism for the correction of a specific mutation. Our data offer prospects for EB treatment in particular cases, since revertant mosaicism seems to occur at a higher frequency than expected. This opens the possibility of applying revertant cell therapy in mosaic EB of the LAMB3 gene by using autologous naturally corrected keratinocytes, thereby bypassing the recombinant gene correction phase. IntroductionReverse mutations in germline or somatic cells bearing an inherited disease-causing mutation can change the phenotype from affected to normal by reexpression of the involved protein. These reverse mutations can be true back mutations -leading to the original wild-type sequence and thus wild-type protein -or can be additional second-site mutations that compensate for the effect of the primary inherited mutation (1). In the latter case, small changes in the amino acid sequence may occur. Revertant mosaicism has been demonstrated for different genetic diseases and in different cell types, as in hepatocytes, lymphocytes, and, in the case of epidermolysis bullosa (EB), keratinocytes (reviewed in refs. 2, 3). EB is a clinically heterogeneous group of heritable blistering disorders leading to fragility of the skin and mucous membranes. The subgroup junctional EB (JEB), characterized by separation at the lamina lucida of the epidermal basement membrane zone (BMZ), is caused by recessive mutations in the genes encoding type XVII collagen (COL17A1), integrin α6β4 (ITGA6 and ITGB4), or laminin-332 (LM-332; LAMA3, LAMB3, and LAMC2) (4). Recently, the first instance of correction of EB skin by transplantation of genetically modified epidermal stem cells was reported for a compound heterozygous carrier of laminin β3 (LAMB3) mutations (5).Revertant mosaicism in EB due to in vivo reversion of somatic cells has been described for the genes COL17A1 and KRT14 (6-10). The first reported case involved the reversion of one of the defective COL17A1 alleles into a wild-type sequence due to a mitotic gene conversion event (6). In a second EB patie...

Revertant mosaicism is a naturally occurring phenomenon involving spontaneous correction of a pathogenic gene mutation in a somatic cell. It has been observed in several genetic diseases, including epidermolysis bullosa (EB), a group of inherited skin disorders characterized by blistering and scarring. Induced pluripotent stem cells (iPSCs), generated from fibroblasts or keratinocytes, have been proposed as a treatment for EB. However, this requires genome editing to correct the mutations, and, in gene therapy, efficiency of targeted gene correction and deleterious genomic modifications are still limitations of translation. We demonstrate the generation of iPSCs from revertant keratinocytes of a junctional EB patient with compound heterozygous COL17A1 mutations. These revertant iPSCs were then differentiated into naturally genetically corrected keratinocytes that expressed type XVII collagen (Col17). Gene expression profiling showed a strong correlation between gene expression in revertant iPSC-derived keratinocytes and the original revertant keratinocytes, indicating the successful differentiation of iPSCs into the keratinocyte lineage. Revertant-iPSC keratinocytes were then used to create in vitro three-dimensional skin equivalents and reconstitute human skin in vivo in mice, both of which expressed Col17 in the basal layer. Therefore, revertant keratinocytes may be a viable source of spontaneously gene-corrected cells for developing iPSC-based therapeutic approaches in EB.

Revertant mosaicism by somatic reversion of inherited mutations has been described for a number of genetic diseases. Several mechanisms can underlie this reversion process, such as gene conversion, crossing-over, true back mutation, and second-site mutation. Here, we report the occurrence of multiple corrections in two unrelated probands with revertant mosaicism of non-Herlitz junctional epidermolysis bullosa, an autosomal recessive genodermatosis due to mutations in the COL17A1 gene. Immunofluorescence microscopy and laser dissection microscopy, followed by DNA and RNA analysis, were performed on skin biopsy specimens. In patient 1, a true back mutation, 3781T-->C, was identified in the specimen from the arm, and a second-site mutation, 4463-1G-->A, which compensated for the frameshift caused by the inherited 4424-5insC mutation, was identified in the 3' splice site of exon 55 in a specimen from the middle finger. Patient 2 showed--besides two distinct gene conversion events in specimens from the arm and hand sites, both of which corrected the 1706delA mutation--a second-site mutation (3782G-->C) in an ankle specimen, which prevented the premature ending of the protein by the 3781C-->T nonsense mutation (R1226X). Thus, both inherited mutations, paternal as well as maternal, reverted at least once by different reversion events in distinct cell clusters in the described patients. The occurrence of multiple correcting mutations within the same patient indicates that in vivo reversion is less unusual than was generally thought. Furthermore, in the male patient, mosaic patterns of type XVII collagen-positive keratinocytes were present in clinically unaffected and affected skin. This latter observation makes it likely that reversion may be overlooked and may happen more often than expected.

