Evaluation of the global association between cholesterol-associated polymorphisms and Alzheimer's disease suggests a role for rs3846662 and HMGCR splicing in disease risk
© Simmons et al; licensee BioMed Central Ltd. 2011
Received: 2 May 2011
Accepted: 25 August 2011
Published: 25 August 2011
Recent genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNP)s that are essentially unequivocally associated with peripheral cholesterol. Since the alleles of the APOE gene, which modulate peripheral cholesterol metabolism, and midlife plasma cholesterol are both associated with Alzheimer's disease (AD) risk, we have evaluated the hypothesis that SNPs associated with plasma cholesterol are also associated with AD.
Seventeen non-APOE SNPs reproducibly associated with cholesterol per GWAS were tested for association with AD in ~2,000 AD and ~4,000 non-AD subjects. As a group, these SNPs are associated with AD. Two SNPs in particular, rs3846662 and rs1532085, are associated with AD risk and age-of-onset. Additionally, rs3846662 was associated with HMGCR exon 13 splicing in human liver but not brain, possibly obscured by CNS cell-type heterogeneity. However, rs3846662 was associated with HMGCR exon 13 splicing in liver- and brain-derived cell lines.
Cholesterol-associated SNPs outside of APOE confer a global risk for AD. Rs3846662 and rs1532085 are associated with both AD risk and age-of-onset. Rs3846662 is associated with HMGCR exon 13 inclusion. Since rs3846662 affects AD risk and age-of-onset as well as statin responsiveness, this SNP may confound clinical trials evaluating the protective effects of statins on AD.
KeywordsAlzheimer genetics cholesterol GWAS SNP HMGCR statin
Late-onset Alzheimer's disease (AD) is a devastating form of dementia with no clear etiology. As much as 80% of age-adjusted AD risk may be genetic based upon studies of monozygotic twins [1, 2]. The primary genetic modulators of AD risk are the alleles of the gene encoding apolipoprotein E (APOE), i.e. APOE-ε2, APOE-ε3 and APOE-ε4. The presence of APOE-ε4 alone accounts for up to 53% of AD risk while the presence of APOE-ε2 is protective against the disease [3–5].
Multiple theories have been proposed to account for the mechanism underlying the association of APOE with AD [6, 7]. One theory suggests that the alleles of APOE modulate AD risk via their effects on cholesterol homeostasis. This is supported by evidence that the APOE-ε4 and APOE-ε2 alleles are associated with increased and decreased levels of plasma cholesterol, respectively . Furthermore, elevated midlife cholesterol is itself a risk factor for AD, raising the possibility that genetic modulators of cholesterol may also modulate AD risk [9, 10]. To this end, prior studies have demonstrated variable associations between polymorphisms in genes related to cholesterol metabolism and AD risk [11–13]. However, ontological analysis indicates that genes involved in cholesterol metabolism are significantly overrepresented as being associated with AD . Genome-wide association studies (GWAS)s have recently uncovered single nucleotide polymorphisms (SNP)s that are robustly and reproducibly associated with total cholesterol, low-density lipoprotein (LDL) and/or high-density lipoprotein (HDL) [15–23]. As such, these SNPs are essentially unequivocally associated with cholesterol and hence serve as outstanding tools to evaluate the genetic relatedness between cholesterol and AD. Thus, based on (i) the significant heritability of AD, (ii) the robust involvement of APOE in AD risk, (iii) a growing body of evidence that cholesterol itself modulates AD risk and (iv) the ontological overrepresentation of cholesterol gene variants in AD GWAS results we hypothesize that SNPs associated with peripheral cholesterol via GWAS also contribute to AD risk.
To test this hypothesis, we evaluated SNPs associated with plasma total cholesterol, LDL and HDL for their association with AD risk. Our results indicate that, as a group, these SNPs are significantly associated with AD. Additionally, rs3846662 and rs1532085 are also associated with AD age-of-onset. In vivo, rs3846662 is associated with HMGCR exon 13 inclusion in human liver but lacks a clear association with HMGCR splicing in human brain. HMGCR staining in brain indicated that the enzyme is expressed in both neurons and glia. Thus, we evaluated the function of rs3846662 in vitro using human liver- and CNS-derived cell lines, where rs3846662 was associated with the splicing efficiency of HMGCR exon 13 in both cell types. In conclusion, cholesterol-associated SNPs identified by GWAS, as a group, are associated with AD and exhibit the potential to elucidate novel mechanisms underlying AD risk and age-of onset.
Cholesterol-associated SNPs are also associated with AD
A review of GWAS via the HuGE Navigator database identified eighteen non-redundant SNPs whose associations with total cholesterol, LDL and/or HDL are highly significant (p < 1 × 10-10) and have been replicated in at least two populations. To assess the contribution of these SNPs to AD risk we queried their association with AD using a three-phase approach.
Phase 1 association testing between these eighteen cholesterol-associated SNPs and AD was performed by using 843 AD and 1,264 non-AD samples. PLINK was used to test for SNP-AD associations per additive models for the eighteen cholesterol-associated SNPs. Of the eighteen SNPs, rs157580 is in linkage with APOE and served as a positive control for AD association. In our Phase I study population, rs157580 and was significantly associated with AD in an additive model (p = 3.0 × 10-22, OR = 0.51). Henceforth only seventeen cholesterol-associated SNPs were considered for the purpose of multiple testing.
Phase 1 analysis of association between cholesterol SNPs and AD.
Three of the SNPs that demonstrated a nominal association or trend with AD (p < 0.1) have also been associated with gene regulation. Rs3846662 has been associated with statin responsiveness via an intermediate effect on HMGCR exon 13 alternative splicing [24, 25]. Rs2338104 is associated with MMAB mRNA and protein levels in human liver . Rs1532085 has been implicated in LIPC expression in human liver .
Phase 2 analysis of association between cholesterol SNPs and AD.
Phase 3 analysis of association between cholesterol SNPs and AD.
