Tau-tubulin Kinase-1 Polymorphisms And Risk Of Late-onset Alzheimer's Disease

Research Background: Alzheimer's disease(AD)is the most common form of dementia in the elderly,with a prevalence of over 60 million worldwide.With the gradual aging of China's population and the gradual increase in the average life expectancy of the human body,the number of AD patients in China is increasing year by year,while patients with advanced AD due to increased cognitive impairment and often associated with various types of mental and behavioral abnormalities,In the future it will be a serious burden on the patient's family,society and the entire public health system.The two main clinical features of AD are progressive memory impairment and progressive impairment of cognitive function.AD mostly occult onset,early can not easily be detected.Early clinical manifestations of AD,often only mild behavioral abnormalities,cognitive function and intelligence levels are often no significant changes,and early imaging of AD patients due to lack of basis,just by its non-specific clinical manifestations is difficult make the right diagnosis.Therefore,the early detection of AD,early diagnosis and treatment is particularly important.So far,the pathogenesis of AD and pathogenesis are not very clear and are considered to be caused by the accumulation of senile plaques or amyloid plaques and neuro-fibrillary tangles.Senile plaques resulting from ?-amyloid(A?)deposition and neurofibrillary tangles(NFTs)derived from aberrant tau protein aggregation.AD can be divided into early-onset familial Alzheimer'sdisease(EOAD)and late-onset Alzheimer's disease(LOAD).The LOAD is more common and its onset age is later than 65 years old,accounting for more than 90% of the total number of AD patients.LOAD is a polygenic disease,and 79% of its risk is attributable to genetic factors.Previous gene studies have identified a number of AD-related mutations,such as CLU,CRI,PICALM,and NEDD9,but these genes are still controversial.Apolipoprotein E(Apo E)gene is associated with the pathogenesis of AD,and Apo E?4 allele is a risk factor for AD.Apo E?4 allele is associated with AD pathogenesis and was found in a dose-dependent manner.Apo E?4 allele is also a genetic risk factor of late-onset Alzheimer's disease,but not all individuals with Apo E?4 allele develop AD,only 20.29% of the late-onset Alzheimer's disease can be attributed to a mutation in Apo E?4.At present,the pathogenesis of AD has not been fully elucidated,and the lack of effective means of early diagnosis and prevention.Therefore,we need to find more possible candidate genes,and provide more important basis for the diagnosis and treatment of AD in the future.After tau protein is found to constitute the main component of AD neurofibrillary tangles,tau protein has become the current research focus.The normal physiological function of tau protein is regulated by phosphorylation.Under physiological condition,the tau protein phosphorylation level of nerve cells is lower.During the development of AD,abnormal tau hyperphosphorylation,resulting in abnormal aggregation,and the final deposition in neurons to form neuronal fiber tangles,the lesion formation and evolution is a long-term development of chronic,progressive process.Tau-tubuline kinase 1(TTBK1)is a recently discovered brain-specific protein kinase involved in tau phosphorylation at AD-related sites.Recently,a study examined the contribution of TTBK1 to the susceptibility for AD,by analyzing SNPs in this gene in a large group of AD patients and controls.They proved that rs2651206 within the TTBK1 gene have association with AD susceptibility in Spanish.TTBK1 gene may participate in the onset process of LOAD.However,the association needs to be confirmed by further replication studies,particularly in other ethnic cohorts.In order to test whether TTBK1 is also the candidate gene of LOAD in Chinese and to define the correlation of TTBK1 with LOAD in north Chinese Han population,we conducted the initial studies of TTBK1 gene polymorphism in north Chinese Han population.Objective: This experiment is to observe the ApoE gene distribution in an ethnically homogeneous Han Chinese population,understand its differences with other ethnic groups,evaluate whether the Apo E gene might influence the risk of LOAD.At the same time,we evaluated whether the two polymorphisms(rs2651206 and rs7764257)of the TTBK1 gene might influence the risk of LOAD in an ethnically homogeneous Han Chinese population.Methods: In this case–control study,400 sporadic LOAD patients and 388 healthy subjects free from any neurodegenerative disorders were recruited for the study.All the above subjects were of Northern Han Chinese in origin.The patients were recruited from the Department of Neurology at Qingdao Municipal Hospital,and several other hospitals in Shandong Province.A clinical diagnosis of probable AD fulfilled the criteria of theNational Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's disease and Related Disorders Association.The control groups were confirmed healthy and neurologically normal by medical history,general examinations,laboratory examinations,and Mini Mental State Examination(MMSE)score?28.Subjects with significant illness were excluded from our study.Clinical characteristics of AD and control subjects were detected and insure no significant association was found.All the subjects were classified according to Apo E gene.DNA was extracted from blood samples.Apo E genotype