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Association of alleles carried at TNFA -850 and BAT1 -22 with Alzheimer's disease Gnjec, Anastazija; D'Costa, Katarzyna J; Laws, Simon M; Hedley, Ross; Balakrishnan, Kelvin; Taddei, Kevin; Martins, Georgia; Paton, Athena; Verdile, Giuseppe; Gandy, Samuel E; Broe, G A; Brooks, William S; Bennett, Hayley; Piguet, Olivier; Price, Patricia; Miklossy, Judith; Hallmayer, Joachim; McGeer, Patrick L; Martins, Ralph N Aug 20, 2008

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ralssBioMed CentJournal of NeuroinflammationOpen AcceResearchAssociation of alleles carried at TNFA -850 and BAT1 -22 with Alzheimer's diseaseAnastazija Gnjec1,2, Katarzyna J D'Costa1,2, Simon M Laws1,2, Ross Hedley1,2, Kelvin Balakrishnan1,2, Kevin Taddei1,2, Georgia Martins1,2, Athena Paton1,2, Giuseppe Verdile1,2, Samuel E Gandy3, G Anthony Broe4, William S Brooks5, Hayley Bennett4, Olivier Piguet4, Patricia Price5,6,7, Judith Miklossy8, Joachim Hallmayer9, Patrick L McGeer8 and Ralph N Martins*1,2Address: 1Centre of Excellence for Alzheimer's Disease Research and Care, Faculty of Computing, Health and Science, School of Exercise, Biomedical and Health Sciences, Edith Cowan University, Joondalup Drive, Joondalup, 6027, WA, Australia, 2Sir James McCusker Alzheimer's Disease Research Unit, School of Psychiatry and Clinical Neurosciences, University of Western Australia, Hollywood Private Hospital, Nedlands, 6009, WA, Australia, 3Mount Sinai School of Medicine, New York, New York, 10029, USA, 4Prince of Wales Medical Research Institute, UNSW, Barker Street, Randwick, NSW 2031, Australia, 5Centre for Education and Research on Aging, University of Sydney and Concord Repatriation General Hospital, Concord, NSW, 2139, Australia, 6School of Surgery and Pathology, University of Western Australia, Nedlands, Australia, 7Department of Clinical Immunology and Biochemical Genetics, Royal Perth Hospital, Perth, WA, 6000, Australia, 8Kinsmen Laboratory of Neurological Research, Department of Psychiatry, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC, V6T 1Z3, Canada  and 9Department of Genetics, and Center for Narcolepsy, Department of Psychiatry, Stanford University, Stanford, CA, 94305, USAEmail: Anastazija Gnjec - agnjec@cyllene.uwa.edu.au; Katarzyna J D'Costa - dcosta@wehi.edu.au; Simon M Laws - simon.laws@lrz.tu-muenchen.de; Ross Hedley - lesley.hedley@bigpond.com; Kelvin Balakrishnan - kelvin.balakrishnan@gmail.com; Kevin Taddei - k.taddei@ecu.edu.au; Georgia Martins - gmartins@cyllene.uwa.edu.au; Athena Paton - apaton@cyllene.uwa.edu.au; Giuseppe Verdile - g.verdile@ecu.edu.au; Samuel E Gandy - samgandy@earthlink.net; G Anthony Broe - broet@sesahs.nsw.gov.au; William S Brooks - w.brooks@unsw.edu.au; Hayley Bennett - hayley.bennett@unsw.edu.au; Olivier Piguet - o.piguet@unsw.edu; Patricia Price - patricia.price@uwa.edu.au; Judith Miklossy - miklossy@astro.tem; Joachim Hallmayer - joachimh@stanford.edu; Patrick L McGeer - mcgeerpl@interchange.ubc.ca; Ralph N Martins* - r.martins@ecu.edu.au* Corresponding author    AbstractBackground: Inflammatory changes are a prominent feature of brains affected by Alzheimer'sdisease (AD). Activated glial cells release inflammatory cytokines which modulate theneurodegenerative process. These cytokines are encoded by genes representing severalinterleukins and TNFA, which are associated with AD. The gene coding for HLA-B associatedtranscript 1 (BAT1) lies adjacent to TNFA in the central major histocompatibility complex (MHC).BAT1, a member of the DEAD-box family of RNA helicases, appears to regulate the production ofinflammatory cytokines associated with AD pathology. In the current study TNFA and BAT1promoter polymorphisms were analysed in AD and control cases and BAT1 mRNA levels wereinvestigated in brain tissue from AD and control cases.Methods: Genotyping was performed for polymorphisms at positions -850 and -308 in theproximal promoter of TNFA and position -22 in the promoter of BAT1. These were investigatedsingly or in haplotypic association in a cohort of Australian AD patients with AD stratified on thePublished: 20 August 2008Journal of Neuroinflammation 2008, 5:36 doi:10.1186/1742-2094-5-36Received: 26 June 2008Accepted: 20 August 2008This article is available from: http://www.jneuroinflammation.com/content/5/1/36© 2008 Gnjec et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 10(page number not for citation purposes)basis of their APOE ε4 genotype. Semi-quantitative RT-PCR was also performed for BAT1 fromRNA isolated from brain tissue from AD and control cases.Journal of Neuroinflammation 2008, 5:36 http://www.jneuroinflammation.com/content/5/1/36Results: APOE ε4 was associated with an independent increase in risk for AD in individuals withTNFA -850*2, while carriage of BAT1 -22*2 reduced the risk for AD, independent of APOE ε4genotype. Semi-quantitative mRNA analysis in human brain tissue showed elevated levels of BAT1mRNA in frontal cortex of AD cases.Conclusion: These findings lend support to the application of TNFA and BAT1 