Association of BMAL1 and CLOCK Gene Polymorphisms with Preeclampsia Risk with Subtype Analysis

Preeclampsia (PE), a multifactorial hypertensive disorder arising after 20 weeks of gestation, is characterized by new-onset hypertension with proteinuria or end-organ dysfunction [1]. It is clinically categorized into early-onset PE (eoPE, diagnosed <34 weeks of gestation) and late-onset PE (loPE, diagnosed ≥34 weeks of gestation), the former of which is linked to considerably worse clinical outcomes [2,3]. Affecting approximately 2% to 8% of pregnancies worldwide, and 4% to 5% in China, PE poses serious risks to both maternal and fetal health [4,5]. It is associated with acute maternal complications such as HELLP syndrome, and elevates the long-term maternal risks of metabolic diseases including cardiovascular disease, chronic kidney disease, and type 2 diabetes mellitus [6,7,8,9,10,11]. Additionally, PE adversely affects the fetus, increasing the risks of growth restriction, preterm birth, and placental abruption [12]. As a leading cause of maternal mortality, PE results in an estimated 76,000 maternal deaths and 500,000 fetal or neonatal deaths globally each year [13]. Despite advances in medical technology, termination of pregnancy remains the only intervention that definitively resolves the underlying pathological cause of PE, namely placental dysfunction [14]. It is imperative to advance etiological research for identifying high-risk populations based on pathogenic mechanisms, thereby facilitating early screening and intervention to prevent PE onset, improve pregnancy outcomes, and reduce societal healthcare burdens.

Although the precise mechanisms underlying PE remain incompletely elucidated, two predominant pathophysiological theories have been proposed: the placental two-stage model and the maternal cardiometabolic maladaptation theory [15,16,17]. According to the two-stage model, the initial stage involves defective placental development due to impaired trophoblast invasion and inadequate remodeling of the spiral arteries, leading to reduced placental perfusion, hypoxia, and oxidative stress [17,18,19]. It is widely recognized that these placental alterations provoke the release of anti-angiogenic factors, such as soluble fms-like tyrosine kinase-1 (sFlt-1), and pro-inflammatory mediators into the maternal circulation, initiating the second stage—systemic endothelial dysfunction, inflammatory activation, and the clinical manifestations of PE [20,21,22,23]. EoPE is closely aligned with this model, characterized by profound placental insufficiency, elevated sFlt-1 levels, decreased vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), and frequent fetal growth restriction [24,25,26,27]. Alternatively, the cardiometabolic theory posits that pre-existing maternal cardiovascular dysfunction or metabolic abnormalities—such as chronic hypertension, obesity, or insulin resistance—impair the hemodynamic adaptations to pregnancy, ultimately leading to placental hypoperfusion and PE [28,29,30,31]. It is observed that loPE is often associated with this etiology, wherein maternal factors predominate over primary placental defects. It is also noted that the clinical presentation is frequently less severe with respect to angiogenic imbalance and placental pathology [32,33,34].

Emerging evidence suggests that disruption of placental circadian rhythms may contribute significantly to the pathogenesis of PE [35,36]. Circadian rhythms are endogenous oscillations with a period of approximately 24 h that regulate numerous biological processes through molecular clockworks [37,38]. In mammals, these rhythms are coordinated by the suprachiasmatic nucleus (SCN) of the hypothalamus, which serves as the central pacemaker that synchronizes peripheral oscillators across various tissues via the hierarchical regulation of clock gene expression [39,40]. The core molecular clock operates through interlocked transcriptional-translational feedback loops involving key genes such as brain and muscle Arnt-like protein 1 (BMAL1, also termed ARNTL), circadian locomotor output cycles kaput (CLOCK), cryptochromes (CRYs) and period homolog proteins (PERs) [41], all of which are expressed in animal and human placental tissue [42,43,44,45,46] and extravillous trophoblast cell lines [47]. Notably, among these placental clock genes, circadian rhythmicity is exclusively observed in the expression of BMAL1 and CLOCK [35,46]. Importantly, dysregulation of the BMAL1 and CLOCK genes has been closely linked to core pathological mechanisms of PE, including imbalances in vascular endothelial dysfunction [48,49,50,51], as well as mitochondrial impairment and oxidative stress [52,53,54,55]. These observations position BMAL1 and CLOCK genes as potential central players in PE development. The BMAL1 gene is located on chromosome 11p15.3, while the CLOCK gene is located on chromosome 4q12. Altered expression of the CLOCK gene has been observed in placental tissue from PE patients [56]. However, given the practical challenges in obtaining placental tissue prenatally, there is a critical need to identify non-invasive biomarkers. Intriguingly, BMAL1 and CLOCK gene polymorphisms have been associated with hypertension in a study of healthcare workers [57], supporting their potential role in PE pathologies. Therefore, we hypothesize that specific single nucleotide polymorphisms (SNPs) detectable in peripheral blood, which regulate BMAL1 and CLOCK expression, could serve as genetic proxies for placental gene function and be associated with PE risk.

