Abstract

We performed HLA-A, -B, and -C antigen and -DR DNA typing in 111 Japanese patients with idiopathic thrombocytopenic purpura (ITP). DRB1*0410 was significantly increased in ITP patients compared with healthy controls (relative risk = 9.52, P < .05), but the other DRB1*04 alleles showed no significant differences. On HLA-DR serotyping, patients with Vogt-Koyanagi-Harada disease (VKH) had a high frequency of DR4, so we compared the frequencies of DRB1*04 suballeles between ITP and VKH. The high frequency of DRB1*04 was dependent on DRB1*0405 in VKH, but on DRB1*0410 in ITP. Plasma autoantibodies were studied in 111 patients using a microtiter well assay. Thirty-six patients had anti-GPIIb/IIIa autoantibodies, and antibody positivity was associated with HLA-DR4 (29 of 36, 80.6% v 28 of 75, 37.3%) but not with DRB1*0410. When HLA-DR4 and DRB1*0410 were compared between patients with a good or poor response to prednisolone, HLA-DR4 was decreased and DRB1*0410 was significantly decreased (χ2 = 11.455, P < .01) in patients with a good response. In conclusion, this study showed that genetically determined factors influence the course of ITP. However, our findings should be considered preliminary because of possible racial differences in HLA status between Japanese and other ITP patients.

IDIOPATHIC THROMBOCYTOPENIC purpura (ITP) is a disease caused by circulating autoantibodies that react with the platelet membrane.1,2 It is thought that platelet-associated IgG is an important factor in the mechanism of ITP, since an increase of such IgG is closely related to a reduced platelet count in this disease.1-4 Although the platelet surface antigens corresponding to the antiplatelet autoantibodies involved in ITP are largely unknown, there have been several recent reports on autoantibodies to glycoprotein (GP)IIb/IIIa and GPIb,5-7and the antigens for these GPs are gradually being elucidated.8-12 Thus, the mechanism related to thrombocytopenia is becoming clear,13,14 but the etiology of ITP remains uncertain, with both genetic and environmental factors apparently involved. Based on serologic studies, associations between certain HLAs and many autoimmune diseases have long been described.15,16 Several groups have tried to establish a relationship between HLA class I or HLA-DR and chronic ITP.17-20 However, the results have been inconsistent, possibly because only a limited number of HLA-DR antigens could be determined serologically in the past.17-20 

With the development of the polymerase chain reaction (PCR), identification of HLA alleles at the DNA level has become possible and has allowed more precise determination of the susceptibility epitopes showing a strong association with various autoimmune diseases.21,22 The HLA class II region encodes the heterodimeric (α and β chains) GPs expressed on the surface of antigen-presenting cells in the immune system. The T-cell receptor interacts with a complex formed by the antigenic peptide fragment bound by HLA molecules on antigen-presenting cells. The genetic polymorphism of class II molecules is known to be clustered in discrete regions of the β chains termed allelic hypervariable regions,23which regulate the variability of immune responses through antigen recognition by HLA-restricted T cells. Thus, the polymorphic amino acid residues in allelic hypervariable regions of the HLA class II molecule have been suggested to have an important role in determining susceptibility or resistance to some autoimmune diseases.24,25 The HLA haplotype is also regarded as a potentially important factor in ITP,26 but its role remains unclear.

Although various methods have been used for the treatment of chronic ITP,27-31 splenectomy and administration of corticosteroids are still the mainstays of therapy.1,2However, no parameters have been found to predict the response to treatment. In this study, we performed HLA-A, -B, and -C antigen and -DR DNA typing of Japanese ITP patients and investigated the HLA-DR4 gene variations in several clinical or pathologic subtypes of ITP. Our purpose was to determine whether anti-GPIIb/IIIa autoantibodies and the response to corticosteroid therapy are associated with specific HLA systems.

MATERIALS AND METHODS

Subjects.

We studied 111 unrelated Japanese patients (29 men and 82 women) with ITP who presented to our hospital from April 1994 to December 1996. The diagnosis of ITP was made according to the standard criteria of thrombocytopenia with a normal or increased number of megakaryocytes and no evidence of any secondary cause of thrombocytopenia. The subjects were aged 22 to 78 years, and none had received a blood transfusion. Table 1 shows a brief clinical profile of the 111 ITP patients. Seventy-one controls were also randomly selected from among healthy unrelated Japanese individuals. Furthermore, 53 Japanese patients with Vogt-Koyanagi-Harada disease (VKH) were studied as disease controls. Approval was obtained from the Institutional Review Board for these studies. Informed consent was provided according to the Declaration of Helsinki.

Table 1.

Clinical Characteristics of the ITP Patients

Characteristic Value
No. of patients  111  
Sex ratio (male:female)  29:82  
Age (yr)  
 Range  22-78  
 Median  53  
Platelet count (104/μL) 3.68 ± 0.39-151 
PAIgG (ng/107)* 286 ± 39-151 
Characteristic Value
No. of patients  111  
Sex ratio (male:female)  29:82  
Age (yr)  
 Range  22-78  
 Median  53  
Platelet count (104/μL) 3.68 ± 0.39-151 
PAIgG (ng/107)* 286 ± 39-151 

*PAIgG was measured by a competitive enzyme-linked immunosorbent assay (normal range, 10 to 25 ng/107 platelets).

F0-151

Mean ± SE.

Treatment.

Prednisolone was administered first at a dose of 0.5 to 1 mg/kg daily. The response of each patient was assessed from the change in the platelet count at 6 months after starting therapy. A good response was defined as an increase in the platelet count of greater than 50 × 109/L, a platelet count greater than 100 × 109/L off therapy, and no more than one relapse during follow-up study. A poor response was defined as an increase in the platelet count of less than 50 × 109/L. Relapse was defined as a decrease in the platelet count to less than 50 × 109/L after a normal count had been reached. The duration of follow-up study was at least 6 months from the start of therapy.

HLA serotyping.

ITP patients and healthy controls were subjected to serotyping for HLA class I and class II antigens using the standard complement-dependent microcytotoxicity method.32 

HLA DNA typing by PCR–restriction fragment length polymorphism.

