Immune Response and Lipid Metabolism Genes Associated with Acute Cerebral Circulatory Failure in Pre-Eclampsia in a Central Asian Population

Study participants were provided with information about the purpose of the study to investigate genetic factors associated with pre-eclampsia and ACVD, and procedures for collecting blood samples and medical data were described. They were assured of confidentiality, voluntary participation, the right to withdraw at any time without repercussions, and the potential risks and benefits were explained and then signed a voluntary consent for the use of their blood samples and medical history data. Permission to conduct the study was obtained from the local ethical committee of the Kazakh National Medical University named after S. D. Asfendiyarov (Almaty, Kazakhstan) under application No. 1189 dated 28 September 2021. The study complied with all ethical norms and international standards.

The control group included 1,800 conditionally healthy participants of Kazakh ethnicity. The inclusion criteria were belonging to the Kazakh ethnic group (including those over 65 years of age), an age from 18 to 75 years and the ability to make an independent decision to agree to participate in the project. All participants underwent a preliminary questionnaire to confirm that they met these criteria. The authors informed the participants about the anonymous and voluntary participation, and the participants provided their consent. DNA samples were stored in the Miras Biobank of the Scientific Centre of Obstetrics, Gynaecology and Perinatology NAO, established within the framework of the project “Genetic studies of pre-eclampsia in Central Asian and European populations” (InterPregGen) under grant agreement No. 282540 under the 7th Framework Programme of the European Commission.

DNA extraction from peripheral blood was performed by magnetic separation of M-PVA particles on a Prepitto automated analyser (PerkinElmer, USA) using the Prepito DNA CytoPure reagent kit (Wallac Finland). This method ensured high purity and concentration of DNA, which was necessary for subsequent genotyping. The isolation process included several stages of purification and concentration, which minimised the risk of contamination and ensured high quality of genetic material.

Genotyping was performed on 2.5 million SNPs at deCODE Genetics using Illumina Omni Chip 2.5–8 chips as part of the InterPregGen project. This approach provided detailed data on genetic polymorphisms associated with immune response and lipid metabolism. PLINK software was used to control the quality of the Miras Biobank genomic database, and SNPs with minor allele frequency (MAF) less than 1%, call rate below 98%, p<5×10⁻⁷ and deviation from Hardy-Weinberg equilibrium (P < 0.05) were excluded. The application of these criteria ensured high accuracy and reliability of the results obtained.

Six polymorphisms of genes related to immune response and lipid metabolism were successfully screened for quality standards. The nonparametric χ² test implemented using PLINK software was used for statistical analysis. Genotype frequencies were checked for compliance with the Hardy-Weinberg law (HWE) using the HWE-test function in PLINK software. Statistical significance of the results was determined at the level of p < 0.05. Additionally, comparative studies were performed to compare the data of our population with the results of studies of other populations. A variety of databases and results of genome-wide association studies (GWAS) of genetic aspects of pre-eclampsia were used to select significant SNPs associated with acute cerebral circulatory failure. The list of resources used included the National Centre for Biotechnology Information (NCBI), Ensembl, Genotype and Phenotype Database (dbGaP), National Human Genome Research Institute (NHGRI), GWAS catalogue, 1000 Genomes Project and others. These resources have provided extensive information on potential genetic markers and their association with disease.

Although initial genotyping encompassed over 2.5 million SNPs using the Illumina Omni Chip 2.5-8 platform, only six SNPs were retained for focused analysis based on stringent filtering and functional relevance. The selection process was guided by both statistical and biological criteria. First, SNPs were filtered for data quality using PLINK software, excluding those with a MAF below 1%, call rate under 98%, and significant deviation from the Hardy-Weinberg equilibrium (p < 0.05). Second, among the high-quality variants, candidate SNPs were prioritised based on their known or hypothesised involvement in the pathophysiological pathways of pre-eclampsia and acute cerebral circulatory failure, specifically within the domains of immune response and lipid metabolism. Selection was informed by a comprehensive review of GWAS, meta-analyses and curated databases such as NCBI, Ensembl, NHGRI GWAS Catalog, dbGaP, and the 1000 Genomes Project. The final set included three SNPs related to immune function (TLR4 rs4986790, PLEKHA1 rs2281673 and PLEKHG1 rs9478812) and three related to lipid metabolism (APOE rs7412, FTO rs1421085, and LPL rs285). These SNPs were chosen due to their established or potential mechanistic roles in inflammatory signalling, endothelial dysfunction, lipid dysregulation and metabolic stress – key processes implicated in the aetiology of pre-eclampsia and its vascular complications. Thus, the focused selection enhances biological interpretability, reduces the risk of type I error associated with multiple testing, and aligns with the study’s goal of identifying population-specific genetic risk markers in a Central Asian cohort.

