053 Novel mutations in the LAMB3 gene in two Japanese families with Herlitz junctional epidermolysis bullosa and their application for prenatal diagnosis

JSID Abstracts

049

052

97-KD LINEAR IgA BULLOUS DBRMATOSIS ANTIGEN LOCALIZES AT LAMINA LUCIDA BETWEEN NC16A DOMAIN .AND C-TERM!;AL &&iko .H. DOMAIN OF IgO-KD ?ULLOU,s Pt?MPHIGOI? ANTIGEN. 4 I C. Yee .K.cey .G. 1. Gim . &&&a!. Department of Dermatology, ’ Keio University School of Medicine, Tokyo, ’ Nippon Kokan Hospital, Kawasaki. Japan. ’ NIH, Bethesda, MD, ’ Medical College of Wisconsin. Milwaukee. WI, ’ University 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 possssses multiple regions of amino acid identity with the extracellular domain of IgO-kD bullous pemphigoid antigen (BPAGZ). To investigate further rslstionship between these autoantigens, ultrauruchtrsl localis&n ws studied by immunogold electron microscopy usine crvoulttsthin sectionsof normal human skin. Pol~clonal antibodies against the NClbA domain of BPAG2 immunolsbcled along the plasma membrsue of the hemidesmosomsl complex. Polyclousl antibodies against C-teninal domain of BPAG2 immunolabeled the lamina denss-lamina lucida interface. In contrast, two distinct monoclonal antibodies against 97- LAD mainly bound to the lamina lucida ssif sandwiched by theNCl6Adomain and C-tertuinal domain of BPAGZ. These results of co-localization in ultrsstntcturnl level suggest the possibility that 97-LAD is closely related to, and may form s complex with, BPAGZ.

RECURRENT MUTATIONS IN COL7Al IN DYSTROPHIC EPIDERYOLYSIS BULLOSA: A STUDY OF 20 PATIENTS REFERRED TO KEIO UNIVERSITY HOSPITAL. T, 1.2 H. hi Dept. of Dermatology. Keio Univ. School of Basic Research Lab., KO&, Corp., Tokyo, Japan, sDept. of Dermatology. Hirosakl Univ. School of Medicine. Hirosaki, Japan, 4Dept. of Dermatology, Jefferson Msdlcal College, Phlladslphia. USA. Dystrophlc epldsrmolysis bullosa (DEB) is caused by mutations in the type VII collagen gene, COUAt. The purpose of this study was to examine the occurrence of three recurrent mutations In COL7Al (581SdalC. 6676+1QtoC. 8572GtoT) previously reported in Japanese DEB patients. Total genomic DNA extracted from peripheral blood was subjected to PCR amplifloation, followed by hslsroduplsx analysis, direct nwleolide sequencing, and restriction endonuclease digestion. As a result. the 5618delC, 6576+1GtoC, and 8572GtoT mulallons were detected In 4, 1, and 3 cases, respecllvely. among the 20 DEB patients studied, while they were not present in three pallenls with degnlte dominant DEB. One patient. a compound heterozygote of 5SlgdelC/S572QtoT, showed positive sxprssslon of type VII collagen at Me epidermal basement membrane. as detennlned by Indirect lmmunoflwrescsnce. In conclusion, cerlaln COL7Al mutations reoccur In DEB, and their ldsntiflcatlon facllltates mutation detection In Japanese DEB patlents.

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111

DISAPPEARANCE OF o I (3 I INTEGRIN IN THE LESIONAL SKIN OF THE PATIENTS WITH BULLOUS PEMPHIGOID WITH CIRCULATING ANTIBODIES AGAINST BPlgO AND/OR BP230. T.lida l ‘.T.Mursmstu ’ , C.Nskstsai ‘,T.Shirsi t.’ Dspartment of Dcmtatulogy, Nsrs Prefectural Hospital, Nsrs,Jspsu. ’ Depsrtment of Demtstology, Nut’s Medical University, Kushihars,

Japan

To know the mechanism of the blister formation in bullous pemphigoid(BP),we exsmittsd the expression of bssement membrane zone (BMZ) cutttpuneuts such BS a I i-n, 8 4 integris lsminid, type IV collagen. type W collagen and BP180 eutigeu in the lesional skin of the patients with BP(n=Zg),atypical BP(n=l) ,snd epidemtolysis bulloss uquisits(EBA,n=l) NaCI-separated normal skin (n=3) were used ss ccmtrul. The expteasion of BMZ components were evaluated by immunutluoresceace (IF) by using monuclonal antibodies. In addition, circulating anti-BMZ antibodies were examined by IF wing normsl skin and by Western immuuoblotting(lB)usiug nonual epidcruml extrsct. In all csses of BP, the cxpressiou of o. integrin and B 4 h&grin completely disappeared in the lesional skin. These BP set’s reacted with IgOkD and/or 230kD antigens by IB. In contrsst. in the skin frum the patients with atypical BP and EBA. and salt-spilt normal skin, linear IF of o a integrin aad B 4 in&grin was observed. These results suggest that anti-BMZ antibodies sre responsible for the damage of trsnsmembrsnc adhesion molecules of hemidesmusumes in the lesional skin of the cases of typical BP.

