Because APOE ε4
and age are important AD risk factors, the characterization of regulatory elements surrounding the APOE
gene that may predict the level of apoE E4 protein, is vital in understanding AD pathogenesis. The influence of APOE
promoter SNPs on apoE levels in plasma and brain has been studied [18
] but, to our knowledge, the influence of APOE
promoter SNPs or APOE
distal regulatory regions on CSF apoE levels has not been characterized. In a previous investigation, SNPs proximal to APOE
were found to be associated with AD risk [46
] leading us to wonder if SNPs in the region proximal to APOE
lay within an un-characterized APOE
regulatory element. Thus, in this investigation we evaluated the differences in healthy subjects CSF apoE levels while taking into account age, gender and APOE ε4
status, as well as associations with 21 SNPs in a large 70 kb region surrounding the APOE
gene. This study was an exploratory investigation to test the hypothesis that multiple genetic loci surrounding APOE
can predict CSF apoE levels. The results indicate that in addition to the APOE
promoter, SNPs in the TOMM40
gene, the ME1 and the BCR regions may also predict CSF apoE levels.
Consistent with the previous reports, CSF apoE levels increased with increasing age (, Panel D); and the levels were not associated with APOE ε4
genotype (, Panel A) or allele (, Panel B) or gender (, Panel C) [4
Backward linear regression models indicate that out of the 21 SNPs entered into the models, six SNPs may predict CSF apoE levels (three SNPs within TOMM40; rs11556505, n17664883, rs157584, one APOE promoter SNP, rs449647, one SNP within ME1; n17684509, and one SNP within the BCR; rs7247551; ). Of these six SNPs only two SNPs predict CSF apoE levels either with APOE ε4 (n17664883) or without APOE ε4 (rs449647) present in the model (data not shown).
Interestingly, previous reports indicate that the APOE
−491 promoter polymorphism (rs449647) is associated with AD risk, although it is unclear whether LD is obscuring these results [6
]. In our investigation the non-variant (AA genotype) carriers of −491 polymorphism appeared to have higher CSF apoE levels than the variant (AT and TT genotypes) carriers both alone and as part of a haplotype (; ) which is consistent with previous reports by Laws et al. where higher brain apoE levels [18
] and plasma apoE levels [19
] are associated with the −491 AA genotype. To our knowledge this is the first investigation of the association between CSF apoE levels and APOE
promoter SNPs. These results reported here, along with previous reports, implicate the −491 promoter polymorphism as an important regulator of apoE levels.
The association with the novel proximal SNP, n17664883, suggests that an APOE
regulatory element exists in the region distantly proximal to APOE
that may predict CSF apoE levels. This region within intron 4 of the TOMM40
gene, may contain a regulatory element that contributes to high CSF apoE levels when the major homozygote genotype (non-variant) is present (, ). Whereas the minor homozygote or the heterozygotes (variant) may contribute to lower CSF apoE levels (, ). Alternatively, CSF apoE levels may be consequences of TOMM40
gene action. The TOMM40
gene encodes the TOM40 protein which is the pore subunit of the mitochondrial outer membrane protein translocator [35
]. Currently, there is no evidence of protein-protein interaction between TOM40 and apoE. However, recent reports suggest that Aβ
PP may be targeted to the mitochondria and translocated across the mitochondrial membrane via the TOM40 protein [2
]; additionally, the translocation arrest of Aβ
PP in the TOM40 pore may lead to mitochondria dysfunction and neuronal loss in AD [7
]. Therefore, it is possible that variants of TOM40 may be more susceptible to the translocation arrest of Aβ
PP and may lead to more profound decline of mitochondrial function and neuronal damage. The biological feedback mechanism would then be activated and produce more apoE for the neuronal repair or regeneration, a well-known function of apoE.
We also assessed whether the APOE
distal regulatory elements (HCR, ME and BCR) can predict CSF apoE levels. Because evidence suggests that the HCR is primarily active in the liver [1
], we hypothesized that only the SNPs within the ME1 (rs483082, n17684509, rs584007) and BCR (rs7247551) but not HCR2 (rs35136575), would predict apoE expression in CSF. Indeed, in our analyses the HCR2 SNP did not have an effect on CSF apoE levels, but SNPs from both ME1 and BCR did (, , ). Interestingly, only one of the three SNPs within ME1, a small region which spans 620 bp, is predicted to be associated with CSF apoE levels (). This result may be attributed to high correlation of other SNPs in the model with SNPs in the ME1 region, thus, leading to the elimination of other ME1 SNPs from the regression model. This result does not necessarily reflect a lack of a biological influence on CSF apoE levels by the other ME1 SNPs.