polymorphisms inearly diagnosis or risk assessment strategies for AD and suggest a potential role for BAT1 in theregulation of inflammatory reactions in AD pathology.BackgroundInflammation is a prominent pathological feature of theAlzheimer's disease (AD) brain, and might be initiated bythe extracellular accumulation of amyloid β (Aβ) peptide[1]. Activated microglia and astrocytes cluster around theAβ deposits and neurofibrillary tangles of AD brains andcan release neurotoxic agents, including complement pro-teins and pro-inflammatory cytokines, such as interleukin(IL)-1β, IL-6 and tumor necrosis factor-alpha (TNFα) [2].Polymorphisms in genes encoding IL-1α, IL-1β, IL-6 andTNFα correlate with heightened risk of AD [3]. For exam-ple, IL1B -511 [4], IL6 -174 [5] and TNFA -308 [6,7] asso-ciate with increased or reduced risk of AD. We showedthat the IL1A -889 T/T and IL1B +3954 T/T genotypesmark increased risk for late-onset Alzheimer's disease(LOAD) in an Australian cohort [8].When investigating potential genetic risk factors for ADpathology it is important to include established geneticrisk factors. The most widely accepted genetic risk factorfor late onset-forms of AD (LOAD) is the ε4 allele of thegene encoding apolipoprotein E (APOE ε4) [9,10]. Tworecent studies have explored a potential associationbetween APOE ε4 and the TNFA -850T (*2) promoter pol-ymorphism in Irish [11] and Spanish [12] cohorts withconflicting outcomes. While in the Irish cohort possessionof the TNFA -850*2 allele significantly increased the riskof dementia associated with APOE ε4 [11], no such syner-gistic effect was detected in the Spanish cohort [12] sug-gesting that the effect could be population specific or thatother genetic or environmental factors may also play acontributing role. The availability of APOE genotype datafrom previous studies conducted by our research group[13,14] enabled us to investigate the potential linkbetween APOE ε4 and TNFA -850*2 in a well character-ised Australian cohort.TNFA -308*2 (A allele) marks susceptibility to severalautoimmune and inflammatory disorders (for a reviewsee [15]) and has higher transcriptional activity thanTNFA -308*1 (G allele) [16,17]. However TNFA -308*2and linked alleles may mark increased risk [6,18] or pro-tection [7,19] against AD, so we investigated TNFA -308tions of these polymorphisms or haplotypiccombinations of the respective alleles with AD pathologyin an Australian cohort.HLA-B associated transcript 1 (BAT1) is implicated in theregulation of several AD-associated cytokines [20,21].BAT1 is a member of the DEAD-box family of RNA heli-cases, encoded in the central major histocompatibilitycomplex (MHC) near to TNFA [22]. Members of this fam-ily are a group of highly conserved proteins involved inunwinding of RNA secondary structures [23]. DEAD-boxproteins have been implicated in a number of differentprocesses involving RNA such as mRNA stabilization [24].Studies of anti-sense transfectants suggest BAT1 may act asa negative regulator of pro-inflammatory cytokines,namely IL-1, IL-6 and TNFα [20]. Furthermore, BAT1 pro-moter polymorphisms located at positions -22 and -348can influence transcription through differential bindingof transcription factors [21]. The C allele at BAT1 -22(BAT1 -22*2) is found on a conserved ancestral haplotypeassociated with an increased risk of immunopathology(HLA-A1, B8, TNFA -308*2, DR3, DQ2) [21]. NeitherTNFA -308*2 nor BAT1 -22*2 are unique to this haplo-type, but when carried together form a haplospecificmarker of a conserved block of the central MHC [25].Here we present data from an investigation of associationsbetween AD, the APOE ε4 genotype and carriage of TNFA-308*2, TNFA -850*2 and BAT1 -22*2 in a well-character-ized Australian cohort. In addition, we report on BAT1mRNA levels examined in frontal cortex (Fc) brain tissuefrom AD and control cases in order to investigate whetherchanges in BAT1 expression are associated with AD.MethodsGenotypingAlleles carried at BAT1 -22 (G→C) and TNFA -308 (G→A)and TNFA -850 (C→T) promoter polymorphisms wasdetermined in 631 individuals from a population ofNorthern European descent (97% Caucasian). There were359 control donors (45.7% females) with age at veni-puncture of 76.7 ± 13.1 years (mean ± SD) and 272 ADcases (59.2% females, age: 77.1 ± 10.5). 391 cases werepatients recruited from a memory clinic in Perth, WesternPage 2 of 10(page number not for citation purposes)alleles singly or in haplotypic combination with polymor-phisms in adjacent candidate genes to elucidate associa-Australia (226 AD cases and 165 controls). The remainderof patients were participants in the Sydney Older PersonsJournal of Neuroinflammation 2008, 5:36 http://www.jneuroinflammation.com/content/5/1/36Study; a random sample of community-dwelling peopleaged 75 and over at recruitment. Of these, 46 were classi-fied as having AD at assessment, while 194 had no cogni-tive impairment and were used as controls for thisanalysis. All studies were conducted with approval fromthe institutional ethics committees and with informedconsent of the