Clinical and functional characterization of a novel heterozygous mutation c.473T > A (p.Leu158Gln) in the SERPINC1 gene causing recurrent arteriovenous thrombophilia

Abstract

Background

Hereditary antithrombin deficiency (HATD) is a rare disease caused by mutations in the SERPINC1 gene characterized with venous thromboembolism and/or arterial thrombotic events. We identified a proband with recurrent arterial and venous thrombosis at multiple anatomical sites and subsequently performed comprehensive thrombophilia screening and genetic analysis within the kindred.

Methods

Mutation screening of the SERPINC1 gene in the proband and family members was conducted using Sanger sequencing. Wild-type and mutant (c.473T > A; p.Leu158Gln) SERPINC1 expression plasmids were constructed and transiently transfected into HEK293T cells. Functional consequences of the variant were assessed through immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and computational structural analysis.

Results

Thrombophilia evaluation revealed significantly reduced antithrombin activity (46.2%) in the proband. Sanger sequencing identified an unreported heterozygous missense variant (c.473T > A; p.Leu158Gln) in SERPINC1. Cellular studies demonstrated that this mutation reduced both the quantity and functional activity of secreted antithrombin. Immunoblot analysis indicated a slightly faster migration of the mutant protein compared to wild-type, while immunofluorescence revealed abnormal cytoplasmic aggregation. Bioinformatics analysis confirmed substitution of leucine-158 by glutamine without disruption of conserved disulfide bonds (Cys40-Cys160, Cys128-Cys182, Cys247-Cys430) or N-glycosylation sites (Asn128, Asn167, Asn187, Asn224).

Conclusion

We establish the c.473T > A (p.Leu158Gln) variant in SERPINC1 as a pathogenic mutation underlying type I HATD, characterized by impaired secretion, cytoplasmic retention, and reduced functional activity of the mutant protein.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12959-025-00801-0.

Introduction

Antithrombin (AT, also designated as AT III) is encoded by the SERPINC1 gene located at chromosome 1q23.1-23.9. The gene comprises seven exons and six introns spanning 13.5 kilobases. AT is a single-chain glycoprotein with 432 amino acids, four N-linked glycosylation sites, three pairs of disulfide bonds, with a molecular mass of approximately 58 kDa [1]. This glycoprotein is primarily synthesized by hepatocytes and vascular endothelial cells, serving as the major physiological inhibitor of coagulantion proteases in human plasma. AT exerts its anticoagulant function through irreversible inhibition of multiple serine proteases in both the intrinsic and extrinsic coagulation pathways. While its primary targets are thrombin (FIIa) and activated factor X (FXa), AT also effectively inactivates FIXa, FXIa, FXIIa, and FVIIa. Notably, the inhibitory activity of AT is potentiated 100- to 1000-fold through its interaction with heparin [2].

HATD is an uncommon condition caused by mutations in the SERPINC1 gene. Its incidence ranges from 1 in 5,000 to 1 in 500 in the general population and approximately from 1 in 100 to 5 in 100 among patients with venous thromboembolism (VTE) [3]. HATD significantly increased risk of venous thromboembolic events compared to other thrombophilia-prone individuals [4]. The first thrombus typically develops early and is prone to recurrence, often occurring in deep vein thrombosis of the lower extremities or pulmonary embolism. Less common affected locations include the cerebral sinus [5], vena cava, mesenteric vein [6], renal vein, and hepatic vein (Budd Chiari syndrome) [7]. Although arterial thrombosis is usually associated with atherosclerosis, which is not characteristic of antithrombin deficiency, there are also documented instances of this occurrence [8]. HATD is primarily classified into type I (characterized by low antigen levels and activity) and type II (characterized by normal antigen levels and reduced activity). Type II HATD is further categorized into three subtypes based on the impaired functional domains: type IIa with a reactive site defect (RS), type IIb with a heparin binding site defect (HBS), and type IIc with a pleiotropic defect (PE). To date, The Human Gene Mutation Database (HGMD) has documented 546 mutations in the SERPINC1 gene (https://www.hgmd.cf.ac.uk/ac/gene.php?gene=SERPINC1). Missense and nonsense mutations are the most prevalent, while deletions, insertions, and repeat variants have also been reported.

