The Epidermal Transcriptome Analysis of a Novel c.639_642dup LORICRIN Variant-Delineation of the Loricrin Keratoderma Pathology.

Loricrin keratoderma (LK) is a rare autosomal dominant genodermatosis caused by LORICRIN gene mutations. The pathogenesis of the disease is not yet fully understood. So far, only 10 pathogenic variants in LORICRIN have been described, with all of them but one being deletions or insertions. The significance of rare nonsense variants remains unclear. Furthermore, no data regarding the RNA expression in affected patients are available. The aim of this study is to describe the two variants in the LORICRIN gene found in two distinct families: the novel pathogenic variant c.639_642dup and a rare c.10C > T (p.Gln4Ter) of unknown significance. We also present the results of the transcriptome analysis of the lesional loricrin keratoderma epidermis of a patient with c.639_642dup. We show that in the LK lesion, the genes associated with epidermis development and keratocyte differentiation are upregulated, while genes engaged in cell adhesion, differentiation developmental processes, ion homeostasis and transport, signaling and cell communication are downregulated. In the context of the p.Gln4Ter clinical significance evaluation, we provide data indicating that LORICRIN haploinsufficiency has no skin consequences. Our results give further insight into the pathogenesis of LK, which may have therapeutic implications in the future and important significance in the context of genetic counseling.

Identification of the novel SDR42E1 gene that affects steroid biosynthesis associated with the oculocutaneous genital syndrome.

Hereditary connective tissue diseases form a heterogeneous group of disorders that affect collagen and extracellular matrix components. The cornea and the skin are among the major forms of connective tissues, and syndromes affecting both organs are often due to mutations in single genes. Brittle cornea syndrome is one of the pathologies that illustrates this association well. Furthermore, sex hormones are known to play a role in the maintenance of the structure and the integrity of the connective tissue including the skin and cornea, and may be involved in pathogenesis of oculocutaneous diseases. Herein, a double consanguineous family of Moroccan origin with two affected siblings, with suspected brittle cornea syndrome, was recruited. Ophthalmic examinations and genetic testing were performed in all the nuclear family individuals. Clinical examinations showed that the two affected boys presented with thinning of the cornea, blue sclera, keratoconus, hyperelasticity of the skin, joint hypermobility, muscle weakness, hearing loss and dental abnormalities that are compatible with the diagnosis of BCS disease. They showed however additional clinical signs including micropenis, hypospadias and cryptorchidism, suggesting abnormalities in endocrine pathways. Using a duo exome sequencing analysis performed in the mother and the propositus, we identified the novel homozygous missense mutation c.461G > A (p.Arg154Gln) in the short-chain dehydrogenase/reductase family 42E member 1 (SDR42E1) gene. This novel mutation, which co-segregated with the disease in the family, was predicted to be pathogenic by bioinformatics tools. SDR42E1 stability analysis using DynaMut web-server showed that the p.Arg154Gln mutations has a destabilizing effect with a ΔΔG value of -1.039 kcal/mol. As this novel gene belongs to the large family of short-chain dehydrogenases/reductases (SDR) thought to be involved in steroid biosynthesis, endocrinological investigations subsequently revealed that the two patients also had low levels of cholesterol. Karyotyping revealed a normal 46,XY karyotype for the two boys, excluding other causes of disorders of sex development due to chromosomal rearrangements. In conclusion, our study reveals that mutation in the novel SDR42E1 gene alters the steroid hormone synthesis and associated with a new syndrome we named oculocutaneous genital syndrome. In addition, this study highlights the role of SDR42E1 in the regulation of cholesterol metabolism in the maintenance of connective tissue and sexual maturation in humans.

Arterial Tortuosity Syndrome: An Ascorbate Compartmentalization Disorder?