A study of SNPs associated with ACVD in pre-eclampsia in the Kazakh population revealed significant differences in the frequencies of minor alleles of genes responsible for immune response and lipid metabolism. The present study has implications for understanding genetic risk factors for the development of pre-eclampsia and its complications in a particular ethnic group.

The study analysed genes associated with immune response and lipid metabolism in the Kazakh population. Immune response genes include TLR4 (rs4986790), PLEKHA1 (rs2281673) and PLEKHG1 (rs9478812). Lipid metabolism genes include APOE (rs7412), FTO (rs1421085) and LPL (rs285). The frequencies of minor alleles in the studied sample of Kazakhs were as follows: for TLR4 gene (rs4986790) the frequency was 3.3%, for PLEKHA1 (rs2281673) – 5.9%, for PLEKHG1 (rs9478812) 27.0%, for APOE (rs7412) 7.8%, for FTO (rs1421085) 28.3%, and for LPL (rs285) the minor allele frequency was at 36.0%.

These data emphasise significant population-specific differences in gene frequencies, which may have important implications for understanding genetic susceptibility to diseases such as pre-eclampsia in the Kazakh population. SNP rs4986790 of TLR4 gene showed the lowest frequency of minor allele – 3.3%, and rs285 of the LPL gene showed the highest – 36.0%. These data indicate the presence of specific genetic characteristics in the Kazakh population that may influence the risk of pre-eclampsia and related complications such as ACVD. It is essential to note that allele frequencies may vary between different ethnic groups, emphasising the need for population-specific genetic studies (Table 1).

The TLR4 gene encoding Toll-like receptor 4 plays a key role in the activation of the innate immune response. This receptor recognises pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecules (DAMPs), initiating an inflammatory response. TLR4 is expressed on the surface of various cells of the immune system, including macrophages, dendritic cells and neutrophils. Upon activation, TLR4 triggers a signalling cascade leading to the production of pro-inflammatory cytokines and chemokines.

The low frequency of the minor allele of the TLR4 gene (3.3%) in the Kazakh population may indicate a reduced probability of excessive inflammatory reactions that may contribute to the development of pre-eclampsia and ACVD. This may be the result of evolutionary adaptation to reduce the risk of autoimmune and inflammatory diseases. However, it may also mean that the body may be less efficient at recognising and responding to certain pathogens or tissue damage, which may have its own negative consequences in the context of other diseases. Low-frequency variants may not have high predictive value for conditions such as pre-eclampsia and SLE due to their rarity. However, they provide valuable information about mechanisms, revealing how genetic variations can influence immune responses and inflammation. In particular, the TLR4 variant may protect against excessive inflammation, contributing to a better understanding of disease mechanisms, even if it is not a reliable predictor in clinical practice.

In contrast, the high frequency of the minor allele of the LPL gene (36.0%) associated with lipid metabolism may indicate a higher predisposition to metabolic disorders in the Kazakh population. Lipoprotein lipase, encoded by the LPL gene, plays a pivotal role in lipoprotein and triglyceride metabolism [11,12]. This enzyme hydrolyses triglycerides in chylomicrons and very low-density lipoproteins (VLDL), thereby allowing delivery of fatty acids to tissues for use or storage. Alterations in lipoprotein lipase activity may affect the lipid profile and consequently the risk of cardiovascular disease, including complications of pre-eclampsia [13,14]. The high frequency of the minor allele of the LPL gene may be associated with altered enzyme activity, which may lead to abnormalities in lipid metabolism. This, in turn, may contribute to the development of dyslipidaemia, obesity and insulin resistance, factors that play an important role in the pathogenesis of pre-eclampsia and cardiovascular complications.

