M. J. Giphart

Secretion of Tumour Necrosis Factor α and Lymphotoxin α in Relation to Polymorphisms in the TNF Genes and HLA-DR Alleles. Relevance for Inflammatory Bowel Disease

The genes for tumour necrosis factor alpha (TNFα) and lymphotoxin alpha (LTα; TNFβ) are tandemly arranged in the central region of the MHC. They may, therefore, be of importance for the aetiology of MHC‐associated diseases. The authors have prospectively studied the secretion of TNFα and LTα in relation to polymorphisms at positions ‐308 and ‐238 in the TNFα gene (TNFA), and two polymorphisms in the first intron of the LTα gene (LTA), as well as HLA‐DR in 30 patients with chronic inflammatory bowel diseases (IBD) and 12 healthy controls. In the Dutch population, the alleles of these four polymorphisms are present in only five combinations, called TNF‐haplotypes: TNF‐C, ‐E, ‐H, ‐I, and ‐P. Significant associations between TNF haplotypes and TNFα and LTα secretion were found when PBMC were cultured with T‐cell activators, irrespective of disease. Mean TNFα secretion of individuals carrying the HLA‐DR3 associated TNF‐E haplotype was significantly higher, as compared to individuals without this haplotype (26u2003441u2003pg/ml versus 19u2003629u2003pg/ml; Pu2003=u20030.014). Individuals carrying the TNF‐C haplotype produced the lowest amount of TNFα (17u2003408u2003pg/ml; Pu2003=u20030.022). The TNF‐C and TNF‐E haplotypes differ only at position ‐308 in the promoter of TNFA. Individuals carrying the HLA‐DR1 associated TNF‐I haplotype produced significantly less LTα when compared to those who lack this haplotype (1979u2003pg/ml versus 3462u2003pg/ml; Pu2003=u20030.006). As the TNF‐I haplotype is also associated with low TNFα secretion, this haplotype thus defines a ‘low secretor phenotype’. In conclusion, this is the first study to show associations between TNF haplotypes and TNFα and LTα secretion when T‐cell stimulators are used. These findings will contribute to define disease heterogeneity in IBD and may be of relevance for understanding the pathogenesis of autoimmune diseases.

Associations with autoimmune disorders and HLA class I and II antigens in inclusion body myositis.

Whether autoimmune mechanisms play a role in the pathogenesis of inclusion body myositis (IBM) is unknown. Human leukocyte antigen (HLA) analysis in 52 patients, including 17 with autoimmune disorders (AIDs), showed that patients were more likely to have antigens from the autoimmune-prone HLA-B8-DR3 ancestral haplotype than healthy control subjects, irrespective of the presence of AIDs. Patients lacked the apparently protective HLA-DR53 antigen. The results provide further support for an autoimmune basis in IBM.

Genotype effects and epistasis in type 1 diabetes and HLA-DQ trans dimer associations with disease

Alleles of HLA class II genes DQB1, DQA1, and DRB1 in the MHC region are major determinants of genetic predisposition to type 1 diabetes (T1D). Several alleles of each of these three loci are associated with susceptibility or protection from disease. In addition, relative risks for some DR-DQ genotypes are not simply the sum or product of the single haplotype relative risks. For example, the risk of the DRB1*03-DQB1*02/DRB1*0401-DQB1*0302 genotype is often found to be higher than for the individual DRB1*03-DQB1*02 and DRB1*0401-DQB1*0302 homozygous genotypes. It has been hypothesized that this synergy or epistasis occurs through formation of highly susceptible trans-encoded HLA-DQ(α1, β1) heterodimers. Here, we evaluated this hypothesis by estimating the disease associations of the range of DR-DQ genotypes and their inferred dimers in a large collection of nuclear families. We determined whether the risk of haplotypes in DRB1*0401-DQB1*0302-positive genotypes relative to the DRB1*03-DQB1*02-positive genotypes is different from that of DRB1*01-DQB1*0501, which we used as a baseline reference. Several haplotypes showed a different risk compared to DRB1*01-DQB1*0501, which correlated with their ability to form certain trans-encoded DQ dimers. This result provides new evidence for the potential importance of trans-encoded HLA DQ molecules in the determination of HLA-associated risk in T1D.

Susceptibility loci for complex regional pain syndrome

&NA; An association between HLA‐DR13 and patients with complex regional pain syndrome (CRPS) who progressed towards multifocal or generalized tonic dystonia was recently reported. We now report on a new locus, centromeric in HLA‐class I, which was significantly associated with a spontaneous development of CRPS, suggesting an interaction between trauma severity and genetic factors conferring CRPS susceptibility. Additionally, an association with the D6S1014 locus was found, supporting the previous finding of an association with HLA‐DR13.

