Brief Communications

Association of primary sclerosing cholanrritis with HLA=DRw52a? v

N. Grunnet, H. H. Rasmussen, U. Tage-Jensen, S. Nerrby Rasmussen. Association of primary sclerosing cholangitis with HLA-DRw52a? Tissue Antigens 1991: 38: 133-136.

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N. Grunnet, H. H. Rasmussen, U. TageJsnsan and S. Warby Rasmussen Regional Center for Blood Transfusion and Clinical Immunology, and Department of Medical Gastroenterology, Aalborg Hospital, Aalborg, Denmark

Key words: primary sclerosing cholangitis RFLP - oligonucleotides-HIA association Received 18 April, revised, accepted for publication 11 June 1991

In a recent report (l), using a serological technique, it was stated that the antigen HLA-DRw52a could be identified in 100% of 29 patients with primary sclerosing cholangitis (PSC) from a population normally presenting this antigen in only 35% of individuals. Fifteen of the 29 patients had a single common haplotype: Al,B8,Cw7,DRw17,DQw2, DRw52a (1). To investigate if this very clear finding could be shown in a homogeneous population of Danes, we decided to HLA type age- and sex-matched groups of patients with ulcerative colitis either with or without PSC. PSC is a chronic cholestatic liver disease characterized by inflammation, obliterative fibrosis, and segmental dilation of the intrahepatic and extrahepatic bile ducts. PSC frequently occurs in association with ulcerative colitis but may occur alone (2). Out of 298 patients with ulcerative colitis, we found 11 to have PSC (3). The diagnoses of PSC were based on clinical, biochemical, histologic and, most important, radiologic criteria with diffuse irregularity and narrowing of the extrahepatic and intrahepatic bile ducts, according to Wiesner et al. (2). Two of the patients with PSC died of cholangiocarcinoma, leaving 9 patients to be included in this study. Nine matched patients with only ulcerative colitis were included as a reference group. HLA typings were done 1) by standard NIH serology methods using the lymphocytotoxicity micromethods for HLA-ABC and HLA-DR,DQ and 2) by HLA typing at the DNA level using the

restriction fragment length polymorphism (RFLP) method performed by a procedure as described by Grunnet et al. (4) and the polymerase chain reaction (PCR) method, in principle as described by Fernandez-Vina et al. (5) according to our modification of the protocols used in the 1lth International Histocompatibility Workshop studies. The genomic DNA was prepared by a salting-out procedure (4)and then amplified. The PCR reaction mixture consisted of 1 pg genomic DNA, PCR buffer (10 mM Tris-HC1 pH 8.4;50 mM KCl; 1.5 mM MgCl,, 0.01% gelatin), 2 mM each of dATP, dCTP, dGTP, and TTP 50 pmol of each of the primers and distilled water to a total volume of 100 p1. The mixture was covered with 100 pl mineral oil to prevent evaporation and heated for 10 min at 99"C, then 2 units Taq DNA Polymerase (Perkin Elmer/Cetus Corp.) was added. The DRB generic amplification and the DRw52 group (DRB3 gene) were both accomplished by 35 cycles of incubation at 94°C for 30 seconds, 64°C for 1 min, and 72°C for 2 min using the primers DRBAMP-A and DRBAMP-B for generic amplification and DRBAMP-52 and DRBAMP-B for group-specific amplification. The primers have these sequences: DRBAMP-A: CCCCACAGCACGTTTGTTG DRBAMP-B:CCGCTGCACTGTGAAGCTCT DRBAMP-52:CCCAGCACGTTTCTTGGAGCT. Two microliters of each PCR product were spotted on positively-charged nylon membrane (Boehringer Mannheim) and dried at room temperature (RT); then re-wet with 0.4N NaOH for 5 min, 133

Grunnet et al. soaked in 10 x SSPE for 10 min, dried at RT and illuminated with UV-lamp. The filters were prehybridized at 54°C in 10 ml of a solution containing 50 mM Tris-HC1pH 8.0; 3.OM tetramethylammonium chloride, 2 mM EDTA pH 8.0; 5 x Denhardt solution, 0.1 % SDS and 100 pg/ml heat denatured herring sperm DNA. After 1 hour the 32P-labelled probe was added to the solution and hybridization allowed to continue for 2 h at 54°C. Then the filters were rinsed twice for 10 min at RT with 2 x SSPE, 0.1 % SDS, then 10 min at RT in tetramethylammonium chloride (TMAC) solution (50 mM Tris-HC1 pH 8.0; 3.0 M tetramethylammonium chloride, 2 mM EDTA, 0.1 YOSDS), and finally the filters were washed five times for 10 min at 59°C in TMAC solution. All washing procedures were performed in a hybridization incubator. The synthetic oligonucleotides used in the experiments were each 18 nucleotides long and were designed to hybridize with the various alleles of the DRw53 and DRw52 group on DRB4 and DRB3 amplified genes (see Table 1 for the nucleotide sequences and specificities of the selected sequence-specific oligonucleotides (SSO)). The probes were 5' 32Pend labelled and set up in the following reaction mixture: 10 pmol SSO; 2.5 pl 10 x kinase buffer (0.5M Tris HCl pH 7.6; 0.1M MgC1,; 50 mM DTT; 1 mM spermidin HCl), 60 pCi ( -"P)-ATP, 20 units T, Polynucleotide kinase and dH,O to bring total reaction volume to 25 p1; mixed and incubated at 37°C for 30 min, then the reaction was stopped by adding 1 pl of 0.5 M EDTA pH 8.0. All HLA typings were done without knowledge of the diagnosis. For comparison, the frequency of HLA-DRw52a was found to be 36.7% in a popula-

tion of 98 healthy Danes determined with the RFLP technique (4). Assignment of HLA-DRw52a, -DRw52b, -DRw52c, and -DRw53 by RFLP was done according to the criteria used in the 10th International Histocompatibility Workshop (6) and later data (7, 8). Assignment of the HLA-DRB3 and -DRB4 alleles (for nomenclature see 9) was possible by using HLA-DRB generic SSO and HLADRB group-specific SSO. The results of the HLA-DR and -DQ typings by RFLP are shown in Table 2. From the RFLP type those individuals being HLA-DRw52a could be assigned. Table 2 also shows the results of the oligonucleotide typings. It is seen that 5 of the 9 PSC patients possess the HLA-DRw52a marker, which gives a percentage of 55.6 positivity. Two of the patients with ulcerative colitis were HLADRw52a. In Table 3 the results of the serological HLA-ABC and DR types are shown. We were not able to detect those individuals having the HLADRw52a antigen by the serological methods. However, from the serological HLA typings it is not possible to find clear evidence for either single antigens or a particular haplotype being present more often in the group of PSC patients. Based on the RFLP and supported by the PCR-SSO, we were not able to find the 100% association between PSC and HLA-DR52a as reported recently (1). Our finding in the group of PSC patients is significantly different from Prochazka et al. (l), 55.6% vs loo%, p

Association of primary sclerosing cholangitis with HLA-DRw52a?

Brief Communications Association of primary sclerosing cholanrritis with HLA=DRw52a? v N. Grunnet, H. H. Rasmussen, U. Tage-Jensen, S. Nerrby Rasmus...
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