Activated partial thromboplastin time prolongation without hemorrhagic dienhesis: a study of a chinese family coexisting with hereditary KNG1 p.Arg240 mutation and --SEA/αα genotype thalassemia

Abstract

High molecular weight kininogen (HK) deficiency is a rare autosomal recessive disorder caused by mutations in the KNG1 gene. This study reports a 66-year-old male Chinese patient who presented with significantly prolonged activated partial thromboplastin time (aPTT) and microcytic hypochromic anemia. Whole-exome sequencing revealed a homozygous nonsense mutation in exon 6 of the KNG1 gene (c.718 C > T, p.Arg240*) in the proband. This mutation results from a cytosine-to-thymine substitution, generating a premature termination codon that leads to truncated HK protein translation and loss of function. Additionally, genetic testing identified a concurrent heterozygous α-thalassemia --^SEAdeletion in the proband, located at 16p13.3 and encompassing the HBA2 and HBA1 genes. Pedigree analysis indicated that both the proband and his sister were homozygous for the KNG1 mutation and exhibited prolonged aPTT, whereas their children and some descendants were heterozygous carriers with normal coagulation function. Within the same family, all heterozygous carriers of the α-thalassemia --^SEAdeletion presented with microcytic hypochromic anemia. This study represents the first documented case of co-occurrence of a homozygous KNG1 p.Arg240* mutation and the --^SEAdeletion α-thalassemia.

Introduction

Activated Partial Thromboplastin Time (aPTT) serves as a critical laboratory parameter for evaluating the functionality of the intrinsic and common coagulation pathways, primarily reflecting the activity levels of coagulation factors VIII, IX, XI, and XII. A prolongation of aPTT exceeding 10 s beyond the normal control value often indicates deficiencies in intrinsic coagulation factors, the presence of pathological anticoagulants (such as lupus anticoagulant), or heparin interference [1]. It is noteworthy that deficiencies in intrinsic coagulation factors (e.g., FVIII/IX) typically manifest as bleeding tendencies, whereas deficiencies in contact system factors (e.g., FXII, prekallikrein [PK], or high-molecular-weight kininogen [HK]), while causing significant aPTT prolongation, are generally not associated with clinical bleeding symptoms. This discrepancy arises because the artificial negatively charged surfaces (e.g., kaolin) in aPTT reagents rely on contact factors to initiate the in vitro coagulation process; deficiencies in these factors interrupt this artificially triggered pathway [2]. However, physiological hemostasis in vivo primarily depends on the tissue factor pathway initiation and thrombin-mediated amplification feedback mechanisms, rendering contact system deficiencies clinically irrelevant in actual coagulation function [3, 4].

As a key cofactor in the contact activation system, HK forms complexes with FXII and PK on negatively charged surfaces exposed after vascular injury, promoting mutual activation and triggering subsequent coagulation cascades [5]. HK is encoded by the KNG1 gene located on human chromosome 3. Mutations in the KNG1 gene can lead to HK deficiency, a rare autosomal recessive disorder characterized by isolated significant aPTT prolongation without clinical bleeding manifestations. Since its first report in 1974, only approximately 30 cases have been confirmed worldwide [6–13]. This study reports a case of HK deficiency caused by the KNG1 gene c.718 C > T (p.Arg240*) variant in a large Chinese pedigree, which also carries an α-thalassemia mutation.

Materials and methods

Case history

On March 6, 2024, a 66-year-old male patient presented to the Qujing Central Hospital of Yunnan Province, China, with a 5-hour history of sudden intermittent colicky abdominal pain. Ultrasonography revealed right-sided hydronephrosis and hydroureter, which were subsequently confirmed by computed tomography (CT) as an upper ureteral calculus complicated by hydronephrosis and infection. The patient was subsequently admitted to the Department of Urology for management of urinary tract calculi.

Coagulant assays

Coagulation profiles including prothrombin time (PT), aPTT with silica activator, thrombin time (TT), fibrinogen, fibrin degradation products (FDPs), lupus anticoagulant (LA), von Willebrand factor (vWF) activity, and intrinsic coagulation factor activities—were assessed using a fully automated coagulation analyzer (ACL-TOP750 LAS, Instrumentation Laboratory, USA). To investigate the prolonged aPTT, mixing studies were performed: an immediate correction test measured aPTT in patient plasma (PP), normal pooled plasma (NPP), and an immediate 1:1 PP/NPP mixture (aPTT₃); an incubated correction test involved measuring aPTT in PP, NPP, and a pre-mixed 1:1 PP/NPP aliquot after a 2-hour incubation at 37 °C (aPTT₄, aPTT₅, aPTT₆), followed by immediate measurement of a new 1:1 mixture prepared from the incubated PP and NPP (aPTT₇).