HLA DNA typing was performed according to the manufacturer's instructions (SMITEST HLA DNA-typing system; Sumitomo Metal, Tokyo, Japan). Genomic DNA from patients and controls was isolated by phenol extraction of sodium dodecyl sulfate–lysed and proteinase K–treated cells. DNA was amplified by the PCR procedure with Taq DNA polymerase and typed by the PCR-restriction fragment length polymorphism (RFLP) method.33 The reaction mixture was subjected to 30 cycles of denaturation for 1 minute at 96° to 97°C, annealing for 1 minute at 55° to 62°C, and extension for 2 minutes at 72°C in an automated PCR thermal sequencer (Iwaki Glass Inc, Tokyo, Japan). After amplification, aliquots of the reaction mixture were digested with allele-specific restriction endonucleases for 3 hours after addition of the appropriate reaction buffer. Samples of the cleaved and amplified DNAs were subjected to electrophoresis on 12% polyacrylamide gel in a minigel apparatus (Mupid-2; Cosmo Bio Co, Tokyo, Japan). Cleavage or noncleavage of amplified fragments was detected by staining with ethidium bromide. Discrimination of genotypes was made on the basis of RFLP band patterns thus generated.

Detection of plasma autoantibodies against GPIIb/IIIa by microtiter well assay.

The assay was previously described by Nomura et al34 and Kokawa et al.35 A suspension of washed platelets (1 × 107/μL) in 100 μg/mL leupeptin and 10 mmol/L EDTA was sonicated on ice and centrifuged at 12,500g for 30 minutes. The supernatant was solubilized in 2% Triton X-100 and centrifuged at 100,000g for 30 minutes to remove the Triton X-100–insoluble fraction. After centrifugation, the supernatant was used as the platelet lysate. Autoantibodies were assayed by a modification of the method of Woods et al.5,6 In brief, microtiter wells were coated with a monoclonal anti-GPIIb/IIIa antibody (NNKY1-32)36,37 by overnight incubation. The platelet lysate was then added to microtiter wells and incubated. After washing, appropriate dilutions of plasma from ITP patients (n = 111), disease controls (n = 30), or normal controls (n = 20) were added and incubated. After washing again, horseradish peroxidase–conjugated rabbit anti-human IgG was added, and the amount of IgG that bound to the platelets was determined by measuring peroxidase activity using a plate reader. Assay results were expressed in terms of the percent change in peroxidase activity above or below the level in control (normal serum) wells, using the following formula: percent change = (OD platelet extract wells − OD control wells)/OD control wells × 100.

Samples with a percent increase greater than 3 SD above the mean of 20 normal plasma samples were considered to be positive.

Assay of platelet-associated IgG.

A competitive enzyme-linked immunosorbent assay was used to quantify platelet-associated IgG (PAIgG) in patients with ITP.38 The upper limit of normal was 25 ng/107 platelets.

Statistical analysis.

The χ2 method with continuity correction and Fisher's exact test were used for data analysis. Relative risk was calculated according to Wolf's method with Holdane's correction. Briefly, it was calculated as (a × d)/(b × c), where a, b, c, and d are the number of marker-positive patients, marker-negative patients, marker-positive controls, and marker-negative controls, respectively.39 

RESULTS

HLA-DR serotyping.

The frequency of the HLA-DR antigens and the relative risk were calculated in 111 unrelated Japanese ITP patients and 71 unrelated Japanese controls (Table 2). DR8, DR9, and DR53 were increased in ITP patients compared with healthy controls (relative risk > 1.50). On the other hand, DR1, DR6, and DR52 were decreased in ITP patients compared with healthy controls (relative risk < 0.50). However, these differences were not statistically significant. The frequencies of DR4 and DR53 were high in both healthy controls and ITP patients. It is thought that DR53 is associated with DR4, DR7, and DR9. Because there are racial differences in HLA frequency, we studied HLA frequencies in VKH patients as Japanese disease controls. VKH is an inflammatory disease affecting multiple organs, causing bilateral panuveitis, meningitis, hearing loss, tinnitus, and vitiligo.40 It was previously reported that VKH is closely associated with DR4 and DR53 by HLA serotyping of Japanese patients.41 In the present study, DR4 and DR53 were also found in almost all VKH patients examined (94.3%).

Table 2.

HLA-DR Antigenic Frequencies of ITP Patients and Controls

HLA Antigen Controls (n = 71) ITP Patients (n = 111)Relative Risk
No. % No. %
DR1  10 14.1  8  7.2  0.47  
DR2  29  40.8  43  38.7 0.92  
DR3  1  1.4  1  0.9  0.64  
DR4  34 47.9  57  51.3  1.15  
DR5  9  12.7  15 13.5  1.08  
DR6  27  38.0  22  19.8  0.36 
DR7  0  0.0  0  0.0  0.64  
DR8  13  18.3 31  27.9  1.73  
DR9  17  23.9  36  32.4 1.52  
DR10  1  1.4  1  0.9  0.70  
DR52  35 49.3  36  32.4  0.49  
DR53  42  59.2  81 73.0  1.86 
HLA Antigen Controls (n = 71) ITP Patients (n = 111)Relative Risk
No. % No. %
DR1  10 14.1  8  7.2  0.47  
DR2  29  40.8  43  38.7 0.92  
DR3  1  1.4  1  0.9  0.64  
DR4  34 47.9  57  51.3  1.15  
DR5  9  12.7  15 13.5  1.08  
DR6  27  38.0  22  19.8  0.36 
DR7  0  0.0  0  0.0  0.64  
DR8  13  18.3 31  27.9  1.73  
DR9  17  23.9  36  32.4 1.52  
DR10  1  1.4  1  0.9  0.70  
DR52  35 49.3  36  32.4  0.49  
DR53  42  59.2  81 73.0  1.86 
HLA DNA typing.

