HLA-DRB1*0401 IS ASSOCIATED WITH SUSCEPTIBILITY TO INSULIN-DEPENDENT DIABETES MELLITUS INDEPENDENTLY OF THE DQB1 LOCUS

The distribution of DRB1*04 alleles and DRB1/DQB1 haplotypes was analysed in 57 DR4⁺ caucasoid subjects with insulin-dependent diabetes mellitus (IDDM) and 96 DR4⁺ healthy controls selected on the basis of DR serology, and the findings were analysed in relation to age at diagnosis of IDDM. DNA samples were amplified using specific DR and DQ primers and hybridized with sequence-specific oligonucleotide probes. A significantly increased combined frequency of DRB 1*0401 and 0402 was observed in IDDM subjects aged ≤12 years at diagnosis (allele frequency 88.4% compared with 62.0% in controls, P < 0.025). There was a non-significant increase in DRB 1*0401 and 0402 in IDDM subjects ≤12 years when compared with IDDM subjects >12 years (P < 0.1). DRB 1 *0404 was decreased in the total IDDM subject group compared with controls (4.8% vs. 19.0%, P < 0.025) but did not reach statistical significance in the individual age at diagnosis groups. In contrast, the frequency of DQB1 *0302 was increased uniformly across both ages at diagnosis groups. In controls DRB 1*0401 occurred in haplotype association with DQB 1*0301 in a significantly greater frequency than with DQB 1*0302. However, 95.0% of DRB 1*0401 IDDM subjects were DQB 1*0302. DRB 1*0404, which was decreased in frequency in IDDM subjects, occurred in association significantly more frequently with DQB 1 *0302 in controls. These results imply that DRB 1 and DQB 1 have independent roles as HLA susceptibility genes in IDDM. DQB1 may have a permissive role whereas DRB1 could influence the rate at which underlying disease progresses to clinical IDDM.     

The HLA-DRB1*0405 haplotype is most strongly associated with IDDM in Algerians.

Insulin-dependent diabetes mellitus (IDDM) in Caucasians is strongly associated with HLA-DR3-DQ2 and DR4-DQ8. In order to investigate the HLA class II associations with IDDM in Algerians, we have used polymerase chain reaction (PCR) and sequence specific oligonucleotide analysis (SSO) to identify DQA1, DQB1, and DRB1 alleles, haplotypes and genotypes in 50 unrelated IDDM patients and 46 controls from a homogeneous population in Western Algeria. Both DRB1*0301-DQA1*0501-DQB1*0201 (DR3-DQ2) and DRB1*04-DQA1*0301-DQB1*0302 (DR4-DQ8) haplotypes were found at increased frequencies among the patients compared to controls (45% vs. 13%, RR = 5.5, Pc < 10(-5) and 37% vs. 4%, RR = 12.9, Pc < 10(-4), respectively). Among the latter, in contrast to other Caucasian populations, only DRB1*0405-DQA1*0301-DQB1*0302 was significantly increased in the Algerian patients (25% vs. 1% in controls, RR = 30.3, Pc < 10(-3). Accordingly, the highest risk of disease was observed in DRB1*0301-DQA1*0501-DQB1*0201/DRB1*0405-DQA1+ ++*0301-DQB1*0302 heterozygotes (34% in patients vs. 0% in controls; RR = 49; Pc < 10(-3). This observation and its comparison with DR-DQ haplotypes in other ethnic groups suggest that the DRB1*0405 allele which encodes an Asp57-negative beta chain may contribute to IDDM susceptibility in a similar way as Asp57-negative DQ beta chains.     

HLA-DR4 SUBTYPE AND -B ALLELES IN DQB1*0302-POSITIVE HAPLOTYPES ASSOCIATED WITH IDDM

We determined the distribution of DR4 subtypes in 309 DQB1*0302-positive haplotypes found in insulin-dependent diabetes mellitus (IDDM) patients and 70 control haplotypes present only in healthy family members. An increased frequency of DRB1*0401 allele (74.4% vs. 55.7%, P = 0.003) and a decrease of DRB1*0404 allele (23.6% vs. 40.0%, P = 0.0064) was revealed. A further analysis of extended haplotypes demonstrated strong linkages between various B alleles and DRB1*04 subtypes. HLA-B39 was more frequent in DRB1*0404–DQB1*0302-positive IDDM haplotypes compared with control ones (37.0% vs. 14.3%, P = 0.049), suggesting an involvement of the region telomeric to HLA-DRB1 in the susceptibility to IDDM.     

