Maternal DRB1*1501, DQA1*0102, DQB1*0602 haplotype in fetomaternal alloimmunization against human platelet alloantigen HPA-6b (GPIIIa-G1n489)

Fetomaternal incompatibility of platelet alloantigens may lead to alloimmunization and neonatal alloimmune thrombocytopenia (NAIT). Human platelet alloantigen (HPA) 6b, which associates with residue Gln 489 of platelet membrane glycoprotein IIIa, has been described as a cause of NAIT. We have studied the MHC genes of all available family members in the six thus far reported families with a thrombocytopenic newborn and fetomaternal HPA-6b incompatibility. Maternal HPA-6b antibodies could be detected in five mothers to the altogether seven thrombocytopenic male infants. The MHC genes HLA-DRB, -DQA1, -DQB1, -DPB1, TAP1,2 and HSP70-Hom were studied by using polymerase chain reaction (PCR)-based DNA analysis methods. All five mothers with detectable circulating HPA-6b antibodies at the time of delivery shared an identical DRB1 *1501, DQA1 *0102, DQB1 *0602 haplotype. The sixth, HPA antibody-negative mother and a HPA-6b-negative mother to a healthy HPA-6b⁺ child were negative for this haplotype. The frequency of DRB1*15-positive haplotype was increased in immunized mothers (100%) as compared with the general Finnish population (27%), but the association was not statistically significant after correction. We conclude that there is a potential association between the MHC haplotype DRB1 *1501, DQA1 *0102, DQB1 *0602 and alloimmunization to the HPA-6b antigen and that this alloimmunization probably involves different HLA class II molecules from immunization to HPA-la.     

Polymorphisms of TAP 1 and TAP2 genes in Graves' disease

Graves' disease is an autoimmune disorder in which HLA DQA1*0501 and DQB1*0201 confer predisposition. The genes for transporters associated with antigen processing (TAP1 and TAP2) locate near to HLA DQ coding regions and display only a limited degree of polymorphism. Since polymorphisms of TAP might influence susceptibility to Graves' disease by a possibly different selection of antigenic peptides, we investigated sequence variants of TAP1 and TAP2 genes in 235 patients with Graves' disease and 218 random healthy controls by polymerase chain reaction (PCR) followed by sequence specific oligonucleotide analysis (SSO), single strand conformational polymorphism (SSCP) analysis and amplification refractory mutation system (ARMS). TAP1*0301 (Val-333/Asp-637: 71% vs. 55% in controls. p< .0008, RR=2.05) and TAP2*0101 (Val-379/Ala-565/Thr-665/stop-687: 83% vs. 69% in controls, p< .003, RR=2.20) showed a positive association with Graves' disease whereas TAP1*0401 a negative (Ile-333/Gly-637: 4% vs. 13% in controls, p< .001, RR=0.25). After selection of patients and controls for HLA DQA1*0501 a similar association was found for TAP1*0301 (72% vs. 50% in controls, p< .002, RR=2.63) and TAP1*0401 (4% vs. 16% in controls, p< .004, RR=0.22), when matching for HLA DQB1*0201 as well as for TAP1*0401 (3% vs. 16% in controls, p< .005, RR=0.18). Our findings indicate that the positive association of TAP1*0301 and the negative of TAP1*0401 with Graves' disease cannot only be explained by linkage disequilibrium between TAP alleles and HLA DQ. Therefore, these TAP alleles contribute to genetic susceptibility in Graves' disease as additional permissive and protective factors.     

The prevalence of HPA-1a alloimmunization and the potential risk of FNAIT depend on both the DRB3*01:01 allele and associated DR-DQ haplotypes