In this study, we identified and characterized a novel missense mutation (c.473T > A) in the SERPINC1 gene from a young male patient with recurrent arteriovenous thrombosis. Furthermore, we conducted comprehensive analyses to elucidate the genotype–phenotype correlation and investigated the structural and functional implications of this mutation at the molecular level.

Methods

SERPINC1 gene analysis

Genomic DNA was extracted from peripheral blood samples of all participants using a commercial DNA extraction kit. For SERPINC1 gene analysis, specific primers were designed to amplify all seven exons and their flanking sequences using NCBI Primer-BLAST. PCR amplification was performed following standard protocols, and the resulting products were purified and sequenced. The obtained sequences were aligned against the SERPINC1 reference (GenBank ID: NG_012462.1) using Chromas software for mutation identification and analysis.

Cell transfection

The wild-type SERPINC1 gene and its p.Leu158Gln mutant were independently cloned into pcDNA3.1 + expression vectors (Proteinbio, China). HEK293T cells were maintained in complete Dulbecco’s Modified Eagle’s Medium (DMEM) containing 5% fetal bovine serum and 1% penicillin-streptomycin antibiotic mixture, cultured at 37 °C in a humidified 5% CO2 atmosphere. When cells reached 80–90% confluency, they were trypsinized and seeded into six-well culture plates at appropriate densities. Transfection was initiated at approximately 70% confluency using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, USA) according to the manufacture’s protocol. Briefly, for each transfection: (1) Tube A contained 5 µl Lipofectamine 3000 reagent diluted in 125 µl serum-free DMEM; (2) Tube B contained 2.5 µg plasmid DNA mixed with 5 µl P3000 enhancer reagent 125 µl serum-free DMEM. The contents of both tubes were combined, gently mixed, and incubated at room temperature for 15 min to allow complex formation before being added dropwise to the cultured cells. After 48 h of incubation under standard conditions, both conditioned media and transfected cells were collected for subsequent analysis. Co-expression studies mimicking the heterozygous state were not performed.

Western blotting

For protein extraction, HEK293T cells were lysed in lysis buffer containing protease and phosphatase inhibitors on ice. Protein concentrations were quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. Equal amounts of total protein lysates (20–30 µg per lane) were resolved by SDS‒polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently electrotransferred onto 0.45 μm polyvinylidene difluoride (PVDF) membranes (Millipore, USA) using a wet transfer system.

The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h at room temperature, followed by incubation with rabbit polyclonal anti-AT primary antibodies (Proteintech, China) at 4 °C overnight with gentle agitation. After three washes with TBST, the membranes were incubated with secondary antibodies (Proteintech, China) for 2 h at room temperature.

Protein bands were detected using an enhanced chemiluminescence (ECL) substrate kit (Keygene, China) and visualized with a ChemiDoc imaging system (Bio-Rad, USA). Band intensities were quantified using ImageJ software and normalized to β-actin as an internal control.

Enzyme-linked immunosorbent assay

Cell culture supernatants were collected and centrifugated at 3000 × g for 10 min at 4 °C to eliminate cellular debris. AT concentrations were quantified using a commercial human AT ELISA kit (FineTest, China) following the manufacturer’s protocol. Briefly, 100 µl of appropriately diluted samples and serially diluted standards were added to their respective wells in duplicate. After 90 min of incubation at 37 °C, plates were washed four times with 1× wash buffer to remove unbound components. Subsequently, 100 µl of biotinylated detection antibody was added to each well and incubated for 60 min at 37 °C. Following another washing step, 100 µl of horseradish peroxidase (HRP)-conjugated streptavidin (SABC) was added and incubated for 30 min at 37 °C. After final washing, 90 µl of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate solution was added and the enzymatic reaction was allowed to proceed for 15 min in the dark before being stopped with 50 µl of stop solution. Absorbance was immediately measured at 450 nm using a microplate reader (BioTek, USA), with 630 nm as the reference wavelength. AT concentrations were determined by interpolating sample optical density values against the standard curve generated from known concentrations.

