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The pdx1−/− zebrafish mutant was recently established as a novel animal model of diabetic retinopathy. In this study, we investigate whether knockout of pdx1 also leads to diabetic kidney disease (DKD). pdx1−/− larvae exhibit several signs of early DKD, such as glomerular hypertrophy, impairments in the filtration barrier corresponding to microalbuminuria, and glomerular basement membrane (GBM) thickening. Adult pdx1−/− mutants show progressive GBM thickening in comparison with the larval state. Heterozygous pdx1 knockout also leads to glomerular hypertrophy as initial establishment of DKD similar to the pdx1−/− larvae. RNA sequencing of adult pdx1+/− kidneys uncovered regulations in multiple expected diabetic pathways related to podocyte disruption and hinting at early vascular dysregulation without obvious morphological alterations. Metabolome analysis and pharmacological intervention experiments revealed the contribution of phosphatidylethanolamine in the early establishment of kidney damage. In conclusion, this study identified the pdx1 mutant as a novel model for the study of DKD, showing signs of the early disease progression already in the larval stage and several selective features of later DKD in adult mutants.

Purpose Infantile Caffey disease is a rare disorder characterized by acute inflammation with subperiosteal new bone formation, associated with fever, pain, and swelling of the overlying soft tissue. Symptoms arise within the first weeks after birth and spontaneously resolve before the age of two years. Many, but not all, affected individuals carry the heterozygous pathogenic COL1A1 variant (c.3040C>T, p.(Arg1014Cys)). Methods We sequenced COL1A1 in 28 families with a suspicion of Caffey disease and performed ultrastructural, immunocytochemical, and biochemical collagen studies on patient skin biopsies. Results We identified the p.(Arg1014Cys) variant in 23 families and discovered a novel heterozygous pathogenic COL1A1 variant (c.2752C>T, p.(Arg918Cys)) in five. Both arginine to cysteine substitutions are located in the triple helical domain of the pro alpha 1(I) procollagen chain. Dermal fibroblasts (one patient with p.(Arg1014Cys) and one with p.(Arg918Cys)) produced molecules with disulfide-linked pro alpha 1(I) chains, which were secreted only with p.(Arg1014Cys). No intracellular accumulation of type I procollagen was detected. The dermis revealed mild ultrastructural abnormalities in collagen fibril diameter and packing. Conclusion The discovery of this novel pathogenic variant expands the limited spectrum of arginine to cysteine substitutions in type I procollagen. Furthermore, it confirms allelic heterogeneity in Caffey disease and impacts its molecular confirmation.

Glyoxalase 2 is the second enzyme of the glyoxalase system, catalyzing the detoxification of methylglyoxal to d-lactate via SD-Lactoylglutathione. Recent in vitro studies have suggested Glo2 as a regulator of glycolysis, but if Glo2 regulates glucose homeostasis and related organ specific functions in vivo has not yet been evaluated. Therefore, a CRISPR-Cas9 knockout of glo2 in zebrafish was created and analyzed. Consistent with its function in methylglyoxal detoxification, SD-Lactoylglutathione, but not methylglyoxal accumulated in glo2

Background: Although patients with multiple arterial dissections in distinct arterial regions rarely present with known connective tissue syndromes, we hypothesized that mild connective tissue abnormalities are common findings in these patients. Methods: From a consecutive register of 322 patients with cervical artery dissection (CeAD), we identified and analyzed 4 patients with a history of additional dissections in other vascular beds. In three patients, dermal connective tissue was examined by electron microscopy. DNA from all four patients was studied by whole-exome sequencing and copy number variation (CNV) analysis. Results: The collagen fibers of dermal biopsies were pathologic in all three analyzed patients. One patient carried a CNV disrupting the COL3A1 and COL5A2 genes (vascular or hypermobility type of Ehlers–Danlos syndrome), and another patient a CNV in MYH11 (familial thoracic aortic aneurysms and dissections). The third patient carried a missense substitution in COL5A2. Conclusion: Three patients showed morphologic alterations of the dermal connective tissue, and two patients carried pathogenic variants in genes associated with arterial connective tissue dysfunction. The findings suggest that genetic testing should be recommended after recurrent arterial dissections, independently of apparent phenotypical signs of connective tissue disorders.

