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aUTOANTIBODIES FROM PATIENTS WITH CICATRICIAL PEMPHIGOID RECOGNIZE LAMININ a3 CHAIN MONOMER.
Chihiro Matsui, Taro Kitagawa, Minoru Ohtsuyama, Masaaki Morohashi.
Department of Dermatology, Toyama Medical and Pharmaceutical University, Toyama, JAPAN.
Recent studies have identified a group of cicatricial pemphigoid patients who have IgG anti-basement membrane autoantibodies that recognize epiligrin, a heterotrimeric protein closely related or identical to laminin 5. To further understand the pathophysiology of blister formation in these patients, we have sought to identify the specific polypeptide(s) and epitope(s) targeted by their auto antibodies. Comparative studies show that sera from these patients and polyclonal antibody immunoprecipitate the same set of disulfide-linked polypeptides from media and cell fraction of biosynthetically radiolabeled human keratinocytes. Moreover, these sera immunoprecipitate a3 subunit monomer of laminin 5 from keratinocyte cell fraction but show no reactivity to b3 and g2 monomers. The same sera precipitate same polypeptides from mucous membrane-derived keratinocytes. The epitope mapping using bacterial fusion protein of a3 subunit is now in progress
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97-KD LINEAR IgA BULLOUS DERMATOSIS ANTIGEN LOCALIZES AT LAMINA LUCIDA BETWEEN NC16A DOMAIN AND C-TERMINAL DOMAIN OF 180-KD BULLOUS PEMPHIGOID ANTIGEN.
Akira Ishiko1, 2, Hiroshi Shimizu1, Takuji Masunaga1, Carole Yee3, Kim B. Yancey3, George J. Giudice4, John J. Zone5, Takeji Nishikawa1.
Department of Dermatology, 1Keio University School of Medicine, Tokyo, 2Nippon Kokan Hospital, Kawasaki, Japan, 3NIH, Bethesda, MD, 4Medical College of Wisconsin, Milwaukee, WI, 5University of Utah School of Medicine, Salt Lake City, UT, USA.
97-kD linear IgA bullous dermatosis antigen (97-LAD), localizing at epidermal basement membrane zone, is the major autoantigen of this autoimmune blistering disease and possesses multiple regions of amino acid identity with the extracellular domain of 180-kD bullous pemphigoid antigen (BPAG2). To investigate further relationship between these autoantigens, ultrastructural localization was studied by immunogold electron microscopy using cryoultrathin sections of normal human skin. Polyclonal antibodies against the NC16A domain of BPAG2 immunolabeled along the plasma membrane of the hemidesmosomal complex. Polyclonal antibodies against C-terminal domain of BPAG2 immunolabeled the lamina densa-lamina lucida interface. In contrast, two distinct monoclonal antibodies against 97- LAD mainly bound to the lamina lucida as if sandwiched by the NC16A domain and C-terminal domain of BPAG2. These results of co-localization in ultrastructural level suggest the possibility that 97-LAD is closely related to, and may form a complex with, BPAG2.
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DISAPPEARANCE OF a6b4 integrin IN THE LESIONAL SKIN OF THE PATIENTS WITH BULLOUS PEMPHIGOID WITH CIRCULATING ANTIBODIES AGAINST BP180 AND/OR BP230.
Takashi Iida1,Tsutomu Muramatsu2,Chie Nakatani2,Toshihiko Shirai2.
1Department of Dermatology, Nara Prefectural Hospital, Nara, 2Department of Dermatology, Nara Medical University, Kashihara, Japan.
To know the mechanism of the blister formation in bullous pemphigoid(BP),we examined the expression of basement membrane zone(BMZ)components such as integrin, £integrin, laminin5, typecollagen, typecollagen and BP180 antigen in the lesional skin of the patients with BP(n=28),atypical BP(n=1),and epidermolysis bullosa aquisita(EBA,n=1). NaCl-separated normal skin(n=3)were used as control. The expression of BMZ components were evaluated by immunofluorescence(IF)by using monoclonal antibodies. In addition, circulating anti-BMZ antibodies were examined by IF using normal skin and by Western immunoblotting(IB)using normal epidermal extract. In all cases of BP, the expression of integrin and £integrin completely disappeared in the lesional skin. These BP sera reacted with 180kD and/or 230kD antigens by IB. In contrast, in the skin from the patients with atypical BP and EBA, and salt-spilt normal skin, linear IF of integrin and £integrin was observed. These results suggest that anti-BMZ antibodies are responsible for the damage of transmembrane adhesion molecules of hemidesmosomes in the lesional skin of the cases of typical BP.
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CARBOXY-TERMINAL STRUCTURE OF PLECTIN-2.
Sakuhei Fujiwara, Naoko Takeo.
Department of Dermatology, Oita Medical University, Oita, Japan.
The serum from an individual with a subepidermal blistering disease was previously shown to recognize a 450-kDa epidermal autoantigen. The molecular structure of this antigen was investigated by screening a HeLa cell cDNA library with the patient's serum. Two different, but closely related, 0.8- and 1.0-kb cDNAs were isolated, the former was almost identical with human plectin, and the latter was similar but not identical with plectin. Epitope mapping showed the cDNA of 450-kDa autoantigen was the latter and this molecule was named plectin-2. It has a unique carboxy-terminal domain which contains three highly homologous repeats of 534 amino acids. This feature was shared with desmoplakin, but not with human or rat plectin. Another splicing variant was also found. This data suggest that plectin-2 might play an important role in interaction with keratins and hemidesmosomal elements.