In summary, this case–control study aimed to investigate the associations of BMAL1 and CLOCK gene polymorphisms with PE risk. Following genotyping and quality control that included testing for Hardy–Weinberg equilibrium (HWE) as well as linkage disequilibrium (LD) pruning, we evaluated the associations of single SNPs, haplotypes, and gene–environment interactions with PE risk. Given the potential etiological differences between early-onset and late-onset PE, the distribution of PE-associated variants was further analyzed across these subtypes. Expression quantitative trait locus (eQTL) analysis was performed to evaluate the correlations between these PE-associated risk loci and BMAL1/CLOCK gene expression levels in whole blood. Furthermore, a protein–protein interaction (PPI) network analysis was employed to uncover functional pathways implicated by PE-associated genes.

In this study, we identified a polymorphism within the BMAL1 gene (rs11022780) that was significantly associated with the risk of PE. Further subgroup analysis revealed that this association was predominantly attributed to eoPE. Functional relevance of this variant was supported by cis-eQTL analysis, indicating its role in modulating BMAL1 expression in whole blood. Additionally, PPI analysis further confirmed the central role of BMAL1 as a hub protein within the circadian rhythm network. In contrast, no significant associations were observed for the tested polymorphism in the CLOCK gene (rs1048004) or the other two BMAL1 variants (rs4757144 and rs969485) with PE risk.

Our study identified a significant association between the BMAL1 gene polymorphism (rs11022780) and PE, which was predominantly driven by the eoPE subtype. This finding lends genetic support to the growing evidence implicating circadian clock disruption, specifically through BMAL1 dysregulation, in PE pathogenesis. The subtype-specificity of this association aligned with the distinct pathophysiological theories: the ‘placental’ theory for eoPE and the ‘maternal’ theory for loPE. The observed genetic risk likely contributed to the placental maldevelopment that characterizes eoPE through several interconnected mechanisms. As a core clock transcription factor, BMAL1 is known to influence cell proliferation and invasion. Given its documented role in modulating pathways like Wingless/Integrated (WNT)/β-catenin [60,61], a reduction in its expression may similarly disrupt key placental processes. This could manifest as impaired trophoblast invasion and spiral artery remodeling [17,62], which are central to the pathophysiology of eoPE. Furthermore, BMAL1 is crucial for maintaining redox homeostasis, and its deficiency is linked to attenuated antioxidant capacity and exacerbated oxidative damage [63,64]—a phenotype highly consistent with the severe placental ischemia–reperfusion injury and oxidative stress observed in eoPE [3,24,65]. Beyond placental and oxidative stress mechanisms, BMAL1 dysfunction may also impair vascular function by negatively influencing VEGF signaling [49] and promoting endothelial inflammation and dysfunction [51,66], thereby contributing to the systemic vascular abnormalities and hypertension in eoPE. In contrast, the lack of a significant association in the loPE subgroup reflected its more heterogeneous etiology, often linked to pre-existing maternal cardiometabolic dysfunction [32,33,34]. However, this interpretation had to be tempered by the results of our post hoc power analysis, which revealed limited statistical power for the loPE subgroup analyses (power = 25.4–29.3%). Consequently, the absence of a significant association for loPE should be viewed as inconclusive due to the high risk of a type II error, and a potential role for BMAL1 in a subset of loPE cases cannot be ruled out. The significant association detected in eoPE, despite a borderline p-value and modest power (55.8–77.5%) for some genotype comparisons, suggests that the underlying genetic effect in this subtype is likely substantial enough to be detectable even in our cohort, yet it still warrants confirmation in larger, specifically powered studies. Collectively, our data suggest that BMAL1 genetic variation may predispose to eoPE by disrupting core placental processes, including invasion, oxidative balance, and vascular function, while its role in loPE remains an open question necessitating future investigation in larger cohorts.

In line with a previous study of healthcare workers that identified the TT genotype of BMAL1 rs11022775 as protective against hypertension (OR = 0.426) [57], our study found a significant associations of the BMAL1 SNP rs11022780 with PE. Notably, under both codominant (TT vs. CC) and recessive (TT vs. CC + CT) genetic models, the TT genotype of rs11022780 conferred a protective effect against PE (aOR = 0.26 and 0.25, respectively). Although this SNP was located within an intronic region, further functional analyses indicated that the C allele of rs11022780 was correlated with decreased BMAL1 mRNA expression in whole blood. Supporting the potential functional relevance of this variant, Burgermeister et al. reported that in metastatic colorectal cancer patients receiving bevacizumab (an anti-VEGF therapy), the TT genotype of rs11022780 was associated with longer overall survival, whereas the C allele predicted poorer clinical outcomes (HR = 1.61, p = 0.014) [67]. This finding gained additional biological plausibility given the known similarities between trophoblast behavior and tumor cell biology. As a core regulator of the molecular circadian clock, BMAL1 was unique in that its single-gene ablation results in a complete loss of circadian rhythmicity [68]. In the present study, the C allele of rs11022780 was associated with reduced BMAL1 expression in whole blood. The lower frequency of the protective genotype (TT for rs11022780) in PE cases suggests that reduced BMAL1 expression may disrupt circadian rhythmicity and lead to downstream effects such as impaired VEGF signaling. These disturbances may represent a potential mechanism contributing to vascular dysfunction and inadequate placental remodeling, thereby possibly playing a role in the pathogenesis of PE [3,49,67]. While our findings suggest a protective mechanism mediated by BMAL1 expression, the extrapolation of this eQTL evidence from whole blood to placental pathophysiology warrants careful consideration.

