APOE E2 Carriers Showed Worse Associative Learning Than E3 Carriers in a Cognitively Normal Aging Han Chinese Population

Background: Polymorphism in the APOE gene has been shown to be associated with cognitive function, however, the related studies are not consistent. To investigate the relationship between APOE gene polymorphism and cognitive function, we conducted the current cross-sectional study specically to investigate the effect of different APOE genotypes on cognitive performance in normal elderly adults Methods: A total of 156 older adults with normal cognitive function were enrolled in the current study. According to different genetic types, they were divided into three groups: 1) E2/2 or E2/3 (APOE E2); 2) E3/3 (APOE E3); and 3) E2/4, E3/4, or E4/4 (APOE E4). Then Montreal Cognitive Assessment (MoCA) and Neuropsychological Test Battery (NTB) were used to assess their global cognitive function and domain-specic cognitive function, respectively. Results: The results of Kruskai-Wallis H test showed that the scores of associative learning in APOE E2 group were lower than that in E3 groups (p<0.05), but there was no statistical difference (p>0.05) in associative learning between E2 group and E4 group, and E3group and E4 group. Similarly, there was no difference (p>0.05) in the global cognitive function among the three groups. Conclusion: APOE E2 is associated with decreased associative learning function than APOE E3 in a cognitively normal aging Han Chinese population.


Introduction
Genetic variance might account for individual differences in adult cognitive function 1 . Apolipoprotein E (APOE), a gene implicated in the transport of cholesterol and other lipids between cellular structures 2 , has been experiencing as the largest contributor to genetic risk for late onset Alzheimer's disease (AD) 3 . It is genetically associated with two single-nucleotide polymorphisms (SNPs) that mark three alleles ε4, ε3, and ε2 4 . And the 4 allele has been demonstrated to increase associations between cerebral Aβ level and cognitive functioning in adults with dementia and healthy older adults 5,6 . Although considerable research has been done on ε4, ε2 has been seriously neglected because of its low allele frequency. Previous studies suggest that possession of a ε2 allele, prevalent in 15% of the population 7 , is associated with a lower risk of AD, less AD neuropathology as well as a slower progression of vascular cognitive impairment 8-10 . However, a study 11 shows that possession of an ε2 allele is associated with poorer cognitive performance and more psychiatric symptoms in chronic, combat-related posttraumatic stress disorder (PTSD) subjects, while another study 12 also suggests that carriers of the e2 allele shows performance disadvantages in sustained attention. What's more, some studies have even identi ed the ε2 allele as a risk factor in dysbetalipoproteinemia 13 , cerebral small-vessel disease 14 , and aggressiveness of certain cancer 15 . Therefore, relevant research conclusions are not consistent.
Until now, only a few studies have been involved in the relationship between APOE gene polymorphism and cognitive function in Chinese normal cognitive elderly. For example, Zhen J 16 et al nd that APOE genotype might modify the risk for cognitive impairment in old age diabetes patients, and Su yy 17 et al prove that ε2 might as a protective factor in Chinese dialysis population since it might reduce the prevalence and of the onset age of depression. However, these above studies only focus on the general cognitive function of the subjects but neglect their speci c cognitive areas, so we conducted this crosssection study to examine the relationship between the APOE ε2 allele and various cognitive elds (composed of global cognitive function and multiple domains of cognitive function) among the elderly with normal cognitive function in China Methods A total of 156 elderly people (male/female = 61/95) with normal cognitive function were included in the study. Sampling methods and processes have been described in detail in our previous study 18 . All participants met the following criteria: Han Chinese, aged 60 and over; 2) normal cognitive ability; 3) without major medical abnormalities (e.g. cancer and infection); 4) without serious mental illness (e.g. schizophrenia, and dementia); 5) willing to cooperate. A standardized questionnaire was utilized to collect these participants' general information (for example, age and education), daily living habits (smoking and drinking) and medical conditions (diabetes and hypertension).What's more, a completion of physical examinations, MRI scans and laboratory tests were also obtained for each subject.
All participants gave written consent to participating in this study. And the study was approved by the Research Ethical Committee of the a liated mental health center of Shanghai jiaotong university school of medicine.

