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Human Molecular Genetics Advance Access originally published online on December 8, 2007
Human Molecular Genetics 2008 17(7):929-935; doi:10.1093/hmg/ddm365
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Sex-dependent association of a common low-density lipoprotein receptor polymorphism with RNA splicing efficiency in the brain and Alzheimer’s disease

Fanggeng Zou1,{dagger}, Rangaraj K. Gopalraj4,{dagger}, Johann Lok1, Haiyan Zhu4, I-Fang Ling4, James F. Simpson4, H. Michael Tucker4, Jeremiah F. Kelly5, Samuel G. Younkin1, Dennis W. Dickson1, Ronald C. Petersen6, Neill R. Graff-Radford2, David A. Bennett5, Julia E. Crook3, Steven G. Younkin1 and Steven Estus4,*

1 Department of Neuroscience 2 Department of Neurology and 3 Biostatistics Unit, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA 4 Department of Physiology and Sanders-Brown Center on Aging, University of Kentucky, 800 S. Limestone Street, Lexington, KY 40536-0230, USA 5 Rush Alzheimer's Disease Center, Rush University Medical Center, Chicago, IL, USA 6 Department of Neurology, Mayo Clinic, Rochester, MN 55905, USA

* To whom correspondence should be addressed. Tel: +1 8593233985; Fax: +1 8593232866; Email: sestus2{at}email.uky.edu

Received October 18, 2007; Accepted December 5, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Since apoE allele status is the predominant Alzheimer's disease (AD) genetic risk factor, functional single nucleotide polymorphisms (SNPs) in brain apoE receptors represent excellent candidates for association with AD. Recently, we identified a SNP, rs688, as modulating the splicing efficiency of low-density lipoprotein receptor (LDLR) exon 12 in female human liver and in minigene-transfected HepG2 cells. Moreover, the rs688T minor allele was associated with significantly higher LDL and total cholesterol in women within the Framingham Offspring Study cohort. Since LDLR is a major apoE receptor in the brain, we hypothesized that rs688 modulates LDLR splicing in neural tissues and associates with AD. To evaluate this hypothesis, we first transfected LDLR minigenes into SH-SY5Y neuroblastoma cells and found that the rs688T allele reduces exon 12 inclusion in this neural model. We then evaluated the association of rs688 allele with exon 12 splicing efficiency in vivo by quantifying LDLR splicing in human anterior cingulate tissue obtained at autopsy; the rs688T allele is associated with decreased LDLR exon 12 splicing efficiency in aged males, but not females. Lastly, we evaluated whether rs688 associates with AD by genotyping DNA from 1457 men and 2055 women drawn from three case-control series. The rs688T/T genotype was associated with increased AD odds in males [recessive model, odds ratio (OR) of 1.49, 95% confidence interval (CI) of 1.13–1.97, uncorrected P = 0.005], but not in females. In summary, these studies identify a functional apoE receptor SNP that is associated with AD in a sex-dependent fashion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Genetic variants that alter protein expression or function represent powerful tools to investigate the role of that particular protein in human disease. Single nucleotide polymorphisms (SNPs) that alter exon splicing enhancers (ESEs) and, thereby exon splicing efficiency, are emerging as functional variants capable of modulating disease susceptibility [(1–3), reviewed in (4)]. We recently identified the minor T allele of rs688 (rs688T) as neutralizing a putative ESE within exon 12 of the low-density lipoprotein receptor (LDLR). Moreover, the rs688T allele associates with inefficient exon 12 splicing in the human female liver in vivo, causes inefficient LDLR splicing in minigene-transfected HepG2 cells and associates with increased LDL and total cholesterol in females in the Framingham Offspring Study (5).

