The Role Of Regulatory Genetic Variation And Gene Expression In Alzheimer 's Disease (AD)

Part I:Development of a PCR and next-generation DNA sequencing-based assay for measuring allele-specific mRNA expression of Alzheimer’s disease candidate genes in human brain and novel mathematical framework for identifying potential cis-acting eQTL’s and regulatory variantsCommon genetic variants that regulate mRNA expression are suspected to contribute to complex disorders, including Alzheimer’s disease (AD), where overproduction of toxic beta-amyloid peptide has been shown to increase the risk of developing Alzheimer’s disease. Single-nucleotide polymorphisms (SNPs) linked to AD in genome-wide association studies (GWASs) have been found to be located primarily in introns or non-coding regions in the neighborhood of genes, rather than protein coding regions, consistent with a role for gene regulation in this disorder. Although high-throughput microarray-based assays have been widely used to measure differences in mRNA expression in independent samples, they usually lack the sensitivity to detect rare mRNAs and accuracy to detect small, genotype-dependent changes in mRNA expression against the typically large background variation in mRNA express due to non-genetic factors.In this section, I describe a novel and efficient polymerase-chain reaction (PCR) and next-generation sequencing (NGS)-based assay to analyze allele-specific mRNA expression of AD candidate genes in human brains, using "marker" SNPs within the mRNAs to distinguish alleles. Using this assay, my colleagues and I succeeded in analyzing allelic expression imbalance (AEI) of mRNA expression for 21 candidate AD genes in 52 independent human brain samples. Among these genes 20/21 (95%) showed AEI ratios greater than 1+0.2 in at least one sample and only 1/21 (5%) showed no AEI. These results demonstrate that subtle allele-specific differences in mRNA expression for candidate AD genes in human brain are common, and open the possibility that genetically determined differences in expression of these gene, singly or in combination, contribute to AD liability.In addition to observing common variation in mRNA expression, we also noticed that arranging log2-transformed AEI ratios obtained for independent brain sample from most-negative to most-positive values revealed highly distinct distributions of log2AEI ratios for each gene/marker SNP combination. Based on these observations, we developed a mathematical framework for analyzing these distributions, which showed that log2AEI distributions can provide important information about the number, location and contribution of potential cis-acting regulatory variants to mRNA expression. We expanded these mathematical models to include eQTLs, thereby creating the first unified framework for analyzing allele-specific and total mRNA expression.In conclusion, we have developed a highly sensitive and accurate method for quantifying differences in allele-specific mRNA expressed in human brain and novel mathematical framework for analyzing these differences. Our assay allowed us to measure different mRNA expression for many AD candidate genes that might have been entirely missed in previous studies in human brains and identify the candidate genetic variants that directly cause, or SNPs that accurately predict, difference in mRNA. Alleles of SNPs that accurately predict high-or low-mRNA expression should be also useful markers in AD association studies to identifying individual genes and gene combinations that contibute to AD liability.Part II:Identification of a common regulatory variant in the promoter region of PION that contributes to Alzheimer’s disease liability independently of APOE4Alzheimer’s disease (AD) is a common and devastating neurodegenerative disorder, currently affecting an estimated 36 million individuals worldwide (Alzheimer’s Disease International:World Alzheimer Report 2009) and bringing untold suffering to patients and their families. As a result of aging of the current population, as many as 115 million individuals worldwide are predicted to develop AD by the year 2050 (World Alzheimer Report 2009). Currently, there is no cure for AD or known methods for its prevention. For this reason, much hope has been placed on elucidating the genetic factors that contribute to AD liability as a first step towards developing new strategies for treating and preventing this disorder.There is a growing consensus among researchers that genetic variants that regulate gene expression play crucial roles in complex disorders including AD. Based on the observations that overproduction of toxic beta-amyloid peptide (A-beta) has been demonstrated to increase the risk of AD diagnosis and many independent proteins directly or indirectly affect the generation of this peptide, we hypothesize that genetically determined differences in expression of the genes encoding the proteins related to A-beta production contribute to the risk of developing AD. Among these genes, we decided to focus on PION, a gene of unknown function that encodes the precursor of gamma-secretase activating peptide (gSAP), which increases the production of toxic A-beta by stimulating the cleavage of amyloid-beta protein precursor (APP) by the protease gamma-secretase.In this section, I describe the use of:1) quantitative PCR-based assays in combination with whole genome genotype data to demonstrate common genetically controlled variation of PION mRNA expression in human brain and 2) AD case-control association studies to identify PION as a novel AD liability gene in both Han Chinese and Caucasian populations. Specifically, we demonstrated that expression of PION mRNA in human brain is regulated by a promoter-region genetic variant that is highly linked with the SNP rs4727380 and that rs4727380 minor G-allele is a low-expression allele and major C-allele a high expression allele. Using rs4727380 as a genetic marker, we carried out a Han Chinese-based association study with 397 AD cases and 474 controls. This study provided the first evidence linking PION to AD risk, with the high-expression rs4727380 allele identified as the liability allele. This result is consistent with our hypothesis that high-expression of PION increases the formation of A-beta and thereby increases AD liability. In addition, we further showed that the rs4727380 liability allele is recessive and that association of this SNP with AD diagnosis is strongest in apolipoprotein, type E, allele 4 (APOE4) non-carriers and in AD cases with age-at-onset (AAO) above age 65. These results imply that PION contributes to AD liability independently of the APOE4 allele, the genetic factor with the largest known impact on AD liability. The results also suggest that the contribution of PION to AD takes many years to become apparent, with the strongest effects seen in late-onset Alzheimer’s disease (LOAD). We obtained similar results or trends using rs4727380 as a genetic marker in a Caucasian population-based AD association study, implying that PION contributes to AD liability in both Han Chinese and Caucasian populations.In conclusion, we found that PION mRNA expression is variable in human brain and identified a promoter region SNP that is highly correlated with this expression. These results allowed us to identify PION as a novel candidate AD susceptible gene, and provide additional evidence that regulatory genetic variants contribute to the risk of developing AD.