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The clinical problem of a non-healing fistula in ano in a child affected with poikiloderma with neutropenia (PN) was the stimulus for an innovative study by Parajuli et al. that sheds light on the pathological mechanisms in this disease. Multiparametric analyses of the patient's blood mononuclear cells by cell culture, flow cytometry and multiplex cytokine assay suggested a block of monocyte differentiation. Monocyte transcriptome profiling revealed a signature consistent with the haematological picture and the clinical presentation. Commentary on: Parajuli et al. Defective monocyte plasticity and altered cAMP pathway characterize USB1-mutated poikiloderma with neutropenia Clericuzio type. Br J Haematol 2024;204:683-693., (© 2023 British Society for Haematology and John Wiley & Sons Ltd.)

Poikiloderma with neutropenia (PN) Clericuzio type (OMIM #604173) is a rare disease with areas of skin hyper- and hypopigmentation caused by biallelic USB1 variants. The current study was spurred by poor healing of a perianal tear wound in one affected child homozygous for c.266-1G>A (p.E90Sfster8) mutation, from a family reported previously. Treatment with G-CSF/CSF3 or GM-CSF/CSF2 transiently increased neutrophil/monocytes count with no effect on wound healing. Analysis of peripheral blood revealed a lack of non-classical (CD14 +/- CD16 + ) monocytes, associated with a systemic inflammatory cytokine profile, in the two affected brothers. Importantly, despite normal expression of cognate receptors, monocytes from PN patients did not respond to M-CSF or IL-34 in vitro, as determined by cytokine secretion or CD16 expression. RNAseq of monocytes showed 293 differentially expressed genes, including significant downregulation of GATA2, AKAP6 and PDE4DIP that are associated with leucocyte differentiation and cyclic adenosine monophosphate (cAMP) signalling. Notably, the plasma cAMP was significantly low in the PN patients. Our study revealed a novel association of PN with a lack of non-classical monocyte population. The defects in monocyte plasticity may contribute to disease manifestations in PN and a defective cAMP signalling may be the primary effect of the splicing errors caused by USB1 mutation., (© 2023 British Society for Haematology and John Wiley & Sons Ltd.)

Desmosomes are dynamic complex protein structures involved in cellular adhesion. Disruption of these structures by loss-of-function variants in desmosomal genes leads to a variety of skin- and heart-related phenotypes. In this study, we report TUFT1 as a desmosome-associated protein, implicated in epidermal integrity. In two siblings with mild skin fragility, woolly hair, and mild palmoplantar keratoderma but without a cardiac phenotype, we identified a homozygous splice-site variant in the TUFT1 gene, leading to aberrant mRNA splicing and loss of TUFT1 protein. Patients' skin and keratinocytes showed acantholysis, perinuclear retraction of intermediate filaments, and reduced mechanical stress resistance. Immunolabeling and transfection studies showed that TUFT1 is positioned within the desmosome and that its location is dependent on the presence of the desmoplakin carboxy-terminal tail. A Tuft1-knockout mouse model mimicked the patients' phenotypes. Altogether, this study reveals TUFT1 as a desmosome-associated protein, whose absence causes skin fragility, woolly hair, and palmoplantar keratoderma., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Start codon variants in ubiquitin ligase KLHL24 lead to a gain-of-function mutant KLHL24-ΔN28, which mediates the excessive degradation of keratin 15, desmin, and keratin 14, resulting in alopecia, cardiopathy, and epidermolysis bullosa syndrome. Patients with alopecia, cardiopathy, and epidermolysis bullosa syndrome normally present atrophic scars after wounds heal, which is rare in KRT14-related epidermolysis bullosa. The mechanisms underlying the formation of atrophic scars in epidermolysis bullosa of patients with alopecia, cardiopathy, and epidermolysis bullosa syndrome remain unclear. This study showed that KLHL24-ΔN28 impaired skin wound healing by excessively degrading vimentin. Heterozygous Klhl24 c .3G>T knock-in mice displayed delayed wound healing and decreased wound collagen deposition. We identified vimentin as an unreported substrate of KLHL24. KLHL24-ΔN28 mediated the excessive degradation of vimentin, which failed to maintain efficient fibroblast proliferation and activation during wound healing. Furthermore, by mediating vimentin degradation, KLHL24 can hinder myofibroblast activation, which attenuated bleomycin-induced skin fibrosis. These findings showed the function of KLHL24 in regulating tissue remodeling, atrophic scarring, and fibrosis., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Brittle cornea syndrome (BCS) is a rare autosomal recessive disorder characterized by extreme thinning and fragility of the cornea, and mutations in ZNF469 cause BCS-1. We aimed to establish a znf469 mutant zebrafish line to explore its roles and possible pathogenic mechanism in cornea development and disorder., Methods: znf4694del/4del mutant zebrafish was generated using the CRISPR/Cas9 technology. Transmission electron microscopy (TEM) was performed to examine the phenotype of the cornea in different developmental stages. RNA sequencing and quantitative real-time polymerase chain reaction were used to reveal the molecular mechanism., Results: Macroscopically, homozygous znf469 mutant zebrafish larvae exhibited a curved body from 72 hours postfertilization, similar to kyphoscoliosis, and a noninflated swimbladder at 7 days postfertilization (dpf). TEM revealed an extreme reduction of corneal stroma thickness in homozygous znf469 mutant zebrafish in both the central and peripheral cornea from the early development stage. RNA-sequencing analysis demonstrated that the znf469 mutation leads to the decreased synthesis of various extracellular matrix (ECM) components, such as collagens and proteoglycans, but increased synthesis of 26S proteasome family members., Conclusions: The results of our work indicate that znf469 is a critical gene that, as a widely considered transcription factor, may regulate the synthesis and degradation of a large number of ECM components that play an important role in corneal development.