Rs3846662 and HMGCR splicing and expression in vivo
Since rs3846662 has been associated in vitro with HMGCR exon 13 splicing and with statin responsiveness, and since the contribution of statin pharmacotherapy to AD risk reduction is controversial, we chose to focus upon the actions of this SNP in further studies [24, 25, 28–30]. We began by testing for an association between rs3846662 and HMGCR exon 13 splicing in vivo using cDNA from human liver and brain, both of which are major cholesterol biosynthetic organs. HMGCR splicing was quantified as the percentage of HMGCRΔ13 out of total HMGCR message (calculated as HMGCRΔ13 /(HMGCRΔ13 + HMGCR_FL)).
Effect of rs3846662 on splicing in-vitro using liver and brain cell lines
The primary findings of our study are that: (1) SNPs unequivocally associated with plasma cholesterol demonstrate a significant global association with AD risk, (2) rs3846662 and rs1532085 are associated with AD age-of-onset, (3) rs3846662 is associated with HMGCR exon 13 splicing in human liver in vivo and in brain and liver-derived cell lines in vitro.
Our understanding of CNS cholesterol homeostasis is in its infancy relative to peripheral cholesterol homeostasis [34–37]. CNS cholesterol, under normal physiologic conditions, is maintained as a separate pool from plasma cholesterol by the blood brain barrier . The SNPs studied here were chosen because they were unequivocally associated with plasma cholesterol per se [12–14, 39]. Hence, these SNPs, which also are globally associated with AD risk, may influence AD via (1) peripheral effects on vasculature, and/or (2) direct effects on CNS cholesterol homeostasis.
Two lines of evidence support the possibility that these SNPs influence AD risk via peripheral or vascular effects. First, the genes associated with the majority of the tested SNPs are expressed at low levels in the brain compared to peripheral tissues, e.g., MMAB/MVK, TIMD4/HAVCR1, LIPC and CETP . Hence, SNPs in these genes are unlikely to influence CNS cholesterol. Second, these SNPs are capable of impacting human disease in the periphery, as they have been associated with coronary artery disease [15, 23, 41]. For example, rs3846662 was associated with myocardial infarction risk in an Asian population . However, SNPs which have the largest effects on plasma cholesterol did not exhibit the most significant associations with AD, as perhaps would be expected if plasma cholesterol is indeed the primary risk factor for AD [15–23].
The primary evidence supporting a role for at least a subset of the SNPs to act centrally is that APOE and HMGCR are both expressed at relatively high levels in the brain . Regarding HMGCR, rs3846662 is associated with HMGCR exon 13 splicing in vivo in human liver and in vitro in brain and liver-derived cell lines, as well as lymphocytes . Rs3846662 is not significantly associated with HMGCR exon 13 splicing in brain, although rs3846662 exhibits a similar trend as in liver. Thus, we speculate that rs3846662 may yet be functional in brain but its effects on HMGCR exon 13 splicing is confounded by cell-type heterogeneity and differential expression of RNA splicing factors in the CNS that our synaptophysin: GFAP model has not captured. Thus rs3846662_G may function in brain to increase HMGCR exon 13 retention, as observed in liver, although this effect is not discernible on the background of variation in HMGCR splicing in multiple brain cell types. Hence, SNPs related to APOE and HMGCR may act within the CNS. However, central and peripheral SNP effects are not necessarily mutually exclusive [43–45]. A future GWAS of CNS cholesterol would further clarify the role of SNPs in brain cholesterol metabolism and AD risk.
In conclusion, we report that SNPs which are robustly associated with peripheral cholesterol are globally associated with AD risk. Two SNPs in particular, rs3846662 and rs1532085, are associated with the age of AD onset. We also propose a model wherein rs3846662 acts to modulate HMGCR exon 13 splicing, plasma cholesterol and AD risk. Essentially, carriers of the rs3846662_G allele retain exon 13 of HMGCR more efficiently than rs3846662_A carriers. This relative increase in production of full-length HMGCR leads to increased basal cholesterol and, over the lifespan of an individual, increased AD risk. Lastly, rs3846662_G individuals appear to be at greater risk for AD and to be more likely to benefit from statin therapy to reduce AD risk.
We performed a review of GWAS literature pertinent to human plasma cholesterol phenotypes by querying the Human Genome (HuGE) Navigator http://www.hugenavigator.net with the search term "cholesterol" and then focusing upon the traits of total cholesterol, LDL and HDL . We identified 77 unique SNPs, located in or near 56 genes, associated with plasma total cholesterol, LDL and HDL. For each of these SNPs, a significant association with plasma cholesterol was observed in an initial study population and at least one confirmatory population. To limit multiple testing issues, we set a cutoff for significance of p < 1 × 10-10, which yielded 50 SNPs. These data were further filtered based on linkage disequilibrium. SNPs found to be in strong LD (r2 > 0.8 according to the CEU HapMap population) were considered to be redundant and only the most significantly cholesterol-associated SNP was retained for further analysis . As a result, our final dataset represented 18 unique SNPs residing in or near a total of 34 different genes. One of these SNPs, rs157580, is located in the gene TOMM40 and resides within a haplotypic block that includes the APOE gene. Rs157580 has been previously associated with AD and thus was considered a positive control for AD association in the present study [60, 61].
Populations and Genotyping
This work was performed in compliance with the Helsinki Declaration, with the approval of the Mayo Clinic and University of Kentucky Institutional Review Boards. Testing for an association between the eighteen cholesterol-associated SNPs and AD was performed by using a case-control population consisting of 1,940 AD and 3,925 non-AD subjects that has been previously described [62, 63]. Briefly, we utilized subjects recruited for the Mayo Clinic case-control series that consisted of Caucasian individuals residing within the United States. Subjects were pooled from three separate series, including two having received clinical diagnoses of probable AD according to NINCDS-ADRDA criteria from Jacksonville, FL (JS) and Rochester, MN (RS) and age-matched controls with a score of 0 on the Clinical Dementia Rating scale. The third population was taken from the Mayo Clinic Jacksonville, FL brain bank (AUT) and had received autopsy-confirmed diagnoses of AD (NINCDS-ADRDA, Braak score > 4.0) or were chosen as controls (Braak < 2.5, not including other unrelated pathology).