Based on the serologic data, we performed DNA typing of DR4, DR8, and DR9. HLA-DRB1 genotyping was performed by the PCR-RFLP method. DRB1*04 (DR4), *08 (DR8), and *09 (DR9) allele frequencies in ITP patients and healthy controls are shown in Table 3.DRB1*0410 was significantly increased in ITP patients compared with healthy controls (relative risk = 9.52, P < .05). However, none of the other alleles (DRB1*04, *08, and *09) showed a significant difference between ITP patients and healthy controls. HLA-DR serotyping showed that VKH patients had a high frequency of DR4, so we compared the frequency of DRB1*04 suballeles between ITP and VKH patients. Using the International Histocompatibility Workshop (1984) definitions, HLA-DR4 was classified as DR4.1 and DR4.2 subgroups with panel sera. We then compared DRB1*04 suballeles in these two subgroups among ITP patients, VKH patients, and controls (Table4). ITP patients showed an increase of DR4.1 subgroup alleles and a decrease of the DR4.2 subgroup. VKH patients also showed an increase of the DR4.1 subgroup. The high frequency of DR4.1 was dependent on DRB1*0405 in VKH patients, but was related to DRB1*0410 in ITP patients. Among 71 healthy controls, 34 had DRB1*04 (DR4). To analyze the suballelic distribution of DRB1*04 (DR4) more precisely in ITP and VKH patients, we investigated the DRB1*04 frequency in DR4-positive patients alone (Table 5). DRB1*0410 was again significantly increased in ITP patients compared with VKH patients and healthy controls.

Table 3.

HLA-DRB1*04, *08, and *09 Frequencies of ITP Patients and Controls

DRB1 Controls (n = 71)ITP Patients (n = 111) Relative Risk P
No. % No. %
*0401  1.4  2  1.8  1.28  
*0403  2  2.8  3  2.7 0.96  
*0404  0  0.0  2  1.8  3.26  
*0405 19  26.8  14  12.6  0.40  
*0406  8  11.3  7.2  0.61  
*0407  2  2.8  2  1.8  0.63 
*0408  0  0.0  2  1.8  3.26  
*0410  2  2.8 24  21.6  9.52  <.05  
*0802  4  5.6  2.7  0.47  
*0803  8  11.3  24  21.6  2.17 
*0804  1  1.4  4  3.6  2.62  
*0901  17 23.9  36  32.4  1.52   
DRB1 Controls (n = 71)ITP Patients (n = 111) Relative Risk P
No. % No. %
*0401  1.4  2  1.8  1.28  
*0403  2  2.8  3  2.7 0.96  
*0404  0  0.0  2  1.8  3.26  
*0405 19  26.8  14  12.6  0.40  
*0406  8  11.3  7.2  0.61  
*0407  2  2.8  2  1.8  0.63 
*0408  0  0.0  2  1.8  3.26  
*0410  2  2.8 24  21.6  9.52  <.05  
*0802  4  5.6  2.7  0.47  
*0803  8  11.3  24  21.6  2.17 
*0804  1  1.4  4  3.6  2.62  
*0901  17 23.9  36  32.4  1.52   
Table 4.

Comparison of HLA-DRB1*04 Frequencies of ITP Patients, VKH Patients, and Controls

DRB1 Controls (n = 71)ITP Patients (n = 111) VKH Patients (n = 53)
No. % No. % No. %
DR4.1 group   31.0   39.6   75.4  
 DRB1*0401 1  1.4  2  1.8  0  0.0  
 DRB1*0404  0  0.0 2  1.8  0  0.0  
 DRB1*0405  19  26.8  14 12.6  27  50.9  
 DRB1*0408  0  0.0  2  1.8 0  0.0  
 DRB1*0410  2  2.8  24  21.6  13 24.5  
DR4.2 group   16.9   11.7   18.9 
 DRB1*0403  2  2.8  3  2.7  3  5.7 
 DRB1*0406  8  11.3  8  7.2  6  11.3 
 DRB1*0407  2  2.8  2  1.8  1  1.9 
DRB1 Controls (n = 71)ITP Patients (n = 111) VKH Patients (n = 53)
No. % No. % No. %
DR4.1 group   31.0   39.6   75.4  
 DRB1*0401 1  1.4  2  1.8  0  0.0  
 DRB1*0404  0  0.0 2  1.8  0  0.0  
 DRB1*0405  19  26.8  14 12.6  27  50.9  
 DRB1*0408  0  0.0  2  1.8 0  0.0  
 DRB1*0410  2  2.8  24  21.6  13 24.5  
DR4.2 group   16.9   11.7   18.9 
 DRB1*0403  2  2.8  3  2.7  3  5.7 
 DRB1*0406  8  11.3  8  7.2  6  11.3 
 DRB1*0407  2  2.8  2  1.8  1  1.9 

DR4.1 and DR4.2 epitopes were confirmed to be 13H,33H, 11V, and 74A to74E.

Table 5.

HLA-DRB1*04 Frequencies of DR4-Positive ITP and VKH Patients Compared With Controls

DRB1 Controls (n = 34)ITP Patients (n = 57) VKH Patients (n = 48)
No. % No. % No. %
DR4.1 group   64.7   77.2   83.4  
 DRB1*0401 1  2.9  2  3.5  0  0.0  
 DRB1*0404  0  0.0 2  3.5  0  0.0  
 DRB1*0405  19  55.9  14 24.6  27  56.3  
 DRB1*0408  0  0.0  2  3.5 0  0.0  
 DRB1*0410  2  5.9  24  42.1  13 27.1  
DR4.2 group   35.3   22.8   20.9 
 DRB1*0403  2  5.9  3  5.3  3  6.3 
 DRB1*0406  8  23.5  8  14.0  6  12.5 
 DRB1*0407  2  5.9  2  3.5  1  2.1 
DRB1 Controls (n = 34)ITP Patients (n = 57) VKH Patients (n = 48)
No. % No. % No. %
DR4.1 group   64.7   77.2   83.4  
 DRB1*0401 1  2.9  2  3.5  0  0.0  
 DRB1*0404  0  0.0 2  3.5  0  0.0  
 DRB1*0405  19  55.9  14 24.6  27  56.3  
 DRB1*0408  0  0.0  2  3.5 0  0.0  
 DRB1*0410  2  5.9  24  42.1  13 27.1  
DR4.2 group   35.3   22.8   20.9 
 DRB1*0403  2  5.9  3  5.3  3  6.3 
 DRB1*0406  8  23.5  8  14.0  6  12.5 
 DRB1*0407  2  5.9  2  3.5  1  2.1 
Characteristics of HLA-DRB1*0410-positive ITP patients.

