Association Of Cholesterol Metabolism Genes With Alzheimer’s Disease In Chinese Han Population And Its Possible Mechanisms

Alzheimer’s disease (AD) is the most common form of senile dementia. It clinically manifests as progressive and irreversible memory loss and cognitive decline, and the overall prevalence increases exponentially with age. The etiology of AD is very complex, and it has been approved that genetic variants may play an important role in the pathogenesis of this disorder. However, the only well-replicated locus for inherited susceptibility to LOAD is the apolipoprotein E (APOE) gene, thus leading to a continuous search for additional susceptibility factors for the LOAD. Recently, two independent large-scale genome-wide association studies (GWAS) identified3novel SNPs associated with LOAD:including rs11136000within CLU on chromosome8p21-p12, rs6656401within CR1on chromosome1q32and rs3851179within PICALM on chromosome11. The studies did not specifically comment on ABCA1but it is difficult to overlook the fact that in one of those, ABCA1rsl2686004was associated at p=5.30E-0.5and OR-1.207. Overall, the representation of identified genes involved in cholesterol metabolism in those two studies is impressive, lending further credibility to the results of the studies suggesting a role for cholesterol metabolism genes in AD pathogenesis.In the present study, we assessed the association between SAD and the four putative AD risk genes that emerged from previous GWAS analyses. And also did some work to elucidate the possible mechanisms.Section1:Association of CLU, CR1, PICALM and ABCA1with Alzheimer’s disease in Chinese Han populationObjective:To study the association of CLU,CR1,PICALM and ABCA1with SAD in Chinese Han population, and to explore whether there was an interaction between known SAD risk factors including age, gender and ApoE s4allele.Methods:We performed a case-control study to collect clinical data and peripheral blood samples. Genomic DNA was isolated from blood samples using the QIAGEN kit. Genotypes were determined using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) assay or direct sequencing. The APOE genotypes were determined as described previously. For each SNP a test for deviation from Hardy-Weinberg equilibrium was performed. Differences in genotype or allele frequencies between cases and controls were analyzed by chi-square or by Fisher’s exact test. Logistic regression models were used to consider such variables as age, gender and APOE ε4dose. Differences in allelic and/or genotypic frequency were considered statistically significant when the p-value was less than0.05.Results:A total1065subjects were recruited for this study, including474SAD patients and591controls. The general data of the participants are compared between the two groups. No statistically significant differences were observed for age and sex (p>0.05). The MMSE score was significant lower in SAD patients than in controls (p <0.0001). The APOE s4allele frequency was also significantly different between SAD patients and control subjects (p <0.0001).All the SNPs studied were in Hardy-Weinberg equilibrium in both the AD and control groups.We found a significant association between the R219K within ABCA1and SAD, with more RK in total AD group (P=0.014), LOAD group (P=0.005), female AD group (P=0.017) and APOE non-ε4ε4group (P=0.008). Logistic regression manifested the risk of AD increased in RK carriers in total AD group (P=0.015, OR=1.536, CI=1.087-2.170), LOAD group (P=0.005, OR=1.921, CI=1.213-3.041), female AD group (P=0.007, OR=1.836, CI=1.179-2.859) and APOE non-ε4ε4group (P=0.013, OR=1.576, CI=1.103-2.252). K allele (RK+KK) also increased the risk of AD compared with RR allele in LOAD group (P=0.029, OR=1.619, CI=1.050-2.497) and female AD group (P=0.021, OR=1.641, CI=1.078-2.498). No discrepancy was found in V825I within ABCAl.(These results were published on American Journal of Geriatric Psychiatry, please see attachment for details)However, we did not observe a difference in the frequencies of the4SNPs within CLU,CR1and PICALM between the SAD and control groups (rsl1136000: P=0.711; rs9331888:P=0.210; rs6656401:P=0.482; rs3851179:P=0.890). In addition, there is no association when stratified by age, gender and APOE ε4allele.(These results were published on Journal of Alzheimer Disease, please see attachment for details)Conclusions:Our results indicated that the RK genotype or K allele (RK+KK) of R219K within ABCA1may increase the risk of SAD in Chinese Han population, and the CLU, CR1,PICALM polymorphisms may not modify the risk of SAD in Chinese Han population.Section2Possible mechanisms underlying the association between ABCA1R219K polymorphism and SADObjective:To compare ABCA1mRNA expression level and plasma lipid levels between AD and control group and the effects of R219K genotypes on the peripheral blood mRNA expression level and plasma lipid levels, and thus to explore the possible mechanisms underlying the association between ABCA1R219K polymorphism and SAD.Methods:We performed a case-control study to collect clinical data and peripheral blood samples. Genomic RNA was extracted using the conventional phenol-chloroform methods. Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was conducted to detect ABCA1mRNA expression in peripheral blood. The independent samples T-test was used to compare mRNA expression and plasma lipid levels.Results:A total67subjects were recruited for this study, including35SAD patients and32controls.The ABCA1mRNA level in AD group was significantly lower than in control group. The mean2-ΔΔCt in AD group was0.029526, while in controls, the mean2-ΔΔCt was0.22345, nearly ten times of the AD group. The difference between the two groups was statistically significant (P=0.022). In addition, the R219allele has a significant impact on mRNA expression level. The mRNA in control group was about ten times higher then in AD group (AD0.034359, Control0.295301, P=0.039).We did not observe a significant difference of the plasma lipid levels between AD group and controls. However, when stratified by R219K genotypes, the triglyceride (TG) level was significant different between the two groups in RK carriers (P=0.003). In addition, the high-density lipoprotein (HDL) level was also higher in AD group among RK carriers (AD group:1.46mmol/1, control group:1.10mmol/1), but the difference did not reach the statistical criteria (P=0.071).Conclusion:The ABCA1R219K polymorphism may reduce ABCA1expression, further contribute to lipid metabolism disorder, and thus alter the risk of SAD.