Screening Of Alzheimer’s Disease Associated Genes In The Southern Chinese Population And Its Possible Mechanisms

Alzheimer’s disease (AD) is the most common form of senior dementia, leading to irreversible decline in both cognition and behavior. To date, the etiology of AD remains obscure, embracing both genetic and environmental risk factors. It has been approved that genetics variants many play an crucial role in the pathogenesis of this disorder, AD has a strong genetic basis with heritability estimates up 58%-79%. According to the previous researches, the genetic factors are certain to contribute to the risk. The mutations in amyloid precursor protein (APP), presenilinl (PSEN1) and presenilin2 (PSEN2) genes have been confirmed to cause the familial autosome dominant inherited AD (FAD), which accounts for less than 1% of all AD patients. The vast majority of AD patients are sporadic AD (SAD). But the possession of this allele isn’t sufficient for the development of AD. Therefore, people have been explored other AD susceptibility genes. In recent years, large genome-wide association studies (GWAS) have identified a number of AD susceptibility loci.These results were emerged from Caucasian population but rare in Chinese populations. Therefore, We aimed to evaluate the association of these loci with AD in Chinese population and explore possible mechanisms.Section 1:Screening of Alzheimer’s disease associated genes in the southern Chinese populationObjective:Recently, large genome-wide association studies (GWAS) have identified a number of AD susceptibility loci. We sought to evaluate the association of these loci with AD in the southern Chinese population, and to study whether there was an interaction between SAD risk factors embracing age, gender, and ApoEε4 allele.Methods:we performed a case-control study to collect clinical date and peripheral blood. Genomic DNA was extracted from peripheral blood using TIANamp blood DNA Kit (TIANGEN), and we evaluated 95 SNPs in 75 genes, most of which were reported to be associated with AD risk in populations of Caucasians. Using Mass ARRAY iPLEX system and Illumina Human Omni Express Bead Chips. Statistical analysis was carried out by Plink 1.07 and SPSS version 14.0. In general information between patients and controls, quantitative values and qualitative values were performed by student’s t test and chi-square test respectively.The Hardy-Weinberg equilibrium (HWE) of the SNPs in both patients and controls was carried out using chi-square test. Genotypes and allele frequencies were compared by chi-square analysis or Fisher’s exact test. The logistic regression analysis was used to examine the association between AD and the polymorphisms as well as the odds radios (OR) and 95% confidence interval (CI) with the covariance of age at onset, gender and ApoEε4 carrying status. The criterion for significant difference is set at p<0.05.Results:A total 849 subjects were recruited for the study, including 422 SAD cases and 427 controls. After a quality control analysis, each of the 89 SNPs was tested for its association with AD in 393 patients and 383 controls. General information of SAD patients and controls were compared. No significant difference was seen in either age or gender between the two groups. The MMSE score was prominently lower in SAD patients than that in control groups. The ApoEε4 allele frequency was statistical higher in SAD patients. Only six SNPs (BIN1 rs7561528; NPC1 rs1788799; CUGBP2 rs2242451; PVRL2 rs6859; TOMM40 rs2075650 and TOMM40 rsl57581)were significantly associated with AD risk.1. The association between BIN1 rs7561528 polymorphism and AD. Our data showed that the allele and the genotype frequency of rs7561528 were between AD patients and controls in total subject (allele p=0.004 OR=0.622 95%CI=0.451-0.858, genotype p=0.017). When stratified by age of onset, gender and, ApoEε4 non-carriers status, the allele and the genotype frequency were significant different in ApoEε4 non-carriers status (allele p=0.003 OR=0.400,95%CI=0.216-0.742, genotype: p=0.020), the difference was still significant in subgroups in EOAD and female group (EOAD:p=0.018 OR=0.622 95%CI=0.419-0.925, female:p=0.035 OR=0.641 95%CI=0.423-0.971). logistic regression manifested that A allele is a protective factor of AD risk in total subject, this effect was also found EOAD(p=0.040 OR=0.494 95%CI=0.252-0.969).2. The association between NPCl rs1788799 polymorphism and AD. Our data showed that the distribution of NPCl rs1789799 in allele frequency (allele p=0.027 OR=0.644 95%CI=0.435-0.954) was significantly different in between AD patients and controls in total subject. After stratification on age of onset, gender and ApoEε4 carrying status, the difference were not found between AD and controls. The further logistic regression revealed that G allele is a protective factor of AD risk in total subjects (p=0.022 OR=0.603 95%CI=0.392-0.792), this effect was also found male (p=0.04 OR=0.488 95%CI=0.246-0.968), after adjusted for on age of onset, gender and ApoEε4 carrying status.(These results were published on American Journal of Psychiatry Gente, please see attachment for details).3. The association between CUGBP2 rs2242451 polymorphism and AD. Our data showed that the distribution of CUGBP2 rs2242451 in allele frequency was significantly different in between AD patients and controls in total subject (p=0.030 OR=0.739 95%CI=0.562-0.972). After stratification on age of onset, gender and ApoEe4 carrying status, In allele frequency was the difference in female (p=0.029 OR=0.663 95%CI=0.458-0.959). logistic regression revealed that A allele is a protective factor of AD risk in ApoEε4 non-carriers, LOAD and male (ApoEε4 non-carriers p=0.004 OR=0.981, LOAD:p=0.019 OR=0.609, male:p=0.029 OR=0.557), after adjusted for on age of onset, gender and ApoEε4 carrying status.4. The other three SNPs were in PVRL2 (rs6859) and TOMM40 (rs2075650 and rs157581), near the ApoE gene. Each of these three SNPs was in strong LD with ApoE.Conclusions:1. BIN1 rs7561528 polymorphism and AD:the minor A allele frequency of the SNP might be a protective factor for SAD in the southern Chinese population.2. NPC1 rs1788799 polymorphism and AD:the minor G allele frequency of the SNP might be a protective factor for SAD in the southern Chinese population.3. CUGBP2 rs2242451 polymorphism and AD:The minor A allele frequency of the SNP might be a protective factor for SAD in the southern Chinese population.4. Most of AD susceptibility loci from GWAS in Caucasians are not replicated with southern Chinese population AD.Section 2:Possible mechanisms underlying the association between BIN1、NPC1 and SADObjective:To compare BIN1 and NPC1 mRNA expression level between AD and control group, and thus to explore the possible mechanisms underlying the association between BIN1 and NPC1 polymorphism and SAD.Methods:We performed a case-control study to collect clinical data and peripheral blood monocyte cells (PBMCs) samples. Genomic RNA was isolated using the conventional phenol-chloroform methods. Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was conducted to detect BIN] and NPC1 mRNA expression in peripheral blood. The independent samples T-test was used to compare mRNA expression levels.Results:A total 104 subjects were recruited for this study, including 48 SAD patients and 56 controls. The BIN1 mRNA level in AD group was significantly lower than in control group. The mean 2-△△Ct in AD group was (0.02857), while in controls, the mean 2-△△Ct was (0.22345). The difference between the two groups was statistically significant (p=0.031). However, there were no statistically significant in different genotypes. We did not observe a significant difference of the NPC1 mRNA expressive level between AD group and in control group and different genotypes.Conclusion:BIN1 mRNA expressive levels was a significant lower between AD patients and controls, and thus protective protective factor of SAD.