ABC Transporters, APOE, CYP46A1 and LRP1 Gene Polymorphisms as Markers of Dementia Development in Patients with Hyperlipidemia

1. Introduction

The World Health Organization (WHO) defines dementia as a set of symptoms of a progressive or chronic nature, in the course of which cognitive functions deteriorate to a greater extent than in the case of the biological aging process. Nearly 10 million new cases were diagnosed every year [1]. In 2021 cardiovascular disease- ischemic heart disease and stroke were respectively first and third cause of death worldwide [2]. It is reviewed in existing literature that shared risk factors such as BMI, hypertension, diabetes, dyslipidemia can be affected by genetics. Numerous convergent genes are involved in disease progress of both morbid conditions [3]. Growing importance of molecular diagnostics in clinical practice might be a useful tool to understand pathogenesis and simultaneously a chance to modify lifestyle before prodromal phases. According to the latest report based on analysis nearly half million cases hyperlipidemia diagnosed in younger age results in higher risk in all-cause dementia (HR 1.46 in diagnosis <50 years) [4]. Moreover, lifestyle modification after CVD diagnosis reduces the risk of developing dementia [5]. Conclusions from observational studies and genotyping trials provide evidence that genetic markers are needed in public health policy for prevention.

Table 1 presents the genes selected for analysis based on their alignment in lipid metabolism processes and the development of dementia.

To follow this line, we conduct ABCA1, ABCB7, ABCB1, APOE, CYP46A1 and LRP1 genotyping to compare allele frequency in three groups of patients diagnosed in 1) dementia with coexisting hyperlipidemia, 2) hyperlipidemia, 3) healthy controls, to clarify their role in dementia development and assess their potential for use as early markers of dementia among patients with dyslipidemia.

2. Results

2.1. Baseline Characteristics

A total of 285 participants, 90 with dementia and hyperlipidemia patients, 94 with hypercholesterolemia and 101 individuals as allele frequency control group were included in the study. No significant difference was observed in sex distribution. Patients with both dementia and hyperlipidemia were older than with hyperlipidemia (70.8 (±11.5) vs. 64.1 (±12.9). Dementia & hyperlipidemia participants had a lower burden of comorbidities. Both diabetes and hypertension were more frequently observed in the hyperlipidemia group (p<0.05). The mean age of dementia diagnosis was 65.8 (±9.8), duration is average 7.8 (±2.4). MMSE and MoCA scores were respectively 18.1 (±4.0) and 19.0 (±4.1). In hyperlipidemia & dementia group laboratory workup shows significantly increased cholesterol, HDL, LDL and triglycerides levels (p<0.05 in all parameters).

Table 2. Attributes of the sample.

Parameter	Dementia & hyperlipidemia (n=109)	Hyperlipidemia (n=94)	p-value
Male / Female, sex	47/62 (43.1%/56.9%)	37/57 (39.4%/60,6%)	p=0.588
Age, y	70.8 (±11.5)	64.1 (±12.9)	p <0.05
Weight, kg	78.0 (70.0÷86.5)	79.5 (68.0÷95.5)	p=0.420
Diabetes	13 (11.9%)	27 (28.7%)	p <0.05
Hypertension	20 (18.4%)	68 (72.3%)	p <0.05
Age of dementia diagnosis, y	65.8 (±9.8)	-
Dementia duration, y	7.8 (±2.4)	-
MMSE score	18.1 (±4.0)	-
MoCA score	19.0 (±4.1)	-
Total cholesterol, mg/dl	231.0 (220.0÷244.0)	146.0 (118.0÷168.0)	p <0.05
HDL cholesterol, mg/dl	55.0 (49.0÷64.0)	43.0 (32.0÷56.0)	p <0.05
LDL cholesterol, mg/dl	140.7 (±21.3)	77.3 (±34.6)	p <0.05
Triglicerydes, mg/dl	195.2 (±53.1)	130.7 (±75.5)	p <0.05

Data expressed as n (%) mean (SD) or median (first÷third quartile). SI conversion: cholesterol to mmol/l, multiplied by 0.0259; triglycerides to mmol/l, by 0.0113.

