Polymorphisms in theSortilin-Related Receptor 1Gene Are Associated with Cognitive Impairment in Filipinos

Background/Aims. Sortilin-related receptor 1 (SORL1) is involved in the neuronal transport processes and plays a role in the formation of amyloid plaques. This study investigated the association of 6 SORL1 single nucleotide polymorphisms (SNPs 8, 9, 10, 13, 19, and 23) with cognitive impairment (CI) in Filipinos. Methods. DNA samples from 484 subjects (100 Alzheimer’s Disease (AD) cases, 109 mild cognitive impairment (MCI) cases, 18 other types of CI, and 257 no dementia controls (NDC)) were genotyped using TaqMan SNP Genotyping Assays. Data Analysis. Our study showed strong linkage disequilibrium in the SNPs 8, 9, and 10 block. Our results showed that CI was significantly associated with SNPs 13 and 23. None of the SORL1 SNPs studied was associated with AD while SNPs 8, 9, 10, and 23 were associated with MCI. Conclusion. The findings had provided evidence that SORL1 may predispose individuals to CI. Further studies are needed to clarify the role of SORL1 in Filipinos with AD.


Introduction
Sortilin-related receptor 1 (SORL1) is genetically linked to Alzheimer's disease (AD) [1][2][3][4][5][6][7]. Recent evidence indicates that it acts as a regulatory gatekeeper for determining the ultimate destination of amyloid precursor protein (APP) [8]. APP processing generates the -amyloid (A ) peptides, which are deposited as the amyloid plaques in brains of individuals with AD [9]. AD was associated with the "C, " "G, " and "C" alleles at single nucleotide polymorphisms (SNP) 8, 9, and 10, in the 5 -end of SORL1, respectively and the "G" and "T" alleles at SNPs 19 and 23 in the 3 -end of SORL1, respectively [2]. Using frozen brain tissue from autopsy-confirmed AD cases, Lee et al. reported that some AD patients displayed low level of SORL1 [1]. These data suggest that inherited or acquired changes in SORL1 expression or function are mechanistically involved in causing AD.
Mild cognitive impairment (MCI) refers to individuals who exhibit cognitive deficit but not dementia [10]. It has an incidence rate of 9.9/1000 person-years and an annual conversion rate of 10% to 12% to AD, in contrast to a conversion rate of 1% to 2% in the normal elderly population [11]. Sager et al. reported that MCI subjects with low SORL1 expression levels were significantly more cognitively impaired than subjects with high levels of expressed SORL1 [10].
Currently, there has been no published data on the genetic variation of SORL1 in the Filipino population. In this study, we sought to investigate the role of SORL1 (SNPs 8, 9, 10, 13, 19, and 23) in Filipinos with cognitive impairment (CI), MCI, and AD as well as its association with sex, age, and its interaction to Apolipoprotein E-4 (APOE-4).  from each subject or from reliable collateral informants who are familiar with the subject's cognitive state.

Methodology
The group who evaluated the subjects consisted of a neurologist, psychologists, and a nurse. Every datagathering session began with briefing to include an intake interview for the demographic data and medical history using the Philippine Neurological Association (PNA) 10item Questionnaire for Common Neurological Diseases. Clinical dementia rating (CDR), assessment of the presence or absence of AD based on the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) Alzheimer's criteria, Montreal Cognitive Assessment (MOCA), Mini-Mental State Exam (MMSE-P), Disability Assessment for Dementia (DAD), Neuropsychiatric Inventory (NPI), and Geriatric Depression Scale (GDS) were individually administered to all the subjects.

Data Analysis.
For each SNP, goodness-of-fit to Hardy-Weinberg equilibrium (HWE) was assessed using the Chi-Square test ( 2 ). Haploview ver. 4.2 software developed by the Broad Institute was used to estimate the degree of linkage disequilibrium (LD), to detect single marker association, and to determine presence of haplotype blocks [12]. The default settings were used in these analyses, which create 95% confidence bounds on D to delineate SNP pairs in strong LD. SPSS ver. 16.0 software was used for the association analysis. Differences between the genotype and allele frequencies between the CI cases and no dementia controls (NDC) in Filipinos were determined by 2 test. Univariate logistic regression was performed to determine the association of the SNPs with the CI cases. The association of the SORL1 SNPs with the CI cases was indicated in odds ratio (OR) with the corresponding 95% confidence intervals. Stratified analyses were performed to determine if associations differed between sex, age, and Apolipoprotein E-4 (APOE-4) groups. All tests were two-tailed and considered significant at < 0.05.

SORL1
Genotypes. The allele and genotype frequencies of the SORL1 SNPs (Table 1) observed in this study population were in HWE, except for SNP 13 ( 2 = 7.2127) which showed a significantly higher frequency for the G allele. When the 2 of SNP 13 was computed for the NDC only, the result was in HWE ( 2 = 0.4084).
The alleles C, G, G, G, G, and A alleles of SNPs 8, 9, 10, 13, 19, and 23, respectively, were detected as the risk alleles for the cognitively impaired population ( Table 2). Haplotype block 1 showed a strong LD in SNPs 8, 9, and 10 ( Figure 2). This block formed a three-locus haplotype, C-G-G, and T-A-A. However, none of the haplotypes were associated with CI, MCI, and AD (data not shown).