Alloimmunization against human platelet antigen (HPA)-1a during pregnancy can cause foetal/neonatal alloimmune thrombocytopenia (FNAIT) and severe bleeding in the foetus or newborn and likely depends on several factors. HPA-1a alloimmunization is associated with DRB3*01:01, which is associated with several DR-DQ haplotypes. However, it is not known to what extent these haplotypes contribute to the prevalence of HPA-1a alloimmunization. HPA-1a–alloimmunized women, identified in a prospective study, and random donors were typed for selected DRB3, DRB4, DRB1, DQA1 and DQB1 alleles to determine allele and DR-DQ haplotype frequencies. DRB3*01:01 was carried by 94% HPA-1a–immunized women compared to 27% in the general population. In the first population, the DR3-DQ2 haplotype was overrepresented (P < .003). The prevalence of HPA-1a alloimmunization was estimated to be about twice as frequent with DR3-DQ2 compared to DR13-DQ6, together accounting for about 90% of DRB3*01:01–positive individuals. Further, we examined DQB1*02 and DRB4*01:01 alleles for their reported association with HPA-1a alloimmunization, in the context of DR-DQ haplotypes. Since ~ 80% of DQB1*02 alleles are linked to the DR3-DQ2 haplotype, the association might be coincidental. However, the DQB1*02:02–associated DR7-DQ2 haplotype was also overrepresented in alloimmunized women, suggesting a role for this allele or haplotype in HPA-1a alloimmunization. As DRB4*01:01 is predominantly associated with the DR7-DQ2 haplotype in HPA-1a–alloimmunized individuals, the reported association with FNAIT may be coincidental. Typing for DR-DQ haplotypes revealed important genetic associations with HPA-1a alloimmunization not evident from typing individual alleles, and the presence of different DRB3-associated DR-DQ haplotypes showed different prevalence of HPA-1a alloimmunization.     

Reactivity of T cells from women with antibodies to the human platelet antigen (HPA)-1a to peptides encompassing the HPA-1 polymorphism

The human platelet antigen-1a (HPA-1a) is the most common alloantigenic target in fetal and neonatal alloimmune thrombocytopenia (NAIT). Treatment currently depends on the outcome in previous pregnancies. HPA-1 specific T cell responses were determined in 14 HPA-1a alloimmunized women during or after pregnancies affected by NAIT. Peripheral blood mononuclear cells were incubated with peptides encompassing the Leu33Pro polymorphism (residues 20-39 and 24-45 in both Leu33 (HPA-1a) and Pro33 (HPA-1b) forms) or control recall antigens in the presence of autologous sera and T cell proliferation was measured by (3)H-thymidine incorporation. Control antenatal and postpartum sera suppressed T cell proliferation and use of such sera was avoided. Most patients (86%) responded to the HPA-1a peptides with 64% also having weaker T cell proliferation to the HPA-1b peptides; 14% had no activity towards any peptide despite responding to control antigens. Administration of IVIG during pregnancy appeared to reduce T cell reactivity to HPA-1 peptides. Postnatal anti-HPA-1a T cell responses from women who had a severe history of NAIT (an intracranial haemorrhage in a previous fetus) were greater than those from women with a mild history. This assay may have the potential to predict disease severity if performed prior to or early in pregnancy.     

Leucine33-proline33 substitution in human platelet glycoprotein IIIa determines HLA-DRw52a (Dw24) association of the immune response against HPA-1a (Zwa/PIA1) and HPA-1b (Zwb/PIA2).

Alloantibody formation against HPA-1a (Zwa/PIA1) has, to date, only been found in HLA-DRw52(a+) (Dw24) individuals. Alloimmunization against the product of the other HPA-1 allele, HPA-1b, is rare. We have been able to evaluate ten cases of HPA-1b alloimmunization in Europe in order to study whether there is an association between HLA phenotype and anti-HPA-1b antibody formation. HLA typing of these patients was performed with particular attention to the DRw52a specificity using specific T-cell clones. No association with DRw52a or any other known HLA phenotype was found. This finding implies that the amino acid substitution leucine33-proline33 in GPIIIa, responsible for HPA-1a/b, is of primary importance for the association of anti-HPA-1a antibody formation with DRw52a. These data show that the amino acid polymorphism affects the presentation of the immunogenic oligopeptides of HPA-1a and -1b in the HLA class-II groove.     

Is there a relationship between anti-HPA-1a concentration and severity of neonatal alloimmune thrombocytopenia?

