INPP5D Rs35349669 Polymorphism With Late-onset Alzheimer’s Disease In Northern Han Chinese: A Replication Study And Meta-analysis

Objective: Previous studies have reported that INPP5 D was implicated in the pathogenesis of LOAD through the regulation of microglial cell function.A recent genome-wide association study discovered a new locus single nucleotide polymorphism(SNP,rs35349669)of INPP5 D which was significantly associated with susceptibility to late-onset Alzheimer‘s disease(LOAD)in Caucasians.Since variants and their frequencies of INPP5 D may vary from diverse ethnic groups,replication is necessary to validate the potential impacts of INPP5 D in non-Caucasian population including Asians.To address this question,the present study was conducted to evaluate the association of rs35349669 SNP with LOAD in Han Chinese population.Methods:We investigated 984 sporadic LOAD patients(mean age at onset: 75.15 ± 6.08 years;578women)and 1352 healthy control subjects(mean age at examination: 75.50 ± 6.49 years;744 women)matched for sex and age.The entire population were unrelated Northern Han Chinese derived from Shandong Province.The diagnosis of probable AD met with the criteria of the NINCDS–ADRDA.No family history of neurodegenerative disorders or other dementias were reported among AD patients.The control subjects wererecognized to be healthy and neurologically normal on the basis of medical history,general examinations,laboratorial examinations and acquired ≥28 points on the Mini-Mental State Examination(MMSE).Informed consent was obtained from all individuals or from the subject‘s guardian,and the protocol of this study was approved by the Ethical Committee of Qingdao Municipal Hospital.Genomic DNA was extracted from peripheral blood leukocytes of AD patients and healthy individuals using the Wizard genomic DNA purification kit(Cat.#A1125,Promega,USA).Genotyping of INPP5D(rs35349669)and APOE polymorphisms was performed by a custom-by-design 48-Plex SNP scan TM Kit.This was high-throughput and cost-saving SNP genotyping methods based on double ligation and multiplex fluorescence PCR.The genotyping of APOE were carried out using the improved multiplex ligase detection reaction(i MLDR)method.Data analysis was accomplished using Gene Mapper Software v4.0(Applied Biosystems).Randomly selected DNA samples from each genotype were analyzed in duplicate using ligation detection reaction and sequence analysis method.Consistent results were achieved by these two methods.We accomplished statistical analysis with SPSS16.0 software.Genotype and allele frequencies were calculated by counting.The Hardy–Weinberg equilibrium(HWE)was tested for each SNP with genotype data from both AD patients and controls.Differences in the characteristics between the two groups were tested using Student‘s t-test or the chi-square test.Differences between cases and controls after stratification for APOEε4 status were also examined by the chi-square test.The association of rs35349669 with LOAD risk was further analyzed using logistic regression adjusting for age,gender,and APOEε4 status under various genetic models that were defined as 1(TT + TC)versus 0(CC)for dominant,1(TT)versus 0(TC + CC)for recessive,and 0(CC)versus 1(TC)versus 2(TT)for additive.The P value,odds ratios(ORs)and 95% confidence intervals(CIs)were calculated.Estimation of the statistical power was performed with the STPLAN 4.3 software.Values of P < 0.05 were deemed to have statistically significance.Additionally,we also combined our data with the results from other studies about rs35349669 and LOAD risk through fixed-effects inverse variance-weighted methods.Meanwhile,we generated I2 estimates with evaluate the possible effect of study heterogeneity on the results.Stata V.12.0 was used to perform all these analyses.Results: The current evidence did not support the correlation between rs35349669 and LOAD in Chinese.The genotype and allele frequencies did not differ between LOAD and controls(genotype P = 0.167,allele P = 0.094).We observed no significant differences between AD and controls in genotype and allele distributions(P > 0.05)when the total group was stratified in by APOE ε4 carriers and APOE ε4 noncarriers.Likewise,the total group was also stratified into male and female subgroup by gender,the frequency of genotypes and allele for rs35349669 did not show any obvious differences between AD and controls in male or female subgroup(P > 0.05).Excluding blending effects in our initial association analyses,we reassessed rs35349669 effects under various genetic models in logistic regression adjusting for age at onset in LOAD patients(age at examination in control group),gender,and APOEε4 status,the result also failed to reveal any significant difference between LOAD and controls.A meta-analysis about the association of rs35349669 with LOAD revealed that rs35349669 was strongly associated with LOAD(OR = 1.08,95%CI = 1.05-1.11)without evident analysis heterogeneity(I2 = 16.7%).In subgroup analysis,the rs35349669 polymorphism was also significantly associated with the risk for LOAD in Caucasian(OR = 1.06,95%CI = 1.01-1.10),however,the results showed that there was no association of rs35349669 with AD risk in Chinese population(OR = 0.77,95%CI = 0.53-1.13).Conclusion:Our study has replicated in Northern Han Chinese about the association between the rs35349669 SNP on INPP5 D and LOAD risk.However,we failed to find significant differences in the genotypic or allelic distributions of rs35349669 polymorphism between LOAD cases and healthy controls in a Han Chinese population,even after stratification for APOEε4 status and statistical adjustment for age,gender and APOEε 4status.INPP5Drs35349669 polymorphism was identified to be strongly associated with the development of LOAD(P < 0.05)in two-stage meta-analysis of GWAS in 74,046 Caucasians.Subsequently,Ruizet al.confirmed the significant association in Spanish,however,our study and Xiao et al.failed to replicate the association in Caucasian population.Although our study failed to find any association between the examined SNP with LOAD,we could not rule out the possibility that other SNPs of INPP5 D associated with LOAD.Additionally,with renewed genetic sequencing about INPP5 D in the near future,it may be helpful to find new loci that related to LOAD.