HLA-Linked Susceptibility and Resistance Genes in Crohn's Disease

Transcription

1 GASTROENTEROLOGY 1995;109: HLA-Linked Susceptibility and Resistance Genes in Crohn's Disease ATSUSHI NAKAJIMA,* NOBUYUKI MATSUHASHI,* TATSUHIKO KODAMA,* YOSHIO YAZAKI,* MASAKAZU TAKAZOE, ~ and AKINORI KIMURA *Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo; ~Division of Gastroenterology, Social Health Insurance Medical Center, Tokyo; and Division of Adult Diseases, Department of Tissue Physiology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan Background & Aims: Previous studies have shown a positive association of HLA-DR4-DQ4 with Crohn's disease in the Japanese population, but the association between Crohn's disease and HLA genes has yet to be fully elucidated. The aim of this study was to determine the Crohn's disease/hla association using a DNA typing method. Methods: A total of 90 unrelated patients with Crohn's disease and 336 healthy controls were typed for HLA class II genes including DP using DNA typing with the polymerase chain reaction-sequencespecific oligonucleotide probes method. Results: AIlelic analysis showed that DRBI*0405, DRB1*0410, DQAI*03, DQBI*0401, and DQBI* 0402 are positively associated and DRB1*1501, DRB1*1302, and DQBI*0602 negatively associated with Crohn's disease. DP genes showed no significant association with Crohn's disease. Haplotype analysis showed positive associations with DRBI*0405- DQAI*03- DQBI*0401, DRB1*O410-DQA1*O3-DQBl* 0402, and DRBI*O802- DQAI*O3-DQBI*0402 haplotypes and negative associations with DRB1*1501-DQA1*O102-DQBl*0602 and DRB1*1302-DQA1*O102-DQB1*0604 haplotypes. Conclusions: In Crohn's disease in the Japanese population, the HLA-linked disease susceptibility gene is primarily associated with DQBI*04, in which leucine at the 56th position is a unique amino acid, and the disease resistance allele is suggested to be DQAI*0102. A great deal of evidence points to an association of various autoimmune diseases with HLA antigens and genes. ~ The major relevant features of HLA genes in this regard are polymorphism, marked differences between ethnic groups, and strong linkage disequilibrium between the alleles of each gene. Only few studies have reported the association of HLA class II with Crohn's disease (CD) in white populations, 2 although a suballele of DQw5 has been suggested to be related to disease susceptibility. However, in the Japanese population, previous studies using a serological method ~ have reported a strong association between HLA class II DR4-DQ4 antigens and CD. Thus, to date, an association between HLA genes and CD has been postulated but not yet established. This may be because of (l) the difficulty in performing an ethnically matched study, (2) the possibility of heterogeneity within CD itself, 4 and (3) the paucity of studies on subtypes of class II antigens and the lack of studies that include DP genes. A recent study by Duerr and Neigut stressed the importance of patient selection in the studies of genetic susceptibility in inflammatory bowel disease) Because the Japanese comprise a very homogeneous population, <7 it is comparatively easy to include strictly ethnicmatched case and control patients. This is a great advantage in a disease association study. However, there may still be heterogeneity within Japanese patients with CD; accordingly, it is necessary to first reconfirm whether DR4-DQ4 is really associated with Japanese CD. Serological methods have been used for HLA typing, but such methods are not adequate for precisely analyzing and classifying the polymorphisms of HLA genes. Recently, molecular genetic technology has been applied to defining HLA polymorphisms more specifically, and a polymerase chain reaction-sequence-specific oligonucleotide probe (PCR-SSOP) method has been used to identify class II gene structure in detail. 