There is uncertainty about the relationship between anti-HPA-1a levels and severity of neonatal alloimmune thrombocytopenia (NAIT). To investigate this relationship further,the concentration of anti-HPA-1a in HPA-1b homozygous women was determined, using a newly developed quantitative ELISA that uses purified anti-HPA-1a to obtain a standard curve. Seventy-eight samples collected from 22 HPA-1b homozygous pregnant women at various stages of pregnancy were tested. These included five women who had delivered babies with severe NAIT. A national HPA-1a antibody standard (NIBSC 93/710), designated as 1 arbitrary unit/mL (AU/mL),was used in each ELISA to calibrate the purified anti-HPA- 1a, enabling the presentation of results as AU/mL. Moreover, selected samples were also assayed by PAK 12 and their reactivity compared with quantity of antibody. The use of the purified HPA- 1a antibody yielded consistent sigmoid curves, enabling the measurement of HPA-1a antibody concentration in the test samples. The antibody concentration was significantly correlated with the antibody titer in the 78 samples studied (R = 0.54, p < 0.001). Furthermore, there was a significant correlation between PAK 12 and the quantitative ELISA in a selected number of cases, with or without NAIT (R = 0.71, n = 10; p < 0.02). On the other hand, there was no correlation of antibody concentration with NAIT incidence (R = -0.046). This study indicates that there is no relationship between anti-HPA-1a concentration and severity of NAIT when ELISA is used, although the correlation between ELISA and other methods, such as monoclonal antibody immobilization of platelet antigens (MAIPA) assay, remains to be determined.     

Gene frequencies of the HPA-1 to HPA-13, Oe and Gov platelet antigen alleles in Taiwanese,Indonesian, Filipino and Thai populations

Human platelet antigen (HPA) systems consist of more than twelve bi-allelic antigen polymorphisms in which a base pair substitution leads to change in an amino acid of a glycoprotein expressed on the platelet. The neonatal alloimmune thrombocytopenia (NAIT), post transfusion purpura, and refractoriness to platelet transfusion can be induced by antibodies against human platelet antigens: e.g. HPA-1a, 3a, 4a, 5a, and Gova. HPA typing is essential for the diagnosis and treatment of a variety of diseases. We developed a PCR-based method to detect HPA-1 to HPA-13, Oe and Gov platelet alloantigens. In this method, the amplified PCR products were used to recognize the polymorphism after restriction enzyme digestions. Among 566 Taiwanese, 107 Indonesian, 100 Filipino and 137 Thai subjects studied, HPA-1a, 2a, 4a, 5a, 6a, 7aW, 8aW, 9a, 10a, 11a, 12a, 13a, Oea genes were present in every sample; while HPA-1b, 2b, 4b, 5b and 6b were rarely found. HPA-7aW, 8aW, 9, 10, 11, 12, 13, and Oea alleles were noted to be monomorphic only. HPA-3a/3b alleles had frequencies of 0.595/0.405, 0.505/0.495, 0.507/0.493, 0.530/0.470, while Gova/Govb of 0.462/0.538, 0.450/0.550, 0.463/0.537, 0.520/0.480 among Taiwanese, Indonesians, Thais and Filipinos respectively. The prevalence rates of HPA-1 to 13 in this study were also consistent with other previous reports using different methods. The alloimmunization due to Gov and HPA-3 antigens need to be emphasized in these populations.     

Neonatal alloimmune thrombocytopenia due to anti-HPA-5b (Bra)

Neonatal alloimmune thrombocytopenia (NAIT) results from maternal immunization against fetal platelet antigens and can occur during the first pregnancy. The most common complications of NAIT are neonatal thrombocytopenia, intracerebral hemorrhage, and fetal death. Most cases of NAIT in Caucasians are caused by anti-HPA-1a (PlA1). Anti-HPA-5b (Bra) accounts for only 4.3 percent of all NAIT cases. NAIT due to anti-HPA-5b is thought to be milder and have fewer complications than NAIT caused by anti-HPA-1a because of the lower number of HPA-5b antigenic sites per platelet. This report describes a severe case of NAIT due to anti-HPA-5b that was treated by intrauterine platelet transfusion.     

HLA microsatellite associations with insulin-dependent diabetes mellitus in Singaporean Chinese.