In this study, the eQTL evidence linking rs11022780 to BMAL1 expression was derived from publicly available whole-blood datasets rather than placenta. We recognize that placental tissue is the most relevant for PE pathophysiology and that placenta-specific eQTLs would offer more direct evidence; indeed, dedicated placental eQTL studies have revealed numerous biologically significant eQTLs, some of which colocalize with loci for birthweight and other perinatal traits [69]. At the same time, large cross-tissue eQTL resources demonstrate that many cis-eQTLs are shared across diverse tissues, providing a practical justification for utilizing whole-blood data when placental eQTLs are unavailable [70]. Additionally, the use of whole-blood eQTLs offers a practical advantage, as blood sampling is non-invasive and more feasible for large-scale population studies compared with placental tissue collection. Moreover, core circadian genes including BMAL1 and CLOCK are expressed in the human placenta and have established roles in placental function and PE pathogenesis [71], supporting the biological plausibility that genetic regulation of BMAL1 may influence PE risk via placental mechanisms. While our genetic and eQTL data suggest that the rs11022780 locus influences PE risk by modulating BMAL1 expression, future work is needed to deepen our understanding of this regulatory relationship through direct transcriptomic and proteomic analyses in disease-relevant models, including placental trophoblasts.

In contrast, our study did not reveal a significant association between PE susceptibility and the evaluated CLOCK SNPs—rs1048004. It is noteworthy that rs1048004 is located within the 3′-UTR region of the CLOCK gene, where sequence variations may influence mRNA stability or translational efficiency. Previous studies conducted in an American population have reported significant associations between this SNP and breast cancer risk [72]. This observation provides indirect evidence supporting the possibility that this loci possess regulatory functions. However, such effects appear not to extend to PE pathogenesis in our cohort. One potential explanation for this discrepancy lies in the phenotypic heterogeneity of PE. Supporting this view, Zhou et al. reported that CLOCK transcript levels were significantly reduced specifically in term PE placentas, but not in preterm cases, pointing to a potential subtype-specific association [36]. Our study population consisted predominantly of eoPE (n = 97) and loPE (n = 105) cases, with eoPE being particularly prone to result in preterm delivery [73]. Thus, the limited number of true term PE cases in our cohort may have attenuated the ability to detect an association with CLOCK gene polymorphism. Additionally, the absence of an association in the present study could be attributed to the possibility that the evaluated SNP may not capture the true functional variants within the CLOCK gene relevant to PE in the Chinese population. Genetic architecture, including allele frequencies and linkage disequilibrium patterns, often differs across ethnic groups. Therefore, risk loci identified in study of American populations may not be directly generalizable to Chinese individuals, owing to divergent genetic backgrounds and environmental influences.

Collectively, our results demonstrated that the rs11022780 locus in the BMAL1 gene was significantly associated with PE, most notably with eoPE, and suggested that it may operate by modulating BMAL1 expression, thereby providing novel molecular insights into the genetic architecture of the disease. Several limitations must be considered when interpreting these findings. As a hospital-based case–control investigation, the study may have been affected by admission bias. Moreover, although no significant association was detected between this SNP and late-onset PE, this absence of association likely reflects the limited sample size in the loPE subgroup rather than a true biological absence, underscoring the need for larger subtype-stratified analyses in the future. In addition, the eQTL analysis was based on whole-blood datasets rather than placental tissue, which is more directly relevant to PE; while cross-tissue studies have shown that many cis-eQTLs are shared across tissues, future validation using placenta-derived data will be important to confirm the observed regulatory relationship. The molecular consequences of this variant remain to be fully elucidated through direct transcriptomic or proteomic studies. Furthermore, data on peripartum management, such as antithrombotic therapy, were not collected, and their potential influence on outcomes was not assessed. Finally, our findings are derived from a single-center Chinese cohort; therefore, both independent validation in external populations and investigation in diverse ethnic groups are necessary to confirm the robustness and generalizability of the observed genetic effect.

This study identifies the BMAL1 rs11022780 polymorphism to be associated with PE, with this association being notably observed in eoPE. Functional evidence indicates a potential role for this variant in mediating disease risk via regulatory effects on BMAL1 expression. Protein interaction data further establish BMAL1 as a central component of the circadian rhythm network. Collectively, these results offer multilevel genetic and mechanistic insights into the role of BMAL1 in PE pathogenesis, with particular relevance to eoPE cases. This work provides vital insights into PE’s etiology and holds promise for developing non-invasive genetic biomarkers for early risk stratification, which is crucial for improving pregnancy outcomes.