HLA and smoking in prediction and prognosis of small cell lung cancer in autoimmune Lambert-Eaton myasthenic syndrome.

Patients with small cell lung cancer (SCLC) survive longer if they have the antibody-mediated Lambert-Eaton myasthenic syndrome (LEMS), making this autoimmune disorder a prototype disease for studying cancer immunosurveillance. Patients with nontumor LEMS (NT-LEMS) never develop SCLC but are otherwise indistinguishable clinically. Therefore, we have compared immunogenetic factors in SCLC-LEMS and NT-LEMS and studied their role in the pathogenesis of LEMS and survival from SCLC. In 48 British and 29 Dutch Caucasian LEMS patients, we studied clinical symptoms, antibody titers, HLA types and alleles at six nearby located microsatellite loci. Highly significant associations were found in NT-LEMS, which appeared strongest with HLA-B8, but also involved HLA-DQ2, -DR3 and six flanking microsatellite alleles. SCLC-LEMS patients were not different from controls. Smoking was a strong predictor of SCLC. In contrast, HLA-B8 positivity correlated with a decreased risk of SCLC even among the smokers. Moreover, in SCLC-LEMS patients, HLA-B8 positivity correlated with prolonged survival after LEMS onset. We propose that two distinct immunopathogenetic routes can lead to one clinically and serologically indistinguishable autoimmune myasthenic syndrome. HLA-DR3-B8 is strongly associated with LEMS in nontumor patients only. In other LEMS patients, SCLC apparently provides a powerful autoimmunogenic stimulus that overrides HLA restrictions in breaking tolerance to calcium channels. Moreover, negativity for HLA-B8 combined with smoking behavior points more strongly to an underlying SCLC and predicts a worse prognosis in SCLC-LEMS patients.

Evidence for genetic heterogeneity in inflammatory bowel disease (IBD); HLA genes in the predisposition to suffer from ulcerative colitis (UC) and Crohn's disease (CD)

Family and epidemiological studies support a genetic susceptibility to UC and CD. Conflicting reports regarding associations between UC and HLA‐DR2 and between CD and various HLA alleles have been published. The aim of this study was to determine whether molecularly defined HLA‐DR genes are associated with these diseases in a Dutch group of patients. Fifty‐nine unrelated Dutch UC patients and 89 CD patients were typed using DNA‐based methods. A total of 2400 healthy local blood donors served as controls. The phenotype frequency of the HLA‐DRB1*15 allele was increased in UC patients compared with controls (42% versus 26% in controls; Pu2003=u20030.006; odds ratio (OR)u2003=u20032.1), and was predominantly found in female patients (53% versus 24%; Pu2003=u20030.001; ORu2003=u20033.5). The DRB1*15 allele was increased in UC patients having a positive family history (Pu2003=u20030.01; ORu2003=u20035.8). Among the 16 patients who showed an increase in extent of disease during follow up, 10 were DRB1*15+ (Pu2003=u20030.002; ORu2003=u20034.8). The frequency of the DRB1*13 allele was decreased in patients with UC (15% versus 28% in controls; Pu2003=u20030.04; ORu2003=u20030.5). In CD, no association was observed between disease or particular clinical subgroups and any allele tested. The present study provides additional evidence for the genetic association between UC and HLA‐DRB1*15, and supports recent findings that the susceptibility gene(s) for CD is not located in the HLA class II region.

Functional genetic polymorphisms in cytokines and metabolic genes as additional genetic markers for susceptibility to develop type 1 diabetes.

Genetic association with type 1 diabetes (T1D) has been established for two chromosomal regions: HLA DQ/DR (IDDM1) and INS VNTR (IDDM2). To identify additional genetic markers, we tested polymorphisms in regulatory regions of several cytokine and important metabolic genes. These polymorphisms exhibit functional consequences for expression and function. Functional genetic polymorphisms of proinflammatory (T-helper-1: IL-2, IL-12 and IFN-γ), anti-inflammatory (T-helper-2: IL-4, IL-6 and IL-10) and metabolic (IGF-I, VDR and INS) genes were determined in 206 Dutch simplex families with juvenile onset T1D and the results were analysed using the transmission disequilibrium test. Significantly increased transmission to T1D probands was observed for the loci IDDM1, IDDM2 and the vitamin D receptor. Although none of the other individual polymorphisms was associated with disease individually, the combination of T-helper-2 and metabolic/growth alleles IL-10*R2, IL-4*C, VDR*C and IGF-I*wt was found to be transmitted more frequently than expected (67%, Pc=0.015). We conclude that additional genetic predisposition to T1D is defined by combinations of markers (eg Th2 and metabolic) rather than by a single marker. The consequences of the increased transmission of a low Th2 expressing genotypes together with a normal Th1 profile may result in a net proinflammatory cytokine expression pattern.