8'9 Using this DNA typing method, a single base nucleotide difference can be detected, and the serologically defined class II genes can be further subtyped at the nucleotide sequence level. Because a strong linkage disequilibrium exists between HLA alleles, the gene of primary responsibility may be identified by analyzing the frequency of haplotypes that contain susceptibility-associated alleles. The association of HLA class II genes with ulcerative colitis in the Japanese population has already been reported in several papers. I -16 Using serological typing, Bw52-DR2 has been reported to be associated with Japa- Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; Pc, corrected P value; PCR-SSOP, polymerase chain reaction-sequence-specific oligonucleotide probe; RFLP, restriction fragment length polymorphism; RR, relative risk; SDS, sodium dodecyl sulfate; TMAC, tetramethylammonium chloride by the American Gastroenterological Association /95/53.00

2 November 1995 RESPOIBLE HLA CLASS II GENES IN CD 1463 nese ulcerative colitis. 14 Using a genetic typing (PCRrestriction fragment length polymorphism [RFLP] method), an increased frequency in alleles Bw52, Dpw9, and DR2 has been observed as well as an increased frequency in haplotype A24-Bw52-tumor necrosis factor (10.5 kilobases)-dr2-dqwl-dpw9 (DPBI*0901). 12 Recently, molecular, DNA-based typing methods have identified DRBI*IS02 as the allele responsible in HLA- DR2-associated Japanese ulcerative colitis. **'~5 How- ever, a DNA-based HLA class II association with Japanese CD has yet to be reported. We therefore performed an investigation into the association between CD and HLA-DR, -DQ, and -DP genes in the Japanese population. We determined the disease susceptibility and resistance alleles in CD, and the responsible class II genes were identified using haplotype analysis. Materials and Methods DNA typing of HLA class II genes was performed according to the Eleventh International Histocompatibility Workshop reference protocol for the HLA DNA-typing techniquej r Patients and Controls Genomic DNA was isolated from unrelated Japanese patients with CD (n = 90) and Japanese healthy controls (n = 336). Among ethnic Japanese, except for the populations of Hokkaido and Okinawa, HLA distribution has been reported to be relatively homogeneous. In fact, HLA class II allele distribution of the control group in this study was essentially equal to that reported in other studies. <r All study subjects were Japanese, and none of the patients or the controls were from Okinawa or Hokkaido. The control group was ethnically matched with the patient group. The diagnosis of CD was established clinically as well as by radiographic, endoscopic, and histological examination of biopsy specimens. The patients with CD were further subclassified into perforating vs. nonperforating types, with or without anal lesions, and with or without history of polysurgery. The perforating type included patients with intestinal perforations, abscesses, and/ or fistulas. In terms of surgical procedures, patients without history of surgery were distinguished from those who had undergone two or more procedures. DNA Extraction and Amplification Genomic DNA was prepared from peripheral blood leukocytes as previously described. 6 Briefly, leukocytes were isolated from 20 ml blood and resuspended in 10 ml red blood cell lysis buffer (10 mmol/l Tris-HC1 [ph 7.6], 5 mmol/ L MgCl2, and 10 mmol/l NaC1). The lysed erythrocytes were removed by centrifugation, and the pellet was resuspended in 3 ml white blood cell lysis buffer (10 mmol/l Tris-HC1 [ph 7.6], 10 mmol/l ethylenediaminetetraacetic acid [EDTA], and 50 mmol/l NaC1) with 1% sodium dodecyl sulfate (SDS) and proteinase K (500 ~g/ml; Boehringer Mannheim, Indianapolis, IN). The reaction solution was incubated overnight at 42 C, followed by phenol-chloroform-isoamylalcohol extrac- tion. The DNA was precipitated with 100% cold ethanol and resuspended in Tris-EDTA buffer (10 mmol/l Tris-HCl and 1 mmol/l EDTA). The second exons of HLA class II genes of genomic DNA samples (0.5 [.tg each) were amplified by PCR with generic primer pairs as follows: (1) for DPA1, 5'-GCGGACCATGTGTCAACTTAT-3' and 5'-GCCTGAGT- GTGGTTGGAACG-3'; (2) for DPB1, 5'-GAGAGTGG- CCCTCACTC-3' and 5'-GCCGGCCCAAAGCCCTCACTC- 3'; (3) for DQA1, 5'-ATGGTGTAAACTTGTACCAGT-3' and 5 '-TTGGTAGCAGCGGTAGAGTTG-3'; (4) for DQB 1, 5'-CATGTGCTACTTCACCAACGG-3' and 5'-CTGGTAG- TTGTGTCTGCACAC-3'; and (5) for DRB1, 5'-CCCC- ACAGCACGTTTCTTG-3' and 