Background/Aims: Extracellular vesicles (EVs), including microvesicles and exosomes, deliver bioactive cargo mediating intercellular communication in physiological and pathological conditions. EVs are increasingly investigated as therapeutic agents and targets, but also as disease biomarkers. However, a definite consensus regarding EV isolation methods is lacking, which makes it intricate to standardize research practices and eventually reach a desirable level of data comparability. In our study, we performed an inter-laboratory comparison of EV isolation based on a differential ultracentrifugation protocol carried out in 4 laboratories in 2 independent rounds of isolation. Methods: Conditioned medium of colorectal cancer cells was prepared and pooled by 1 person and distributed to each of the participating laboratories for isolation according to a pre-defined protocol. After EV isolation in each laboratory, quantification and characterization of isolated EVs was collectively done by 1 person having the highest expertise in the respective test method: Western blot, flow cytometry (fluorescence-activated cell sorting [FACS], nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). Results: EVs were visualized with TEM, presenting similar cup-shaped and spherical morphology and sizes ranging from 30 to 150 nm. NTA results showed similar size ranges of particles in both isolation rounds. EV preparations showed high purity by the expression of EV marker proteins CD9, CD63, CD81, Alix, and TSG101, and the lack of calnexin. FACS analysis of EVs revealed intense staining for CD63 and CD81 but lower levels for CD9 and TSG101. Preparations from 1 laboratory presented significantly lower particle numbers (p < 0.0001), most probably related to increased processing time. However, even when standardizing processing time, particle yields still differed significantly between groups, indicating inter-laboratory differences in the efficiency of EV isolation. Importantly, no relation was observed between centrifugation speed/k-factor and EV yield. Conclusions: Our findings demonstrate that quantitative differences in EV yield might be due to equipment- and operator-dependent technical variability in ultracentrifugation-based EV isolation. Furthermore, our study emphasizes the need to standardize technical parameters such as the exact run speed and k-factor in order to transfer protocols between different laboratories. This hints at substantial inter-laboratory biases that should be assessed in multi-centric studies.

The pdx1-/- zebrafish mutant was recently established as a novel animal model of diabetic retinopathy. Here, we investigate whether knockout of pdx1 also leads to diabetic kidney disease (DKD). pdx1-/- larvae exhibit several signs of early DKD such as glomerular hypertrophy, impairments in the filtration barrier corresponding to microalbuminuria and glomerular basement membrane (GBM) thickening. Adult pdx1-/- mutants show progressive GBM thickening in comparison to the larval state. Heterozygous pdx1 knockout also leads to glomerular hypertrophy as initial establishment of DKD similar to the pdx1-/- larvae. RNA sequencing (RNA-seq) of adult pdx1+/- kidneys uncovered regulations in multiple expected diabetic pathways related to podocyte disruption and hinting at early vascular dysregulation without obvious morphological alterations. Metabolome analysis and pharmacological intervention experiments revealed the contribution of phosphatidylethanolamine (PtdE) in the early establishment of kidney damage. In conclusion, this study identified the pdx1 mutant as a novel model for the study of DKD showing signs of the early disease progression already in larval stage and several selective features of later DKD in adult mutants.

Reactive carbonyl species (RCS) are spontaneously formed in the metabolism and modify and impair the function of DNA, proteins and lipids leading to several organ complications. In zebrafish, knockout of the RCS detoxifying enzymes glyoxalase 1 (Glo 1), aldehyde dehydrogenase 3a1 (Aldh3a1) and aldo-ketoreductase 1a1a (Akr1a1a) showed a signature of elevated RCS which specifically regulated glucose metabolism, hyperglycemia and diabetic organ damage. aldh2.1 was compensatory upregulated in glo1

Elevated levels of methylglyoxal (MG) and its associated post-translational modifications have been reported to be associated with progression and development of numerous pathological conditions. Despite such extensive evidence, it still remains unclear what the specific effects of MG are other than that induction of cytotoxicity. Here we evaluated the effects of MG in cardiac endothelial cells in vitro. We found that MG leads to a non-proliferative state and endothelial dysfunction, which is reversible as MG-H1, a major post-translational modification induced by MG, is turned over by lysosomal degradation. MG-induced cellular stunning/paralysis describes a new hallmark for cellular dysfunction which could lead to alterations in tissue homeostasis as well as cell-to-cell interactions, thereby contributing to the pathogenesis of diseases.