DNA from these individuals was used to evaluate an association between cholesterol-associated SNPs and AD in three phases. Phase 1 was designed to identify cholesterol-associated SNPs that are nominally associated with AD. Phase 2 was designed to follow up nominally significant Phase 1 SNPs, focusing upon SNPs that had also been associated with gene regulation. Phase 3 tested for SNP association with AD in a combined population of both Phase 1 and Phase 2 subjects to maximize statistical power.
Phase 1 included subjects pooled from the JS, RS and AUT series and, after quality control screening, consisted of 843 AD and 1,264 non-AD subjects. Average ages of AD and non-AD individuals were 74.3 (SD = 4.5) and 72.4 (SD = 4.6) years, respectively. In the event that a cholesterol-associated SNP from GWAS was not available by using this platform an appropriate proxy SNP was selected using SNAPproxy, a HapMap based proxy search (http://www.broadinstitute.org/mpg/snap/; ). Phase 2 consisted of 1,097 AD and 2,261 non-AD subjects pooled from the JS, RS and AUT series with average ages of 80.2 (SD = 7.9) and 82.4 (SD = 5.6) years, respectively.
Phase 1 samples were genotyped by using HumanHap300-Duo Genotyping BeadChips processed with an Illumina BeadLab station (Illumina, San Diego, CA) at the Mayo Clinic Genotyping Shared Resource center (Rochester, MN). Phase 2 samples were genotyped by using SEQUENOM MassARRAY iPLEX Platform (Sequenom, San Diego, CA). All samples were subject to strict quality control including elimination of samples with call rates < 90%, MAF < 0.01, H-W p < 0.001, discrepancy between reported and genotyped sex, cryptic relatedness and discordance upon visual inspection of genotype clusters.
AD Association Testing
Association testing for Phase 1 was performed using PLINK software to generate odds ratios (OR)s, 95% confidence intervals (CI)s and uncorrected p-values based on additive logistic regression models http://pngu.mgh.harvard.edu/purcell/plink. Of the eighteen cholesterol-associated SNPs we identified in GWAS literature, rs157580 was considered to be a positive control for association with AD and thus was not considered regarding the number of tests performed. The overall association between these seventeen SNPs and AD was tested for global significance using multivariable logistic regression.
Phase 2 AD-SNP association testing was performed by using three SNPs identified in Phase 1 that were nominally associated or trended with AD (p < 0.1) and that had also been reported as having an association with gene regulation [24–27]. PLINK software was again used to generate additive logistic regression models for SNP-AD association testing.
For phase 3, we maximized statistical power by testing for association between AD and rs3846662, rs1532085 or rs7298565 in the Phase 1 and Phase 2 populations combined. Association testing was done using additive logistic regression models generated in PLINK. Rs3846662 and rs1532085 were also tested for their effect on AD age-of-onset using Cox regression models in SPSS v.18 software (IBM, Somers, NY). For each SNP, genotypes with similar effects were combined and tested against the third genotype for association with AD age-of onset using SPSS.
With a total of ~2,000 AD cases and ~4,000 non-AD controls, we have greater than 80% power to detect an association between AD and rs3846662 or rs1532085. Power calculations were performed by using the method developed by Purcell S et al. http://pngu.mgh.harvard.edu/~purcell/gpc/cc2.html.
Human brain tissue from the anterior cingulate was generously provided by the Sanders-Brown Alzheimer's Disease Center Neuropathology Core and have been described elsewhere . The samples were from deceased individuals with an average age at death of 82.4 ± 8.7 (mean ± SD) years for non-AD and 81.7 ± 6.2 years for AD subjects. The average postmortem interval (PMI) for non-AD and AD subjects was 2.8 ± 0.8 and 3.4 ± 0.6 hours, respectively. Non-AD and AD samples were comprised of 48% and 55% female subjects. MMSE scores were, on average, 28.4 ± 1.6 for non-AD subjects and 11.9 ± 8.0 for AD subjects.
Human liver samples were obtained from the Brain and Tissue Bank for Developmental Disorders (Baltimore, MD) and have been previously described . The samples were from deceased individuals with an average overall age at death of 26.9 ± 9.0 years of age. The average age at death was similar for women (27.3 ± 8.5 years, n = 17) and for men (26.6 ± 9.4 years, n = 23). The average PMI for women was 12.9 ± 4.5 hours while the average PMI for men was 10.0 ± 3.0 hours.
Evaluation of rs3846662 effect on HMGCR exon 13 splicing efficiency in vivo
To evaluate the association between rs3846662 and HMGCR exon 13 splicing in human brain and liver tissue, genomic DNA and RNA were prepared from human tissue samples; cDNA was then reverse transcribed as reported previously [68, 69]. Isoform specific primers were designed to amplify either HMGCR that contains exon 13 (HMGCR_FL) or lacks exon 13 (HMGCRΔ13). To accomplish this we designed a forward primer specific to HMGCR exon 12 (5'-TGAGCGTGGTGTATCTATTCG-3') and reverse primers specific to either HMGCR exon 13 (5'-GGCCACAAGACAACCTTCTG-3') or the junction of HMGCR exons 12 and 14 (5'-CCTCCACCAAGCAAGGAGTA-3') to amplify HMGCR_FL and HMGCRΔ13, respectively.
Quantitative real-time PCR was performed on an MJ Opticon 4 thermal cycler (Biorad, Hercules, CA) by using 20 ng of subject cDNA together with 10 μl of SYBR green reagent (Quanta Biosciences, Gaithersburg, MD), 10 μl of H2O and forward and reverse primers, each at 1 μM final concentration. Cycling conditions consisted of a 3 minute denaturation step at 95°C followed by 40 cycles of denaturation for 15 seconds at 95°C and annealing/extension for 45 seconds at 60°C. A final melting curve was used to assess amplification fidelity in conjunction with subsequent inspection of PCR products on 8% TBE-PAGE gel (Sigma) stained with SYBR gold (Invitrogen, Carlsbad, CA).
Isoform-specific PCR products were generated, quantified at A260 and used to generate standard curves consisting of 10-fold serial dilutions that were amplified alongside samples with each PCR run. These standard curves allowed for monitoring reaction efficiency and absolute quantification of HMGCR isoforms. HMGCR expression was normalized to the geometric mean of internal reference genes, HPRT and RPL32, for each individual sample as we have described previously [70–72] and compared to the expression of synaptophysin and GFAP, which were quantified as described previously .