5'-CCGCTGCACTGTGAA- GCTCT-3'. After PCR, aliquots of amplified DNA (about 50 pg) were spotted onto a nylon membrane filter (Hybond N Plus; Amersham, Little Chalfont, England). Hybridization With a2p-labeled Probe Dot blot hybridization was performed using 32P-labeled SSOP using a tetramethylammonium chloride (TMAC) solution (50 mmol/l Tris-HC1 [ph 8.0], 3 mol/l TMAC, 2 mmol/i EDTA, and 0.1% SDS). It should be emphasized that with the TMAC solution, hybridization and washing can be performed at a certain temperature independent of the GC content of the probe. Thus, the temperature for hybridization and washing depends only on the length of the oligonucleotides. For example, in this study, when using an 18mer probe, hybridization was performed at 54 C for 2 hours and washing at 58 C for 10 minutes twice. Then the membrane was exposed to an x-ray film (XAR-5; Eastman Kodak, Rochester, NY) with an intensifying screen at room temperature for 1 hour. DRB1 genes were further subtyped following the procedure of group-specific amplification and dot blot hybridization with various kinds of SSOP as described, s Haplotype Analysis Haplotype analysis was based on population studies referring to the haplotype frequencies and linkage disequilibrium values for three-locus haplotypes (DRB1-DQA1-DQB1) in the Japanese)* Statistical Analysis Statistical analysis of the frequencies of HLA class II alleles and haplotypes between the patient group and the control group was performed using the Z2 statistic or Fisher's Exact Test depending on the sample size. Corrected P values (P~) were obtained by multiplying the P values with the numbers of alleles tested for each locus. Relative risk (RR) is expressed as the strength of association between the disease and the HLA alleles according to Woolfs formula) 9 Sequence Analysis PCR products were directly sequenced using an automatic sequencer (ABI 373; Perkin-Elmer Applied Biosystems, Foster City, CA).

3 1464 NAKAJIMA ET AL. GASTROENTEROLOGY Vol. 109, No. 5 Results Allelic Analysis The frequencies of HLA class II alleles in Japanese patients with CD and controls are shown in Tables 1-3. For the DPA and DPB alleles, no significant difference in distribution between CD and control was noted (Table 1). In the case of the DQA and DQB alleles, frequencies of DQAl*03, DQB 1"0401, and DQB 1 *0402 were significantly increased in patients with CD. On the other hand, the frequency of DQAI*0102 was significantly decreased (Table 2). The frequency of DQB 1"0602 was decreased in patients with CD, but the difference was not significant. Analysis of the DRB 1 allele showed that DRB 1"0405 and DRB 1"0410 were significantly associated with CD. The frequencies of DRBl*1501 and DRBI*1302 were decreased in CD, but the difference was not significant (Table 3). These results show that DRBI*0405, DRBI*0410, DQAI*03, DQBI*0401, and DQBI*0402 are positively associated with CD, whereas DQA 1 *0102, DQB 1" 0602, DRB 1 * 1302, and DRB 1 * 1501 are negatively associated with CD. Haplotype Analysis The frequencies of the DRB 1-DQA 1-DQB 1 haplotypes containing the disease-associated alleles are shown in Table 4. The frequency of all haplotypes containing the DQB*04 allele ('0401 and *0402) was increased, and the frequency of all haplotypes containing the DQA*0102 allele was decreased in the patient group. Table 1. HLA-DP Alleles in Patients With CD and Controls CD Control DPA1 n = 45 % n = 227 % P " , " " DPB1 n = 45 % n = 315 % P " * ,3 " " * " " " " " " " " These results suggest that the HLA-linked disease susceptibility gene and disease resistance gene are primarily associated with DQB*04 and DQAI*0102, respectively. HLA Class II and Disease Subtypes The frequencies of HLA class II alleles in the perforating or nonperforating types, patients with or without anal lesions, and patients without previous surgery or with polysurgery (two or more) are shown in Table 5. No significant differences were noted among these subtypes. Sequence Analysis Dot blot hybridization of DQB1 PCR products with SSO5708 indicated the presence of Leu at the 56th position, which was further confirmed by sequence analysis. Discussion The association between class II genes and CD in the Japanese was analyzed at the DNA level in this study. The results are noteworthy in several aspects. First, a negative association of class II alleles with CD is shown. Such negative association has not been reported pre- Table 2. HLA-DQ Alleles in Patients With CD and Controls CD Control DQA1 n = 90 % n = 336 % RR P ,34 <0.005 a < b , DQB1 n = 9 % n = 336 % RR P " * , * " * <0.02 c * * * ,8 " " * * " <0,002 d * , < apc = ~Pc = Po = Opo = epo =