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recurrent MUTATIONS IN COL7A1 IN DYSTROPHIC EPIDERMOLYSIS BULLOSA:a STUDY OF 20 PATIENTS REFERRED TO KEIO UNIVERSITY HOSPITAL.
Takuji Masunaga1, 2, Hiroshi Shimizu1, Yasuko Takizawa1, Takeji Nishikawa1, Katsuto Tamai3, Isao Hashimoto3, Jouni Uitto4.
1Department of Dermatology, Keio University School of Medicine, 2Basic Research Laboratory, KOS Corporation, Tokyo, 3Department of Dermatology, Hirosaki University School of Medicine, Hirosaki, Japan. 4Department of Dermatology, Jefferson Medical Colleg
Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the type VII collagen gene, COL7A1. The purpose of this study was to examine the occurrence of three recurrent mutations in COL7A1 (5818delC, 6576+1GtoC, 8572GtoT) previously reported in Japanese DEB patients. Total genomic DNA extracted from peripheral blood was subjected to PCR amplification, followed by heteroduplex analysis, direct nucleotide sequencing, and restriction endonuclease digestion. As a result, the 5818delC, 6576+1GtoC, and 8572GtoT mutations were detected in 4, 1, and 3 cases, respectively, among the 20 DEB patients studied, while they were not present in three patients with definite dominant DEB. One patient, a compound heterozygote of 5818delC/8572GtoT, showed positive expression of type VII collagen at the epidermal basement membrane, as determined by indirect immunofluorescence. In conclusion, certain COL7A1 mutations re-occur in DEB, and their identification facilitates mutation detection in Japanese DEB patients.
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NOVEL MUTATIONS IN THE LAMB3 GENE IN TWO JAPANESE FAMILIES WITH HERLITZ JUNCTIONAL EPIDERMOLYSIS BULLOSA AND THEIR APPLICATION FOR PRENATAL DIAGNOSIS.
Yasuko Takizawa1, Hiroshi Shimizu1, Leena Pulkkinen3, Yoshiki Hiraoka2, John A. McGrath4, Kaoru Suzumori5, Sadakazu Aiso2, Jouni Uitto3, Takeji Nishikawa1.
1Department of Dermatology, 2Department of Anatomy, Keio University School of Medicine, Tokyo, Japan. 3Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, PA, U.S.A. 4Department of Cell Pathology, St. John's Institute
The LAMB3 gene encoding the b3 chain of laminin 5 is a candidate gene in the autosomal recessive disorder, junctional epidermolysis bullosa (JEB). In this study, we performed genetic analyses in two unrelated Japanese families with Herlitz junctional epidermolysis bullosa (H-JEB) and identified two novel nonsense mutations in the LAMB3 gene. The Q166X mutation (CAG>TAG), was found in the maternal allele of family 1 and the paternal allele of family 2. The W610X mutation (TGG>TGA), was found in the paternal allele of family 1 and the maternal allele of family 2. Thus, the probands of both families were compound heterozygotes for both nonsense mutations and their parents were heterozygous carriers of each mutation. Both mutations cause premature termination of translation resulting in truncated b3 chain resulting in the absence expression of laminin 5 in the epidermal basement membrane zone. Based on these data, DNA-based prenatal diagnosis was performed by chorionic villus sampling for the subsequent pregnancies in both families. Both fetuses were heterozygous for W610X mutation but had no Q166X mutation indicating that they were heterozygous carriers of one of the mutations and were predicted to be phenotypically normal in terms of this blistering disorder.
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VARIABLE EXPRESSION OF PLECTIN EPITOPES IN PATIENTS WITH EPIDERMOLYSIS BULLOSA SIMPLEX ASSOCIATED WITH MUSCULAR DYSTROPHY (EBS/MD).
Hiroshi Shimizu1, Yukio Kurihara1, Katsushi Owaribe2, Gerhard Wiche3, Satoru Murata4, Hideo Yaoita4, Hiroshi Hachisuka5, Masako Udono6, Atsushi Kawai7, Leena Pulkkinen8, Jouni Uitto8, Takeji Nishikawa1.
1Keio University School of Medicine, Japan, 2Nagoya University, 3University of Vienna, 4Jichii Medical School, 5Kurume University School of Medicine, 6Nagasaki University, 7Shimoshizu Hospital, 8Jefferson Medical College, USA.
We recently identified homozygous deletion mutations in the plectin gene in 2 Japanese families with EBS/MD (Hum Mol Genet 5:1539,1996). In this study, we have studied the expression pattern of plectin and HD1, a probable isoform of plectin, in the skin of 4 unrelated patients with EBS/MD using a panel of MoAbs against plectin (5B3, 10F6) or HD1 (121, E2, K15, 156). All MoAbs bound to the epidermal basement membrane (BM) of normal human skin by IF and inner plaque of hemidesmosomes by immunoelectron microscopy. In addition, cell surface labeling of keratinocyte was detected with 5B3, 10F6, K15 and 156. . Basement membrane labeling of all MoAbs was completely absent in 3 patients with severe muscular dystrophy but only slightly reduced in 1 patient with milder clinical severity who was homozygous for a 9-bp inframe deletion mutation of the plectin gene. Keratinocyte cell surface labeling of 5B3 and 10F6 was also altered similar to the basement membrane labeling, whereas that of K15 and 156 was clearly present in spite of disappearance of basement membrane labeling. These data suggest that the expression of plectin epitopes is variable in patients with EBS/MD, and HD1 might be an isoform of plectin.