participants. Methods of recruitment, diag-nostic criteria and APOE genotyping were as described[13,14,26,27].Genomic DNA was extracted from peripheral lym-phocytes using a standard protocol [28]. BAT1 -22 alleleswere determined by PCR amplification in a total volumeof 20 μL, containing 1.0 U of Taq polymerase (Fisher Bio-tec, Australia), 0.2 mM each dNTP and 3.0 mM MgCl2, ona Mastercycler Gradient thermal cycler (Eppendorf, Ger-many) as follows: 1 cycle of 95°C for 5 minutes, 44 cyclesof 95°C for 30 seconds, 56°C for 35 seconds and 72°Cfor 40 seconds, followed by 1 cycle of 72°C for 10 min-utes. The oligonucleotide primers, (P1) 5'-CAACCG-GAAGTGAGTGCA -3' and (P2) 5'-CAGACCATCGCCTGTGAA-3', were purchased fromGenset Pacific Pty. Ltd (Lismore, Australia). Ampliconswere digested at 37°C using 5 U Alw44I (restrictionsequence GTGCAC), separated on 8% non-denaturingpolyacrylamide gel at 110 V for 1.5 hours and stained withethidium bromide to reveal DNA fragments with migra-tion patterns specific for each allele (Allele 1 (G) = 170base pairs (bp); Allele 2 (C) = 152 bp and 18 bp; Figure 1).TNFA -308 alleles were determined via PCR amplificationin a total volume of 20 μL, containing 0.6 U TAQti (FisherBiotec, Australia), 0.2 mM each dNTP, 1.5 mM MgCl2 and0.5 mg/ml BSA amplified as follows: 1 cycle of 94°C for 2minutes, 35 cycles of 94°C for 30 seconds, 63°C for 30seconds and 72°C for 30 seconds, followed by 1 cycle of72°C for 5 minutes. Primers, (P1) 5'-AGGCAATAG-GTTTTGAGGGCCAT-3' (underline denotes mismatch)and (P2) 5'-TCCTCCCTGCTCCGATTCCG-3', were pur-chased from Proligo Pty. Ltd (Lismore, Australia). Ampli-cons were digested at 37°C using 3 U NcoI (restrictionsequence C▲CATGG), separated on 5% high resolutionagarose gels at 280 V (12 minutes) and stained with ethid-ium bromide to reveal fragments with migration patternsspecific for each allele (Allele 1 (G) = 88 bp and 19 bp;Allele 2 (A) = 107 bp).TNFA -850 alleles were determined via PCR amplificationin a total volume of 20 μL, containing 0.6 U of TAQtipolymerase (Fisher Biotec, Australia), 0.2 mM each dNTP,1.5 mM MgCl2 and 0.5 mg/ml BSA as follows: 1 cycle of94°C for 3 minutes, 35 cycles of 94°C for 45 seconds,60°C for 30 seconds and 72°C for 45 seconds, followedby 1 cycle of 72°C for 5 minutes. Primers were modifiedfrom those initially published [27]. (P1) 5'-TCGAG-TATCGGGGACCCCCCGTT-3' (underline denotes mis-match) and (P2) 5'-CCAGTGTGTGGCCATATCTTCTT-3'were purchased from Proligo Pty. Ltd (Lismore, Australia).Amplicons were digested at 37°C using 3 U HincII(restriction sequence GTT▲AAC), separated on a 5% highresolution agarose gels at 280 V (12 minutes) and stainedwith ethidium bromide to reveal DNA fragments withmigration patterns specific for each allele (Allele 1 (C) =105 bp and 23 bp; Allele 2 (T) = 128 bp) [29].Brain tissue samplesTotal RNA and protein was isolated from brain tissue(frontal cortex) samples from subjects with histopatho-logically confirmed definite AD and control cases withoutany AD pathology. Autopsy was performed within 48hours after death. Subjects with PS1 mutations and anumber of familial AD cases with APOE ε4 genotypeswere from local pedigrees and from the brain tissue bankof Drexel University College of Medicine (Philadelphia,PA, USA). Control brain tissue was obtained locally(Western Australia) and tissues were also received fromthe New South Wales (NSW) Tissue Resource Centre (Syd-ney, NSW, Australia), which is supported by The Univer-sity of Sydney, Neuroscience Institute of Schizophreniaand Allied Disorders, National Institute of Alcohol Abuseand Alcoholism and NSW Department of Health.RNA extraction and semi-quantitative RT-PCRTotal RNA was isolated using Trizol® (Gibco BRL, GrandIsland, New York, USA) according to manufacturer'sinstructions. RNA was extracted from 100 mg of frontalcortex brain tissue from 12 cases with familial AD eitherBAT1 -22 G/C promoter polymorphism genotypingFigure 1BAT1 -22 G/C promoter polymorphism genotyping. A representation of a typical -22 C/G genotyping gel produced after digested PCR product was run on an 8% non-denatur-ing PAGE gel. M = Marker (100 base pair marker – arrows represent 400, 300 and 200 bp fragments). Black arrowheads correspond to allele fragments: -22 C = 152 bp & 18 bp, and -22 G = 170 bp. Lane 1 = -22 CC genotype. Lanes 2,4,5,7,8,9,10 and 11 = -22 CG genotype. Lanes 3 and 6 = -22 Page 3 of 10(page number not for citation purposes)with PS1 mutations or linked to inheritance of the APOE-ε4 allele (mean age at time of death: 63 years, range: 50 –GG genotype.Journal of Neuroinflammation 2008, 5:36 http://www.jneuroinflammation.com/content/5/1/3677) and from 16 control cases without AD pathology(mean age at time of death: 50.25 years, range: 18 – 74years). RNA concentrations were determined spectropho-tometrically and 1 μg aliquots were reverse transcribedusing the Omniscript™ Reverse Transcriptase Kit (QIA-GEN; Victoria, Australia).Primers required to assess the expression of BAT1 and β-ACTIN mRNA were purchased from Genset Pacific Pty.Ltd (Lismore, Australia): BAT1(F): 5'-AGAGGCTCTCTCG-GTATCA-3', BAT1(R): 5'-GCTGATGTTGACCTCGAAA-3',BACTIN(F): 5'-TGGAATCCTGTGGCATCCATGAAAC-3',BACTIN(R): 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'.Primers for glyceraldehyde-3-phosphate dehydrogenase(GAPDH) were as previously described [30]. 