**6.** *APOE* **in a healthy Polish population under 60 years of age**

Despite the many years of research, AD remains a disease that is difficult to predict and diagnose, with few blood-derived biomarkers possible for use in routine clinical setting. As was stated before, *APOE* remains the most significant genetic risk factor of AD. This creates a need for the development of a novel, quick, and reliable method of analyzing the *APOE* genotype and the apoE plasma concentration. The role of *APOE* in the development of dementia and its influence on longevity in elderly people has been studied by [53]. As per our knowledge, *APOE* studies on a younger population in Poland have been neglected and there are no literature data on association of the *APOE* genotype and the apoE plasma level in nondemented Polish adults.

#### **6.1. Aim of the study**

In this study, we tried to assess the influence of the *APOE* genotype and the effect of demo‐ graphic factors on the apoE level in a subset of Polish non-demented volunteers less than 60 years of age.

#### **6.2. Subjects**

A total of 83 healthy adults (70 females, mean age: 51.9 ± 7.2; 13 males, mean age: 44.9 ± 11.7) under 60 years of age with no signs of dementia or other neurological disorders were enrolled in the study. All participants provided signed, written consent. The research project was approved by the Bioethical Committee at the Poznan University of Medical Sciences, decision no. 1031/13, dated May 5, 2013.

#### **6.3. Materials**

Each volunteer's blood was collected on an anticoagulant—K3EDTA (Monovette™ vacuum system, Sarstedt, USA). A total of 3 ml of blood was immediately aliquoted, then frozen and stored at −80°C upon nucleic acid isolation. Subsequently, the remaining blood was centri‐ fuged (1400 relative centrifugal force [RCF], 10 min) and the collected plasma was aliquoted and stored at −80°C.

#### **6.4. Methodology**

#### *6.4.1. APOE genotyping*

First, a subject's DNA was extracted from frozen blood using gravity flow microcolumns (Genomic Micro AX Blood Gravity, A&A Biotechnology, Poland). The DNA concentration was measured by a microplate spectrophotometer (Take3, Epoch, BioTek, USA) and adjusted to 20 ng/μL with Milli-Q® water. Subsequently, genotyping was performed according to a modified mismatch primer method [87]. Briefly, three quantitative polymerase chain reaction (qPCR) specific to each *APOE* allele were performed with the use of four different primers (as shown in **Table 2**). The qPCR included two steps: primary pre-amplification (15 cycles) with annealing at 64°C followed by 30 cycles of secondary amplification with annealing at 62°C. The reactions were performed on a CFX Connect™ Real-Time PCR Detection System (Bio-rad, USA) in 10 μL volumes, with 250 nM primers and 50 ng of genomic DNA using 1× SsoFast™ EvaGreen® Supermix (Bio-rad, USA). The cycling conditions were: 30 s initial denaturation at 98°C followed by cycles of 98°C for 5 s and 64 and 62°C for 10 s. The reaction was considered positive once the products appeared before the 10th cycle of secondary qPCR. The method was validated by Sanger sequencing in an external laboratory.


**Table 2.** Starters used for genotyping of *APOE*.

#### *6.4.2. ApoE quantification*

Determination of the plasma apoE concentration was performed by the enzyme-linked immunosorbent assay (ELISA) method. The analysis was performed according to the manu‐ facturer's protocol (Human apoE ELISA Kit, Mabtech, Sweden) using 10,000× diluted plasma samples. Absorbance was measured by an EPOCH microplate reader (BioTek, USA). The concentrations were calculated from a four-parametric standard curve (*R* = 0.998) by Gen5 ver. 2.01 software (provided with the reader).

#### **6.5. Results**

the *APOE/TOMM40* locus holds the key for healthy senescence without a pathological memory

Despite the many years of research, AD remains a disease that is difficult to predict and diagnose, with few blood-derived biomarkers possible for use in routine clinical setting. As was stated before, *APOE* remains the most significant genetic risk factor of AD. This creates a need for the development of a novel, quick, and reliable method of analyzing the *APOE* genotype and the apoE plasma concentration. The role of *APOE* in the development of dementia and its influence on longevity in elderly people has been studied by [53]. As per our knowledge, *APOE* studies on a younger population in Poland have been neglected and there are no literature data on association of the *APOE* genotype and the apoE plasma level in non-

In this study, we tried to assess the influence of the *APOE* genotype and the effect of demo‐ graphic factors on the apoE level in a subset of Polish non-demented volunteers less than 60

A total of 83 healthy adults (70 females, mean age: 51.9 ± 7.2; 13 males, mean age: 44.9 ± 11.7) under 60 years of age with no signs of dementia or other neurological disorders were enrolled in the study. All participants provided signed, written consent. The research project was approved by the Bioethical Committee at the Poznan University of Medical Sciences, decision

