ㄡㄡ莢ざ呿蒂菜⻆扡粗喀莘鍚莘’噤狤

ㄡㄡ莢ざ呿蒂菜⻆扡粗喀莘鍚莘’噤狤

-127- Genetic Disease/Genetherapy 9/5 Room-B
THE MOLECULAR DEFECT IN HUMAN ERYTHROPOIETIC PROTOPORPYRIA
Tokihiko Shimada1, Seita Fukumaru1, Sinichi Yotsumoto1, Mina Masuda2, Keiko Kobayashi2, Shigeru Taketani3, Takeyori Saheki2, Tamotsu Kanzaki1.
1Department of Dermatolgy, 21st Department of Biochemistory, Kagoshima University Faculty of Medicine, Kagoshima, 3Department of Hygiene, Kansai Medical University, Morichuchi, Osaka, Japan.
Erythropoietic protoporphyria (EPP) is characterized an elevated protporphyrin level, photosensitivity and sometimes hepatobiliary disease. EPP is caused by ferrochelatase (EC4.99.1.1) deficiency and is considered to be inherited in an aoutsomal-dominant manner. Since the ferrochelatase cDNA and gene were cloned and sequenced, detailed analysis of the molecular defects underlying EPP has become possible . In this study, we report the molecular basis of ferrochelastase in 5 Japanese patients from 3 non-consanguineous families with exon 9 skipping in ferrochelatase mRNA.

-128- Genetic Disease/Genetherapy 9/5 Room-B
LINKAGE ANALYSIS OF JAPANESE HAILEY-HAILEY DISEASE (HHD) KINDREDS WITH DNA MARKERS AT CHROMOSOME 3Q.
Shigaku Ikeda1, Takako Shigihara1, Mika Koike1, Chizuru Watanabe1, Ervin H. Epstein Jr2, Hideoki Ogawa1.
1Departments of Dermatology, Juntendo University, School of Medicine, Tokyo, Japan. 2University of California, San Francisco, USA.
We have previously mapped HHD gene to ch3q21-24, between DNA markers D3S1589 and D3S1541. That localization was obtained by linkage analysis of European and Middle-Eastern ancestry. In an effort to extend the universality of these findings, we now have analyed Japanese HHD kindreds by DNA markers in the region of these loci. Although the limited number of available family members has prevented the attainment of a statistically significant LOD score, no recombinants were observed at these tested loci. These results indicate that locus heterogeneity is at least uncommon even in Japanese (and also probably in other Agian) kindreds and suggest that mutations in a single gene underlie the Hailey-Hailey phenotype in all human population.

-129- Genetic Disease/Genetherapy 9/5 Room-B
SEQUENCES OF EXON-INTRON JUNCTIONS AND GENOMIC ORGANIZATION OF SERINE/THREONINE PROTEIN PHOSPHATASE (PPP1CC) AS A CANDIDATE GENE FOR DARIER'S DISEASE.
Nobuyasu Mayuzumi1, Shigaku Ikeda1, Epstein H.Epstein Jr.2, Hideoki Ogawa1.
1Department of Dermatology, Juntendo University, School of Medicine, Tokyo, Japan. 2University of California aSan Francisco, San Francisco, USA.
Work from several laboratories has localized the gene whose mutations cause Darier's Disease to small region at chromosome 12q23-24.1. As part of our efforts to identify the actual gene, we are screening genes that have been localized to this region. One such gene is PPP1CC, the protein product of which is a phosphatase that potentially dephosphorylates several molecules and controles cellular functions, We report here the genomic organization and intron-exon junction of this gene. Our next goal is to utilize the intron sequences to design primers that will allow us to amplify each exon and to use single strand conformation polymorphism (SSCP) analysis to test for PPP1CC mutations in patients with Darier's disease.

