Analysis of the Phenotype and Gene Mutations of Two Families With Combined Mutations of Anticoagulant Protein Genes

ABSTRACT

Background

AT and PC are key components of the anticoagulant system. Mutations in their encoding genes, SERPINC1 and PROC, can lead to insufficient protein levels or impaired function, thereby increasing an individual's susceptibility to venous thromboembolism (VTE). In this study, we describe the clinical characteristics and functional effects of compound heterozygous mutations in SERPINC1 and PROC in two pedigrees.

Methods

Anticoagulant protein activity and antigen levels were evaluated in family members. Targeted sequencing was performed using next‐generation sequencing (NGS) and CNVplex technology. The identified variants were assessed for evolutionary conservation, pathogenic potential, and their impact on protein structure. Thrombin generation was measured using the calibrated automated thrombogram (CAT) assay.

Results

The PC:A and PC:Ag of proband 1 were decreased to 57% and 59.2%, and PC:A of proband 2 was decreased to 68%. The SERPINC1 (OMIM#:613118) and PROC (OMIM#:176860) gene analysis indicated that there were c.400+5G>A and c.883G>A in proband 1, c.811C>T and c.880C>T in proband 2, respectively. These mutation sites are highly conserved across homologous species. Bioinformatics analysis predicts their potential pathogenicity, suggesting that they may alter the three‐dimensional structures of both AT and PC proteins, thereby compromising their functional integrity. Thrombin generation assays revealed that two AT mutation carriers exhibited varying degrees of elevated ETP. In the presence of sTM, two PC mutation carriers showed significantly impaired plasma anticoagulant function without a significant reduction in thrombin generation.

Conclusion

In both pedigrees, we identified two distinct mutations in AT and PC. Dual mutations in SERPINC1 and PROC confer markedly increased VTE susceptibility.

In these two pedigrees, we identified distinct mutations in the AT and PC genes. Individuals harboring both mutations exhibited a significantly higher incidence of venous thrombosis.

1. Introduction

Venous thromboembolism (VTE) represents a significant global health burden. Deep vein thrombosis (DVT) is the primary source of thromboemboli, which can dislodge and travel to the lungs, leading to pulmonary embolism (PE)—a potentially life‐threatening complication. The annual incidence of VTE in Asia is 0.13 to 0.57 cases per 1000 people (Omesiete et al. 2018; Thachil et al. 2022), and it shows a steady upward trend (Wendelboe and Weitz 2024). Venous thromboembolism (VTE) is a multifactorial disorder resulting from the interplay of acquired and genetic risk factors (Connors 2017). The presence of genetic abnormalities in conjunction with any thrombogenic factors—such as diabetes, obesity, smoking, oral contraceptive use, hormone replacement therapy, malignancy, antiphospholipid syndrome, fractures, surgical procedures, and pregnancy can significantly enhance blood coagulability and increase the risk of venous thromboembolism (VTE). Current evidence suggests that genetic factors contribute to over 60% of the risk for venous thromboembolism (VTE) pathogenesis, primarily through complex interactions between inherited thrombophilia traits and acquired environmental risk factors (Seligsohn and Lubetsky 2001). In China, deficiencies in antithrombin (AT), protein C (PC), and protein S (PS) represent among the most significant genetic risk factors for venous thromboembolism (VTE) (Zhang, Gui, et al. 2020; Zhang et al. 2022). Clinically, thromboembolic events associated with deficiencies in anticoagulant proteins typically present early in life, are characterized by a high risk of recurrence, and often manifest with sudden onset. A significant number of patients have a positive family history of thrombosis. Several studies have demonstrated considerable variability in the clinical manifestations of patients with anticoagulant protein deficiency, which is influenced by the number of mutations, the functional consequences of these mutations, and associated gene polymorphisms (Li et al. 2019). Animal model studies have demonstrated that impaired synthesis of hepatic antithrombin and protein C can precipitate acute, life‐threatening venous thrombosis (Kovac et al. 2017). In this study, we conducted comprehensive phenotypic and clinical evaluations of two patients with severe venous thromboembolism (VTE) associated with combined mutations in hereditary anticoagulant proteins, as well as their family members. Bioinformatics tools were employed to investigate the underlying molecular pathogenesis and its functional consequences on coagulation.

2. Materials and Methods

2.1. Ethical Compliance

This study has been approved by the Ethics Committee of Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine, and all participants have signed informed consent forms.