THE HLA-DRB1*0101 ALLELE IS RESPONSIBLE FOR HLA SUSCEPTIBILITY TO LICHEN RUBER PLANUS

Serological studies have demonstrated that lichen ruber planus is associated with the HLA-DR1 antigen. This association was also confirmed by us in the Sardinian population. To establish which DRB1 molecular alleles are involved, we studied a selected group of 14 DR1 positive patients affected by cutaneous idiopathic lichen planus and a group of DR1 positive healthy controls using PCR with sequence-specific primers (PCR-SSP). Comparisons between the allele frequencies in patients and controls showed a positive association with cutaneous idiopathic lichen planus for the DRB 1*0101 allele (RR = 5.8, P= 0.0097). DRB1*0101 and DRB1*0102 are associated with the same DQA1 and DQB1 alleles and are different only for two amino acids in positions 85 and 86 of the DRB 1 gene. In our case report predisposition to cutaneous idiopathic lichen planus is correlated with a valine in position 85 and a glycine in position 86 at the second exon of the DRB 1 gene.     

VARIATION IN THE VITAMIN D RECEPTOR GENE IS ASSOCIATED WITH MULTIPLE SCLEROSIS IN AN AUSTRALIAN POPULATION

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) resulting in accumulating neurological disability. The disorder is more prevalent at higher latitudes. To investigate VDR gene variation using three intragenic restriction fragment length polymorphisms (Apa I, Taq I and Fok I) in an Australian MS case-control population. One hundred and four Australian MS patients were studied with patients classified clinically as Relapsing Remitting MS (RR-MS), Secondary Progressive MS (SP-MS) or Primary Progressive MS (PP-MS). Also, 104 age-, sex-, and ethnicity-matched controls were investigated as a comparative group. Our results show a significant difference of genotype distribution frequency between the case and control groups for the functional exon 9 VDR marker Taq I (p_Gen = 0.016) and interestingly, a stronger difference for the allelic frequency (p_All = 0.0072). The Apa I alleles were also found to be associated with MS (p_All = 0.04) but genotype frequencies were not significantly different from controls (p_Gen = 0.1). The Taq and Apa variants are in very strong and significant linkage disequilibrium (D′ = 0.96, P < 0.0001). The genotypic associations are strongest for the progressive forms of MS (SP–MS and PP–MS). Our results support a role for the VDR gene increasing the risk of developing multiple sclerosis, particularly the progressive clinical subtypes of MS.     

T cell epitopes of type II collagen in HLA-DRB1*0101 or DRB1*0405-positive Japanese patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a T cell-mediated autoimmune disease, but target antigens (autoantigens) responsible for T cell activation remain unclear. Type II collagen (CII) is a candidate autoantigen that is largely confined to the articular cartilage. To investigate whether CII is an important antigen in patients with RA, we examined peripheral blood T cell reactivity to CII in HLA-DRB1*0101 and DRB1*0405-positive RA patients. Reactivities to candidate T cell epitopes of CII were also examined. Peripheral blood T cell reactivity to CII and CII peptides (256-271, 429-442, 593-610, 1064-1081) were detected by measurement of IL-2, IFN-gamma, and IL-4 in culture supernatant of PBMC after in vitro antigen stimulation. Cytokine concentration was measured by ELISA. In DRB1*0101-positive patients, T cell reactivity to CII as detected by measurement of IL-2 production in culture supernatant, was present in 4 out of 9 patients. IL-2 production upon stimulation with CII 256-271 peptide was found in all of these 4 patients. In DRB1*0405-positive patients, high frequency of positive T cell response to CII was detected in 9 out of 11 patients. IFN-gamma production was also detected in 4 out of 6 patients producing IL-2 by stimulation with CII. T cell response to CII 256-271 and/or CII 1064-1081 was detected in these patients. In DRB1*0101-positive RA patients, CII 256-271 peptide might function as a T cell epitope, whereas either CII 256-271 or CII 1064-1081 peptide may be a major T cell epitope in DRB1*0405-positive RA patients. In DRB1*0405-positive RA patients, CII reactive T cells might play a crucial role in the development of RA through IFN-gamma production.     

