Maki Goto

Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer

Harlequin ichthyosis (HI) is a devastating skin disorder with an unknown underlying cause. Abnormal keratinocyte lamellar granules (LGs) are a hallmark of HI skin. ABCA12 is a member of the ATP-binding cassette transporter family, and members of the ABCA subfamily are known to have closely related functions as lipid transporters. ABCA3 is involved in lipid secretion via LGs from alveolar type II cells, and missense mutations in ABCA12 have been reported to cause lamellar ichthyosis type 2, a milder form of ichthyosis. Therefore, we hypothesized that HI might be caused by mutations that lead to serious ABCA12 defects. We identify 5 distinct ABCA12 mutations, either in a compound heterozygous or homozygous state, in patients from 4 HI families. All the mutations resulted in truncation or deletion of highly conserved regions of ABCA12. Immunoelectron microscopy revealed that ABCA12 localized to LGs in normal epidermal keratinocytes. We confirmed that ABCA12 defects cause congested lipid secretion in cultured HI keratinocytes and succeeded in obtaining the recovery of LG lipid secretion after corrective gene transfer of ABCA12. We concluded that ABCA12 works as an epidermal keratinocyte lipid transporter and that defective ABCA12 results in a loss of the skin lipid barrier, leading to HI. Our findings not only allow DNA-based early prenatal diagnosis but also suggest the possibility of gene therapy for HI.

Humanization of autoantigen

Transmissibility of characteristic lesions to experimental animals may help us understand the pathomechanism of human autoimmune disease. Here we show that human autoimmune disease can be reproduced using genetically engineered model mice. Bullous pemphigoid (BP) is the most common serious autoimmune blistering skin disease, with a considerable body of indirect evidence indicating that the underlying autoantigen is collagen XVII (COL17). Passive transfer of human BP autoantibodies into mice does not induce skin lesions, probably because of differences between humans and mice in the amino acid sequence of the COL17 pathogenic epitope. We injected human BP autoantibody into Col17-knockout mice rescued by the human ortholog. This resulted in BP-like skin lesions and a human disease phenotype. Humanization of autoantigens is a new approach to the study of human autoimmune diseases.

Epidermolysis Bullosa Simplex Associated with Pyloric Atresia Is a Novel Clinical Subtype Caused by Mutations in the Plectin Gene (PLEC1)

Epidermolysis bullosa (EB) is an inherited mechano-bullous disorder of the skin, and is divided into three major categories: EB simplex (EBS), dystrophic EB, and junctional EB (JEB). Mutations in the plectin gene (PLEC1) cause EBS associated with muscular dystrophy, whereas JEB associated with pyloric atresia (PA) results from mutations in the alpha6 and beta4 integrin genes. In this study, we examined three EB patients associated with PA from two distinct families. Electron microscopy detected blister formation within the basal keratinocytes leading to the diagnosis of EBS. Surprisingly, immunohistochemical studies using monoclonal antibodies to a range of basement membrane proteins showed that the expression of plectin was absent or markedly attenuated. Sequence analysis demonstrated four novel PLEC1 mutations. One proband was a compound heterozygote for a nonsense mutation of Q305X and a splice-site mutation of 1344G-->A. An exon-trapping experiment suggested that the splice-site mutation induced aberrant splicing of the gene. The second proband harbored a heterozygous maternal nonsense mutation, Q2538X and homozygous nonsense mutations R1189X. Analysis of the intragenic polymorphisms of PLEC1 suggested that R1189X mutations were due to paternal segmental uniparental isodisomy. These results indicate that PLEC1 is a possible causative gene in this clinical subtype, EBS associated with PA. Furthermore, two patients out of our three cases died in infancy. In terms of clinical prognosis, this novel subtype is the lethal variant in the EBS category.

Epidermolysis bullosa simplex in Japanese and Korean patients: genetic studies in 19 cases

Background  Epidermolysis bullosa simplex (EBS) comprises a group of hereditary bullous diseases characterized by intraepidermal blistering caused by mutations in either keratin gene, KRT5 or KRT14. Significant correlation between the position of mutations within these proteins and the clinical severity of EBS has been noted. A recent report showed EBS cases in Israel had unique genetic features compared with European or U.S.A. associated families, which suggests that the ethnic and geographical features of EBS patients may be different.

Genetic studies of 20 Japanese families of dystrophic epidermolysis bullosa

AbstractDystrophic EB (DEB) is clinically characterized by mucocutaneous blistering in response to minor trauma, followed by scarring and nail dystrophy, and is caused by mutations in the COL7A1 gene encoding type VII collagen. DEB is inherited in either an autosomal dominant (DDEB) or recessive (RDEB) fashion. DDEB basically results from a glycine substitution mutation within the collagenous domain on one COL7A1 allele, while a combination of mutations such as premature stop codon, missense, and splice-site mutations on both alleles causes RDEB. In this study, mutation analysis was performed in 20 distinct Japanese DEB families (16 RDEB and four DDEB). The result demonstrated 30 pathogenic COL7A1 mutations with 16 novel mutations, which included four missense, five nonsense, one deletion, two insertion, one indel, and three splice-site mutations. We confirmed that Japanese COL7A1 mutations were basically family specific, although three mutations, 5818delC, 6573+1G>C, and E2857X, were recurrent based on previous reports. Furthermore, the Q2827X mutation found in two unrelated families would be regarded as a candidate recurrent Japanese COL7A1 mutation. The study furthers our understanding of both the clinical and allelic heterogeneity displayed in Japanese DEB patients.