To elucidate mechanisms of cancer progression, we generated inducible human neoplasia in 3-dimensionally intact epithelial tissue. Gene expression profiling of both epithelia and stroma at specific time points during tumor progression revealed sequential enrichment of genes mediating discrete biologic functions in each tissue compartment. A core cancer progression signature was distilled using the increased signaling specificity of downstream oncogene effectors and subjected to network modeling. Network topology predicted that tumor development depends upon specific ECM-interacting network hubs. Blockade of one such hub, the β1 integrin subunit, disrupted network gene expression and attenuated tumorigenesis in vivo. Thus, integrating network modeling and temporal gene expression analysis of inducible human neoplasia provides an approach to prioritize and characterize genes functioning in cancer progression. Significance Investigating tumor progression in patient samples is complicated by etiologic heterogeneity, genetic instability, and an overabundance of precursor lesions that fail to progress. These complexities obscure construction of a dynamic picture of progression from normal tissue to invasive cancer. Here, we generate inducible human neoplasia driven by conditionally active Ras and characterize the sequence of gene expression programs engaged in epithelial tumor tissue and adjacent stroma during carcinogenesis. We show that tumor-intrinsic gene expression can be refined by sufficient downstream oncogene effectors and apply a generalizable network modeling strategy to prioritize targets based upon local interconnectivity. This analysis highlights the importance of tumor-stroma interaction during tumorigenesis and identifies β integrin as a potential oncotherapeutic that distinguishes normal and neoplastic tissue.

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Background Epidermolysis bullosa (EB) is a heterogeneous group of rare and incurable genetic disorders characterized by fragility of the skin and mucosae, resulting in blisters and erosions. Several epidemiological studies in other populations have been carried out, reporting varying and sometimes inconclusive figures, highlighting the need for standardized epidemiological analyses in well‐characterized cohorts. Objectives To evaluate the epidemiological data on EB in the Netherlands, extracted from the molecularly well‐characterized cohort in the Dutch EB Registry. Methods In this observational study all EB‐patients that were based in the Netherlands and captured in the Dutch EB Registry between 1988 and 2018 were included. The epidemiological outcomes were based on complete diagnostic data (clinical features, immunofluorescence, electron microscopy and mutation analysis), with longitudinal follow‐up. Results A total of 464 EB‐patients (287 families) were included. The incidence and point‐prevalence of EB in the Netherlands were 41.3 per million live births and 22.4 per million population, respectively. EB Simplex (EBS), Junctional EB (JEB), Dystrophic EB (DEB) and Kindler EB were diagnosed in 45.7%, 18.8%, 34.7% and 0.9% of the EB‐patients, respectively, with an incidence and point‐prevalence of 17.5 and 11.9 (EBS), 9.3 and 2.1 (JEB), 14.1 and 8.3 (DEB), 0.5 and 0.2 (Kindler EB). In 90.5% of the EB‐patients the diagnosis was genetically confirmed. During the investigated time period 73 EB‐patients died, 72.6% of whom as a direct consequence of their EB. Conclusion The epidemiological outcomes of EB in the Netherlands are high, attributed to a high detection rate in a well‐organized set‐up, indicating that EB might be more common than previously assumed. These epidemiological data help to understand the extensive need for (specialized) medical care of EB‐patients and is invaluable for the design and execution of therapeutic trials. This study emphasizes the importance of thorough reporting systems and registries worldwide.