Recent large-scale LOAD GWAS have identified SNPs in ABCA7
, and PICALM
. To determine if gene expression is altered in AD, mRNA levels for each gene were measured by real-time PCR in the parietal lobe of AD case and age-matched, cognitively normal, control brains. All gene expression values were normalized to GAPDH
, a housekeeping gene that accounts for total cell number. Because AD brains are characterized by neuronal loss, reactive gliosis, and microglial activation, we also corrected gene expression levels for specific subpopulations of cells (neurons [MAP2
], microglia [AIF1
], and astrocytes [GFAP
]) to determine if there were cell specific effects on gene expression. ABCA7
expression was associated with CDR (p
0.0304), where higher expression levels are correlated with elevated CDR (). CDR scores increase with cognitive and functional decline 
. This association remained significant after correcting for subpopulations of cells (). After correcting expression for neuronal number, BIN1
expression was associated with age at onset (p
0.0407) and disease duration (p
0.0407), where higher expression levels are correlated with later age at onset and shorter disease duration (). The expression of the neuronal isoform of BIN1
n) was also associated with disease duration after correcting for total, neuronal, and microglial cell populations (). Correcting expression levels for neuronal and microglial cell populations produced significant associations between disease status and CDR with CD33 and CR1 expression (). Correcting CLU
expression levels for neuronal number resulted in the association of CLU
expression with disease status after correcting for neuronal cell populations (p
0.0159) (). CLU
is alternatively spliced into two isoforms 
isoforms containing exon 5 (CLU1
) produced similar association patterns after correcting for neuronal and microglia cell populations (). Additionally, MS4A6A
expression levels were weakly associated with Braak tangle and Braak plaque scores (p
0.0564 and p
0.0559, respectively), where higher expression levels are correlated with higher Braak scores (). Higher Braak scores are indicative of more extensive tau and amyloid pathology in the brain 
. The association between MS4A6A
expression and Braak tangle and Braak plaque scores was slightly stronger after correcting for neuronal expression (p
0.0437 and 0.0215, respectively; ). Accounting for microglia number revealed an association between MS4A6A
expression and CDR (p
0.0311) and Braak tangle score (p
0.0453). BIN1, CD2AP, EPHA1
, and PICALM
expression levels, however, were not associated with AD status or AD pathology (). Together, we demonstrate that in the absence of strong statistical associations between gene expression and clinical/neuropathological AD outcomes, accounting for subpopulations of cells reveals additional gene expression effects that are likely related to gene function and/or AD-specific cell loss.
Gene expression is associated with AD pathology.
The top LOAD risk genes fall into three functional categories: immune response (CLU, CR1, ABCA7, MS4A, CD33, and EPHA1), cholesterol metabolism (CLU and ABCA7), and synaptic function (PICALM, BIN1, CD33, CD2AP, and EPHA1). We used the expression data for these genes to test whether expression levels of genes in a similar functional class are correlated. Expression of CD33 and MS4A6A, both of which function in immune response, were highly correlated (). Furthermore, expression of CD33 and MS4A6A were highly correlated with AIF1 expression, a marker for microglia, the immune cell of the brain (). Expression of genes related to synaptic function, BIN1, BIN1n, CD2AP, and PICALM, were highly correlated (). BIN1 and PICALM expression were also highly correlated with GFAP expression, an astrocytic marker (). ABCA7 expression, involved in immune response and cholesterol metabolism, was highly correlated with BIN1 and CD2AP expression, which are involved in synaptic function (). Together, these results demonstrate that genes that fall into the same functional category are related at the RNA level. Thus, their dysfunction may be linked in AD.
Expression of genes involved in immune response and synaptic function are highly correlated in brain tissue.
To determine if the LOAD GWAS SNPs influence gene expression, we analyzed the association of SNP genotype with gene expression using an ANCOVA and testing for association with an additive model, the model utilized when originally reporting association between these SNPs and risk for AD 
. We failed to detect an association between GWAS SNPs and cis-acting expression quantitative trait loci (eQTL) after correcting for the total cell population () or specific cell types (Table S3
AD GWAS SNPs do not modify gene expression in the parietal lobe of human brains.
LOAD GWAS SNPs were identified based on their association with disease status. To determine if these SNPs contribute to AD pathology, independent of gene expression, we analyzed the association of each SNP with clinical (disease status, age at onset, disease duration, and CDR) and neuropathological (Braak tangle and Braak plaque score) measures of AD. The minor allele of rs3764650 in ABCA7
was associated with a later age at onset and shorter disease course (p
0.0040, respectively; ; ). The minor allele of rs670139 in MS4A6E
was associated with Braak tangle and Braak plaque score (p
0.0581, respectively; ). We failed to detect an association between the remaining GWAS SNPs and the clinical/neuropathological measures of AD ().
Gene SNPs do not significantly influence AD brain pathology.
Rs3764650 in ABCA7 is associated with age at onset.
To replicate our findings, we analyzed a publically available AD dataset 
, in which RNA was measured by the Illumina Human Refseq-8 Expression Bead Chip System. Of the nine genes analyzed in our cohort, only five survived quality control measures in the replication dataset: ABCA7, BIN1, CLU, MS4A6A
, and PICALM
. We analyzed residual expression levels for association with disease status. MS4A6A
expression levels were significantly associated with disease status (p
0.0346 and p
0.0334, respectively), where MS4A6A
expression was up regulated in the AD brains compared with controls (). BIN1
expression levels were marginally associated with disease status (p
0.0540), where expression was also up regulated in AD brains compared with controls ().
CLU and MS4A6A expression are associated with AD status in a replication dataset.