HLA-DRB1* GENE SEQUENCES IN HLA-DR4 POSITIVE AND NEGATIVE PATIENTS WITH RHEUMATOID ARTHRITIS

The second exon of the DRB1 gene encoding for the first domain of the HLA-DR β-chain was sequenced in 16 patients (10 DR4/DR1 positive, 6 DR4/DR1 negative) with seropositive rheumatoid arthritis (RA). We could confirm the strong association of susceptibility to RA with functionally equivalent conformations on otherwise distinct MHC molecules. At least one HLA-DR allele in all of the analysed DR4 or DR1 positive patients showed such an epitope with a minimal variability limited to residue 71. However, in HLA-DR4 and -DR1 negative patients such a similar epitope could not be detected.     

Two novel HLA-DRB1 alleles identified using a sequence-based typing: HLA-DRB1*1443 and HLA-DRB1*1351*

Two novel HLA-DRB1 alleles, DRB1*1443 and DRB1*1351, were identified using a sequence-based typing protocol. DRB1*1443 differed from DRB1*140501 by one single-nucleotide substitution in exon-2 (codon 77, ACC-->GCC), which corresponded to an amino acid change of threonine to alanine. DRB1*1351 was identical to DRB1*1301 but differed by a single-nucleotide substitution at codon 50 (GTG-->TTG), resulting in an amino acid change of valine to leucine. Both new alleles present unique polymorphisms, which have not been seen among other DRB1 alleles and which have no known effect on peptide binding.     

血管紧张素原基因M235T分子变异与心肌梗塞的关系

目的为探讨血管紧张素原（ＡＧＴ）基因Ｍ２３５Ｔ分子变异与心肌梗塞（ＭＩ）的关系。方法采用聚合酶链反应、限制性片段长度多态性分析，对５７例ＭＩ患者和７６例无冠心病证据的对照组进行ＡＧＴ基因Ｍ２３５Ｔ等位基因检测。结果ＭＩ患者ＡＧＴ基因２３５ＴＴ型（０．７０）和Ｔ２３５等位基因（０．８２）的频率显著高于对照组（分别为０．４２和０．６３，Ｐ＝０．０１３和Ｐ＜０．０２５）。经校正冠心病的主要危险因素后，ＡＧＴ基因２３５ＴＴ型仍可显著增加心肌梗塞发生的危险性（比数比３．６５，Ｐ＝０．０１６）。结论ＡＧＴ基因２３５ＴＴ型可能是中国人群ＭＩ发病的重要危险因素之一。     

Identification of sequence errors in HLA-DRB1*0801 and HLA-DRB1*12011

We report the identification of previously unrecognised errors in the nucleotide sequences of two long established HLA-DRB1 alleles, DRB1*0801 and DRB1*12011. The errors were detected during development of sequencing based typing (SBT) methods for the HLA-DRB1 locus and were confirmed by sequencing cell lines from the 10th International Histocompatibility Workshop (IHW).     

BETA-CELL APOPTOSIS IS RESPONSIBLE FOR THE DEVELOPMENT OF IDDM IN THE MULTIPLE LOW-DOSE STREPTOZOTOCIN MODEL

Although insulin-dependent diabetes mellitus (IDDM) results from irreversible loss of beta cells, the mode of cell death responsible for this loss has not previously been categorized. In this study, the multiple low-dose streptozotocin (stz) model (intraperitoneal injection of stz at a concentration of 40 mg/kg body weight per day for five consecutive days) was used to investigate beta-cell death during the development of IDDM in male C57B1/6 mice. Apoptotic cells were evident by light microscopy within the islets of Langerhans of treated animals from day 2 (the day of the second stz injection) until day 17. Immunohistochemical localization of insulin to the dying cells confirmed the beta-cell origin of the apoptosis. Two peaks in the incidence of beta-cell apoptosis occurred: the first at day 5, which corresponded to an increase in blood glucose concentration, and the second at day 11, when lymphocytic infiltration of the islets (insulitis) was maximal. Insulitis did not begin until day 9, by which time treated animals had developed overt diabetes as revealed by blood glucose and pancreatic immunoreactive insulin (IRI) measurements. Beta-cell apoptosis preceded the appearance of T-cells in the islets and continued throughout the period of insulitis. Thus, whether induced by stz or a subsequent immune response, apoptosis is the mode of cell death responsible for beta-cell loss in the multiple low-dose stz model of IDDM.     