Molecular analysis

This study has been approved by the Medical Ethics Committee of Qujing Central Hospital of Yunnan Province (Approval No.: 2024-048(Science)-01, Qujing, Yunnan, China). In accordance with the ethical principles set forth in the Declaration of Helsinki, written informed consent was obtained from each participant prior to inclusion in the study. Peripheral blood samples were collected from the probands and their third-generation family members.

Genomic DNA was extracted from the proband’s peripheral blood samples. Whole-exome sequencing (WES) was performed by KingMed Diagnostics (Guangzhou, China). Briefly, libraries were prepared and captured to enrich the exonic regions and their flanking sequences, followed by sequencing on an Illumina platform to ensure a mean coverage depth of > 90x across the targeted regions, with over 98% of the bases achieving a minimum depth of 20x.Bioinformatic analysis of the sequencing data was conducted using a standardized pipeline. Raw sequencing reads were aligned to the human reference genome (UCSC hg19) using Burrows-Wheeler Aligner (BWA). Variant calling for single nucleotide variants (SNVs) and small insertions/deletions (Indels) was performed using the Genome Analysis Toolkit (GATK) suite. Detected variants were annotated and filtered using Variant Effect Predictor (VEP) software, with a focus on variants reported in public databases including ClinVar, OMIM, HGMD, and gnomAD. The pathogenicity of the prioritized variants was predicted using a combination of in silicoalgorithms and interpreted according to the guidelines established by the American College of Medical Genetics and Genomics (ACMG). Copy number variation (CNV) analysis was also performed on the WES data using an internally developed bioinformatic tool, and any identified CNVs were assessed based on the joint standards and guidelines from ACMG and the Clinical Genome Resource (ClinGen).

Molecular modeling

To assess pathogenicity and predict the impact of mutations on high-molecular-weight kininogen, we constructed three-dimensional structural models of mutated HMWK proteins using the AlphaFold2 platform, followed by visualization analysis with PyMOL software to elucidate spatial conformational changes.

Result

Proband study

A 66-year-old male patient scheduled for urolithiasis surgery underwent routine preoperative laboratory tests, which revealed a markedly prolonged aPTT of 120 s. In contrast, prothrombin time (PT), thrombin time (TT), fibrinogen (Fib), and D-dimer levels all remained within normal ranges. The aPTT correction test using a 1:1 mixture with normal pooled plasma showed complete correction both immediately and after a 2-hour incubation. Calculated Rosner indices strongly suggested an intrinsic coagulation factor deficiency rather than the presence of an inhibitor. Coagulation factor assays demonstrated markedly elevated factor VIII activity, with the remaining factors either normal or slightly reduced, effectively excluding classical hemophilia and acquired coagulation disorders. Further immunological testing yielded negative results for lupus anticoagulant (dRVVT ratio: 0.93) and anti-cardiolipin antibodies, with no detectable factor VIII inhibitors (Table 1). Despite therapeutic interventions including vitamin K administration and fresh frozen plasma transfusion (600 mL), persistent aPTT prolongation was observed. The patient also presented with microcytic hypochromic anemia, characterized by mild hemoglobin reduction (106 g/L), decreased mean corpuscular volume (67.0 fL; reference ≥ 80 fL), reduced mean corpuscular hemoglobin (21 pg; reference ≥ 27 pg), and lowered mean corpuscular hemoglobin concentration (296 g/L; reference ≥ 320 g/L). Wright-stained peripheral blood smears confirmed these findings, demonstrating enlarged central pallor of erythrocytes—a hallmark feature of hypochromic anemia.

Table 1.