Nonhealing wounds are a major area of unmet clinical need remaining problematic to treat. Improved understanding of prohealing mechanisms is invaluable. The enzyme arginase1 (ARG1) is involved in prohealing responses, with its role in macrophages best characterized. ARG1 is also expressed by keratinocytes; however, ARG1 function in these critical wound repair cells is not understood. We characterized ARG1 expression in keratinocytes during normal cutaneous repair and reveal de novo temporal and spatial expression at the epidermal wound edge. Interestingly, epidermal ARG1 expression was decreased in both human and murine delayed healing wounds. We therefore generated a keratinocyte-specific ARG1-null mouse model (K14-cre;Arg1 fl/fl ) to explore arginase function. Wound repair, linked to changes in keratinocyte proliferation, migration, and differentiation, was significantly delayed in K14-cre;Arg1 fl/fl mice. Similarly, using the arginase inhibitor N(omega)-hydroxy-nor-L-arginine, human in vitro and ex vivo models further confirmed this finding, revealing the importance of the downstream polyamine pathway in repair. Indeed, restoring the balance in ARG1 activity through the addition of putrescine proved beneficial in wound closure. In summary, we show that epidermal ARG1 plays, to our knowledge, a previously unreported intrinsic role in cutaneous healing, highlighting epidermal ARG1 and the downstream mediators as potential targets for the therapeutic modulation of wound repair., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose: Erlotinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, causes skin disorders such as dry skin, which impairs the skin barrier function. Stratum corneum (SC) lipids play an important role in skin barrier function; therefore, this study aimed to investigate the relationship between erlotinib-related dry skin and changes in the intercellular lipid composition and structure of the SC., Methods: Overall, 21 patients with non-small lung cancer were enrolled in this study. All patients received 150 mg/day erlotinib orally. A SC sample of each patient was collected from the inner forearm using the tape stripping method on days 0, 7, 14, 28, and 56 after erlotinib administration. The intercellular lipid components of ceramide (CER), free fatty acid (FFA), and cholesterol sulfate (CS) in samples extracted from the tape were analyzed using liquid chromatography/time-of-flight/mass spectrometry. SC samples from six healthy subjects were collected as controls on days 0, 28 and 56 and analyzed similarly., Results: Although total CER and FFA levels were not changed after erlotinib administration, the levels of CER subclasses [AP] and [AH] and hydroxy FFA, which are structural components of CER subclass [A], decreased. In contrast, the CS levels increased after erlotinib administration. Moreover, higher CS levels in the SC correlated with the clinical condition of dry skin. No changes were observed in the SC lipid composition in healthy subjects., Conclusion: Erlotinib-related dry skin was associated with a higher CS level in the SC.

Post-transcriptional modification of snRNA is central to spliceosome function. Usb1 is an exoribonuclease that shortens the oligo-uridine tail of U6 snRNA, resulting in a terminal 2',3' cyclic phosphate group in most eukaryotes, including humans. Loss of function mutations in human Usb1 cause the rare disorder poikiloderma with neutropenia (PN), and result in U6 snRNAs with elongated 3' ends that are aberrantly adenylated. Here, we show that human Usb1 removes 3' adenosines with 20-fold greater efficiency than uridines, which explains the presence of adenylated U6 snRNAs in cells lacking Usb1. We determined three high-resolution co-crystal structures of Usb1: wild-type Usb1 bound to the substrate analog adenosine 5'-monophosphate, and an inactive mutant bound to RNAs with a 3' terminal adenosine and uridine. These structures, along with QM/MM MD simulations of the catalytic mechanism, illuminate the molecular basis for preferential deadenylation of U6 snRNA. The extent of Usb1 processing is influenced by the secondary structure of U6 snRNA.

Background: Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a malformation of the eyelids. Forkhead Box L2 (FOXL2) is the only gene known to be associated with BPES., Methods: We identified two Han Chinese BPES families with premature ovarian insufficiency (POI). Sanger sequencing and in vitro functional analysis were performed to identify the genetic cause., Results: Sanger sequencing identified two novel mutations (c.462_468del, c.988_989insG) in FOXL2, one in each family. The in vitro functional analysis confirmed that both novel mutations were associated with impaired transactivation of downstream genes. Specifically, the single-base insertion, c.988_989insG, led to subcellular mislocalization and aggregation of the encoded protein, which validated the hypothesis that the two novel FOXL2 mutations are deleterious and associated with POI in the two BPES families., Conclusion: The novel mutations identified in the present study will enhance the present knowledge of the mutation spectrum of FOXL2. The in vitro experiments provide further insights into the molecular mechanism by which the two new variants mediate disease pathogenesis and may contribute to elucidating the genotype-phenotype correlation between the two novel FOXL2 mutations and POI., (© 2018 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Classical Ehlers-Danlos syndrome (cEDS) is characterized by marked cutaneous involvement, according to the Villefranche nosology and its 2017 revision. However, the diagnostic flow-chart that prompts molecular testing is still based on experts' opinion rather than systematic published data. Here we report on 62 molecularly characterized cEDS patients with focus on skin, mucosal, facial, and articular manifestations. The major and minor Villefranche criteria, additional 11 mucocutaneous signs and 15 facial dysmorphic traits were ascertained and feature rates compared by sex and age. In our cohort, we did not observe any mandatory clinical sign. Skin hyperextensibility plus atrophic scars was the most frequent combination, whereas generalized joint hypermobility according to the Beighton score decreased with age. Skin was more commonly hyperextensible on elbows, neck, and knees. The sites more frequently affected by abnormal atrophic scarring were knees, face (especially forehead), pretibial area, and elbows. Facial dysmorphism commonly affected midface/orbital areas with epicanthal folds and infraorbital creases more commonly observed in young patients. Our findings suggest that the combination of ≥1 eye dysmorphism and facial/forehead scars may support the diagnosis in children. Minor acquired traits, such as molluscoid pseudotumors, subcutaneous spheroids, and signs of premature skin aging are equally useful in adults., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Classic Ehlers-Danlos syndrome (cEDS) is characterized by fragile, hyperextensible skin and hypermobile joints. cEDS can be caused by heterozygosity for missense mutations in genes COL5A2 and COL5A1, which encode the α2(V) and α1(V) chains, respectively, of collagen V, and is most often caused by COL5A1 null alleles. However, COL5A2 null alleles have yet to be associated with cEDS or other human pathologies. We previously showed that mice homozygous null for the α2(V) gene Col5a2 are early embryonic lethal, whereas haploinsufficiency caused aberrancies of adult skin, but not a frank cEDS-like phenotype, as skin hyperextensibility at low strain and dermal cauliflower-contoured collagen fibril aggregates, two cEDS hallmarks, were absent. Herein, we show that ubiquitous postnatal Col5a2 knockdown results in pathognomonic dermal cauliflower-contoured collagen fibril aggregates, but absence of skin hyperextensibility, demonstrating these cEDS hallmarks to arise separately from loss of collagen V roles in control of collagen fibril growth and nucleation events, respectively. Col5a2 knockdown also led to loss of dermal white adipose tissue (WAT) and markedly decreased abdominal WAT that was characterized by miniadipocytes and increased collagen deposition, suggesting α2(V) to be important to WAT development/maintenance. More important, Col5a2 haploinsufficiency markedly increased the incidence and severity of abdominal aortic aneurysms, and caused aortic arch ruptures and dissections, indicating that α2(V) chain deficits may play roles in these pathologies in humans., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