Genetic Association Analysis.
Individual SNPs were analyzed in the CI, MCI, and AD cases (Table 3). SNP 13 ( value  Association of SORL1 with sex and age was also evaluated ( Table 4). The 6 SORL1 SNPs were not associated with the CI, AD, and MCI cases in the male group. In the female group, SNPs 13 ( value = 0.045) and 23 ( value AA = 0.017; value TA = 0.017) were associated with CI, SNP 23 was associated with MCI ( value = 0.033; value AA = 0.011; value TA = 0.013), and no SORL1 SNP was associated with AD.
Based on the mean age, subjects were divided into two: the  (Table 3).

Discussion
When two or more alleles are in strong LD, it means that these alleles are inherited together. There are millions of polymorphisms in the human genome, many of which are in LD with each other; thus, it is not necessary to test all polymorphisms [12]. In this study, the block of SNPs 8, 9, and 10 was in strong equilibrium. However, none of the haplotypes were associated with CI, MCI, and AD. This result did not replicate the findings from the Caribbean-Hispanic, Israeli-Arab, and North European datasets [2], but a similar result was reported for the Han Chinese population [13].
Our data showed SORL1 was associated with CI. But when restricted to AD and MCI, SORL1 was associated with MCI but not with AD. SORL1 has been reported to affect AD in different population but replication studies have given inconsistent results [3][4][5][6][7]14]. The meta-analysis study by Reitz and colleagues suggested that negative findings were likely related to underpowered studies with small sample   sizes, allelic heterogeneity, or both. Because AD is a complex disease, it is also likely that interaction between two or more genes contributes to disease's phenotype.
The relationship between SORL1 and MCI remains unclear but may suggest that MCI can be modulated by SORL1 like how it is modulated by APOE. Whitehair et al. reported the influence of APOE-4 genotype on rates of cognitive decline in MCI. They found out that APOE-4 carriers had significantly more rapid decline in cognitive and functional performance [15]. On the other hand, SORL1 expression in individuals with MCI was heterogeneous, such that some MCI cases had SORL1 expression similar to healthy controls while it was reduced in others to levels similar to those seen in AD [10,16]. These reports indicate that SORL1 and APOE have similar effect on the cognition of individuals with MCI. SORL1 (SNP 23) was significantly associated with the female sex in this study. A previous report showed a similar 6 Asian Journal of Neuroscience result wherein SORL1 (SNP 4) was also associated with the female sex [6]. For individuals over the age of 80, women are at slightly higher risk of AD, while men may be at higher risk of vascular dementia [17]. Prevalence of MCI in males was higher than that in females; however, the transition in women from normal cognition to dementia is more abrupt in women [18]. The importance of estrogen receptor-mediated neuroprotection in females was cited as a plausible factor in cognition [19]. These reports suggest the possible sex-specific interplay of risk factors, disease course, and survival in this disease.
As we grow older, our cognitive abilities gradually decline. For AD, age is a major risk factor [2]. Around 50% of all people more than 85 years old suffer from AD [20]. Interestingly, no association was observed in the >70 y/o group with CI, MCI, and AD. Interestingly, the ≤70 y/o group was associated with SORL1 in the CI and MCI cases. We hypothesized that the sample size, subject heterogeneity, and age range in our groupings may be the reasons why we were not able to replicate the positive results of Rogaeva et al.
The association of SORL1 with CI, female sex, and ≤70 y/o group elucidated in this study may indicate the early effects of this gene in the development of CI leading to dementia.
It has been suggested that SORL1 activity is influenced by APOE [20]. Alterations in the three-dimensional structure and binding properties of APOE in the APOE-4 isoform may result in changes in the interaction between SORL1 and APOE that could affect the capacity of SORL1 to bind to APP. Thus, the APOE isoform may have a modifying effect on the association of SORL1 and APOE; specifically, effects of distinct SORL1 SNPs may be weakened by the APOE-4 allele. Several studies showed association of SORL1 SNPs with AD in APOE-4 noncarriers [7,21].
In this study, significant association of the SORL1 SNPs was detected with MCI and CI in APOE-4 noncarriers but not with AD. To determine possible interaction of SORL1 and APOE, we also tested the association of APOE-4 allele with CI, MCI, and AD. The result showed that APOE-4 allele was associated with AD ( value = 0.017). APOE-4 allele was not associated with CI ( value = 0.140) and MCI ( value = 0.870). Based on this result, it could be possible that the presence of APOE-4 allele may have masked the effect of the SORL1 in AD. These data support the role of SORL1 in AD pathogenesis, contributing to an increased risk to CI even when the APOE-4 allele is not present.
Because SORL1 plays a crucial role in APP processing, it is a reliable gene that could help fill up missing links in the complex genetics of AD and other CIs. This study provides baseline information on the possible contribution of SORL1 to increased risk of CI in Filipinos. Although no association was observed between the SORL1 and AD, our findings provide evidence that SORL1 may signal a predisposition to cognitive impairment in Filipinos.