Testing for an association between rs3846662 and HMGCR expression and splicing was performed by using linear regression. Furthermore, the covariates age, sex and PMI were included in the analysis to correct for confounding differences in these demographics between brain and liver samples. All tests were performed using SPSS v.18 software.
Immunofluorescent staining of HMGCR
Paraffin-embedded blocks of human anterior cingulate were sliced at a thickness of 5 μm. Sections were fixed to glass slides prior to deparaffinization in a series of xylene/EtOH/H2O. Deparaffinized sections then submersed in pH 6 citrate buffer containing emulsifiers (Cell Marque, Cat # CMX633) and subject to antigen retrieval in a decloaking chamber (Biocare Medical, Cat # DC2002) for 30 min at 80C then by 10 min at 75C. Following antigen retrieval, slides were rinsed with H2O and then 1 × TBS (pH 7.4) before blocking with 5% goat serum in 1 × TBS for one hour. Slides were then rinsed again with 1 × TBS prior to incubation with rabbit α-HMGCR (1:25 dilution, Millipore Cat. # 07-457, Lot # 28743) and either mouse α-MAP2 (1:100 dilution, Sigma) or mouse α-GFAP (1:25 dilution, ICN Biomedicals, Cat # 691102) in a solution of 2% goat serum (Sigma, Cat #G6767) overnight at 4°C. Slides were rinsed with 1 × TBS and then treated with Alexa Fluor 488 goat α-mouse (Mol. Probes, Cat # 11029) and Alexa Fluor 568 goat α-rabbit (Mol. Probes, Cat # 11036) secondary antibodies (1:200 dilution) in 0.15% goat serum and 1 × TBS for one hour at room temperature. Labeled slides were then washed in 1 × TBS before treatment with Autofluorescence Eliminator Reagent (Chemicon, Cat # 2160) per the manufacturer's instructions. Slides were mounted using an anti-fading solution containing DAPI (Vectashield, Cat # H-1200), coverslipped, and visualized on a Nikon Diaphot fluorescence microscope.
In vitro minigene studies
HMGCR exon 12-14 mini-gene vectors harboring either the A or G allele of rs3846662 were a kind gift of Dr. Jan L. Breslow (The Rockefeller University) and have been previously described . The sequence of the HMGCR inserts differs only at rs3846662 (A/G).
To evaluate the effect of rs3846662 in homogenous cell types, as compared to the heterogeneous cellular environment of human brain and liver, we chose brain and liver-derived cell lines for transfection with HMGCR mini-genes. H4 human neuroglioma cells were maintained in Opti-MEM (Invitrogen) supplemented with a final concentration of 10% fetal bovine serum (FBS), 50 U/ml penicillin and 50 μg/ml streptomycin (P/S). HepG2 human hepatocellular carcinoma cells were grown in DMEM (Invitrogen) supplemented with a final concentration of 10% FBS and 1% P/S. Both cell lines were incubated at 37°C in a humidified 5% CO2 environment.
Both H4 and HepG2 cells were seeded in 6-well plates (2 × 105 cells/well) containing either antibiotic-free Opti-MEM or DMEM media and 10% FBS, respectively. Cells were allowed to grow for 24 hours prior to transfection with 1 μg of either allele-specific HMGCR mini-gene vector in 6 μl of FuGENE6 reagent (Roche Applied Sciences, Switzerland) and 94 μl of Opti-MEM (for H4 cells) or DMEM (for HepG2 cells), per the manufacturer's recommendations. Total RNA was prepared from cells twenty-four hours after transfection (RNeasy Mini Kit, QIAGEN) and quantified at A260 before being reverse transcribed using random hexamers per the manufacture's directions (SuperScript III, Invitrogen). Transfections were performed in triplicate for each allele.
Conventional PCR amplification of cDNA transcripts from HMGCR mini-genes was used to assess the splicing efficiency of HMGCR exon 13 in vitro as a function of rs3846662. A reverse primer specific to HMGCR exon 14 (5'-AGTGCTGTCAAATGCCTCCT-3') and a forward primer specific to the expressed, upstream portion of the pSPL3 exon-trapping vector (5'-TCTGAGTCACCTGGACAACC-3') were used to amplify vector-derived HMGCR_FL and HMGCRΔ13. Conventional PCR and gel-based methods were used to quantify vector-derived HMGCR isoforms because the large PCR product size (440 bp for HMGCRΔ13 and 599 bp for HMGCR_FL) precluded real time PCR analysis. Amplification was performed for 32 cycles following an initial denaturation at 95°C for 5 minutes followed by cycling of 95°C denaturation for 30 seconds, 60°C annealing for 30 seconds and 72°C extension for 1 minute, with a final extension at 72°C for 2 minutes. PCR products were resolved using 8% TBE-PAGE gel electrophoresis and visualized following staining with SYBR-gold and fluorescent image capture (Fuji FLA2000). Vector-derived HMGCR_FL and HMGCRΔ13 were identified by size and confirmed by sequencing (Davis Sequencing, Davis, CA). The amounts of HMGCR_FL and HMGCRΔ13 were quantified by fluorescent densitometry, corrected for background and normalized for size as described previously . The ratios of HMGCR_FL and HMGCRΔ13 for each transfection were then calculated to assess splicing efficiency and analyzed for their association with rs3846662 by using a paired Student's t-test for each transfection experiment.
List of abbreviations
genome wide association study
single nucleotide polymorphism
Funding of this work was provided by the National Institutes of Health and National Institute on Aging grants P01-AG030128 (SE), RO1-AG18023 (SGY), T32-AG000242 (CRS) as well as the Mayo Alzheimer's Disease Research Center P50-AG16574 (SGY). We would like to thank the University of Kentucky AD Center, which is supported by P30-AG028383, for providing human tissue. We also thank Dave Fardo for assistance with statistical methods.
