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1.
Blood ; 127(8): 997-1006, 2016 Feb 25.
Article in English | MEDLINE | ID: mdl-26744459

ABSTRACT

Genetic disorders affecting biogenesis and transport of lysosome-related organelles are heterogeneous diseases frequently associated with albinism. We studied a patient with albinism, neutropenia, immunodeficiency, neurodevelopmental delay, generalized seizures, and impaired hearing but with no mutation in genes so far associated with albinism and immunodeficiency. Whole exome sequencing identified a homozygous mutation in AP3D1 that leads to destabilization of the adaptor protein 3 (AP3) complex. AP3 complex formation and the degranulation defect in patient T cells were restored by retroviral reconstitution. A previously described hypopigmented mouse mutant with an Ap3d1 null mutation (mocha strain) shares the neurologic phenotype with our patient and shows a platelet storage pool deficiency characteristic of Hermansky-Pudlak syndrome (HPS) that was not studied in our patient because of a lack of bleeding. HPS2 caused by mutations in AP3B1A leads to a highly overlapping phenotype without the neurologic symptoms. The AP3 complex exists in a ubiquitous and a neuronal form. AP3D1 codes for the AP3δ subunit of the complex, which is essential for both forms. In contrast, the AP3ß3A subunit, affected in HPS2 patients, is substituted by AP3ß3B in the neuron-specific heterotetramer. AP3δ deficiency thus causes a severe neurologic disorder with immunodeficiency and albinism that we propose to classify as HPS10.


Subject(s)
Adaptor Protein Complex 3/genetics , Adaptor Protein Complex delta Subunits/genetics , Hermanski-Pudlak Syndrome/classification , Hermanski-Pudlak Syndrome/genetics , Immunologic Deficiency Syndromes/genetics , Seizures/genetics , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique , Humans , Mutation , Transfection
2.
Nanomedicine ; 11(5): 1179-87, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25791808

ABSTRACT

Genetic skin diseases caused by mutations resulting in diminished protein synthesis could benefit from local substitution of the missing protein. Proteins, however, are excluded from topical applications due to their physicochemical properties. We prepared protein-loaded thermoresponsive poly(N-isopropylacrylamide)-polyglycerol-based nanogels exhibiting a thermal trigger point at 35°C, which is favorable for cutaneous applications due to the native thermal gradient of human skin. At≥35°C, the particle size (~200nm) was instantly reduced by 20% and 93% of the protein was released; no alterations of protein structure or activity were detected. Skin penetration experiments demonstrated efficient intraepidermal protein delivery particularly in barrier deficient skin, penetration of the nanogels themselves was not detected. The proof of concept was provided by transglutaminase 1-loaded nanogels which efficiently delivered the protein into transglutaminase 1-deficient skin models resulting in a restoration of skin barrier function. In conclusion, thermoresponsive nanogels are promising topical delivery systems for biomacromolecules. FROM THE CLINICAL EDITOR: Many skin disorders are characterized by an absence of a specific protein due to underlying gene mutation. In this article, the authors described the use of a thermoresponsive PNIPAM-dPG nanogel for cutaneous protein delivery in a gene knock-down model of human skin. The results may have implication for nano-based local delivery of therapeutic agents in skin.


Subject(s)
Acrylic Resins/chemistry , Delayed-Action Preparations/chemistry , Gels/chemistry , Glycerol/chemistry , Polymers/chemistry , Skin/metabolism , Transglutaminases/administration & dosage , Administration, Cutaneous , Animals , Asparaginase/administration & dosage , Asparaginase/pharmacokinetics , Cattle , Delayed-Action Preparations/metabolism , Gels/metabolism , Gene Knockdown Techniques , Glycerol/metabolism , Humans , Polymers/metabolism , Serum Albumin, Bovine/administration & dosage , Serum Albumin, Bovine/pharmacokinetics , Skin/ultrastructure , Skin Absorption , Swine , Temperature , Testosterone/administration & dosage , Testosterone/pharmacokinetics , Transglutaminases/genetics , Transglutaminases/pharmacokinetics
3.
J Control Release ; 185: 45-50, 2014 Jul 10.
Article in English | MEDLINE | ID: mdl-24727058