polymorphism was determined by multiplex allele-specific polymerase chain reaction(Multi-ARMS).Understand the distribution of Apo E gene in the Han Chinese population and the difference with other ethnic groups.After stratified by gender,we also analyze the Apo E genotype and allele frequency in the LOAD group and the control group.We also analysis whether Apo E?4 allele has influence in different gender.The polymorphisms at positions rs2651206 and rs7764257 within TTBK1 were genotyped using allele specific-polymerase chain reaction-restriction fragment length polymorphism(ASP-PER-RFLP)technique and matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI–TOF-MS)technique.We then stratified by the Apo E?4 alleles for further analysis.Data were analyzed using a commercially available statistical package(SPSS version 11.5).Differences in study population between two groups were examined using the Student t-test.Hardy-Weinberg equilibrium was assessed using the?2-test.Genotype and allele distributions were compared using the ?2-test.Differences in allele and genotype distribution between casesand controls were analyzed using logistic regression adjusted for age,gender,and APOE?4 status.Haplotypic frequencies were estimated using SHEsis software.The criterion for significant difference was P < 0.05.Result:1.There was no statistically significant difference in the gender(P = 0.27)and age distribution(P = 0.37)of AD and control subjects.There was no statistically significant difference in body mass index,systolic blood pressure,diastolic blood pressure,fasting blood glucose,total cholesterol,triglyceride,high density lipoprotein and low density lipoprotein between the two groups(P> 0.05).The genotype of the two groups was consistent with Hardy-Weinberg equilibrium.2.The distributions of Apo E allele ?2,?3 and ?4 in LOAD group and normal control group were significantly different(P <0.001),the distribution of Apo E gene polymorphism and its correlation with LOAD showed that there were significant differences.The frequency of ?4 allele in LOAD group was significantly higher than that in control group(P <0.001).There was no significant difference between the LOAD group and the control group.The Apo E?4 allele carriers had a higher risk of LOAD than those of the Apo E?4(-)group(P <0.001).3.After stratification by sex,the frequency of Apo E genotype and allele frequency in LOAD group and control group were still significantly different(P <0.001).The?4 allele was significantly different between LOAD group and control group.The frequency of distribution in the two subgroups was significantly higher than that in the control group(P<0.001).There was no significant difference in ?2 between LOAD subgroup and control group.4.The effects of ?4 on gender were analyzed in the LOAD group.LOAD group of male group of 168 cases,of which 84 cases of ?4(+),84 cases of ?4(-),LOAD group of232 cases of women,including ?4(+)111 cases,?4(-)121 cases,There was no significant difference between the two groups(P = 0.670).5.No significant differences were detected for the TTBK1 rs7764257 polymorphism between AD patients and controls(genotype P = 0.138,allele P = 0.054).When these data were stratified by the APOE?4 status,no significant association was found in subjects with APOE?4 allele(P = 0.069),however,significant association was found in subjects without APOE?4 allele(P = 0.026).6.The genotype and allelic frequencies of TTBK1 rs2651206 polymorphism were significantly different between LOAD group and control group,the P value between different genotypes was 0.031,and the difference between different alleles P value was0.011.The genotype and allele frequencies of rs2651206 were not significantly different between the AD group and the control group(P = 0.738,P = 0.765)in the Apo E?4(+)group and the Apo E?4 allele.There were significant differences in genotype frequency and allele frequency of rs2651206 between Apo E?4(-)group and the two genotypes.The difference between the two genotypes was 0.001,and the difference between different alleles was 0.001.7.Multivariate logistic regression analysis revealed that rs2651206 polymorphismwas still strongly associated with LOAD after adjusted for age,gender,and the APOE?4status(OR = 0.72,95%CI,0.52–0.99,P = 0.05),T(TT+TC)was 0.72 times higher than that in non-carriers of rs2651206 alleles T(CC).For rs7764257,there was no significant difference between the(GG+GA)genotypes and the AA genotype(P = 0.37).There was no significant correlation between rs7764257 polymorphism and LOAD.8.The haplotypes of TTBK1 single nucleotide polymorphisms were analyzed by the maximum expectation algorithm(EM),and the results showed that there were significant differences between LOAD and control haplotypes.Compared with other haplotypes,T/G haplotype and LOAD were significantly correlated,T/G haplotype can reduce the risk of LOAD.Conclusions:1.The results of this study showed that the polymorphism of Apo E gene was associated with the risk of LOAD in Han Chinese population.2.The results showed that Apo E?4 allele could increase the risk of LOAD in Han Chinese population.3.The results of this study show that TTBK1 gene polymorphism is associated with the risk of LOAD in Han Chinese population.4.The present study showed that TTBK1 gene SNP rs2651206 allele T and haplotype TG could significantly reduce the risk of LOAD in Han Chinese population.There was no significant correlation between the polymorphism of rs7764257 and LOAD.