Association between LRP1 C766T polymorphism and Alzheimer’s disease susceptibility: a meta-analysis

Low density lipoprotein receptor-related protein 1 (LRP1) C766T polymorphism (rs1799986) has been extensively investigated for Alzheimer’s disease (AD) susceptibility. However, results in different studies have been contradictory. Therefore, we conducted a meta-analysis containing 6455 AD cases and 6304 controls from 26 independent case–control studies to determine whether there was an association between the LRP1 C766T polymorphism and AD susceptibility. The combined analysis showed that there was no significant association between LRP1 C766T polymorphism and AD susceptibility (TT + CT versus CC: OR = 0.920, 95% CI = 0.817–1.037, P = 0.172). In subgroup analysis, significant decreased AD susceptibility was found among Asian population in allele model (T versus C: OR = 0.786, 95% CI = 0.635–0.974, P = 0.028) and dominant model (TT + CT versus CC: OR = 0.800, 95% CI = 0.647–0.990, P = 0.040). Moreover, T allele of LRP1 C766T was statistically associated with late onset of AD (LOAD) (T versus C: OR = 0.858, 95% CI = 0.748–0.985, P = 0.029; TT + CT versus CC: OR = 0.871, 95% CI = 0.763–0.994, P = 0.040). In conclusion, our meta-analysis suggested that LRP1 C766T polymorphism was associated with lower risk of AD in Asian, and could reduce LOAD risk especially. Considering some limitations of our meta-analysis, further large-scale studies should be done to reach a more comprehensive understanding.

and subsequently degraded by endopeptidase 12 . Therefore, LRP1 are involved in the bulk transport, primary production, brain and systemic clearance of AD toxin Aβ, and thus plays a critical role in AD pathogenesis.
The silent C766T polymorphism in exon 3 of LRP1 gene (rs1799986) has attracted extensive attention since first reported as a risk factor for AD 13 . However, results in different studies have been contradictory. The inconsistency is likely to relate with insufficient statistical power, racial differences or other demographic variables. Therefore, we conducted a comprehensive meta-analysis to determine whether there was an association between the LRP1 C766T polymorphism and AD susceptibility.

Results
Eligible studies. A total of 167 relevant studies were identified from initial database searching, of which 35 publications were included based on titles and abstracts (Fig. 1). Furthermore, 4 reviews, 1 duplicated publication and 3 studies with inadequate information were excluded after careful reading of the full text. Besides, manual search of references revealed 3 more articles. After primary data extracted from the 30 independent studies, 4 studies were excluded for genotype distribution of controls was not in Hardy-Weinberg equilibrium (HWE) [14][15][16][17] . Finally, 26 eligible studies containing 6455 AD cases and 6304 controls were included in our meta-analysis. The characteristics of the 26 studies on LRP1 C766T polymorphism and AD susceptibility was summarized in Table 1. The ethnicities of these subjects involved in the comparisons were diverse, including Caucasian (n = 16), Asian (n = 6), African (n = 1) and mixed (n = 3). Besides, LRP1 C766T genotype and allele distribution among AD cases and controls was summarized in Table 2, and the control group in all studies was in HWE.
In subgroup analysis by ethnicity, T allele of LRP1 C766T was found to be associated with decreased AD susceptibility among Asian population (T versus C: OR = 0.786, 95% CI = 0.635-0.974, P = 0.028; TT + CT versus CC: OR = 0.800, 95% CI = 0.647-0.990, P = 0.040) (Fig. 3). However, we did not observe any association for all comparisons in Caucasians. When stratified by time of AD onset, we found T allele of LRP1 C766T may act as a protective factor for late onset of AD (LOAD) (T versus C: OR = 0.858, 95% CI = 0.748-0.985, P = 0.029; TT + CT versus CC: OR = 0.871, 95% CI = 0.763-0.994, P = 0.040) (Fig. 4), but no significant association was observed for early onset of AD (EOAD). Furthermore, no significant interaction was observed for APOE ε4 status (P > 0.05). The results of univariate and multivariate meta-regression analyses showed that age, MMSE and/or APOE ε4 were not potential factor(s) for heterogeneity among those studies, but gender might contributed to the heterogeneity (as shown in Table 4).
Publication bias. Begg's test and Egger's test were performed to evaluate the publication bias of the included studies. The shape of Begg's funnel plot appeared to be approximately symmetrical (Fig. 5). Besides, statistical significance was also not observed according to Egger's test (P > 0.05, Table 3). In general, there was no publication bias in our included studies.