Significance: Cardiovascular disorders are the most important cause of morbidity and mortality in the Western world. Monogenic developmental disorders of the heart and vessels are highly valuable to study the physiological and pathological processes in cardiovascular system homeostasis. The arterial tortuosity syndrome (ATS) is a rare, autosomal recessive connective tissue disorder showing lengthening, tortuosity, and stenosis of the large arteries, with a propensity for aneurysm formation. In histopathology, it associates with fragmentation and disorganization of elastic fibers in several tissues, including the arterial wall. ATS is caused by pathogenic variants in SLC2A10 encoding the facilitative glucose transporter (GLUT)10. Critical Issues: Although several hypotheses have been forwarded, the molecular mechanisms linking disrupted GLUT10 activity with arterial malformations are largely unknown. Recent Advances: The vascular and systemic manifestations and natural history of ATS patients have been largely delineated. GLUT10 was identified as an intracellular transporter of dehydroascorbic acid, which contributes to collagen and elastin cross-linking in the endoplasmic reticulum, redox homeostasis in the mitochondria, and global and gene-specific methylation/hydroxymethylation affecting epigenetic regulation in the nucleus. We revise here the current knowledge on ATS and the role of GLUT10 within the compartmentalization of ascorbate in physiological and diseased states. Future Directions: Centralization of clinical, treatment, and outcome data will enable better management for ATS patients. Establishment of representative animal disease models could facilitate the study of pathomechanisms underlying ATS. This might be relevant for other forms of vascular dysplasia, such as isolated aneurysm formation, hypertensive vasculopathy, and neovascularization. Antioxid. Redox Signal. 34, 875-889.

The deregulation of NOTCH pathway, inflammatory cytokines, and keratinization genes in two Dowling-Degos disease patients with hidradenitis suppurativa.

Dowling-Degos disease (DDD) is a rare autosomal-dominant genodermatosis and it has been associated with hidradenitis suppurativa (HS). Deregulation of NOTCH pathway has been linked to the development of HS in DDD context (DDD-HS). However, molecular alterations in DDD-HS, including altered gene expression of NOTCH and downstream effectors that are involved in the follicular differentiation and inflammatory response, are poorly defined. We report two cases of patients diagnosed with DDD-HS, one of those, under Adalimumab treatment. Our results have shown downregulation of NOTCH1/NCSTN pathway, distinct molecular profiles of inflammatory cytokines (IL23A and TNF), and a novel aberrant upregulation of genes involved in the cornified envelope (CE) formation (SPRR1B, SPRR2D, SPRR3, and IVL) in paired HS lesions of two DDD patients.

Neurovascular manifestations in connective tissue diseases: The case of Marfan Syndrome.

Patients with connective tissue diseases (CTDs) are suspected to be at higher risk for cerebrovascular involvement, such as intracranial aneurysms, dissections and strokes, than the general population. Particularly, Marfan Syndrome (MFS) has been reported as associated with an increased risk of cerebrovascular alterations. Literature data report different prevalence of intracranial aneurysms in MFS, ranging from 4 % to 29 %, suggesting a role of genetic cause that involves the regulation of the TGF-ß signaling. Ischemic and hemorrhagic strokes have been also reported in MFS, but with an estimated prevalence from 3 % to 4 %. However, the aetiology of both events appears to be reliable more to a cardiac source than to the primary connective tissue defect. Finally, the available literature suggests that MFS patients have a higher prevalence of arterial tortuosity of neck and head vessels and these findings may be related to an enhanced chance of dissection. Overall, despite of the lack of studies, we could affirm that it may exists an increased prevalence of some neurovascular findings in MFS patients. Nevertheless, further studies are required to determine the true prevalence of these features and investigate specific gene mutations involved in MFS.

Pro-Fibrotic Phenotype in a Patient with Segmental Stiff Skin Syndrome via TGF-ß Signaling Overactivation.

Transforming growth factor ß (TGF-ß) superfamily signaling pathways are ubiquitous and essential for several cellular and physiological processes. The overexpression of TGF-ß results in excessive fibrosis in multiple human disorders. Among them, stiff skin syndrome (SSS) is an ultrarare and untreatable condition characterized by the progressive thickening and hardening of the dermis, and acquired joint limitations. SSS is distinct in a widespread form, caused by recurrent germline variants of FBN1 encoding a key molecule of the TGF-ß signaling, and a segmental form with unknown molecular basis. Here, we report a 12-year-old female with segmental SSS, affecting the right upper limb with acquired thickening of the dermis evident at the magnetic resonance imaging, and progressive limitation of the elbow and shoulder. To better explore the molecular and cellular mechanisms that drive segmental SSS, several functional studies on patient's fibroblasts were employed. We hypothesized an impairment of TGF-ß signaling and, consequently, a dysregulation of the associated downstream signaling. Lesional fibroblast studies showed a higher phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK1/2), increased levels of nuclear factor-kB (NFkB), and a nuclear accumulation of phosphorylated Smad2 via Western blot and microscopy analyses. Quantitative PCR expression analysis of genes encoding key extracellular matrix proteins revealed increased levels of COL1A1, COL3A1, AGT, LTBP and ITGB1, while zymography assay reported a reduced metalloproteinase 2 enzymatic activity. In vitro exposure of patient's fibroblasts to losartan led to the partial restoration of normal transforming growth factor ß (TGF-ß) marker protein levels. Taken together, these data demonstrate that in our patient, segmental SSS is characterized by the overactivation of multiple TGF-ß signaling pathways, which likely results in altered extracellular matrix composition and fibroblast homeostasis. Our results for the first time reported that aberrant TGF-ß signaling may drive the pathogenesis of segmental SSS and might open the way to novel therapeutic approaches.