ABSTRACT

A growing intended or accidental exposure to nanoparticles asks for the elucidation of potential toxicity linked to the penetration of normal and lesional skin. We studied the skin penetration of dye-tagged dendritic core-multishell (CMS) nanotransporters and of Nile red loaded CMS nanotransporters using fluorescence microscopy. Normal and stripped human skin ex vivo as well as normal reconstructed human skin and in vitro skin disease models served as test platforms. Nile red was delivered rapidly into the viable epidermis and dermis of normal skin, whereas the highly flexible CMS nanotransporters remained solely in the stratum corneum after 6h but penetrated into deeper skin layers after 24h exposure. Fluorescence lifetime imaging microscopy proved a stable dye-tag and revealed striking nanotransporter-skin interactions. The viable layers of stripped skin were penetrated more efficiently by dye-tagged CMS nanotransporters and the cargo compared to normal skin. Normal reconstructed human skin reflected the penetration of Nile red and CMS nanotransporters in human skin and both, the non-hyperkeratotic non-melanoma skin cancer and hyperkeratotic peeling skin disease models come along with altered absorption in the skin diseases.


Subject(s)
Drug Carriers/metabolism , Nanoparticles/metabolism , Oxazines/administration & dosage , Skin/metabolism , Drug Carriers/analysis , Female , Humans , Microscopy, Fluorescence , Nanoparticles/analysis , Oxazines/pharmacokinetics , Particle Size , Skin/pathology , Skin Absorption , Skin Diseases/metabolism , Skin Diseases/pathology
4.
Exp Dermatol ; 23(4): 286-8, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24612062

ABSTRACT

The aim of this study was to assess a recently established 3D model of congenital ichthyosis, representing severe epidermal barrier function defects, for skin penetration and permeation. We have generated disease models by knock-down of either TGM1 or ALOXE3 in primary human keratinocytes, and using keratinocytes and fibroblasts from patients with congenital ichthyosis. The results indicate disturbed barrier function as demonstrated by increased permeation of testosterone and caffeine particularly in TGM1 knock-down models compared to control models. In addition, enhanced penetration of the model dye nile red incorporated into solid lipid nanoparticles and core-multishell nanotransporters, respectively, was evident in disease models. Thus, in vitro skin disease models reproduce differences in barrier permeability and function seen in congenital ichthyosis and pave the way to personalised disease models. Furthermore, our findings indicate that nanocarriers may be useful in new, topical therapeutic approaches for the currently very limited treatment of congenital ichthyosis.


Subject(s)
Ichthyosiform Erythroderma, Congenital/metabolism , Skin Absorption , Tissue Engineering , 3T3 Cells , Aged , Animals , Child , Fibroblasts , Humans , Keratinocytes , Male , Mice
5.
J Invest Dermatol ; 133(9): 2202-11, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23549421

ABSTRACT

The barrier function of the human epidermis is supposed to be governed by lipid composition and organization in the stratum corneum. Disorders of keratinization, namely ichthyoses, are typically associated with disturbed barrier activity. Using autozygosity mapping and exome sequencing, we have identified a homozygous missense mutation in CERS3 in patients with congenital ichthyosis characterized by collodion membranes at birth, generalized scaling of the skin, and mild erythroderma. We demonstrate that the mutation inactivates ceramide synthase 3 (CerS3), which is synthesized in skin and testis, in an assay of N-acylation with C26-CoA, both in patient keratinocytes and using recombinant mutant proteins. Moreover, we show a specific loss of ceramides with very long acyl chains from C26 up to C34 in terminally differentiating patient keratinocytes, which is in line with findings from a recent CerS3-deficient mouse model. Analysis of reconstructed patient skin reveals disturbance of epidermal differentiation with an earlier maturation and an impairment of epidermal barrier function. Our findings demonstrate that synthesis of very long chain ceramides by CerS3 is a crucial early step for the skin barrier formation and link disorders presenting with congenital ichthyosis to defects in sphingolipid metabolism and the epidermal lipid architecture.


Subject(s)
Ceramides/biosynthesis , Ichthyosis, Lamellar/genetics , Ichthyosis, Lamellar/pathology , Sphingosine N-Acyltransferase/genetics , Sphingosine N-Acyltransferase/metabolism , Animals , Cells, Cultured , Ceramides/chemistry , Child , Child, Preschool , Disease Models, Animal , Epidermal Cells , Epidermis/pathology , Exome/genetics , Family Health , Female , Fibroblasts/cytology , Genes, Recessive , Homozygote , Humans , Infant, Newborn , Keratinocytes/cytology , Male , Mice , Molecular Weight , Mutation, Missense , Pedigree , Phenotype
6.
Altern Lab Anim ; 39(5): 471-80, 2011 Oct.
Article in English | MEDLINE | ID: mdl-22103940