AT activity detection

AT activity was quantitatively measured using the BIOPHEN™ AT (Anti-IIa) chromogenic assay kit (Hyphen BioMed, France). This assay principle relies on the heparin-catalyzed inhibition of a fixed, excess concentration of thrombin (Factor IIa) by AT present in test samples. Briefly, when heparin is present in excess, it accelerates the formation of thrombin-AT complexes, thereby reducing free thrombin activity. The remaining uncomplexed thrombin then cleaves a specific chromogenic substrate, releasing para-nitroaniline (pNA). The pNA release is spectrophotometrically quantified at 405 nm, with the absorbance being directly proportional to residual thrombin activity. Since thrombin inhibition is stoichiometrically related to AT concentration, the measured absorbance shows an inverse correlation with functional AT levels in the sample.

Immunofluorescence

After 24 h of transfection, HEK293T cells were replated onto 12 well plates (with cell slides; with consistent cell numbers) and cultured for another 24 h before immunofluorescence staining. Cells were fixed with formaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5%BSA. After overnight incubation with primary antibody, cells were washed with PBS and incubated with secondary antibody (Proteintech, China). Finally, DAPI containing antifade reagent (Beyotime, China) was applied and samples were incubated with 4 ℃. Samples were captured with a confocal microscope (Leica, Germany) using emission wavelengths of 488 nm and 520 nm within 72 h (maintain consistent parameters during shooting).

Bioinformatic analysis

The evolutionary conservation of Leu158 was assessed using PhyloP and PhastCons scores. The potential pathogenicity of the mutation was predicted through online bioinformatics tools. Structural alterations induced by the site-directed mutation were generated based on the crystal structure of native AT in its monomeric form (PDB ID: 1t1f), then visualized and analyzed using PyMOL 2.6.0. Additionally, potential changes in glycosylation patterns and disulfide bond formation were evaluated using web-based prediction platforms.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.5 (GraphPad Software). Intergroup comparisons were conducted using unpaired Student’s t-test for two-group comparisons and one-way analysis of variance (ANOVA) for multi-group comparisons. Statistical significance was defined as a two-tailed p-value < 0.05.

Results

Case presentation

A 37-year-old male presented to the Department of Cardiovascular Medicine of our hospital on October 17, 2022, with chief complaint of chest pain. His medical history included cerebral venous thrombosis at age 19, for which he discontinued aspirin therapy without medical supervision. At age 29, he developed superior mesenteric vein thrombosis requiring partial small intestine resection, after which he maintained daily aspirin therapy (100 mg orally).

Initial evaluation showed elevated cardiac biomarkers (cTnT 0.415 µg/L, CK-MB 13.91 ng/ml). Electrocardiogram demonstrated sinus rhythm (72 bpm) with T-wave abnormalities. Echocardiography revealed preserved left ventricular function (ejection fraction 62.7%) with moderate tricuspid regurgitation. Coronary angiography identified proximal right coronary artery occlusion with substantial thrombus burden (RCA, Fig. 1A). Thrombectomy yielded multiple dark red, block-shaped thrombi, followed by administration of recombinant human TNK tissue-type plasminogen activator and successful RCA recanalization via balloon angioplasty.

Fig. 1.

Clinical characteristics and gene analyse. A, coronary arteriography comparison before and after Percutaneous Coronary Intervention (PCI). The white arrow points to right coronary artery. B, pedigree map of the proband. C, gene sequencing of proband and his family. D, prediction of deleterious of gene mutation with different websites

The patient was diagnosed with myocardial infarction. Post-procedural management included dual antiplatelet therapy (clopidogrel + aspirin) combined with anticoagulation (low molecular weight heparin transitioning to warfarin, then rivaroxaban). At discharge, medication included clopidogrel bisulfate (75 mg qd) and rivaroxaban (15 mg qd). However, three-month follow-up angiography revealed recurrent proximal RCA occlusion with thrombus, and he was treated with balloon dilation. Due to suboptimal response to rivaroxaban in HATD, anticoagulation was switched to dabigatran etexilate (150 mg bid). The patient remains on clopidogrel (75 mg qd) and dabigatran (150 mg bid) without subsequent thrombotic events during follow-up.