Table 6 shows the clinical characteristics of HLA-DRB1*0410-positive ITP patients. All of them were female, and six had the homotype DRB1*0410; however, these six patients did not have any common distinguishing characteristics. The sex distribution of HLA-DRB1*0410 in our laboratory showed a slight female predominance (61.2% female), but there were no family members with HLA-DRB1*0410 and ITP. Furthermore, the frequencies of HLA-A or HLA-B antigens were not different from those in the controls. Plasma autoantibodies were studied in 111 ITP patients using a microtiter well assay, and 36 patients (32.4%) had anti-GPIIb/IIIa autoantibodies. Table 7 shows the association of HLA-DR4 with autoantibodies to GPIIb/IIIa. There was a positive association between anti-GPIIb/IIIa antibodies and HLA-DR4 (29 of 36, 80.6% v 28 of 75, 37.3%), but there was no association with DRB1*0410. HLA-DR4 and DRB1*0410 were compared in patients with a good or poor response to prednisolone (Table 7). HLA-DR4 was slightly decreased in patients with a good response to prednisolone (poor v good, 22 of 54, 59.3%v 25 of 57, 43.9%), and DRB1*0410 was significantly decreased (poor v good, 21 of 54, 38.9% v 3 of 57, 5.3%, χ2 = 11.455, P < .01). However, anti-GPIIb/IIIa antibody was not correlated with the response to steroid therapy.

Table 6.

HLA-DRB1*0410-Positive Patients With ITP

Patient No. Sex/Age (yr) HLA-A HLA-BHLA-DRB1*
1  F/60  24, -  54, 61  0410/0410  
F/44  2, 26  51, 60  0410/1201  
3  F/25  24, 11 35, 55  0410/0410  
4  F/76  2, 24  7, 46 0410/0803  
5  F/20  2, 11  51, 54  0410/1405  
F/31  11, 24  7, 59  0410/0410  
7  F/52  11, 24 35, 67  0410/1406  
8  F/59  24, 33  44, 60 0410/1502  
9  F/61  24, -  52, 54  0410/1502  
10 F/68  2, 11  54, 67  0410/0804  
11  F/48  2, - 61, 46  0410/0803  
12  F/51  11, 24  59, 62 0410/0406  
13  F/65  11, 24  61, 62  0410/1201 
14  F/68  11, -  54, -  0410/0410  
15  F/25 2, 24  56, 61  0410/0901  
16  F/24  24, 31  46, 55  0410/0410  
17  F/25  11, -  51, -  0410/1501 
18  F/66  11, 24  54, -  0410/0901  
19  F/19 11, 31  51, 75  0410/0802  
20  F/57  2, 31  67, 75  0410/0101  
21  F/71  24, -  52, 54  0410/0802 
22  F/50  2, -  35, 54  0410/1201  
23  F/34 24, 31  39, 61  0410/0410  
24  F/40  24, -  61, 62  0410/0901 
Patient No. Sex/Age (yr) HLA-A HLA-BHLA-DRB1*
1  F/60  24, -  54, 61  0410/0410  
F/44  2, 26  51, 60  0410/1201  
3  F/25  24, 11 35, 55  0410/0410  
4  F/76  2, 24  7, 46 0410/0803  
5  F/20  2, 11  51, 54  0410/1405  
F/31  11, 24  7, 59  0410/0410  
7  F/52  11, 24 35, 67  0410/1406  
8  F/59  24, 33  44, 60 0410/1502  
9  F/61  24, -  52, 54  0410/1502  
10 F/68  2, 11  54, 67  0410/0804  
11  F/48  2, - 61, 46  0410/0803  
12  F/51  11, 24  59, 62 0410/0406  
13  F/65  11, 24  61, 62  0410/1201 
14  F/68  11, -  54, -  0410/0410  
15  F/25 2, 24  56, 61  0410/0901  
16  F/24  24, 31  46, 55  0410/0410  
17  F/25  11, -  51, -  0410/1501 
18  F/66  11, 24  54, -  0410/0901  
19  F/19 11, 31  51, 75  0410/0802  
20  F/57  2, 31  67, 75  0410/0101  
21  F/71  24, -  52, 54  0410/0802 
22  F/50  2, -  35, 54  0410/1201  
23  F/34 24, 31  39, 61  0410/0410  
24  F/40  24, -  61, 62  0410/0901 
Table 7.

Anti-GPIIb/IIIa Autoantibody Status and Response to Prednisolone in Patients With DR4 or DRB1*0410

Anti-GPIIb/IIIaSteroid Response
Positive (n = 36)Negative (n = 75) Poor (n = 54) Good (n = 57)
No. % No.% No. % No. %
DR4  29  80.6* 28 37.3  32  59.36-151 25  43.9  
DRB1*0410  11 30.66-152 13  17.3  21  38.96-153 3  5.3 
Anti-GPIIb/IIIaSteroid Response
Positive (n = 36)Negative (n = 75) Poor (n = 54) Good (n = 57)
No. % No.% No. % No. %
DR4  29  80.6* 28 37.3  32  59.36-151 25  43.9  
DRB1*0410  11 30.66-152 13  17.3  21  38.96-153 3  5.3 

2 = 6.954, P < .05.

F6-151

χ2 = 1.862, not significant.

F6-152

χ2 = 2.098, not significant.

F6-153

χ2 = 11.455, P < .01.