- Gatz M, Pedersen NL, Berg S, Johansson B, Johansson K, Mortimer JA, Posner SF, Viitanen M, Winblad B, Ahlbom A: Heritability for Alzheimer's disease: the study of dementia in Swedish twins. J Gerontol A Biol Sci Med Sci. 1997, 52: M117-125.PubMedView ArticleGoogle Scholar
- Gatz M, Reynolds CA, Fratiglioni L, Johansson B, Mortimer JA, Berg S, Fiske A, Pedersen NL: Role of genes and environments for explaining Alzheimer disease. Archives of general psychiatry. 2006, 63: 168-174. 10.1001/archpsyc.63.2.168.PubMedView ArticleGoogle Scholar
- Corder EH, Saunders AM, Risch NJ, Strittmatter WJ, Schmechel DE, Gaskell PC, Rimmler JB, Locke PA, Conneally PM, Schmader KE, et al: Protective effect of apolipoprotein E type 2 allele for late onset Alzheimer disease. Nature genetics. 1994, 7: 180-184. 10.1038/ng0694-180.PubMedView ArticleGoogle Scholar
- Strittmatter WJ, Roses AD: Apolipoprotein E and Alzheimer disease. Proceedings of the National Academy of Sciences of the United States of America. 1995, 92: 4725-4727. 10.1073/pnas.92.11.4725.PubMedPubMed CentralView ArticleGoogle Scholar
- Davidson Y, Gibbons L, Pritchard A, Hardicre J, Wren J, Stopford C, Julien C, Thompson J, Payton A, Pickering-Brown SM, et al: Apolipoprotein E epsilon4 allele frequency and age at onset of Alzheimer's disease. Dementia and geriatric cognitive disorders. 2007, 23: 60-66. 10.1159/000097038.PubMedView ArticleGoogle Scholar
- Verghese PB, Castellano JM, Holtzman DM: Apolipoprotein E in Alzheimer's disease and other neurological disorders. Lancet Neurol. 2011, 10: 241-252. 10.1016/S1474-4422(10)70325-2.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim J, Basak JM, Holtzman DM: The role of apolipoprotein E in Alzheimer's disease. Neuron. 2009, 63: 287-303. 10.1016/j.neuron.2009.06.026.PubMedPubMed CentralView ArticleGoogle Scholar
- Wilson PW, Myers RH, Larson MG, Ordovas JM, Wolf PA, Schaefer EJ: Apolipoprotein E alleles, dyslipidemia, and coronary heart disease. The Framingham Offspring Study. JAMA: the journal of the American Medical Association. 1994, 272: 1666-1671. 10.1001/jama.272.21.1666.PubMedView ArticleGoogle Scholar
- Kivipelto M, Helkala EL, Hanninen T, Laakso MP, Hallikainen M, Alhainen K, Soininen H, Tuomilehto J, Nissinen A: Midlife vascular risk factors and late-life mild cognitive impairment: A population-based study. Neurology. 2001, 56: 1683-1689.PubMedView ArticleGoogle Scholar
- Kivipelto M, Helkala EL, Laakso MP, Hanninen T, Hallikainen M, Alhainen K, Soininen H, Tuomilehto J, Nissinen A: Midlife vascular risk factors and Alzheimer's disease in later life: longitudinal, population based study. Bmj. 2001, 322: 1447-1451. 10.1136/bmj.322.7300.1447.PubMedPubMed CentralView ArticleGoogle Scholar
- Papassotiropoulos A, Wollmer MA, Tsolaki M, Brunner F, Molyva D, Lutjohann D, Nitsch RM, Hock C: A cluster of cholesterol-related genes confers susceptibility for Alzheimer's disease. J Clin Psychiatry. 2005, 66: 940-947. 10.4088/JCP.v66n0720.PubMedView ArticleGoogle Scholar
- Wollmer MA: Cholesterol-related genes in Alzheimer's disease. Biochim Biophys Acta. 2010, 1801: 762-773.PubMedView ArticleGoogle Scholar
- Wollmer MA, Sleegers K, Ingelsson M, Zekanowski C, Brouwers N, Maruszak A, Brunner F, Huynh KD, Kilander L, Brundin RM, et al: Association study of cholesterol-related genes in Alzheimer's disease. Neurogenetics. 2007, 8: 179-188. 10.1007/s10048-007-0087-z.PubMedView ArticleGoogle Scholar
- Jones L, Holmans PA, Hamshere ML, Harold D, Moskvina V, Ivanov D, Pocklington A, Abraham R, Hollingworth P, Sims R, et al: Genetic evidence implicates the immune system and cholesterol metabolism in the aetiology of Alzheimer's disease. PloS one. 2010, 5: e13950-10.1371/journal.pone.0013950.PubMedPubMed CentralView ArticleGoogle Scholar
- Aulchenko YS, Ripatti S, Lindqvist I, Boomsma D, Heid IM, Pramstaller PP, Penninx BW, Janssens AC, Wilson JF, Spector T, et al: Loci influencing lipid levels and coronary heart disease risk in 16 European population cohorts. Nature genetics. 2009, 41: 47-55. 10.1038/ng.269.PubMedPubMed CentralView ArticleGoogle Scholar
- Heid IM, Boes E, Muller M, Kollerits B, Lamina C, Coassin S, Gieger C, Doring A, Klopp N, Frikke-Schmidt R, et al: Genome-wide association analysis of high-density lipoprotein cholesterol in the population-based KORA study sheds new light on intergenic regions. Circ Cardiovasc Genet. 2008, 1: 10-20. 10.1161/CIRCGENETICS.108.776708.PubMedView ArticleGoogle Scholar
- Kathiresan S, Melander O, Guiducci C, Surti A, Burtt NP, Rieder MJ, Cooper GM, Roos C, Voight BF, Havulinna AS, et al: Six new loci associated with blood low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or triglycerides in humans. Nat Genet. 2008, 40: 189-197. 10.1038/ng.75.PubMedPubMed CentralView ArticleGoogle Scholar
- Kathiresan S, Willer CJ, Peloso GM, Demissie S, Musunuru K, Schadt EE, Kaplan L, Bennett D, Li Y, Tanaka T, et al: Common variants at 30 loci contribute to polygenic dyslipidemia. Nat Genet. 2009, 41: 56-65. 10.1038/ng.291.PubMedPubMed CentralView ArticleGoogle Scholar