Although the etiology of late onset Alzheimer's disease (AD) is unclear, several lines of evidence suggest that LDLR represents an excellent candidate gene for AD association. First, LDLR is a primary brain receptor for apoE, alleles of which are widely accepted to modulate AD risk (6,7). Second, LDLR deficiency associates with increased amyloid-beta deposition in Tg2576 APP transgenic mice, although not PDAPP transgenic mice, suggesting a possible linkage between LDLR and this AD hallmark (6,7). Third, murine LDLR deficiency has been associated with learning deficits (7). Fourth, LDLR mutations are a primary cause of familial hypercholesterolemia, suggesting that other LDLR family members do not compensate adequately for insufficient LDLR function [reviewed in (8–10)]. Lastly, both apoE alleles and rs688 associate with cholesterol homeostasis, which itself has been implicated in Aß production and risk for AD [(5), reviewed in (11,12)]. Here, we report evaluation of rs688 function in neural cells as well as rs688 association with splicing differences in the human brain and risk for AD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
To evaluate whether rs688 modulates LDLR exon 12 splicing in neural cells, we transfected human neuroblastoma SH-SY5Y cells with LDLR minigenes consisting of LDLR exons 9–14 as well as the intervening introns. Since the minigenes were cloned from rs688C/C and rs688T/T individuals and varied at additional SNPs (5), we also investigated the role of rs688 specifically, i.e. we used site-directed mutagenesis to convert the rs688 site within the minigenes from rs688C to rs688T (rs688C–T), and, conversely, rs688T to rs688C (rs688T–C). Splicing efficiency resulting from the parent and mutant constructs was analyzed by preparing RNA at 24 h after transfection, converting to cDNA and PCR amplifying with a sense primer corresponding to sequence derived from the vector and an antisense primer corresponding to sequence within exon 14. This analysis found that the rs688C allele minigene consistently produced a greater proportion of RNA that contained exon 12 relative to the rs688T minigene (Fig. 1). Moreover, converting rs688C to the minor allele T reduced the efficiency of exon 12 inclusion. Similarly, when rs688T was converted to rs688C, a greater proportion of the RNA contained exon 12 (Fig. 1). In summary, rs688 modulates LDLR exon 12 splicing in neural cells, extending our prior observation that this SNP is functional within HepG2 cells (5).


Figure 1
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  		Figure 1. Rs688 modulates LDLR splicing efficiency in neuroblastoma cells in vitro. LDLR minigenes containing rs688C and rs688T alleles, or the same haplotypes wherein rs688C and rs688T were specifically converted by site-directed mutagenesis, were transfected into SH-SY5Y cells in parallel and RNA isolated for analyses 24 h later. These results depict representative images of the splicing results with PCR products corresponding to LDLR exons 10–14, as well as LDLR exons 10, 11, 13 and 14 (delta 12 LDLR), and LDLR exons 10–13–14 (delta 11–12 LDLR). Quantification of rs688 effects on splicing is also depicted; data points represent separate analyses beginning with cell transfection. The rs688T allele was associated with decreased splicing efficiency regardless of background haplotype, as reflected by Kruskal–Wallis statistical analyses (P = 0.026).

 
To investigate whether rs688 is associated with exon 12 splicing efficiency in the human brain in vivo, we analyzed 41 RNA samples prepared from the human anterior cingulate, a brain area moderately affected in AD. We focused on exon 12 splicing per se by using appropriate PCR primers in two separate reactions. The first reaction identified the proportion of LDLR mRNA containing exon 12, i.e. the sense primer corresponded to the LDLR exon 11–12 junction, whereas the antisense primer corresponded to exon 12 per se (150 bp PCR product). The second reaction identified the proportion of LDLR mRNA lacking exon 12, i.e. a sense primer corresponding to exon 10 was used in conjunction with an antisense primer specific to the sequence at the junction of exons 11 and 13. This latter reaction generated a 170 bp product. This assay revealed that rs688T allele is associated with a significantly lower proportion of exon 12-containing LDLR mRNA in males, but not females (Fig. 2A and B). Interestingly, these results in the aged brain are the mirror image of our prior findings regarding human liver, where rs688T is associated with lower exon 12 splicing efficiency in pre-menopausal women but not in age-matched males (5); we are unclear as to the mechanisms underlying these sex differences in SNP-associated splicing in young adult liver versus aged adult brain. In the brain, the apparent difference in the proportion of exon 12-deficient LDLR between rs688T/T and rs688C/C males was 8%. Since exon 12 deletion causes a frameshift and a premature stop codon, these LDLR isoforms are predicted to encode truncated, non-functional LDLR proteins (5). In summary, rs688 associates with LDLR exon 12 splicing efficiency in males, representing a sex-dependent decrease in the proportion of LDLR mRNA encoding functional LDLR.