Table 2. Attributes of the sample.

Parameter	Dementia & hyperlipidemia (n=109)	Hyperlipidemia (n=94)	p-value
Male / Female, sex	47/62 (43.1%/56.9%)	37/57 (39.4%/60,6%)	p=0.588
Age, y	70.8 (±11.5)	64.1 (±12.9)	p <0.05
Weight, kg	78.0 (70.0÷86.5)	79.5 (68.0÷95.5)	p=0.420
Diabetes	13 (11.9%)	27 (28.7%)	p <0.05
Hypertension	20 (18.4%)	68 (72.3%)	p <0.05
Age of dementia diagnosis, y	65.8 (±9.8)	-
Dementia duration, y	7.8 (±2.4)	-
MMSE score	18.1 (±4.0)	-
MoCA score	19.0 (±4.1)	-
Total cholesterol, mg/dl	231.0 (220.0÷244.0)	146.0 (118.0÷168.0)	p <0.05
HDL cholesterol, mg/dl	55.0 (49.0÷64.0)	43.0 (32.0÷56.0)	p <0.05
LDL cholesterol, mg/dl	140.7 (±21.3)	77.3 (±34.6)	p <0.05
Triglicerydes, mg/dl	195.2 (±53.1)	130.7 (±75.5)	p <0.05

Data expressed as n (%) mean (SD) or median (first÷third quartile). SI conversion: cholesterol to mmol/l, multiplied by 0.0259; triglycerides to mmol/l, by 0.0113.

2.2. Polymorphisms

First, we assessed differences in genotype frequency in all experiment groups: dementia& hyperlipidemia, non-demented with hyperlipidemia and controls (Table 3). The distribution of ABCA1 (rs2422493), ABCA7, ABCB1, APOE, CYP46A1 and LRP1 genotypes were not significantly different. The ABCA1 (rs2230806) genotypes were not evenly distributed between groups (p=0,004). Further analysis confirmed the existence of differences in the distribution of genotypes between dementia & hyperlipidemia versus hyperlipidemia, with a more frequent prevalence of the GG genotype (p=0.0002, RR=3.22, CI=1.63÷ 6.37) and G allele (p=0.0007, RR=1.53, CI=1.19÷1.97) in patients with dementia. This genotype was also related to an increased risk of Alzheimer disease diagnosis (p=0.006). We also compared the occurrence of genotypes in males and females, but no differences were observed.

Next, we conducted an analysis to evaluate the influence of investigated genotypes on age of dementia diagnosis and duration of disease. Patients with ABCA1 (rs2230806) GG variant presented shorter dementia duration compared to AA and GA genotypes (p=0.0001 and p=0.0004 respectively). Moreover, individuals with ABCA1 (rs2230806) GG genotype achieved lower scores on MoCA scale during first examination (p=0.01) (Figure 1).

An analysis of the impact of the polymorphisms studied on total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides levels was not conducted due to the long-term use of statins by patients in the hyperlipidemia group.

3. Discussion

The aim of this study was to assess the usefulness of ABCA1, ABCB7, ABCB1, APOE, CYP46A1 and LRP1 genotyping as promising dementia risk factors among a wide group of patients with hyperlipidemia diagnosed first. Our findings indicate that ABCA1 (rs2230806) is a suitable candidate. Analysis of other investigated polymorphisms did not reveal differences between the compared groups.

ABCA1 transporter is ATP binding cassette family protein involved in cholesterol efflux and metabolism. Its expression is regulated by several factors, including cholesterol itself, which provides ligands for liver X receptors that stimulate ABCA1 transcription [15]. This transporter plays a crucial role in various diseases including dyslipidemia, coronary heart disease, type 2 diabetes, and potential implications for neurological disorders [16].

Apart from critical role in HDL formation and lipid distribution at the plasma membranes, dysregulation of apolipoprotein lipidation associated with the occurrence of genetic polymorphism increases the risk of dementia developing by promoting the formation of Aβ through the activation of secretase action on the transmembrane amyloid precursor protein [17,18]. At the neurovascular unit without deficits, ABCA1 works alongside ABCB1 to facilitate Aβ clearance from the brain. While ABCB1 directly transports Aβ into the blood circulation, ABCA1 neutralizes Aβ aggregation capacity in an ApoE-dependent manner [19].