The PLEKHG1 gene, which showed a minor allele frequency of 27.0%, is involved in the regulation of cell signalling and cytoskeleton reorganisation. The protein encoded by this gene is a guanine nucleotide exchange factor (GEF) for small GTPases of the Rho family. These GTPases play important roles in a variety of cellular processes including actin cytoskeleton reorganisation, cell adhesion, migration, and polarisation. These processes are important for normal vascular endothelial function, the disruption of which is one of the key mechanisms for the development of pre-eclampsia. The endothelial dysfunction characteristic of pre-eclampsia may be due in part to dysregulation of the cytoskeleton and intercellular contacts, in which PLEKHG1 is involved [15,16]. The intermediate frequency of the minor allele of this gene (27.0%) may indicate a balance between protective and risk factors in relation to the development of vascular complications in the Kazakh population.

The PLEKHA1 gene, the minor allele of which has a frequency of 5.9% in the study population, encodes a protein containing a pleckstrin homologous domain. This protein is involved in cell signalling and vesicular transport. Although its exact role in the development of pre-eclampsia and ACVD is not completely clear, alterations in its function may affect intracellular signalling pathways, potentially contributing to pathological processes.

The APOE gene encoding apolipoprotein E plays a key role in lipoprotein and cholesterol metabolism. The frequency of minor allele SNP rs7412 of this gene in the Kazakh population was 7.8%. Apolipoprotein E is involved in transport and metabolism of lipids, as well as in the repair and regeneration of nervous tissue. Different APOE isoforms (E2, E3, E4) are associated with different risk of cardiovascular and neurodegenerative diseases. In the context of pre-eclampsia and ACVD, variations in the APOE gene may influence the lipid profile and susceptibility of the vascular system to damage.

The FTO gene, known for its association with obesity and metabolic syndrome, showed a minor allele frequency of SNP rs1421085 28.3% in the Kazakh population. The FTO protein functions as a nucleic acid demethylase and is involved in the regulation of metabolism and energy balance. Variations in the FTO gene may influence the risk of obesity, which is a known risk factor for pre-eclampsia and cardiovascular complications.

The distribution of expected and observed genotype frequencies of the genes under study in the Kazakh population was analysed for compliance with the Hardy-Weinberg law. This law describes the stability of allele and genotype frequencies in an ideal population in the absence of evolutionary factors such as mutations, natural selection, gene drift and migration.

The Hardy-Weinberg law is based on several key assumptions. First, the population must be infinitely large, which minimises the effect of random variation in allele frequencies. This condition eliminates the effect of gene drift, which can significantly alter allele frequencies in small populations. Second, mating in the population must occur randomly (panmixia), which means that all individuals have an equal chance to reproduce regardless of their genotype. Third, there should be no natural selection in the population, meaning that all genotypes should have the same fitness and probability of survival and reproduction. In addition, there should be no mutations in the population that could alter allele frequencies. This assumption rules out genetic variability that could arise from new mutations. Finally, for the Hardy-Weinberg law to hold, there must be no migration of genes into or out of the population, which prevents the inflow or outflow of genetic material that could alter allele frequencies. All these assumptions together create ideal conditions under which allele and genotype frequencies remain constant from generation to generation, allowing Hardy-Weinberg’s law to be used to predict population genetic structure in the absence of evolutionary forces.

All the SNPs studied were in Hardy-Weinberg equilibrium (p > 0.05), indicating genetic equilibrium in the population. This is an important result, as it confirms that the study sample was representative and was not subjected to significant external influences that could alter allele and genotype frequencies (Table 2).

This also indicates the absence of significant evolutionary pressures on the studied genes in the population, which allows for the consideration of obtained frequencies as a basic for the Kazakh population. The data obtained in this study can be used for further genetic studies and comparisons with other populations, which contributes to the expansion of knowledge about the genetic structure and adaptations of different ethnic groups.