Figure 2
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  		Figure 2. Rs688 is associated with LDLR splicing efficiency in the male human brain in vivo. This figure depicts representative splicing patterns corresponding to the indicated PCR products as well as quantitation of exon 12 splicing efficiency in the brains of males (A) and females (B). Rs688 was associated significantly with splicing in males (P = 0.041) but not females (P = 0.43) as determined by Jonckheere–Terpstra ranked sum tests.

 
Since LDLR is a primary apoE receptor in the brain (6,7), we next investigated in three AD case–control series whether rs688 associates with AD; these series were derived from clinically diagnosed subjects at the Mayo Clinic Jacksonville, FL (JS) and the Religious Orders Studies (ROS), as well as clinically diagnosed subjects at the Mayo Clinic Rochester, MN, which were combined with a series of AD autopsies (RS-AUT) (13–15). We genotyped these series and performed analysis of the association of AD with rs688 (recessive model) using logistic regression adjusted for series, presence of apoE4, age and sex. We also considered interaction terms for rs688 with series, and apoE4, finding that there was strong evidence of an interaction of rs688 with sex (P = 0.001), but no evidence of heterogeneity in rs688 effects across series (P = 0.46) or of interactions with age (P = 0.93) or the presence of apoE4 (P = 0.45). Separate logistic regression models were then used to analyze male and female data further. In males, there was strong evidence of increased risk for AD with rs688T/T (OR = 1.49, P = 0.005, Fig. 3). However, in females, the estimated OR was 0.79 (P = 0.056), consistent with the sex-dependent splicing in the brain. There was no significant difference in ORs among the three series for either males or females, but the RS-AUT and ROS populations displayed a more robust association in males than the JS series. The statistical significance of this finding in the male series overall was maintained even after correction for multiple testing, i.e. conservative Bonferroni correction for testing allelic, recessive and dominant models would adjust the overall P-value to 0.015. Hence, the rs688T allele that is associated with decreased splicing efficiency in males is, when homozygous, also associated with increased risk for AD in males. Since the level of significance in females was marginal, especially after any correction for multiple testing, we interpret the results in females as suggestive evidence that rs688T may be associated with reduced AD risk in females. In summary, rs688T/T is associated with increased risk for AD in males, but not in females.


Figure 3
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  		Figure 3. Rs688 is associated with AD in males. This Forest plot depicts the OR and 95% CI for the association of rs688 with AD in each series, separately for males and females. The bar width reflects the 95% CI whereas the symbol size reflects the relative size of the series. The open diamonds represent the overall OR combining all three series with the diamond width corresponding to 95% CI. The sample sizes for each genotype for males were: rs688C/C (476), rs688C/T (704) and rs688T/T (277) whereas those for females were rs688C/C (656), rs688C/T (1021) and rs688T/T (278).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
The primary findings of this report are that rs688 is a functional SNP within neural cells, associates with LDLR exon 12 splicing efficiency significantly in the male brain, and associates with significantly increased risk for AD in males. Hence, the overall significance of this study is 2-fold. First, these results support the hypothesis that a functional SNP in LDLR, a brain apoE receptor, is associated with AD. Secondly, the unusual sex-dependent association of rs688 with AD may be relevant to other genetic variants, suggesting that AD genetic studies may benefit by analyzing males and females separately.

Given that rs688 is a common SNP within an excellent AD candidate gene, several prior studies have evaluated the association between AD and rs688 or linked SNPs (16–20). When these studies were undertaken, the investigators were not aware of the sex dependence of rs688 function and therefore did not parse their series by sex. Importantly, when we analyzed rs688 in males and females together by logistic regression adjusting for series, the presence of apoE4 and sex, the lack of association in females obscured the association, i.e. the overall OR for rs688 in the recessive model was 1.04 (95% CI, 0.86–1.25; P = 0.69). Hence, stratifying for sex was critical to detect the association described here. The use of a recessive model contributed modestly to the detection of the association, because even an allelic dosage model showed that while AD risk for rs688C/T male individuals was equivalent to that of rs688C/C males (OR = 1.00; 95% CI, 0.78–1.27), rs688T/T males had an increased AD risk (OR = 1.49; 95% CI, 1.09–2.04; P = 0.013). In summary, additional case–control series must be analyzed to confirm the association of rs688T/T with an increased risk for AD in male, but not female subjects. The correlation between this association and functional alteration in splicing, which also occurs in male but not female subjects, strongly suggests, however, that replication is likely in confirmatory series of sufficient size.