Laboratory results of clinical phenotype analysis of proband

Study title	Result	Unit	Reference interval
Conventional coagulation tests
PT	13.1	s	10.00–15.00
aPTT	120.1	s	28.00–43.00
TT	18	s	14.00–21.00
Fib	5.69	g/L	2.00–4.00
D-Dimer	1.97	µg/mL	0-0.50
antithrombin	87.7	%	80.00-120.00
FDP	21.4	µg/mL	0–5.00
drvvt-SN	1.02		≦ 1.2
drvvt-NR	0.93		≦ 1.2
SCT-SN	0.82		≦ 1.2
SCT-NR	0.96		Negative
ACA	Negative		Negative
LA	Negative		Negative
Quantitative assay of factor VIII inhibitors
Factor II activity	0.735	%	79–131
Factor V activity	0.799	%	62–139
Factor VII activity	0.826	%	50–129
Factor VIII activity	2.035	%	50–150
Factor IX activity	1.309	%	65–150
Factor X activity	0.791	%	77–131
Factor XI activity	0.701	%	50–150
Factor XII activity	1.09	%	50–150
vWF activity	109%	%	50–190.00
vWF Ag	81%	%	50–160.00
Prolonged aPTT mixing study
aPTT1	121.9	s	28.00–43.00
aPTT2	35.7	s	28.00–43.00
aPTT3	36.4	s
aPTT4	129.3	s
aPTT5	38.3	s
aPTT6	38.7	s
aPTT7	38	s
Rosner index	7.14		< 11.0: Corrected​ > 11.0–15.0: Not Corrected​
			> 15.0: Suggests Coagulation Inhibitors​

aPTT¹, patient plasma; aPTT², normal pooled plasma, NPP; aPTT³, 1:1 mix immediately; aPTT⁴, patient plasma incubated for two hours; aPTT⁵, normal pooled plasma incubated for two hours; aPTT⁶, 1:1 mix incubated for two hours; aPTT⁷, 1:1 mix after two hours of incubation; Rosner index1(aPTT³-aPTT²)×100/aPTT¹

Following informed consent obtained in accordance with the Helsinki Declaration, plasma and cellular samples were collected for DNA preparation. Whole exome sequencing identified a homozygous nonsense mutation (NM_001102416.3:c.718 C > T, p.Arg240*) in exon 6 of the KNG1 gene (chr3:186449379). This cytosine-to-thymine substitution generated a premature termination codon, resulting in truncated HK protein translation. Sanger sequencing confirmed the homozygous c.718 C > T nucleotide variant at the identical genomic locus in the proband (Fig. 1A). In addition, whole-exome sequencing initially indicated a heterozygous copy number variation (CNV) in the 16p13.3 region. Subsequent third-generation sequencing confirmed that this variant is the --^SEAdeletion (genotype: --^SEA/αα), which encompasses the HBA2 and HBA1 genes.

Fig. 1.

Genetic analysis of the KNG1 c.718 C > T variant and co-inherited α-thalassemia. (A) Sanger sequencing validation of the homozygous c.718 C > T (p.Arg240*) mutation in the proband (lower panel) compared to wild-type (upper panel); (B) Pedigree segregation of the KNG1 variant: filled symbols (homozygous mutant), half-filled symbols (heterozygous carriers), open symbols (wild-type); circles represent females, squares represent males. Arrow indicates the proband; (C) Pedigree segregation of the --SEA/αα genotype. Heterozygous carriers (half-filled symbols) and wild-type individuals (open symbols) are indicated.

Following diagnosis, the patient underwent transurethral flexible ureteroscopic laser lithotripsy. The procedure was completed successfully with an intraoperative blood loss of approximately 50 milliliters, and no postoperative hemorrhage was observed.

Family analysis

The KNG1 gene (chromosome 3) and the genes responsible for α-thalassemia (chromosome 16) are located on different chromosomes, therefore their inheritance in the pedigree was analyzed independently. The parents of the proband were first cousins with a history of consanguineous marriage; both are now deceased. The proband has an older brother and an older sister. The sister also exhibited similarly prolonged aPTT, while the other 12 family members showed normal values. Pedigree analysis indicated that the proband’s sister was also homozygous for the KNG1 mutation. All children of the proband and his sister were heterozygous carriers of the KNG1 mutation. Among six fourth-generation family members, three carried the variant heterozygously (Fig. 1B). All seven heterozygous KNG1 carriers displayed normal coagulation function. Additionally, third-generation sequencing covering 621 α- and β-thalassemia loci confirmed that the proband’s sister was a heterozygous carrier of the --^SEA/αα genotype. All descendants of the proband and his sister were identified as heterozygous carriers of the --^SEAdeletion. Among the six fourth-generation individuals, two inherited the --^SEAdeletion​ heterozygously (Fig. 1C), and all heterozygous carriers exhibited microcytic hypochromic anemia (Table 2). Although the KNG1 gene (located at 3q27.3) and the α-globin gene cluster (located at 16p13.3) are genomically distant and their mutations follow unlinked autosomal recessive inheritance patterns, pedigree analysis in this family reveals a similarity in their modes of inheritance: mutant alleles of both defects exhibit a tendency toward co-segregation and clustering within specific branches of the pedigree.

