Yasuko Yamanaka

CGI-58 Is an α/β-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes

CGI-58 is the causative molecule underlying Dorfman-Chanarin syndrome, a neutral lipid storage disease exhibiting apparent clinical features of ichthyosis. CGI-58, associated with triacylglycerol hydrolysis, has an alpha/beta-hydrolase fold and is also known as the alpha/beta-hydrolase domain-containing protein 5. The purpose of this study was to elucidate the function of CGI-58 and the pathogenic mechanisms of ichthyosis in Dorfman-Chanarin syndrome. Using an anti-CGI-58 antibody, we found CGI-58 to be expressed in the upper epidermis, predominantly in the granular layer cells, as well as in neurons and hepatocytes. Immunoelectron microscopy revealed that CGI-58 was also localized to the lamellar granules (LGs), which are lipid transport and secretion granules found in keratinocytes. CGI-58 expression was markedly reduced in the epidermis of patients with harlequin ichthyosis, demonstrating defective LG formation. In cultured keratinocytes, CGI-58 expression was mildly up-regulated under high Ca(2+) conditions and markedly up-regulated in three-dimensional, organotypic cultures. In the developing human epidermis, CGI-58 immunostaining was observed at an estimated gestational age of 49 days, and CGI-58 mRNA expression was up-regulated concomitantly with both epidermal stratification and keratinocyte differentiation. CGI-58 knockdown reduced expression of keratinocyte differentiation/keratinization markers in cultured human keratinocytes. Our results indicate that CGI-58 is expressed and packaged into LGs during keratinization and likely plays crucial role(s) in keratinocyte differentiation and LG lipid metabolism, contributing to skin lipid barrier formation.

Annular Lichen planus: Study of the Cellular Mechanisms of Annularity

Annular lichen planus is a rare, unique subtype of lichen planus. We report a 34-year-old Japanese male who had multiple, dark red to purple, annular macules with slightly raised borders. Histopathological examination of a skin biopsy specimen from the peripheral region of the macule showed the typical features of lichen planus. Immunohistochemical stainings revealed that a number of CD1a-positive, S-100-protein-positive Langerhans cells were present at the border zone of the annular lesion and most of the infiltrating cells were CD4-positive, CD8-negative lymphocytes. Conversely, inside the annular lesion, the number of Langerhans cells was decreased, and an equal number of CD4-positive cells and CD8-positive cells was present in the sparse infiltration. These results suggest that activation of Langerhans cells initiated a lichenoid tissue reaction but a subsequent depletion of Langerhans cells suppressed the lymphocyte infiltration. These sequential events might be involved in the formation of the unique annular structure in this condition.

A novel PTPN11 missense mutation in a patient with LEOPARD syndrome

LEOPARD syndrome (LS) is an autosomal dominant, multiple congenital anomaly syndrome so named because it is characterized by multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonic stenosis, abnormal genitalia, retardation of growth and sensorineural deafness. We report a Japanese boy with LS harbouring the novel PTPN11 (protein-tyrosine-phosphatase nonreceptor type 11) gene mutation c.1381G>T (p.Ala461Ser). An 8-year-old boy had lentigines at birth which gradually increased in number with age. Physical examination revealed multiple light or dark brown lentigines of various sizes scattered over the whole body, including the face (Fig. 1a–c). He also had pigeon breast (Fig. 1d) and cryptorchidism. There was no sensorineural deafness, ocular hypertelorism, growth retardation or cardiology abnormalities including electrocardiographic conduction defect (Fig. 1e–g). A diagnosis of LS was made based on multiple lentigines, skeletal anomalies and genitourinary abnormalities. The patient’s parents gave their written informed consent as the patient’s legal guardians. Direct sequencing of the entire coding regions, exon 2 to exon 15, of PTPN11 (GenBank accession number NT123456) revealed a single nucleotide substitution at codon 461 in exon 12 on one allele of PTPN11 (TGC to TTC; alanine to serine; p.Ala461Ser) (Fig. 2a). The

Keratinocyte lipid transporter ABCA12 is highly expressed In the late stages of human epidermal development