ABSTRACT

Loss-of-function mutations in the filaggrin gene (FLG) are a strong predisposing factor for atopic dermatitis, although their relevance to the disease pathomechanism needs further elucidation. The generation of an in vitro model of atopic skin would not only permit further evaluation of the underlying pathogenetic mechanisms and the testing of new treatment options, but would also allow toxicological studies to be performed in a simple, rapid and inexpensive manner. In this study, we have knocked down FLG expression in human keratinocytes and created three-dimensional skin models, which we used to investigate the impact of FLG on epidermal maturation and on skin absorption and its response to irritation. Histopathological evaluation of the skin models showed impaired epidermal differentiation in the FLG knock-down model. In addition, skin irritation induced by an application of sodium dodecyl sulphate resulted in significantly higher lactate dehydrogenase leakage, and interleukin (IL)-6 and IL-8 levels, than in the control model. To assess the effect of filaggrin deficiency on skin absorption of topically applied agents, we quantified the percutaneous absorption of lipophilic and hydrophilic model drugs, finding clinical relevance only for lipophilic drugs. This study clearly demonstrates that important clinical characteristics of atopic skin can be mimicked by using in vitro skin models. The FLG knock-down construct is the first step toward an in vitro model that allows clinical and toxicological studies of atopic-like skin.


Subject(s)
Dermatitis, Atopic/pathology , Fibroblasts/metabolism , Intermediate Filament Proteins/genetics , Intermediate Filament Proteins/metabolism , Keratinocytes/metabolism , Animals , Cells, Cultured , Fibroblasts/cytology , Filaggrin Proteins , Gene Silencing , Humans , Keratinocytes/cytology , Models, Biological , Mutation , Tissue Culture Techniques
7.
Am J Hum Genet ; 89(4): 564-71, 2011 Oct 07.
Article in English | MEDLINE | ID: mdl-21944047

ABSTRACT

Autosomal-recessive exfoliative ichthyosis presents shortly after birth as dry, scaly skin over most of the body with coarse peeling of nonerythematous skin on the palms and soles, which is exacerbated by excessive moisture and minor trauma. Using whole-genome homozygosity mapping, candidate-gene analysis and deep sequencing, we have identified loss-of-function mutations in the gene for protease inhibitor cystatin A (CSTA) as the underlying genetic cause of exfoliative ichthyosis. We found two homozygous mutations, a splice-site and a nonsense mutation, in two consanguineous families of Bedouin and Turkish origin. Electron microscopy of skin biopsies from affected individuals revealed that the level of detachment occurs in the basal and lower suprabasal layers. In addition, in vitro modeling suggests that in the absence of cystatin A protein, there is a cell-cell adhesion defect in human keratinocytes that is particularly prominent when cells are subject to mechanical stress. We show here evidence of a key role for a protease inhibitor in epidermal adhesion within the lower layers of the human epidermis.


Subject(s)
Cystatin A/genetics , Ichthyosis/genetics , Mutation , Protease Inhibitors/metabolism , Amino Acid Sequence , Base Sequence , Cell Adhesion , Epidermis/metabolism , Family Health , Female , Foot/pathology , Genome , Homozygote , Humans , Male , Models, Genetic , Molecular Sequence Data , Pedigree , Sequence Homology, Amino Acid , Stress, Mechanical
9.
Am J Hum Genet ; 87(2): 274-81, 2010 Aug 13.
Article in English | MEDLINE | ID: mdl-20691404

ABSTRACT

Generalized peeling skin disease is an autosomal-recessive ichthyosiform erythroderma characterized by lifelong patchy peeling of the skin. After genome-wide linkage analysis, we have identified a homozygous nonsense mutation in CDSN in a large consanguineous family with generalized peeling skin, pruritus, and food allergies, which leads to a complete loss of corneodesmosin. In contrast to hypotrichosis simplex, which can be associated with specific dominant CDSN mutations, peeling skin disease is characterized by a complete loss of CDSN expression. The skin phenotype is consistent with a recent murine Cdsn knockout model. Using three-dimensional human skin models, we demonstrate that lack of corneodesmosin causes an epidermal barrier defect supposed to account for the predisposition to atopic diseases, and we confirm the role of corneodesmosin as a decisive epidermal adhesion molecule. Therefore, peeling skin disease will represent a new model disorder for atopic diseases, similarly to Netherton syndrome and ichthyosis vulgaris in the recent past.