Pulmonary capillary microthrombosis has been proposed as a major pathogenetic factor driving severe COVID-19. Autopsy studies reported endothelialitis but it is under debate if it is caused by SARS-CoV-2 infection of endothelial cells. In this study, RNA in situ hybridization was used to detect viral RNA and to identify the infected cell types in lung tissue of 40 patients with fatal COVID-19. SARS-CoV-2 Spike protein-coding RNA showed a steadily decreasing signal abundance over a period of three weeks. Besides the original virus strain the variants of concern Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529) could also be detected by the assay. Viral RNA was mainly detected in alveolar macrophages and pulmonary epithelial cells, while only single virus-positive endothelial cells were observed even in cases with high viral load suggesting that viral infection of endothelial cells is not a key factor for the development of pulmonary capillary microthrombosis.

Increased acrolein (ACR), a toxic metabolite derived from energy consumption, is associated with diabetes and its complications. However, the molecular mechanisms are mostly unknown, and a suitable animal model with internal increased ACR does not exist for in vivo studying so far. Several enzyme systems are responsible for acrolein detoxification, such as Aldehyde Dehydrogenase (ALDH), Aldo‐Keto Reductase (AKR), and Glutathione S‐Transferase (GST). To evaluate the function of ACR in glucose homeostasis and diabetes, akr1a1a−/− zebrafish mutants are generated using CRISPR/Cas9 technology. Accumulated endogenous acrolein is confirmed in akr1a1a−/− larvae and livers of adults. Moreover, a series of experiments are performed regarding organic alterations, the glucose homeostasis, transcriptome, and metabolomics in Tg(fli1:EGFP) zebrafish. Akr1a1a−/− larvae display impaired glucose homeostasis and angiogenic retina hyaloid vasculature, which are caused by reduced acrolein detoxification ability and increased internal ACR concentration. The effects of acrolein on hyaloid vasculature can be reversed by acrolein‐scavenger l‐carnosine treatment. In adult akr1a1a−/− mutants, impaired glucose tolerance accompanied by angiogenic retina vessels and glomerular basement membrane thickening, consistent with an early pathological appearance in diabetic retinopathy and nephropathy, are observed. Thus, the data strongly suggest impaired ACR detoxification and elevated ACR concentration as biomarkers and inducers for diabetes and diabetic complications., Acrolein (ACR) plays a critical role in diabetes processing. This work generates an akr1a1a knockout zebrafish model and shows that the loss of Akr1a1a leads to an impaired detoxification and accumulation of ACR in vivo, illustrating that internally increased ACR causes altered glucose homeostasis, which finally induces pathological retinal angiogenesis and thickening of the glomerular basement membrane (GBM).

Heterozygous missense mutations in the human COL7A1 gene – coding for collagen VII – lead to the rare, dominantly inherited skin disorder dominant dystrophic epidermolysis bullosa (DDEB), which is characterised by skin fragility, blistering, scarring and nail dystrophy. To better understand the pathophysiology of DDEB and develop more effective treatments, suitable mouse models for DDEB are required but to date none have existed. We identified the two most common COL7A1 mutations in DDEB patients (p.G2034R and p.G2043R) and used CRISPR-Cas9 to introduce the corresponding mutations into mouse Col7a1 (p.G2028R and p.G2037R). Dominant inheritance of either of these two alleles results in a phenotype that closely resembles that seen in DDEB patients. Specifically, mice carrying these alleles show recurrent blistering that is first observed transiently around the mouth and paws in the early neonatal period and then again around the digits from 5-10 weeks of age. Histologically, the mice show micro-blistering and reduced collagen VII immunostaining. Biochemically, collagen VII from these mice displays reduced thermal stability, which we also observed to be the case for DDEB patients carrying the analogous mutations. Unlike previous rodent models of epidermolysis bullosa, which frequently show early lethality and severe disease, these mouse models, which to our knowledge are the first for DDEB, show no reduction in growth and survival, and – together with a relatively mild phenotype – represent a practically and ethically tractable tool for better understanding and treating epidermolysis bullosa. This article has an associated First Person interview with the first author of the paper., Summary: We developed mouse models for the blistering genetic skin disorder dominant dystrophic epidermolysis bullosa (DDEB) by introducing mutations into mouse Col7a1. These models should help to improve the understanding and treatment of DDEB.