- Ridker PM, Pare G, Parker AN, Zee RY, Miletich JP, Chasman DI: Polymorphism in the CETP gene region, HDL cholesterol, and risk of future myocardial infarction: Genomewide analysis among 18 245 initially healthy women from the Women's Genome Health Study. Circ Cardiovasc Genet. 2009, 2: 26-33. 10.1161/CIRCGENETICS.108.817304.PubMedPubMed CentralView ArticleGoogle Scholar
- Sabatti C, Service SK, Hartikainen AL, Pouta A, Ripatti S, Brodsky J, Jones CG, Zaitlen NA, Varilo T, Kaakinen M, et al: Genome-wide association analysis of metabolic traits in a birth cohort from a founder population. Nature genetics. 2009, 41: 35-46. 10.1038/ng.271.PubMedPubMed CentralView ArticleGoogle Scholar
- Sandhu MS, Waterworth DM, Debenham SL, Wheeler E, Papadakis K, Zhao JH, Song K, Yuan X, Johnson T, Ashford S, et al: LDL-cholesterol concentrations: a genome-wide association study. Lancet. 2008, 371: 483-491. 10.1016/S0140-6736(08)60208-1.PubMedPubMed CentralView ArticleGoogle Scholar
- Wallace C, Newhouse SJ, Braund P, Zhang F, Tobin M, Falchi M, Ahmadi K, Dobson RJ, Marcano AC, Hajat C, et al: Genome-wide association study identifies genes for biomarkers of cardiovascular disease: serum urate and dyslipidemia. American journal of human genetics. 2008, 82: 139-149. 10.1016/j.ajhg.2007.11.001.PubMedPubMed CentralView ArticleGoogle Scholar
- Willer CJ, Sanna S, Jackson AU, Scuteri A, Bonnycastle LL, Clarke R, Heath SC, Timpson NJ, Najjar SS, Stringham HM, et al: Newly identified loci that influence lipid concentrations and risk of coronary artery disease. Nature genetics. 2008, 40: 161-169. 10.1038/ng.76.PubMedView ArticleGoogle Scholar
- Medina MW, Gao F, Ruan W, Rotter JI, Krauss RM: Alternative splicing of 3-hydroxy-3-methylglutaryl coenzyme A reductase is associated with plasma low-density lipoprotein cholesterol response to simvastatin. Circulation. 2008, 118: 355-362. 10.1161/CIRCULATIONAHA.108.773267.PubMedPubMed CentralView ArticleGoogle Scholar
- Burkhardt R, Kenny EE, Lowe JK, Birkeland A, Josowitz R, Noel M, Salit J, Maller JB, Pe'er I, Daly MJ, et al: Common SNPs in HMGCR in micronesians and whites associated with LDL-cholesterol levels affect alternative splicing of exon13. Arterioscler Thromb Vasc Biol. 2008, 28: 2078-2084. 10.1161/ATVBAHA.108.172288.PubMedPubMed CentralView ArticleGoogle Scholar
- Fogarty MP, Xiao R, Prokunina-Olsson L, Scott LJ, Mohlke KL: Allelic expression imbalance at high-density lipoprotein cholesterol locus MMAB-MVK. Human molecular genetics. 2010, 19: 1921-1929. 10.1093/hmg/ddq067.PubMedPubMed CentralView ArticleGoogle Scholar
- Teslovich TM, Musunuru K, Smith AV, Edmondson AC, Stylianou IM, Koseki M, Pirruccello JP, Ripatti S, Chasman DI, Willer CJ, et al: Biological, clinical and population relevance of 95 loci for blood lipids. Nature. 2010, 466: 707-713. 10.1038/nature09270.PubMedPubMed CentralView ArticleGoogle Scholar
- Aisen PS, Andrieu S, Sampaio C, Carrillo M, Khachaturian ZS, Dubois B, Feldman HH, Petersen RC, Siemers E, Doody RS, et al: Report of the task force on designing clinical trials in early (predementia) AD. Neurology. 2011, 76: 280-286. 10.1212/WNL.0b013e318207b1b9.PubMedPubMed CentralView ArticleGoogle Scholar
- Haag MD, Hofman A, Koudstaal PJ, Stricker BH, Breteler MM: Statins are associated with a reduced risk of Alzheimer disease regardless of lipophilicity. The Rotterdam Study. J Neurol Neurosurg Psychiatry. 2009, 80: 13-17. 10.1136/jnnp.2008.150433.PubMedView ArticleGoogle Scholar
- Feldman HH, Doody RS, Kivipelto M, Sparks DL, Waters DD, Jones RW, Schwam E, Schindler R, Hey-Hadavi J, DeMicco DA, Breazna A: Randomized controlled trial of atorvastatin in mild to moderate Alzheimer disease: LEADe. Neurology. 2010, 74: 956-964. 10.1212/WNL.0b013e3181d6476a.PubMedView ArticleGoogle Scholar
- Johnson JM, Castle J, Garrett-Engele P, Kan Z, Loerch PM, Armour CD, Santos R, Schadt EE, Stoughton R, Shoemaker DD: Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science. 2003, 302: 2141-2144. 10.1126/science.1090100.PubMedView ArticleGoogle Scholar
- Cooper TA: Use of minigene systems to dissect alternative splicing elements. Methods. 2005, 37: 331-340. 10.1016/j.ymeth.2005.07.015.PubMedView ArticleGoogle Scholar
- Singh G, Cooper TA: Minigene reporter for identification and analysis of cis elements and trans factors affecting pre-mRNA splicing. Biotechniques. 2006, 41: 177-181. 10.2144/000112208.PubMedView ArticleGoogle Scholar
- Fan J, Donkin J, Wellington C: Greasing the wheels of Abeta clearance in Alzheimer's disease: the role of lipids and apolipoprotein E. Biofactors. 2009, 35: 239-248. 10.1002/biof.37.PubMedView ArticleGoogle Scholar
- Yu C, Youmans KL, LaDu MJ: Proposed mechanism for lipoprotein remodelling in the brain. Biochimica et biophysica acta. 2010, 1801: 819-823.PubMedPubMed CentralView ArticleGoogle Scholar