p23 is a small acidic protein with intrinsic molecular chaperone activity. It is best known as a co-chaperone of the major cytosolic molecular chaperone Hsp90. p23 binds the N-terminus of Hsp90 and stabilizes the ATP-bound and N-terminally closed Hsp90 dimer. It is in this configuration that many Hsp90 clients are most stably bound. Considering the important role of p23 in the Hsp90 cycle, it came as a surprise that it is not absolutely essential for viability in the budding yeast or for mouse development. Mice without p23 develop quite normally until birth and then all die perinatally because of immature lungs. The only other apparent phenotype of late stage embryos and newborns is a skin defect, which we have further characterized here. We found that skin differentiation is impaired, and that both apoptosis and cell proliferation are augmented in the absence of p23; the consequences are a severe thinning of the stratum corneum and reduced numbers of hair follicles. The altered differentiation, spontaneous apoptosis and proliferation are all mimicked by isolated primary keratinocytes indicating that they do require p23 functions in a cell-autonomous fashion. Since the phenotype of p23-null embryos is strikingly similar to that of embryos lacking the glucocorticoid receptor, a paradigmatic Hsp90-p23 client protein, we investigated glucocorticoid signaling. We discovered that it is impaired in vivo and for some aspects in isolated keratinocytes. Our results suggest that part of the phenotype of p23-null embryos can be explained by an impact on this particular Hsp90 client, but do not exclude that p23 by itself or in association with Hsp90 affects skin development and homeostasis through yet other pathways.

Fibroblast growth factor receptors (FGFRs) and FGF ligands are highly expressed in the developing kidney and lower urinary tract. Several classic studies showed many effects of exogenous FGF ligands on embryonic renal tissues in vitro and in vivo. Another older landmark publication showed that mice with a dominant negative Fgfr fragment had severe renal dysplasia. Together, these studies revealed the importance of FGFR signaling in kidney and lower urinary tract development. With the advent of modern gene targeting techniques, including conditional knockout approaches, several publications have revealed critical roles for FGFR signaling in many lineages of the kidney and lower urinary tract at different stages of development. FGFR signaling has been shown to be critical for early metanephric mesenchymal patterning, Wolffian duct patterning including induction of the ureteric bud, ureteric bud branching morphogenesis, nephron progenitor survival and nephrogenesis, and bladder mesenchyme patterning. FGFRs pattern these tissues by interacting with many other growth factor signaling pathways. Moreover, the many genetic Fgfr and Fgf animal models have structural defects mimicking numerous congenital anomalies of the kidney and urinary tract seen in humans. Finally, many studies have shown how FGFR signaling is critical for kidney and lower urinary tract patterning in humans.

Circumferential skin creases Kunze type (CSC-KT) is a specific congenital entity with an unknown genetic cause. The disease phenotype comprises characteristic circumferential skin creases accompanied by intellectual disability, a cleft palate, short stature, and dysmorphic features. Here, we report that mutations in either MAPRE2 or TUBB underlie the genetic origin of this syndrome. MAPRE2 encodes a member of the microtubule end-binding family of proteins that bind to the guanosine triphosphate cap at growing microtubule plus ends, and TUBB encodes a β-tubulin isotype that is expressed abundantly in the developing brain. Functional analyses of the TUBB mutants show multiple defects in the chaperone-dependent tubulin heterodimer folding and assembly pathway that leads to a compromised yield of native heterodimers. The TUBB mutations also have an impact on microtubule dynamics. For MAPRE2, we show that the mutations result in enhanced MAPRE2 binding to microtubules, implying an increased dwell time at microtubule plus ends. Further, in vivo analysis of MAPRE2 mutations in a zebrafish model of craniofacial development shows that the variants most likely perturb the patterning of branchial arches, either through excessive activity (under a recessive paradigm) or through haploinsufficiency (dominant de novo paradigm). Taken together, our data add CSC-KT to the growing list of tubulinopathies and highlight how multiple inheritance paradigms can affect dosage-sensitive biological systems so as to result in the same clinical defect., (Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Brittle cornea syndrome (BCS) is a rare, generalized connective tissue disorder associated with extreme corneal thinning and a high risk of corneal rupture. Recessive mutations in transcription factors ZNF469 and PRDM5 cause BCS. Both transcription factors are suggested to act on a common pathway regulating extracellular matrix genes, particularly fibrillar collagens. We identified bilateral myopic choroidal neovascularization as the presenting feature of BCS in a 26-year-old-woman carrying a novel PRDM5 mutation (p.Glu134*). We performed immunohistochemistry of anterior and posterior segment ocular tissues, as expression of PRDM5 in the eye has not been described, or the effects of PRDM5-associated disease on the retina, particularly the extracellular matrix composition of Bruch's membrane., Methods: Immunohistochemistry using antibodies against PRDM5, collagens type I, III, and IV was performed on the eyes of two unaffected controls and two patients (both with Δ9-14 PRDM5). Expression of collagens, integrins, tenascin and fibronectin in skin fibroblasts of a BCS patient with a novel p.Glu134* PRDM5 mutation was assessed using immunofluorescence., Results: PRDM5 is expressed in the corneal epithelium and retina. We observe reduced expression of major components of Bruch's membrane in the eyes of two BCS patients with a PRDM5 Δ9-14 mutation. Immunofluorescence performed on skin fibroblasts from a patient with p.Glu134* confirms the generalized nature of extracellular matrix abnormalities in BCS., Conclusions: PDRM5-related disease is known to affect the cornea, skin and joints. Here we demonstrate, to the best of our knowledge for the first time, that PRDM5 localizes not only in the human cornea, but is also widely expressed in the retina. Our findings suggest that ECM abnormalities in PRDM5-associated disease are more widespread than previously reported.

Poikiloderma with neutropenia (PN, Clericuzio-type poikiloderma with neutropenia) is a rare autosomal recessive disorder caused by biallelic mutations in the USB1 gene (Alias C16orf57 and MPN1). To date, there have been only 37 reported cases worldwide of this disorder that presents with neutropenia, early onset poikiloderma, respiratory infections, palmo-plantar hyperkeratosis, and skeletal defects. Here we described the generation of human induced pluripotent stem cell lines (PN1 and PN2) from the peripheral blood of a 1-year-old patient using the dox-inducible STEMCCA vector. This patient presented with bacteremia, pneumonia, and neutropenia. Analysis of bone marrow demonstrated normal cellularity with trilineage hematopoiesis and neutropenia., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Purpose: To describe corneal cross-linking (CXL) as a treatment option for brittle cornea syndrome (BCS)., Methods: Case report., Results: Ethical decision making enabled bilateral sequential transepithelial CXL in an 11-year-old girl with BCS. Postoperative courses were uneventful with a bilateral stromal demarcation line, unchanged corneal transparency, constant endothelial cell density, and stable topography 2 years after intervention., Conclusions: Modified CXL can safely be performed in patients with BCS. Ethical review may be helpful for interventions deviating from standard practice.