054

051 CARBOXY-TEFMIN

AL STRUCTURE OF PLECTIN-2. Taknn. Department of Dermatology, Oita Medical Oita, Japan

University,

The serum from an individual prsviously

shown to recognize

The molecttlar cell cDNA related.

smtcme

library

with s subspidsrmal

a 450-kDs

with the patient’s

O.S- and l.O-kb

cDNAs

serum.

were

latter

terminal

domain which

disease wss

by screening a HeLa different,

but closely

the former

was almost

was similar but not identical with of 450-kDa

was named plectin-2. contains

VARIABLE EXPRESSION OF PLECTIN EPITOPES IN PATIENTS WITH EPIDERMOLYSIS BULLOSA SIMPLEX ASSOCIATED WITH

autoantigen

Two

isolated,

plectin. Epitope mapping showed the cDNA and this molecule

blistering

epidsrmal

of this antigen wss investigated

identical with human plectin. and the

latter

nutosomnl recessive disorder. junctionuJ epidemwlysis bullosa (JEB). In this study, we p.erfonuedgenetic analyses in tw uurelatedJapanesehmilies with Herlitz junctional epidemwlysis bullcsa (H-JEB) and identified we novel nunsen~emutations in the LAMB3 gene. The Ql66X mutation (CAG->TAG). was found in the matemnl allele of family I sud the paternal allele of family 2. The W6lOX mutation (TGG->TGAJ. was found in the patemsl allele of family I uud the maternal allele of family 2. Thus! the probands of both families were compound hcteruzygotes for both u~useuse mutuuuus and their pwents were heteruzygous carriers of each mutation. Both mutations cause premature temtination of trsuslatiou resulting in truncated 83 chain resulting in the absenceexpression of laminin 5 in the epidermul basemeut membranezone. Basedon these data, DNA-bed prenataldiagnosis wus perfomted by churionie villus sampling for the subsequeutpregusucies in both families. Both fetuses were heterozygous for W6lOX mutation but had no Ql66X mutation indicating that they were betemryguus carriers of one of the mutations and were predicted to be phenotypically normal in teruu of this blistering disorder.

three highly

autoantigen was the

It has a unique homologous

amino acids. This feature was shared with desmoplakin,

csrboxy-

repeats of

534

but not with human

or rat plectin. Another splicing variant was also found. This data suggest that plectio-2

might

play an important

role in interaction

with

keratins

and

w5~6~7~g~g,~l lKeio Univ. School of Med., Japan.2Nugoya Univ., 3Uuiv. of Viennn. 4Jichii Medical School, 5Kururue Univ. School of Med. 6Nagasoki Univ. 7Shimoshiru Hosp., glefferson Medical College, USA. We recently identified homozygous deletion mutations in the plectin gene in 2 Japanesefamilies with EBSiMD (Hum Mol Geuet 5: 1539.1996). In this study, we have studied the expression puttem of pie& and HDI, a probable isofortu of plectin. in the skin of 4 unrelatedpatieuts with EBSiMD using u panel of MoAbs ngoinst plectin (583. lOF6l or HDI (121. E2, KI5, 156). All MoAbs bound to the epidermol basement membraue (BM) of normsl human skin by IF and inner plaque of kmidesmoaomes by humunueleetmn micmrcopy. In addition. cell surfan labeling of kaatinocyte was detected with 583. lOF6, KIS uud 156.. Basement membranelabeling of ull MoAbs was completely abseut in 3 ptients with sews muscular dystmphy but only slightly reduced in I patieut with milder clinical scvetily who wus homoryguus for u 9-hp inframe deletion mutation of tk plectin gene. Kaatinocyte cell surface labeling of 583 and IOF wus also altered situhar to the bwement membrane Isbeling, whereas that of KI5 nnd I56 was clearly present in spite of disuppesrsuce of basementmembranelabeling. These datn suggest that the expression of plectin epitopes is varinbk in patients with EBS/MD, and HDI might be au isoform of plectin.