Low prevalence of lipid metabolism abnormalities in APOE ε2-genotype and male patients 60 years or older with schizophrenia

The study was conducted at three mental health centres in Shanghai, China (the Shanghai Mental Health Center, the Mental Health Center of Jiading District in Shanghai, and the Mental Health Center of Fengxian District in Shanghai) between July 1, 2015 and December 31, 2015 (Fig. 1). Information regarding sex, age, height, weight, body mass index (BMI), and current prescribed medicines was recorded for all patients. Obesity was classified according to the health of the People’s Republic of China industry standard WS/T 428–2013, which defines overweight as a BMI ≥ 24.0. Schizophrenia was diagnosed by a senior psychiatrist according to the International Classification of Diseases 10 diagnostic standard. Diabetes was previously diagnosed by an endocrinologist according to the World Health Organization 1999 criteria [22]. High blood pressure was defined as high average measured blood pressure (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg) or previous diagnosis by a clinical specialist.

Research subjects fasted from 20:00 in the evening prior to blood sampling and were prohibited from drinking water for 4 h prior to blood sampling. A total of 5 mL of venous blood was collected from each patient in the fasting state between 06:30 and 08:30. Samples were incubated at room temperature for 30 min and then centrifuged at 3000 rpm for 15 min to collect the serum. All samples were tested for serum glucose, triglyceride content, cholesterol content, high-density lipoprotein, and low-density lipoprotein.

A total of 5 mL of peripheral blood was slowly injected into an EDTA-coated anticoagulant tube, shaken, and then centrifuged at 3000 rpm for 20 min at 4 °C. DNA in 1 mL of blood was extracted with a whole blood genome DNA isolation kit (spin column, Tiangen Biochemical Science and Technology Co., Ltd., Beijing, China).

Multiplex PCR reactions on the basis of the two SNP cores of the APOE gene were conducted using the rs429358 and rs7412 design primers as follows: P1:5′-GCCTACAAATCGGAACTGGA CAGCTCCTCGGTGCTCTG-3′ and P2:5′-TAAGCGGCTCCTCCGCGATGCCCCGGCCTGGTACACTG-3′. The total reaction volume was 20 μL and included the following: 1 μL (50 ng) genomic DNA, 2 μL 1e reaction buffer, 0.6 μL 3 mM Mg²⁺, 2 μL of 2 mM of each dNTP, 0.2 μL 1 U Taq enzyme, 12.2 μL double-distilled H₂O (ddH₂O), and 2 μL 0.5p Primer Mix. PCR products were examined using 3.0% agarose gel electrophoresis.

Details of the multiplex LDR reaction can be found in Table 1. The reaction volume was 10 μL and included the following: 1 μL reaction buffer, 1 μL 2 pmol/μL Probe Mix (each), 0.05 μL 2 U Taq DNA ligase, 4 μL ddH₂O, and 4 μL PCR product. Finally, APOE genotypes were assigned according to product fragment sizes on 3.0% agarose gel electrophoresis (Table 2). All genotyping was performed in duplicate.

All data were inputted using the EpiData3.1 software and analysed with the SPSS 17.0 software package (SPSS Inc., Chicago, IL). APOE genotypes were calculated by alignment inspection according to the Hardy–Weinberg law. Continuous data (age, chlorpromazine equivalent dose, triglycerides, cholesterol, high-density lipoprotein, low-density lipoprotein, and fasting plasma glucose) are represented with descriptive statistics as means ± standard deviation. Categorical data (sex, overweight status, diabetes, high blood pressure, atypical antipsychotics use, lipid-lowering medication use, and glucose-lowering medication use) are represented as percentages of the total population. APOE genotypes were divided according to three categories: APOE ε2 (ε2/ε2 and ε2/ε3), APOE ε3 (ε3/ε3), and APOE ε4 (ε3/ε4 and ε4/ε4). Patients with the ε2/ε4 genotype were excluded from analysis because of known opposite effects of ε2 and ε4 on lipid levels. Inpatients who were ε2 carriers or non-ε2 carriers were labelled as APOE ε2+ or APOE ε2-, respectively. Continuous data were analysed between two groups using independent sample t tests and among three groups using a one-way analysis of variance. Categorical data were analysed using chi-squared (χ ² ) tests; however, atypical antipsychotics use and lipid-lowering medication use were analysed using Fisher’s exact test. Low-density lipoprotein was examined with pairwise comparisons using Hochberg’s GT2(H) method, while cholesterol was examined with pairwise comparisons using the Games-Howell(A) method. Finally, a regression analysis was performed on cholesterol, low-density lipoprotein, and potential influencing factors. The significance level was set at P < 0.05.