Each volunteer's blood was collected on an anticoagulant—K3EDTA (Monovette™ vacuum system, Sarstedt, USA). A total of 3 ml of blood was immediately aliquoted, then frozen and stored at −80°C upon nucleic acid isolation. Subsequently, the remaining blood was centri‐ fuged (1400 relative centrifugal force [RCF], 10 min) and the collected plasma was aliquoted

First, a subject's DNA was extracted from frozen blood using gravity flow microcolumns (Genomic Micro AX Blood Gravity, A&A Biotechnology, Poland). The DNA concentration was measured by a microplate spectrophotometer (Take3, Epoch, BioTek, USA) and adjusted to 20

**6.** *APOE* **in a healthy Polish population under 60 years of age**

decline.

260 Update on Dementia

demented Polish adults.

no. 1031/13, dated May 5, 2013.

**6.1. Aim of the study**

years of age.

**6.2. Subjects**

**6.3. Materials**

and stored at −80°C.

**6.4. Methodology**

*6.4.1. APOE genotyping*

Our study on Polish subjects showed that the observed genotype frequencies of *APOE* are in line with the Hardy-Weinberg equilibrium (*p* = 0.9365). The dominating allele was *APOE* E3 (83.7%) and the least common allele was *APOE* E2 (3.0%), as is shown in **Figure 1**. Interestingly, we did not observe any *APOE* E2/E2 homozygotes, as is shown in **Table 3**.

Our results indicate that the apoE plasma concentration depends on the *APOE* genotype (oneway analysis of variance [ANOVA], *p* = 0.021). Generally, in *APOE* E3/E3 carriers we recorded the highest mean concentrations of apoE, while in the *APOE* E4/E4 homozygotes we recorded the lowest mean concentrations. In females with the *APOE* E2/E3 allele, the concentration of apoE was slightly lower than in the E3 homozygotes. Interestingly, in a single case of an E2/E4 carrier we observed an increased level of plasma apoE. Subsequently, the plasma apoE concentration in *APOE* E3/E3 carriers was higher in males than in females. Conversely, in *APOE* E3/E4 carriers the recorded apoE concentration was higher in females. Hence, the decrease in apoE due to the *APOE* E4 genotype was more pronounced in males than in females (41% vs 16%), as shown in **Table 3**. Overall, the apoE concentration was insignificantly higher in males than in females (2.54 vs 2.24 mg/dL; *p* = 0.194, Student's *t*-test). The observed positive trend of increasing apoE in older individuals did not reach statistical significance (*r* = 0.201, *p* = 0.0687; Pearson correlation coefficient). However, after stratification according to gender, we observed significant correlation of the apoE plasma level and age in females (*r* = 0.348, *p* = 0.00128; Pearson correlation coefficient). The concentrations of apoE stratified according to *APOE* status, gender, and age are shown in **Table 4** and **Figure 2**, respectively.

**Figure 1.** Frequencies of *APOE* alleles in Polish, cognitively normal volunteers under 60 years of age.


*Note:* Hardy-Weinberg equilibrium calculations, *p* = 0.9365, *n* = 83.

**Table 3.** Hardy-Weinberg equilibrium calculations of *APOE* variants in Polish, cognitively normal volunteers less than 60 years of age.


*Note:* Mean concentration ± SD (mg/dL) or (single result).

**Table 4.** Mean plasma apoE concentration (mg/dL) in Polish, cognitively normal volunteers under 60 years of age stratified according to gender and *APOE* genotype.

**Figure 2.** Mean apoE plasma concentration (mg/dL) in Polish, cognitively normal volunteers under 60 years of age stratified according to gender and age.

#### **6.6. Discussion**

E2/E4 carrier we observed an increased level of plasma apoE. Subsequently, the plasma apoE concentration in *APOE* E3/E3 carriers was higher in males than in females. Conversely, in *APOE* E3/E4 carriers the recorded apoE concentration was higher in females. Hence, the decrease in apoE due to the *APOE* E4 genotype was more pronounced in males than in females (41% vs 16%), as shown in **Table 3**. Overall, the apoE concentration was insignificantly higher in males than in females (2.54 vs 2.24 mg/dL; *p* = 0.194, Student's *t*-test). The observed positive trend of increasing apoE in older individuals did not reach statistical significance (*r* = 0.201, *p* = 0.0687; Pearson correlation coefficient). However, after stratification according to gender, we observed significant correlation of the apoE plasma level and age in females (*r* = 0.348, *p* = 0.00128; Pearson correlation coefficient). The concentrations of apoE stratified according to

*APOE* status, gender, and age are shown in **Table 4** and **Figure 2**, respectively.

