Analysis of phenotype and gene mutation in three pedigrees with inherited antithrombin deficiency

Abstract

Background

Inherited AT deficiency is an autosomal‐dominant thrombophilic disorder usually caused by various SERPINC1 defects associated with a high risk of recurrent venous thromboembolism. In this article, the phenotype, gene mutation, and molecular pathogenic mechanisms were determined in three pedigrees with inherited AT deficiency.

Methods

Coagulation indices were examined on STAGO STA‐R‐MAX analyzer. The AT:Ag was analyzed by ELISA. All exons and flanking sequences of SERPINC1 were amplified by PCR. AT wild type and three mutant expression plasmids were constructed and then transfected into HEK293FT cells. The expression level of AT protein was analyzed by ELISA and Western blot.

Results

The AT:A and AT:Ag of probands 1 and 3 were decreased to 49% and 52 mg/dL, 38% and 44 mg/dL, respectively. The AT:A of proband 2 was decreased to 32%. The SERPINC1 gene analysis indicated that there was a p.Ile421Thr in proband 1, a p.Leu417Gln in proband 2, and a p.Met252Thr in proband 3, respectively. The AT mRNA expression level of the three mutants was not significantly different from AT‐WT by qRT‐PCR. The results of ELISA and Western blot tests showed that the AT‐M252T and AT‐I421T mutants had a higher AT expression than the AT wild type (AT‐WT), and the AT protein expression of AT‐L417Q mutants had no significant difference compared with AT‐WT in the cell lysate. The AT expression levels of AT‐M252T and AT‐I421T mutants were lower than that of AT‐WT, and there was no significant difference between AT‐L417Q mutant and AT‐WT in the supernatant.

Conclusion

The p.I421T and p.M252T mutations affected the secretion of AT protein leading to type I AT deficiency of probands 1 and 3. The p.Leu417Gln mutation was responsible for the impaired or ineffective activity AT protein in proband 2 and caused type II AT deficiency.

In this article, the results showed that three mutations did not affect the expression of AT gene transcription level, but the p.M252T and p.I421T mutations reduced the secretion of AT protein and leading to type I AT deficiency. The p. L417Q mutation leading to type II AT deficiency by synthesized dysfunctional AT molecules to reducing AT:A.

1. INTRODUCTION

Antithrombin (AT) is the major serine protease inhibitor (SERPIN) in plasma that regulates the proteolytic activities of coagulation proteases of both intrinsic and extrinsic pathways. AT is synthesized predominantly by hepatocytes, has a half‐life of approximately 2.4 days and a molecular weight of 58 kDa, and contains 432 amino acids. ¹ AT is the major inhibitor of thrombin, activated coagulation factor IX (FIXa), and activated coagulation factor Х (FXa) in plasma, and is also able to inactivate the other serine proteases of the intrinsic coagulation pathway, activated factors XI and XII (FXIa and FXIIa). ² In the physiologic state, AT circulates in a relatively inefficient inhibitor form. However, the anticoagulant effect of AT is greatly increased (at least 1000‐fold) by the presence of heparin and other heparin‐like glycosaminoglycans. This effect is the basis for the use of heparin and low‐molecular‐weight heparins (LMWH) as anticoagulants in patients with venous thromboembolism (VTE). ³

The AT gene (SERPINC1) which encodes antithrombin is mapped on chromosome 1q23‐25 and comprises 7 exons encompassing 13.5 kb of genomic DNA. ⁴ The 1392 bp mRNA codes for a 464 amino acid polypeptide chain that undergoes several posttranslational modifications including cleavage of the signal peptide (32 amino acids) and N‐glycosylation. ⁵ , ⁶ Inherited AT deficiency is an autosomal‐dominant disorder caused by a defect in the SERPINC1 gene. It was first described in 1965 as one of the important risk factors for hemostatic diseases characterized by hereditary and recurrent thrombosis. In healthy people, the prevalence of inherited AT deficiency is around 0.02%–0.25%, but the incidence in patients with venous thrombosis is increased around 1%–2%. ⁷ , ⁸ , ⁹ Inherited AT deficiency is divided into type І with a reduction in the antigen levels and functional activity and type II deficiency with reduced antithrombin activity but almost normal antigen level. ¹⁰ , ¹¹

