232 Genetics o f MHC 24 June 1997 - Poster presentations

Examples are the CD1 and FCRN genes, as well as the recently describad human MRI gone which maps to chromosome lq25 (Hashimoto et el. Science 269: 693). In an attempt to identify novel Mhc class I-like genes of the rat we applied the PCR strategy originally designed to isolate class I genes of bony fishes (Hashimoto et el. PNAS 91: 6863).

Material and Methods: For amplification of class I-like sequences, degon- erete primers derived from conserved regions of the fourth exon were used. Rat Mr1 cDNA was established by RACE techniques. Expression of the rat Mr1 gone was examined by northem blot using total RNA from=aplean, lymphoblasts, thymus, liver, kidney, heart, brain, lung, small intestine, placenta and testis. Total lung RNA was used for RT-PCR analysis.

Results: PCR revealed products of expected length of 120 bp which were cloned and sequenced. One clone showed a high degree of homology to the human MRI gone. The corresponding complete cDNA sequence of 1.4 kb was isolated by RACE techniques and showed an open reading frame with sequence similarity of 780 at the nucleotlde and 74% at the amino acid level to the human MRI gone. Thus the rat homolog of the human MRI gone was identified. According to Southam blot data, Mr1 represents a single copy gane. Preliminary evidence indicates that this gone is not linked to the rat MHC. Expression analysis by non'hem blotting showed two Mr1 transcripts of about 1.6 kb and 5 kb. RT-PCR revealed the expected amplificate and an additional one the sequence of which lacked the information for the complete a2 domain. Thus, alternative splicing of the Mr1 transcript occurs.

Conclusions: A novel class I-like gane of the rat has been identified which is the homolog of the human MR1 gone. The rat Mrl gone is broadly expressed, and alternatively spliced transcripts occur.

lP .1 .03 .231 A cel l l ine wi th defect o f new l ymphob l ss to i d e I

c lass II HLA exp ress ion but de r i ved f rom c lass I I -poei t ive pat ient

M. Maficzak, J. Dubis, A. Ko~, K. Baldy-Chudzik, M. Mechnicki, R. Pacholczyk, M. Prussak, B. Nowakowska, P. Ku~niemzyk. L. Hirszfeld Institute of Immunology and Experimental Theral~, Weigla 12, 53-114 Wroolaw, Poland

Introduction: Several lymphoblastoid cell lines with various defects of HLA class II expression had been described, shedding light on regulation of class II gone expression. Here, we present a new lymphoblastoid cell line (LCL) with such a defect, derived from a class II-positive patient.

Material and Methods: Lymphoblastoid cell line (LCL) HAJ was derived from peripheral blood lymphocytes of female patient with renal insufficiency. HLA class II expression was tasted by: (a) flow cytofluorimetry using a panel of monoclonal antibodies; (b) RT-PCR using HLA-DQA1 and -DPA1 locus-specific primers. The presence of HLA-DRB1, -DQA1 and -DPA1 genes was also looked for using amplification from gonomic DNA with locus-specific primers. Another LCL, PAJ, expressing cell surface HLA class II antigens, served as positive control.

Results: Flow cytofluorimetry revealed presence of HLA class I antigens on both HAJ and PAJ cells. In contrast, class II antigens were absent from the cell surface of HAJ cells while they were abundant on PAJ cells. Permeabilization and fixation of cells with acetone/formaldehyde solution revealed intracallular Ki-67 antigen but no class II molecules. The genes HLA-DRB1, -DQA1 and -DPA1 were present in the HAJ ganome. In RT-PCR, transcripts of DQA1 and DPA1 genes were easily detectable in PAJ but not in HAJ cells.

Conclusion: Results suggest a defect of transcription of genes for all class II antigens in HAJ cells. Fusion with cells with already described defects of transcription factors should revealed the nature of regulatory defect in HAJ cells.

P.1.03.241 Ev idence for gene t i c he te rogene i t y I

in IBD. HLA genes in the p red lspos lUon to suf fer f rom u lcerat ive co l i t i s and Crohn 's d l sssss

G. Bourne I J.B.A. Crusius 1 G.M.Th. Schreuder 3, H.P.R. Hsilemans 1, B.U.G.A. Meijer I , P.J. Kostense 2, M.J. Giphart 3, S.G.M. Meuwissen 1 , A.S. Pefia 1.1 Department of Gastroenterology, Free University Hospital Amsterdam, The Netherlands, 2Department of Epidamiology and Biostatistics, Free University Hospital Amsterdam, The Netherlands, 3Department of Immunohaematology, Academic Hospital Leiden, Leiden, The Netherlands

Introduction: Family and epidemiological studies support a genetic suscepti- bility to suffer from ulcerative colitis and Crohn's disease. Conflicting reports regarding associations between ulcerative colitis and HLA-DR2 and between Crohn's disease and various HLA alleles have been published. The aim of this study was to determine whether molecularly defined HLA-DR genes are associated with these diseases in a Dutch group of patients.