A Novel Humanized Neonatal Autoimmune Blistering Skin Disease Model Induced by Maternally Transferred Antibodies

All mammal neonates receive maternal Abs for protection against pathogenic organisms in the postnatal environment. However, neonates can experience serious adverse reactions if the Abs transferred from the mother recognize self-molecules as autoAgs. In this study, we describe a novel model for autoimmune disease induced by transferred maternal Abs in genetically transformed Ag-humanized mice progeny. Bullous pemphigoid is the most common life-threatening autoimmune blistering skin disease that affects the elderly, in which circulating IgG autoAbs are directed against epidermal type XVII collagen (COL17). We have established a genetically manipulated experimental mouse model in which maternal Abs against human COL17 are transferred to pups whose skin expresses only human and not mouse COL17, resulting in blistering similar to that seen in patients with bullous pemphigoid. Maternal transfer of pathogenic Abs to humanized neonatal mice is a unique and potential experimental system to establish a novel autoimmune disease model.

Direct injection of plasmid DNA into the skin induces dermatitis by activation of monocytes through toll‐like receptor 9

Direct injection of naked DNA into skin can be efficiently used to transfer genes into keratinocytes in vivo. However, bacterial DNA is known to be a potent stimulus for vertebrate immune cells and immune systems. Towards the clinical use of this method, this study examined whether the application of plasmid DNA by direct injection induces any adverse skin effects.

Keratinocyte-/Fibroblast-Targeted Rescue of Col7a1-Disrupted Mice and Generation of an Exact Dystrophic Epidermolysis Bullosa Model Using a Human COL7A1 Mutation

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe hereditary bullous disease caused by mutations in COL7A1, which encodes type VII collagen (COL7). Col7a1 knockout mice (COL7(m-/-)) exhibit a severe RDEB phenotype and die within a few days after birth. Toward developing novel approaches for treating patients with RDEB, we attempted to rescue COL7(m-/-) mice by introducing human COL7A1 cDNA. We first generated transgenic mice that express human COL7A1 cDNA specifically in either epidermal keratinocytes or dermal fibroblasts. We then performed transgenic rescue experiments by crossing these transgenic mice with COL7(m+/-) heterozygous mice. Surprisingly, human COL7 expressed by keratinocytes or by fibroblasts was able to rescue all of the abnormal phenotypic manifestations of the COL7(m-/-) mice, indicating that fibroblasts as well as keratinocytes are potential targets for RDEB gene therapy. Furthermore, we generated transgenic mice with a premature termination codon expressing truncated COL7 protein and performed the same rescue experiments. Notably, the COL7(m-/-) mice rescued with the human COL7A1 allele were able to survive despite demonstrating clinical manifestations very similar to those of human RDEB, indicating that we were able to generate surviving animal models of RDEB with a mutated human COL7A1 gene. This model has great potential for future research into the pathomechanisms of dystrophic epidermolysis bullosa and the development of gene therapies for patients with dystrophic epidermolysis bullosa.

Response of intractable skin ulcers in recessive dystrophic epidermolysis bullosa patients to an allogeneic cultured dermal substitute.

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited skin disorder caused by mutations in the COL7A1 gene, which encodes collagen VII (COL7). Skin ulcers in RDEB patients are sometimes slow to heal. We describe here the therapeutic response of intractable skin ulcers in two patients with generalized RDEB to treatment with an allogeneic cultured dermal substitute (CDS). Skin ulcers in both patients epithelialized by 3-4 weeks after this treatment. Immunohistochemical studies demonstrated that the COL7 expression level remained reduced with respect to the control skin and that it did not differ significantly between graft-treated and untreated areas. Electron microscopy showed aberrant anchoring fibrils beneath the lamina densa of both specimens. In conclusion, CDS is a promising modality for treatment of intractable skin ulcers in patients with RDEB, even though it does not appear to increase COL7 expression.

Compound Heterozygous ABCA12 Mutations Including a Novel Nonsense Mutation Underlie Harlequin Ichthyosis

Recently, it has been reported that several harlequin ichthyosis (HI) patients survive the neonatal period and their condition subsequently improves. Here we describe a 2-year-old Japanese boy who exhibited typical clinical features of HI at birth. He survived beyond the neonatal period after oral retinoid treatment and, at the age of 2 years, showed moderately thick, lamellar scales and erythroderma over his whole body. The patient is a compound heterozygote for 2 ABCA12 mutations, a paternal deletion mutation c.2021_2022del (p.Lys674ArgfsX63) and a novel maternal nonsense mutation c.7444C → T (p.Arg2482X). Electron microscopic observation of a skin biopsy specimen from the perinatal period revealed epidermal ultrastructural features consistent with HI. Immunofluorescence labeling using antiserum against a C-terminal ABCA12 epitope showed loss of expression in the patient’s epidermis. The present patient demonstrates that rapid diagnosis of HI by ABCA12 expression analysis and mutation detection, and early commencement of systemic retinoid therapy are crucial to significantly improving an HI patient’s prognosis.