Background: Haploinsufficiency of the FOXL2 transcription factor in humans causes Blepharophimosis/Ptosis/Epicanthus Inversus syndrome (BPES), characterized by eyelid anomalies and premature ovarian failure. Mice lacking Foxl2 recapitulate human eyelid/forehead defects and undergo female gonadal dysgenesis. We report here that mice lacking Foxl2 also show defects in postnatal growth and embryonic bone and cartilage formation., Methods: Foxl2 (-/-) male mice at different stages of development have been characterized and compared to wild type. Body length and weight were measured and growth curves were created. Skeletons were stained with alcian blue and/or alizarin red. Bone and cartilage formation was analyzed by Von Kossa staining and immunofluorescence using anti-FOXL2 and anti-SOX9 antibodies followed by confocal microscopy. Genes differentially expressed in skull vaults were evaluated by microarray analysis. Analysis of the GH/IGF1 pathway was done evaluating the expression of several hypothalamic-pituitary-bone axis markers by RT-qPCR., Results: Compared to wild-type, Foxl2 null mice are smaller and show skeletal abnormalities and defects in cartilage and bone mineralization, with down-regulation of the GH/IGF1 axis. Consistent with these effects, we find FOXL2 expressed in embryos at 9.5 dpc in neural tube epithelium, in head mesenchyme near the neural tube, and within the first branchial arch; then, starting at 12.5 dpc, expressed in cartilaginous tissue; and at PO and P7, in hypothalamus., Conclusions: Our results support FOXL2 as a master transcription factor in a spectrum of developmental processes, including growth, cartilage and bone formation. Its action overlaps that of SOX9, though they are antagonistic in female vs male gonadal sex determination but conjoint in cartilage and skeletal development.

Ultradian oscillators are cyclically expressed genes with a period of less than 24h, found in the major signalling pathways. The Notch effector hairy and enhancer of split Hes genes are ultradian oscillators. The physiological signals that synchronise and entrain Hes oscillators remain poorly understood. We investigated whether cellular metabolism modulates Hes1 cyclic expression. We demonstrated that, in mouse myoblasts (C2C12), Hes1 oscillation depends on reactive oxygen species (ROS), which are generated by the mitochondria electron transport chain and by NADPH oxidases NOXs. In vitro, the regulation of Hes1 by ROS occurs via the calcium-mediated signalling. The modulation of Hes1 by ROS was relevant in vivo, since perturbing ROS homeostasis was sufficient to alter Medaka (Oryzias latipes) somitogenesis, a process that is dependent on Hes1 ultradian oscillation during embryo development. Moreover, in a Medaka model for human microphthalmia with linear skin lesions syndrome, in which mitochondrial ROS homeostasis was impaired, we documented important somitogenesis defects and the deregulation of Hes homologues genes involved in somitogenesis. Notably, both molecular and developmental defects were rescued by antioxidant treatments. Our studies provide the first evidence of a coupling between cellular redox metabolism and an ultradian biological oscillator with important pathophysiological implication for somitogenesis., (Copyright © 2015. Published by Elsevier Ltd.)

Poikiloderma with neutropenia (PN) is a rare inherited disorder characterized by poikiloderma, facial dysmorphism, pachyonychia, short stature and neutropenia. The molecular testing of PN patients has identified mutations in the C16orf57 gene, which encodes a protein referred to as USB1 (U Six Biogenesis 1). In this study, we developed a zebrafish model of PN by the microinjection of morpholino antisense oligos to suppress usb1 gene function. Severe morphological defects, including a bent tail, thin yolk extension and reduced body length, were predominant in the Usb1-suppressed embryos (morphants). We also observed significantly decreased number of neutrophils in the morphants by Sudan Black staining. Interestingly, the splicing of genes involved in neutrophil differentiation and development, such as mpx, ncf1, ela3l and npsn, was aberrant in the morphants. However, the splicing of haematopoietic precursors and erythroid-specific genes was unaltered. Importantly, the neutrophil defects were almost completely rescued by co-injection of ela3l mRNA, the most markedly affected gene in the morphants. Our study demonstrated a possible role of USB1 in modulating the tissue-specific gene splicing that eventually leads to the impaired development of neutrophils. This zebrafish model could serve as a valuable tool to investigate the causative role of USB1 in PN pathogenesis.

Lamin A is a key component of the nuclear lamina produced through post-translational processing of its precursor known as prelamin A.LMNA mutations leading to farnesylated prelamin A accumulation are known to cause lipodystrophy, progeroid and developmental diseases, including Mandibuloacral dysplasia, a mild progeroid syndrome with partial lipodystrophy and altered bone turnover. Thus, degradation of prelamin A is expected to improve the disease phenotype. Here, we show different susceptibilities of prelamin A forms to proteolysis and further demonstrate that treatment with rapamycin efficiently and selectively triggers lysosomal degradation of farnesylated prelamin A, the most toxic processing intermediate. Importantly, rapamycin treatment of Mandibuloacral dysplasia cells, which feature very low levels of the NAD-dependent sirtuin SIRT-1 in the nuclear matrix, restores SIRT-1 localization and distribution of chromatin markers, elicits release of the transcription factor Oct-1 and determines shortening of the prolonged S-phase. These findings indicate the drug as a possible treatment for Mandibuloacral dysplasia.

FOXL2 is an essential transcription factor that is required for proper development of the ovary and eyelid. Mutations in FOXL2 cause an autosomal dominant genetic disorder, blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). BPES type I patients have eyelid malformation and premature ovarian failure leading to infertility, whereas women with type II BPES are fertile or subfertile. In the present study, we evaluated and compared apoptotic and antiproliferative activities of wild-type (WT) and mutant FOXL2 proteins found in BPES type I and II in human granulosa cell tumor-derived KGN cells. Ectopic expression of WT FOXL2 induced apoptosis and inhibited cell cycle progression in human granulosa cells. In contrast, mutated FOXL2s found in BPES type I significantly reduced these activities, whereas mutated FOXL2s in BPES type II showed intermediate activities. Furthermore, mutant FOX L2 proteins were defective in activating transcription of target genes including Caspase 8, TNF-R1, FAS, p21, and BMP4, which regulate apoptosis, proliferation, and differentiation of granulosa cells. Thus, decreased apoptotic and antiproliferative activities caused by mutant forms of FOXL2 found in BPES patients may at least partially contribute to the pathophysiology of ovarian dysfunction.