2.2. Patients

2.2.1. Patient 1

Male, diagnosed with left lower extremity venous and external iliac vein thrombosis, which resulted in pulmonary embolism at an outside institution, sought further evaluation of the underlying etiology at Ruijin Hospital. Currently, the patient is undergoing oral anticoagulant therapy with Rivaroxaban (15 mg twice daily) and wearing compression stockings, maintaining a stable condition. No history of thrombosis was identified among other family members. The pedigree is illustrated in Figure 1A.

FIGURE 1.

Family pedigrees of two patients.

2.2.2. Patient 2

Male, experienced sudden left lower extremity DVT at the age of 27 without standardized anticoagulant treatment. At the age of 35, he developed recurrent left lower extremity DVT and PE, for which he received intermittent anticoagulant therapy with Rivaroxaban. He was subsequently admitted to Ruijin Hospital due to sudden onset of chest tightness, and PE was confirmed via CT imaging. The patient underwent thrombolytic therapy, inferior vena cava filter placement, and was treated with low molecular weight heparin (LMWH) for 2 weeks. Following discharge, maintenance therapy with Rivaroxaban 20 mg once daily was initiated, and his condition has remained stable since then. No history of thrombosis was identified among other family members. The pedigree is shown in Figure 1B.

The control group: In order to establish a laboratory reference range, one hundred healthy subjects were randomly selected from our hospital as controls to exclude genetic polymorphisms consisted of 100 healthy individuals undergoing physical examination, including 52 males and 48 females, aged between 22 and 46 years.

2.3. Collection and Processing of Specimens

Collect 2.7 mL of peripheral venous blood from the study subjects, anticoagulate with a 0.109 mol/L sodium citrate solution at a ratio of 1:9, and centrifuge at 3000 r/min for 15 min. Subsequently, collect the upper platelet‐poor plasma for phenotypic indicator detection. The lower blood cell fraction is used for genomic DNA extraction and stored at −40°C for subsequent testing. The Blood Genomic DNA Extraction Kit (centrifugal column type, batch number Y2117) was purchased from Beijing Tiangen Biochemical Technology Co. Ltd., and the extraction PROCedure was strictly performed according to the manufacturer's instructions.

2.4. Laboratory Phenotyping

Clinical examination data were collected from the subjects and their relevant family members. Antithrombin activity (AT:A) and protein C activity (PC:A) were measured using chromogenic substrate assays, while protein S activity (PS:A) was determined via a coagulation‐based method. All assays were performed on an ACL‐TOP fully automated coagulation analyzer (Instrumentation Laboratory, USA) using the manufacturer's reagent kits. Antithrombin antigen (AT:Ag) and protein C antigen (PC:Ag) levels were quantified using enzyme‐linked immunosorbent assay (ELISA) with commercial reagent kits (Enzyme Research Laboratories, USA). All procedures were performed in strict accordance with the manufacturer's protocols.

2.5. Whole Exome Sequencing and Sanger Sequencing Validation

The thrombophilia gene testing panel, designed by Ruijin Hospital affiliated with Shanghai Jiao Tong University School of Medicine, includes a total of 35 candidate genes associated with thrombosis. Next‐generation sequencing (NGS) and CNVplex technology were employed to detect point mutations, small deletions or insertions, and copy number variations (CNVs) in the coding and regulatory regions of these genes. The identified variations were confirmed by Sanger sequencing through comparison with the reference PC (GenBank NG_016323) and AT (GenBank X68793.1) gene sequences deposited in the American NCBI GenBank. Identical mutation sites were subsequently detected in additional family members, supporting the familial inheritance pattern.