The transfer of normal genes into somatic cells is one strategy to treat patients with genetic diseases. However, this strategy has still encounters technical problems including effi cacy of gene transfer rate and practical clinical safety. Thus, other strategies including pharmacological therapy or gene correction, are receiving increasing attention. Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the COL7A1 encoding type VII collagen. This study examined the feasibility of antisense oligoribonucleotide (AON) therapy for DEB. AON was designed to induce skipping of a targeted exon containing a premature termination codon mutation, resulting in restoration of the open reading frame. We targeted exon 70 of COL7A1 since a recurrent mutation 5818delC was localized to exon 70 in Japanese DEB patients. We initially designed and synthesized two AONs to modulate splicing of exon 70 and found that one AON induced effective skipping of normal exon 70 containing the 16 amino acids. Attachment and migration analyses showed that recombinant collagen without contribution of exon 70 had similar effect to normal type VII collagen. Next, we synthesized mutation-specifi c AON by deleting cytosine at 5818. Introduction of this AON into DEB keratinocytes harboring 5818delC without expression of type VII collagen showed that the AON induced skipping of exon 70 in the abnormal 5818delC allele. Furthermore, 6.2 % of the DEB keratinocytes started to express type VII collagen in vitro after application of the mutation-specifi c AON. Injection of the AON into rat model grafted the DEB keratinocytes and fi broblasts induced detectable of type VII collagen expression at the basement membrane zone. We conclude that skipping of targeted exons using mutation-specifi c AON may show potential for future gene therapy for DEB patients.We recently reported that ABCA12 works as an epidermal keratinocyte lipid transporter, and that defective ABCA12 results in a loss of the skin lipid barrier, leading to harlequin ichthyosis (HI), one of the most devastating genodermatoses. In the present study, precise expression pattern of ABCA12 was studied in human embryonic and fetal skin of 7-22 weeks estimated gestational age (EGA) and newborn skin samples. For controls, we also studied the expression of transglutaminase 1 (TGase1) that is known to cross-link several precursor proteins in the formation of the cornifi ed cell envelope during keratinocyte terminal differentiation. In twolayered epidermis (about 6-9 weeks EGA), both ABCA12 and TGase1 were only expressed in periderm cells. In three-layered epidermis (10-13 weeks EGA), ABCA12 staining was seen not only in periderm, but also throughout the entire epidermis, while TGase1 staining was restricted to the periderm. A similar pattern was observed during four- or more-layered epidermal development (14-22 weeks EGA). In newborn skin, ABCA12 and TGase1 were seen only in the upper epidermal layers, mainly the granular layer. These staining patterns were similar to those in normal adult skin. Next, we studied ABCA12 mRNA expression in extracts of the fetal skin (at 10, 14, 15 weeks EGA). In 15 weeks EGA, the expression level of ABCA12 mRNA was signifi cantly increased compared with that in the early development (10 and 14 weeks EGA). This increasing pattern of ABCA12 mRNA expression is consistent with ABCA12 immunofl uorescent staining during human epidermal development. The unique pattern of ABCA12 expression during human epidermal development might imply severe symptoms of HI patients with ABCA12 mutations around the birth. 2006 ESDR ABSTRACTS www.Mutations in ABCA12 lead to harlequin ichthyosis and lamellar ichthyosis. The keratinocyte lipid transporter ABCA12 is a member of the ATP-binding cassette transporter family, and members of the ABCA subfamily have closely related functions as lipid transporters. Previously, we reported that the pathomechanism of harlequin ichthyosis involves the defective function of the lipid transporter ABCA12.To further elucidate the precise distribution pattern and function of ABCA12, we performed double-labeling immunofl uorescence staining for ABCA12 and for Golgi-associated or lamellar granule-associated molecules both on normal human epidermis and cultured normal human keratinocytes. We studied the precise localization of ABCA12 and other molecules using confocal laser scanning microscope. In normal human epidermis, ABCA12 was observed mainly in the granular layers with glucosylceramide (one of the major lamellar granule contents) and transglutaminase 1 (a cornifi ed cell envelope-associated keratinization marker), but not always colocalized with GM130 and TGN46 (Golgi-related molecules) that were expressed from the lower epidermis. In normal human keratinocytes cultured in high Ca++ concentration medium, ABCA12 colocalized with GM130 (a cis-Golgi- associated molecule), TGN46 (a trans-Golgi-associated molecule) and glucosylceramide. Transglutaminase 1 was restricted to the cell membrane and ABCA12 localization was within the cytoplasm distinct from transglutaminase 1 localization. The present results suggest that ABCA12 is mainly expressed in differentiated, granular layer keratinocytes with glucosylceramide and transglutaminase 1, and, at the subcellular level, ABCA12 is distributed from the cisside of the Golgi apparatus to trans-Golgi network, lamellar granules at the cell periphery. Our results suggest that ABCA12 may play an important role in lipid transport from the cis-side of the Golgi apparatus through the trans-Golgi network, to the cell periphery via lamellar granules in human epidermal granular layer keratinocytesHarlequin ichthyosis (HI) is a devastating genodermatosis that is often fatal during the neonatal period. Until the identifi cation of ABCA12-encoding a keratinocyte lipid transporter, as the causative gene for HI, prenatal diagnosis (PNDx) had been performed for more than 20 years by electron microscopic examination of fetal skin biopsy samples. We report here the fi rst case of DNA-based PNDx for HI. The proband, the fi rst child of healthy non-consanguineous French parents, showed a typical HI phenotype and died soon after birth. ABCA12 immunostaining was markedly reduced in the proband’s skin. Direct sequence analysis of ABCA12 revealed that the proband was a compound heterozygote for two novel mutations: a maternal nonsense mutation p.Ser1249X in exon 26 and a paternal missense mutation p.Arg2479Lys occurring at the last codon of exon 50. p.Ser1249X leads to an approximate 52 % truncation of the ABCA12 protein losing both ATP-binding cassette active sites. p.Arg2479Lys involves a highly conserved codon among diverse species in the second ABCA12 ATP-binding cassette. For their third pregnancy, the parents requested PNDx. Direct sequence analysis using fetal genomic DNA from amniotic fl uid cells at 17 weeks of pregnancy revealed that the fetus was a compound heterozygote for both mutations. The fetus was predicted to be affected and the parents requested the pregnancy to be terminated. The aborted fetus showed typical signs of HI. Analysis of ABCA12 transcripts of cultured keratinocytes from the abortus showed the presence of six abnormally spliced products arising from the allele carrying the missense mutation. Four of them lead to premature termination codons while the two others produced deleted proteins missing 21 and 31 amino acids in the second ATP-binding cassette. These results indicated residual expression of ABCA12. The present report paves the way for molecular PNDx of HI in the earlier stages of pregnancy