Table 2.

Laboratory test results for clinical phenotypic analysis of pedigree members

Normal Range	Age (years)	KNG1 c.718 C > T genotype	--SEA/αα genotype	PT (s)	APTT (s)	TT (s)	Fib (g/L)	RBC (×10¹²/L)	HB (g/L)	MCV fL	MCH pg	MCHC (g/L)
Ⅱ1	70	C/C	αα/αα	12.3	32.3	17.1	3.20	4.93	157	92.5	31.8	344
Ⅱ2	66	T/T	--SEA/αα	13.1	121.9	18.0	5.69	5.04	106	67.0	21.0	296
Ⅱ3	73	T/T	--SEA/αα	13.3	99.1	16.3	4.19	6.23	135	66.8	21.7	325
Ⅲ1	38	C/C	αα/αα	12.5	33.5	16.5	3.21	5.26	139	90.3	26.4	349
Ⅲ2	35	C/T	--SEA/αα	13.6	34.9	16.0	2.75	5.18	111	68	21.4	315
Ⅲ3	48	C/T	--SEA/αα	12.7	34.7	16.3	2.88	6.29	136	62.3	21.6	337
Ⅲ4	51	C/T	--SEA/αα	12.3	32.8	16.0	3.04	6.66	141	60.3	21.2	331
Ⅲ5	51	C/T	--SEA/αα	12.8	36.2	15.8	3.42	6.66	119	64.0	17.9	334
Ⅳ1	12	C/C	αα/αα	13.1	39.3	16.3	2.54	5.11	150	86.5	29.4	339
Ⅳ2	3	C/C	αα/αα	12.8	31.9	15.4	3.93	4.87	134	83.2	27.5	331
Ⅳ3	7	C/T	--SEA/αα	13.4	35.9	16.5	3.14	5.56	116	61.3	20.9	340
Ⅳ4	8	C/T	--SEA/αα	13.2	42.0	15.6	3.56	5.57	120	65.5	21.5	329
Ⅳ5	7	C/T	αα/αα	13.0	31.6	15.6	2.87	4.69	135	82.7	28.8	348
Ⅳ6	12	C/C	αα/αα	12.7	37.0	16.3	3.22	5.27	149	86.7	28.3	326

Bioinformatics analysis and molecular modeling

To elucidate the impact of the c.718 C > T (p.Arg240*) mutation on transcript stability, this study employed the online tool NMDEscPredictor to assess the likelihood of this nonsense mutation triggering nonsense-mediated mRNA decay (NMD). The prediction results demonstrated that the premature termination codon (PTC) introduced by this mutation meets NMD activation criteria (specifically, the PTC is located more than 50 nucleotides upstream of the last exon-exon junction), indicating a high probability of mutant transcript degradation, which would lead to significantly reduced or completely absent allelic expression.

Given the potential for partial escape of mutant transcripts from NMD, we also predicted the structural consequences of the 240-amino acid truncated protein resulting from the c.718 C > T nonsense mutation. Using AlphaFold2 [14, 15], we modeled the three-dimensional structure of the mutant protein. Structural analysis revealed that the truncated protein not only lacks the C-terminal sequence but, more critically, loses the functional domain essential for the binding of HK to PK and FXII. This structural defect is predicted to completely disrupt HK’s core cofactor function in the contact system.

Discussion

The KNG1 gene is located on human chromosome 3 (3q27.3) and encodes two major kininogen isoforms through alternative splicing: HK and low-molecular-weight kininogen (LK) [16, 17]. These two isoforms share identical first four domains (D1–D4) but differ in their C-terminal regions. Domain 1 contains a low-affinity calcium-binding site; Domain 2 exhibits inhibitory activity against calpains; Domain 3 serves as the primary binding site for platelets and endothelial cells; Domain 4 (D4, amino acids 358–481) encompasses the major binding site for FXII. HK includes a unique D5H domain (encoded by exon 10), which specifically binds negatively charged surfaces (e.g., heparan sulfate, endothelial cells, platelets, kaolin), thereby initiating the downstream coagulation cascade [18]. Domain 6 contains a binding site for both PK (S583–K613) and FXI (P574–M631) [19]. In contrast, LK contains only a shorter C-terminal domain, D5L, encoded by exon 11, and is not involved in coagulation processes [18].