Autosomal recessive congenital ichthyosis (ARCI) belongs to a heterogeneous group of disorders of keratinization. To date, 10 genes have been identified to be causative for ARCI. NIPAL4 (Nipa‐Like Domain‐Containing 4) is the second most commonly mutated gene in ARCI. In this study, we present a large cohort of 101 families affected with ARCI carrying mutations in NIPAL4. We identified 16 novel mutations and increase the total number of pathogenic mutations in NIPAL4 to 34. Ultrastructural analysis of biopsies from six patients showed morphological abnormalities consistent with an ARCI EM type III. One patient with a homozygous splice site mutation, which leads to a loss of NIPAL4 mRNA, showed additional ultrastructural aberrations together with a more severe clinical phenotype. Our study gives insights into the frequency of mutations, a potential hot spot for mutations, and genotype–phenotype correlations.

Summary Regulation of glucose homeostasis is a fundamental process to maintain blood glucose at a physiological level, and its dysregulation is associated with the development of several metabolic diseases. Here, we report on a zebrafish mutant for Aldo-keto-reductase 1a1b (akr1a1b) as a regulator of gluconeogenesis. Adult akr1a1b−/− mutant zebrafish developed fasting hypoglycemia, which was caused by inhibiting phosphoenolpyruvate carboxykinase (PEPCK) expression as rate-limiting enzyme of gluconeogenesis. Subsequently, glucogenic amino acid glutamate as substrate for gluconeogenesis accumulated in the kidneys, but not in livers, and induced structural and functional pronephros alterations in 48-hpf akr1a1b−/− embryos. Akr1a1b−/− mutants displayed increased nitrosative stress as indicated by increased nitrotyrosine, and increased protein-S-nitrosylation. Inhibition of nitrosative stress using the NO synthase inhibitor L-NAME prevented kidney damage and normalized PEPCK expression in akr1a1b−/− mutants. Thus, the data have identified Akr1a1b as a regulator of gluconeogenesis in zebrafish and thereby controlling glucose homeostasis., Graphical Abstract, Highlights • Adult akr1a1b−/− mutant zebrafish develop fasting hypoglycemia • Loss of Akr1a1b inhibits renal phosphoenolpyruvate carboxykinase (PEPCK) expression • Accumulation of glucogenic amino acid glutamate alters the kidney in akr1a1b mutants • Akr1a1b regulates gluconeogenesis via protein-S-nitrosylation, Human Metabolism; Molecular Genetics

