AD Etiology Associated Cholesterol-regulating Gene Variants And Involved Pathogenic Mechanisms

Background:Alzheimer s disease(AD)is the most common type of dementia among the oldest,which is featured with aging-related progressive cognitive impairment and memory loss.As a complex neurodegenerative disease with polygenic background,the pathogenetic mechanisms of AD remains largely elusive.Cholesterol metabolism homeostasis has been demonstrated to participate in AD etiology and variants of cholesterol metabolism regulating genes have been reported to modify AD risk,in which mechanisms involved warrant intensive exploration.Systemic and extensive studies at genomic and transcriptomic level separately help to explore on AD pathology as well as biomarkers,which would have potential clinical application.Objective:1)To screen and validate new genetic variations of cholesterol homeostasis-regulating genes,and to validate,in our AD samples,candidate AD risk-related genetic variants reported in Western countries while has not been reported in Chinese AD patients.2)To establish in vitro cholesterol-regulating gene models,with which to study on the potential of cholesterol metabolism homeostasis-related genes on promoting cholesterol metabolism homeostasis and involved mechanisms,thus to explore preliminarily on the pathogenetic mechanisms of AD.3)To analyze AD-specific differential expression profile.Methods:1)We performed the targeted-sequencing of 12 nuclear receptor genes plus APOE,which were involved in cholesterol metabolism homeostasis modulation,in 854 AD patients,1059 mild cognitive impairment due to AD(MCI-AD)patients and 1254 cognitively normal elderly controls to screen new AD susceptibility genetic variants of nuclear receptors encoding genes,and to explore preliminarily on the latent pathogenetic mechanism.2)We genotyped and analyzed subsequently 93 AD risk-related genetic variants,which had been reported in Western countries while not been reported in Chinese AD patients between 2015-2017 period,in 183 AD patients,255 MCI-AD patients and 1544 controls.3)We conducted APOC1 gene overexpression and knock-down by use of RNAi,established MRC-5 cell models with decreased APOC1 expression and APOC1 overexpression,respectively,and tested classic cell proliferation and apoptosis related indicators like apoptosis body,Cytochrome c,cleaved Caspase 3 and Caspase 8,after treatment of H2O2.We also established HepG2 cell models with APOC1 expression derceased and overexpressed,respectively,and tested mitochondrial function related indicators like Complex I and mitochondrial transmembrane potential,after high glucose level stimulation.4)We conducted strand-specific RNA-seq of blood RNA samples belonging to 29 AD patients,12 MCI-AD patients and 4 controls from Guangxi district,together with bioinformatic analyses,to analyze preliminarily AD specific blood transcriptome expression profile.Results:1)We discovered a new low-frequency variant,ESR1 rs9340803 at 6q25.1(MAF:0.38%),which s both AD and MCI-AD risk related[OR(95%CI):3.20(1.84?422),p<0.001;OR(95%CI):3.08(1.75-3.89),p<0.001].This variant also correlated with disease phenotypes(p<0.05).Preliminary functional study on it revealed decreased the transcription regulating activity of ESR1 in vitro.2)We discovered,for the first time,that CLU rs117389184,a low-frequency SNP(MAF:0.5%),would increase the risk of Chinese AD as well as MCI-AD patients and associated with cognitive impairment[OR(95%CI):3.10(1.14?3.30),p=0.02].This variant s predicted to interfere with the binding of transcription factor and thus affect CLU gene transcription.3)We established cell models with APOC1 overexpression and knock-down.Preliminary results indicated that abnormal expression of APOC1 would effect on mitochondrial function and cell apoptosis.4)We also discovered,for the first time,622 differentially expressed genes(272 upregulated,350 downregulated)and 889 differentially expressed long non-coding RNAs(IncRNAs;341 upregulated,548 downregulated)in AD and MCI-AD patients from Guangxi district by comparing with those of controls,within which immune response and multiple factors related to senile diseases were involved.Conclusions:1)Cholesterol homeostasis-regulating genes variants would impact on both AD and MCI-AD disease occurrence risk.At the gene level,we discovered,for the first time,that ESR1 rs9340803 A>G and CLU rs1 17389184 C>T could increase AD and MCI-AD risk.These two variants would lead to decreased gene transcription and expression,separately.2)Cholesterol homeostasis-regulating gene APOC1 would have effect on cell apoptosis and mitochondrial dysfunction,which would result from disordered cholesterol metabolism.Results of our study would offer evidence for explorations targeting potential clinical application.3)At the transcriptome level,we also discovered,for the first time,that AD and MCI-AD patients from Guangxi district featured differential expression profile of blood.Multiple factors including abnormal lipids metabolism related disease/phenotypes and neurodegenerative diseases related risk factors.