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The Ehlers-Danlos Syndromes (EDS) are a heterogenous group of heritable connective tissue disorders, characterised by joint hypermobility, skin hyperextensibility and generalised tissue fragility. In all types of EDS skin wound healing is impaired to a variable degree. Additional support through wound management plans may help to improve these outcomes, however, there is paucity of evidence regarding clinical management of skin fragility and wounds in EDS. This paper aims to review current evidence and provide recommendations for management of skin wounds in EDS types. Preventative measures to avoid skin injury are strongly recommended, including avoidance of high impact sport and use of appropriate protection such as shin guards. Bruising is common and some types of EDS are associated with haematoma formation with management including compression bandages and consideration of pharmacological therapy. Skin fragility and tears should be managed with a focus on protection of remaining tissue, avoidance of wound tension and low adherence dressings to avoid further injury. This paper provides clear recommendations to address skin management for this group of patients. It highlights the lack of good quality published data to support treatment decisions.
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The identification of monogenic causes for cornification disorders has enhanced our understanding of epidermal differentiation and skin barrier function. Autosomal dominant lamellar ichthyosis is a rare condition, and ASPRV1 was the only gene linked to autosomal dominant lamellar ichthyosis to date. We identified a heterozygous variant (ENST00000686631.1:c.1372G>T, p.[Val458Phe]) in the NKPD1 gene in 7 individuals from a 4-generation German pedigree with generalized lamellar ichthyosis by whole-exome sequencing. Segregation analysis confirmed its presence in affected individuals, resulting in a logarithm of the odds score of 3.31. NKPD1 encodes the NKPD1 protein, implicated in the plasma membrane; its role in human disease is as yet unknown. Skin histology showed moderate acanthosis and compact orthohyperkeratosis, and the ultrastructure differed clearly from that in ASPRV1-autosomal dominant lamellar ichthyosis. Although NKPD1 mRNA expression increased during keratinocyte differentiation, stratum corneum ceramides exhibited no significant changes. However, affected individuals showed an elevated ratio of protein-bound ceramides to omega-esterified ceramides. This highlights NKPD1's role in autosomal dominant lamellar ichthyosis, impacting ceramide metabolism and skin lipid barrier formation, as demonstrated through functional characterization.
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Omega-6 fatty acids are the primary polyunsaturated fatty acids in most Western diets, while their role in diabetes remains controversial. Exposure of omega-6 fatty acids to an oxidative environment results in the generation of a highly reactive carbonyl species known as trans, trans-2,4-decadienal (tt-DDE). The timely and efficient detoxification of this metabolite, which has actions comparable to other reactive carbonyl species, such as 4-hydroxynonenal, acrolein, acetaldehyde, and methylglyoxal, is essential for disease prevention. However, the detoxification mechanism for tt-DDE remains elusive. In this study, the enzyme Aldh9a1b is identified as having a key role in the detoxification of tt-DDE. Loss of Aldh9a1b increased tt-DDE levels and resulted in an abnormal retinal vasculature and glucose intolerance in aldh9a1b-/- zebrafish. Transcriptomic and metabolomic analyses revealed that tt-DDE and aldh9a1b deficiency in larval and adult zebrafish induced insulin resistance and impaired glucose homeostasis. Moreover, alterations in hyaloid vasculature is induced by aldh9a1b knockout or by tt-DDE treatment can be rescued by the insulin receptor sensitizers metformin and rosiglitazone. Collectively, these results demonstrated that tt-DDE is the substrate of Aldh9a1b which causes microvascular damage and impaired glucose metabolism through insulin resistance.