Chromosome 8p is frequently deleted in various cancer entities and has been shown to correlate with poor patient survival. SH2D4A is located on chromosome 8p and prevents the nuclear translocation of the pro-tumorigenic transcription factor STAT3. Here, we investigated the interaction of SH2D4A and STAT3 to shed light on the non-canonical functions of STAT3 in cooperation with the tumor suppressor SH2D4A. Using an immunoprecipitation-mass spectrometry (IP-MS) approach, we identified the mitochondrial scaffold proteins prohibitin 1 (PHB1) and prohibitin 2 (PHB2) among other proteins to potentially bind to SH2D4A. Co-immunoprecipitation and proximity ligation assays confirmed direct interactions of STAT3, PHB1, and SH2D4A in situ and in vitro. In addition, cell fractionation and immunofluorescence staining revealed co-localization of these proteins with mitochondria. These interactions were selectively interrupted by the small molecule and PHB ligand FL3. Furthermore, FL3 led to a reduction of STAT3 protein levels, STAT3 transcriptional activity, and HIF1α protein stabilization upon dimethyloxalylglycine (DMOG) treatment. Besides, mitochondrial fusion and fission markers, L-OPA1, Mfn1, and FIS1, were dysregulated upon FL3 treatment. This dysregulated morphology was accompanied by significant reduction of mitochondrial respiration, thus, FL3 significantly diminished mitochondrial respirational capacity. In contrast, SH2D4A knockout increased mitochondrial respiration, whereas FL3 reversed the effect of SH2D4A knockout. The here described results indicate that the interaction of SH2D4A and PHB1 is involved in the mitochondrial function and integrity. The demonstrated interaction with STAT3, accompanied by its reduction of transcriptional activity, further suggests that SH2D4A is linking STAT3 to its mitochondrial functions, and inhibition of PHB-interaction may have therapeutic effects in tumor cells with STAT3 activation. journal article research support, non-u.s. gov't 2020 11 30 2020 11 30 imported

In light of the limited treatment options of diabetic polyneuropathy (DPN) available, suitable animal models are essential to investigate pathophysiological mechanisms and to identify potential therapeutic targets. In vivo evaluation with current techniques, however, often provides only restricted information about disease evolution. In the study of patients with DPN, magnetic resonance neurography (MRN) has been introduced as an innovative diagnostic tool detecting characteristic lesions within peripheral nerves. We developed a novel multicontrast ultra high field MRN strategy to examine major peripheral nerve segments in diabetic mice non-invasively. It was first validated in a cross-platform approach on human nerve tissue and then applied to the popular streptozotocin(STZ)-induced mouse model of DPN. In the absence of gross morphologic alterations, a distinct MR-signature within the sciatic nerve was observed mirroring subtle changes of the nerves’ fibre composition and ultrastructure, potentially indicating early re-arrangements of DPN. Interestingly, these signal alterations differed from previously reported typical nerve lesions of patients with DPN. The capacity of our approach to non-invasively assess sciatic nerve tissue structure and function within a given mouse model provides a powerful tool for direct translational comparison to human disease hallmarks not only in diabetes but also in other peripheral neuropathic conditions.

Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of rare disorders of keratinization characterized by generalized abnormal scaling of the skin. Ten genes are currently known to be associated with ARCI: TGM1, ALOXE3, ALOX12B, NIPAL4 (ICHTHYIN), ABCA12, CYP4F22, PNPLA1, CERS3, SDR9C7, and SULT2B1. Over a period of 22 years, we have studied a large patient cohort from 770 families with a clinical diagnosis of ARCI. Since the first report that mutations in the gene CYP4F22 are causative for ARCI in 2006, we have identified 54 families with pathogenic mutations in CYP4F22 including 23 previously unreported mutations. In this report, we provide an up-to-date overview of all published and novel CYP4F22 mutations and point out possible mutation hot spots. We discuss the molecular and clinical findings, the genotype-phenotype correlations and consequences on genetic testing.

Dystrophic epidermolysis bullosa (DEB) is a blistering skin disease caused by mutations in the gene COL7A1 encoding collagen VII. DEB can be inherited as recessive DEB (RDEB) or dominant DEB (DDEB) and is associated with a high wound burden. Perpetual cycles of wounding and healing drive fibrosis in DDEB and RDEB, as well as the formation of a tumor-permissive microenvironment. Prolonging wound-free episodes by improving the quality of wound healing would therefore confer substantial benefit for individuals with DEB. The collagenous domain of collagen VII is encoded by 82 in-frame exons, which makes splice-modulation therapies attractive for DEB. Indeed, antisense oligonucleotide–based exon skipping has shown promise for RDEB. However, the suitability of antisense oligonucleotides for treatment of DDEB remains unexplored. Here, we developed QR-313, a clinically applicable, potent antisense oligonucleotide specifically targeting exon 73. We show the feasibility of topical delivery of QR-313 in a carbomer-composed gel for treatment of wounds to restore collagen VII abundance in human RDEB skin. Our data reveal that QR-313 also shows direct benefit for DDEB caused by exon 73 mutations. Thus, the same topically applied therapeutic could be used to improve the wound healing quality in RDEB and DDEB.