The Escobar variant of multiple pterygium syndrome (OMIM #265000) is a rare, autosomal recessive disorder associated with mutations in the γ-subunit of the nicotinic acetylcholine receptor (CHRNG). CHRNG is expressed in fetal muscle during motor development and contributes to the formation of neuromuscular junctions (NMJs). Anomalies in NMJ structure and function have not been investigated in patients with Escobar syndrome. We report five patients identified as having Escobar syndrome, from four families. In three families, the same mutation (c.459dupA) was identified in CHRNG. A biopsy from brachioradialis muscle was collected from a patient from one of these families and analyzed for NMJ organization using fluorescence microscopy. Compared to spinalis muscle from control patients with idiopathic scoliosis or cerebral palsy (CP), the patient with Escobar syndrome had a significantly higher degree of acetylcholine receptor present outside acetylcholinesterase and significantly less acetylcholinesterase outside acetylcholine receptors. Given the role of the acetylcholine receptor γ-subunit in fetal neuromuscular signal transduction and in establishing the primary encounter of muscle and motor nerve terminal, the CHRNG mutations described in Escobar syndrome may cause a broader disruption of postsynaptic proteins and result in aberrant development of the NMJ due to impaired prenatal neuromuscular transmission and/or abnormal neuromuscular synaptogenesis., (© 2013 Wiley Periodicals, Inc.)

In recent years, targeted agents have rapidly evolved as effective tools in the clinical management of a broad range of malignant diseases. These agents disrupt molecular mechanisms and signaling modules that drive the malignant phenotype in defined subsets of malignancies. Beyond the intended cellular targets crucial to tumor growth and progression, these agents also affect signal transduction in normal cells and tissues. The resulting adverse events and their clinical management continue to change, as newer agents with an ever-increasing target spectrum are developed. We provide a succinct overview of dermatologic toxicities arising from the targeting of receptor tyrosine kinases and downstream effectors. Emergent insights into the pathomechanisms involved and the use of this knowledge base to alleviate cutaneous adverse events are discussed.

Background: Cutaneous lymphatic malformations represent a group of heterogeneous diseases caused by developmental defects of lymphatic system., Objective: The purpose of this study was to report the clinical, histopathological and immunohistochemical features of a distinctive lymphatic malformation., Methods: Twelve patients with similar clinical and histopathological features were included in this study. Immunohistochemical staining of CD31, D2-40, Prox1 and Wilms tumor 1 (WT-1) were performed on all lesions., Results: All cases were either congenital lesions or developed during the first 2 years of life. All presented as red to brown papules or nodules on acral sites. Histopathologically, the lesions consisted of a dermal proliferation of flat or slit-like vessels lined with a single layer of endothelial cells. Hemosiderin or extravascular red blood cells were present in all cases. The constituent vessels expressed CD31, D2-40 and Prox1 and lacked expression of WT-1., Conclusion: On the basis of the clinical, histopathological and immunohistochemical findings, our cases represent a unique type of lymphatic malformation that we believe is distinct from previously reported vascular proliferations. We propose the name of acral hemosideric lymphatic malformation for this entity., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The recessive disorder poikiloderma with neutropenia (PN) is caused by mutations in the C16orf57 gene that encodes the highly conserved USB1 protein. Here, we present the 1.1 Å resolution crystal structure of human USB1, defining it as a member of the LigT-like superfamily of 2H phosphoesterases. We show that human USB1 is a distributive 3'-5' exoribonuclease that posttranscriptionally removes uridine and adenosine nucleosides from the 3' end of spliceosomal U6 small nuclear RNA (snRNA), directly catalyzing terminal 2', 3' cyclic phosphate formation. USB1 measures the appropriate length of the U6 oligo(U) tail by reading the position of a key adenine nucleotide (A102) and pausing 5 uridine residues downstream.We show that the 3' ends of U6 snRNA in PN patient lymphoblasts are elongated and unexpectedly carry nontemplated 3' oligo(A) tails that are characteristic of nuclear RNA surveillance targets. Thus, our study reveals a novel quality control pathway in which posttranscriptional 3'-end processing by USB1 protects U6 snRNA from targeting and destruction by the nuclear exosome. Our data implicate aberrant oligoadenylation of U6 snRNA in the pathogenesis of the leukemia predisposition disorder PN.

Mutations in TPM2, encoding β-tropomyosin, have recently been found to cause a range of muscle disorders. We review the clinical and morphological expression of the previously reported mutations illustrating the heterogeneity of β-tropomyosin-associated diseases and describe an additional case with a novel mutation. The manifestations of mutations in TPM2 include non-specific congenital myopathy with type 1 fibre predominance, nemaline myopathy, cap disease and distal arthrogryposis. In addition, Escobar syndrome with nemaline myopathy is a manifestation of homozygous truncating β-tropomyosin mutation. Cap disease appears to be the most common morphological manifestation. A coarse intermyofibrillar network and jagged Z lines are additional frequent changes. The dominant β-tropomyosin mutations manifest either as congenital myopathy or distal arthrogryposis. The various congenital myopathies are usually associated with moderate muscle weakness and no congenital joint contractures. The distal arthrogryposis syndromes associated with TPM2 mutations include the less severe forms, with congenital contractures mainly of the hands and feet and mild or no muscle weakness. The dominant TPM2 mutations include amino acid deletions/insertions and missense mutations. There is no clear relation between the type of mutations or the localisation of the mutated residue in the β-tropomyosin molecule and the clinical and morphological phenotype., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Study Question: What are the implications of multiple alterations of the forkhead box L2 (FOXL2) gene in blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) patients?, Summary Answer: A multi-mutation of FOXL2, consisting of the expansion of the polyalanine tract from 14 to 24 residues (FOXL2-Ala24), an novel Y186C substitution from c.557A>G, and a synonymous variant (c.505G>A), had a cumulative effect on ovarian phenotypes in BPES patients., What Is Known Already: Mutations in FOXL2, a gene encoding a forkhead transcription factor cause BPES. Overall, the expansion of the polyalanine tract of FOXL2 from 14 to 24 residues (FOXL2-Ala24) accounts for 30% of intragenic mutations., Study Design, Size, Duration: In this study, patients from seven BPES families and six sporadic cases were included., Participants/materials, Setting, Methods: We conducted an extensive clinical, hormonal and functional study in 20 patients carrying the expansion of the polyalanine tract of FOXL2 associated with BPES. A multi-mutation of FOXL2 was detected in one BPES family that showed more severe BPES symptoms. Subcellular localization and transactivation studies were performed for the constructs of FOXL2-Ala24, Y186C and FOXL2-Ala24-Y186C., Main Results: We described the first multi-mutation of FOXL2 (c. [672_701dup30; 557A>G]) that leads to the polyalanine expansion of +10 residues (FOXL2-Ala24) combined with an Y186C substitution and a synonymous variant in a Chinese BPES family. This multi-mutation genotype was associated with more serious BPES clinical manifestations and the development of esotropia in the right eye. In in vitro studies, the multi-mutation affected the function of FOKL2 on the StAR promoter and DK3, and induced more aggressive aggregation and mislocalization of FOXL2 protein. The synonymous variant, while not affecting amino acid coding, causes a change in the RNA stem-loop structure., Limitations, Reasons for Caution: The multi-mutation of FOXL2 was detected in one BPES family and it needs to be validated further by more BPES subjects., Wider Implications of the Findings: The results of our study contribute new insights into the research field of BPES caused by the multi-mutation of FOXL2., Study Funding/competing Interests: This study was supported by Shanghai Leading Academic Discipline Project (Grant number S30205) and Shanghai Jiao Tong University School of Medicine Doctor Innovation Fund (Grant number 201131). The authors have no competing interests to declare.