5 μL cDNAwas amplified in a 20 μL reaction on a LightCycler™(Roche, USA). Each 20 μL PCR reaction contained 1.25mM dNTP, 20 pmol each primer, 0.25 mg/mL BSA, 1.5units Taq Platinum polymerase and 0.5 × SYBR Green(Invitrogen, USA). Amplifications of cDNA were per-formed as follows: Denaturation at 95°C for 5 minutes,followed by amplification with 44 cycles at 94°C for 30seconds, annealing (62°C for BAT1, 64°C for β-ACTIN,and 65°C for GAPDH) for 15 seconds and 72°C for 40seconds. Amplicons were separated on 1% TBE agarosegels and visualised by ethidium bromide staining. Thequantification of cDNA was achieved with SYBR Green Idye (Sigma, USA).Standard curves were generated using 10-fold dilutions ofa previously purified bulk cDNA PCR product (stored at aconcentration of 1 ng/μL) and analysed using a 'fit points'method with the LightCycler™ run software, version 4.0.Melting curve analyses were used to confirm the genera-tion of a single product. This was further confirmed byagarose gel electrophoresis. The amplified BAT1 PCRproducts were sequenced using big-dye terminator chem-istry on an ABI automated DNA sequencer (ABI, USA) toconfirm the specific amplification of BAT1. The housekeeping genes β-ACTIN and GAPDH were used for nor-malization of BAT1 mRNA expression. Statistical signifi-cance analysis was performed using the Mann-Whitney Utest.The Statistical Package for Social Sciences (SPSS version11.5; SPSS Inc., Chicago, Illinois, USA) was used to estab-lish genotype and allele frequencies and to check forHardy-Weinberg equilibrium (HWE). Initial data compar-ison involved Pearson's χ2 and odds ratio (OR) analysis oftwo by two contingency tables to compare the relativegenotype frequencies in AD and control groups. SPSS wasfurther employed to perform Cochran Armitage testing fortrends where assumptions of HWE were not met. Thethe equation simultaneously to determine the overall con-tribution of each genotype on AD in this cohort, whilstcontrolling for established AD risk factors (age and gen-der). Estimation of linkage disequilibrium and analysis ofhaplotypes was performed using Thesias [31].GenBank codes for genes investigated in this studyinclude APOE (MIM: 107741, GeneID: 348), TNFA (MIM:191160, GeneID: 7124) and BAT1 (MIM: 142560,GeneID: 7919).ResultsPearson's chi-square (χ2) and Odds ratio (OR) analysis ofthe BAT1 -22 1/1 and 1/2 genotypes revealed a significantassociation between a complete absence of the BAT1 -22*2 allele and AD (Table 1). However, this apparentlevel of protection afforded by the BAT1 -22*2 allelerevealed no gene dosage effect and was limited tohomozygosity of this allele (Table 1). Pearson's χ2 and ORanalysis of the TNFA -308 single nucleotide polymor-phism (SNP) revealed a weak yet mildly significant trendwhereby possession of the -308*2 allele conferred protec-tion from the development of AD. However, this was onlysignificant when allele frequencies were analysed (Table1). No significant protective effect was observed whengenotype frequencies were analysed. Pearson's χ2 and ORanalysis of genotype and allele frequencies from data gen-erated through the genotyping of the TNFA -850 SNPrevealed a strong association of the TNFA -850*2/2 geno-type and the TNFA -850*2 allele with an increased risk forAD (Table 1).By convention Pearson's χ2 and OR analysis are com-monly used to evaluate data generated from large geno-typing studies and explore frequency distributions.However, in order for such analysis to produce meaning-ful outcomes strict conditions of HWE must be met. In thecurrent study the distributions of APOE and BAT1 -22 alle-les were in HWE (χ2, P = .54 and p = .97, respectively)within the control populations. However significant devi-ation from HWE within the control group populationswas observed for TNFA -850 and TNFA -308 (χ2 test, P <.005). Therefore, subsequent analyses employed Armit-age's trend test (rather than Pearsons's χ2 analysis), to cor-rect for potential type I errors associated with departurefrom HWE [32].Armitage's testing for trends revealed a significant associ-ation between APOE ε4 and AD (χ2 = 108.91, P < 0.0001).TNFA -850*2 was also significantly associated withincreased risk for AD while a significant protective trendwas observed for BAT1 -22*2 (Table 2). The protectiveeffect initially observed for TNFA -308*2 in the genotypePage 4 of 10(page number not for citation purposes)same programme was also used to perform direct logisticregression analysis, where all variables were entered intoand allele frequency distribution analysis (Table 1) didnot reach significance using Armitage's test for trendJournal of Neuroinflammation 2008, 5:36 http://www.jneuroinflammation.com/content/5/1/36(Table 2). This may reflect a haplotypic association withBAT1 -22*2 since the alleles are in linkage disequilibrium(LD) in