The most common context for rs2030806 polymorphism analysis is the risk of coronary artery disease (CAD). Studies have shown that the G allele of rs2230806 is significantly more frequent in coronary artery disease patients, potentially increasing CAD risk [20]. Moreover a meta-analysis revealed that rs2230806 A allele carriers had higher HDL-C levels and lower LDL-C and triglyceride levels compared to non-carriers [21]. Recent research highlights a strong association between cardiovascular disease (CVD) and Alzheimer’s disease (AD). Individuals with subclinical CVD are at higher risk for dementia and AD. Common risk factors for both conditions include hypertension, high LDL cholesterol, low HDL cholesterol, and diabetes [22]. In light of these evidence analysis in relation of neurodegeneration is required. Studies using mice lacking brain ABCA1 have shown increased neuroinflammation, astrogliosis, and cortical neuronal death [23]. Other survey on mouse models confirmed that ABCA1 haplodeficency results in increased Aβ levels and cognitive deficits, but the scale is significantly greater in individuals with the ApoE4 variant [24]. It is worth to be noted that ABCA1 is transcriptionally regulated by Liver X Receptors (LXR) and Retinoic X Receptors (RXR), presenting potential therapeutic targets for neurodegenerative disorders [25,26,27].

Contrary to the presented results of studies on mice findings from published genetic association studies between ABCA1 and Alzheimer’s disease are inconclusive. In Hungarian report the minor A allele of rs2230806 has been linked to a modest protective effect against Alzheimer’s disease [28]. Similarly, in the Chinese Han population, individuals with the A allele achieved better scores on the MMSE cognitive impairment assessment scale and have significantly higher concentration of HDL fraction than non-carriers [29]. According to Wavrant-De Vrièze et al. AG and GG genotypes are associated with the onset of Alzheimer disease, however coincidence with ApoE variant is noteworthy [30]. Sundar et al. indicate a gender-specific association between the ABCA1 rs2230806 polymorphism and late-onset Alzheimer’s disease (LOAD), because women with A allele exhibited 1,75- fold higher risk of the disease [31].

We found significant difference in ABCA1 (rs2230806) genotype distribution between the involved groups hyperlipidemia & dementia and hyperlipidemia. Moreover, GG genotype is related to AD incidence. Patients with GG genotype in comparison to other variants had shorter dementia duration and lower scores on MoCA test during the first examination. Until now, many publications have confirmed that the best risk marker for dementia is the APOE E4 variant; however, the vast majority have relied on comparative studies of healthy individuals and those with dementia. In our study, both groups of participants included patients with hyperlipidemia as an underlying condition, and the differentiating criterion was the presence or absence of dementia. Considering the essential role of APOE as a primary lipid transporter, this enhanced the credibility of the conducted experiment.

Currently, neuropathological examination of brain, neuroimaging and biomarkers widely accepted for the diagnosis of dementia, including the measurement of tau protein, phosphorylated tau protein, and Aβ 1-42 which requires the performance of an invasive procedure to collect cerebrospinal fluid, can provide high diagnostic accuracy in diagnosis of dementia [32]. Some promising plasma phosphorylated tau protein and Aβ42/Aβ40 ratio biomarkers were also reported recently [33,34].

However, molecular testing lately has become a basic diagnostic tool in primary care and has a promising potential to be used in different clinical cases. It is significant to consider the usefulness of genetic markers as screening factors in the targeted group of patients.

Limitations

The conducted study is characterized by several limitations, primarily related to the sample size and the polymorphisms selected based on a review of global literature, which have been reported as involved in both lipid metabolism disorders and dementia-related diseases. However, the study was intended as a preliminary analysis of the potential for identifying gene polymorphisms that should be considered as markers for dementia development in a dedicated patient group. Increasing the number of cases, conducting the analysis across different populations, and significantly expanding the number of polymorphisms tested, for example, through panel tests, would substantially enhance the reliability of the obtained data. In our study, we also did not take into account laboratory test results, particularly those related to lipid metabolism, due to differences in statin use between the two study groups. Cautious planning and implementation of extended lipid parameter diagnostics would undoubtedly add value by narrowing the patient group in which molecular test results could directly influence clinical decisions.

