APOE4 STATUS AND COGNITIVE FUNCTION IN MIDDLE-AGED AND ELDERLY PEOPLE
Pavel N.A.1, Paun M.R.2, Matei P.V.1,2, Dutu I.1, Tudose C.1,2
*Corresponding Author: Valentin P. Matei, Associate Proffesor, MD, PhD. 2nd Ward of “Prof. Dr. Alexandru Obregia” Hospital, Berceni Street no 10, Bucharest, Romania. E-mail: valipmatei@yahoo.com
page: 6

MATERIAL AND METHODS

We conducted a cross-sectional study on patients from a primary care clinic who were recruited while performing their clinical routine checkup at their General Practitioners (GPs). The study was conducted in accordance with the Declaration of Helsinki [11] and was approved by the Local Ethics Committee (no. 11/06/03.2020). Patients included in the study had to understand and sign the Informed Consent Form and fulfill all the described criteria: (a) age between 50-80 years, (b) Mini-Mental State Examination (MMSE) score over 24, (c) Functional Assessment Questionnaire (FAQ) below 9, (d) Hamilton Depression total score below 12, (e) Hamilton Anxiety total score below 17 and (f) no substance use in the previous 6 months other than caffeine or tobacco. Exclusion criteria were: (a) diagnosis of major or mild neurocognitive disorder according to DSM 5,[12] (b) presence of cerebro-vascular disease translated as Hachinski score over 4, (c) current diagnosis of neurodevelopmental disorder, major depressive disorder, anxiety disorder according to DSM 5, (d) severe somatic disorders such as epilepsy, organ failure or other disease that could impair collection of data from the patient such as severe hearing/seeing impairment, motor deficit). We collected social, demographic, and clinical information from the participants, as well as their full psychiatric history. For genetic assessment of APOE status, we collected 3 milliliters of venous blood using EDTA coated tubes, which were then kept refrigerated at 4 degrees Celsius until DNA extraction, which was conducted using DNA extraction kits (Wizzard© Genomic DNA Purification Kit, Promega) form a volume of 300 μL white blood cells. The genotyping was done using the Taqman SNP Genotyping Assay method from Thermo Fished Scientific, according to the manufacturer’s instructions. The assay was conducted by PCR amplification of two oligonucleotide primers, rs429358 and rs7412. Patients with at least one ApoE4 allele were considered ApoE4 positive while the rest were coded as ApoE4 negative and were considered as control group. Cognitive examination was conducted using the MMSE [13], Rey Auditory-Verbal Learning Test (RAVLT) [14], Rey-Osterrieth Complex Figure Test [15], Verbal Fluency [16] and Trail Making Test (TMT) [14]. The Mini-Mental State Examination [13] is a short questionnaire that evaluates attention, memory, calculation, visuo-spatial ability and executive functioning. The maximum score is 30 and scores under 24 are considered suggestive for cognitive impairment. The RAVLT [14] consists of a list of 15 words, and is designed to evaluate the ability to assess verbal memory. The evaluator reads a list of 15 words after which the participant is asked to recall as many as he can; this is done for a total of 5 times (Trials 1-5). Afterwards, the same exercise is done with a different list of 15 words for a single trial and the participant is then asked to recall the words from the first list (Trial 6). Trial 7 (Delay) is done after a 5-minute pause, without the list being reread to the patient. The last examination of RAVLT (Recognition) consists of a text which includes the first list of words, and the patient is asked to recognize them. The Rey-Osterrieth Complex Figure Test [15] consists of 2 trials which examine memory, visuo-spatial ability and executive function. For the first trial (copying), the patient is asked to copy a complex figure, after which the figure is put away. Following a 3-minute break the patient is asked to draw the figure from memory. The maximum score is 36 with higher scores representing better cognitive functioning. The Verbal Fluency [16] test examines verbal ability and executive function. We used the letter fluency variant, consisting of 3 successive 1-minute trials, in which the patient must produce as many words as possible that begin with a given letter. Each trial used a different letter, and all patients received the same letters in the same order. In our analysis we summed all the correct words from all trials into a single score. The Trail Making Test [14] examines a variety of cognitive functions such as attention, visual and spatial ability, sequencing and shifting, psychomotor speed, abstraction, flexibility and executive function [14]. It consists of two timed trials (A and B). Patient is considered to be deficient in the presented cognitive domains if he finishes Trial A after 78 second and Trial B after 273 seconds.[17] The ApoE4 and control groups were matched for age, sex and education and there were no patients with current depressive or anxiety disorder. Descriptive statistics were used to present the sample characteristics. We used Chi-square test for the categorical data such as (ApoE4 status, demographic characteristics) and Student t-test (for normal distributed variables) and Mann-Whitney Test (for non-parametric variables) for continuous variables (such as age, cognitive evaluation test scores). Results are presented with mean (standard deviation) for normally distributed data and median (Inter Quartile Range - IQR) for non-parametric distribution. Statistical significance was considered at alpha below .05.



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