Materials end Methods: Unrelated Dutch ulcerative colitis patients (n = 59) and Crohn's disease patients (n = 89) were typed using molecular, DNA-based methods. 2400 healthy local blood donors served as controls.

Results: The phenotype frequency of the HLA-DRB1*15 allele was in- creased in UC patients as compared to controls (42% versus 26% in controls; p = 0.006; odds ratio (OR) = 2.1), and was predominantly found in female UC patients (53% versus 24%; p = 0.001; OR = 3.5). The HLA-DRB1*15 allele was increased in UC patients having a positive family history (p = 0.01; OR 5.8). Among the sixteen patients who showed an increase in extent of disease during follow-up, ten were HLA-DRB1*15 positive (p = 0.002; OR 4.8). The frequency of HLA-DRB1*15 was significantly increased in those patients who developed arthritis as 6/11 patients with arthritis were HLA-DRB1*15 positive (p = 0.04; OR = 3.5). In CD, no association was obsewed between disease or particular clinical subgroups and any allele tested.

Conclusion: The present study provides additional evidence for the genetic association between ulcerative colitis and HLA-DRB1*15 and supports recent findings that the susceptibility gone(s) for Crehn's disease is not located in the HLA-class II region.

I P.1.03.251 Exp ress ion o f the Ea gene is hep lo type-spec l f l ca l l y I I modu la ted by • p o l y m o r p h l c t ranscr ip t iona l

enhancer

M. Janitz, L. Reiners-Schramm, R. Lauster. Deutsches Rheumaforschungszentrum, Berlin, Germany

Introduction: The truncetlon of the 5' flanking sequence of the Ea gone results in reduced in B lymphocytes. Sequence analysis of this B-cell specific Ea distal control region revealed the presence of regulatory motifs celled the X' and Y' box, which are located in reverse orientation to the X and Y box found in the promoter region. Similar motifs have been identified in the Eb and Ab genes. Uttle is known whether distal regulatory elements of MHC class II genes exhibit any degree of sequence polymorphism. Moreover, previous functional studies of the distal region have not concemed its influence on the allele-specific vadants of the MHC class II promoters. To address this question we here analyse promoter function under the influence of allalic variations of the distal regulatory fragment in four mouse class II haplotypee.

Materials and Methods: Distal enhancer elements of the Ea, Eb and Ab genes were amplified by PCR from gonomic DNA isolated from four mouse strains homozygous for b, d, k and q haplotypa of H-2 complex. Amplified fragments were sequenced and cloned into transient expression plasmids, 3 kb upstream from the respective MHC class II promoters and luciferase gone. Plasmid constructs were transfected to non- or stimulated B cells by alectroporetlon and after 24 h the luciferase activity was measured.

Results: We sequenced enhancer elements of the Ea, Eb and Ab loci from four mouse habiotypes and found silelic polymorphism in the case of the Ea locus, which was confined to the region surrounding the Y' box. The enhancers of Eb and Ab were non-polymorphic. This observation is in contrast to our previous promoter sequence analysis, where the Ea gone was shown to be conserved between haplotypes, but Eb and Ab were polymorphic. Transfection of non-stimulated and stimulated B cells with reporter gone constructs containing both the promoter and the distal regulatory regions of the Ea, Eb and Ab genes from four hablotypes resulted in enhancement of the basic promoter activity when compared with the constructs containing the promoter alone. However this effect can be observed only in non-stimulated B cells. The range of enhancement was haplotype specific.

Conclusion: Differential MHC class II promoter activities may result in a haplotype-apecific number of MHC class II molecules on the surface of antigen presenting cells, thus influencing their interaction with Th0 lymphocytas. Since antigen presentation by B cells is suggested to result in generation of the Th2 cells whereas Th cells interacting with macrophagas develop into Thl cells, dif- ferential levels of the MHC class gone expression may create a haplotypa-spe- cific ability of the individual to develop either Thl of Th2 response. This might be achieved by favouring antigen presentation by the cell type with stronger MHC class II expression (e.g. B cells versus macrophagos). The consequence might be the susceptibility or resistance of certain MHC class II haplotypes to autoimmune processes. Moreover, the level of MHC class II expression can also regulate the number of the B cells as it was shown in the recent studies on the Ab transgone.