Although apoE and LDLR SNPs associate with both cholesterol and AD, we do not interpret our results as supporting a positive correlation between peripheral cholesterol and AD risk generally. Both apoE4 and rs688T associate with increased LDL–cholesterol and increased AD risk, but these associations have clear differences in their sex dependence. The presence of an apoE4 allele in females is associated with a 5 mg/dl increase in LDL–cholesterol (21) and an increased AD risk with an OR of 5.15 (95% CI of 4.23–6.27, data from this study). Males are similar in that apoE4 is associated with a 3 mg/dl increase in LDL–cholesterol (21) and a 3.81 AD OR (95% CI, 3.05–4.75). In contrast, rs688T is associated with a 6 mg/dl increase in LDL–cholesterol in females (5) but an AD OR of 0.79 (95% CI, 0.62–1.01). Moreover, the rs688T allele in males was associated with an insignificant trend towards decreased cholesterol (5) but had an OR of 1.49 (95% CI, 1.13–1.97) for AD risk. Thus, the effects of apoE4 and rs688T on peripheral cholesterol do not correlate with their effects on AD risk. For this reason, we interpret our data as suggesting that rs688 modulates LDLR function with respect to cholesterol in the periphery and possibly with apoE metabolism in the CNS. In the former, the relationship between LDLR and cholesterol has been well substantiated; individuals with impaired LDLR function have less LDL–cholesterol removed from the extracellular space, resulting in increased intracellular cholesterol synthesis, further exacerbating plasma cholesterol levels [reviewed in (10)]. Hence, LDLR in the periphery is intimately linked with cholesterol homeostasis per se. In contrast, LDLR deficiency in the brain does not alter brain cholesterol, at least as modeled by the LDLR-deficient murine brain (6). Rather, LDLR deficiency increases murine brain apoE levels, as well as the levels of apoE3 and apoE4 in mice expressing these human alleles (6,7). Since increased apoE expression associates with increased Aß pathology in the mouse (22), the most parsimonious interpretation of our data in males is a model wherein rs688T decreases exon 12 splicing efficiency, leading to reduced functional LDLR and reduced apoE clearance, and thereby, increased amyloid pathology and AD risk. This model is supported by several additional lines of reasoning. First, the rs688T allele increases the proportion of LDLR that lacks exon 12, which shifts the LDLR reading frame, leading to a premature stop codon in exon 13 and an LDLR isoform lacking the transmembrane domain encoded by exons 16–17 (5); consistent with this possibility, a similarly truncated LDLR produced by a nonsense mutation in exon 13 is retained within the endoplasmic reticulum and causes familial hypercholesterolemia (23). Second, LDLR deficiency in murine models of amyloidogenesis may exacerbate amyloid accumulation and memory deficits (6,7). Third, the association between rs688 and increased AD risk was consistent with a recessive model; we interpret this result as suggesting a threshold effect, i.e. LDLR expression must be reduced below a critical threshold for AD risk to be increased. Hence, while a partial reduction in LDLR because of a single rs688T allele is insufficient to increase the AD risk, a homozygous rs688T/T genotype is sufficient to increase the risk, consistent with the rs688T allele acting in a loss-of-function fashion. In summary, the parsimonious interpretation of these results is a recessive model wherein LDLR protein encoded by the exon 12-deficient LDLR isoform is not functional, representing a loss of apoE receptor, reduced apoE clearance and increased risk for AD in males.

We are unclear as to the mechanism(s) restricting rs688 effects on splicing to male brain tissue and female liver tissue (5). This restriction may be more quantitative than qualitative as close inspection of the opposite sex data, i.e. splicing efficiency in female brain tissue and male liver tissue reveals similar, albeit very modest trends. We have identified two related, estrogen-centric hypotheses that may account for these sex-dependent rs688 effects. First, since (i) rs688T is predicted to neutralize an SRp40-binding site (5), (ii) SRp40 may be estrogen regulated (24) and (iii) estrogen levels in the aged male are higher than those in the aged female because of the action of aromatase upon testosterone (25), we speculate that at least in the brain, estrogen-modulated SRp40 may account for rs688 sex-dependent actions. Our second model proposes that since estrogen acts upon the estrogen-response element in the LDLR promoter to enhance LDLR expression (26), estrogen may also modulate the LDLR splicing by concurrent recruiting of splicing factors such as CAPERalpha, as has been reported for estrogen actions on the transcription and splicing of other genes (27,28). To address these possibilities, we have experiments underway evaluating estrogen actions and ectopic SR protein expression in minigene-transfected cells. We note that transfected cells have consistently demonstrated a role for rs688 in modulating splicing, regardless of the ‘sex’ of the cell line, e.g. the neuroblastoma SH-SY5Y cells were derived from a female individual but showed a male brain splicing pattern. This may again reflect apparent rs688 actions in a permissive, estrogenic environment because the culture medium includes the phytoestrogen phenol red as well as estrogen-containing fetal calf serum. In summary, we hypothesize that rs688 association with splicing in vivo in a sex-dependent fashion reflects estrogen actions on SRp40 activity and/or splicing factor recruitment concurrent with transcription.