2.6. Thrombin Generation Tests

Due to ongoing anticoagulation therapy in both probands, which rendered thrombin generation testing unreliable, we performed thrombin generation assays using platelet‐poor plasma (PPP) obtained from the probands' parents (four mutation carriers). This approach allowed assessment of the mutations' effects on thrombin generation potential. Thrombin generation was measured using a calibrated automated thrombogram (CAT) system (Diagnostica Stago, Asnières, France) according to the manufacturer's specifications. The assay was initiated by adding PPP reagent containing 5 pM tissue factor (Diagnostica Stago). Thrombin generation profiles were then quantitatively compared between plasma samples from mutation carriers and healthy controls. To evaluate thrombomodulin‐mediated protein C pathway function, we compared thrombin generation in healthy controls versus protein C mutation carriers following addition of 10 nM soluble recombinant human thrombomodulin (sTM; Sekisui Diagnostics, Stamford, CT). All assays were performed in duplicate. Baseline levels of AT, PC, and PS activity were comparable between control and mutation carrier plasma samples. Thrombin generation parameters analyzed included: Endogenous Thrombin Potential (ETP)—the integral of thrombin activity over time (area under the curve), representing total thrombin‐generating capacity; Peak thrombin—the maximal concentration of thrombin achieved during the assay; and Start Tail Time (STT), which is the total duration of the reaction. The kits and software were provided by Thrombinoscope BV, and all procedures were strictly followed according to the instructions provided with the reagent kits (Hemker and Giesen 2003).

2.7. In Silico Analysis

Sequence Conservation Analysis and Structural Modeling Sequence conservation analysis was performed using ClustalX‐2.1‐win to assess the evolutionary conservation of the target regions across Homo sapiens and eight homologous species (HomoloGene database; http://www.ncbi.nlm.nih.gov/homologene). For structural characterization, we utilized experimentally resolved three‐dimensional protein models of the PROC (PDB: AF‐P04070‐F1) and SERPINC1 (PDB: AF‐P01008‐F1) genes obtained from the UniProt database (https://www.uniprot.org/). PyMol (version 3.1) was employed to analyze mutant protein models, with particular focus on local conformational changes and alterations in intermolecular interactions.

3. Results

3.1. Phenotyping Results

In pedigree 1, the PC:A and PC:Ag of proband (II1), father (I1), slightly decreased; the values were 57%, 59.2% and 54%, 65.8%. However, All other related parameters in family members were within normal limits. In pedigree 2, the PC:A and AT:A of proband (II1), mother (I2) were slightly decreased, with values 68%, 78%, respectively. While all other measured parameters in family members remained within normal reference intervals (see Table 1).

TABLE 1.

Results of coagulation tests of family members.

Family members	Age (years)	Blood coagulation indicators
		AT:A (%)	PC:A (%)	PS:A (%)	AT:Ag (mg/L)	PC:Ag (%)
1‐II1	17	109	57	106	294.4	59.2
1‐I1	49	112	54	79	267.0	65.8
1‐I2	46	89	126	79	320.9	95.1
2‐II1	42	116	68	93	253.3	107.3
2‐I1	63	104	72	113	253.3	132.5
2‐I2	60	78	129	105	276.0	129.0
Reference range	—	84–120	70–140	60–130	250.0–360.0	75.0–150.0

Abbreviations: AT:A, antithrombin activities; AT:Ag, antithrombin antigen; PC:A, protein C activity; PC:Ag, protein C antigen; PS:A, protein S activity.

3.2. Results of Genetic Analysis

Genetic analysis found that proband 1 carried the c.400+5G>A splice site mutation of PROC and c.883G>A (p.Val295Met) heterozygous mutation of SERPINC1. The proband 2 carried c.811C>T (p.Arg271Trp) heterozygous mutation of PROC and c.880C>T (p.Arg294Cys) heterozygous mutation of SERPINC1. The PROC and SERPINC1 mutations in the two probands were inherited from their father and mother, respectively.

3.3. Conservation Analysis of Homologous Species

Using ClustalX‐2.1‐win software, we assessed amino acid conservation across homologous species. The analysis revealed: Strong conservation of PROC residue Arg271 in seven homologous species (Figure 2A); High conservation of SERPINC1 residues Arg294 and Val295 across eight homologous species (Figure 2B). These evolutionarily preserved positions suggest critical structural and functional roles in their respective proteins.

FIGURE 2.

Conservation analysis of homologous species. (A) Arg271 of PROC; (B) Arg294 and Val295 of SERPINC1.

3.4. In Silico Analysis

Computational analyses consistently predicted deleterious effects for the identified mutations: The SERPINC1 c.883G>A variant was classified as deleterious/probably damaging by consensus across four prediction tools (SIFT, CADD, FATHMM, PolyPhen‐2); The SERPINC1 c.880C>T variant similarly received deleterious/probably damaging predictions from multiple algorithms (VEST3, CADD, FATHMM, PolyPhen‐2); The PROC c.811C>T variant was unanimously assessed as pathogenic by seven prediction tools (MutationTaster, PolyPhen‐2, PROVEAN, and four others) (Table 2).