Mutations in the HLA class II genes leading to loss of expression of HLA-DR and HLA-DQ in diffuse large B-cell lymphoma

Loss of expression of human leukocyte antigen (HLA) classxa0II molecules on tumor cells affects the onset and modulation of the immune response through lack of activation of CD4+ T lymphocytes. Previously, we showed that the frequent loss of expression of HLA classxa0II in diffuse large B-cell lymphoma (DLBCL) of the testis and the central nervous system (CNS) is mainly due to homozygous deletions in the HLA region on chromosome band 6p21.3. A minority of cases showed hemizygous deletions or mitotic recombination, implying that mutation of the remaining copy of the classxa0II genes might be involved. Here, we studied three DLBCLs with loss of HLA-DQ expression for mutations in the DQB1 and DQA1 genes and three tumors with loss of HLA-DR expression for mutations in the DRB1 and DRA genes. In one case, a point mutation in exonxa02 of the DQB1 gene, leading to the formation of a stop codon, was detected at position 47. In a second case, a stop codon was found at position 11 due to a deletion of 19xa0bp in exonxa01 of the DRA gene. No mutations were found in the promoter sequences of the DRA, DQA1 and DQB1 genes. We conclude that both homozygous deletions and hemizygous deletions or mitotic recombination with mutations of the remaining allele may lead to loss of expression of the HLA classxa0II genes, which is comparable to the mechanisms affecting HLA classxa0I expression in solid cancers.

Oligonucleotide typing is a perfect tool to identify antigens stimulatory in the mixed lymphocyte culture

An important criterion for the selection of donors for bone marrow transplantation is the grade of matching for HLA between donor and recipient. For patients that lack an HLA-identical sibling, an extending pool of unrelated volunteers for bone marrow donation is available. From these donors the best matched candidate can be selected by serological typing, followed by a mixed lymphocyte culture (MLC). Oligonucleotide genotyping for HLA class II antigens is considered to be valuable for the prediction of MLC reactivity. We investigated whether this typing method, in combination with serological typing, would cover the recognition of all MLC stimulatory determinants. One hundred thirty-six combinations of HLA-A, -B, and -DR serologically identical individuals were tested in the MLC. Additional typing for HLA-DRB and HLA-DPB by oligonucleotide genotyping made it possible to evaluate the influence of these genes on MLC reactivity. Combinations that were matched for HLA-DRB gave significantly lower responses than those that were mismatched. Nevertheless, in the matched combinations responses were observed to 94% relative response index. These responses could all be attributed to HLA-DP, since all combinations that were identical by HLA-DPB genotyping were negative in the MLC. In conclusion, with the combined use of serology and oligonucleotide genotyping, responder-stimulator combinations can be selected that are identical for all MLC stimulatory determinants.

Increasing complexity of HLA-DR2 as detected by serology and oligonucleotide typing

Serological and oligonucleotide typing was performed on a number of HLA-DR2-positive cells from different ethnic origin, including DR2 haplotypes with various DQ associations. Exons 2 of DRB1 and DRB5 of DR2-positive individuals were locus-specific amplified and hybridized with a number of different oligonucleotides capable of discriminating between the various Dw2, Dw12, Dw21, and Dw22 associated sequences. The linkage of DRB with DQA1 and DQB1 in these haplotypes was analyzed. Among the DR2- positive cells we could define 10 different DR DQ haplotypes by serology and 13 by oligonucleotide typing. The DR2.ES specificity is a serological DRw15 variant which could not be discriminated by oligonucleotide typing from a DRw15 DQw5 haplotype. The DR2.JA variant represents a unique DRB1*1602 DRB5*0101 haplotype. The DR1+2s haplotype consists of a DRB1 DQ region from a Dw1 and a DRB5 gene from a Dw2 haplotype. Its short DR2 serum pattern can be explained by the absence of a DR2 DRB1 gene product. DRB5*0101 sequences were found in association with DRB1*1501, *1502, *1602, and *0101 alleles. Since the DRB5 gene is capable of such different associations it is comparable to the DRB3 and DRB4 genes. This may have implications for the definition of the broad DR2 specificity which is predominantly encoded by the DRB5 gene product. New DR2 haplotypes included the following DQ combinations: DQw2-positive DQA1/B1*0301/0201 and DQw6-positive DQA1/B1*0102/0601 and *0102/0603 haplotypes.