A NOVEL DRB1*0801 HAPLOTYPE CARRYING DRB3*0202 FOUND IN A GERMAN PEDIGREE

DRB1*08 haplotypes have not been known to carry a DRB3 gene. We have found a patient suffering from liver disease who has a novel HLA haplotype of DRB1*0801 with DRB3*0202 as established by family segregation. These two genes were confirmed by sequencing. DR8 and DR52 antigen specificities were serologically detected, indicating expression of these genes.     

COMPLETE NUCLEOTIDE SEQUENCE OF THE HLA-DRB1*1302 ALLELE

Sequencing studies of HLA class II molecules have focused almost exclusively on its exon 2. The complete cDNA sequence of the DRB1*1302 allele is reported here for the first time. A conservative polymorphism was detected outside the exon 2 at residue 206; these changes can only be recorded if complete cDNA sequences are studied, and may be of value in drawing evolutive inferences.     

C5 DEFICIENCY IN A/J MICE IS NOT ASSOCIATED WITH RESISTANCE TO THE DEVELOPMENT OF SECONDARY AMYLOIDOSIS

The aim of the study was to determine whether C5 deficiency in the mouse is associated with resistance to the development of secondary amyloidosis. Chronic inflammation was induced in the F₂ progeny, derived from matings between amyloid-susceptible and amyloid-resistance mice, by daily injections of azocasein for thirty days. Using a restriction fragment length polymorphism generated by digestion of genomic DNA with the restriction enzyme HindIII, C5 sufficient and deficient DNA can be clearly differentiated. Eight mice were found to be C5 sufficient, 32 were heterozygotes and 14 were found to be C5 deficient. Grading of the splenic amyloid load from negative to 4+ was performed after staining tissue squashes with Congo red and viewing them under a polarizing microscope. Seventeen mice were noted to have negative to trace, 18 had moderate (1+-2+) and 19 had heavy (3 H+-4+) amyloid deposition. There was no correlation between splenic amyloid load and C5 deficiency. Based on these results it is clear that C5 deficiency and resistance to secondary amyloidosis are not associated.     

MUTATION OF THE VHL GENE IS ASSOCIATED EXCLUSIVELY WITH THE DEVELOPMENT OF NON-PAPILLARY RENAL CELL CARCINOMAS

To define the possible role of the VHL gene in the development of sporadic renal cell carcinomas, 91 different parenchymal tumours of the kidney have been investigated for mutation of the VHL gene by single strand conformation polymorphism (SSCP) and/or heteroduplex (HD) techniques. Chromosome 3p deletion was detected in 98 per cent of non-papillary renal cell carcinomas and in 25 per cent of chromophobe renal cell carcinomas. In 22 of the 43 non-papillary renal cell carcinomas, abnormally migrating DNA bands were detected by SSCP and/or HD analysis. No mobility shift was seen in any of the 23 chromophobe renal cell carcinomas. In addition, 15 papillary renal cell tumours and ten renal oncocytomas, which are characterized by genetic changes other than loss of chromosome 3p sequences, were analysed for mutation of the VHL gene. None of these tumours showed abnormal migration patterns. The results indicate that mutation of the VHL gene is associated exclusively with the development of non-papillary renal cell carcinoma.     