The results of a comparative analysis of minor allele frequencies of genes related to immune response and lipid metabolism in the Kazakh population and in populations of South Asian and European countries revealed significant differences. During the comparison, adjustment for the false discovery rate was used. This method allowed for a more balanced approach, preserving statistical power while minimising the likelihood of false discoveries in large-scale SNP analyses. The detected differences may be due to multiple factors, including geographic isolation, historical migration routes, founder effects, and various evolutionary pressures acting on the populations over time. For example, the frequency of the minor allele SNP rs4986790 of the TLR4 gene in the Kazakh population was 3.3%. This is significantly lower than in European populations, where this indicator reaches 5.7%, and in South Asian populations, where it is 12.0%. Such differences may indicate possible ethnic variations in genetic susceptibility to inflammatory processes. This, in turn, may influence the risk of developing diseases such as pre-eclampsia and ACVD.

The lower frequency of the TLR4 minor allele in the Kazakh population may indicate a lower genetic predisposition to an excessive inflammatory response, which may be a protective factor against certain inflammatory diseases. This may be the result of adaptation to certain environmental or pathogenic factors specific to the region. However, it may also mean a reduced ability to recognise certain pathogens, which may have its own negative consequences in the context of infectious diseases.

The frequency of the minor allele SNP rs2281673 of the PLEKHA1 gene was 5.9%, which is not significantly different from the frequency in South Asian populations (8.2%) and significantly higher compared to European populations (0%). These data suggest a possible role of this gene in the pathogenesis of pre-eclampsia and ACVD in Asian populations, but not in European populations. The PLEKHA1 gene is involved in the regulation of intracellular signalling and may influence the processes associated with the development of vascular complications. Differences in allele frequencies of this gene between populations may partly explain differences in the prevalence and course of pre-eclampsia in different ethnic groups.

The presence of this minor allele in Asian populations, including the Kazakh population, and its absence in European populations may indicate specific adaptations to environmental or dietary conditions characteristic of the Asian region. It may also indicate different mechanisms of development of pre-eclampsia and its complications in different ethnic groups, highlighting the importance of population-specific studies and personalised approach to diagnosis and treatment.

The minor allele SNP rs9478812 of the PLEKHG1 gene had a frequency of 27.0% in the Kazakh population, which was significantly lower than in East Asian populations (52.0%) and higher than in European populations (21.0%). These differences may reflect different genetic mechanisms involved in the development of pre-eclampsia and its complications in different ethnic groups. The PLEKHG1 gene is involved in the regulation of cell signalling and cytoskeleton reorganisation, which is important for normal vascular endothelial function. The intermediate frequency of the minor allele of this gene in the Kazakh population may indicate a unique balance between risk and protective factors with respect to the development of vascular complications. This frequency distribution may be the result of the complex history of the Kazakh population, including the mixing of different ethnic groups. It may also reflect specific adaptations to environmental and social factors characteristic of the region (Table 3).

Regarding lipid metabolism genes, the frequency of the minor allele SNP rs285 of the LPL gene in the Kazakh population (36.0%) was significantly lower compared to the European (48.0%) and East Asian (66.0%) populations. This may indicate a potentially more favourable lipid profile in the Kazakh population, which may serve as a protective factor against the development of ACVD in pre-eclampsia.

Lipoprotein lipase, encoded by the LPL gene, plays a key role in lipid metabolism by hydrolysing triglycerides in lipoproteins. Differences in the allele frequency of this gene may influence the efficiency of lipid metabolism and therefore the risk of cardiovascular disease. The lower frequency of the minor allele in the Kazakh population may be associated with adaptation to a traditional diet and lifestyle, potentially reducing the risk of metabolic disorders.

The frequency of the minor allele SNP rs7412 of the APOE gene in the Kazakh population was 7.8%, which is not significantly different from the frequencies in European and East Asian populations. The APOE gene plays an important role in lipoprotein metabolism and is associated with the risk of cardiovascular disease. The similarity of frequencies of this allele between populations may indicate a universal role of this gene in lipid metabolism and the development of vascular complications.

Apolipoprotein E, encoded by the APOE gene, is involved in lipid transport and metabolism and plays a role in neural tissue regeneration. Different APOE isoforms (E2, E3, E4) are associated with different risks of cardiovascular and neurodegenerative diseases. The similarity of minor allele frequencies between populations may indicate the conservation of an important functional role of this gene during human evolution.

The frequency of the minor allele SNP rs1421085 of the FTO gene in the Kazakh population was 28.3%. The FTO gene is known for its association with obesity and metabolic syndrome. Variations in this gene may affect eating behaviour and energy balance of the body. The intermediate frequency of the minor allele in the Kazakh population may reflect a balance between obesity risk factors and adaptation to traditional lifestyle and diet.