**Figure 1.** Frequencies of *APOE* alleles in Polish, cognitively normal volunteers under 60 years of age.

4 4.8%

4.19 5.0%

Observed frequencies 0

Expected frequencies 0.08

60 years of age.

262 Update on Dementia

0.0%

0.1%

*Note:* Mean concentration ± SD (mg/dL) or (single result).

stratified according to gender and *APOE* genotype.

*Note:* Hardy-Weinberg equilibrium calculations, *p* = 0.9365, *n* = 83.

**Genotypes** *APOE* **E2/E2** *APOE* **E2/E3** *APOE* **E3/E3** *APOE* **E3/E4** *APOE* **E4/E4** *APOE* **E2/E4**

58 69.9%

58.20 70.1%

**Table 3.** Hardy-Weinberg equilibrium calculations of *APOE* variants in Polish, cognitively normal volunteers less than

**Gender** *APOE* **E2/E3** *APOE* **E2/E4** *APOE* **E3/E3** *APOE* **E3/E4** *APOE* **E4/E4**

**Table 4.** Mean plasma apoE concentration (mg/dL) in Polish, cognitively normal volunteers under 60 years of age

Female 1.98 ± 0.67 2.91 2.35 ± 0.78 2.02 ± 0.52 0.69 Male – – 2.91 ± 0.63 1.72 ± 0.22 – Combined 1.98 ± 0.67 2.91 2.43 ± 0.79 1.95 ± 0.49 0.69

19 22.9%

18.42 22.2% 1 1.2%

1.40 1.8% 1 1.2%

0.66 0.8% As was stated before, the *APOE* E4 allele is associated with increased risk of developing dementia.

According to our results, the *APOE* E3 genotype was the most prevalent genotype in the studied group, while the E2 genotype was the least common. Similar results were reported in the Polish population [53].

Our study shows that *APOE* E4 variant is associated with a decreased concentration of plasma apoE in cognitively normal Polish volunteers less than 60 years of age. Our results are supported by the results of other authors [77], who analyzed plasma apoE concentrations and *APOE* status in a cohort of 75,708 participants in the Copenhagen General Population Study and the Copenhagen City Heart Study. The authors also showed that apoE is dependent on the *APOE* genotype, as they found substantial differences in plasma apoE concentrations between carriers of distinct *APOE* genotypes. However, contrary to our results, in their study the highest level of apoE was observed in *APOE* E2 homozygotes and decreased in E4 carriers in a dose-dependent manner: E2/E2 > E2E3 > E2/E4 > E3/E3 > E3/E4 > E4/E4. The plasma concentration of apoE in E4/E4 homozygotes was up to 65% lower as compared to *APOE* E2/E2 carriers. This partial incompatibility with our results may be explained by the utilization of various methods: the authors used the nephelometry and turbidimetry methods, whereas we used the well-established ELISA method. In another study [88], the authors showed that apoE concentrations in plasma apoE increased with age in a healthy population. We observed a similar trend; however, it was significant only in the female group.

The plasma concentration of apoE may be a valuable dementia biomarker because it is easily available and, according to literature data, decreased apoE may be a risk factor for developing dementia. The above-mentioned Australian follow-up cohort study, comprising mostly Caucasian subjects, showed that the reduced apoE plasma level may be a predictor of a transition from MCI to AD. Moreover, the plasma apoE concentration correlates positively with cognitive function, and patients with a lower apoE level tend to perform worse in neuropsychological tests assessing spatial memory and language abilities [89].

Hence, the assessment of the plasma apoE concentration and the *APOE* status may give valuable information to physicians trying to predict the rate of cognitive decline in the course of dementive disease as well as in normally ageing adults and elderly persons.

#### **7. Summary**

The appearance of dementia in old age is influenced by both biochemical and genetic factors leading to structural disorders in the brain of elderly persons. The level of Aβ is mentioned among the other biochemical factors associated with dementia. The deposition of Aβ in the brain is controlled by *APOE* and by genes associated with the amyloid cascade (*APP, PSEN1*, and *PSEN2*). Subsequently, Aβ toxicity is modified by the *TOMM40*. In the elderly, also abnormal cholesterol, glucose levels, and the weakening of protective and repair mechanisms leading to the generation of ROS (mediated, e.g. by *PON1*) may cause a reduction in cognitive functions. However, the role of genes associated with longevity (e.g. *FOXO3A, CETP*) and normal aging (e.g. *SIRT1, AKT1, CDKN1A*) is not clearly defined in the occurrence of diseases typical for this age group, as shown in **Figure 3**.

Finding a way to control the genetic factors and their protein products may contribute to the prevention of diseases of old age, including depression and dementia, and to improve the quality of life of elderly people.

**Figure 3.** The genetic and biochemical factors associated with normal aging and dementia; β-amyloid—Aβ.