Gene sequencing and analysis

Abnormal laboratory findings for the patient are presented in Table 1. The patient exhibited markedly decreased AT activity (46.2%). Other thrombophilia risk factors such as anticardiolipin antibodies, autoantibodies, anti-neutrophil cytoplasmic antibodies, complement, immunoglobulin, blood homocysteine, lupus anticoagulant, and fibrinolytic function were all within the normal range (Supplementary Table 1). Genetic sequencing identified a heterozygous missense mutation in SERPINC1 gene c.473T > A (p.Leu158Gln), representing a novel variant unreported in the HGMD. Familiar analysis showed similarly reduced AT activity in his mother (51.9%) and daughter (55%), both of whom carried the same SERPINC1 mutation (Fig. 1B&C). In contrast, his father and wife displayed normal AT levels without this genetic alteration. Notably, no thrombotic events were reported among family members.

Table 1.

Coagulation test results of the proband

	Result	Reference range
FVIII: A (%)	116	77.3-128.7
FXII: A (%)	59.4	50–120
AT: A (%)	46.2	103.2-113.8
PC: A (%)	106.8	60–130
PS: A (%)	113.7	70–140
vWF: A (%)	196.5	49.5–187

Evolutionary conservation analysis revealed high conservation at this locus (PhyloP score: 0.101; PhastCons score: 0.995). The variant was classified as pathogenic (PS1) according to the American College of Medical Genetics and Genomics (ACMG) guidelines [9]. Multiple in silico prediction tools consistently supported the mutation’s pathological significance, with classifications including “disease-causing (MutationTaster, https://www.mutationtaster.org/)”, “deleterious (JCVi, https://www.jcvi.org/)”, “damaging (FATHMN, http://fathmm.biocompute.org.uk/)” and “probably damaging (PolyPhen, http://genetics.bwh.harvard.edu/pph2/index.shtml)” (Fig. 1D).

Determination of secretion and distribution in HEK-293T cells

We successfully constructed SERPINC1-Wild type and SERPINC1-(p.Leu158Gln ) expression plasmids. Transfection efficiency in HEK-293T cells was confirmed by green fluorescence visualization. Because cells were transfected with either wild-type or mutant plasmid individually, each lane contained only the corresponding single isoform. Western blot analysis of cell lysates showed comparable AT protein expression levels between wild-type and mutant groups (Fig. 2A & Supplementary Fig. 1). However, the mutant AT protein displayed a slight faster migration across different gel concentrations (Fig. 2B). Functional assessment revealed significantly decreased AT expression and activity in culture supernatants from SERPINC1-(p.Leu158Gln) transfected cells (Fig. 2C&D).

Fig. 2.

Determination of expression and secretion in HEK-293T cells. A, western blotting shown AT expression in cell lysates. B, western blotting results with gels of different concentrations (8%, 10% and 12%). C, ELISA detected AT expression in cell supernatants. D, AT activity in cell supernatants. CON (control: pcDNA3.1-EGFP empty vector)

Immunofluorescence studies demonstrated cytoplasmic localization of AT in both groups with similar overall fluorescence intensity. Notably, the mutant AT protein showed distinct cytoplasmic aggregation patterns compared to the homogeneous distribution observed with wild-type AT (Fig. 3).

Fig. 3.

Immunofluorescent staining of wild type and p.Leu158Gln mutant antithrombin (AT). AT was labeled with green fluorescence, and cell nuclei were counterstained with DAPI (blue)

Bioinformatic analysis

PyMOL 2.6.0 was used to observe the tertiary structure of the AT protein. In the SERPINC1-(p.Leu158Gln) mutant, the Leu158 residue of the D-helix was substituted with Gln (Fig. 4A). Prior to structural analysis, glycosylation alterations were anticipated and subsequently assessed using the NetOGlyc-4.0 service (https://services.healthtech.dtu.dk/services/NetOGlyc-4.0/). Results indicated that this mutation did not impact glycosylation patterns, as depicted in Fig. 4B. Additionally, the integrity of the three pairs of disulfide bonds was confirmed to remain unaffected by the mutation, as shown in Fig. 4C.

Fig. 4.

Bioinformatic analysis. A, the tertiary structure of wild type and mutant AT. B, predict glycosylation sites of AT. C, predict disulfide bonds of AT

Discussion

The study focuses on the pathogenic mechanism of HATD and systematically explores the pathogenicity and underlying molecular mechanism of the SERPINC1 gene mutation p.Leu158Gln in a familial case of recurrent multi-site arteriovenous thrombosis. This investigation encompasses clinical case analysis, gene testing, in vitro functional experiments, and bioinformatics prediction.