The association of junctional epidermolysis bullosa with pyloric atresia (JEB-PA) and aplasia cutis congenita (ACC) was described by El Shafie et al. (J Pediatr Surg 14(4):446–449, 1979) and Carmi et al. (Am J Med Genet 11:319–328, 1982). Most patients die in the first weeks of life, and no curative treatment options are available so far. We describe a patient with JEB-PA and ACC (OMIM # 226730) who was treated for extensive areas of ACC by Integra®-Dermal Regeneration Template and split-thickness skin grafting (STSG). Clinically, the dermal template changed into well-vascularized neodermis, and after STSG, full take of the transplants was detected. No infections of the huge ACC areas were seen. Further studies must validate this treatment option in severe and acute cases of JEB-PA with ACC. Based on clinical findings, we postulate that placement of Integra®-Dermal Regeneration Template with STSG could be a new treatment option for patients having JEB-PA with ACC to prevent severe infection, compartment-syndrome-like conditions, and deformities. Based on literature findings, we assume that Integra®-Dermal Regeneration Template with STSG could even be able to prevent new blistering and thereby be a treatment option in cases of ACC and JEB.

Background Autosomal recessive congenital ichthyosis (ARCI) is a genetically heterogeneous group of rare Mendelian skin disorders characterized by cornification and differentiation defects of keratinocytes. Mutations in nine genes including PNPLA1 are known to cause non-syndromic forms of ARCI. To date, only ten distinct pathogenic mutations in PNPLA1 have been reported. Objectives To identify new causative PNPLA1 mutations, we screened genetically unresolved cases including our ARCI collection comprising more than 700 families. Here, we report on 16 novel mutations present in patients from 17 families. Methods The screening for mutations was performed either by direct Sanger sequencing or in combination with a multi gene panel, followed by sequence and mutation analysis. Results While all previously reported mutations and most of our novel mutations are located within the core patatin domain, here we report on five novel PNPLA1 mutations, which are downstream of this domain. Thus, as recently described for PNPLA2, we hypothesize that a region larger than core domain is required for full enzymatic activity of PNPLA1 in human skin barrier formation. Conclusions We estimate the frequency of PNPLA1 mutations amongst ARCI patients to around 3%. Most of our patients were born as collodion babies and showed a relatively mild ichthyosis phenotype. In four unrelated patients we observed a cyclic scaling course, which seems to be a potential phenotype variation in a small percentage of patients with PNPLA1 mutations. The variability of the clinical manifestations as well as the lack of typical clinical features are specific for patients with PNPLA1 mutations, and emphasize the importance of DNA sequencing for differential diagnosis of ARCIs. This article is protected by copyright. All rights reserved.

X-linked ichthyosis (XLI) is a keratinization disorder caused by deficient activity of steroidsulfatase. In contrast, ichthyosis vulgaris is due to semidominant mutations of the filaggrin gene ( FLG ). In view of phenotypic variations of these ichthyoses we speculated that XLI may be influenced by additional FLG mutations in a significant number of patients. We characterized a group of 51 patients with XLI and systematically analyzed them for additional FLG mutations (R501X, 2282del4, R2447X, S3247X). The study was complemented by morphological analyses. Full FLG sequencing for rare mutations was performed in special cases. Interestingly, prevalence of FLG mutations was significantly increased compared to a population-based control cohort of 1,377 individuals (17.6% vs. 8.4%, p=0.038). Palmoplantar hyperlinearity was significantly associated with the FLG mutation status. Ichthyosis severity score seemed to be increased in XLI with FLG mutations, but the difference was not significant (p=0.124). To our surprise, percentages of atopic manifestations were highly prevalent in both subgroups, 40% and 33% in XLI without and with filaggrin deficiency, respectively. Of note, reduction of filaggrin staining or keratohyalin could not be explained by FLG mutations in all patients. However, we conclude that FLG mutations represent a significant genetic modifier of XLI. [196 words]