the West Australian population [25].Logistic regression analysis including age and gender asso-ciated BAT1 -22*2/2 with protection against AD, whileTNFA -850*1/2 and TNFA -850*2/2 conferred risk (Table3). These findings support Armitage's test for trend resultsand suggest a possible gene dosage effect for the presenceof the TNFA -850*2 allele.Additional logistic regressions analysis of interactionterms between APOE ε4 and the TNFA and BAT1 SNPsshowed no interactions between the effects marked byAPOE ε4, and BAT1 -22*2/2, TNFA -850*1/2 or TNFA -850*2/2. Furthermore, a stratified analysis based onAPOE genotype using the Mantel-Haenszel techniqueshowed no significant differences in Odds ratios whenestimating effects on AD risk of individual SNPs versus acombination of these SNPs with APOE ε4. This suggeststhat the observed protective effect of BAT1 – 22*2/2 andthe increased risk associated with TNFA -850*2 are inde-pendent of APOE ε4 genotype.BAT1 and TNFA are located in close proximity within theMHC [21,22] and their alleles are in marked LD [25].Therefore, the computer programme Thesias [31] wasused to generate LD matrices for analysis of LD and forhaplotype analysis. BAT1 -22, TNFA -308 and TNFA -850were all in LD, so haplotype frequencies were estimatedunder LD for all three markers and combinations of twomarkers. The only significant result was obtained for BAT1-22*1 in combination with TNFA -850*2 (OR = 1.54, P <0.05). However, the individual Odds ratios for TNFA -850*1/2 and TNFA -850*2/2 were higher than for theabove haplotype (i.e. individual OR for TNFA -850*1/2 =1.8 and for TNFA -850*2/2 = 2.7). This indicates that thepresence of BAT1 -22*1 in haplotypic association withTNFA -850*2 cannot explain the risk effects conferred byTNFA -850*2. Therefore, both the protective effect associ-ated with BAT1 -22*2 and the increased risk associatedwith TNFA -850*2 are more likely due to the individualTable 1: Analysis of Genotype and Allele frequencies of the BAT1 -22, TNFA -308 and TNFA -850 polymorphismsMarker Genotype or allele Ctrl numbers (%) AD numbers (%)BAT1 -22 1/1 144 (40.1) 117 (43.0)1/2 167 (46.5) 138 (50.7)2/2 48 (13.4) 17 (6.3)a1 455 (63.4) 372 (68.4)2 263 (36.6) 172 (31.6)TNFA -308 1/1 226 (63.0) 188 (69.1)1/2 104 (29.0) 70 (25.7)2/2 29 (8.0) 14 (5.1)1 556 (77.4) 446 (82.0)2 162 (22.6) 98 (18.0)bTNFA -850 1/1 287 (79.9) 183 (67.3)1/2 61 (17.0) 70 (25.7)2/2 11 (3.1) 19 (7.0)c1 635 (88.4) 436 (80.1)2 83 (11.6) 108 (19.9)dCtrl = Control cases without AD pathologyAD = Alzheimer's disease casesa BAT1 -22*2/2 versus non-2/2 in AD, P < .005 (Pearson χ2 = 8.49) OR = 0.43 (95% CI = 0.24 – 0.77).b TNFA -308*2 allele in AD, P = .048 (Pearson χ2 = 3.91) OR = 0.75 (95% CI = 0.57 – 1.00).c TNFA -850*(2/2, 1/2) versus 1/1 in AD, P < .001 (Pearson χ2 = 13.06) OR = 1.94 (95% CI = 1.35 – 2.78.0).d TNFA -850*2 allele in AD, P < .001 (Pearson χ2 = 16.57) OR = 1.90 (95% CI = 1.39 – 2.59).Table 2: Armitage test for trend for BAT1 and TNFA genotypesMarker Genotype trend χ2-value P-valueBAT1 -22 1/1 < 1/2 < 2/2 7.26 <.05TNFA -308 1/1 < 1/2 < 2/2 5.28 .07Table 3: Direct logistic regression analysisVariable Odds ratio P-value 95.0% C.I.BAT1 -22*2/2a 0.436 <.01 0.238 – 0.798TNFA -850*1/2b 1.8 <.005 1.218 – 2.669TNFA -850*2/2c 2.709 <.05 1.260 – 5.824Direct logistic regression model with Odds ratios representing risk assessment for AD.a Homozygosity of BAT1 -22*2 allele (with absence of allele as reference).b Heterozygosity of TNFA -850*2 allele (with absence of allele as Page 5 of 10(page number not for citation purposes)TNFA -850 1/1 < 1/2 < 2/2 20.17 <.00005reference).c Homozygosity of TNFA -850*2 allele (with absence of allele as reference).Journal of Neuroinflammation 2008, 5:36 http://www.jneuroinflammation.com/content/5/1/36SNPs themselves or a potential haplotypic associationwith other genes.In order to test whether transcription of BAT1 and thehomologous gene DDXL was altered in AD, mRNA levelsof both BAT1 and DDXL were examined in brain frontalcortex tissue of AD and control cases. Analysis of BAT1mRNA levels (Figure 2) revealed significantly elevatedmRNA levels for BAT1 normalized against β-ACTIN (a)while normalization with GAPDH (b) showed marginalsignificance for increased BAT1 mRNA levels in the ADbrains (Mann-Whitney U test: P = .037 and P = .057respectively).DiscussionAD is a multifactorial disorder with a number of altera-tions in the immune profile occurring during disease pro-gression in both the brain [33] and the periphery [34,35].Recently studies have reported links between risk for ADand polymorphisms in the promoter regions of TNFA atpositions -308 [6,18] and -850 [11]. The current study uti-lized a well characterised sample to investigate thesepotential associations in an Australian cohort. In addi-tion, BAT1 has been implicated in modulation of inflam-matory cytokines [20]. Therefore, the current studyinvestigated alleles of the BAT1 -22 promoter polymor-phism as a potential risk factor for AD, singly or in haplo-typic association with the TNFA promoterpolymorphisms.Analysis of individual SNPs