4 November 1995 RESPOIBLE HLA CLASS II GENES IN CD 1465 Table 3. HLA-DRB1 Alleles in Patients With CD and Controls C0 Control DRB1 n = 90 % n = 336 % RR P " " <0.02 a ' " " " * * <0.002 * * " < " " " " " <0.016 " " " " " " * * * " " % = ~& = c& = d~ = viously; in this study, a significant negative association with DQAI*0102 was shown using the DNA typing method. DQBI*0602, DRBl*1501, and DRBI*1302, which are in linkage disequilibrium with DQAI*0102, also showed decreased frequencies in patients with CD, although the negative association was not significant. Second, there had been no reported data about an association of DP genes with CD. Such association was investigated in this study; no significant association was found between DP alleles and CD in the Japanese population. The association of HLA class II genes with Japanese CD is relatively strong compared with that for other autoimmune diseases. For example, insulin-dependent diabetes mellitus (IDDM) in Japan is known to be associated with DQBI*0401, and the RR of DQBI*0401 in Japanese IDDM is The RR of DPBI*0501 in Japanese Graves' disease is 5.3, 21 the RR of DRBI*0501 in Japanese systemic lupus erythematosus is 3.0, 22 the RR of DR2 in Japanese ulcerative colitis is 4.2,12 and the RR of DRBI*0405 in Japanese rheumatoid arthritis is The RR of DQBI*0402 in Japanese patients with CD is as high as 3.9 (Table 2). Upon haplotype analysis, the HLA-linked disease susceptibility gene was identified. In terms of disease resistance genes, it is clear that the resistance-associated allele is DQAI*0102 because DQAI*0102 is the only common allele among all the haplotypes that include the negatively associated class II alleles (Table 4). There are three candidates for the disease susceptibility gene: DRB1*0405/*0410, DQAI*03, and DQBI*04 (*0401/ *0402). IfDRB1 *0405/*0410 were the primary susceptibility gene, the increased frequency of DRBI*0802- DQAI*0401-DQBI*0402 haplotype could not be explained. Similarly, if the DQAI*03 were the primary susceptibility gene, it could not be explained why the frequencies of other DQAl*03-1inked haplotypes, such as DRBI*0901-DQAI*03-DQB*10303, DRBI*0401- Table 4. Haplotype Analysis of Susceptible and Resistant HLA Class II Alleles CD (n = 90) Control (n = 336) DRB DQA DQB n % n % RR P Haplotype including positively associated class II allele " *03---* " "03---' * *03---* * "03---' <0.0021" " *03---* < b "0802--' ' <0.031 Haplo~peincluding negatively associa~d classllallele,1501--* * ,1302--' ' "1302--' ' < <0.031 e a~ = ~ = c~ = d~ = e~ =

5 1466 NAKAJIMA ET AL. GASTROENTEROLOGY Vol. 109, No. 5 Table 5. HLA Class II and CD Disease Types Perforating Nonperforating Class II alleles n = 21 % n = 69 % P DQAI* DQAI* DQ81" DQBI* DQB1* DRB1* DRBl* DRB1* DRB1* Anal lesion (+) Anal lesion (-) DQB1 *0501RAVT P QG R PVA E YWN S Q KE V L E DQBI * S DQB1 * D DQB1 " D D I - - DQB1 * D DQB1 * D DQB1 * O DQB1 * OQB1 " L L - L -A D I - - DQB1 " L - P -D DQB1 * L - P - A DQB1 * L - P - D DQB1 " L - - LD D DQB1 * L - - L D D I - - Figure I. Amino acid sequences of various DQB1 alleles. Leu at the 56th position is unique in the disease susceptibility HLA class II gene in CD, DQB*0401/0402. Class II alleles n = 47 % n = 43 % DQAI* DQAI* DQB1* DQBI* DQB1* DRB1* DRB1* DRB1* DRB1* No surgery Polysurgery (>1) Class II alleles n = 34 % n = 16 % DQAI* DQAI* DQB1* DQBI* DQB1* DRB1* DRB1* DRB1* ,3 DRB1* DQAI*03-DQBI*0301, and DRBI*0406-DQAI*03- DQBl*0302, are not increased (Table 4). The positive association of the various haplotypes listed in Table 4 can only be explained if DQB 1"04 (*0401/*0402) is the primary gene for susceptibility. It should be noted that the frequency of DQBI*04 