Singaporean Chinese with insulin-dependent diabetes mellitus (IDDM) have previously been shown to be associated with the DRB1*0301 haplotype and the joint occurrence of DRB1*0301/*0901 and DRB1*0301/*04. The present study extended previous HLA associations by investigating the HLA region using four microsatellites (TNFa, D6S273, TAP1, DQCARII). Seventy-five IDDM patients and 80 healthy controls were studied. TNFa*3 (RR = 2.26), TNFa*12 (RR = 3.30), TAP1*9 (RR = 2.55) showed increased frequencies while TNFa*11 (RR = 0.29), TAP1*4 (RR = 0.50) showed decreased frequencies in patients compared to controls. Linkage analysis suggested that the positive associations of TNFa*3 and TAP1*9 were secondary to that of DRB1*0301. However, TNFa*12 appeared to provide additional risks to IDDM besides the DRB1*0301 haplotype, whereas TNFa*11 and TAP1*4 conferred an independent protective effect against IDDM. Our findings reinforce the notion that susceptibility to and protection against IDDM may include TNF region. In the present study, TNFa*12 seemed to be the primary association in the DRB1*0405 haplotype and may play an independent role in the pathogenesis of IDDM through TNF-alpha function.     

Neonatal alloimmune thrombocytopenia due to HPA-3a antibodies: a case report

A healthy infant was born at term by elective cesarean section to a 32-year-old para 4, gravida 4, mother. Within 24 hours, the infant was noted to have fairly extensive bruising on the back and shoulders. A full blood count evaluation was remarkable for severe thrombocytopenia (platelet count of 29 x 10(9)/L). Other hematologic parameters were normal. Human leukocyte antigen (HLA) class-1 antibodies but not platelet-specific antibodies were detectable in the maternal serum using a commercial antigen-capture ELISA (GTI-PakPlus kit). Anti-HPA-3a antibodies, while weakly reactive in the monoclonal antibody immobilization of platelet antigens (MAIPA) assay in the immediate postpartum serum, were readily detectable using this assay in a sample taken 4 weeks later. Genotyping for human platelet antigens (HPA) 1-5 by the polymerase chain reaction technique with sequence-specific primers (PCR-SSP) revealed the infant's platelet genotype to be HPA-1a/1a, 3a/3b, while that of the mother was HPA-1a/1a, 3b/ 3b, consistent with a diagnosis of anti-HPA-3a neonatal alloimmune thrombocytopenia (NAIT). This case illustrates the increased sensitivity of the MAIPA technique for the detection of platelet-specific antibodies. We believe this to be the first serologically confirmed case of NAIT due to anti-HPA-3a to be reported in the republic of Ireland.     

Transporter associated with antigen-processing (TAP) genes and susceptibility to tuberculoid leprosy and pulmonary tuberculosis

We have studied TAP polymorphism in a panel of 40 healthy individuals, 57 patients with pulmonary tuberculosis (PTB) and 50 with tuberculoid (TT) leprosy from North India. Only TAP2-A/F occurred with a significantly increased frequency in PTB patients as compared to controls (82.5% vs. 52.5%, P<0.002, Pc<0.01) giving a high relative risk of 4.3. On the other hand, TAP2-B was significantly increased in TT leprosy as compared to controls (76% vs. 47.5%, Pc<0.003, RR 3.5) particularly in patients positive for HLA-DR15 than controls carrying DR15 (77.5% vs. 50%, P<0.03, RR=3.4). Further, TAP2-B allele was positively associated with DR15 negative PTB patients as compared to the DR15 positive group (43.8% vs. 17.1%, P<0.04, RR=0.3), This study along with our earlier studies on HLA association in mycobacterial diseases suggests that in addition to HLA-DR15, alleles in the TAP2 region influence susceptibility to PTB and TT leprosy.     

Human platelet antigen genotypes in Turkish and Caucasian blood donors in Germany