Clericuzio-type poikiloderma with neutropenia (PN) is a rare genodermatosis associated with mutations in the C16orf57 gene, which codes for the uncharacterized protein hMpn1. We show here that, in both fission yeasts and humans, Mpn1 processes the spliceosomal U6 small nuclear RNA (snRNA) posttranscriptionally. In Mpn1-deficient cells, U6 molecules carry 3' end polyuridine tails that are longer than those in normal cells and lack a terminal 2',3' cyclic phosphate group. In mpn1Δ yeast cells, U6 snRNA and U4/U6 di-small nuclear RNA protein complex levels are diminished, leading to precursor messenger RNA splicing defects, which are reverted by expression of either yeast or human Mpn1 and by overexpression of U6. Recombinant hMpn1 is a 3'-to-5' RNA exonuclease that removes uridines from U6 3' ends, generating terminal 2',3' cyclic phosphates in vitro. Finally, U6 degradation rates increase in mpn1Δ yeasts and in lymphoblasts established from individuals affected by PN. Our data indicate that Mpn1 promotes U6 stability through 3' end posttranscriptional processing and implicate altered U6 metabolism as a potential mechanism for PN pathogenesis., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Prelamin A processing impairment is a common feature of a restricted group of rare genetic alterations/disorders associated with a wide range of clinical phenotypes. Changes in histone posttranslational modifications, alterations in non-histone chromatin proteins and chromatin disorganization have been specifically linked to impairment of specific, distinct prelamin A processing steps, but the molecular mechanism involved in these processes is not yet understood . In this study, we show that the accumulation of wild-type prelamin A detected in restrictive dermopathy (RD), as well as the accumulation of mutated forms of prelamin A identified in familial partial lipodystrophy (FPLD) and mandibuloacral dysplasia (MADA), affect the nuclear localization of barrier-to-autointegration factor (BAF), a protein able to link lamin A precursor to chromatin remodeling functions. Our findings, in accordance with previously described results, support the hypothesis of a prelamin A involvement in BAF nuclear recruitment and suggest BAF-prelamin A complex as a protein platform usually activated in prelamin A-accumulating diseases. Finally, we demonstrate the involvement of the inner nuclear membrane protein emerin in the proper localization of BAF-prelamin A complex.

FOXL2 transcription factor is responsible for the Blepharophimosis Ptosis Epicantus inversus Syndrome (BPES), a genetic disease involving craniofacial malformations often associated with ovarian failure. Recently, a somatic FOXL2 mutation (p.C134W) has been reported in >95% of adult-type granulosa cell tumors. Here, we have identified 10 novel FOXL2 partners by yeast-two-hybrid screening and co-immunoprecipitation. Most BPES-inducing mutated FOXL2 proteins display aggregation in cultured cells. Here, we show that two of the partners (NR2C1 and GMEB1) can be sequestered in such aggregates. This co-aggregation can contribute to the pathogenesis of FOXL2 mutations. We have also measured the effects of FOXL2 interactants on the transcriptional regulation of a series of target promoters. Some of the partners (CXXC4, CXXC5, BANF1) were able to repress FOXL2 activity indistinctively of the promoter. Interestingly, CREM-τ2α, which acted as a repressor on most promoters, increased wild-type (WT) FOXL2 activity on two promoters (PTGS2 and CYP19A1), but was unable to increase the activity of the oncogenic mutant p.C134W. Conversely, GMEB1, which also acted as a repressor on most promoters and increased WT FOXL2 activity on the Per2 promoter, increased to a greater extent the activity of the p.C134W variant. Interestingly, partners with intrinsic pro-apoptotic effect were able to increase apoptosis induction by WT FOXL2, but not by the p.C134W mutant, whereas partners with an anti-apoptotic effect decreased apoptosis induction by both FOXL2 versions. Altogether, these results suggest that the p.C134W mutated form fails to integrate signals through protein-protein interactions to regulate target promoter subsets and in particular to induce cell death.

Hereditary mucoepithelial dysplasia (HMD) is a rare, autosomal dominantly inherited, multisystem disease thought to be caused by abnormalities in desmosomes and gap junctions. HMD affects the skin, mucosae, hair, eyes and lungs. Nearly all patients clinically display the triad of non-scarring alopecia, well-demarcated mucosal erythema and erythematous intertriginous plaques. Although histopathological findings of mucous membrane epithelium have been well characterized, only three reports have described histopathologic findings in the skin. We report a case with unique histopathological findings not previously reported in HMD., (Copyright © 2012 John Wiley & Sons A/S.)

Lamin A and lamin C (A-type lamins, both encoded by the LMNA gene) are major components of the mammalian nuclear lamina, a complex proteinaceous structure that acts as a scaffold for protein complexes that regulate nuclear structure and function. Abnormal accumulation of farnesylated-progerin, a mutant form of prelamin A, plays a key role in the pathogenesis of the Hutchinson-Gilford progeria syndrome (HGPS), a devastating disorder that causes the death of affected children at an average age of 13.5 years, predominantly from premature atherosclerosis and myocardial infarction or stroke. Remarkably, progerin is also present in normal cells and appears to progressively accumulate during aging of non-HGPS cells. Therefore, understanding how this mutant form of lamin A provokes HGPS may shed significant insight into physiological aging. In this review, we discuss recent advances into the pathogenic mechanisms underlying HGPS, the main murine models of the disease, and the therapeutic strategies developed in cellular and animal models with the aim of reducing the accumulation of farnesylated-progerin, as well as their use in clinical trials of HGPS.

We performed an immunomorphological study of mast cells from undamaged skin in women with phenotypical evidence of undifferentiated connective tissue dysplasia syndrome, patients of cosmetological clinics. It was found that the numerical density of mast cells containing chymase granules in this condition 1.7-fold surpassed the corresponding parameter in patients without signs of connective tissue dysplasia syndrome, which probably was a result of compensatory and adaptive reaction aimed at activation of the synthesis of the connective tissue extracellular matrix components. It was hypothesized that increased content of chymase-positive mast cells in the skin of patients with connective tissue dysplasia syndrome contributed to the formation of associated arterial hypertension.