NRF2 Augments Epidermal Antioxidant Defenses and Promotes Atopy.

Atopic dermatitis is a chronic form of allergic contact dermatitis that is closely associated with a compromised epidermal barrier. Immunogenicity of a given electrophilic hapten after penetration of this barrier depends directly on biochemical reactions in the thiol-rich layer in the stratum granulosum. In response to electrophilic hapten, NF-erythroid 2-related factor 2 (NRF2) in keratinocytes efficiently induces the production of antioxidants. In this study, we show that the immunogenicity of a given hapten depends directly on the extent to which it induces antioxidant host defenses within the epidermal tissue. We found that allergic contact dermatitis did not develop in NRF2-deficient mice because of compromise of the epidermal innate immune responses that upregulate IL-1α. We also analyzed epidermal NRF2 in association with congenital disorders with features similar to atopic dermatitis in humans. Epidermal samples from patients with Netherton syndrome and peeling skin syndrome exhibited elevated levels of NRF2 and also elevated levels of its downstream target, small proline-rich protein 2. Taken together, these results suggest that the thiol-mediated biochemical responses in the stratum granulosum provide a critical link between defective epidermal barrier function and the development of atopy. Likewise, our results suggested that NRF2 may have a profound impact on the generation of cutaneous immunological memory.

Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock­in mouse model.

The SAM and SH3 domain­containing 1 (SASH1) genes have been identified as the causal genes of dyschromatosis universalis hereditaria (DUH); these genes cause the pathological phenotypes of DUH, and SASH1 variants have been shown to regulate the abnormal pigmentation phenotype in human skin in various genodermatoses. However, investigations into the mutated SASH1 gene have been limited to in vitro studies. In the present study, to recapitulate the molecular pathological phenotypes of individuals with DUH induced by SASH1 mutations, a heterozygous BALB/c mouse model, in which the human SASH1 c.1654 T>G (p. Tyr 551Asp, Y551D) mutation was knocked in was first generated. The in vivo functional experiments on Y551D SASH1 indicated that the increased expression of microphthalmia­associated transcription factor (Mitf) was uniformly induced in the tails of heterozygous BALB/c mice, and an increased quantity of Mitf­positive epithelial cells was also detected. An increased expression of Mitf­ and Mitf­positive cells was also demonstrated in the epithelial tissues of Y551D­SASH1 affected individuals. In the present study, Mitf expression was also found to be increased by Y551D SASH1 in vitro. Taken together, these findings indicate that the upregulation of Mitf is the bona fide effector of the Y551D SASH1­mediated melanogenesis signaling pathway in vivo. SASH1 may function as a scaffold molecule for the assembly of a SASH1­Mitf molecular complex to regulate Mitf expression in the cell nucleus and thus to promote the hyperpigmented phenotype in the pathogenesis of DUH and other genodermatoses related to pigment abnormalities.

Functional Fibrinolysis Assays Reveal Different Mechanisms underlying Plasminogen Dysfunction in Ligneous Conjunctivitis.