Clinical Assessment and Cognitive Assessment
The Neuropsychological Test Battery [consists of Digit span 19 (assess attention, working memory and executive function), Verbal uency 20 (measure language ability related to executive function), Auditory verbal learning test 21 (assess learning ability, recognition memory and delayed free recall), Associative learning and visual identi cation test 22 (assess visual attention and processing speed), Webster picture completion 23 (evaluate executive function) and Webster block design 24 (assess visuospatial and executive function)] and the Montreal Cognitive Assessment (MoCA) 25 were used as tools to assess their speci c cognitive domains and global cognitive ability, respectively.

Data analysis
Continuous variables were expressed as mean ±SD and categorical variables were expressed as frequencies (%). One sample Kolmogorov-Smirrnov test was used to test whether the data conform to a normal distribution. Chi square test was utilized to compare categorical variables. One-way analysis of variance (ANOVA) Least-Signi cant Difference (LSD) was used to compare the differences among the APOE E2 group, APOE E 3 group, and APOE E 4 group (normal distribution data); while Kruskai-Wallis H test was used to compare data of non-normal distribution among three groups. Two-tailed tests were utilized in a signi cance level of P<0.05 for all analyses. All statistical analyses were performed using

Results
Characteristic of subjects with different APOE genotypes (as the data did not conform to the normal distribution) showed that there were statistical differences (p<0.05) in Associative Learning among the three groups. Further comparisons revealed that the scores (6.240±3.163) of Associative Learning in APOE E2 group were lower than those (8.433±3.924) in APOE E3 group (p<0.05), while there was no signi cant difference (p>0.05) between APOE E2 group and APOE E4 group, and between APOE E3 and E4. Figure 1 and Table 2 show the results.

Discussion
In the present study, we investigated the effect of APOE gene polymorphism on cognitive performance in Chinese elderly with normal cognitive function. And found that E2 carriers had worse visual attention and processing speed ability than E3 carriers, while there was no difference between E2 and E4 carriers or E3 and E4 carriers.
There was no statistical difference in age, gender and education among the three groups. By using the Neuropsychological Test Battery and MoCA, we found that scores of association learning test in APOE E2 group (6.240±3.163) were signi cantly lower than that (8.433±3.924) in E 3 group. However, there was no statistical difference (p>0.05) between the E2 group and E4 group (orE3 and E4 group). What's more, there was also no signi cant difference in global cognitive function among the three groups.
Sinclair LI 27 et al found that E2 carriers had slightly better episodic memory and executive functioning than E3 and E4 carriers in early to mid-adult, but Palmer Allred ND 28 et al found E2 carriers had worse global cognitive function than E3 carriers. What's more, a large study 29 (total n=2013) of APOE genotype and cognitive decline conduct in 2014 found no association between either E2 or E4 status and cognitive change in ve separate tests, even when split by age group. So these relevant research conclusions were not consistent, and the discrepancy may be explained by ethnic differences.
There are several mechanisms may explain why APOE E2 is associated with decreased associative learning function than APOE E3. First, APOE ε2 status may in uence the risk and progression of tauopathy 30 . Second, APOE ε2 may increase the likelihood of vascular disease and lead to cognitive decline in speci c areas 31 . Third, under metabolic stress, APOE E2 homozygote may cause dysbetalipoproteinaemia in adults owing to impaired binding of remnant lipoproteins to heparan sulphate proteoglycans as well as the (low density lipoprotein) LDL receptor and related proteins 32 . Fourth, APOE E2 is correlated with increasing brain white matter hyperintensities (WMHs) 14 , which is associated with neurologic decompression sickness, lower neurocognitive test performances as well as repetitive nonhypoxic hypobaric exposure 3334 .
We have to admit that there are some limitations in our research. First, this is only a cross-sectional study, and we cannot establish a causal link between APOE E2 and associative learning function. Second, relatively small sample size reduces the reliability of research. Therefore, a large sample of longitudinal research is needed to further verify the above conclusion

Conclusions
In conclusion, APOE E2 is associated with reduced associative learning function than APOE E3 in healthy elderly. However, this conclusion needs to be veri ed by a larger sample of longitudinal study.

Declarations Ethics approval and consent to participate
This study was conducted in accordance with the principles of Declaration of Helsinki, and approved by the Research Ethical Committee of the a liated mental health center of Shanghai Jiaotong University School of Medicine. All participants had signed the informed consent written informed consent before the start of the study.

Consent for publication
Not applicable.

Availability of data and materials
The data base generated and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.