Subject(s)
Glycoproteins/deficiency , Glycoproteins/genetics , Pruritus/complications , Pruritus/genetics , Base Sequence , Child , Chromosome Mapping , DNA Mutational Analysis , Epidermis/pathology , Family , Humans , Intercellular Signaling Peptides and Proteins , Male , Models, Biological , Molecular Sequence Data , Pedigree , Skin/pathology , Skin/ultrastructure
10.
J Invest Dermatol ; 129(6): 1421-8, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19131948

ABSTRACT

In recent years several new genes for autosomal recessive congenital ichthyosis (ARCI) have been identified. However, little is known about the molecular epidemiology and pathophysiology of this genetically and clinically heterogeneous group of severe disorders of keratinization. ARCI is characterized by intense scaling of the whole integument often associated with erythema. We and others have shown that mutations in ALOX12B and ALOXE3, coding for the lipoxygenases 12R-LOX and eLOX-3 predominantly synthesized in the epidermis, can underlie this rare condition. Here we have surveyed a large group of 250 patients with ARCI for mutations in these two genes. We have identified 11 different previously unreported mutations in ALOX12B and ALOXE3 in 21 ARCI patients from 19 unrelated families and demonstrated that mutations in the two genes are the second most common cause for ARCI in this cohort of patients. Examination of the molecular data revealed allelic heterogeneity for ALOX12B and two mutational hotspots in ALOXE3. Functional analysis of all missense mutations and a splice site mutation demonstrated that complete loss of function of the enzymes underlies the phenotype. Our findings further establish the pivotal role of the 12-lipoxygenase pathway during epidermal differentiation.


Subject(s)
Arachidonate 12-Lipoxygenase/genetics , Arachidonate 12-Lipoxygenase/metabolism , Ichthyosiform Erythroderma, Congenital/genetics , Lipoxygenase/genetics , Lipoxygenase/metabolism , Mutation , Alleles , Cohort Studies , DNA Mutational Analysis , Exons , Genes, Recessive , Haplotypes , Humans , Models, Genetic , Phenotype , Protein Structure, Tertiary , Recombinant Proteins/chemistry
11.
Prostaglandins Other Lipid Mediat ; 82(1-4): 128-34, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17164140

ABSTRACT

12R-lipoxygenase (12R-LOX) and epidermis-type LOX-3 (eLOX-3) are novel members of the multigene family of mammalian LOX. A considerable gap exists between the identification of these enzymes and their biologic function. Here, we present evidence that 12R-LOX and eLOX-3, acting in sequence, and eLOX-3 in combination with another, not yet identified LOX are critically involved in terminal differentiation of keratinocytes and adipocytes, respectively. Mutational inactivation of 12R-LOX and/or eLOX-3 has been found to be associated with development of an inherited ichthyosiform skin disorder in humans and genetic ablation of 12R-LOX causes a severe impairment of the epidermal lipid barrier in mice leading to post-natal death of the animals. In preadipocytes, a LOX-dependent PPARgamma activating ligand is released into the cell supernatant early upon induction of differentiation and available evidence indicates that this ligand is an eLOX-3-derived product. In accordance with this data is the observation that forced expression of eLOX-3 enhances adipocyte differentiation.


Subject(s)
Arachidonate 12-Lipoxygenase/physiology , Epidermis/enzymology , Lipoxygenase/physiology , Skin Physiological Phenomena , Adipocytes/cytology , Adipocytes/enzymology , Animals , Cell Differentiation , Gene Expression , Humans , Ichthyosis/genetics , Ichthyosis/physiopathology , Keratinocytes/cytology , Lipoxygenase/genetics , Mice , Permeability
12.
Hum Mutat ; 26(4): 351-61, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16116617

ABSTRACT

Autosomal-recessive congenital ichthyosis (ARCI) is a clinically and genetically heterogeneous group of severe hereditary keratinization disorders characterized by intense scaling of the whole integument, and differences in color and shape. It is often associated with erythema. To date, six loci for ARCI have been mapped. Mutations in ALOXE3 and ALOX12B on chromosome 17p13, which code for two different epidermal lipoxygenases, were recently found in patients with ichthyosiform erythroderma from Turkey, France, and North Africa. Here we describe molecular and clinical findings in 17 families with ARCI originating from Central Europe, Turkey, and the Indian subcontinent, with mutations in ALOXE3 or ALOX12B. We identified 11 novel point mutations in ALOX12B (one nonsense mutation and 10 missense mutations) and four different inactivating mutations in ALOXE3. The gene products of ALOX12B and ALOXE3, the epidermal lipoxygenases 12R-LOX and eLOX3, respectively, are preferentially synthesized in the skin. They act in sequence to convert arachidonic acid via 12(R)-HPETE to the corresponding epoxyalcohol, 8(R)-hydroxy-11(R),12(R)-epoxyeicosatrienoic acid. To assess the impairment of enzyme activity, we expressed the mutated genes in vitro and determined the activity of the recombinant proteins toward their genuine substrates. All but one of the recombinant mutants were enzymatically inactive. The characterization of disease-causing mutations in ALOXE3 and ALOX12B and the resulting ARCI phenotypes did not result in clear diagnostic criteria; however, we found a first correlation between the genetic findings and the clinical presentation of ichthyosis.


