Isoform of APOE with retained intron 3; quantitation and identification of an associated single nucleotide polymorphism
© Dieter and Estus; licensee BioMed Central Ltd. 2010
Received: 23 July 2010
Accepted: 7 September 2010
Published: 7 September 2010
Alleles of apolipoprotein E (APOE) are the major genetic risk factor for late onset Alzheimer's Disease (LOAD). Recently, an APOE splice variant that retains intron 3 (APOE-I3) was identified. To gain insight into the possible role of this isoform in LOAD, we quantified its expression in a cohort of 56 human brain specimens by using quantitative RT-PCR.
We found that APOE-I3 generally represents a low percentage (< 0.5%) of overall APOE expression. However, in one specimen, the proportion of APOE-I3 was increased about ~13 fold. This specimen was unique in the cohort for possessing the minor allele of an intron 3 single nucleotide polymorphism (SNP), rs12982192. Additionally, an allelic expression imbalance study indicated that the rs12982192 minor allele was associated with increased APOE-I3 expression.
Overall, we interpret our results as suggesting that APOE-I3 represents a minor portion of APOE expression and that rs12982192 is associated with APOE intron 3 retention. Since the minor allele of this SNP is on the same haplotype as the minor allele of rs429358, which defines the APOE4 allele, we speculate that rs12982192 may reflect a modest loss of mRNA encoding functional APOE4.
APOE variants are the single largest genetic factors impacting late onset Alzheimer Disease (LOAD), the most common neurodegenerative disease. In particular, the APOE4 allele confers increased risk, being present at a ~15% frequency in the general population and at 50% in LOAD [1, 2]. APOE consists of 4 exons and 3 introns encoding a total of 317 amino acids. After cleavage of the signal peptide, the mature APOE protein is 299 amino acids. Protein translation begins in exon 2 and ends in exon 4. In the brain, APOE transports cholesterol and modulates Aß clearance [1, 2]. APOE is primarily secreted by astrocytes [3, 4] with microglia  and, possibly, stressed neurons also contributing to APOE production . Overall, insights into APOE expression will provide further information on AD risk.
Recently, Xu et al reported an APOE isoform that retained intron 3 (APOE-I3) and was increased with neuronal stress in murine models . To gain insights into this novel APOE isoform in humans, we quantified APOE-I3 as well as the normal length APOE (APOE-NL) in a cohort of 56 AD and non-AD brain samples. We report that APOE-I3 represents a low proportion of overall APOE expression. In addition, we identified an individual with significantly increased expression of the APOE-I3 isoform. Further analysis revealed that this individual was unique in the cohort for an intron 3 single nucleotide polymorphism (SNP), rs12982192. Moreover, APOE-I3 was in allelic expression imbalance (AEI) in this individual such that the minor allele of rs12982192 correlated with increased expression of the APOE-I3 isoform. Overall, we interpret our results as suggesting that APOE-I3 represents a minor portion of APOE expression and that rs12982192 is associated with APOE intron 3 retention.
Materials and methods
Human autopsy tissue
The human autopsy tissue specimens used here have been described elsewhere [8, 9]. Briefly, anterior cingulate tissue was provided by the University of Kentucky AD Center. Diagnoses of AD and non-AD were based upon evaluation of both cognitive status, i.e., Clinical Dementia Rating and Mini-Mental State Examination scores, as well as neuropathology . The age at death for individuals that were cognitively intact, i.e., non-AD, was 82 ± 9 years (mean ± SD, n = 29) while age at death for AD individuals was 82 ± 6 (n = 27). The average post-mortem interval for non-AD and AD individuals was 2.8 ± 0.8 hours (mean ± SD, n = 29) and 3.4 ± 0.6 hours (n = 27), respectively. The non-AD and AD individuals were well-balanced for sex, i.e., 15 of the 29 non-AD individuals were women while 14 of the 27 AD individuals were women.
Allelic expression imbalance
To determine whether each allele of rs12982192 and rs429358 contributed equally to APOE-I3 and APOE-NL, respectively, we cloned the APOE-I3 and APOE-NL PCR products from specimen #31 (TA-Cloning pcDNA3.1, Invitrogen). The identity of each clone was confirmed by direct sequencing (Davis Sequencing). We then estimated the proportion of each allele present in specimen #31 APOE-I3 and APOE-NL cDNA by comparing the relative sequencing electropherogram peak areas of each allele of rs12982192 and rs429358, respectively, essentially as described ; the results of this assay were normalized relative to standard curves derived from varying proportions of each allelic clone and validated by analysis of genomic DNA (gDNA).
The association between APOE-I3 and/or APOE-NL and sex, age, PMI and APOE4 genotype was analyzed by linear regression (SPSS v 17.0). Differences in AEI in gDNA and cDNA were evaluated by a two-tailed T-test (SPSS, V17). A p < 0.05 was considered to be significant.
To identify SNPs that may underlie the increased APOE-I3 in specimen #31, we generated genomic APOE clones from exon 2 to exon 4 of this individual. These clones included the intervening 1,092 bp intron 2 and 580 bp intron 3. Sequencing revealed that this gDNA was heterozygous for (i) rs12982192, which is a rare T/C SNP located 50 bp into intron 3 (dbSNP Build 131), (ii) rs769449, which has a 6.5% minor allele frequency and is located 78 bp into intron 2, (iii) rs769450, which has a 39.9% minor allele frequency and is also located within intron 2, and (iv) rs429358, which is within exon 4 and determines APOE3/4 status (Figure 1A). The APOE4 haplotype (rs429358C) contained the minor rs12982192C and rs769449A alleles and the major rs769450G allele. Since rs12982192 is within the retained intron 3, we hypothesized that the minor rs12982192C allele may be associated with APOE-I3. To test this possibility, we first assessed whether the minor allele of rs12982192 was present in any of the other 55 DNA samples in this study; only specimen #31 possessed the minor rs12982192C allele. This finding supports the possibility that intron 3 retention is associated with rs12982192.