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Aldehídos , Resistencia a la Insulina , Insulina , Animales , Pez Cebra , Gluconeogénesis , Ácidos Grasos Omega-6RESUMEN
Collagen VII forms anchoring fibrils that are essential for the stability of the skin and other epithelial organs. In addition to such structural functions, it is emerging that collagen VII fills instructive functions. Collagen VII is synthesized by both epithelial cells and fibroblasts. Genetic loss of collagen VII causes dystrophic epidermolysis bullosa, which manifests with chronic skin fragility and fibrosis. Significant progress has been made in developing therapies for dystrophic epidermolysis bullosa; however, such work has also raised questions on the importance of the cellular source of collagen VII for maintenance of tissue integrity and homeostasis. Toward this end, we engineered mice that kept the physiological expression of collagen VII only in epithelial cells or in fibroblasts. Our study revealed that production of collagen VII either by keratinocytes or fibroblasts alone is sufficient for creation of mechanically robust skin. Importantly, we also show tissue-diverse dependence on epithelial and mesenchymal production of collagen VII and provide support for limited amounts of collagen VII being sufficient for tissue protection. Furthermore, a disconnect between collagen VII abundance and anchoring fibril numbers supports the concept that restoration of fully physiological collagen VII levels may not be needed to achieve complete mechanical protection of dystrophic epidermolysis bullosa skin.
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Introduction: The Ehlers-Danlos syndromes (EDS) comprise a group of inherited connective tissue disorders presenting with variable fragility to skin, soft tissue, and certain internal organs, which can cause significant complications, particularly arterial rupture, bowel perforation and joint difficulties. Currently, there are 14 proposed subtypes of EDS, with all except one subtype (hypermobile EDS) having an identified genetic etiology. An understanding of the extracutaneous features and complications within each subtype is key to maximizing clinical care and reducing the risk of further complications. Methods: A systematic review of EDS-related extracutaneous features and complications was undertaken. Results: We identified 839 EDS cases that met the inclusion criteria. We noted a high prevalence of joint hypermobility amongst kyphoscoliotic (39/39, 100%), spondylodysplastic (24/25, 96.0%), and hypermobile (153/160, 95.6%) EDS subtypes. The most common musculoskeletal complications were decreased bone density (39/43, 90.7%), joint pain (217/270, 80.4%), and hypotonia/weakness (79/140, 56.4%). Vascular EDS presented with cerebrovascular events (25/153, 16.3%), aneurysm (77/245, 31.4%), arterial dissection/rupture (89/250, 35.5%), and pneumothorax/hemothorax. Chronic pain was the most common miscellaneous complication, disproportionately affecting hypermobile EDS patients (139/157, 88.5%). Hypermobile EDS cases also presented with chronic fatigue (61/63, 96.8%) and gastrointestinal complications (57/63, 90.5%). Neuropsychiatric complications were noted in almost all subtypes. Discussion: Understanding the extracutaneous features and complications of each EDS subtype may help diagnose and treat EDS prior to the development of substantial comorbidities and/or additional complications. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022308151, identifier CRD42022308151.
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BACKGROUND: The Ehlers-Danlos syndromes (EDSs) comprise a group of connective tissue disorders that manifest with skin hyperextensibility, easy bruising, joint hypermobility and fragility of skin, soft tissues, and some organs. A correct assessment of cutaneous features along with the use of adjunct technologies can improve diagnostic accuracy. OBJECTIVES: To systematically review the cutaneous features and adjunct investigations of EDS. METHODS: A search of PubMed and Web of Science for EDS-related cutaneous features and additional investigations was undertaken from publication of the 2017 International Classification of EDS until January 15, 2022. RESULTS: One-hundred-and-forty studies involved 839 patients with EDS. The EDS female-to-male ratio was 1.36:1 (P < .001). A high prevalence of skin hyperextensibility, bruising, and soft skin were noted. Most patients with vascular Ehlers-Danlos syndrome showed venous visibility, skin fragility, and acrogeria. Classical EDS showed subcutaneous spheroids and molluscoid pseudotumours. In patients that underwent skin biopsies, only 30.3% and 71.4% showed features suggestive of EDS using light microscopy and transmission electron microscopy, respectively. LIMITATIONS: Retrospective study and small cases numbers for some EDS-subtypes. CONCLUSIONS: An accurate clinical diagnosis increases the chances of a molecular diagnosis, particularly for rarer EDS subtypes, whilst decreasing the need for genetic testing where there is a low clinical suspicion for a monogenic EDS-subtype.