In conclusion, rs688 is a common and functional LDLR SNP that modulates LDLR exon splicing efficiency in vitro in neural cells and in vivo in the male brain. Moreover, rs688 associates significantly with increased AD risk in males. Since LDLR expression in the CNS could be manipulated, e.g. by statins that penetrate the blood-brain barrier, these results suggest that LDLR modulation may represent a therapeutic target in at-risk populations. Overall, we anticipate that these studies may prove useful for understanding the role of sex and apoE receptors in AD. Moreover, these studies provide a model for identifying AD susceptibility alleles by focusing on well-characterized functional SNPs in genes likely to influence risk for AD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCES
 
Evaluation of LDLR minigene splicing in vitro
The role of rs688 in LDLR exon 12 splicing efficiency in neural cells was evaluated by transfecting SH-SY5Y cells with LDLR minigenes consisting of LDLR exons 9–14 along with their intervening introns. These minigenes were cloned from rs688C/C and rs688T/T individuals into a pcDNA3.1 backbone (5). The specific effects of rs688 were evaluated by comparing splicing efficiency in clones wherein site-directed mutagenesis (Quik-Change, Stratagene) was used to specifically mutate rs688C to rs688T, and vice versa. LDLR splicing efficiency was evaluated by transfecting the clones into SH-SY5Y cells by using Nucleofection as directed by the manufacturer (Amaxa, Inc., Gaithersberg, MD, USA). Twenty-four hours after transfection, total RNA was isolated and analyzed for LDLR splicing patterns by RT–PCR as described previously (5). Briefly, RNA was converted to cDNA (SuperScript II, Invitrogen) and sequences corresponding to LDLR minigene splice products were PCR amplified (Platinum Taq, Invitrogen) by using a sense primer corresponding to vector sequence at the beginning of the transcription product (5'-ACTAGTCCAGTGTGGTGGAATTGCC-3') and an antisense primer corresponding to sequence within LDLR exon 14 (5'-CATCGTGGTGGATCCTGTTC). PCR profiles consisted of pre-incubation at 94°C for 60 s, followed by cycles of 94°C for 30 s, 60°C for 45 s, and 72°C for 90 s (Perkin Elmer 9600). The minimal number of PCR cycles necessary to discern products were performed, i.e. 30 cycles. PCR products were separated by polyacrylamide gel electrophoresis (PAGE) and visualized by SYBR-gold fluorescence on a fluorescence imager (Fuji FLA-2000). PCR product identities were determined by gel purification and direct sequencing (Davis Sequencing). The amount of full length and inefficiently spliced LDLR isoforms were quantified by fluorescence intensity. For each sample, fluorescence values were corrected for background and normalized for length differences among amplicons. Sample splicing efficiency was then quantified as the amount of LDLR PCR product containing exons 10, 11, 12, 13 and 14 divided by the total LDLR PCR product for that sample. Statistical significance of the results was analyzed by Kruskal–Wallis non-parametric tests.