Background: Musladin-Lueke Syndrome (MLS) is a hereditary disorder affecting Beagle dogs that manifests with extensive fibrosis of the skin and joints. In this respect, it resembles human stiff skin syndrome and the Tight skin mouse, each of which is caused by gene defects affecting fibrillin-1, a major component of tissue microfibrils. The objective of this work was to determine the genetic basis of MLS and the molecular consequence of the identified mutation., Methodology and Principal Findings: We mapped the locus for MLS by genome-wide association to a 3.05 Mb haplotype on canine chromosome 9 (CFA9 (50.11-54.26; p(raw) <10(-7))), which was homozygous and identical-by-descent among all affected dogs, consistent with recessive inheritance of a founder mutation. Sequence analysis of a candidate gene at this locus, ADAMTSL2, which is responsible for the human TGFβ dysregulation syndrome, Geleophysic Dysplasia (GD), uncovered a mutation in exon 7 (c.660C>T; p.R221C) perfectly associated with MLS (p-value=10(-12)). Murine ADAMTSL2 containing the p.R221C mutation formed anomalous disulfide-bonded dimers when transiently expressed in COS-1, HEK293F and CHO cells, and was present in the medium of these cells at lower levels than wild-type ADAMTSL2 expressed in parallel., Conclusions/significance: The genetic basis of MLS is a founder mutation in ADAMTSL2, previously shown to interact with latent TGF-β binding protein, which binds fibrillin-1. The molecular effect of the founder mutation on ADAMTSL2 is formation of disulfide-bonded dimers. Although caused by a distinct mutation, and having a milder phenotype than human GD, MLS nevertheless offers a new animal model for study of GD, and for prospective insights on mechanisms and pathways of skin fibrosis and joint contractures.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1)/serine protease inhibitor, Kunitz type 1 (SPINT1) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte growth factor activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal differentiation and function. In this study, skin and hair phenotypes of mice lacking the Hai-1/Spint1 gene were characterized. Previously, we reported that the homozygous deletion of Hai-1/Spint1 in mice resulted in embryonic lethality attributable to impaired placental development. To test the role of Hai-1/Spint1 in mice, the placental function of Hai-1/Spint1-mutant mice was rescued. Injection of Hai-1/Spint1(+/+) blastocysts with Hai-1/Spint1(-/-) embryonic stem cells successfully generated high-chimeric Hai-1/Spint1(-/-) embryos (B6Hai-1(-/-High)) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that embryonic lethality of Hai-1/Spint1(-/-) mice was caused by placental dysfunction. However, newborn B6Hai-1(-/-High) mice showed growth retardation and died by 16 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-1/Spint1-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-1/Spint1 has critical roles in the regulated keratinization of the epidermis and hair development.

Naegeli-Franceschetti-Jadassohn syndrome (NFJS) is a rare autosomal dominant disorder characterized by loss of dermatoglyphics, reticulate hyperpigmentation of the skin, palmoplantar keratoderma, abnormal sweating, and other developmental anomalies of the teeth, hair, and skin. We recently demonstrated that NFJS is caused by heterozygous nonsense or frameshift mutations in the E1/V1-encoding region of KRT14, but the mechanisms for their deleterious effects in NFJS remain elusive. In this study, we further expand the spectrum of NFJS-causing mutations and demonstrate that these mutations result in haploinsufficiency for keratin 14 (K14). As increased apoptotic activity was observed in the epidermal basal cell layer in NFJS patients and as previous data suggested that type I keratins may confer resistance to tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in epithelial tissues, we assessed the effect of down-regulation of KRT14 expression on apoptotic activity in keratinocytes. Using a HaCaT cell-based assay, we found that decreased KRT14 expression is associated with increased susceptibility to TNF-alpha-induced apoptosis. This phenomenon was not observed when cells were cultured in the presence of doxycycline, a known negative regulator of TNF-alpha-dependant pro-apoptotic signaling. Collectively, our results indicate that NFJS results from haploinsufficiency for K14 and suggest that increased susceptibility of keratinocytes to pro-apoptotic signals may be involved in the pathogenesis of this ectodermal dysplasia syndrome.

The transcription factor, Runx2, is known to play crucial roles in skeletal and tooth morphogenesis. Here we document that Runx2 has a regulatory role in skin and hair follicle development. The expression of Runx2 is restricted to hair follicles and is dynamic, pari passu with follicle development. Follicle maturation is delayed in the absence of Runx2 and overall skin and epidermal thickness of Runx2 null embryos is significantly reduced. The Runx2 null epidermis is hypoplastic, displaying reduced expression of Keratin 14, Keratin 1 and markers of proliferation. The expression pattern of Runx2 in the bulb epithelium of mature hair follicles is asymmetric and strikingly similar to that of Sonic hedgehog. This suggests that Runx2 may be a regulator of hedgehog signaling in skin as it is in bones and teeth. Supporting this possibility, we demonstrate that Sonic hedgehog, Patched1 and Gli1 transcripts are reduced in the skin of Runx2 null embryos. Moreover, we document Patched1 expression in epidermal basal cells and show that the skin of Sonic(+/-) embryos is thinner than that of wild-type littermates. These observations suggest that Runx2 and hedgehog signaling are involved in the well known, but unexplained, coupling of skin thickness to hair follicle development.

Cardio-facio-cutaneous (CFC) syndrome is a sporadic developmental disorder characterized by distinctive craniofacial features, heart defects, mental retardation and ectodermal abnormalities. We recently reported missense germline mutations in the genes MEK1 and MEK2 in patients with CFC. These mutations, including F53S and Y130C MEK1, and F57C MEK2, are the first naturally occurring mutations to be identified in these genes. This study reports data concerning the biochemical functions of the novel mutants, as well as the roles of these MEK genes in the MAPK signaling cascade. Our CFC MEK variants cannot induce ERK unless they are phosphorylated by RAF at two key serine residues in the regulatory loop. When we replaced the serine residues with alanines, ERK phosphorylation was significantly reduced in the presence of RAF. We did find that F57C MEK2 activation was less dependent on RAF signaling than the other mutants. This difference results in F57C MEK2 being resistant to the selective RAF inhibitor SB-590885. All three mutants are sensitive to the MEK inhibitor U0126. The majority of CFC cases result from mutations in B-RAF. A recent report indicates the possibility that cancer cells with activated B-RAF have enhanced, selective sensitivity to MEK inhibitors. Thus, regardless of mutations identified in an individual with CFC, MEK inhibition is a potential therapeutic approach for this population.