The major findings of this paper are (i) APOE-I3 appears to be a rare APOE isoform compared to APOE-NL in the aged human brain, (ii) APOE-I3 was increased ~13-fold in one individual, (iii) rs12982192, an intron 3 SNP, was uniquely present in the specimen with high APOE-I3, and (iv) APOE-I3 expression showed significant AEI in this sample, with the minor rs12982192 allele contributing a greater proportion of APOE-I3 expression. Hence, rs12982192 appears to be associated with increased expression of a rare APOE isoform.
Although the role of apoE-I3 in the brain is unclear, this isoform is infrequent relative to APOE-NL. While APOE is expressed largely in astrocytes [3, 4, 15], Xu et al found that APOE-I3 expression was restricted to neurons in mice . We did not discern a correlation between the expression of APOE-I3 and synaptophysin, an mRNA restricted to neurons, although this correlation may have been precluded by the generally low APOE-I3 expression such that only 13 of the 56 samples with detectable APOE-I3 contributed to this analysis. While APOE-NL encodes a 317 amino acid secreted protein, the sequence at the exon 3 - intron 3 junction results in a stop codon. Hence, APOE-I3 encodes a truncated 79 amino acid, amino-terminal APOE fragment that is apparently not detectable in transfected cells or brain . Although we considered that APOE-I3 may be a candidate for nonsense-mediated decay, APOE-I3 levels were independent of PMI (data not shown), suggesting that APOE-I3 is not rapidly degraded. In summary, the role of APOE-I3 in the brain is unclear but, given that this isoform is rare and that its encoded protein is not detectable, we speculate that APOE-I3 represents a loss of APOE mRNA encoding functional protein.
Several lines of evidence suggest that rs12982192 modulates the proportion of APOE-I3, including (i) rs12982192 resides within intron 3 consistent with its possible role as a cis-acting SNP, (ii) this SNP was uniquely present in the specimen with relatively high APOE-I3 among the 56 brain specimens analyzed, (iii) rs12982192 was associated with significant AEI for APOE-I3 with the rare rs12982192C minor allele contributing a greater proportion of APOE-I3 expression and (iv) the APOE-NL isoform did not display AEI similar to the APOE-I3 isoform, ruling out the possibility of an overall APOE AEI in specimen #31 due to other mechanisms, e.g., a promoter SNP. While this reasoning appears robust to support a role for rs12982192 in APOE-I3 splicing, two lines of evidence suggest that rs129382192 does not act independently to modulate intron 3 splicing. First, rs129382192 did not alter APOE splicing in a transfected cell, minigene paradigm (Dieter and Estus, unpublished observations), suggesting in vivo elements may be necessary to discern the actions of the SNP. Second, while the main finding of the AEI study was that the minor allele of rs12982192 was associated with increased APOE-I3 expression, a secondary finding was that the ~3.5:1 allelic ratio was quantitatively insufficient to account for the ~13-fold increase in overall APOE-I3 expression in specimen #31. We interpret this result as possibly reflecting additional variables specific to specimen #31 that influence APOE-I3 or, more likely, that the difference is attributable to currently unclear technical differences between the RT-qPCR and AEI assays. In summary, although APOE-I3 levels may be modulated by additional variables, several lines of evidence support the association of rs12982192 with APOE-I3 expression.
Since APOE4 is the primary genetic risk factor for AD, SNPs that alter APOE4 expression are likely to modulate the association of APOE4 with AD. In this regard, we note that the minor rs12982192C allele is on the same haplotype as the minor rs429358C allele, which defines the APOE4 allele. Since our AEI study indicates that rs12982192 is associated in cis with retention of APOE intron 3, rs12982192 may emerge as a modulator of APOE4 allelic association with AD. Hence, although APOE-I3 represents only ~3% of total APOE mRNA in the rs12982192 heterozygous individual, the AEI study indicates that the majority of this APOE-I3 is derived from the APOE4 allele, and therefore could approach 5-6% of APOE4 expression. The extent that rs12982192 may be associated with AD risk will require a very large study population, given the rarity of this SNP, i.e., this SNP is listed within dbSNP Build 131 without a true frequency. We note that we have genotyped an additional ~800 DNA samples and have not identified another rs12982192 carrier.
In summary, our major findings are that APOE-I3 is a low abundance APOE isoform. Moreover, the minor C allele of rs12982192 appears associated with increased APOE-I3. Since the minor rs12982192C allele is on the same haplotype as APOE4, and since APOE-I3 appears to represent a loss of functional APOE4 mRNA, rs12982192 may modulate the association of APOE4 with AD risk. Future large case-control association studies are necessary to evaluate this possibility.
late onset Alzheimer's Disease
- (APOE-I3) APOE:
splice variant retaining intron 3
single nucleotide polymorphism
normal length APOE
allelic expression imbalance
real-time quantitative PCR
The authors gratefully acknowledge tissue supplied by the University of Kentucky AD Center, which is supported by P30AG028383, as well as NIH for grant support (R01AG026147 and P01AG030128).
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