revealed no significant associ-ation between AD and TNFA -308*2. This contrasts withreports in the literature that associate the TNFA -308*2allele with either increased risk for AD [6,18] or protec-tion against this disorder [7,19]. While data from the cur-rent study appears to be more supportive of a potentialprotective role for TNFA -308*2 against AD (Table 1), noconclusions can be drawn solely based on genotype andallele frequency analysis due to control group deviationsfrom HWE that might affect the rate of type I error. How-ever, it is possible that the inconclusive result obtained forTNFA -308*2 may be due to haplotypic associations ofthis polymorphism with other MHC markers such as theBAT1-22*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 increasedrisk for AD. The literature shows association of the TNFA-850*2 with vascular dementia [11] and individuals athigh risk for dementia, such as those with Down's Syn-drome [36]. However, a clear association of TNFA -850*2with AD has only previously been reported as a synergisticfrom Northern Spain failed to produce evidence in sup-port of a synergistic effect between TNFA -850*2 andAPOE ε4 [12]. The authors suggested that this discrepancymight reflect true genetic differences between the popula-Semi-quantitative RT-PCR of BAT1 and DDXL mRNA in fron-tal cortex of AD (n = 12) and control cases (n = 16)Figure 2Semi-quantitative RT-PCR of BAT1 and DDXL mRNA in frontal cortex of AD (n = 12) and control cases (n = 16). Data is represented as Box-plots showing median values and quartiles. (A) BAT1 mRNA levels normalized against β-ACTIN (Mann-Whitney U test: *P = .037), (B) BAT1 mRNA levels normalized against GAPDH (Mann-Whitney U test: **P = .057).Page 6 of 10(page number not for citation purposes)effect in combination with APOE ε4 in a Northern Irishpopulation [11], while a similar study in a populationtions and pointed out that differences in allele frequencydistributions between the two different European popula-Journal of Neuroinflammation 2008, 5:36 http://www.jneuroinflammation.com/content/5/1/36tions might indicate linkage disequilibrium between theTNFA -850 and another marker that might represent thetrue disease causing gene [12].The current study presents data in support of the notionthat TNFA -850*2 contributes to the risk of AD independ-ently of the APOE ε4 allele. Furthermore, logistic regres-sion analysis revealed a possible gene dosage effect withincrease in copy numbers of the TNFA -850*2 allele lead-ing to higher Odds ratios. It is, however, possible that agene linkage with TNFA -850*2 would show a parallel ORpattern, and might account for the apparent gene dosageeffect attributed to the TNFA -850*2 allele. Since all threemarkers investigated exerted their effects independently ofAPOE ε4 but were found to be in LD with one another,haplotype frequencies, taking into account LD betweenmarkers, were estimated for all three MHC markers andalso for combinations of two markers in order to investi-gate whether an AD risk or protection associated haplo-type could be responsible for the effects observed.Only one haplotype (BAT1 -22*1 in combination withTNFA -850*2) appeared to be significantly associatedwith risk for AD, but the observed Odds ratio was lowerfor this haplotype (OR = 1.54) than the OR for the singlepolymorphisms associated with AD risk (TNFA -850*1/2,OR = 1.8 and TNFA -850*2/2, OR = 2.7). This indicatesthat, although in LD with the other two markers TNFA -850*2 did not exert its risk for AD through a haplotypicassociation with these polymorphisms. While it cannot beentirely ruled out that linkage disequilibrium with otheras yet not identified markers may be responsible for theeffect observed in this investigation, the current studyidentifies the TNFA -850*2 allele as a candidate markerthat may confer risk for AD in the Australian population.Further investigation with larger participant numbers andin other populations is clearly warranted.While the polymorphisms in the promoter regions ofTNFA are likely to directly affect transcription of the TNFAgene, ultimate levels of TNFα protein in tissues can also beinfluenced by other regulating factors such as BAT1. In thecurrent study BAT1-22*2/2 was significantly associatedwith protection against the development of AD. Similar tothe association between increased risk for AD and thepresence of the TNFA -850*2 allele, the protective effect ofBAT1-22*2/2 was found to be independent of APOE ε4status. Furthermore, none of the estimated haplotypicassociations with the two TNFA markers that are in link-age disequilibrium with BAT1 have provided evidence tosuggest that the effect observed for BAT1-22*2/2 is due toa haplotypic association with these markers. While thepossibility remains that the protective BAT1 effect mightis also possible that BAT1 might assert an independenteffect on AD risk.A potential independent role for BAT1 in AD pathology issupported by the notion that the BAT1 -22 polymorphismmay not only have the potential to affect transcription ofBAT1 but, through the role BAT1 plays in