bcl-2 PROTEIN IS STRONGLY EXPRESSED IN POST-TRANSPLANT LYMPHOPROLIFERATIVE DISORDERS

The aim of this study was to examine a series of Epstein–Barr virus (EBV)-driven post-transplant lymphoproliferative disorders (PTLDs), in order to ascertain the level of bcl-2 immunostaining; to explore the relationship between bcl-2 and p53 protein expression; and to see if any correlation exists between bcl-2 and EBV-latent membrane protein 1 (LMP-1). Seventeen renal and 11 heart/heart–lung PTLD cases were stained with antibodies to EBV-LMP-1, bcl-2 and p53, using paraffin-embedded tissue. All cases of PTLD strongly co-expressed bcl-2 and EBV-LMP. Positive staining was present in small lymphoid and larger immunoblastic cells. These two antibodies showed parallel staining intensity. p53 expression was noted in 13 of 17 renal PTLDs, but in ten of the positive cases only 5–10 per cent of cells were stained. Seven of the 11 heart/heart–lung cases showed 50–60 per cent of cells to be p53-positive; in the remaining four cases, 10–20 per cent of cells were positive. bcl-2 protein, as detected by immunohistochemistry, is markedly overexpressed in all cases of PTLD. This study also demonstrates a strongly positive correlation between bcl-2 expression and EBV-LMP-1 detection in PTLDs. An inverse pattern of p53 and bcl-2 immunoexpression is noted in PTLDs with ‘high grade’ histology: these show marked expression of bcl-2, while p53 is downregulated. A Fisher's exact test yielded a P value of 0·12 when comparing p53-positive renal PTLDs with p53-positive heart/heart–lung PTLDs, indicating that any difference seen is not statistically significant. The postulated mechanism for the positive correlation between bcl-2 and EBV-LMP-1 is that EBV upregulates bcl-2, either directly or indirectly, thus promoting cell survival and ultimately successful viral replication.     

Identification of two new HLA-DRB1 alleles: HLA-DRB1*1350 and DRB1*140502

Two new HLA-DRB1 alleles have been found by using high-resolution sequence-based typing. The two sequences have been officially named DRB1*1350 (HWS10001327-AY048687) and DRB1*140502 (HWS10001790-AY129430). DRB1*1350 differs from DRB1*110101 by two amino acids at positions 37 (Y-->N) and 58 (A-->E). This allele may arise from gene conversion between DRB1*110101 and DRB1*130201 or DRB1*030101, which are commonly found in Taiwan populations. The other allele, DRB1*140502, obtained from a patient with rheumatoid arthritis, differs from DRB1*140501 at codon 58 (GCC-->GCT). However, it causes no change in amino acid sequence and would therefore not have direct clinical implications.     

SEQUENCE OF HLA-DRB1*0818 IN A BONE MARROW DONOR

Exon 2 of a new HLA-DR8 subtype, DRB1*0818, was cloned and sequenced in a volunteer bone marrow donor. This new allele differs from DRB1*0805 by one single nucleotide in codon 67 resulting in an amino acid substitution from phenylalanine to isoleucine. Codon 67 dimorphism apppears to be more frequent in DR8 and DR11 haplotypes. The HLA typing of the donor was A*0201/33 B*51/4403 Cw*0202/14 DRB1*0408/0818 DQB1*0302/0402. This new HLA-DR8 variant is very rare, at least in Caucasoids.     

Identification of a new HLA-DRB1 allele, HLA-DRB1*0436

Anovel allele, HLA-DRB1*0436, was identified in a Taiwan indigenous individual by sequence-based typing. DRB1*0436 was identical to DRB1*0403 in exon2 but differed at codons 67-74 with five nucleotide substitutions. This corresponded to three amino acid changes within the P4 peptide-binding pocket of the DR molecule. These substitutions constitute a motif that is also seen in other DRB1*11 alleles. It is possible that the new allele resulted from a gene conversion event.     

FOAMY HISTIOCYTES IN THE SPLEEN ASSOCIATED WITH IDIOPATHIC THROMBOCYTOPENIC PURPURA

Six spleens removed from patients with idiopathic thrombocytopenic purpura (ITP) have been examined light and electron microscopically.
Foamy histiocytes were found either scatterlngly or in clusters in two cases, and myelin structures were demonstrated in these foamy cells. Various stages of platelet phagocytosis and their intracellular degradation were recognized in the macrophages. These findings support the concept that the appearance of foamy cells in ITP spleens is a result of enhanced destruction of platelet in the spleen.