DISCUSSION

ITP is a clinically well-defined autoimmune disease caused by antiplatelet antibodies, and the antigenic epitope has recently been studied in detail, revealing that the main epitope contains GPIIb/IIIa.7-12 Various autoimmune diseases are associated with HLA class I and/or class II, and thus several groups have investigated the role of HLA class I and HLA-DR in ITP.18-20 However, the findings have been inconsistent, possibly because only a limited number of HLA-DR antigens could be determined serologically in the past.18,20 The polymorphisms of class II genes (HLA-DR, -DQ, and -DP) in the major histocompatibility complex can now be defined precisely by typing using the PCR-RFLP method. It was previously reported that patients with aplastic anemia who possess HLA-DR2 are more likely to respond to immunosuppressive therapy.42,43 Thus, analysis of HLA antigens and alleles may provide useful information for the treatment of autoimmune diseases. In the present study, we performed HLA typing of Japanese ITP patients to determine whether positivity for anti-GPIIb/IIIa antibodies and the response to corticosteroid therapy are associated with specific HLA systems.

We observed an increase of DR4, DR8, DR9, and DR53 in ITP patients compared with healthy controls, although these differences were not statistically significant. Since HLA frequencies show racial differences, the frequencies of DR4 and DR53 were only compared among Japanese patients. As a result, we found an increase of DR53 in both ITP and VKH patients, with a particularly marked increase in VKH. It was previously found that VKH was closely associated with DR4 and DR53 by serotyping of Japanese VKH patients.41 However, the association of DR4 and DR53 was not significant in our ITP patients. Furthermore, no increase of DR9 or DR7, which is known to be tightly linked to HLA-DR53, was observed in ITP patients. These results suggest that DR53 itself is unlikely to confer susceptibility to ITP.

The serologically defined HLA-DR antigen can be divided into many alleles reflecting polymorphism of the amino acid sequence in the allelic hypervariable regions of the β chain. For example, DRB1*04 can be divided into 11 alleles (DRB1*0401 to DRB1*0411). When we analyzed the frequency of HLA-DR*04 alleles, the most important finding was that DRB1*0410 was significantly increased in ITP patients compared with healthy controls (relative risk = 9.52, P < .05). Of further interest was the observation that all patients with DRB1*0410 were females. The immune response is stronger in females than in males, and there is a greater prevalence of autoimmune disease in the female population.44 Additionally, Cavan et al45 have reported strong evidence for a sex difference in the effect of HLA markers on disease susceptibility. However, ITP is a female-predominant disease,1,2 and the sex distribution of HLA-DRB1*0410 in our laboratory showed slight female predominance (61.2% female). Thus, the relationship between DRB1*0410-positive ITP and female hormones seems to deserve further study. DRB1*04 alleles have been suggested to have an important role in determining susceptibility to several autoimmune diseases such as insulin-dependent diabetes mellitus,24 rheumatoid arthritis,25 and VKH.46,47 In particular, VKH appears to be strongly associated with DRB1*04,46,47 so we compared the frequencies of DRB1*04 alleles between our ITP and VKH patients.

We obtained the same results as in previous reports,46,47detecting a high percentage of DRB1*0405 and DRB1*0410 (Table 4). However, DRB1*0405 was low in ITP. To confirm the significance of DRB1*0405 and DRB1*0410 in ITP and VKH patients, we compared DRB1*04 suballelic frequencies in ITP, VKH, and controls with DR4 positivity (Table 5). DRB1*0410 was also significantly increased in ITP patients compared with controls and VKH patients. The published amino acid sequences of the polymorphic β domains of DRB1 genes show that the amino acid specific for both DRB1*0405 and DRB1*0410 is the serine at position 57, instead of aspartic acid as in the other DRB1 alleles (Table 8). Amino acid sequence differences among DR4-associated alleles are confined to the COOH-terminal portion of the β1 domain, primarily within the three allelic hypervariable regions of the DRB1 gene.48,49 Hence, the polymorphic residues in this region are believed to be critical for both T-cell recognition and antigenic peptide binding. However, despite both DRB1*0405 and DRB1*0410 possessing the same amino acid (serine) at position 57, the frequency of DRB1*0405 was low in ITP patients compared with controls. Thus, the serine at position 57 of DRB1*0410 may not play a crucial role in the immunopathology of ITP in Japanese patients.

Table 8.

Comparison of Amino Acid Sequences in the Hypervariable Region of the DRB1 Chain

DRB1 Residues
37577486
*0403 -------- Y -------- D -------- E -------- V  -------- 
*0404 -------- Y -------- D -------- E -------- V  -------- 
*0405 -------- Y -------- S -------- A -------- G7-151 -------- 
*0406 -------- S -------- D -------- E -------- V  -------- 
*0407 -------- Y -------- D -------- E -------- G  -------- 
*0410 -------- Y -------- S -------- A -------- V7-151 -------- 
DRB1 Residues
37577486
*0403 -------- Y -------- D -------- E -------- V  -------- 
*0404 -------- Y -------- D -------- E -------- V  -------- 
*0405 -------- Y -------- S -------- A -------- G7-151 -------- 
*0406 -------- S -------- D -------- E -------- V  -------- 
*0407 -------- Y -------- D -------- E -------- G  -------- 
*0410 -------- Y -------- S -------- A -------- V7-151 -------- 

Amino acid sequences were translated from the nucleotide sequences and are shown as the standard 1-letter codes. Dashes indicate identity with the DRB1*04 sequence.

F7-151

Different sequences in DRB1*0405 and DRB1*0410.

Next, we investigated the significance of DRB1*0410 in ITP patients. First, plasma autoantibodies were studied in 111 patients using a microtiter well assay, which showed that 36 patients (32.4%) had anti-GPIIb/IIIa autoantibodies. Many antiplatelet autoantibodies bind to platelet GPIIb/IIIa.7 In particular, there have recently been investigations on the precise localization of antigenic epitopes, such as the carboxy-terminal region (C-terminus) of GPIIIa,8 the Ca2+-dependent epitope of GPIIb/IIIa complex,9,10 and the 50-kD cysteine-rich region of GPIIIa.11 Bowditch et al12 concluded that a limited number of shared epitopes on platelet GPIIb/IIIa were recognized in ITP. In the present study, we observed a positive association of anti-GPIIb/IIIa antibody with HLA-DR4, although we could not analyze the epitope. Thus, it seems likely that the response to the antigenic epitope of GPIIb/IIIa is restricted by HLA-DR4.

