Abstract

INTRODUCTION

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin producing beta cells in the pancreas (1). Although multiple genes have been implicated, the HLA region genes encoding the DR and DQ proteins are well established as the major T1D susceptibility loci. Other HLA loci contribute to T1D susceptibility, including an additional HLA class II locus, DPB1, and the HLA class I loci A, B, and C [1–3]. The region that lies between the HLA class II and HLA class I regions, sometimes referred to as “class III,” or the “central MHC,” contains a number of genes related to inflammatory and immune responses, including tumor necrosis factor (TNF) and lymphotoxin alpha (LTA), which has also been referred to as TNF-β. TNF and LTA are closely spaced and tandemly arranged on chromosome 6p21. Their protein products have related functions and both bind the same receptor. Associations of polymorphisms in the TNF and LTA loci have been reported for a number of diseases, including, but not limited to, asthma [4], sarcoidosis [5], rheumatoid arthritis [6], Inflammatory Bowel Disease [7], and T1D (see below).

TNF is a major proinflammatory cytokine implicated in islet beta-cell destruction, which results in T1D [8]. Results in animal models [9] indicate that CD4+ T lymphocytes, a major source of IFN-γ, act in collaboration with macrophages, as a major source of TNF, to induce pancreatic beta cell death. LTA is a cytokine produced by lymphocytes that mediates a large variety of inflammatory, immunostimulatory, and antiviral responses. LTA is also involved in the formation of secondary lymphoid organs during development and plays a role in apoptosis. Variations at several TNF and LTA polymorphisms have been reported to correlate with expression levels [7, 10], although the functional significance of these reports is somewhat controversial [11].

Genetic associations of TNF and LTA polymorphisms with T1D have been extensively investigated. Because of their proximity to and linkage disequilibrium (LD) with the HLA class II genes, polymorphisms in the TNF and LTA loci frequently appear to be associated with T1D. For example, an apparent genetic association of TNF with T1D in patients with severe disease was reported in a Chilean study [12]. However, adjustment of the data for LD with HLA class II alleles can change the apparent association of these polymorphisms with T1D. Examples of this include a report of a study of Finnish patients in which no association between LTA restriction fragment length polymorphisms (RFLPs) and T1D was seen after adjustment for DRB1-DQB1 genotype [13]. In a Japanese population [14], after adjustment for class II haplotypes, no significant association of LTA (intron 1 “NcoI”) or TNF (−1037, −857) single nucleotide polymorphisms (SNPs) with T1D was detected. On the other hand, Bouqbis and coworkers [15] reported that TNF (−307) -LTA (+252) haplotypes defined protective HLA class II haplotypes in a Moroccan sample. Separating T1D risk conferred by HLA alleles and that conferred by TNF or LTA alleles can be difficult but is necessary to determine whether or not apparent associations with T1D are real.

In this study, we examined a collection of 283 Caucasian, multiplex T1D families from the Human Biological Data Interchange (HBDI) repository, a substantially larger sample set than those utilized in previous reports of T1D association with polymorphisms in TNF and LTA loci. In addition to its size, this collection of family-based samples has the added advantage of having been completely characterized for the classical HLA-class II and HLA-class I genes in our laboratory. We utilized a genotyping assay that was originally designed to test a set of inflammation-associated polymorphisms (see Methods) and analyzed the genotyping data from the following two TNF SNPs and one LTA SNP: TNF G(-308)A, TNF G(-238)A, and LTA A1069G (intron A). These polymorphisms have been extensively studied in other populations not only for disease association but also for TNF expression levels. We examined the transmission proportions of these polymorphisms first in the total sample, then with data adjusted for LD with HLA DR- and DQ-encoding genes, and, finally, in the following individual, highly-predisposing haplotypes: DRB1*0301-DQB1*02, DRB1*0401-DQB1*0302, DRB1*0404-DQB1*0302, and DRB1*0405-DQB1*0302 or *02.