In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inactivating frameshift mutations of transforming growth factor β receptor type 2 (TGFBR2). TGFBR2 deficiency is considered to drive MSI tumor progression by abrogating downstream TGF‑β signaling. This pathway can alter the expression of coding and non‑coding RNAs, including microRNAs (miRNAs), which are also present in extracellular vesicles (EVs) as post‑transcriptional modulators of gene expression. In our previous study, it was shown that TGFBR2 deficiency alters the protein composition and function of EVs in MSI tumors. To investigate whether mutant TGFBR2 may also affect the miRNA cargo of EVs, the present study characterized miRNAs in EVs and their parental MSI tumor cells that differed only in TGFBR2 expression status. The HCT116‑TGFBR2 MSI cell line model enables the doxycycline (dox)‑inducible reconstituted expression of TGFBR2 in an isogenic background (‑dox, TGFBR2 deficient; +dox, TGFBR2 proficient). Small RNA sequencing of cellular and EV miRNAs showed that the majority of the miRNAs (263/471; 56%) were shared between MSI tumor cells and their EVs. Exploratory data analysis revealed the TGBFR2‑dependent cluster separation of miRNA profiles in EVs and MSI tumor cells. This segregation appeared to result from two subsets of miRNAs, the expression of which were regulated in a TGFBR2‑dependent manner (EVs: n=10; MSI cells: n=15). In the EV subset, 7/10 miRNAs were downregulated and 3/10 were upregulated by TGFBR2 deficiency. In the cellular subset, 13/15 miRNAs were downregulated and 2/15 miRNAs were upregulated in the TGFBR2‑deficient cells. The present study emphasizes the general overlap of miRNA profiles in MSI tumor cells and their EVs, but also highlights the impact of a single tumor driver mutation on the expression of individual miRNAs, as exemplified by the downregulation of miR‑381‑3p in TGFBR2‑deficient MSI tumor cells and their secreted EVs.

Kyphoscoliotic Ehlers&ndash, Danlos Syndrome (kEDS) is a rare genetic heterogeneous disease clinically characterized by congenital muscle hypotonia, kyphoscoliosis, and joint hypermobility. kEDS is caused by biallelic pathogenic variants in either PLOD1 or FKBP14. PLOD1 encodes the lysyl hydroxylase 1 enzyme responsible for hydroxylating lysyl residues in the collagen helix, which undergo glycosylation and form crosslinks in the extracellular matrix thus contributing to collagen fibril strength. FKBP14 encodes a peptidyl-prolyl cis&ndash, trans isomerase that catalyzes collagen folding and acts as a chaperone for types III, VI, and X collagen. Despite genetic heterogeneity, affected patients with mutations in either PLOD1 or FKBP14 are clinically indistinguishable. We aim to better understand the pathomechanism of kEDS to characterize distinguishing and overlapping molecular features underlying PLOD1-kEDS and FKBP14-kEDS, and to identify novel molecular targets that may expand treatment strategies. Transcriptome profiling by RNA sequencing of patient-derived skin fibroblasts revealed differential expression of genes encoding extracellular matrix components that are unique between PLOD1-kEDS and FKBP14-kEDS. Furthermore, we identified genes involved in inner ear development, vascular remodeling, endoplasmic reticulum (ER) stress, and protein trafficking that were differentially expressed in patient fibroblasts compared to controls. Overall, our study presents the first transcriptomics data in kEDS revealing distinct molecular features between PLOD1-kEDS and FKBP14-kEDS, and serves as a tool to better understand the disease.

Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations in COL7A1 causing recessive dystrophic epidermolysis bullosa are amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeted deletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved very efficient targeted deletion of COL7A1 exon 80 in recessive dystrophic epidermolysis bullosa (RDEB) patient keratinocytes carrying a highly prevalent frameshift mutation. This ex vivo non-viral approach rendered a large proportion of corrected cells producing a functional collagen VII variant. The effective targeting of the epidermal stem cell population enabled long-term regeneration of a properly adhesive skin upon grafting onto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sites did not reveal any unintended nuclease activity. Our strategy could potentially be extended to a large number of COL7A1 mutation-bearing exons within the long collagenous domain of this gene, opening the way to precision medicine for RDEB., Highly efficient and safe approaches for precise correction of pathogenic mutations are required to realize the full potential of gene-editing protocols for clinical practice. Here we describe a single-step NHEJ-based CRISPR/Cas9 strategy of COL7A1 mutation-bearing exon deletion with unprecedented efficacy for ex vivo correction of recessive dystrophic epidermolysis bullosa.

Individuals affected with autosomal recessive cutis laxa type 2B and 3 usually show translucent skin with visible veins and abnormal elastic fibers, intrauterine and/or postnatal growth restriction and a typical triangular facial gestalt. Here we describe three unrelated individuals in whom such a cutis laxa syndrome was suspected, especially after electron microscopy revealed immature and less dense dermal elastic fibers in one of them. However, one of these children also displayed optic atrophy and two hypogammaglobulinemia. All had elevated liver enzymes and acute liver failure during febrile episodes leading to early demise in two of them. The only surviving patient had been treated with immunoglobulins. Through exome sequencing we identified mutations in NBAS, coding for a protein involved in Golgi-to-ER transport. NBAS deficiency causes several rare conditions ranging from isolated recurrent acute liver failure to a multisystem disorder mainly characterized by short stature, optic nerve atrophy and Pelger-Huet anomaly (SOPH). Since we subsequently verified Pelger-Huet anomaly in two of the patients the diagnosis SOPH syndrome was unequivocally proven. Our data show that SOPH syndrome can be regarded as a differential diagnosis for the progeroid forms of cutis laxa in early infancy and that possibly treatment of the hypogammaglobulinemia can be of high relevance for the prognosis.

Data on vitamin D status of patients with inherited ichthyosis in Europe is scarce and unspecific concerning the genetic subtype. This study determined serum levels of 25-hydroxyvitamin D3 (25(OH)D3) in 87 patients with ichthyosis; 69 patients were additionally analysed for parathyroid hormone. Vitamin D deficiency was pronounced in keratinopathic ichthyosis (n = 17; median 25(OH)D3: 10.5 ng/ml), harlequin ichthyosis (n = 2;7.0 ng/ml) and rare syndromic subtypes (n = 3; 7.0 ng/ml). Vitamin D levels were reduced in TG1-proficient lamellar ichthyosis (n = 15; 8.9 ng/ml), TG1-deficient lamellar ichthyosis (n = 12; 11.7 ng/ml), congenital ichthyosiform erythroderma (n = 13; 12.4 ng/ml), Netherton syndrome (n = 7; 10.7 ng/ml) and X-linked ichthyosis (n = 8; 13.9 ng/ml). In ichthyosis vulgaris 25(OH)D3 levels were higher (n = 10; 19.7 ng/ml). Parathyroid hormone was elevated in 12 patients. Low 25(OH)D3 levels were associated with high severity of scaling (p = 0.03) implicating scaling as a risk factor for vitamin D deficiency. Thus, this study supports our recent guidelines for ichthyoses, which recommend screening for and substituting of vitamin D deficiency., This study was supported by the Selbsthilfe Ichthyose e.V., the Medical Faculty of the University of Münster (OJI120817 & OJ111409) and of the German Research Foundation (DFG OJ 53/3-1). It is part of the medical thesis of Mi-Ran Kim. The work was supported by the Selbsthilfe Ichthyose e.V., the Medical Faculty of the University of Muenster (OJI120817 & OJ111409) and by the German Research Foundation (DFG OJ 53/3-1). Furthermore, we cooperated with members of the European Reference Network (ERN, subgroup ERN-skin). The cohort of this paper was also part of a project funded by the programme “Innovative Medizinische Forschung” (IMF); project number: SÜ212007.