Association between PSEN1 p.E318G Variant and APOE Polymorphism and Alzheimer Disease in Turkish Patients

Abstract

Objective: Mutations in the Presenilin-1 (PSEN1) gene have been associated with early-onset familial Alzheimer disease (AD) and these mutations usually exhibit full penetrance. However, the p.E318G variant located at exon 9 of PSEN1 is an exception. This variant is also seen in non-demented controls other than patients with AD suggesting that it may be a rare polymorphism or a mutation with low penetrance. In addition, results from studies conducted in different populations investigating the role of p.E318G variant in AD were conflicting. In this study, we aimed to determine the frequency of the PSEN1 p.E318G variant and APOE genotypes in a Turkish cohort and to investigate whether they were associated with the risk of AD.

Materials and Methods: The study included 217 patients with familial AD, 153 patients with sporadic AD, and 402 controls. The PSEN1 p.E318G and APOE genotypes were determined using real-time polymerase chain reaction with hydrolysis probes.

Results: The p.E318G variant was found in five patients with familial AD, three patients with sporadic AD, and 11 control subjects. There was no significant difference in the distribution of the p.E318G variant between patients and controls in familial and sporadic forms. APOE ε4 allele carriers had an increased risk for AD compared with non-carriers both in familial [odds ratio (OR): 3.67, 95% confidence interval (CI): (2.69-4.99); p<0.001] and sporadic cases [OR: 2.91, 95% CI: (2.06-4.10); p<0.001]. No significant difference was found in the distribution of the p.E318G variant with either the absence or presence of the APOE ε4 allele.

Conclusion: Our results showed that PSEN1 p.E318G variation, either alone or together with the APOE ε4 allele, is not associated with AD risk in Turkish patients with AD. However, the APOE ε4 allele constitutes a significant risk factor for AD both in familial and sporadic forms.

Introduction

Alzheimer’s disease (AD) is the most common cause of dementia seen in older adults. A common approach currently accepted according to the age of onset is to categorize AD into early-onset Alzheimer’s disease [(EOAD); age at onset <65 years], and lateonset Alzheimer’s disease [(LOAD; age at onset >65 years]. EOAD is mostly familial, and 10-15% of patients with familial EOAD exhibit an autosomal dominant transmission (1). Autosomal dominantly inherited EOAD has been associated with pathogenic variants in the “Amyloid precursor protein (APP)”, “Presenilin 1 (PSEN1)”, and “Presenilin 2 (PSEN2)” genes. Pathogenic variants in PSEN1 gene are found in about 30-70% of familial EOAD cases and are associated with the most aggressive forms of the disease (2). The PSEN1 gene encodes a transmembrane protein containing 467 amino acids, which cleaves APP into beta-amyloid peptides (Aβ) through gamma-secretase activity. Mutations in the PSEN1 gene cause an increase in the Aβ42/Aβ40 ratio by impairing proteolytic cleavage of APP by gamma-secretase, and as a result, accumulation of Aβ plaques (3). To date, more than 300 mutations in PSEN1 have been reported as pathogenic in the Human Gene Mutation Database (HGMD; www.hgmd.org).

One of the PSEN1 variations considered to be associated with AD is the p.E318G variation (rs17125721) (4,5). This variation occurs due to an A to G transition at codon 318 in PSEN1 exon 9 and leads to a non-conserved glutamic acid to glycine substitution. Results from studies conducted in different populations suggested that the pathogenicity of p.E318G in AD is controversial and it could be either an incompletely penetrant mutation or a rare polymorphism not associated with AD (6). It has been shown that the p.E318G variant was associated with high levels of total tau and phospho-tau in cerebrospinal fluid (4). Furthermore, coexistence of p.E318G variant with the APOE ε4 allele was associated with amyloid deposition and faster episodic memory decline (4). In the present study, our aim was to investigate the frequency of the p.E318G variant both in patients with sporadic and familial AD and controls and to evaluate its association with the APOE ε4 allele in a Turkish cohort.

Material and Methods

Patients and Controls

The study population comprised 370 patients with AD and 402 controls without any history of major, systemic, psychiatric, and neurologic disease. The participants were recruited in the department of neurology, behavioral neurology and movement disorders unit, and underwent comprehensive clinical and neuropsychological examinations and neuroimaging. AD was diagnosed according to the National Institute of Neurological and Communicative Disorders and Stroke and AD (7). Approval was obtained from the Ethics Committee of İstanbul Universityİstanbul Faculty of Medicine, Clinical Research Ethics Committee (decision no: 1209, date: 17/10/2016). The study was performed in line with the principles of the Declaration of Helsinki. Written and signed informed consent was obtained from all participants or legal guardians for subjects unable to consent.

