Takeo Juji

Detection of Platelet Antibodies by a Newly Developed Mixed Agglutination with Platelets

Abstract. We have developed a mixed passive haemagglutination test for the detection of platelet antibodies. This test is essentially a combination of a modified test of mixed agglutination and reversed passive haemagglutination. Using this test, platelet allo‐antibodies (HLA antibodies and non‐HLA antibodies) were found to be detected with very high sensitivity in the sera of pregnant women and transfused patients. The platelet crossmatch test was performed for patients who had to receive massive platelet transfusion. There was a good correlation between the result of the crossmatch and clinical platelet recovery.

High Frequency of Aspartic Acid at Position 57 of HLA-DQ β-Chain in Japanese IDDM Patients and Nondiabetic Subjects

The HLA-DQ β-chain (DQB1) genes of 72 Japanese patients with insulin-dependent diabetes mellitus (IDDM) and 85 control subjects were studied with polymerase chain-reaction (PCR) amplification and allele-specific oligonucleotide hybridization. DQw4 (DQBBIank) and DQw9 (DQB3.3) were increased in IDDM patients compared with the control subjects, and DQB1.2, DQB1.9, and DQw7 (DQB3.1) were decreased. Thirty-five (48.6%) IDDM patients had both alleles carrying an aspartic acid at position 57 of the DQ β-chain (Asp 57), 35 (48.6%) were Asp 57/non-Asp 57 heterozygous, and 2 (2.8%) had non-Asp 57 alleles only. Of 85 control subjects, the respective values for these three genotypes were 49 (57.6%), 29 (34.1%), and 7 (8.2%), respectively. The high frequency of Asp 57 alleles in both IDDM and control subjects contrasts with data for Whites. Therefore, the Asp 57 hypothesis that the presence of an aspartic acid at position 57 of DQ β-chain provides protection against developing IDDM is not tenable for Japanese IDDM patients. The DRB1 gene, particularly position 57 of the DR β-chain, may contribute to IDDM susceptibility in Japanese.

Strong association of insulin autoimmune syndrome with HLA-DR4

Insulin autoimmune syndrome is characterised by spontaneous hypoglycaemia without evidence of exogenous insulin administration, a high serum concentration of total immunoreactive insulin, and the presence of insulin autoantibodies in high titre. HLA typing of 27 patients with insulin autoimmune syndrome showed that all had DR4, which was present in only 43% of 51 healthy controls (odds ratio 72.1, p less than 2 x 10(-6), and 19 (70%) of the patients were positive for the allelic combination, Cw4, Bw62, and DR4. Analysis of the nucleotide sequences of the DRB1, DQA1, and DQB1 genes showed that all the patients had DRB1*0406, DQA1*0301, and DQB1*0302, compared with only 14% of the controls (odds ratio 281, p less than 1 x 10(-10). We conclude that the development of insulin autoimmune syndrome is associated with a strong genetic predisposition.

A study of the association between schizophrenia and the dopamine D3 receptor gene

A study of the genetic association between schizophrenia and aBalI polymorphism in exon 1 of the dopamine D3 (DRD3) gene, a candidate gene for schizophrenia, was conducted. The polymorphism was examined in 91 patients whose symptoms satisfied DSM-III-R for schizophrenia and 90 controls. There were no significant differences between the groups in allele frequencies or genotype counts. Contrary to a previous report, the patients were no more likely to be homozygous than controls. Moreover, no association with the presence of illness could be demonstrated when the patients were grouped according to sex, age of onset, history of admission to psychiatric institutions or positive family history.