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Enfermedades del Tejido Conjuntivo , Síndrome de Ehlers-Danlos , Humanos , Femenino , Masculino , Estudios Retrospectivos , Síndrome de Ehlers-Danlos/diagnóstico , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/patologíaRESUMEN
The Ehlers-Danlos syndromes (EDS) comprise a group of inherited connective tissue disorders presenting with features of skin hyperextensibility, joint hypermobility, abnormal scarring and fragility of skin, blood vessels and some organs. The disease is generally diagnosed through the cluster of clinical features, though the addition of genetic analysis is the gold standard for diagnosis of most subtypes. All subtypes display skin manifestations, which are essential to the accurate clinical diagnosis of the condition. Furthermore, cutaneous features can be the first and/or only presenting feature in some cases of EDS and thus understanding these signs is vital for diagnosis. This review focuses on particular cutaneous features of each EDS subtype and their clinical importance. Provision of a specific diagnosis is important for management, prognosis and genetic counselling, often for family members beyond the individual.
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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-/- larvae, without altering the glutathione metabolism or affecting longevity. Adult glo2-/- livers displayed a reduced hexose concentration and a reduced postprandial P70-S6 kinase activation, but upstream postprandial AKT phosphorylation remained unchanged. In contrast, glo2-/- skeletal muscle remained metabolically intact, possibly compensating for the dysfunctional liver through increased glucose uptake and glycolytic activity. glo2-/- zebrafish maintained euglycemia and showed no damage of the retinal vasculature, kidney, liver and skeletal muscle. In conclusion, the data identified Glo2 as a regulator of cellular energy metabolism in liver and skeletal muscle, but the redox state and reactive metabolite accumulation were not affected by the loss of Glo2.
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Lactoilglutatión Liasa , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/metabolismo , Lactoilglutatión Liasa/genética , Lactoilglutatión Liasa/metabolismo , Piruvaldehído/metabolismo , Ácido Láctico , Glucosa , Tioléster Hidrolasas/metabolismoRESUMEN
The ELMO protein family consists of the homologues ELMO1, ELMO2 and ELMO3. Several studies have shown that the individual ELMO proteins are involved in a variety of cellular and developmental processes. However, it has poorly been understood whether the Elmo proteins show similar functions and act redundantly. To address this question, elmo1 -/- , elmo2 -/- and elmo3 -/- zebrafish were generated and a comprehensive comparison of the phenotypic changes in organ morphology, transcriptome and metabolome was performed in these mutants. The results showed decreased fasting and increased postprandial blood glucose levels in adult elmo1 -/- , as well as a decreased vascular formation in the adult retina in elmo1 -/- , but an increased vascular formation in the adult elmo3 -/- retina. The phenotypical comparison provided few similarities, as increased Bowman space areas in adult elmo1 -/- and elmo2 -/- kidneys, an increased hyaloid vessel diameter in elmo1 -/- and elmo3 -/- and a transcriptional downregulation of the vascular development in elmo1 -/- , elmo2 -/- , and elmo3 -/- zebrafish larvae. Besides this, elmo1 -/- , elmo2 -/- , and elmo3 -/- zebrafish exhibited several distinct changes in the vascular and glomerular structure and in the metabolome and the transcriptome. Especially, elmo3 -/- zebrafish showed extensive differences in the larval transcriptome and an impaired survivability. Together, the data demonstrated that the three zebrafish Elmo proteins regulate not only similar but also divergent biological processes and mechanisms and show a low functional redundancy.
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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.
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COVID-19 , Trombosis , Células Endoteliales/metabolismo , Humanos , Pulmón/patología , ARN Viral , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Trombosis/patología , TropismoRESUMEN
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.
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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-/- animals and therefore this study aimed to investigate the detoxification ability for RCS by Aldh2.1 in zebrafish independent of ethanol exposure. aldh2.1 knockout zebrafish were generated using CRISPR/Cas9 and subsequently analyzed on a histological, metabolomic and transcriptomic level. aldh2.1-/- zebrafish displayed increased endogenous acetaldehyde (AA) inducing an increased angiogenesis in retinal vasculature. Expression and pharmacological interventional studies identified an imbalance of c-Jun N-terminal kinase (JNK) and p38 MAPK induced by AA, which mediate an activation of angiogenesis. Moreover, increased AA in aldh2.1-/- zebrafish did not induce hyperglycemia, instead AA inhibited the expression of glucokinase (gck) and glucose-6-phosphatase (g6pc), which led to an impaired glucose metabolism. In conclusion, the data have identified AA as the preferred substrate for Aldh2.1's detoxification ability, which subsequently causes microvascular organ damage and impaired glucose metabolism.