Exposition to allogenic human platelet antigens (HPAs) can lead to antibody formation causing neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura or platelet (PLT) transfusion refractoriness. The frequencies of HPA differ between ethnical groups which could be associated with different potential alloimmunization risk. The Turkish population is the largest ethnic minority group living in Germany. However, no data are available about the HPA frequency among Turkish population. We compared the frequency of HPA between Caucasian and Turkish blood donors. DNA from blood samples of 119 Caucasian and 117 Turkish blood donors was isolated. The genotype of HPA-1, -2, -3 -4, -5 and -15 was determined using a commercialized polymerase chain reaction kit with sequence-specific primers. In Turkish blood donors, the gene frequencies of HPA-1a/1b, -2a/2b, -3a/3b, -4a/4b, -5a/5b and -15a/15b were 0.863/0.137, 0.868/0.133, 0.607/0.393, 0.996/0.004, 0.893/0.107 and 0.474/0.256, respectively. In Caucasians, we observed 0.798/0.202, 0.908/0.092, 0.567/0.432, 1.000/0.000, 0.916/0.084 and 0.517/0.483 for HPA-1a/1b, -2a/2b, -3a/3b, -4a/4b, -5a/5b and -15a/15b, respectively. No statistically significant difference between genotypes in these populations could be observed. Due to the similar distribution of HPA genotypes in both ethnical groups, no increased risk of NAIT for children of mixed couples or of post-transfusion purpura or PLT transfusion refractoriness secondary to antibodies to HPAs for recipients of PLT concentrates from blood donors of the other ethnicity is given.     

广州汉族配偶人类血小板抗原基因分型的调查

目的调查配偶之间人类血小板抗原（HPA）的不配合率并评估其在新生儿同种免疫性血小板减少症（NAIT）中的作用。方法采用SSP法对200对广州汉族配偶进行了HPA-1～-16基因分型。结果配偶双方HPA-1～-6及HPA-15均在同-HPA位点具有不同基因型．HPA-7～-14及HPA-16均为a／a纯合子。得到HPA-1～-16的等位基因频率。HPA．15、HPA-3、HPA-2、HPA-6、HPA-5、HPA-1和HPA一4的不配合率为别为37．42％、37．02％、8．02％、3．84％、2．44％、0．75％和0．24％,其余HPA位点的不配合率为0。结论HPA-5可能是广州汉族人群NAIT最重要的一个HPA系统。HPA-15、HPA-3、HPA-2、HPA-6和HPA-4也分别具有其免疫学意义,这为HPA的抗体检测以及NAIT的诊断治疗提供了实验依据。     

Gene frequencies of the HPA-1 to 6 and Gov human platelet antigens in Thai blood donors

Human platelet alloantigens (HPA) are important in neonatal alloimmune thrombocytopenia (NAIT), posttransfusion purpura (PTP), platelet transfusion refractoriness, passive alloimmune thrombocytopenia, and transplantation-associated alloimmune thrombocytopenia. Thus, HPA genotyping is essential in diagnosis and treatment. We analyzed HPA-1 to 6 and Gov alleles, using PCR with sequence specific primers (PCR-SSP) in 500 Thai blood donors who had been HLA class I antigen typed. HPA-4a was present in all samples. HPA-1b, -2b, -5b, and -6b were rare, and HPA-4b was not found. HPA-3a and -3b showed frequencies of 56.0 percent and 44.0 percent, respectively. Gova and Govb showed frequencies of 49.1 percent and 50.9 percent, respectively. The prevalence rates of HPA-1 to 6 gene frequencies (GFs) were consistent with those of other Asian populations rather than those of Caucasians. We also report on the GFs of Gova and Govb, which also are comparable to those of Asian populations. Our results could establish a useful HPA- and HLA-matched plateletpheresis donor file and provide an improvement of platelet alloantibody detection in alloimmune thrombocytopenic patients, and, therefore, a more effective platelet transfusion program.     

Neonatal alloimmune thrombocytopenia due to anti-HPA-2b (anti-Koa)

Most severe cases of neonatal alloimmune thrombocytopenia (NAIT) are due to anti-HPA-1a (anti-PlA1) antibodies. We report a case of NAIT due to anti-HPA-2b that resulted in in utero intracranial hemorrhage.A 33-year-old G2P1A0 Caucasian woman had a routine ultrasound at 34 weeks. The fetus appeared to have a left hemispheric hematoma. IVIG, 1g/kg, was started immediately and administered weekly until delivery. One day after receiving the first dose of IVIG, fetal platelet count was 18 x 10(9)/L, and Hb was 116 g/L. Eleven mL of matched platelets compatible by monoclonal antibody immobilization of platelet antigens (MAIPA) assay were transfused in utero, raising the platelet count to 62 x 10(9)/L. Repeat transfusions were done later that week and 1 week later, with pretransfusion counts of 19 x 10(9)/L and 16 x 10(9)/L, respectively. Delivery by C section was done at 35.5 weeks, after the third platelet transfusion. Platelet count at birth was 77 x 10(9)/L. Drainage of the hematoma was performed after transfusion. Testing with a solid phase ELISA revealed reactivity against GP1b/IX. MAIPA testing after platelet treatment with the protease inhibitor leupeptin demonstrated the presence of anti-HPA-2b. On PCR-SSP the mother was HPA-2a homozygous, the father was HPA-2a/2b. Antibodies against the HPA-2b antigen located on the GP1b/IX complex have been reported in rare cases of NAIT. Testing is complicated by proteolytic degradation of the antigen-bearing fragment. Compatible platelets are easily found since approximately 85 percent of donors are HPA-2a/2a.     