The results of this study showed that SNPs of immune response genes and lipid metabolism can be used as prognostic genetic markers for the development of pre-eclampsia and its complications in the form of ACVD in the Kazakh population. These markers can be used to develop personalised approaches to the prevention and treatment of pre-eclampsia and its complications.

SNP rs285 of the LPL gene, the minor allele of which has a frequency of 36.0% in the Kazakh population, can be used as a protective marker of the risk of ACVD development in pre-eclampsia. Its lower frequency compared with European and East Asian populations may indicate a potentially more favourable lipid profile in the Kazakh population. This may be associated with a lower risk of atherosclerosis and other vascular complications that may aggravate the course of pre-eclampsia and increase the risk of ACVD. The use of SNP rs285 of the LPL gene as a genetic marker may help in the risk stratification for the development of ACVD in patients with pre-eclampsia. Patients with a protective allele may have a lower risk of developing severe vascular complications, which may influence the management tactics of pregnancy and labour. The frequencies of other SNPs such as rs7412 of the APOE gene and rs1421085 of the FTO gene also indicate the possibility of their use as prognostic markers. The APOE gene, known for its role in lipid metabolism and cardiovascular disease risk, showed a minor allele frequency of 7.8% in the Kazakh population, which is not significantly different from frequencies in European and East Asian populations. This may indicate a universal role of this gene in the development of vascular complications in pre-eclampsia.

Different APOE isoforms (E2, E3, E4) are associated with different risk of cardiovascular disease. Determination of APOE genotype may help in assessing individual risk of vascular complications in pre-eclampsia and ACVD. For example, carriers of the E4 allele may have an increased risk of atherosclerosis and other vascular pathologies, which may require more intensive monitoring and preventive measures.

The FTO gene, associated with obesity and metabolic syndrome, may also play an important role in the development of pre-eclampsia and its complications. The frequency of minor allele SNP rs1421085 of this gene in the Kazakh population was 28.3%, which may reflect an intermediate risk of metabolic disorders associated with pre-eclampsia. Variations in the FTO gene may influence the risk of obesity, which is a known risk factor for pre-eclampsia and cardiovascular complications. Determination of the FTO genotype may help to identify patients at increased risk of developing metabolic disorders and hence pre-eclampsia. This may be particularly important for the development of personalised prevention strategies, including dietary recommendations and physical activity programmes.

Pre-eclampsia and acute cerebrovascular disease are complex diseases, indicating that their onset is influenced by both hereditary and several non-genetic factors related to the environment and lifestyle [18,19,20]. These factors include maternal age, comorbidities (including hypertension, diabetes, and obesity), socioeconomic status, and access to healthcare. These factors may interact with genetic predisposition identified in the study, thereby altering the clinical manifestations of pre-eclampsia and ACVD in complex ways. Environmental factors, including dietary habits (e.g., excessive sodium intake or insufficient calcium intake) and lifestyle (e.g., low physical activity, smoking), may exacerbate the influence of genetic risk factors, especially those related to lipid metabolism and immune function. In addition, the presence of comorbidities such as obesity, metabolic syndrome, or chronic hypertension, which are common in populations with an increased genetic predisposition to metabolic disorders, can significantly alter the expression of genetic variants, thereby increasing the risk of developing pre-eclampsia or HELLP syndrome.

The relationship between genes and the environment in relation to pre-eclampsia and HELLP syndrome is important for understanding the etiology of the disease and improving its treatment. Genetic variations in lipid metabolism genes, such as LPL or APOE, which play a role in lipid homeostasis and vascular function, can be altered by environmental factors such as diet or physical activity, thereby influencing the onset of dyslipidemia and its subsequent vascular consequences. Similarly, immune response genes, such as TLR4, may interact with inflammatory environmental stimuli, including infections or stress, thereby exacerbating immune system dysfunction and vascular damage. In addition, access to medical care and prenatal care is crucial in the treatment of these diseases, as timely monitoring and interventions can reduce the risks associated with both genetic predisposition and environmental exposure. Therefore, understanding these relationships is important for developing individualised strategies for the prevention and treatment of pre-eclampsia and HELLP syndrome.