BACKGROUND: Ligneous conjunctivitis (LC) is a rare disorder associated with plasminogen deficiency characterized by chronic fibrin deposits in the eyelids. All patients with plasminogen deficiency do not develop LC, whose underlying mechanisms remain unknown. OBJECTIVE: We investigated whether fibrinolytic activity was correlated with phenotype and/or genotype in patients suffering from LC and their relatives. METHODS: Plasminogen activity/antigen levels and PLG mutations were determined in 10 patients with LC, 17 of their asymptomatic relatives, and 10 healthy individuals used as a control group. Plasma fibrinolytic activity was evaluated using three different assays: (1) tissue-plasminogen activator (t-PA) front lysis, (2) cell-based urokinase-dependent euglobulin clot lysis (ECLT) at the surface of corneal cells, and (3) urokinase-dependent plasminogen activation. RESULTS: Plasminogen activity varied from <10 to 40% in patients, 36 to 105% in relatives, and >80% in control healthy individuals. Homozygous K19E mutation was associated with normal antigenic plasminogen levels. In front-lysis experiments, all patients had a lower fibrinolysis rate as compared with their relatives and to control individuals. The cell-based ECLT and plasminogen activation assay demonstrated that urokinase-mediated fibrinolysis was not impaired in patients with homozygous K19E mutation compared with the other mutants. CONCLUSION: We confirm that plasminogen levels fail to predict LC occurrence. In these conditions, t-PA clot lysis front is useful to predict clinical outcome in plasminogen deficiency. Moreover, we provide evidence that occurrence of LC overlaps quantitative and qualitative plasminogen deficiencies. The homozygous K19E mutation is associated with isolated impaired t-PA-mediated fibrinolysis compared with other mutants.

Slc2a10 knock-out mice deficient in ascorbic acid synthesis recapitulate aspects of arterial tortuosity syndrome and display mitochondrial respiration defects.

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicated GLUT10 in the transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations did not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in mouse. Gulo;Slc2a10 double knock-out mice showed mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 double knock-out mice did not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. Transforming growth factor beta signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 double knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined.

Epidermal Langerhans cells resistance to solar-simulated radiation in actinic prurigo patients with low minimal erythema dose.

BACKGROUND: Actinic prurigo is a chronic photodermatosis of unclear pathogenesis. Epidermal Langerhans cell resistance to migration after ultraviolet radiation exposure has been proposed as a possible mechanism, as occurs in polymorphic light eruption patients. The purpose of this study was to evaluate the effect of solar-simulated radiation (SSR) on epidermal Langerhans cells in the uninvolved skin of actinic prurigo patients. PATIENTS AND METHODS: Fifteen patients with actinic prurigo participated in the study. A biopsy from the uninvolved and unirradiated skin of the left buttock was performed, and another from the uninvolved skin of the right buttock, 72 hours after exposure to two MEDs of SSR. Immunohistochemistry staining was used to identify Langerhans cells (CD1a) in all samples. RESULTS: In actinic prurigo patients with normal MED, there was a significant decrease in the number of epidermal Langerhans cells on the buttock skin exposed to two MED of SSR compared with the unirradiated buttock skin (P = .02 and .035 respectively). On the contrary, in patients with low MED there were no significant differences in the number of epidermal Langerhans cells between irradiated and unirradiated skin (P = .39). CONCLUSION: Epidermal Langerhans cells migration after ultraviolet radiation exposure is decreased in actinic prurigo patients with low MED as has been reported in PLE patients, especially, those with low MED or positive UVB provocation tests. Langerhans cells resistance could be part of a common pathogenic mechanism in these two photodermatoses.

Diacylglycerol Kinase ζ Regulates Macrophage Responses in Juvenile Arthritis and Cytokine Storm Syndrome Mouse Models.

Dysregulation of monocyte and macrophage responses are often observed in children with systemic juvenile idiopathic arthritis (sJIA) and cytokine storm syndrome (CSS), a potentially fatal complication of chronic rheumatic diseases. Both conditions are associated with activation of TLR signaling in monocyte and macrophage lineage cells, leading to overwhelming inflammatory responses. Despite the importance of TLR engagement in activating proinflammatory macrophages, relatively little is known about activation of intrinsic negative regulatory pathways to attenuate excessive inflammatory responses. In this study, we demonstrate that loss of diacylglycerol (DAG) kinase (Dgk) ζ, an enzyme which converts DAG into phosphatidic acid, limits inflammatory cytokine production in an arthritic mouse model dependent on TLR2 signaling and in a CSS mouse model dependent on TLR9 signaling. In vitro, Dgkζ deficiency results in reduced production of TNF-α, IL-6, and IL-1ß and in limited M1 macrophage polarization. Mechanistically, Dgkζ deficiency decreases STAT1 and STAT3 phosphorylation. Moreover, Dgkζ levels are increased in macrophages derived from mice with CSS or exposed to plasma from sJIA patients with active disease. Our data suggest that Dgkζ induction in arthritic conditions perpetuates systemic inflammatory responses mediated by macrophages and highlight a potential role of Dgkζ-DAG/phosphatidic acid axis as a modulator of inflammatory cytokine production in sJIA and CSS.