All patients were of Han Chinese descent. There were 160 men (53.3%) and 140 women (46.7%). The mean age was 67.3 ± 6.66 years (range, 60–92 years). Two of the participants had the ε2/ε2 APOE genotype, 38 participants were ε2/ε3, 6 were ε2/ε4, 205 participants (were ε3/ε3, 43 were ε3/ε4, and 6 were ε4/ε4; Fig. 2). According to the Hardy-Weinberg law, there was χ ²  = 243.79, df = 3, and P > 0.05. This indicates that the distribution of the genotype was in accordance with Hardy-Weinberg equilibrium. The allele frequencies were as follows: ε2 = 48 (8.0%), ε3 = 491 (81.8%), and ε4 = 61 (10.2%).

Statistically significant differences were observed for lipid-lowering medication use and low-density lipoprotein according to the APOE genotype (P < 0.05). Near statistically significant differences were observed for cholesterol according to the APOE genotype (P = 0.052). There were no statistically significant differences in sex, age, high blood pressure, diabetes, glucose-lowering medication use, atypical antipsychotics use, plasma glucose, triglycerides, or high-density lipoprotein according to genotype (P > 0.05) (Table 3).

With lipid-lowering medication used as a covariate, cholesterol was significantly lower in elderly patients with schizophrenia with the APOE ε2 genotype than in those with the APOE ε3 and APOE ε4 genotypes (P < 0.05). No statistically significant differences were identified between the APOE ε3 and APOE ε4 genotypes (P > 0.05) (Fig. 3a). Low-density lipoprotein was also lower in patients with the APOE ε2 genotype than in those with the APOE ε3 and APOE ε4 genotypes (P < 0.01). No statistically significant differences were identified between the APOE ε3 and APOE ε4 genotypes (P > 0.05) (Fig. 3b). Cholesterol and low-density lipoprotein were lower in ε2 allele carriers with APOE ε2+ genotypes than in those with APOE ε2- genotypes (independent sample t tests, P < 0.05). No statistically significant differences were identified for triglycerides, plasma glucose, or high-density lipoprotein in ε2 allele carriers (P > 0.05) (Figs. 3c–g).

Women had higher cholesterol levels (5.02 ± 1.10 vs. 4.47 ± 0.90 nmol/L, P < 0.01) and higher low-density lipoprotein levels (2.88 ± 0.81 vs. 2.66 ± 0.77 nmol/L, P < 0.05) than men. Compared to patients administered typical antipsychotics, those administered atypical antipsychotics had higher triglyceride levels (1.43 ± 0.86 vs. 1.09 ± 0.43 nmol/L, P < 0.01), higher cholesterol levels (4.78 ± 1.05 vs. 4.33 ± 0.85 nmol/L, P < 0.05), and higher low-density lipoprotein levels (2.80 ± 0.80 vs. 2.46 ± 0.72 nmol/L, P < 0.05).

Stepwise linear regressions were conducted with cholesterol, low density lipoprotein as the dependent variable and age, sex (0 = women, 1 = men), overweight status (0 = no, 1 = yes), APOE ε2 genotype (0 = no, 1 = yes), APOE ε3 genotype (0 = no, 1 = yes), APOE ε4 genotype (0 = no, 1 = yes), atypical antipsychotic drug use (0 = no, 1 = yes), diabetes (0 = no, 1 = yes), high blood pressure (0 = no, 1 = yes), and lipid-lowering medication use (0 = no, 1 = yes) as independent variables. The results showed that cholesterol and low-density lipoprotein were influenced by sex, APOE ε2 genotype, and atypical antipsychotics use (Table 4).