4. Materials and Methods

4.1. Study Population

The study protocol included 203 patients: 109 diagnosed with both dementia and hyperlipidemia hospitalized in Department of Psychiatry Wrocław Medical University and 94 with hyperlipidemia from 4 Military Hospital with Polyclinic in Wrocław. Moreover 101 healthy volunteers as genotype frequency control group were enrolled. Dementia was diagnosed in line with Alzheimer’s Association criteria while hypercholesterolemia according to defined LDL-C levels and risk of cardiovascular disease. Exclusion criteria were: 1) brain injuries 2) acquired causes of dementia 3) liver or kidney disease 4) secondary causes of dyslipidemia 5) long-term fat-elimination diet. Patients were informed about the details of the study and signed fixed consent forms for participation in molecular tests and clinical data processing without personalities. Questionnaires completed by authorized representatives in each department include demographic data (sex, age), anthropometric data (weight), laboratory results (cholesterol, LDL, HDL, triglycerides), comorbidities (diabetes, hypertension) age of diagnosis, duration of dementia, neuropsychological assessment including MMSE (Mini-Mental State Examination and MoCA (Montreal Cognitive Assessment) results collected at the early stage of diagnosing cognitive impairment, which subsequently progressed to dementia.. The study protocol was approved by the Local Ethics Committee of the Wrocław Medical University(141/2019).

4.2. Genotyping

One blood EDTA sampling was completed to extract DNA in the commercial kit (Omega Bio-tek, Georgia) for the genetic survey. The participants were genotyped for the ABCA1 (rs2230806, rs2422493), ABCA7 (rs4147929, rs3752246 rs3764650), ABCB1 (rs1045642, rs1128503, rs2032582) APOE, (rs429358, rs7412), CYP46A1 (rs754203), LRP1 (rs1799986) using AmpliSNiP Dementia Screening Panel (qPCR) (Amplicon, Poland) on Biometra TOptical 96 system (Analytic Jena, Germany).

4.3. Laboratory Markers

Total cholesterol, triglycerides, HDL cholesterol were measured during standard diczasagnostic process in hospital using the enzymatic colorimetric method on a biochemical analyzer using commercially available kits (Roche Diagnostics, Germany). LDL-C concentrations were calculated using the Friedewald formula (in case of triglycerides >= 400 mg/dl measured directly).

4.4. Statistical Analysis

The statistical analysis was performed using the Statistica Ver. 13.3. (TIBCO Software Inc.). The normality of the distribution was evaluated using the Shapiro-Wilk test. The homogeneity of variance was assessed with Levene’s test. Variables with a normal distribution were presented as the mean ± standard deviation, while different distribution as the median along with the first (Q1) and third (Q3) quartile. Qualitative data were expressed as an average percentage. Chi square or the Fisher exact test were performed to compare categorical data. ANOVA, Kruskal-Wallis or Mann-Whitney test were settled to compare continuous variables. P value below 0.05 was considered as significant.

5. Conclusions

The results of our research confirm the impact of the ABCA1 gene polymorphism rs2230806 on an increased risk of developing dementia among patients with hyperlipidemia. This polymorphism is also associated with the duration of the disease and MoCA scale results in the early stages of diagnosing mild cognitive impairment, which over time progressed to dementia. The ABCA1 gene is involved in the regulation of cholesterol transport and phospholipid balance in cells, while rs2230806 polymorphism leading to downregulation of gene activity play a significant role in the development of atherosclerosis and other age-related diseases, it may suggest that changes also occur within the central nervous system, promoting the onset of dementia. However, due to the relatively small sample size in this experiment, it is necessary to expand the study group in order to confirm the utility of ABCA1 rs2230806 genotyping as a predictive marker for the onset of dementia in individuals with hyperlipidemia.