Most cases with type I defects are heterozygous; homozygous patients have a higher risk of severe venous thromboembolism (VTE) in childhood and may even be fatal in utero. ⁹ For type II AT deficiency, the level of AT in the patient's plasma is not significantly affected; therefore, we speculate that type II has a slight thrombotic effect. Related studies have shown that patients with type II deficiency exhibit extensive and heterogeneous clinical phenotype. ¹²

Inherited AT deficiency is widely considered to be one of the causes of thrombotic diseases. However, there are a few large cohort studies reported. More than 400 different mutations have been registered in the AT Mutation Database (The Human Gene Mutation Database, http://www.hgmd.cf.ac.uk/ac/all.php). Most are missense/nonsense mutations, the remaining being small insertion/deletion, large insertion/deletion, shear mutation, and complex mutation. The SERPINC1 gene mutations are heterogeneous in the population, and there are not many race‐specific mutations. ¹³ , ¹⁴ , ¹⁵

Inherited AT deficiency is the main genetic risk factor for deep vein thrombosis (DVT) patients in China. ¹⁶ Sporadic cases with SERPINC1 gene mutations were reported in the population; however, no mutation hotspots were recognized. In this article, we presented three Chinese pedigrees with inherited AT deficiency, the phenotype, gene mutations, and expression of mutation sites in vitro. The molecular bases of the three pedigrees were analyzed.

2. MATERIALS AND METHODS

2.1. Patients

Proband 1 was a 27‐year‐old woman who was admitted to the emergency department of our hospital for a sudden headache during puerperium, accompanied by nausea and vomiting. Magnetic resonance venography (MRV) showed thrombosis in the straight sinus, confluence of sinuses, and left transverse sinus. Seven family members of three generations had no relevant clinical manifestations. The pedigree is shown in Figure 1A.

FIGURE 1.

Three pedigrees of inherited AT deficiency

Proband 2 was a 74‐year‐old man, sent to our hospital for sudden left limb weakness and no movement of the upper and lower limbs. B‐ultrasound examination of the blood vessels of the lower extremities showed that there was thrombosis in the myenteric venous plexus of the legs. The pedigree is shown in Figure 1B.

Proband 3 was a 30‐year‐old pregnant woman, admitted to our hospital because her left leg was swollen and painful. She had undergone a cesarean section 4 years ago and developed pulmonary embolism (PE) after that delivery. Her mother and uncle had the same history of thrombosis, and her grandfather died of PE at the age of 64, while the other family members had no relevant clinical manifestations. The pedigree is shown in Figure 1C.

In order to establish a laboratory reference range, one hundred healthy subjects were randomly selected from our hospital as controls to exclude genetic polymorphisms; they included 50 males and 50 females with an average age of 28 years (from 16 to 52 years), without liver or kidney disease, no history of bleeding and thrombosis. Our study was reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (China). All participants signed a written informed consent.

2.2. Plasma AT assays

Peripheral blood samples of three family members and 100 healthy subjects were collected in anticoagulant tubes and centrifuged at 2100 g for 10 min, to obtain upper platelet‐poor plasma (PPP) and lower blood cells. The upper plasma was examined by coagulation test, and the lower blood cells were used for genomic DNA extraction.

Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (FIB), and protein S activity (PS:A) were analyzed using the STAGO STA‐R‐Max automatic blood coagulometer (Diagnostica Stago). D‐dimers were measured by the immunoturbidimetric method, AT:A, and protein C activity (PC:A) by the chromogenic substrate method. All laboratory tests were performed in accordance with the manufacturer's instructions (STAGO Company). The AT:Ag were detected by enzyme‐linked immunosorbent assay (ELISA), using the Human Antithrombin‐III (MIM #613118) ELISA kit (Hangzhou Lianke Biotechnology Co., Ltd). The AT protein content was processed and analyzed by Western blot.