TABLE 2.

The pathogenicity prediction results of 3 mutation sites.

Gene	SERPINC1		PROC
Amino acid variations	p.Val295Met	p.Arg294Cys	p.Arg271Trp
MutationTaster	Polymorphism	Polymorphism	Diseasecausing
SIFT	Damaging	Tolerable	—
Polyphen‐2	Probably damaging	Probably damaging	Disease‐causing
PROVEAN	Tolerable	Tolerable	Damaging
VEST3	Tolerable	Damaging	Damaging
CADD	Damaging	Damaging	Damaging
FATHMM	Damaging	Damaging	Damaging
MetaLR	Tolerable	Tolerable	Damaging

Structural modeling and predictive analysis using PyMol indicate that the Arg294 → Cys294 mutation may lead to a significant shortening of the amino acid side chain, thereby disrupting the original hydrogen bond interaction with the adjacent Gly298 residue. For the Val295 → Met295 mutation, the extended side chain of methionine may alter the intermolecular spatial forces. In addition, the substitution of Arg271 with tryptophan results in the disappearance of three hydrogen bonds previously formed with the adjacent Asp269 residue, and the presence of two benzene rings in Trp271 may increase the intermolecular steric hindrance. According to the model's prediction, the alterations of the above three amino acids are very likely to eventually lead to a decrease in the stability of the corresponding protein structure and a change in its function (Figure 3).

FIGURE 3.

Antithrombin and protein C Mutation model analysis. Model building was performed by using the PyMol programs of AF‐P01008‐F1.pdb and AF‐P04070‐F1 (https://www.uniprot.org/). (A1) Wild‐type and (A2) PROC p.Arg271Trp; (B1) Wild‐type and (B2) SERPINC1 p.Arg294Cys; (C1) Wild‐type and (C2) SERPINC1 p.Val295Met; the hydrogen bond is depicted in yellow.

3.5. Thrombin Generation Assay

Compared with the plasma of normal controls, the ETP and Peak of 4 mutation carriers showed varying degrees of increase, suggesting that 2 PC (Protein C) mutations and 2 AT (Antithrombin) mutations may lead to a hypercoagulable state of the blood (Figure 4). Upon treatment with 5 nM soluble thrombomodulin (sTM), PC was activated and exerted its anticoagulant effect, leading to a marked reduction in thrombin generation in the plasma of healthy controls. Specifically, ETP and peak thrombin levels decreased to 23.8% and 37.6% of baseline, respectively, yielding an ETP inhibition rate of 76%. In contrast, carriers of the c.400+5G>A (1‐I1) and p.Arg271Trp (2‐I1) mutations exhibited minimal suppression of thrombin generation, with ETP inhibition rates of only 13% and 40%, respectively. These findings suggest that these genetic mutations may impair the anticoagulant function of the protein C pathway (Table 3).

FIGURE 4.

Thrombin generation tests of plasma sample. (A) Thrombin generation profiles in plasma samples from protein C and antithrombin mutation carriers and normal controls in pedigree 1 (red, green, and blue curves). The dashed line indicates thrombin generation in the presence of soluble thrombomodulin (sTM). (B) Thrombin generation profiles in plasma samples from protein C and antithrombin mutation carriers and normal controls in pedigree 2 (orange, purple, and blue curves). The dashed line indicates thrombin generation in the presence of sTM.

TABLE 3.

Results of the thrombin generation and inhibition test.

Family members	Mutants type	ETP (nM*min)	Peak (nM)	STT (min)	Rate of inhibition (%)
1‐I1	PC‐400+5G>A	1599.31	313.67	21.33	14
	PC‐400+5G>A+sTM	1376.1	303.81	19.67
1‐I2	AT‐V295M	2272.69	316.01	27.5	—
2‐I1	PC‐R271W	1512.21	233.19	23.33	40
	PC‐R271W+sTM	898.75	196.8	18.67
2‐I2	AT R294C	1720.06	400.9	19.67	—
	NC	1518.9	239.01	22.68	76
	NC+sTM	360.8	89.79	17.16

Abbreviations: AT, antithrombin; ETP, the endogenous thrombin potential; NC, normal control; PC, protein C; sTM, soluble thrombomodulin; STT, the start tail time.

