AD is a multifactorial disorder with a number of alterations in the immune profile occurring during disease progression in both the brain  and the periphery [34, 35]. Recently studies have reported links between risk for AD and polymorphisms in the promoter regions of TNFA at positions -308 [6, 18] and -850 . The current study utilized a well characterised sample to investigate these potential associations in an Australian cohort. In addition, BAT1 has been implicated in modulation of inflammatory cytokines . Therefore, the current study investigated alleles of the BAT1 -22 promoter polymorphism as a potential risk factor for AD, singly or in haplotypic association with the TNFA promoter polymorphisms.
Analysis of individual SNPs revealed no significant association between AD and TNFA -308*2. This contrasts with reports in the literature that associate the TNFA -308*2 allele with either increased risk for AD [6, 18] or protection against this disorder [7, 19]. While data from the current study appears to be more supportive of a potential protective role for TNFA -308*2 against AD (Table 1), no conclusions can be drawn solely based on genotype and allele frequency analysis due to control group deviations from HWE that might affect the rate of type I error. However, it is possible that the inconclusive result obtained for TNFA -308*2 may be due to haplotypic associations of this polymorphism with other MHC markers such as the BAT1-22*2 allele.
In contrast to the ambiguous result obtained for TNFA -308*2, analysis of individual SNPs revealed that TNFA -850*2 was clearly significantly associated with increased risk for AD. The literature shows association of the TNFA -850*2 with vascular dementia  and individuals at high risk for dementia, such as those with Down's Syndrome . However, a clear association of TNFA -850*2 with AD has only previously been reported as a synergistic effect in combination with APOE ε4 in a Northern Irish population , while a similar study in a population from Northern Spain failed to produce evidence in support of a synergistic effect between TNFA -850*2 and APOE ε4 . The authors suggested that this discrepancy might reflect true genetic differences between the populations and pointed out that differences in allele frequency distributions between the two different European populations might indicate linkage disequilibrium between the TNFA -850 and another marker that might represent the true disease causing gene .
The current study presents data in support of the notion that TNFA -850*2 contributes to the risk of AD independently of the APOE ε4 allele. Furthermore, logistic regression analysis revealed a possible gene dosage effect with increase in copy numbers of the TNFA -850*2 allele leading to higher Odds ratios. It is, however, possible that a gene linkage with TNFA -850*2 would show a parallel OR pattern, and might account for the apparent gene dosage effect attributed to the TNFA -850*2 allele. Since all three markers investigated exerted their effects independently of APOE ε4 but were found to be in LD with one another, haplotype frequencies, taking into account LD between markers, were estimated for all three MHC markers and also for combinations of two markers in order to investigate whether an AD risk or protection associated haplotype could be responsible for the effects observed.
Only one haplotype (BAT1 -22*1 in combination with TNFA -850*2) appeared to be significantly associated with risk for AD, but the observed Odds ratio was lower for this haplotype (OR = 1.54) than the OR for the single polymorphisms associated with AD risk (TNFA -850*1/2, OR = 1.8 and TNFA -850*2/2, OR = 2.7). This indicates that, although in LD with the other two markers TNFA -850*2 did not exert its risk for AD through a haplotypic association with these polymorphisms. While it cannot be entirely ruled out that linkage disequilibrium with other as yet not identified markers may be responsible for the effect observed in this investigation, the current study identifies the TNFA -850*2 allele as a candidate marker that may confer risk for AD in the Australian population. Further investigation with larger participant numbers and in other populations is clearly warranted.
While the polymorphisms in the promoter regions of TNFA are likely to directly affect transcription of the TNFA gene, ultimate levels of TNFα protein in tissues can also be influenced by other regulating factors such as BAT1. In the current study BAT1-22*2/2 was significantly associated with protection against the development of AD. Similar to the association between increased risk for AD and the presence of the TNFA -850*2 allele, the protective effect of BAT1-22*2/2 was found to be independent of APOE ε4 status. Furthermore, none of the estimated haplotypic associations with the two TNFA markers that are in linkage disequilibrium with BAT1 have provided evidence to suggest that the effect observed for BAT1-22*2/2 is due to a haplotypic association with these markers. While the possibility remains that the protective BAT1 effect might be due to LD with another gene as yet not investigated, it is also possible that BAT1 might assert an independent effect on AD risk.
A potential independent role for BAT1 in AD pathology is supported by the notion that the BAT1 -22 polymorphism may not only have the potential to affect transcription of BAT1 but, through the role BAT1 plays in mRNA stabilization, this protein may also affect translation of a number of inflammatory cytokines linked to AD pathology, including TNFA. It has previously been reported that BAT1 plays a potential role in the regulation of inflammatory cytokines, including TNFA [20, 21] and the BAT1 -22 allele has been associated with certain autoimmune disease susceptible ancestral haplotypes such as the 8.1 MHC AH amongst others . Since BAT1 appears to regulate a number of inflammatory cytokines for which alterations are observed in AD pathology the current study is the first to provide evidence to show that a BAT1 promoter polymorphism is significantly associated with AD pathology.