mRNA stabiliza-tion, this protein may also affect translation of a numberof inflammatory cytokines linked to AD pathology,including TNFA. It has previously been reported thatBAT1 plays a potential role in the regulation of inflamma-tory cytokines, including TNFA [20,21] and the BAT1 -22allele has been associated with certain autoimmune dis-ease susceptible ancestral haplotypes such as the 8.1 MHCAH amongst others [21]. Since BAT1 appears to regulate anumber of inflammatory cytokines for which alterationsare observed in AD pathology the current study is the firstto provide evidence to show that a BAT1 promoter poly-morphism is significantly associated with AD pathology.It is of interest to note that for the TNFA -850 polymor-phism the less frequent allele conferred risk for AD whilethe opposite was found for the less frequent allele (C) ofthe BAT1 -22 polymorphism which was associated with adecreased risk for AD. This finding that the BAT1 -22*2(C) allele is associated with protection against AD is incontrast to the findings for autoimmune disorders wherethe less common number 2 allele is implicated withancestral haplotypes that confer increased risk [20,21]. Inorder to explain this phenomenon it is important to gaina better understanding of the function of BAT1. The yeasthomolog of BAT1, Sub2p, has been shown to be requiredfor mRNA export through nuclear pores [37,38]. Previousfindings have shown that the -22 C BAT1 allele, associatedwith the autoimmune disease susceptible 8.1 MHC ances-tral haplotype, may result in reduced BAT1 transcription[21]. However, it has also been demonstrated that bothinjection of excess UAP56 (BAT1) into Xenopus oocytes aswell as depletion of HEL, the Drosophila homologue ofUAP56, by RNAi resulted in defects in mRNA export fromthe nucleus [39,40]. This indicates that both excess levelsof BAT1 and a lack of this protein can lead to abnormali-ties in mRNA export and splicing. Hence, the presence ofdifferent alleles of BAT1 -22 may potentially lead to arange of different aberrations in mRNA processing result-ing in a variety of different phenotypic manifestations ofpathology. It is, therefore, possible that the BAT -22*2allele per se may be protective against AD but still also bepart of an array of SNPs that may confer risk for certainautoimmune disorders. The complexity of potential phe-notypical effects as well as possible haplotypic associa-tions of BAT1 -22 with other genes indicate that furtherstudies are warranted to explore whether the BAT1-22*1Page 7 of 10(page number not for citation purposes)be due to LD with another gene as yet not investigated, it allele may confer an independent risk for AD other thanJournal of Neuroinflammation 2008, 5:36 http://www.jneuroinflammation.com/content/5/1/36just in haplotypic combination with TNFA -850*2 asobserved in the current study.Therefore, while the possibility of LD with other genescannot be ruled out the current study provides evidence insupport for a potential role for BAT1 in AD pathology.BAT1 -22 and TNFA -850 in combination with other bio-chemical and cognitive markers might serve as geneticmarkers for diagnostic purposes or AD risk assessmentstrategies. Moreover, in light of current international drugdevelopment research in the AD field, establishment ofgenetic profiles may help to identify individuals morelikely to experience benefits from certain treatments ormay prevent individuals genetically unfavourably predis-posed from receiving costly, yet ineffective treatment.Since the SNPs investigated could also lead to functionaldifferences it is of great importance to investigate pheno-typical characteristics conferred by these polymorphisms.Considering that BAT1 has a potential regulatory role forinflammatory cytokines [20,21] analysis of BAT1 mRNAand protein levels in AD brain tissue may reveal a func-tional role for the BAT1 protein in AD pathology. Toinvestigate whether transcription of BAT1 was affected inAD, levels of BAT1 mRNA were determined in brain tissuefrom confirmed AD and control cases. This revealed sig-nificantly elevated levels of BAT1 and DDXL mRNA in Fcof AD cases and suggests a potential functional role forBAT1 in AD pathogenesis. It is not implausible to suggestthat levels of BAT1 may rise as a response mechanism tocounteract the inflammatory reactions that occur inregions of AD pathology. However, a repetition of thisstudy with a larger sample size to enable parametric anal-ysis of results may help to confirm the significance ofthese findings.These data are of particular interest in light of recent find-ings that oligonucleotides spanning the promoter poly-morphism -22 to -348 region of BAT1 autoimmunedisease resistant 7.1 AH bind DNA/protein complexes asshown by electrophoretic mobility shift assays [41]. Atposition -22 these complexes appear to include theoctamer binding protein family member, transcriptionfactor Oct1 [39]. Oct1 has been shown to bind TNFA atposition -857T and can interact with the pro-inflamma-tory NF-κB transcription factor p65 subunit [42]. As TNFαhas been implicated in inflammation