The hypothesis generated by these results is that there may be additional regulators of APOE
in the proximal region as far upstream as 15 kb. The proximal region may act in combination with regulatory elements in the distal region, such as the ME1 and the BCR to increase the activity of the APOE
promoter. An example of distant proximal enhancers can be demonstrated by the APOB
gene, which has a cis
-element enhancer located 54 to 62 kilobases 5′ to the structural gene [29
]. Further support of our hypothesis is demonstrated by our finding whereby multiple loci together have effects on CSF apoE levels (, , ) implicating an influence on CSF apoE levels by a large haplotype. Such a concept is in line with studies suggesting that promoter haplotypes of APOE
can influence plasma apoE levels [40
]. But, our study goes beyond previous studies by investigating contributions by distal regulatory regions, such as ME1, HCR2 and BCR, on CSF apoE levels.
There were limitations of this exploratory investigation. First, our study sample size (n = 134) may be too small to detect small effects contributed by a few of the SNPs tested that have low minor allele frequencies. Even though we required all minor allele frequencies to be equal to or greater than 2%, and collapsed genotypes into variant and non-variant groups, some of the haplotype numbers were low (). Second, the statistical results should be approached with caution because stepwise linear regression models do not take into account multiple comparisons so that p-values do not represent true significance until corrected for multiple comparisons (). Thus, it is important to note that this is an exploratory investigation intended to generate further hypotheses.
In summary, linear regression models were used to search for APOE regulatory SNPs that predict CSF apoE levels. These SNPs are located within a large region surrounding the APOE gene. Six SNPs were found to predict CSF apoE levels; three TOMM40 SNPs (rs11556505, n17664883, rs157584), one APOE promoter SNP (rs449647; −491) and two APOE distal SNPs (ME1; n17684509, BCR; rs7247551). For two of these SNPs, the novel SNP within the TOMM40 gene (n17664883) and the APOE promoter SNP (rs449647; −491), there is a significant difference in CSF apoE levels between genotypes and these two SNPs also appear to contribute to haplotype CSF apoE levels. These results support the hypothesis that modestly penetrant SNPs within APOE regulatory elements may explain part of the variation in CSF apoE protein levels. Our data indicate that a multigenetic approach may be more powerful in explaining the variation in apoE levels than a monogenetic approach. However, the total contribution of several SNPs together was modest, with a large proportion of the variation remaining unaccounted for (R2 value, 0.19; ), which may suggest that future evaluation of molecular haplotypes in the APOE gene region in a larger study population is required to explain more specifically the variation in apoE levels. In addition, given that APOE proximal SNPs within the TOMM40 gene predict CSF apoE levels, a possible independent influence on CSF apoE levels by the TOM40 protein may exist.
The mechanism whereby APOE ε4
increases the risk of AD is uncertain. APOE ε4
carriers show substantial variance for age at onset of AD. Some individuals who are ε
4 homozygotes may be spared from AD even if they live into their 9th
]. Factors that alter apoE protein expression, such as an APOE ε4
haplotype that includes proximal and distal regulatory elements, may help to explain this variance. In addition to aging, we have now shown that certain SNPs appear to be associated with levels of apoE protein in CSF in cognitively normal individuals. If these SNPs are also associated with younger age of onset in AD, this would implicate levels of apoE as a factor in AD pathogenesis. An extension of this study would be an investigation of familial AD cases to evaluate the influence of family history of AD on AD age-at-onset and associations between SNPs within and around APOE, APOE ε4
status, CSF apoE levels as well as other AD biomarkers such as CSF Aβ40
, and tau.
In conclusion, this exploratory investigation has generated further hypotheses regarding the possible influence on CSF apoE levels by multiple genetic loci within and surrounding the APOE gene suggesting that the actual effect is likely to be determined by these loci’s haplotype structure.