Lyme-Borreliosis and possible association with HLA-antigens*

The frequencies of HLA A, B, C and DR antigens were evaluated in 220 persons from West Germany with inapparent and manifest Borrelia burgdorferi infections. Thirty-seven forest workers showing elevated antibody titres against Borrelia burgdorferi had asymptomatic infection, and are described as stage 0. One hundred and eighty-three patients presented with the clinical stages 1-3 of the infection. Control persons (n = 655) were typed in the same time period and by identical staff. HLA CW3 was present in 36.3% of patients as compared to 23.2% of the controls (RR = 1.88, pcorr = 0.03) and was significantly associated with manifest infection. In addition, the antigen A2 was found slightly but not significantly more frequent in the patients (55.2% vs 44%; pcorr = 0.41). The phenotype combination HLA A2 and Cw3, however, was significantly elevated in patients (24.6% vs 10.8%; pcorr = 0.0005). In contrast to these class 1 antigens, HLA DR3 showed a tendency of negative association with manifest infection. But this finding was not yet found to be significant (15.3% vs 25.3%; RR = 0.53, pcorr = 0.26). The frequency of HLA DR2 showed a constant decrease from stage 0 to stage 3 (inapparent infection to late complications). Using the rank correlation coefficient of Spearman, this association was found to be significant (-1.00, p less than or equal to 0.05). All other tested HLA antigens and antigen combinations showed no significant differences. The data suggest that HLA CW3 may be associated with Borrelia burgdorferi infection, whereas HLA DR2 and DR3 may be associated with less incidence of severe courses and less complications in this disorder.     

Protection from insulin-dependent diabetes mellitus is linked to a peptide transporter gene

HLA class II association with insulin-dependent diabetes mellitus (IDDM) is well established but is still difficult to map to a particular locus. Polymorphism of the genes coding for transporter associated with antigen processing (TAP1 and TAP2), and located in the HLA class II region, was studied in 167 IDDM patients (116 adult-onset and 51 childhood-onset patients) and 98 normal controls using oligotyping after genomic amplification. A dominant protective effect was observed for theTAP2*0201 allele [relative risk (RR)=0.3, corrected probability (pc) < 0.001]. Conversely, susceptibility to IDDM was associated with apparent homozygosity for the TAP2*0101 allele (RR=3.4, pc < 0.001). Protection was independent from but additive to the protection conferred by the DRB1*02 DQB1*0602 haplotype (RR=0.06, pc<0.05), and antagonistic to the DRB1*03 DQB1*0201 and DRB1*04 DQB 1*0302 haplotypes predisposing effect (RR=1.1, not significant), arguing in favor of an absence of linkage disequilibrium between TAP2 and HLA class II genes. This was assessed by x² analysis. TAP1 allelic distribution was not different among diabetics and controls. A significant association was observed between the presence of TAP2*0101 and that of islet cell antibodies (p < 0.05). These data suggest that the TAP2 gene, which encodes protein required for delivery of antigen peptides to class I molecules in the endoplasmic reticulum, could modulate the autoimmune response leading to β cell destruction. From a practical point of view, they make the combined screening of HLA class II and TAP2 loci a highly valuable tool in IDDM prediction.     