Functional verification and mechanism analysis

HATD was initially documented in 1965 by Egeberg, establishing an association between reduced plasma AT levels and familial thrombotic predisposition [10]. HATD manifests distinct clinical phenotypes based on mutation subtypes. Null mutations exhibit significantly higher venous thromboembolism (VTE) incidence and earlier disease onset compared to missense mutations [11]. Type I deficiencies, representing the most severe clinical manifestations, predominantly result from disruptive genetic alterations including premature termination codons, splicing variants, and frameshift mutations in the SERPINC1 gene [12, 13]. While type II deficiencies typically stem from missense mutations with milder structural consequences, certain missense variants can include type I deficiency through mechanisms affecting RNA processing or protein trafficking [14, 15].

Missense mutations are the predominant type of SERPINC1 gene mutations, with first-onset ages distributed in young to middle adulthood and influenced by multiple factors (Supplementary Table 2). Intra-familial variability exists, such as the antithrombin Debrecen mutation (p. Leu205Pro), where patients experienced thrombosis at ages 15 and 65, respectively [16]. Additional risk factors like pregnancy may further lower the onset age [17]. Homozygous exhibit earlier onset and higher mortality than heterozygotes [18]. However, due to limited large-scale studies and individual clinical variability, the impact of specific mutation sites on onset age remains unclear.

In this study, the proband developed cerebral venous thrombosis at 19, a younger age than reported cases. This may be attributed to the mutation’s location in the D helix, impairing AT secretion and function, compounded by obesity and smoking as synergistic thrombotic risk factors. The mature AT protein, composed of 432 amino acids, features two critical functional domains: an N-terminal heparin-binding region and a C-terminal protease inhibitory active site [19]. Structural analysis demonstrated that the Leu158 residue is located within the D-helix of AT’s tertiary structure, which consists of three β-sheets (A, B, C) and nine α-helices (A-I) [20]. This region constitutes the heparin-binding domain, where interaction with the pentameric polysaccharide of heparin induces a conformational change that enhances AT’s inhibitory activity against FXa and FIXa by several orders of magnitude [21]. While previous studies have described the SERPINC1-p.Leu158Pro mutation causing type I HATD, no comprehensive literature is available and our study represents the first comprehensive characterization of the p.Leu158Gln variant [22]. The proline substitution is known to disrupt α-helix structure due to its unique cyclic conformation and inability to form hydrogen bonds. In contrast, our findings demonstrate that the glutamine substitution results in a slightly faster migration and abnormal intracellular aggregation patterns, suggesting structural alterations and impaired secretion. Furthermore, although the p.Leu158Gln substitution is predicted to increase mass, a slightly faster migration was consistently observed under reducing conditions. This discrepancy likely reflects altered SDS-binding or subtle conformational changes rather than a true mass difference.

Intramolecular disulfide bonds are essential for the correct folding, structural stability, and secretion of the AT protein [23]. Fuyong Zhang et al. revealed that the missense mutation (Cys462Tyr) disruptsthe 279Cys-462Cys disulfide link, resulting in type I HATD [24]. The 160Cys residue established a disulfide link with 40Cys, however bioinformatics research indicated that the p.Leu158Gln mutation did not interfere with the formation of the 40Cys-160Cys bond.

N-linked glycosylation is an essential post-translational modification that plays a significant role in protein folding, functionality, and degradation [25]. A prior study indicated that hypoglycosylation can lead to antithrombin deficiency in the absence of a SERPINC1 gene mutation [26]. Furthermore, a genetic abnormality may provide an extra N-glycosylation site, so disrupting secretion and function [27]. The amino acid sequence of AT has four N-glycosylation sites: Asn128, Asn167, Asn187, and Asn224. We utilized a professional website to assess the impact of gene mutations on the four glycosylation sites of AT and determined that there was no effect.

Notably, D-helix mutations including p.Phe77Leu [28] and p.Thr147Ala [29] are known to affect heparin binding. We hypothesized that p.Leu158Gln might similarly influence heparin affinity through structure perturbations, though definitive confirmation requires high-resolution structural analysis via cryo-electron microscopy.