is 61.1% (55 of 90) in patients with CD compared with 31.8% (107 of 336) in the control (P = ). Using DNA typing, the amino acid sequence of the second exon of HLA class II gene can be determined. A comparison of the amino acid sequences of the DQB1 alleles is shown in Figure 1. It is obvious that Leu at the 56th position is a unique amino acid. In fact, using SSO DQB5708, a single amino acid substitution, Pro to eu at position 56, is detected. Moreover, direct sequencing of DQB 1*04-positive PCR products confirmed that Leu was present at position 36 (Figure 2). The 36th position is located at the antigen-binding groove of the DQ mole- P cule and may play an important role in the pathogenesis of the disease. This is extremely interesting in light of the fact that amino acid substitutions at positions 56 and 57 are characteristic in IDDM. 24 Perforating or not, with or without anal lesions, and N8 with or without polysurgery are clinically relevant features of CD. Therefore, differences in HLA class II distribution related to these features were examined. However, as de- scribed in Table 5, no significant difference was noted. How do DQA 1 *0102 and/or DQB 1 *04 participate in the development of CD? One possible interpretation of p the results of this study is that certain disease susceptibility genes are linked to DQAI*0102 and/or DQBI*04. Duerr and Neigut speculated that the disease-associated HLA allele in UC that is linked with actual susceptibility genes may be different in different ethnic groupss; addi- N8 tional ethnically matched studies would be necessary to elucidate the primary susceptibility genes near the HLA locus. Nevertheless, considering the result that both the disease susceptibility gene and the disease resistance gene have been found to exist at close loci, and because there is a unique amino acid substitution in DQBI*04, we speculate that the HLA gene itself is the primary respon- 50 $SO A V R A V T P I. G R L O R E Y W N S Q + "+ B. C a + I / e., r Figure 2. Sequence analysis of DQBI*04 allele. (A) Direct sequencing of DQBl*O4-positive PCR product. Leu at the 56th position is in the SSO 5708 sequence. (B and C) Every DQB1 PCR product was dot blot hybridized with SSO Only DQBI*04 (0401/0402) gives a positive signal.

6 November 1995 RESPOIBLE HLA CLASS II GENES in CD 1467 sible gene in CD at least in the Japanese population. The HLA class II gene product itself, DQB04 and/or DQA0102, may determine disease susceptibility. Considering that class II molecules work as antigen-presenting molecules, the antigen-presenting pattern of those class II molecules may be unique, and this altered antigen presentation may be the cause of the disease onset. Futami et al. proposed that Gly at the 86th position in the HLA class II ~ chain may play an important role in determining the susceptibility to Japanese UC, 15 and Watanabe et al. also speculated that the composition of amino acid residues at the 70th-74th positions may play a crucial role in the pathogenesis of RA. 25 Therefore, it is especially interesting in light of the amino acid sequence analysis that the 56th amino acid was shown to be unique in the disease-related allele DQB04. In conclusion, in the Japanese population, (1) DRBI* 0405, DRBI*0410, DQAI*03, DQBI*0401, and DQBI* 0402 are positively associated with CD; (2) DQAI* 0102, DQBI*0602, DRBl*1302, and DRBl*1501 are negatively associated with CD; (3) the HLA-linked disease susceptibility gene is primarily associated with DQBI*04 ('0401/'0402), in which Leu at the 56th position is unique; (4) the HLA-linked disease resistance gene is primarily associated with DQAI*0102; and (5) no HLA class II-related disease subtype was noted. References 1. Tiwari JL, Terasaki PI. HLA and disease associations. Heidelberg, Germany: Springer Verlag, 1989: Toyoda H, Wang S, Yang H, Redford A, Magalong D, Tyan D, McLeen CK, Pressman SR, Shanahan F, Targan SR, Rotter JI. Distinct associations of HLA class II genes with inflammatory bowel disease. Gastroenterology 1993; 104: Matake H, Okabe N, Naito S, Yao T. An HLA study on 149 Japanese patient with Crohn's disease. 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