SUBJECTS, MATERIALS, AND METHODS

RESULTS

Table 1 shows the transmission proportions for the LTA A1069G, TNF G(-308)A, and TNF G(-238)A SNPS in the total set of 283 families. The LTA A1069G, and TNF-α G(-308)A SNPs appear to have strong positive association with T1D (p<0.011 and p<1x 10⁻⁵, respectively), while the TNF G(-238)A SNP shows no apparent association. Notably, the two SNPs that showed significant association with T1D in the initial analysis were also found to be in strong linkage disequilibrium (LD) with DRB1*0301-DQB1*02 haplotypes (referred to as “DR3 haplotypes”), which are highly-predisposing for T1D, while the SNP that showed no apparent association also showed no LD with DR3 haplotypes. DR3 is common in T1D patients. Haplotype frequency of DR3 was 31.4% for affected sibs but only 9.5% for the affected family based control (AFBAC) haplotypes [19], and 328 of 566 patients (58.0%), representing 185 families, had at least one DR3 haplotype (not shown). Table 2 shows the transmission proportions for the three polymorphisms after the expected values have been adjusted to account for the LD between the TNF loci and HLA class II haplotypes. Therefore, expected values are no longer 50% for each allele of the biallelic SNPs, but, rather, are estimated based on the LD values observed between the TNF SNP alleles and HLA class II haplotypes in the AFBAC haplotypes [19] in this data set. The results in Table 2 are in striking contrast to those in Table 1. After adjustment for LD, the associations observed in Table 1 for the LTA A1069G and TNF G(-308)A SNPs lose significance, while the TNF G(-238)A SNP is now positively associated with T1D (p<0.006).

Because the haplotypes DR3 and DR4 are most strongly associated with T1D in Caucasians, we examined the transmission proportion of the TNF SNPs on those haplotypes. For DR3 haplotypes, all of which include the alleles DRB1*0301 and DQB1*02, Table 3 shows that the A allele of the TNF G(-238)A SNP is significantly overtransmitted (81.0%; p<0.009). This demonstrates risk heterogeneity of DR3 haplotypes and is consistent with the hypothesis that this polymorphism, or another locus in LD with it, may affect T1D susceptibility independently of HLA class II loci. However, risk heterogeneity of DR3 haplotypes has been previously demonstrated, with those DR3 haplotypes that carry B18 alleles having higher T1D risk than those DR3 haplotypes that carry B8 alleles [20]. We found that the putative predisposing allele “A” of the TNF G(-238)A SNP is found on B18-DR3 haplotypes in this data set but is absent from B8-DR3 haplotypes. We examined the transmission proportion of all three TNF SNPs on B18-DR3 and on B8-DR3 haplotypes (Table 4). No significant transmission distortion was present on B18-DR3 haplotypes for any of the three TNF polymorphisms (Table 4). These results are consistent with the notion that the TNF-α (-238)A may simply be a marker for a highly predisposing B18-DR3 haplotype rather than having a susceptibility effect that can be attributed to the TNF polymorphism itself. This hypothesis is supported by the fact that the transmission proportion of B18-DR3 haplotypes is nearly identical between haplotypes carrying TNF (-238)A and those carrying TNF (-238)G (Table 4).

B8-DR3 haplotypes that include the A allele of the LTA A1069G SNP were undertransmitted with borderline significance (Table 4, p < 0.05); however, the numbers are small, and the p value was not adjusted for multiple testing. Neither of the other TNF polymorphisms exhibited transmission distortion on B8-DR3 haplotypes. Undertransmission of the A allele of the LTA A1069G SNP was also observed for DR4 haplotypes that carry the DRB1*0405 allele, but, again the result is based on small numbers (9 not transmitted vs. 5 transmitted, p<0.05, data not shown). No other DR4 haplotypes had significant transmission distortion of any of the three SNPs tested (data not shown). While the observation that the same LTA allele appears protective on two different DR haplotypes is notable, the hypothesis that the allele is protective (or is in LD with a protective allele at another locus) requires further testing in a much larger sample set.

DISCUSSION

The data reported here illustrate the need for caution in interpretation of T1D association studies for SNPs in the HLA region. The apparent T1D association of each of the LTA and TNF SNPs examined in this study changes following adjustment for LD with the DRB1-DQB1 haplotypes. A similar change in apparent effect on T1D susceptibility was previously reported in our analysis of HLA-A genotyping data from the same set of families [1]. Before adjustment for LD with DRB1-DQB1 haplotypes, the allele A*0101 appeared significantly predisposing for T1D (p<0.007), but after adjustment, A*0101 appeared significantly protective (p<0.007). The A*0101 allele on that haplotype is clearly not a contributing factor to genetic risk for T1D and, in fact, may contribute to the observation that A1-B8-DR3 haplotypes are less predisposing than are B18-DR3 haplotypes. The data presented here show that the TNF (-238)A allele is present on B18-DR3 haplotypes and not on B8-DR3 haplotypes. Although the hypothesis could be made that the TNF (-238)A allele contributes to the higher T1D risk of B18-DR3 vs. B8-DR3 haplotypes, the similar transmission proportions of B18-DR3 haplotypes with and without the TNF-α (-238)A allele argue against this. In summary, these data show no evidence of a causal role in T1D susceptibility for polymorphisms in the LTA and TNF loci.

Acknowledgments

ABBREVIATIONS

Footnotes

References