It is of interest to note that for the TNFA -850 polymorphism the less frequent allele conferred risk for AD while the opposite was found for the less frequent allele (C) of the BAT1 -22 polymorphism which was associated with a decreased risk for AD. This finding that the BAT1 -22*2 (C) allele is associated with protection against AD is in contrast to the findings for autoimmune disorders where the less common number 2 allele is implicated with ancestral haplotypes that confer increased risk [20, 21]. In order to explain this phenomenon it is important to gain a better understanding of the function of BAT1. The yeast homolog of BAT1, Sub2p, has been shown to be required for mRNA export through nuclear pores [37, 38]. Previous findings have shown that the -22 C BAT1 allele, associated with the autoimmune disease susceptible 8.1 MHC ancestral haplotype, may result in reduced BAT1 transcription . However, it has also been demonstrated that both injection of excess UAP56 (BAT1) into Xenopus oocytes as well as depletion of HEL, the Drosophila homologue of UAP56, by RNAi resulted in defects in mRNA export from the nucleus [39, 40]. This indicates that both excess levels of BAT1 and a lack of this protein can lead to abnormalities in mRNA export and splicing. Hence, the presence of different alleles of BAT1 -22 may potentially lead to a range of different aberrations in mRNA processing resulting in a variety of different phenotypic manifestations of pathology. It is, therefore, possible that the BAT -22*2 allele per se may be protective against AD but still also be part of an array of SNPs that may confer risk for certain autoimmune disorders. The complexity of potential phenotypical effects as well as possible haplotypic associations of BAT1 -22 with other genes indicate that further studies are warranted to explore whether the BAT1-22*1 allele may confer an independent risk for AD other than just in haplotypic combination with TNFA -850*2 as observed in the current study.
Therefore, while the possibility of LD with other genes cannot be ruled out the current study provides evidence in support for a potential role for BAT1 in AD pathology. BAT1 -22 and TNFA -850 in combination with other biochemical and cognitive markers might serve as genetic markers for diagnostic purposes or AD risk assessment strategies. Moreover, in light of current international drug development research in the AD field, establishment of genetic profiles may help to identify individuals more likely to experience benefits from certain treatments or may prevent individuals genetically unfavourably predisposed from receiving costly, yet ineffective treatment. Since the SNPs investigated could also lead to functional differences it is of great importance to investigate phenotypical characteristics conferred by these polymorphisms.
Considering that BAT1 has a potential regulatory role for inflammatory cytokines [20, 21] analysis of BAT1 mRNA and protein levels in AD brain tissue may reveal a functional role for the BAT1 protein in AD pathology. To investigate whether transcription of BAT1 was affected in AD, levels of BAT1 mRNA were determined in brain tissue from confirmed AD and control cases. This revealed significantly elevated levels of BAT1 and DDXL mRNA in Fc of AD cases and suggests a potential functional role for BAT1 in AD pathogenesis. It is not implausible to suggest that levels of BAT1 may rise as a response mechanism to counteract the inflammatory reactions that occur in regions of AD pathology. However, a repetition of this study with a larger sample size to enable parametric analysis of results may help to confirm the significance of these findings.
These data are of particular interest in light of recent findings that oligonucleotides spanning the promoter polymorphism -22 to -348 region of BAT1 autoimmune disease resistant 7.1 AH bind DNA/protein complexes as shown by electrophoretic mobility shift assays . At position -22 these complexes appear to include the octamer binding protein family member, transcription factor Oct1 . Oct1 has been shown to bind TNFA at position -857T and can interact with the pro-inflammatory NF-κB transcription factor p65 subunit . As TNFα has been implicated in inflammation observed in AD brains  the above studies together with the current findings suggest an important association between BAT1 expression and regulation of inflammatory cytokines in the AD brain. The exact mechanisms of this link between BAT1 -22 promoter polymorphism and inflammatory reactions in the AD brain remain to be explored in future studies.
To establish the role of BAT1 in AD pathology it is imperative to examine levels of BAT1 in AD affected tissues in a larger number of cases. Apart from its presence in brain tissue, BAT1 mRNA transcripts have been detected in pancreas, kidney, skeletal muscle, liver, lung and heart . The presence of BAT1 in hematopoietic cells  makes this protein a potential biomarker in early diagnosis or monitoring of progression of disorders with inflammatory responses, such as AD.