4. Discussion

Through comprehensive characterization and clinical evaluation of two unrelated VTE probands and their family pedigrees, this study identified multiple thrombophilia‐associated pathogenic mutations. Specifically, we identified amino acid substitutions at Val295 and Arg294 in AT and at Arg271 in PC, as well as the c.400+5G>A splice‐site mutation in PC. All four variants have been previously reported in multiple populations, including those from Japan, China, and Saudi Arabia (Alsultan et al. 2016; Miyata et al. 2009; Tang et al. 2022; Zeng et al. 2015).

To elucidate the molecular mechanisms underlying anticoagulant protein gene mutations, we performed coagulation assays, bioinformatics analysis, and thrombin generation tests on mutation carriers. Notably, the antithrombin activity levels in individuals carrying the c.880C>T (p.Arg294Cys) and c.883G>A (p.Val295Met) variants were within the normal range, consistent with prior reports (Miyata et al. 2009; Zeng et al. 2015). However, thrombin generation assays demonstrated significantly elevated ETP, suggesting these mutation carriers exhibit enhanced thrombin generation and consequently higher thrombotic risk relative to healthy controls. Furthermore, bioinformatics analyses confirmed these mutations affect evolutionarily conserved residues and are predicted to compromise protein stability, potentially leading to either impaired secretion or altered stability of the affected proteins.

AT is a single‐chain glycoprotein primarily synthesized in the liver and vascular endothelial cells, and it is the key physiological anticoagulant protein in humans. As a serpin (serine protease inhibitor) family member, AT potently inactivates key coagulation serine proteases—most notably thrombin (FIIa) and activated factor X (FXa) through irreversible complex formation, thereby mediating its primary anticoagulant function (Zhang, Sun, et al. 2020). The SERPINC1 gene, encoding AT, spans approximately 15 kb on chromosome 1q23‐25 and comprises seven exons interrupted by six introns. In its native state, AT exhibits limited thrombin‐binding capacity; however, heparin binding induces a critical conformational transition that dramatically enhances (by ~1000‐fold) its inhibitory activity against target proteases. Genetic mutations in SERPINC1 commonly result in inherited antithrombin deficiency, a condition associated with a dramatically elevated (up to 20‐fold higher) risk of thromboembolic events compared to the general population (Bates et al. 2008).

PC, a vitamin K‐dependent plasma glycoprotein, is predominantly synthesized in hepatocytes. The gene encoding PC (PROC) spans 11.6 kilobases on chromosome 2q13‐q14, with a genomic structure comprising nine exons interspersed by eight introns (Hepner and Karlaftis 2013). In circulation, PC predominantly exists as an inactive zymogen. Proteolytic activation requires cleavage of a Lys‐Arg dipeptide at the activation peptide region, converting the single‐chain precursor into a two‐chain active serine protease. This maturation process enables the activated protein C (APC) to execute its potent anticoagulant function through proteolytic degradation of coagulation factors Va and VIIIa (Perera et al. 2000). Hereditary PC deficiency is usually inherited in an autosomal dominant pattern. Heterozygous individuals typically develop the disease in adulthood, with deep vein thrombosis being a common manifestation. However, homozygous or compound heterozygous mutations result in more severe phenotypes, typically manifesting as life‐threatening neonatal purpura fulminans and frequently progressing to disseminated intravascular coagulation (DIC) with associated pulmonary embolism (Winther‐Larsen et al. 2020; Tang et al. 2022).

In our study, both patients presenting with severe VTE exhibited compound heterozygous mutations in anticoagulant protein genes. This genetic configuration is exceptionally rare in autosomal dominant thrombophilic disorders, with only sporadic case reports documented in the literature (Tomomasa et al. 2013; Wang et al. 2018; Miyata et al. 2009). Notably, Patient 1 was an adolescent who developed extensive lower extremity DVT involving the external iliac vein, which subsequently progressed to PE, in the absence of any identifiable acquired thrombotic risk factors. Routine phenotypic screening revealed only mildly decreased PC:A (57%) and PC:Ag levels (59.2%). Genetic testing identified a heterozygous missense mutation in the SERPINC1 gene (c.883G>A) and a splice‐site mutation in the PROC gene (c.400+5G>A). The second patient developed recurrent VTE, with four distinct thrombotic events (including DVT and PE) documented over a 15‐year clinical course, despite intermittent anticoagulant prophylaxis. The phenotypic screening likewise showed only mildly decreased PC:A (68%). Molecular characterization revealed the presence of two heterozygous missense variants: SERPINC1 c.880C>T and PROC c.811C>T. Both patients demonstrated initial clinical improvement with thrombolytic and anticoagulant therapy; however, they exhibited persistent susceptibility to recurrent thrombotic events. Familial genetic analysis confirmed the inherited nature of these pathogenic variants.