Discussion
AD, as a continuum, bring about serious threat to human health. Considering early detection and intervention at the asymptomatic stage may offer better chance of therapeutic success, it is urgent to identify early diagnostic biomarkers 18,19 . LRP1, a member of the LDL receptor family, is an endocytic receptor for more than 40 structurally diverse ligands. The findings of previous studies indicate that LRP1 and many of its ligands (eg. APOE and A2M) are co-deposited with Aβ in senile plaques in AD brains 20,21 . Subsequent studies demonstrated that LRP1 modulates the clearance of Aβ via receptor-mediated pathway in central nervous system [22][23][24] . Besides, soluble LRP1 provides an endogenous peripheral 'sink' activity for Aβ by preventing plasma free Aβ access to the brain 25 . It has also been reported that LRP1 is responsible for a rapid peripheral uptake of Aβ by the liver, which plays a key role in systemic clearance of Aβ 26 . On the other hand, endocytosis of LRP1 could modulate APP trafficking, and contribute to Aβ generation 27 . Interestingly, LRP1 can regulate Aβ metabolism in two contrary sides. The association between LRP1 polymorphisms and AD susceptibility also has been described extensively, especially exon 3 C766T polymorphism. Kang et al. first reported the LRP1 C766T polymorphism, and found a positive association between C allele and AD susceptibility 13 . This finding was replicated in some following studies [28][29][30] , but Kolsch et al. found the opposite result that carriers of a C allele were at lower risk of AD 31 , while some failed to show any association between LRP1 C766T polymorphism and AD [32][33][34][35][36][37][38][39][40][41][42][43][44][45] . Previously, three meta-analysis have tried to clarify the relationship between LRP1 C766T polymorphism and AD susceptibility, which one revealed a weak correlation of LRP1 CC genotype with AD 40 , but other two separately studies showed that no positive evidence was involved in the relationship between this polymorphism and AD risk among overall 36 and Chinese population 46 . Since several factors could be responsible for these discrepancies, such as inadequate sample size, variability in phenotype definition and allele frequency polymorphisms in different ethnic backgrounds 47 , we conducted a comprehensive meta-analysis with different genetic models in this study, to better clarify the association between LRP1 C766T polymorphism and AD susceptibility.
New results from our research did not show any association of LRP1 C766T polymorphism with AD susceptibility from 6455 AD cases and 6304 controls in overall population. This result is consistent with two published meta-analyses 36,46 . Compared with the results from previous studies, our data from meta-analysis was relatively reliable to illustrate the association between LRP1 C766T polymorphism and AD susceptibility, because we used different genetic models with a larger number of case-controls.
Due to that people in different ethnic populations may have different allele frequency, and can affect the heterogeneity, we additionally conducted subgroup analysis by ethnicity, time of AD onset and APOE ε4 status. The outcomes by subgroups revealed that T allele of LRP1 C766T could reduce the risk of AD in allele model (T versus C) and dominant model (TT + CT versus CC) among Asian population, no significant role was found in Caucasian group. In terms of onset age, the results from subgroup analysis showed that T allele of LRP1 C766T could act as a protective factor for late onset of AD, but no significant association with early onset of AD. This is also consistent with previous report 13 . It's recognized that APOE ε4 is an important pathogenic factor for the development of AD. Several studies have revealed a possible protective effect of TT genotypes in carriers of APOE ε4 alleles 48,49 . However, APOE ε4 status did not show that the influence of the association between LRP1 C766T polymorphism and AD susceptibility in our study. Moreover, our meta-regression analysis also showed that APOE ε4 status, age, and MMSE were not responsible for heterogeneity.  Table 3. Meta-analysis of LRP1 C766T polymorphism and AD susceptibility. OR: odds ratio; CI: Confidence interval; EOAD: early onset of AD; LOAD: late onset of AD. a Number of comparisons.
LRP1 C766T polymorphism is a silent mutation, which does not change the amino acid sequence or splice site. Therefore, it is unlikely to alter the biological function by a direct causal effect with the polymorphism. Some studies consider that the LRP1 C766T polymorphism might be responsible for susceptibility to AD by interact with other genes, such as APOE [48][49][50][51] , MAPT 52 , and MAPK8IP1 53 . In addition, some speculated that LRP1 C766T may be in linkage disequilibrium with a deleterious mutation in the LRP1 gene, or with other biologically relevant mutation on neighbouring genes, which affected LRP1 expression 44,50 . Besides, several studies have a hypothesis that the LRP1 C766T polymorphism might alter the secondary structure of the LRP mRNA to affect the translation and stability of the protein 13,48 . To date, the conclusion with LRP1 C766T polymorphism with AD susceptibility is conflicting, further genetic analyses of this locus are needed to illuminate the potential mechanism and the functional interactions with AD.
Some limitations of our meta-analysis should be acknowledged. The sample size in some subgroup analysis was small, which may increase the risk of false negatives or false positives. Besides, we did not perform subgroup analysis based on other factors participated in the progression of AD, such as educational background, due to a lack of sufficient information. Larger and broader independent investigations are required to better understand the role of LRP1 C766T polymorphism in AD pathogenesis.
In conclusion, our meta-analysis suggested that LRP1 C766T polymorphism was associated with lower risk of AD in Asian, and could reduce LOAD risk especially. Furthermore, large-scale studies should be performed to reach more understanding of this association.