Platelet antibody screening by flow cytometry is more sensitive than solid phase red cell adherence assay and lymphocytotoxicity technique: a comparative study in Thai patients

The objective of this study was to compare the sensitivity and specificity of lymphocytotoxicity test (LCT), solid phase red cell adherence assay (SPRCA) and flow cytometry in detecting platelet reactive antibodies against human leukocyte antigens (HLA) class I and human platelet antigens (HPA). Sera from 38 thrombocytopenic patients and 5 mothers of thrombocytopenic newborns were screened for platelet reactive antibodies by these three methods using screening platelets and/or lymphocytes panels derived from six subjects. The sensitivity and specificity of each method and levels of agreement were analysed. HLA antibodies were found in 18, 17 and 19 out of 43 patients' sera tested by LCT, SPRCA and flow cytometry, respectively. Four out of 43 patients' sera were reactive against HPA by flow cytometry, but were reactive to only 2 sera by SPRCA. Using flow cytometry as the reference method, the sensitivities/specificities of SPRCA and LCT in HLA antibody detection were 84.21/95.83% and 94.73/100%, respectively, with a good strength of agreement. SPRCA had 50% sensitivity and 100% specificity in HPA antibody detection compare to flow cytometry. Flow cytometry appeared to be the most sensitive technique compared with SPRCA and LCT for both HPA and HLA antibody screening. SPRCA sensitivity was too low for HPA antibody detection, but this might be because of the small number of samples. There was one serum from the mother of a baby suffering neonatal alloimmune thrombocytopenia (NAIT), in whom SPRCA could not detect HPA antibodies, while flow cytometry came out positive. Therefore, SPRCA should not be used in NAIT investigation and flow cytometry should be employed instead.     

Association of transporter associated with antigen processing genes with Behçet's disease in Japanese

Contribution of transporter associated with antigen processing (TAP) genes to the pathogenesis of Beh?et's disease (BD) was studied. Restriction fragment length polymorphic analysis of TAP genes was carried out in 46 Japanese patients with BD and 95 healthy subjects. There were no significant differences in allele frequencies of TAP1 and TAP2 genes between whole patients with BD and control population. No significant differences in the frequencies of TAP alleles were observed, when patients of BD with complete type or incomplete type were compared with control population, respectively. An allele frequency of TAP2C was, however, slightly but significantly high in patients with BD who had symptom of erythema nodosum (24.1%) as compared to the control group (11.6%). [p < .05, RR = 2.4]. The allele frequency of TAP2C was slightly high in HLA*B5101 positive patients with BD (28.6%) as compared to HLA*B5101 negative patients (10.9%), but the difference did not reach statistical significance. The absence of genotype TAP2B/C was observed in whole patients group, though it was present in control subjects (14.7%). [p = 0.003, RR = 0.06]. A genotype frequency of TAP2C/H was high in patients with BD who had symptom of skin lesions (7.5%) as compared to the control group (0.0%). [p = 0.03, RR = 15.4]. These results suggest the possibility that TAP molecule play some part in formation of skin lesion, such as erythema nodosum in BD in Japanese.     

POLYMORPHISMS OF TAP1 AND TAP2 GENES IN GERMAN PATIENTS WITH TYPE 1 DIABETES MELLITUS

Type 1 diabetes mellitus (IDDM) is an autoimmune disorder in which the alleles HLA DQA1*0501–DQB1*0201 and DQA1*0301–DQB1*0302 confer strong susceptibility. The genes for transporters associated with antigen processing (TAP1 and TAP2) are located near HLA DQ and display only a limited degree of polymorphism. Since polymorphisms of TAP might influence susceptibility to IDDM possibly by selection of different antigen peptides, we investigated sequence variants of TAP1 and TAP2 genes in 120 German patients with IDDM and 218 random healthy German controls by polymerase chain reaction (PCR) followed by sequence-specific oligonucleotide analysis (SSO), single-strand conformation polymorphism (SSCP) analysis and amplification refractory mutation system (ARMS). TAP1*02011 (16% vs. 4% in controls, P = 0.001, RR = 5.0) and TAP2*0101 (96% vs. 69% in controls, P < 0.0001, RR = 10.6) showed a positive association with IDDM. However, these associations disappeared when patients and controls were matched for predisposing HLA DQA1 or DQB1 alleles as well as for DRB1*0401. In conclusion, our findings indicate that the observed association of TAP variants with IDDM in German patients is due to linkage disequilibrium with HLA DQ alleles/DRB1*04 subtypes.