In this case, the patient has a nonsense mutation in the KNG1 gene at c.718 C > T, which is expected to cause premature termination of protein translation, resulting in a truncated protein of only 222 amino acids for both HK and LK (excluding the 18 amino acid signal peptide), completely lacking all key functional domains at the C-terminal (D4, D5H, D5L). Due to the lack of binding sites for PK and FⅪ in the mutant protein, it cannot support the normal activation of the contact system, and the patient presents with isolated prolonged aPTT (120s). The homozygous c.718 C > T mutation identified in this family is consistent with the mutation reported by Panchione Y et al. in Italy, and the laboratory test results are highly similar [20]. KGN p.Arg240* variant was classified as pathogenic based on the PVS1 (very strong) level of evidence, as it is a null variant (nonsense) in a gene where loss of function is a known disease mechanism. Among the 13 reported cases of HK deficiency, 10 cases exhibited homozygous mutations, with 6 of these involving consanguineous marriages [5]. The investigation of this family found that the proband’s parents were consanguineously married, and the proband’s sister was a homozygous carrier of the KNG1 mutation. These findings are consistent with the reported genetic characteristics of HK deficiency. Consistent with the reported cases of HK deficiency, the proband and her sister, both homozygous for the KNG1 mutation, have no bleeding symptoms. This confirms the modern coagulation theory: the core of physiological hemostasis is the extrinsic pathway initiated by tissue factor (TF). When a blood vessel is injured, TF is exposed and binds to FVIIa, directly activating FX to generate initial thrombin. Subsequently, a small amount of thrombin efficiently completes hemostasis through a powerful positive feedback amplification mechanism (activating platelets, FV, FVIII, and FXI), a process that is completely independent of the contact system (FXII, PK, HK). Therefore, although defects in contact system factors lead to abnormal aPTT in vitro, they do not affect physiological hemostasis in vivo [21].

It is worth noting that this mutation may have simultaneously affected the synthesis of LK. LK mainly serves as a substrate for tissue kallikrein and a precursor of bradykinin, participating in inflammation and immune regulation [18]. Theoretically, the absence of LK may affect the generation of bradykinin, thereby potentially influencing vasodilation, permeability, and innate immune responses. However, the patient in this case did not exhibit any related clinical symptoms (such as abnormal blood pressure or immune abnormalities), suggesting that there may be other compensatory mechanisms in the body (such as the renin-angiotensin system) maintaining stable blood pressure, and there is significant redundancy in the inflammation and immune regulation pathways. In the future, further observation and basic research are needed to prove that the KNG1 gene mutation is limited to laboratory abnormalities in coagulation function only.

Genomic sequencing analysis revealed a 21.4 kb heterozygous deletion in the 16p13.3 region of the proband, which covered the α-globin gene cluster including HBA2 and HBA1, and was closely related to the pathogenesis of α-thalassemia. This deletion followed an autosomal recessive inheritance pattern and exhibited a typical gene dosage effect: a single copy deletion was an asymptomatic carrier, a double copy deletion led to the characteristics of α-thalassemia (mild anemia), a triple copy deletion caused HbH disease (moderate anemia with hepatosplenomegaly), and a quadruple copy deletion resulted in the fatal Hb Bart’s hydrops fetalis syndrome [22, 23]. The 21.4 kb deletion found in this case was of the Southeast Asian (SEA) type, with clinical manifestations of characteristic microcytic hypochromic anemia (significantly reduced MCV and MCH) [24]. Among the eight --^SEA/αα heterozygous mutation carriers in this pedigree, six individuals (II-2, II-3, III-3, III-4, III-5, and IV-4, all harboring a compound heterozygous genotype of the KNG1 c.718 C > T mutation) reported symptoms including fatigue, despite having hemoglobin levels at the lower limit of the normal range. This finding contrasts with the conventional clinical perception that the --^SEA/αα heterozygous genotype, a form of α-thalassemia trait, is typically characterized as mild or silent, without anemia-related symptoms. The discrepancy may be attributed to the subjective nature of symptoms, potential exacerbating factors (such as transient infections exemplified by the proband’s urinary tract infection), and comorbid conditions like unaddressed iron deficiency. Regrettably, systematic data on iron metabolism parameters and other potential predisposing factors were not comprehensively collected in this study, representing a limitation of this retrospective analysis.