Genotyping

Genomic DNA was isolated from peripheral blood leukocytes according to standard procedures. Screening of the PSEN1 E318G variant was performed using quantitative real-time polymerase chain reaction (RT-qPCR) with a Taqman SNP Genotyping Assay (assay ID: C____589147_10, Thermo Fischer Scientific Inc.). A total of 10 μL qPCR reaction mix was composed of 0.5 μL TaqMan Genotyping Assay (Applied Biosystems), 5 μL LightCycler 480 Probes Master (Roche), 2.5 μL RNase-free water and 2 μL DNA (50 ng/μL). The qPCR was performed on a LightCycler480 system (Roche) under the following conditions: 95 °C 10 min, 45 cycles of 95 °C 15 s, and 60 °C 1 min. APOE genotypes were determined using RT-PCR with hydrolysis probes. The APOE RT amplifications included 10 min at 95 °C; 45 cycles of 10 sec at 95 °C; 30 sec at 56 °C and 1 sec at 72 °C. The end-point analysis was assessed using the LightCycler 480 genotyping software.

Statistical Analysis

Student’s t-test was used for normally distributed continuous variables and Fisher’s exact test for categorical variables. Maximum likelihood estimates of odds ratios (OR) and the 95% confidence intervals (CI) were calculated using binary logistic regression analysis. The SPSS software was used for all statistical analyses (IBM Corp., USA, version 21.0). P values below 0.05 were regarded as statistically significant.

Results

Patients with AD were divided into two groups: familial AD (at least one first-degree relative with a history of dementia) and sporadic AD (no family history of dementia). The characteristics of the study population are given in Table 1. A total of 370 patients with AD including 217 patients with familial (mean age: 64.6±10.7 years) and 153 patients with sporadic AD (mean age: 66.6±11.4 years).

As shown in Table 2, the p.E318G variant was found in five (2.3%) patients with familial AD and three (2%) with sporadic AD and in 11 (2.7%) control subjects. The p.E318G variant was detected only in the heterozygous state and no homozygous carriers were found. No significant difference in distribution of p.E318G was found for familial AD vs. controls (p=0.746) and sporadic AD vs controls (p=0.767).

In all groups, the distribution of allele frequencies and genotypes of APOE was significantly different (p<0.001) between patients and controls (Table 3). The ε3/ε4 and ε4/ε4 genotypes were found to be more common in the patient groups than in the controls. The APOE ε2, ε3, ε4 allele frequencies were 3.9%, 74.1%, and 22% in patients with familial AD and 2.6%, 78.6%, and 18.8% in patients with sporadic AD, and in the controls, the frequencies were found as 5%, 87.3% and 7.7%, respectively. Individuals with sporadic AD with at least one copy of APOE ε4 allele had a 2.91-fold increased risk [OR: 2.91, 95% CI: (2.06- 4.10); p<0.001] for AD than individuals without the ε4 allele, and in patients with familial AD, risk increased up to 3.67 fold [OR: 3.67, 95% CI: (2.69-4.99); p<0.001].

To examine the possible interaction of the APOE ε4 allele and p.E318G variant, we investigated the distribution of p.E318G variant in ε4 allele carriers and non-carriers. In both the presence and absence of the APOE ε4 allele, no significant difference was found in the distribution of p.E318G variant between patients and controls in all groups (Table 4). However, there was a weak trend (p=0.268) for the coexistence of the APOE ε4 allele and the p.E318G variant in patients with familial AD. Of the five familial p.E318G carriers, three carried at least one copy of the APOE ε4 allele, but this trend was lacking in controls, as none of the p.E318G carriers were carrying the ε4 allele.

General characteristics of patients with the PSEN1 p.E318G variant were given in Table 5. Six of the patients were diagnosed with EOAD, one with LOAD and one with atypical dementia. Of the three patients carrying APOE ε4 allele, two were homozygous and one was heterozygous for ε4 allele. The mean age at onset of patients was 56.2±11.2, range 36-76 years. The mean age of patients with the PSEN1 p.E318G variant was 60.2±11 while controls with the p.E318G variant were younger (mean age ± standard deviation: 48.6±11.3) than the patients.

Discussion

In this study, we evaluated the frequency of the p.E318G variant and APOE genotypes and their contribution to AD in familial and sporadic forms. Also, we investigated whether there was an association between the p.E318G variant and the APOE ε4 allele.

The p.E318G variant is located on the hydrophilic loop between transmembrane domain VI and VII of PSEN1 protein (8). This region of the PSEN1 protein is less conserved between its homologues in other species and predicted to be functionally less important (9). The pathogenicity of the p.E318G variant is still under debate. In initial studies, it was considered as pathogenic in patients with EOAD (10,11); however, its presence in controls and no segregation with disease made its pathogenicity questionable (2,12,13,14,15,16,17,18). Even so, results from studies conducted in different populations investigating the role of p.E318G variant in AD were conflicting. Evidence from some case-control studies indicated p.E318G as a risk variant for patients with familial AD (2,12,13,18), but other studies found no significant association of p.E318G and AD (14,16,17,19). Therefore, the association between the PSEN1 p.E318G variant and AD remains controversial.