Unlabelled: The RAS-MAPKinase pathway is a signal transduction cascade which has been studied extensively during the last decades for its role in human oncogenesis. Activation of this cascade is controlled by cycling of the RAS protein between an inactive and an active state and by phosphorylation of downstream proteins. The signalling cascade regulates cell proliferation, differentiation and survival. Disturbed RAS signalling in malignancies is caused by acquired somatic mutations in RAS genes or other components of this pathway. Recently, germline mutations in genes coding for different components of the RAS signalling cascade have been recognized as the cause of several phenotypically overlapping disorders, recently referred to as the neuro-cardio-facial-cutaneous syndromes. Neurofibromatosis type 1, Noonan, LEOPARD, Costello and cardiofaciocutaneous syndromes all present with variable degrees of psychomotor delay, congenital heart defects, facial dysmorphism, short stature, skin abnormalities and a predisposition for malignancy. These findings point to important roles for this evolutionary conserved pathway in oncogenesis, development, cognition and growth., Conclusion: it has become obvious in recent years that the neuro-cardio-facial-cutaneous syndromes all share a common genetic and pathophysiologic basis. Dysregulation of the RAS-MAPKinase pathway is caused by germline mutations in genes involved in this pathway. Undoubtedly more genes causing related syndromes will be discovered in the near future since there are still a substantial number of genes in the pathway that are not yet associated with a known syndrome.

Hedgehog (Hh) signaling is conserved from flies to humans and is indispensable in embryogenesis and adulthood. Patched (Ptc) encodes a receptor for Hh ligands and functions as a tumor suppressor. PTCH1 mutations in humans are found in basal cell carcinoma (BCC) and irradiated Ptc1(+/-) mice recapitulate this phenotype. However, due to embryonic lethality associated with the Ptc1 null mutation, its normal function in embryonic and adult skin remains unknown. Here we describe the epidermal phenotypes of a spontaneous and viable allele of Ptc1, Ptc1(mes), in which the C-terminal domain (CTD) is truncated. Ptc1(mes/mes) embryos display normal epidermal and hair follicle development. Postnatal Ptc1(mes/mes) skin displays severe basal cell layer hyperplasia and increased proliferation, while stratification of the suprabasal layers is mostly normal. Interestingly, truncation of the Ptc1 CTD did not result in skin tumors. However, long term labeling studies revealed a greater than three-fold increase in label-retaining cells in the interfollicular epidermis of Ptc1(mes/mes) adults, indicating possible expansion of the epidermal stem cell compartment. Increased expression of regulators of epidermal homeostasis, c-Myc and p63, was also observed in Ptc1(mes/mes) adult skin. These results suggest that the CTD of Ptc1 is involved in regulating epidermal homeostasis in mature skin.

The genetic diseases Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD) arise from accumulation of farnesylated prelamin A because of defects in the lamin A maturation pathway. Both of these diseases exhibit symptoms that can be viewed as accelerated aging. The mechanism by which accumulation of farnesylated prelamin A leads to these accelerated aging phenotypes is not understood. Here we present evidence that in HGPS and RD fibroblasts, DNA damage checkpoints are persistently activated because of the compromise in genomic integrity. Inactivation of checkpoint kinases Ataxia-telangiectasia-mutated (ATM) and ATR (ATM- and Rad3-related) in these patient cells can partially overcome their early replication arrest. Treatment of patient cells with a protein farnesyltransferase inhibitor (FTI) did not result in reduction of DNA double-strand breaks and damage checkpoint signaling, although the treatment significantly reversed the aberrant shape of their nuclei. This suggests that DNA damage accumulation and aberrant nuclear morphology are independent phenotypes arising from prelamin A accumulation in these progeroid syndromes. Since DNA damage accumulation is an important contributor to the symptoms of HGPS, our results call into question the possibility of treatment of HGPS with FTIs alone.

Plectin, a typical cytolinker protein, is essential for skin and skeletal muscle integrity. It stabilizes cells mechanically, regulates cytoskeleton dynamics, and serves as a scaffolding platform for signaling molecules. A variety of isoforms expressed in different tissues and cell types account for this versatility. To uncover the role of plectin 1, the major isoform expressed in tissues of mesenchymal origin, against the background of all other variants, we raised plectin isoform 1-specific antibodies and generated isoform-deficient mice. In contrast to plectin-null mice (lacking all plectin isoforms), which die shortly after birth because of severe skin blistering, plectin isoform 1-deficient mice were viable at birth, had a normal lifespan, and did not display the skin blistering phenotype. However, dermal fibroblasts isolated from plectin 1-deficient mice exhibited abnormalities in their actin cytoskeleton and impaired migration potential. Similarly, plectin 1-deficient T cells isolated from nymph nodes showed diminished chemotactic migration in vitro. Most strikingly, in vivo we found that leukocyte infiltration during wound healing was reduced in the mutant mice. These data show a specific role of a cytolinker protein in immune cell motility. Single isoform-deficient mice thus represent a powerful tool to unravel highly specific functions of plectin variants.

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) transduces signals from members of the TNFR superfamily and the Toll/IL-1R family, leading to activation of transcription factors such as NFkappaB and AP-1. Genetic disruption of the TRAF6 gene in mice results in various developmental abnormalities during embryogenesis, including osteopetrosis, failure of neural tube closure, defective formation of skin appendices, absence of lymph nodes, and absence of mature thymic epithelial cells. To clarify the effect of TRAF6 in development, we previously identified a TRAF-interacting protein with a forkhead-associated domain (TIFA), which binds and activates TRAF6 upon extracellular stimulation. To understand the physiological roles of TRAF6 and TIFA in early development, we studied these genes in Xenopus laevis. Here, we describe identification of X. laevis homologs of mammalian TRAF6 (XTRAF6) and TIFA (XTIFA). As was the case for the mammalian homologs, overexpression of XTRAF6 or XTIFA activated NFkappaB, whereas XTIFA carrying a mutation that abolishes XTRAF6 binding failed to activate NFkappaB, suggesting that XTIFA activates NFkappaB by binding to XTRAF6. XTIFA and XTRAF6 mRNAs were expressed at similar levels in zygotes from the neurula stage and then increased. Whole-mount in situ hybridization revealed that XTRAF6 mRNA was expressed in the head region and neural tube during the neurula stage, and the expression expanded to the pharyngeal apparatus during the tailbud stage. This localization is consistent with the defective neural tube closure and abnormal thymus organogenesis observed in TRAF6-deficient mice. Our results suggest possible cooperation between XTRAF6 and XTIFA during embryogenesis.

The human MrgX3 gene, belonging to the mrgs/SNSRs (mas related genes/sensory neuron specific receptors) family, was overexpressed in transgenic rats using the actin promoter. Two animal lines showed cataracts with liquification/degeneration and swelling of the lens fiber cells. The transient epidermal desquamation was observed in line with higher gene expression. Histopathology of the transgenic rats showed acanthosis and focal parakeratosis. In the epidermis, there was an increase in cellular keratin 14, keratin 10, and loricrin, as well as PGP 9.5 in innervating nerve fibers. These phenotypes accompanied an increase in the number of proliferating cells. These results suggest that overexpression of the human MrgX3 gene causes a disturbance of the normal cell-differentiation process.