Clinical treatment insights

AT is the most important natural anticoagulant substance in the body, which maintains coagulation anticoagulant balance by inhibiting thrombin and factor Xa. HATD caused by SERPINC1 gene mutation is an autosomal dominant genetic disease, accounting for 4–5% of hereditary thrombophilia. In this case, the patient had recurrent venous thrombosis (cerebral vein, mesenteric vein) in early adulthood followed by coronary artery thrombosis in middle age, underscoring the potential for both venous and arterial thrombotic manifestations in HATD patients. While historical data primarily associated HATD with venous thromboembolism, emerging evidence indicates a substantially elevated risk of arterial thrombotic events in certain patients, particularly when coexisting with atherosclerotic risk factors. The current patient’s long-standing smoking habit and obesity likely synergized with the underlying AT deficiency to promote endothelial dysfunction, inflammatory activation, and subsequent plaque rupture. These observations emphasize the importance of comprehensive thrombophilia screening, including AT activity assays and SERPINC1 genetic testing, for young patients presenting with recurrent thrombosis (especially mixed arteriovenous events) or positive family histories. Notably, this case illustrates the critical consequences of anticoagulation discontinuation, as the patient experienced thrombotic recurrence following self-cessation of therapy, thereby reinforcing the necessity for early diagnosis and lifelong antithrombotic management in confirmed HATD cases.

The patient carries a heterozygous missense mutation of SERPINC1 c.473T >A. Although his mother and daughter were confirmed as mutation carriers, neither exhibited thrombotic events, indicating the incomplete penetrance characteristic of this gene variant. This observed gender disparity in phenotypic expression may be attributed to several protective mechanisms in female carriers: (1) estrogen-mediated effects including platelet aggregation [30] and enhanced expression of anticoagulant proteins; and (2) reduced exposure to prothrombotic environmental factors such as smoking and surgical interventions. However, given the potential for late-onset thrombosis in HATD, longitudinal clinical surveillance of asymptomatic carriers remains essential for comprehensive risk assessment.

The anticoagulant therapy in this case was sequentially modified from warfarin to rivaroxaban and ultimately to dabigatran etexilate, reflecting the therapeutic challenges in HATD. The principal clinical dilemma concerns the appropriateness of direct oral anticoagulants (DOACs) in HATD management [31]. While rivaroxaban (a factor Xa inhibitor) is effective for most thrombophilia, its mechanism of action remains partially dependent on AT-mediated indirect inhibition of factor Xa [32]. The patient’s suboptimal AT level (46.2%) likely contributed to the inadequate therapeutic response observed with rivaroxaban, as evidenced by persistent RCA occlusion on follow-up imaging. In contrast, dabigatran etexilate’s direct thrombin inhibition mechanism [33] circumvents the AT-dependent pathway, potentially offering superior efficacy in severe AT deficiency. The absence of recurrent thrombosis following the transition to dabigatran etexilate supports this pharmacological rationale, though vigilant monitoring for hemorrhagic complications remains imperative during long-term therapy.

Limitations

While cellular and bioinformatics analyses characterize the SERPINC1 p.Leu158Gln mutation’s functional effects, key gaps remain: (1) atomic-level structural changes require cryo electron microscopy resolution; (2) heparin-binding impairment needs surface pasmon resonance validation; (3) expanded pedigree analysis is needed to clarify genotype-phenotype correlations. Addressing these will enhance mechanistic understanding and clinical management.

Conclusion

In summary, our study establishes the SERPINC1 p.Leu158Gln mutation as the molecular basis for type I HATD in this clinically severe case presenting with both arterial (myocardial infarction) and venous thrombotic events. Structural and functional analyses suggest this missense mutation likely induces conformational changes that impair both AT secretion and anticoagulant activity. While these findings provide important mechanistic insights, further validation through structural biology approaches and expanded clinical studies will be essential to fully elucidate the mutation’s pathophysiological consequences and therapeutic implications.

Electronic Supplementary Material

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Author contributions

ZCX performed the experiments and was a major contributor in writing the manuscript. GXM and XYN analyzed and interpreted the patient data. LYH conducted the bioinformatic analysis. KLN and XB organized the project. LZ, ZMJ, YL and WY provided guidance. All authors read and approved the final manuscript.

Funding

Not applicable.

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.