The frequency of the p.E318G variant in our total patients with AD (2.1%) was lower than that described for European populations, actually it was the lowest reported to date (Dutch: 2.7%, Italian: 3.3%, French: 3.5%, Finnish: 6.6%, Spanish: 6.7%, Australian: 3.6%, and Brazilian: 4%) (13,14,15,16,19). Similarly, the frequency of the variant in our control group (2.7%) was lower than the reported frequencies for European populations (Finnish: 6.8%, Spanish: 4.5%, Dutch: 4.1%, French: 4.1%, Polish: 4.0%) (14,15,16,17,19), but has similar frequency with the Australian population (2.2%) (12). Comparing the frequency of the p.E318G variant in patients with familial AD with those of other populations, our population has the lowest frequency (2.3%), but the frequency in patients with sporadic AD (2%) was consistent with the results from previous studies (2,12,13). Despite the larger number of patients included in our study, our results did not validate the previously reported significant associations of the p.E318G variant with AD. The lack of association between p.E318G variant and AD in our study is in agreement with previous studies that showed no significant associations either in patients with sporadic or familial AD in different populations (14,16,17,19). Combined with our results, these data suggested that the association between the p.E318G variant and AD might vary among different populations.

The association between APOE gene and AD risk has been studied in a great number of populations. Studies showed that the frequencies of APOE ε2, ε3 and ε4 alleles varied between populations due to geographic locations and different ethnicities, but in most of them, the ε4 allele was considered as a risk factor for AD. In our study, the APOE ε allele frequencies in patients with AD and controls were very similar to those reported in the Turkish population (20,21,22,23) but were lower than in other populations (23). The low frequency of the APOE ε4 allele both in our study and previous studies is in line with the observation that ε4 frequency is low in Mediterranean countries. Our data confirm a clear association between APOE ε4 allele and AD; we found that the ε4 allele increased AD risk in patients with AD with a higher OR in patients with familial AD. The combined effect of PSEN1 polymorphisms and APOE ε alleles on AD risk has been investigated previously in Turkish patients with LOAD; however, no significant difference was found (24).

The coexistence of the APOE ε4 allele and the p.E318G variant is associated with higher amyloid plaque deposition, faster episodic memory decline, and subsequent increased AD risk (4). In their study, Benitez et al. (4) showed that p.E318G variant modified AD risk in APOE ε4 carriers and increased the AD risk equally to ε4 homozygous in ε4 heterozygous; they showed that the PSEN1 p.E318G carriers who also carried the APOE ε4 allele were at higher risk of developing AD than carriers of p.E318G variant alone, and had twice the risk of AD than those carrying the APOE ε4 allele alone (4). Therefore, to investigate the association of APOE ε4 allele and PSEN1 p.E318G, we examined the distribution of p.E318G variant in APOE ε4 allele carriers. Although no significant association was found, our study highlighted the tendency for the coexistence of at least one copy of the APOE ε4 allele and the p.E318G variant in patients with familial AD.

Study Limitations

This study has limitations as follows. First, due to the small number of p.E318G carriers in this study, it is difficult to assess the significance of tendency for the coexistence of the APOE ε4 allele and the p.E318G variant in patients with familial AD. Further investigations in larger case-control series are necessary to confirm this association in the Turkish population because PSEN1 p.E318G variant and APOE ε4 interaction is an important modifier of AD risk. Secondly, controls carrying the p.E318G variant were aged younger than 60 years and might later develop the disease; therefore, prospective follow-up examinations should be considered in controls. Another limitation of this study was that the control group was significantly younger than the patient group; this may not affect the results of the frequency of PSEN1 p.E318G variation or APOE ε4 allele, but Mini-Mental State Examination scores may have been affected. Finally, another limitation was our relatively small sample size; the results of this study will be strengthened by studies with larger study groups.

Conclusion

In our study, we do not provide evidence for the association of the PSEN1 p.E318G variant with AD risk and its interaction with the APOE ε4 allele. In addition, our results claim that the APOE ε4 allele is a significant risk factor in AD in the Turkish population. As far as we know, our study is the first to investigate the role of E318G variant in Turkish patients with AD. Therefore, further analyses in larger case-control cohorts are necessary to fully understand the effect of p.E318G on AD risk in the Turkish population. We suggest that special consideration should be taken in the interpretation of PSEN1 p.E318G variant in AD, especially in familial forms.

Approval was obtained from the Ethics Committee of İstanbul University-İstanbul Faculty of Medicine, Clinical Research Ethics Committee (decision no: 1209, date: 17/10/2016).

Written and signed informed consent was obtained from all participants or legal guardians for subjects unable to consent.

Externally and internally peer-reviewed.

Surgical and Medical Practices: H.H., E.L., H.G., B.B., Concept: G.G., N.E.Ü., B.B., Design: G.G., N.E.Ü., R.A., Ç.D., Data Collection or Processing: G.G., H.H., E.L., H.G., R.A., Ç.D., Analysis or Interpretation: G.G., H.H., E.L., N.E.Ü., H.G., B.B., Literature Search: G.G., R.A., Ç.D., Writing: G.G.

No conflict of interest was declared by the authors.

This work was supported by the Research Fund of Istanbul University (project no: 35352).

The authors would like to thank the patients and families for their generous participations.

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