- Mauch DH, Nagler K, Schumacher S, Goritz C, Muller EC, Otto A, Pfrieger FW: CNS synaptogenesis promoted by glia-derived cholesterol. Science. 2001, 294: 1354-1357. 10.1126/science.294.5545.1354.PubMedView ArticleGoogle Scholar
- Koudinov AR, Koudinova NV: Cholesterol's role in synapse formation. Science. 2002, 295: 2213-PubMedView ArticleGoogle Scholar
- Bjorkhem I, Meaney S: Brain cholesterol: long secret life behind a barrier. Arterioscler Thromb Vasc Biol. 2004, 24: 806-815. 10.1161/01.ATV.0000120374.59826.1b.PubMedView ArticleGoogle Scholar
- Papassotiropoulos A, Wollmer MA, Tsolaki M, Brunner F, Molyva D, Lutjohann D, Nitsch RM, Hock C: A cluster of cholesterol-related genes confers susceptibility for Alzheimer's disease. The Journal of clinical psychiatry. 2005, 66: 940-947. 10.4088/JCP.v66n0720.PubMedView ArticleGoogle Scholar
- de la Grange P, Gratadou L, Delord M, Dutertre M, Auboeuf D: Splicing factor and exon profiling across human tissues. Nucleic acids research. 2010, 38: 2825-2838. 10.1093/nar/gkq008.PubMedPubMed CentralView ArticleGoogle Scholar
- Waterworth DM, Ricketts SL, Song K, Chen L, Zhao JH, Ripatti S, Aulchenko YS, Zhang W, Yuan X, Lim N, et al: Genetic variants influencing circulating lipid levels and risk of coronary artery disease. Arterioscler Thromb Vasc Biol. 2010, 30: 2264-2276. 10.1161/ATVBAHA.109.201020.PubMedPubMed CentralView ArticleGoogle Scholar
- Hiura Y, Tabara Y, Kokubo Y, Okamura T, Goto Y, Nonogi H, Miki T, Tomoike H, Iwai N: Association of the functional variant in the 3-hydroxy-3-methylglutaryl-coenzyme a reductase gene with low-density lipoprotein-cholesterol in Japanese. Circ J. 2010, 74: 518-522. 10.1253/circj.CJ-09-0790.PubMedView ArticleGoogle Scholar
- Grundberg E, Adoue V, Kwan T, Ge B, Duan QL, Lam KC, Koka V, Kindmark A, Weiss ST, Tantisira K, et al: Global analysis of the impact of environmental perturbation on cis-regulation of gene expression. PLoS Genet. 2011, 7: e1001279-10.1371/journal.pgen.1001279.PubMedPubMed CentralView ArticleGoogle Scholar
- Ding J, Gudjonsson JE, Liang L, Stuart PE, Li Y, Chen W, Weichenthal M, Ellinghaus E, Franke A, Cookson W, et al: Gene expression in skin and lymphoblastoid cells: Refined statistical method reveals extensive overlap in cis-eQTL signals. American journal of human genetics. 2010, 87: 779-789. 10.1016/j.ajhg.2010.10.024.PubMedPubMed CentralView ArticleGoogle Scholar
- Dimas AS, Deutsch S, Stranger BE, Montgomery SB, Borel C, Attar-Cohen H, Ingle C, Beazley C, Gutierrez Arcelus M, Sekowska M, et al: Common regulatory variation impacts gene expression in a cell type-dependent manner. Science. 2009, 325: 1246-1250. 10.1126/science.1174148.PubMedPubMed CentralView ArticleGoogle Scholar
- Wolozin B, Kellman W, Ruosseau P, Celesia GG, Siegel G: Decreased prevalence of Alzheimer disease associated with 3-hydroxy-3-methyglutaryl coenzyme A reductase inhibitors. Archives of neurology. 2000, 57: 1439-1443. 10.1001/archneur.57.10.1439.PubMedView ArticleGoogle Scholar
- Jick H, Zornberg GL, Jick SS, Seshadri S, Drachman DA: Statins and the risk of dementia. Lancet. 2000, 356: 1627-1631. 10.1016/S0140-6736(00)03155-X.PubMedView ArticleGoogle Scholar
- Li G, Higdon R, Kukull WA, Peskind E, Van Valen Moore K, Tsuang D, van Belle G, McCormick W, Bowen JD, Teri L, et al: Statin therapy and risk of dementia in the elderly: a community-based prospective cohort study. Neurology. 2004, 63: 1624-1628.PubMedView ArticleGoogle Scholar
- Rea TD, Breitner JC, Psaty BM, Fitzpatrick AL, Lopez OL, Newman AB, Hazzard WR, Zandi PP, Burke GL, Lyketsos CG, et al: Statin use and the risk of incident dementia: the Cardiovascular Health Study. Archives of neurology. 2005, 62: 1047-1051. 10.1001/archneur.62.7.1047.PubMedView ArticleGoogle Scholar
- Sparks DL, Connor DJ, Sabbagh MN, Petersen RB, Lopez J, Browne P: Circulating cholesterol levels, apolipoprotein E genotype and dementia severity influence the benefit of atorvastatin treatment in Alzheimer's disease: results of the Alzheimer's Disease Cholesterol-Lowering Treatment (ADCLT) trial. Acta Neurol Scand Suppl. 2006, 185: 3-7.PubMedView ArticleGoogle Scholar
- Rockwood K: Epidemiological and clinical trials evidence about a preventive role for statins in Alzheimer's disease. Acta Neurol Scand Suppl. 2006, 185: 71-77.PubMedView ArticleGoogle Scholar
- Zhou B, Teramukai S, Fukushima M: Prevention and treatment of dementia or Alzheimer's disease by statins: a meta-analysis. Dementia and geriatric cognitive disorders. 2007, 23: 194-201. 10.1159/000099037.PubMedView ArticleGoogle Scholar
- Krauss RM, Mangravite LM, Smith JD, Medina MW, Wang D, Guo X, Rieder MJ, Simon JA, Hulley SB, Waters D, et al: Variation in the 3-hydroxyl-3-methylglutaryl coenzyme a reductase gene is associated with racial differences in low-density lipoprotein cholesterol response to simvastatin treatment. Circulation. 2008, 117: 1537-1544. 10.1161/CIRCULATIONAHA.107.708388.PubMedView ArticleGoogle Scholar