Significant ethnic disparities exist in the genetic architecture of VTE. Predominant thrombophilic variants in Caucasian populations—including Factor V Leiden, prothrombin G20210A, and antithrombin Cambridge II, demonstrate markedly lower prevalence across Asian demographics, with reported allele frequencies < 0.5% compared to 3%–8% in European populations (Seligsohn and Lubetsky 2001; Corral et al. 2007; Zhang et al. 2010). Conversely, mutations such as c.565C>T and c.574_576del in PROC appear to be exclusive to Asian populations (Tang, Guo, et al. 2012; Tang, Lu, et al. 2012). The PROC c.400+5G>A splice‐site variant (identified in Family 1) occurs at the highly conserved +5 position of intron 5's donor splice site. This variant is predicted to disrupt canonical splicing mechanisms, potentially causing: exon 5 skipping during mRNA processing, and consequent quantitative and functional protein C deficiencies through either impaired secretion or accelerated degradation of the aberrant protein product (Reitsma et al. 1991). Structurally, Arg271 resides within protein C's catalytic triad domain (residues 212–461), a region that encompasses the characteristic serine protease fold essential for proteolytic activity. This evolutionarily conserved residue plays a critical role in maintaining the structural integrity of the active site (Yamamoto et al. 1992). Molecular modeling demonstrated that the c.811C>T substitution creates steric clashes between adjacent residues, leading to distortion of the catalytic domain architecture. This structural perturbation likely impairs protein C's proteolytic function by either disrupting the active site geometry or interfering with substrate binding.

Recent population genetics research (Zeng et al. 2017) has identified three mutational hotspots in SERPINC1 c.883G>A, c.881G>T and c.880C>T with elevated prevalence in Chinese cohorts. Carriers of these variants exhibit a striking 20‐fold increased risk of VTE despite maintaining normal‐range antithrombin activity levels—a phenotypic pattern that precisely matches our clinical observations in both probands. Although recombinant modeling suggests these mutations may have only a minor effect on SERPINC1 expression and AT activity (Zeng et al. 2017), our thrombin generation assays revealed that these mutations impair anticoagulant capacity, leading to increased thrombin generation and heightened thrombotic risk. We hypothesize that SERPINC1 conventional functional assays, which focus on individual protease inhibition under specific conditions, may not fully capture the impact of mutations at the s1B terminus, which affects antithrombin's interaction with target proteases, particularly thrombin. Since the closely positioned amino acid residues Tyr285 and Glu287 belong to an exogenous site that enhances the protease interaction through the variable activation of antithrombin by heparin (Izaguirre et al. 2014). Therefore, the mutations in Arg294 or Val295 identified in this study may also impair the interaction of natural antithrombin with certain proteases (especially thrombin).

5. Conclusion

This study conducted comprehensive clinical and genetic analyses of two pedigrees with combined hereditary antithrombin and protein C deficiency. The identified variants—PROC c.400+5G>A and c.811C>T, along with SERPINC1 c.883G>A and c.880C>T—may contribute to the severe VTE observed in both probands. Nevertheless, comprehensive in vitro functional studies are required to elucidate the precise molecular mechanisms underlying these variants. While acknowledging the technical constraints of existing assays, we strongly advocate for implementing genetic screening in cases of idiopathic thrombophilia. Such proactive diagnostic approaches, coupled with timely preventive interventions, may substantially enhance clinical management and patient prognosis.

Author Contributions

Yueli Guo provided the research materials and conducted the gene assays. Tingting Shan was responsible for recruiting healthy participants. Xiaojing Zou designed the study protocol. Yueli Guo drafted the manuscript and prepared all figures. Wanzheng Kong and Chun Zhao performed the statistical analyses. All authors critically reviewed and approved the final version of the manuscript.

Funding

This work was supported by the Department of Science and Technology of Wenzhou (YC20250200).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.