In our case-control study of homosexual men in the MACS, carriage of LTA (+252G) and TNF (−308A), which closely tag the conserved extended MHC haplotype CEH 8.1, were associated with an approximately two-fold higher risk of AIDS-NHL. These LTA
variants have repeatedly been reported in association with NHL unrelated to HIV infection 2–5, 7
. We detected associations of similar magnitude with SNPs tagging a nearby segment of the CEH 8.1 that contains a complement gene cluster. Alleles of HLA genes present on CEH 8.1 and less frequently studied in association with non-AIDS NHL 9–11
also showed comparable relationships among the subset of MACS subjects with available HLA typing. This first study of MHC effects in AIDS-NHL thus succeeded in its purpose of replicating the established association with non-AIDS NHL.
The multiple previous positive studies of the associated SNPs may have focused attention on TNF
because its encoded protein is involved in a range of neoplastic processes 39, 40
and because its promoter variant −308A has been implicated, albeit not invariably, in relatively high TNF production 15, 16
. However, that TNF
marker and its companion in LTA
are well documented elements of the most extensively conserved haplotype in the genome yet observed. CEH 8.1 stretches for at least 2 Mb between HLA class I and class II loci 20, 21
. Although this inordinately strong conservation of the haplotypic relationships of those two SNPs to CEH 8.1 is not in doubt, we nevertheless verified that our observed associations did not mainly reflect unusual recombinant events in that central MHC region. We demonstrated that more cases than controls who displayed the LTA-TNF G-A combination carried other alleles in the neighboring loci from LTA
that are also recognized CEH 8.1 variants 38
. While these associations increase the likelihood that one or more causal loci in this extended haplotype will be implicated in NHL, they also diminish the probability that any population association with a given marker in that haplotype will actually signify a causal relationship. In order to emphasize the CEH 8.1-wide effect, we have reported associations with haplotypes rather than with SNPs.
Our study tested a specific hypothesis, and it is most unlikely that our detection of the identical associations seen multiple times before in non-AIDS NHL represents a chance finding. On the other hand, the small size of our study sample may have led to unstable and potentially somewhat inflated risk estimates; replication in another HIV cohort would not only confirm but also better quantify the risk.
We analyzed different groups of cases and controls and applied alternative statistical models to ensure that our results were not method-dependent. We have emphasized the unmatched case-control design with adjustment for the CD4+
count because it provided greater statistical power and probably yielded a less conservative statistical test. Further, the CEH 8.1 was earlier thought to be associated with a rapid decline of CD4+
cells in HIV-1 infection 26
. It was therefore important that an analysis of cases and controls matched by duration of HIV infection effectively eliminated the possibility of confounding by differential decline of CD4+
cells among pairs with CEH 8.1-positive NHL cases.
The extraordinary conservation in CEH 8.1 will make identification of the precise causal determinant on this haplotype by typical genetic approaches more difficult. By the same token, we documented that one-third of the G and A alleles were carried on non-HLA-B*08 lineages. Smaller numbers of subjects appear to carry both G and A alleles either on the same or the opposite chromosome but in the absence of the full CEH 8.1. If these cases represent recombinant chromosomes carrying CEH 8.1 with reduced conservation, they could be highly informative for fine mapping of the candidate region of the central MHC. However, since the EM-inferred haplotypes may not be entirely accurate for LTA +252 and TNF −308 double heterozygotes, direct experimental assessment of the phase in such subjects is warranted for future studies.
NHL is a neoplasm for which the strongest risk factors identified to date reflect dysregulation of the immune system. However, regardless of whether immune deficiency is congenital, iatrogenic in the setting of post-transplant immunosuppression, or acquired as a consequence of HIV infection, its presence could complicate the evaluation of true causal factor(s) whose function is altered in NHL. In the context of HIV/AIDS, factors other than the decline of CD4+
count with disease progression are suggested by 1) a disproportionally higher risk of NHL (10–100-fold higher than the population risk) in HIV-infected individuals even in the setting of moderate immune deficiency 41, 42
and 2) a less dramatic decline of systemic NHL incidence as compared with that of KS and primary CNS lymphoma in the era of highly active antiretroviral therapy43–45
. Prolonged immune deficiency and low CD4+
count one year prior to the time of NHL diagnosis have also been reported as independent predictors of NHL outcome46
. As noted above, we controlled for possible confounding of the observed genetic relationships by the underlying decline in immunity.
Inclusion of a substantial proportion of subjects who also developed KS did not distort the association with CEH 8.1 because the HLA alleles of that haplotype have not been associated with HIV-related KS in the MACS 47
. Clinical information on the histological type was available for only a subset of our cases; that limitation precluded statistically meaningful tests by tumor type. Likewise, the study showed a clear association with systemic AIDS-NHL but that with central nervous system AIDS-NHL could not be accurately assessed because of the small sample size; however the trend was similar to that observed with the unstratified sample (Supplementary Table S2
Recent reports 1, 6, 8
have described a protective association with TNF −857T, a SNP that was not included in our genotyping scheme. Although no earlier study detected significant associations with both the TNF −308A - LTA +252G haplotype and the TNF −857 SNP, we cannot exclude a protective role for the latter in our AIDS-NHL. The documented occurrence of the two TNF SNPs on distinct MHC lineages 8
suggests that they are differentially distributed in different Caucasian populations. That may explain the consistency in the findings for the haplotype containing −308A in our study and the others including relatively heterogeneous European populations, and the contrasting –857T association in subjects of predominantly British ancestry. Moreover, the –857T association has appeared stronger with follicular lymphoma 1, 8
, while the −308A association has appeared stronger with diffuse large B cell disease 4, 6
, the form more frequently represented in our population as well.
The pathogenetic mechanisms underlying the increased susceptibility of CEH 8.1 carriers to NHL and to a number of other conditions of autoimmunity and immune dysfunction 21
, are poorly understood. Adaptive and demographic considerations have been invoked to account for the apparent contrast between increased disease susceptibility and high population frequency (selective advantages) of CEH 8.1 48
. Our data show that, except for TNF −308A and CFB m70, the remaining four SNPs associated with AIDS-NHL have elevated minor allele frequencies in both the controls (MAF ≥ 0.27) and the cases (MAF≥ 0.38), emphasizing the necessity to focus future mapping efforts on those MHC markers in strong LD with the presumably less extended TNF −308(A)-CFB m70(G) subhaplotype.
To summarize, the present study extends previously reported association of LTA +252G and TNF −308A from non-AIDS to AIDS-NHL and, as with the recent observations in non-AIDS NHL 10, 11
, shows that the positive association with these two polymorphisms extends across CEH 8.1. In light of the extraordinary allelic invariance across several megabases of central and extended MHC of CEH 8.1-bearing chromosomes 20, 38, 49–51
, our results as well as those reported for non-AIDS NHL strongly support an association with one or more genetic variants somewhere in the G-A-bearing haplotypes rather than suggesting an LTA-TNF
-specific causal relationship. Fortunately, few large HIV-infected populations now go untreated for long enough to permit NHL to develop in numbers sufficient for an investigation similar to ours. It could therefore be difficult to use another population study to confirm our observation of an MHC haplotype determinant common to the pathogenesis of AIDS- and non-AIDS NHL alike.