-130- Genetic Disease/Genetherapy 9/5 Room-B
THE ANALYSIS OF a-GALACTOSIDASE IN A PATIENT WITH FABRY'S DISEASE.
Shinnosuke Ishida , Akio Yamakage , Soji Yamazaki, Kaoru Ichimura, Shigeru Matuzaki.
Department of Dermatology, Dokkyo University School of Medicine, Tochigi, Japan.
THE ANALYSIS OF 汐-GALACTOSIDASE IN A PATIENT WITH FABRY'S DISEASE. S.Ishida*1, A.Yamakage1, S.Yamazaki1, K.Ichimura2, S.Matuzaki2. 1Department of Dermatology and 2Biochemistry, Dokkyo University School of Medicine, Tochigi, Japan. Fabry's disease is an X-chromosome-linked recessive disorder of glycosphingolipid catabolism due to a deficiency of 汐-galactosidase A (Gal A). Recently we have experienced a mild phenotype of Fabry's disease. We measured his Gal A activity and analyzed the gene mutation. We also did family study. Gal A activity was measured with digestion of p-nitrophenyl-汐-D-galactoside by leukocyte extract. Two genomic fraguments of Gal A were amplified by long PCR and bilateral sides of each fragment containing exons were cut by the restriction enzymes. The fragments were constructed to the plasmid vector. The nucleotide sequence was determined by the method of Sanger et al. Family study was performed by the method of restriction fragment length polymorphisms. The patient had a residual activity of this enzyme (about 9.7% of control levels), and father, mother and brother had normal activity. The sequence analysis of the genomic DNA of Gal A revealed the mutation, R112C (112Arg◥Cys, 334C◥T) in exon 2. Patient's father and brother showed normal pattern but mother was heterozygote.

-131- Genetic Disease/Genetherapy 9/5 Room-B
PROMOTER/ENHANCER CASSETTES FOR KERATINOCYTE GENE THERAPY: CONTROL OF TRANSGENE EXPRESSION IN KERATINO- CYTES IN VIVO.
Xianmin Meng, Daisuke Sawamura, Sinsuke Ina, Hajime Nakano, Katsuto Tamai, Katumi Hanada, Isao Hashimoto.
Department of Dermatology, Hirosaki University School of Medicine, Hirosaki, Japan.
Successful keratinocyte gene therapy requires the promoter/enhancercassette to regulate the expression of the theraputic gene properly inkeratincyets in vivo. Plasmids, which were constructed with variouspromoter/enhancer cassette fused with the lacZ, were in vivo transferred into keratinocytes using the naked DNA method. Activities of those promoter/enhancer cassettes were evaluated by measuring 汕-galactosidase activity in keratinocytes. The CMV-IE promoter exhibited the highest activity, whereas the chicken 汕-actin and the human keratin 10 promoter showed relati vely strong expression in cellular promoters. The CMV-IE enhancer stronglyincreased the activity of chicken 汕-actin by 15-fold. Another interesting result is that the activity of transferred metallothionein promoter can be increased 2.5-fold or 8-fold by systemic or topical application of zinc and dexamethasone and 3-fold by UVB irradiation. When a 69% fragment of bovine papilloma virus was connected with metallothionein and K10 promoters, the 汕-gal activity was increased and the 汕-gal gene expression was extended. These results indicate the importance of promoter/enhancer cassette inkeratinocyte gene therapy.

-132- Genetic Disease/Genetherapy 9/5 Room-B
IN VIVO GENE TRANSFER TO KERATINOCYTE-USEFULLNESS OF HIGH MOBILITY GROUP-1 PROTEIN AND GENE INTRODUCTION OF TGF-a AND TGF-b2-.
Shinsuke Ina, Daisuke Sawamura, Xianming Meng, Katsuto Tamai, Katsumi Hanada, Isao Hashimoto.
Department of Dermatology, Hirosaki University School of Medicine, Hirosaki, Japan.
For keratinocyte gene therapy, we must develop an efficient introduction method. As the method by which gene is introduced to keracinocyte directly, we have reported effectiveness of hemmaggluting virus of Japan ( HVJ ) -liposome method ( Kaneda et al, 1989, Sawamura et al, 1996 ), in which gene and high mobility group-1 ( HMG-1 ) are introduced simultaneously. Recently, it has been shown that the method that naked DNA is injected directly into the skin( naked DNA method ) is also effective ( Henngge et al﹞1995 ). Taking advantages of HVJ-liposome method and naked DNA method, we injected the naked DNA bound with HMG-1 in the skin, which resulted in an increase of introducing efficiency. Using this method, we introduced TGF-汐 and TGF-汕2 expression vectors and observed histological changes in the skin.