Study of HLA class I, class II and complement genes (C2, C4A, C4B and BF) in Japanese psoriatics and analysis of a newly-found high-risk haplotype by pulsed field gel electrophoresis

SummaryGenetic polymorphisms of HLA antigens and HLA-linked serum complement components (C2, C4A, C4B and BF) were investigated in 79 Japanese patients suffering from psoriasis. HLA typing revealed increased frequencies of HLA-A1, A2, B39, Bw46, Cw6, Cw7 and Cw11. Among complement components, positive associations were obtained with C4A4 and C4B2 and a negative association with BFF. The major histocompatibility complex haplotype (supratype), HLA-A2-Cw11-Bw46-C2C-BFS-C4A4-C4B2-DRw8 is purported to be a new high-risk haplotype in Japanese patients with psoriasis. Analysis of patients with this supratype via pulsed field gel electrophoresis showed the existence of specific, extensive DNA deletions near HLA-DR genes, but no disease-specific patterns could be observed by means of this technique. The newly-found high-risk haplotype indicates racial and ethnic differences among psoriatic patients.

ROUTINE LOW AND HIGH RESOLUTION TYPING OF THE HLA‐DRB GENE USING THE PCR‐MPH (MICROTITRE PLATE HYBRIDIZATION) METHOD

We describe HLA‐DRB1 typing using polymerase chain reaction‐based microtitre plate hybridization (PCR‐MPH), which can process large numbers of samples. MPH typing is similar to an enzyme‐linked immunosorbent assay (ELISA), in which a tandemly ligated sequence‐specific oligonucleotide is immobilized on microtitre wells. The typing procedure consisted of two steps. In the first, PCR‐MPH with 16 probes was performed to determine the specificities of the serological levels (DR1, DR2, DR3, DR4, DR11, DR12, DR13, DR14, DR7, DR8, DR9 and DR10) after generic amplification (‘low resolution typing’). In the second step, DR1, DR2, DR4, DR 12/8 and DR3/11/13/14 were group‐specifically amplified based on the results of the first PCR‐MPH, and microtitre plate hybridization proceeded in a similar manner to the first step (‘high resolution typing’). Low resolution typing was completed within 2 h after generic amplification, and the results of high resolution typing were obtained in another 3.5 h after amplification. The allelic types classified using PCR‐MPH were completely concordant with those obtained by PCR‐ single‐strand conformation polymorphism or PCR‐restriction fragment length polymorphism.

Haplotype study on C4 polymorphism in Japanese. Associations with MIHC Alleles, complotypes, and HLA-complement haplotypes

Genetic polymorphism of the fourth component of human complement (C4) was investigated in 83 Japanese families which have been typed for HLA-A, -B, -C, -DR, C2, and BF. Four common C4A alleles and four common C4B alleles were observed. The allele frequencies estimated from unrelated parents were as follows: C4A3, 0.686; A4, 0.132; A2, 0.106; AQ0, 0.067; ARares, 0.009; C4B1, 0.587; B2, 0.167; B5, 0.088; and BQ0, 0.158. Eight different C4 haplotypes were observed with frequencies of more than 0.01. The estimated haplotype frequencies were as follows: C4A3-B1, 0.513; A4-B2, 0.114; A2-BQ0, 0.106; A3-B5, 0.088; AQ0-B1, 0.059; A3-BQ0, 0.047; A3-B2,0.038; A4-B1, 0.015; and Rares, 0.021. Strong positive gametic associations were found in the following C4-HLA haplotypes: C4A2BQ0-A24, C4A2BQ0-Bw52, C4A3B5-Bw54, C4A3B5-Bw59, C4A4B2-Bw46, C4A3B5-Cw1, C4A2BQ0-DR2, and C4A3B5-DR4. Eleven complotypes were observed with frequencies of more than 0.01. C4A2BQ0 and C4A3B5 were exclusively associated with BFS-C2C. BFF was associated with C4A3B1, C2AT, C2B, and C2BH were associated with C4A3B1, A4B2, and C4A3B1, respectively. Eight different HLA-complement haplotypes were found to be characteristic of Japanese. These combinations are considerably different from those reported in Caucasoid populations.

HLA DR4 and rheumatoid arthritis in Japanese people.