- Chasman DI, Posada D, Subrahmanyan L, Cook NR, Stanton VP, Ridker PM: Pharmacogenetic study of statin therapy and cholesterol reduction. JAMA: the journal of the American Medical Association. 2004, 291: 2821-2827. 10.1001/jama.291.23.2821.PubMedView ArticleGoogle Scholar
- Istvan E: Statin inhibition of HMG-CoA reductase: a 3-dimensional view. Atheroscler Suppl. 2003, 4: 3-8.PubMedView ArticleGoogle Scholar
- Istvan ES: Structural mechanism for statin inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Am Heart J. 2002, 144: S27-32. 10.1067/mhj.2002.130300.PubMedView ArticleGoogle Scholar
- Istvan ES, Deisenhofer J: Structural mechanism for statin inhibition of HMG-CoA reductase. Science. 2001, 292: 1160-1164. 10.1126/science.1059344.PubMedView ArticleGoogle Scholar
- Yu W, Gwinn M, Clyne M, Yesupriya A, Khoury MJ: A navigator for human genome epidemiology. Nature genetics. 2008, 40: 124-125. 10.1038/ng0208-124.PubMedView ArticleGoogle Scholar
- Frazer KA, Ballinger DG, Cox DR, Hinds DA, Stuve LL, Gibbs RA, Belmont JW, Boudreau A, Hardenbol P, Leal SM, et al: A second generation human haplotype map of over 3.1 million SNPs. Nature. 2007, 449: 851-861. 10.1038/nature06258.PubMedView ArticleGoogle Scholar
- Kim S, Swaminathan S, Shen L, Risacher SL, Nho K, Foroud T, Shaw LM, Trojanowski JQ, Potkin SG, Huentelman MJ, et al: Genome-wide association study of CSF biomarkers Abeta1-42, t-tau, and p-tau181p in the ADNI cohort. Neurology. 2011, 76: 69-79. 10.1212/WNL.0b013e318204a397.PubMedPubMed CentralView ArticleGoogle Scholar
- Feulner TM, Laws SM, Friedrich P, Wagenpfeil S, Wurst SH, Riehle C, Kuhn KA, Krawczak M, Schreiber S, Nikolaus S, et al: Examination of the current top candidate genes for AD in a genome-wide association study. Mol Psychiatry. 2010, 15: 756-766. 10.1038/mp.2008.141.PubMedView ArticleGoogle Scholar
- Carrasquillo MM, Belbin O, Hunter TA, Ma L, Bisceglio GD, Zou F, Crook JE, Pankratz VS, Dickson DW, Graff-Radford NR, et al: Replication of CLU, CR1, and PICALM associations with alzheimer disease. Arch Neurol. 2010, 67: 961-964. 10.1001/archneurol.2010.147.PubMedPubMed CentralView ArticleGoogle Scholar
- Carrasquillo MM, Zou F, Pankratz VS, Wilcox SL, Ma L, Walker LP, Younkin SG, Younkin CS, Younkin LH, Bisceglio GD, et al: Genetic variation in PCDH11X is associated with susceptibility to late-onset Alzheimer's disease. Nat Genet. 2009, 41: 192-198. 10.1038/ng.305.PubMedPubMed CentralView ArticleGoogle Scholar
- Johnson AD, Handsaker RE, Pulit SL, Nizzari MM, O'Donnell CJ, de Bakker PI: SNAP: a web-based tool for identification and annotation of proxy SNPs using HapMap. Bioinformatics. 2008, 24: 2938-2939. 10.1093/bioinformatics/btn564.PubMedPubMed CentralView ArticleGoogle Scholar
- Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ, Sham PC: PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet. 2007, 81: 559-575. 10.1086/519795.PubMedPubMed CentralView ArticleGoogle Scholar
- Purcell S, Cherny SS, Sham PC: Genetic Power Calculator: design of linkage and association genetic mapping studies of complex traits. Bioinformatics. 2003, 19: 149-150. 10.1093/bioinformatics/19.1.149.PubMedView ArticleGoogle Scholar
- Zou F, Gopalraj RK, Lok J, Zhu H, Ling IF, Simpson JF, Tucker HM, Kelly JF, Younkin SG, Dickson DW, et al: Sex-dependent association of a common low-density lipoprotein receptor polymorphism with RNA splicing efficiency in the brain and Alzheimer's disease. Hum Mol Genet. 2008, 17: 929-935.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhu H, Tucker HM, Grear KE, Simpson JF, Manning AK, Cupples LA, Estus S: A common polymorphism decreases low-density lipoprotein receptor exon 12 splicing efficiency and associates with increased cholesterol. Hum Mol Genet. 2007, 16: 1765-1772. 10.1093/hmg/ddm124.PubMedPubMed CentralView ArticleGoogle Scholar
- Ling IF, Gopalraj RK, Simpson JF, Estus S: Expression and regulation of a low-density lipoprotein receptor exon 12 splice variant. J Neurochem. 2010Google Scholar
- Ling IF, Estus S: Role of SFRS13A in low-density lipoprotein receptor splicing. Hum Mutat. 2010, 31: 702-709. 10.1002/humu.21244.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhang X, Ding L, Sandford AJ: Selection of reference genes for gene expression studies in human neutrophils by real-time PCR. BMC Mol Biol. 2005, 6: 4-10.1186/1471-2199-6-4.PubMedPubMed CentralView ArticleGoogle Scholar
- Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002, 3: RESEARCH0034-PubMedPubMed CentralView ArticleGoogle Scholar
- Grear KE, Ling IF, Simpson JF, Furman JL, Simmons CR, Peterson SL, Schmitt FA, Markesbery WR, Liu Q, Crook JE, et al: Expression of SORL1 and a novel SORL1 splice variant in normal and Alzheimers disease brain. Mol Neurodegener. 2009, 4: 46-10.1186/1750-1326-4-46.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to 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.