Although the KNG1 gene (3q27.3) and the α-globin gene cluster (16p13.3) are genetically unlinked, the inheritance patterns of these two defects observed in this pedigree exhibit a high degree of similarity, primarily attributable to consanguineous marriage in the family’s founders (Generation I). The consanguineous couple in Generation I were likely both heterozygous for the two mutations, transmitting both the KNG1 c.718 C > T variant and the α-thalassemia mutation to multiple offspring in Generation II (e.g., II-2, II-3, II-7), rendering these descendants double heterozygotes and thereby establishing a genetic background carrying both mutations within the family. Although no subsequent consanguinity occurred in later generations (Generations II and III), starting from these double-heterozygous ancestors, their descendants had a probability of independently inheriting the two mutations through routine Mendelian inheritance. This led to the coincidental cosegregation and clustering of both defects in specific branches of the pedigree (e.g., III-3, III-4, IV-2). Consequently, the observed similarity in inheritance patterns does not stem from physical linkage but rather results from the combined effects of founder inheritance and subsequent genetic contingency.

Although this study achieved a definitive diagnosis of a rare case of HK deficiency caused by compound heterozygous KNG1 mutations through genetic sequencing and underscored the importance of comprehensive genetic screening, several limitations remain. First, hemoglobin electrophoresis was not performed for the proband to obtain key parameters such as HbA2 and HbF levels, which would have enabled further quantification of the phenotypic manifestations of the compound heterozygous α-thalassemia. The absence of this data constrained our ability to more precisely evaluate the contribution of thalassemia to the observed hematological abnormalities, such as microcytic hypochromic anemia. Second, while the study relied primarily on bioinformatic tools to predict the NMD effect induced by the p.Arg240 mutation and the consequent protein truncation, it lacked functional in vitro validation—such as utilizing patient plasma or constructed expression vectors—to directly assess the mutation’s impact on HK protein expression, degradation, and its interactions with PK and FXII.

In conclusion, this case provides clinical and molecular evidence from the Chinese population that underscores the inclusion of contact system factor deficiencies in the differential diagnosis of isolated aPTT prolongation. Definitive genetic testing is crucial to avoid unnecessary interventions and alleviate patient anxiety. Additionally, the proband’s co-occurring compound heterozygous α-thalassemia deletion adds a layer of complexity to familial genetic counseling, highlighting the critical importance of comprehensive genetic screening for such patients.

Author contributions

P.J. and ZY.B. wrote the main case report text, JY.H. prepared Fig. 2. All authors including YL.J, XD.L., JQ.S., F.Z., D.W., and FX.Z. reviewed the case report.

Fig. 2.

Bioinformatics analysis and molecular modeling of the KNG1 c.718C>T (p.Arg240*) mutation. (A)​ Assessment of nonsense-mediated NMD likelihood for the KNG1 c.718C>T mutation using the NMDEsc Predictor online tool. The analysis was based on the relative position of the premature termination codon to the final exon-exon junction. The prediction indicated a high probability​ of NMD activation for the mutant transcript, suggesting its potential degradation. (B, C) Ribbon diagrams of the HK protein three-dimensional structures as predicted by AlphaFold2. The truncation leads to a complete loss of the C-terminal functional domain. (D-G)​ Predicted molecular interaction interfaces between HK and its binding partners PK (D, E) and FⅫ (F, G), as modeled by AlphaFold3. Comparative analysis reveals that the truncated HK protein (E, G) exhibits a severely impaired interface​ compared to the wild-type protein (D, F), predicting a loss of critical binding interactions.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 82002223 to Ping Ji); the Shanghai Natural Science Foundation General Program (Grant No. 24ZR1463300 to Ping Ji); and the Yunnan Province-Level Research Project of Qujing Central Hospital (Grant No. 2022YJKTY07 to Zhiyao Bai).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

This study has been approved by the Medical Ethics Committee of Qujing Central Hospital in Yunnan Province (Approval No.: 2024-048(Science)-01, Qujing, Yunnan, China).

Informed consent

Informed consent was obtained from the subject involved in the study.

Consent to publish

The consent to publish was obtained from the subject involved in the study.

Competing interests

The authors declare no competing interests.