Materials and Methods
Search strategy. We searched electronic databases PubMed, Embase and CNKI (up to August 2016) using the following keywords: ("Alzheimer's disease" or "Alzheimer disease" or "AD") and ("low density lipoprotein receptor-related protein 1" or "LDL receptor-related protein 1" or "LRP1") and ("polymorphism" or "SNP" or "variant" or "genotype") without language restriction. The bibliographies of the retrieved studies were also screened to identify relevant publications.
Inclusion and exclusion criteria. The eligible studies had to meet all the following criteria: (1) a casecontrol study to evaluate the association between LRP1 C766T polymorphism and risk of AD; (2) useful data including sample size, allele or genotype distribution were given; (3) genotype distribution of controls followed the HWE. Accordingly, the exclusion criteria were as follows: (1) reviews, meta-analysis or editorial articles; (2) studies were provided with inadequate information; (3) for the studies with overlapping data, only the most relevant articles with the largest dataset were included in the final analysis.
The literature retrieval and inclusion were carried out in duplication by two independent reviewers.  Data extraction. Two reviewers independently extracted the following information: first author, year of publication, country, ethnicity, total number of cases and controls, mean age of cases and controls, proportion of female in cases and controls, AD diagnosis criteria, genotyping method, source of controls, time of AD onset, genotype or/and allele distribution in cases and controls. If conflicting results produced, two reviewers would review the publications again and reached a consensus by discussion.
Quality assessment. Two reviewers independently assessed the quality of each included studies in the meta-analysis according to the criteria of quality assessment (as referred in the Reference of 54,55 ), and the disagreements were judged by the third reviewer to ensure a consistent outcome. Quality scores of studies ranged from 0 (the lowest) to 15 (the highest). Studies with quality scores among 10 to 15 were grouped into high quality studies and other studies scored between 0 and 9 were categorized into low quality studies.
Statistical analysis. HWE in controls was tested by a chi-square test. Summary odds ratio (OR) with confidence interval (95% CI) for genotypes and alleles were used to evaluate the strength of association between LRP1 C766T polymorphism and AD susceptibility. The significance of the pooled OR was measured using the Z-test. Four genetic models were performed in our meta-analysis: allele model (T versus C), codominant model [homozygote comparison (TT versus CC) and heterozygote comparison (CT versus CC)], dominant model (TT + CT versus CC), and recessive model (TT versus CC + CT). The heterogeneity was also quantified with I 2 statistics. If no significant heterogeneity was found between the studies, the pooled OR was calculated by using the fixed effects model (the Mantel-Haenszel method) 56 . Otherwise, the random effects model (the DerSimonian and Laird method) was applied 57 . Both of univariate and multivariate meta-regression analyses were also carried out to explore potential sources of heterogeneity among studies. The log of the ORs from involved studies was using as dependent variables, and age, gender, Mini-Mental State Exam (MMSE) and/or APOE ε4 status as covariates. Publication bias was tested by Begg's test and Egger's test 58,59 . We also performed subgroup analysis according to ethnicity, time of AD onset and APOE ε4 status, respectively. Statistical analyses were conducted with Stata Version 11.0 (College Station, TX, USA), and a two-sided P < 0.05 was considered statistically significant.  Table 4. The potential sources of heterogeneity between LRP1 polymorphism and AD risk were evaluated by both of univariate and multivariate meta-regression analyses. SE = standard error; 95%CI = 95% confidence interval.