Self-reactive T cells are controlled either by elimination in the thymus or by induction of tolerance in the periphery.50,51Both mechanisms depend on the ability of antigen-presenting cells to process and present self-peptides in the context of HLA molecules, although it is now apparent that not all possible peptides from a self-antigen are presented.52,53 These limitations in processing suggest that T cells specific for some determinants on self-proteins may escape tolerance and therefore remain part of the normal T-cell repertoire. Such autoreactive T cells may only cause pathologic conditions when the presentation of normally hidden self-peptides occurs or when the immune system is confronted with foreign molecular mimics. Interestingly, autoreactive T cells to GPIIb/IIIa have been isolated from ITP patients,54,55whereas Filion et al56 reported that autoreactive T cells to GPIIb/IIIa are present in the peripheral blood of healthy individuals (this may represent the condition of “anergy”). However, there was no association of anti-GPIIb/IIIa autoantibody with DRB1*0410 in the present study. There are many epitopes recognized by anti-GPIIb/IIIa autoantibodies,8-10 but the clinical significance of these autoantibodies is not always clear.34The lack of a correlation between anti-GPIIb/IIIa antibody and the DRB1*0410 allele may suggest that this antibody does not cause ITP.

Next, we compared HLA-DR4 and DRB1*0410 distribution in patients with a good or poor response to prednisolone. HLA-DR4, particularly DRB1*0410, was significantly decreased in patients with a good response. There have been various reports on the mechanism of action of steroid hormones in ITP.1,57,58 Treatment with corticosteroids appears to shorten the duration of thrombocytopenia by inhibiting phagocytosis and thereby increasing the lifespan of platelets.1,57 Steroids may also directly inhibit IgG production and increase IgG catabolism.1,59 The inhibition of IgG production may result from an effect of steroids on lymphocytes, but few reports have been published on the antibodies in patients responding poorly to steroid therapy. In the present study, anti-GPIIb/IIIa was not correlated with the response to steroids. A relationship between the HLA system and the outcome of therapy for ITP was previously reported by Gratama et al.19 Their results suggested that HLA-DR4 may predict the response to treatment, ie, a poor response to corticosteroids and a favorable outcome of splenectomy. However, they did not study DRB1*04 alleles. On the other hand, Islam et al47 reported that DRB1*0405 and/or DRB1*0410 were responsible for the chronic type of VKH. The sequences of DRB1*0405 and DRB*0410 are identical except for amino acid 86: glycine in DRB1*0405 and valine in DRB1*0410 (Table 8).60Three hypervariable regions determine HLA antigenicity and possibly most of the peptide-binding capacity of HLA proteins61; these regions are identical between DRB1*0405 and DRB1*0410. DR4 specificity is determined primarily by the first and second hypervariable regions (amino acids 8 to 14 and 26 to 37) of the DRB1* peptide,60 so these regions are likely to determine susceptibility to anti-GPIIb/IIIa antibody in patients with ITP. Although it is difficult to conclude that amino acid 86 is the ITP-susceptibility determinant, there is a possibility that this amino acid of HLA-DRB1*0410 plays an important role in resistance to prednisolone therapy. However, other DRB1*0410-related genes may also participate in steroid resistance, so further studies of this issue are needed.

In conclusion, this was the first study to compare HLA-DR4–related alleles in pathologic and clinical subgroups of ITP. Some antigenic epitopes of GPIIb/IIIa seem likely to be partially restricted by HLA-DR4, but there was no association of anti-GPIIb/IIIa autoantibody with DRB1*0410. On the other hand, DRB1*0410 was significantly decreased in patients with a good response to prednisolone. Our findings indicate that genetic factors influence the clinical course of ITP, but this study should be considered preliminary because of possible racial differences in the HLA status of ITP patients from Japan and other countries.

ACKNOWLEDGMENT

The authors thank Prof Masanobu Uyama (Department of Ophthalmology, Kansai Medical University, Osaka, Japan) for his helpful suggestions.

Supported in part by a Research Grant for Advanced Medical Care from the Ministry of Health and Welfare of Japan.

Address reprint requests to Shosaku Nomura, MD, The First Department of Internal Medicine, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi Osaka 570, Japan.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.