observed in ADbrains [2] the above studies together with the current find-ings suggest an important association between BAT1expression and regulation of inflammatory cytokines inthe AD brain. The exact mechanisms of this link betweenBAT1 -22 promoter polymorphism and inflammatoryreactions in the AD brain remain to be explored in futureTo establish the role of BAT1 in AD pathology it is imper-ative to examine levels of BAT1 in AD affected tissues in alarger number of cases. Apart from its presence in braintissue, BAT1 mRNA transcripts have been detected in pan-creas, kidney, skeletal muscle, liver, lung and heart [43].The presence of BAT1 in hematopoietic cells [20] makesthis protein a potential biomarker in early diagnosis ormonitoring of progression of disorders with inflamma-tory responses, such as AD.ConclusionThe current study has revealed an APOE ε4 independentassociation of TNFA -850*2 with increased risk for AD,and an APOE ε4 independent association of BAT1 -22*2/2 with decreased risk for AD. These findings were notenhanced by haplotype analysis of polymorphisms inlinkage disequilibrium suggesting that the observedeffects may have resulted from the single SNPs. Hence,these SNPs may represent valuable markers in risk assess-ment, prognosis and therapeutic approaches for AD. Inaddition, the current study has provided evidence for anovel role for BAT1 in AD pathogenesis. BAT1 may play arole in regulating the inflammatory response in ADthrough influencing mRNA export and translation. Inves-tigations of BAT1 promoter polymorphisms and mRNAand protein levels in other populations are clearly war-ranted to confirm this initial finding. Inflammatory proc-esses form important underlying mechanisms in ADpathology. Elucidating the role of the currently investi-gated SNPs in AD pathology may contribute towards anunderstanding of the regulatory mechanisms of theseevents, and may provide new targets for drug develop-ment to combat AD.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsAG has isolated RNA from AD and control brain tissueand has been drafting and writing the manuscript, hasperformed data analysis for the mRNA work, and has beeninvolved in interpretation of data and revising the manu-script critically for important intellectual content. KD hasperformed the semi-quantitative RT-PCR and data analy-sis and has made substantial contributions towards draft-ing the manuscript. SML has made substantialcontributions towards genotyping, data analysis andinterpretation and drafting of the manuscript. RH, KB andKT contributed towards the genotyping process. GM andAP have been involved in the sample acquisition and/orthe DNA extraction process. GV and SEG have made sub-stantial intellectual contributions towards the manu-script. GAB, WSB, HB and OP were involved in samplePage 8 of 10(page number not for citation purposes)studies. acquisition and processing. PP has made substantial con-tributions to the concept and design of the study and theJournal of Neuroinflammation 2008, 5:36 http://www.jneuroinflammation.com/content/5/1/36manuscript as expert adviser, and has contributed towardsdata interpretation. JM contributed towards analysingbrain tissue from a substantial proportion of the cases forhistopathological diagnosis. JH has been criticallyinvolved in statistical analyses and interpretation of data,including genotype and haplotype analyses. PM has pro-vided substantial expert advice with regard to analysis andinterpretation of data and manuscript drafting. RNM hasmade the most substantial contributions towards the con-ception and design of the study and has given finalapproval of the version to be published. All of the authorshave read and approved the final manuscript.AcknowledgementsThis project was supported by the McCusker Foundation for Alzheimer's Disease Research, Edith Cowan University and Hollywood Private Hospital, Department of Veteran Affairs and the NHMRC. The authors would also like to acknowledge the excellent help in form of statistical analysis contrib-uted by Dr Karen Josebury. Furthermore, the authors would like to acknowledge the Sir Zelman Cowen Universities' Fund which provided funding for collection of blood samples. We thank Dr Noel Tan for dissec-tion and histopathological examination of brains. We also extend our thanks to Dr Clive Cooke (Queen Elizabeth Medical Centre, Perth, WA, Australia) for dissection and macroscopic examination of brains. Further-more, we would like to thank Professor Glenda Halliday (Prince of Wales Medical Research Institute, Randwick, NSW, Australia) for valuable discus-sion with regard to the brain samples used.References1. McGeer EG, McGeer PL: The importance of inflammatorymechanisms in Alzheimer disease.  Exp Gerontol 1998,33:371-378.2. Gonzalez-Scarano F, Baltuch G: Microglia as mediators of inflam-matory and degenerative diseases.  Annu Rev Neurosci 1999,22:219-240.3. McGeer PL, McGeer EG: Polymorphisms in inflammatorygenes and the risk of Alzheimer disease.  Arch Neurol 2001,58:1790-1792.4. 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