Eighty-eight Japanese patients with rheumatoid arthritis and 104 normal Japanese persons were typed for HLA A, B, C, and DR antigens. The frequency of HLA DR4 was 70.5% in patients compared with 46.1% in normal controls (P less than 0.001). However, a sex difference in the frequency of HLA DR4 in patients was noted. HLA DR4 was found in 80.6% of male patients, which was highly significant compared with controls (P less than 0.0005), while only a borderline increase of 60.5% was found in female patients (P less than 0.05). In addition, the frequency of HLA DR2 was remarkably low in male patients. These suggest the possible heterogeneity of rheumatoid arthritis in Japanese.

HLA-DQβ chain DNA restriction fragments can differentiate between healthy and narcoleptic individuals with HLA-DR2

The human HLA-D histocompatibility region encodes for polymorphic cell-surface glycoproteins called Ia or class II antigens (DR, DQ, and DP antigens). The antigens consist of a noncovalently associated c~ chain with an Mr of 33 000 and a/~ chain with an M r of 28 000, the later carrying the majority of the polymorphic determinants (Owerbach et al. 1983, Cohen et al. 1984). These class II antigens are essential for cellular recognition and cooperation in the immune response and they control susceptibility to a wide variety of human diseases (Dausset and Svejgaard 1975, Benacerraf 1981, Thorsby et al. 1982). Narcolepsy is a sleep disorder characterized by excessive periods of sleep during the daytime, irresistible attacks of sleep, and sudden loss of tone in the striated muscles which typically occurs at moments of increased emotion. A remarkably high level of association between HLA-DR2 and narcolepsy was first reported by Honda and co-workers (1984). All of 111 narcoleptic patients tested were found to be DR2positive (ig 2 = 141, P < 10 -29) (Juji et al. 1984, Matsuki et al., in press). This high level of association was also observed for all the 37 Caucasoid patients studied by Langdon and co-workers (1984). A large number of the cDNA and genomic clones for the class II antigens were isolated and characterized (Inoko et al. 1984, 1985a, b). In this paper, the DQp cDNA probe was used to define polymorphisms at the genomic level in order to differentiate between narcoleptic patients and healthy individuals with DR2 homozygotes or heterozygotes. Marked differences were found between narcoleptic patients and normal healthy individuals. All the narcoleptic patients shared DQp Eco RI 2.4 kb, Barn HI 2.9 kb, and Pst I 12 kb fragments, which were detected in healthy controls only at decreased frequencies (37.9%, 55.1%, and 82.9%, respectively). In contrast, the narcoleptic patients showed a decreased frequency of an Eco

Polymorphisms of transporter associated with antigen processing genes in atopic dermatitis

We investigated polymorphisms of transporter associated with antigen process (TAP) genes in atopic dermatitis. We developed a polymerase chain reaction-restriction fragment length polymorphism method for discriminating TAP alleles. Genomic DNA was obtained from 29 Japanese patients with atopic dermatitis and 35 control subjects. Dimorphic regions of TAP1 and TAP2 genes were amplified by polymerase chain reaction. Amplified products were digested with restriction endonucleases to determine TAP alleles: Sau3A1 for TAP1 codon 333 (Ile-Val), AccI for TAP1 codon 637 (Asp-Gly), and EcoRII for TAP2 codon 687 (Gln-Stop). We observed four alleles for TAP1 and dimorphism for TAP2 codon 687. Six of 35 controls had the TAP1 D allele, which has been reported to be a rare allele in Caucasian populations. Gene frequency of TAP1 637Asp exhibited a tendency to increase in the patients with atopic dermatitis. TAP1 637Asp and TAP1 A alleles were estimated to constitute a haplotype with DRB1* 1302-DQB1*0604 and DRB1*0803-DQB1*0601 in the Japanese population. Because TAP1 and TAP2 genes are located between HLA-DQB1 and -DPB1 loci, analysis of TAP gene polymorphisms will be useful for a better understanding of susceptibility loci in HLA class II-associated disease.