Subject(s)
Genes, Recessive , Ichthyosiform Erythroderma, Congenital/metabolism , Lipoxygenase/physiology , Point Mutation , Arachidonate 12-Lipoxygenase/genetics , Arachidonate 12-Lipoxygenase/physiology , Catalysis , Chromatography, High Pressure Liquid , Epidermal Cells , Epidermis/enzymology , Epidermis/metabolism , Homozygote , Humans , Ichthyosiform Erythroderma, Congenital/genetics , Lipoxygenase/genetics , Lipoxygenase/metabolism , Loss of Heterozygosity , Microsatellite Repeats , Phenotype , Population Groups/genetics , Protein Structure, Tertiary , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
13.
Arch Dermatol Res ; 296(12): 585-7, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15875205

ABSTRACT

A 56 year old man presented with ichthyosis vulgaris since early childhood, clinically characterised by fine scaling of the trunk and hyperkeratotic scales on the exterior surfaces of the upper and lower extremities. The patient also showed hypothyroidism due to hypoplastic thyroid, cataract, hypercholesterinemia with concommitant arcus cornealis and biliary concrements. Renal lithiasis caused by calcio-oxalate was additionally present. Endocrinological screening revealed growth hormone deficiency in the 1.55 m tall man-(secondary) osteoporosis was observed. The clinical symptomatology indicates that this case cannot be considered as a subtype of the inherited ichthyosis group, but suggests a new syndrome as a separate nosologic entity.


Subject(s)
Cataract/etiology , Corneal Opacity/etiology , Human Growth Hormone/deficiency , Ichthyosis Vulgaris/complications , Keratosis/etiology , Thyroid Gland/pathology , Humans , Hypothyroidism/etiology , Ichthyosis Vulgaris/genetics , Male , Middle Aged , Syndrome
14.
Hum Genet ; 112(1): 50-6, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12483299

ABSTRACT

Mal de Meleda (MDM) or keratosis palmoplantaris transgrediens of Siemens is an autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma (PPK) and transgressive keratosis with an onset in early infancy. There is no associated involvement of other organs; however, a spectrum of clinical presentations with optional and variable features has been described. Mutations in the ARS (component B)-81/s gene ( LY6LS) on chromosome 8q24-qter, which encodes SLURP-1, have recently been identified in patients with MDM. Here, we have analyzed four MDM families for mutations in SLURP-1. In a large Palestinian pedigree with multiple consanguinity, patients are homozygous for a new mutation that substitutes an arginine for a conserved glycine residue at position 86. A different mutation in Turkish patients results in the same amino acid exchange. Some remarkable similarities are seen in the clinical picture of patients from both families. Patients of an Emirati Bedouin family have a homozygous alteration of the translation initiation codon. In a German family with no known consanguinity, we have shown pseudodominant inheritance. Three affected children and their affected mother are homozygous for the missense mutation W15R. Our findings indicate that the MDM type of transgressive PPK is caused by SLURP-1 mutations in patients from various origins and demonstrate allelic heterogeneity for mutations in SLURP-1.


Subject(s)
Antigens, Ly/genetics , Keratoderma, Palmoplantar/genetics , Mutation , Urokinase-Type Plasminogen Activator/genetics , Adult , Amino Acid Substitution , Arginine/metabolism , Chromosomes, Human, Pair 8 , Consanguinity , Female , Genes, Recessive , Genetic Heterogeneity , Germany , Haplotypes , Homozygote , Humans , Infant , Keratoderma, Palmoplantar/complications , Keratoderma, Palmoplantar/etiology , Keratoderma, Palmoplantar/pathology , Male , Mutation, Missense , Pedigree , Point Mutation , Turkey/ethnology , United Arab Emirates
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