REFERENCES

1
Karpatkin
S
Autoimmune thrombocytopenic purpura.
Blood
56
1980
329
2
McMillan
R
Chronic idiopathic thrombocytopenic purpura.
N Engl J Med
304
1981
1135
3
Arnott
J
Horsewood
P
Kelton
JG
Measurement of platelet-associated IgG in animal models of immune and nonimmune thrombocytopenia.
Blood
69
1987
1294
4
Oyaizu
N
Yasumizu
R
Miyama-Inaba
M
Nomura
S
Yoshida
H
Miyawaki
S
Shibata
Y
Mitsuoka
S
Yasunaga
K
Morii
S
Good
RA
Ikehara
S
(NZWxBXSB) F1 mouse. A new animal model of idiopathic thrombocytopenic purpura.
J Exp Med
167
1988
2017
5
Woods
VL
Oh
EH
Mason
D
McMillan
R
Autoantibodies against the platelet glycoprotein IIb/IIIa complex in patients with chronic ITP.
Blood
63
1984
368
6
Woods
VL
Kurata
Y
Montgomery
RR
Tani
P
Mason
D
Oh
EH
McMillan
R
Autoantibodies against platelet glycoprotein Ib in patients with chronic immune thrombocytopenic purpura.
Blood
64
1984
156
7
McMillan
R
Tani
P
Millard
F
Berchtold
P
Renshaw
L
Woods
VL
Platelet-associated and plasma anti-glycoprotein autoantibodies in chronic ITP.
Blood
70
1987
1040
8
Fujisawa
K
O'Toole
TE
Tani
P
Lofus
JC
Plow
EF
Ginsberg
MH
McMillan
R
Autoantibodies to the presumptive cytoplasmic domain of platelet glycoprotein IIIa in patients with chronic immune thrombocytopenic purpura.
Blood
77
1991
2207
9
Fujisawa
K
Tani
P
O'Toole
TE
Ginsberg
MH
McMillan
R
Different specificities of platelet-associated and plasma autoantibodies to platelet GPIIb-IIIa in patients with chronic ITP.
Blood
79
1992
1441
10
Fujisawa
K
McMillan
R
Platelet-associated antibody to glycoprotein IIb/IIIa from chronic immune thrombocytopenic purpura patients often binds to divalent cation-dependent antigens.
Blood
81
1993
1284
11
Kekomaki
R
Dawson
B
McFarland
J
Kunicki
TJ
Localization of human platelet autoantigens to the cysteine-rich region of glycoprotein IIIa.
J Clin Invest
88
1991
847
12
Bowditch
RD
Tani
P
Fong
KC
McMillan
R
Characterization of autoantigenic epitopes on platelet glycoprotein IIb/IIIa using random peptide libraries.
Blood
88
1996
4579
13
Ballem
PJ
Segal
GM
Stratton
JR
Gernsheimer
T
Adamson
JW
Slichter
SJ
Mechanisms of thrombocytopenia in chronic autoimmune thrombocytopenic purpura: Evidence of both impaired platelet production and increased platelet clearance.
J Clin Invest
80
1987
33
14
George
JN
Woolf
SH
Raskob
GE
Wasser
JS
Aledort
LM
Ballem
PJ
Blanchette
VS
Bussel
JB
Cines
DB
Kelton
JG
Lichtin
AE
McMillan
R
Okerbloom
JA
Regan
DH
Warrier
I
Idiopathic thrombocytopenic purpura: A practice guideline developed by explicit methods for the American Society of Hematology.
Blood
88
1996
3
15
Bodmer WF, Bodmer J (eds): Proceedings of the Seventh International Histocompatibility Workshop, 1977. Histocompatibility Testing. Copenhagen, Denmark, Munksgaard, 1978
16
(suppl)
Gibofsky
A
Winchester
R
Hansen
J
Patarroyo
M
Dupont
B
Paget
S
Lahita
R
Halper
J
Fotino
M
Yunis
E
Kunkel
HG
Contrasting patterns of newer histocompatibility determinants in patients with rheumatoid arthritis and systemic lupus erythematosus.
Arth Rheum
21
1978
S133
17
Karpatkin
S
Association of HLA-DRw2 with autoimmune thrombocytopenic purpura.
J Clin Invest
63
1979
1085
18
Helmerhorst
FM
Nijenhuis
LE
De Lange
GG
Van den Berg-Loonen
PM
Jansen
MFM
Von dem Borne
AEGKR
Engelfriet
CP
HLA antigens in idiopathic thrombocytopenic purpura.
Tissue Antigens
20
1982
372
19
Gratama
JW
D'Amaro
J
De Koning
J
Den Ottolander
GJ
The HLA-system in immune thrombocytopenic purpura: Its relation to the outcome of therapy.
Br J Haematol
56
1984
287
20
Porges
A
Bussel
J
Kimberly
R
Schulman
I
Pollack
M
Pandey
J
Barandun
S
Hilkartner
M
Elevation of platelet associated antibody levels in patients with chronic idiopathic thrombocytopenic purpura expressing the B8 and/or DR3 allotypes.
Tissue Antigens
26
1985
132
21
Saiki
RK
Bugawan
TL
Horn
GT
Mullis
KB
Erlich
HA
Analysis of enzymatically amplified β-globin and HLA-DQβ DNA with allele-specific oligonucleotide probes.
Nature
324
1986
163
22
Mullis
KB
Fallona
F
Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction.
Methods Enzymol
144
1987
335
23
Bell
JI
Denny
DD
Foster
L
Belt
T
Todd
JA
McDevitt
HO
Allelic variation in the DR subregion of the human major histocompatibility complex.
Proc Natl Acad Sci USA
84
1987
6234
24
Todd
JA
Bell
JA
McDevitt
HO
HLA-DQβ gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus.
Nature
329
1987
599
25
Watanabe
Y
Tokunaga
K
Matsuki
K
Takeuchi
F
Matsuta
K
Maeda
H
Omoto
K
Juji
T
Putative amino acid sequence of HLA-DRB chain contributing to rheumatoid arthritis susceptibility.
J Exp Med
169
1989
2263
26
Gaiger
A
Neumeister
A
Heinzl
H
Pabinger
I
Panzer
S
HLA class-I and -II antigens in chronic idiopathic autoimmune thrombocytopenia.
Ann Hematol
68
1994
299
27
Ahn
YS
Harrington
WJ
Treatment of idiopathic thrombocytopenic purpura.
Annu Rev Med
28
1977
299
28
Imbach
P
Barandun
S
D'Appuzo
V
Baumgartner
C
Hirt
A
Morell
A
Rossi
E
Schoni
M
Vest
M
Wagner
HP
High dose intravenous gamma globulin for idiopathic thrombocytopenic purpura in childhood.
Lancet
2
1981
1228
29
Ahn
YS
Harrington
WJ
Simon
SR
Mylvaganam
R
Pall
LM
So
AG