GORAB scaffolds COPI at the trans-Golgi for efficient enzyme recycling and correct protein glycosylation.

COPI is a key mediator of protein trafficking within the secretory pathway. COPI is recruited to the membrane primarily through binding to Arf GTPases, upon which it undergoes assembly to form coated transport intermediates responsible for trafficking numerous proteins, including Golgi-resident enzymes. Here, we identify GORAB, the protein mutated in the skin and bone disorder gerodermia osteodysplastica, as a component of the COPI machinery. GORAB forms stable domains at the trans-Golgi that, via interactions with the COPI-binding protein Scyl1, promote COPI recruitment to these domains. Pathogenic GORAB mutations perturb Scyl1 binding or GORAB assembly into domains, indicating the importance of these interactions. Loss of GORAB causes impairment of COPI-mediated retrieval of trans-Golgi enzymes, resulting in a deficit in glycosylation of secretory cargo proteins. Our results therefore identify GORAB as a COPI scaffolding factor, and support the view that defective protein glycosylation is a major disease mechanism in gerodermia osteodysplastica.

Inactivating PTH/PTHrP Signaling Disorders.

Pseudohypoparathyroidism (PHP), pseudo-PHP, acrodysostosis, and progressive osseous heteroplasia are heterogeneous disorders characterized by physical findings, differently associated in each subtype, including short bones, short stature, a stocky build, ectopic ossifications (features associated with Albright's hereditary osteodystrophy), as well as laboratory abnormalities consistent with hormone resistance, such as hypocalcemia, hyperphosphatemia, and elevated parathyroid hormone (PTH) and thyroid-stimulating hormone levels. All these disorders are caused by impairments in the cAMP-mediated signal transduction pathway and, in particular, in the PTH/PTHrP signaling pathway: the main subtypes of PHP and related disorders are caused by de novo or autosomal dominantly inherited inactivating genetic mutations, and/or epigenetic, sporadic, or genetic-based alterations within or upstream of GNAS, PRKAR1A, PDE4D, and PDE3A. Here we will review the impressive progress that has been made over the past 30 years on the pathophysiology of these diseases and will describe the recently proposed novel nomenclature and classification. The new term "inactivating PTH/PTHrP signaling disorder," iPPSD: (1) defines the common mechanism responsible for all diseases, (2) does not require a confirmed genetic defect, (3) avoids ambiguous terms like "pseudo," and (4) eliminates the clinical or molecular overlap between diseases.

Activography reveals aberrant proteolysis in desquamating diseases of differing backgrounds.

The role of epidermal proteolysis in overdesquamation was revealed in Netherton syndrome, a rare ichthyosis due to genetic deficiency of the LEKTI inhibitor of serine proteases. Recently, we developed activography, a new histochemical method, to spatially localize and semiquantitatively assess proteolytic activities using activity-based probes. Activography provides specificity and versatility compared to in situ zymography, the only available method to determine enzymatic activities in tissue biopsies. Here, activography was validated in skin biopsies obtained from an array of distinct disorders and compared with in situ zymography. Activography provides a methodological advancement due to its simplicity and specificity and can be readily adapted as a routine diagnostic assay. Interestingly, the levels of epidermal proteolysis correlated with the degree of desquamation independent of skin pathology. Thus, deregulated epidermal proteolysis likely represents a universal mechanism underlying aberrant desquamation.

Expression of E-cadherin, syndecan 1, Ki-67, and maintenance minichromosome 3 in tissue lesions of actinic prurigo obtained by incisional biopsy.

Actinic prurigo (AP) is an idiopathic photodermatosis; the initial manifestations usually occur during the first decades of life but can appear at any age. Cases are usually diagnosed late once the lesions have exacerbated; due to the extensive involvement of the vermilion border and the etiology, it has been confused with and related to a potentially malignant process. Syndecan-1 and E-cadherin were positive in the epidermis, with moderate-to-intense staining in 100% of samples. Ki67 and MCM3 were expressed in the lower third of the epidermis and showed greater immunolabeling in samples that contained lymphoid follicles (Ki 67: epidermis [17.7% ± 6.79%] and dermis [7.73% ± 6.69%]; MCM3: epidermis [22.92% ± 10.12%] and dermis [6.13% ± 6.27%]). In conclusión AP is a disease in which there is no evidence that the lesions are potentially cancerous. AP cheilitis should not be confused with actinic cheilitis because they are separate entities.