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Acetaldehído , Neovascularización Retiniana , Pez Cebra , Acetaldehído/metabolismo , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa Mitocondrial/genética , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Animales , Glucosa/metabolismo , Vasos Retinianos , Pez Cebra/metabolismoRESUMEN
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.
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Diabetes Mellitus , Nefropatías Diabéticas , Podocitos , Animales , Diabetes Mellitus/metabolismo , Nefropatías Diabéticas/metabolismo , Femenino , Membrana Basal Glomerular , Humanos , Hipertrofia/metabolismo , Masculino , Fenotipo , Fosfatidiletanolaminas , Podocitos/metabolismo , Pez CebraRESUMEN
Neonatal collodion baby or ichthyosis can pose a diagnostic challenge, and in many cases, only additional organ involvement or the course of the disease will help differentiate between non-syndromic and syndromic forms. Skin abnormalities are described in about 20% of the congenital disorders of glycosylation (CDG). Among those, some rare CDG forms constitute a special group among the syndromic ichthyoses and can initially misdirect the diagnosis towards non-syndromic genodermatosis. DOLK-CDG is such a rare subtype, resulting from a defect in dolichol kinase, in which the congenital skin phenotype (often ichthyosis) is later associated with variable extracutaneous features such as dilatative cardiomyopathy, epilepsy, microcephaly, visual impairment, and hypoglycemia and may lead to a fatal course. We report two neonatal cases of lethal ichthyosis from the same family, with distal digital constrictions and a progressive course leading to multi-organ failure and death. Postmortem trio whole-exome sequencing revealed the compound heterozygous variants NM_014908.3: c.1342G>A, p.(Gly448Arg) and NM_014908.3: c.1558A>G, p.(Thr520Ala) in the DOLK gene in the first affected child, which were confirmed in the affected sibling. Reduced staining with anti-α-Dystroglycan antibody was observed in frozen heart tissue of the second child as an expression of reduced O-mannosylation due to the dolichol kinase deficiency. In addition to the detailed dermatopathological changes, both cases presented hepatic and extrahepatic hemosiderosis on histological examination. Our patients represent an early and fatal form of DOLK-CDG with a striking presentation at birth resembling severe collodion baby. Both cases emphasize the phenotypic variability of glycosylation disorders and the importance to broaden the differential diagnosis of ichthyosis and to actively search for organ involvement in neonates with ichthyosis.
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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.
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Ictiosis Lamelar , Ictiosis , Deficiencia de Vitamina D , Humanos , Ictiosis/diagnóstico , Ictiosis/genética , Ictiosis Lamelar/diagnóstico , Ictiosis Lamelar/genética , Hormona Paratiroidea , Vitamina D , Deficiencia de Vitamina D/diagnóstico , Deficiencia de Vitamina D/epidemiología , Deficiencia de Vitamina D/genéticaRESUMEN
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.
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Acroleína/metabolismo , Diabetes Mellitus Experimental/metabolismo , Glucosa/metabolismo , Hígado/metabolismo , Receptor de Insulina/metabolismo , Animales , Modelos Animales de Enfermedad , Homeostasis , Larva/metabolismo , Metabolómica/métodos , Transducción de Señal , Transcriptoma , Pez Cebra/metabolismoRESUMEN
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 proα1(I) procollagen chain. Dermal fibroblasts (one patient with p.(Arg1014Cys) and one with p.(Arg918Cys)) produced molecules with disulfide-linked proα1(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.
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Cadena alfa 1 del Colágeno Tipo I/genética , Cisteína , Hiperostosis Cortical Congénita , Arginina/genética , Preescolar , Colágeno Tipo I , Cisteína/genética , Humanos , Mutación , Procolágeno/genéticaRESUMEN
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 5-10â 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.
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Epidermólisis Ampollosa Distrófica/genética , Mutación Puntual , Animales , Sistemas CRISPR-Cas , Colágeno Tipo VII/genética , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
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.