Danazol for treatment of idiopathic thrombocytopenic purpura.
N Engl J Med
308
1983
1396
30
Berchtold
P
McMillan
R
Therapy of chronic idiopathic thrombocytopenic purpura in adults.
Blood
74
1989
2309
31
Andersen
JC
Response of resistant idiopathic thrombocytopenic purpura to pulsed high-dose dexamethasone therapy.
N Engl J Med
330
1994
1560
32
Terasaki
PI
McClelland
JD
Microdroplet assay of human serum cytotoxins.
Nature
204
1964
998
33
Ota
M
Seki
T
Fukushima
H
Tsuji
K
Inoko
H
HLA DRB1 genotyping by modified PCR-RFLP method combined with group-specific primers.
Tissue Antigens
39
1992
187
34
Nomura
S
Yanabu
M
Soga
T
Kido
H
Fukuroi
T
Yamaguchi
K
Nagata
H
Kokawa
T
Yasunaga
K
Analysis of idiopathic thrombocytopenic purpura patients with antiglycoprotein IIb/IIIa or Ib autoantibodies.
Acta Haematol
86
1991
25
35
Kokawa
T
Nomura
S
Yanabu
M
Yasunaga
K
Detection of platelet antigen for antiplatelet antibodies in idiopathic thrombocytopenic purpura by flow cytometry, antigen-capture ELISA, and immunoblotting: A comparative study.
Eur J Haematol
50
1993
74
36
Nomura
S
Nagata
H
Oda
K
Kokawa
T
Yasunaga
K
Effects of EDTA on the membrane glycoprotein IIb-IIIa complex—Analysis using flow cytometry.
Thromb Res
47
1987
47
37
Nomura
S
Suzuki
M
Kido
H
Yamaguchi
K
Fukuroi
T
Yanabu
M
Soga
T
Nagata
H
Kokawa
T
Yasunaga
K
Differences between platelet and microparticle glycoprotein IIb/IIIa.
Cytometry
13
1992
621
38
Tsubakio
T
Kurata
Y
Yonezawa
T
Kitani
T
Quantification of platelet-associated IgG with a competitive solid-phase enzyme-immunoassay.
Acta Haematol
66
1981
251
39
Thomson
G
A review of theoretical aspects of HLA and disease association.
Theor Popul Biol
20
1981
168
40
Synder
DA
Tessler
HH
Vogt-Koyanagi-Harada syndrome.
Am J Ophthalmol
90
1980
69
41
Ohno
S
Immunological aspects of Behcet's and Vogt-Koyanagi-Harada's disease.
Trans Ophthalmol Soc UK
101
1981
335
42
Nimer
SD
Ireland
P
Meshkinpour
A
Frane
M
An increased HLA DR2 frequency is seen in aplastic anemia patients.
Blood
84
1994
923
43
Nakao
S
Takamatsu
H
Chuhjo
T
Ueda
M
Shiobara
S
Matsuda
T
Kaneshige
T
Mizoguchi
H
Identification of a specific HLA class II haplotype strongly associated with susceptibility to cyclosporine-dependent aplastic anemia.
Blood
84
1994
4257
44
Ahmed
SA
Penhale
WJ
Talal
N
Sex hormones, immune responses, and autoimmune diseases.
Am J Pathol
121
1985
531
45
Cavan DA, Penny MA, Jacobs KH, Kelly MA, Jenkins D, Mijpvic C, Chow C, Cockram CS, Hawkins BR, Barnett AH: The HLA association with Graves' disease is sex-specific in Hong Kong Chinese subjects. Clin Endocrinol (Oxf) 40:63, 1994
46
Shindo
Y
Inoko
H
Yamamoto
T
Ohno
S
HLA-DRB1 typing of Vogt-Koyanagi-Harada's disease by PCR-RFLP and the strong association with DRB1*0405 and DRB1*0410.
Br J Ophthalmol
78
1994
223
47
Islam
SMM
Numaga
J
Fujino
Y
Hirata
R
Matsuki
K
Maeda
H
Masuda
K
HLA class II genes in Vogt-Koyanagi-Harada disease.
Invest Ophthalmol Vis Sci
35
1994
3890
48
Petesdorf
EW
Smith
AG
Mickelson
EM
Martin
PJ
Hansen
JA
Ten HLA-DR alleles defined by sequence polymorphisms within the DRB1 first domain.
Immunogenetics
33
1991
267
49
Lanchbury
JSS
Hall
MA
Welsh
KI
Panayi
GS
Sequence analysis of HLA-DR4B1 subtypes: Additional first domain variability is detected by oligonucleotide hybridization and nucleotide sequencing.
Hum Immunol
27
1990
136
50
Kappler
JW
Roehm
N
Marrack
P
T cell tolerance by clonal elimination in the thymus.
Cell
49
1987
273
51
Schwartz
RH
Acquisition of immunologic tolerance.
Cell
57
1988
1073
52
Schild
H
Rotzschke
O
Kalbacher
H
Rammensee
HG
Limit of T cell tolerance to self proteins by peptide presentation.
Science
247
1990
1587
53
Nagy
Z
Lehmann
PV
Falcioni
F
Muller
S
Adorini
L
Why peptides? Their possible role in the evolution of MHC-restricted T cell recognition.
Immunol Today
10
1989
132
54
Semple
JW
Freedman
J
Increased antiplatelet T helper lymphocyte reactivity in patients with autoimmune thrombocytopenia.
Blood
78
1991
2619
55
Ware
RE
Howard
TA
Phenotypic and clonal analysis of T lymphocytes in childhood immune thrombocytopenic purpura.
Blood
82
1993
2137
56
Fillion
MC
Proulx
C
Bradley
AJ
Devine
DV
Sekaly
R-P
Decary
F
Chartrand
P
Presence in peripheral blood of healthy individuals of autoreactive T cells to a membrane antigen present on bone marrow–derived cells.
Blood
88
1996
2144
57
Cines
DB
Schreiber
AD
Immune thrombocytopenia: Use of a Coombs antiglobulin test to detect IgG and C3 on platelets.
N Engl J Med
300
1979
106
58
Sartorius
JA
Steroid treatment of idiopathic thrombocytopenic purpura in children: Preliminary results of a randomized cooperative study.
Am J Pediatr Hematol Oncol
6
1984
165
59
Saxon
A
Stevens
RH
Ramer
SJ
Clements
PL
Yu
DTY
Glucocorticoids administered in vivo inhibit human suppressor T lymphocyte function and diminish B lymphocyte responsiveness in in vivo immunoglobulin synthesis.
J Clin Invest
61
1977
922
60
Marsh
S
Bodmer
J
HLA class II nucleotide sequences, 1992.
Hum Immunol
35
1992
1
61
Fremont
DH
Matsumura
M
Stura
EA
Peterson
PA
Wilson
IA
Crystal structures of two viral peptides in complex with murine MHC class I H-2Kb.
Science
257
1992
919