Evaluation of LDLR splicing in vivo
Human anterior cingulate brain samples were generously provided by the Sanders-Brown Alzheimers Disease Center Neuropathology Core. The samples were from deceased individuals with an average age at death for females of 82 ± 8 years (mean ± SD, n = 20) and for males of 81 ± 8 (n = 21). The average postmortem interval (PMI) for females was 3.0 ± 0.8 h (mean ± SD, n = 20) whereas for males it was 3.0 ± 0.9 h (n = 21). Available samples included four rs688C/C, ten rs688C/T, and seven rs688T/T males, as well as seven rs688C/C, seven rs688C/T, and six rs688T/T females. Total RNA was prepared and converted to cDNA in 1 µg aliquots with random hexamers and reverse transcriptase (Invitrogen, SuperScriptII) as we described previously (5,29,30). LDLR mRNAs containing exon 12 were identified by subjecting 1/60th of the cDNA to PCR with a sense primer corresponding to the LDLR exon 11–12 junction (5'-AATGGCATCACCCTAGATCTC) and an antisense primer that recognized exon 12 (5'-GGTGGGCCAGCCTCTTTTCATC). LDLR isoforms lacking exon 12 were identified by using a sense primer that recognized exon 10 (5'-CATCGTGGTGGATCCTGTTC) and an antisense primer specific to the LDLR exon 11–13 junction (5'-CAAAATACTTTGTCCTAGGGTGA). PCR products directed by these two pairs of primers were amplified separately under conditions of 94°C for 4 min followed by 31 cycles of 94°C for 30 s; 60°C for 30 s and 72°C for 30 s. After amplification, the PCR products from each individual were pooled, separated by PAGE, and visualized and quantified by SYBR-gold fluorescence as described above. Male and female tissues were processed simultaneously, and splicing efficiency data obtained without regard to sex of the donor. For statistical analyses of splicing efficiency, data from males and females were separated and analyzed by two-tailed, non-parametric tests for three ordered groups, i.e. Jonckheere-Terpstra tests (SPSS).

Genetics studies
Three different series (JS, RS-AUT and ROS) were analyzed. The JS series was collected through the Mayo Clinic Jacksonville memory disorders clinic (13). This study included 215 AD males (mean age ± SD, 79 ± 8 years) and 227 non-AD males (age, 78 ± 7 years) as well as 362 females with AD (age, 78 ± 6 years) and 339 non-AD females (age, 78 ± 8 years). The RS series was collected through the prospective, community-based Mayo Clinic AD registry, as well as the Mayo Clinic Rochester memory disorders clinic (13). For this cohort, we obtained data for 188 males with AD (age at diagnosis: 81 ± 8 years) and 519 non-AD males (age, 78 ± 6 years), as well as 330 females with AD (age, 81 ± 8 years) and 607 non-AD females (age, 79 ± 6 years). The AD cases in the AUT series, which were combined with the RS series, were obtained from brains with neuropathologically confirmed AD that were collected at autopsy from Caucasians in Jacksonville, FL, Rochester, MN and many additional sites, and included 235 males (age at death, 81 ± 6 years) as well as 337 females (age, 85 ± 7 years) The ROS series represents the deceased members of the larger, prospective, community-based ROS series and have been described elsewhere (20). For this project, we used DNA from 37 males with AD (age, 86 ± 7 years), and 36 non-AD males (age, 80 ± 7 years), as well as 47 females with AD (age, 89 ± 6 years), and 33 non-AD females (age, 83 ± 6). Clinical AD diagnoses on all series were made with NINCDS-ADRDA criteria (31). Genomic DNA was extracted from peripheral blood leukocytes or autopsied tissue by using routine methods. Samples were genotyped for rs688 and/or rs5925, a surrogate which is in near perfect linkage disequilibrium with rs688, by using unlabeled PCR primers and TaqMan MGB probes (FAM and VIC dye labeled) obtained via the ‘Assays-by-Design’ service from Applied Biosystems (Foster City, CA, USA) on an ABI-7000 or ABI-7900 (Applied Biosystems). The association of AD with rs688 was assessed by using logistic regression as adjusted for series, age, sex and presence of apoE4. Models were fit separately for males and females after strong evidence of an interaction of sex with the effect of rs688 was found in a single model. Tests of interaction of rs688 by series, apoE4 and age were also conducted to evaluate whether there was evidence of heterogeneity across series. For apoE and LDLR, the genotype frequencies in males and females from each series were consistent with Hardy Weinberg equilibrium.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCES
 
NIH: R01AG026147 (SE), P30AG10161, R01AG15819 (DAB) and AG16574 (RCP, SGY), and P30AG028383 (University of Kentucky Alzheimers Disease Center).


   ACKNOWLEDGEMENTS
 
The authors acknowledge the University of Kentucky Alzheimers Disease Center which supplied tissue for study.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger}The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 

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