2.3. AT gene analysis

Peripheral blood genomic DNA of all subjects was extracted from the anticoagulant blood samples using a DNA Extraction Kit (TIANGEN) according to the manufacturer's protocol. Primers for all 7 exons and their flanking sequences of SERPINC1 were designed with NCBI Primer. The specific amplification conditions were performed as previously described. ¹⁷ The amplified products were identified and sequenced (Shanghai Personal Biotechnology Co., Ltd.). The sequencing results were compared with the SERPINC1 standard sequence published in NCBI (GenBank ID: NG_012462.1), using Chromas software for mutation sites. Primers for R560Q and G20210A mutations which are common risk factors for hereditary venous thromboembolism were designed, and corresponding sites of the probands and their family members were screened.

2.4. Bioformatics analysis

The bioinformatics tools, including Mutation Taster (http://www.mutationtaster.org/), PROVEAN (http:// provean.jcvi.org/index.php), LRT (http://varcards.biols.ac.cn/), SIFT (http://sift.jcvi.org), and PolyPhen‐2 (http://genetics.bwh.harvard.edu/pph2/index.shtml), were applied to predict and analyze the possible impact of novel mutation on the structure and function of proteins.

Conservation analysis relied on ClustalX‐2.1‐win (Science Foundation Ireland), a multiple sequence alignment software used to analyze the conservation of affected amino acids with nine homologous species (HomoloGene, http://www.ncbi.nlm.nih. gov/homologene).

The PyMol software package (DeLano Scientific) was used to assess local structural effects of mutation in the AT gene, to determine the exact type and position of amino acid substitution in the three‐dimensional structural model. The AT protein crystal structure data were obtained from the Protein Data Bank (PDB, http://www.rcsb.org/pdb/home/home.do, PDB ID: 1ANT).

2.5. Construction, transfection, and purification

The vector used in this experiment was pCDH‐copGFP‐T2A‐Puro vector. Wild ‐type AT plasmid (AT‐WT) and empty vector were purchased from Nanjing Tsingke Biotechnology Co., Ltd. The AT‐WT was used as a template to construct AT‐M252T (p.Met252Thr), AT‐I421T (p.Ile421Thr), and AT‐L417Q (p.Leu417Gln) expression plasmids, according to manufacturer's instructions (QuikChange Lightning Site‐Directed Mutagenesis Kit). Mutant primers were designed using PrimerX online software (http://www.bioinformatics.org/primerx/) and synthesized by Nanjing Tsingke Biotechnology Co., Ltd.

HEK293FT cells were cultured in Dulbecco's modified Eagle medium (DMEM), which was supplemented with 10% fetal bovine serum (FBS). The wild‐type and mutant plasmids were transfected into human embryonic kidney 293 cells (HEK293FT). After 48 h transfection, total mRNA was extracted from the cells using TRIzol reagent and then reversely transcribed to cDNA. AT mRNA levels were determined using Magic SYBR Green qPCR Mix (Maibo, M223), according to manufacturer's instructions. The AT:Ag expression in cell culture supernatant and cell lysate was collected and analyzed by ELISA. Western blot was used to detect AT protein level in the cell supernatant and lysate.

3. RESULTS

3.1. Phenotype and genotype

In pedigree 1, the AT:A and AT:Ag of proband (II₃), father (I₁), and daughter (III₁) all decreased; the values were 49%, 52 mg/dl, 48%, 42 mg/dl 40%, and 45 mg/dl, respectively, representing a type I AT deficiency. The phenotype results of the three pedigrees with inherited AT deficiency is shown in Table 1. Genetic analysis found that the above three members all carry the c.1358 T > C (Ile421Thr) heterozygous mutation in SERPINC1. In pedigree 2, the AT:A of proband (I1), and the eldest son (II2), second daughter (II4), and granddaughter (III3) decreased, with values 32%, 43%, 52%, and 48%, respectively; however, AT:Ag was within normal range, indicating a type II AT deficiency. They were all heterozygous for the c.1346 T > A (Leu417Gln) mutation. In pedigree 3, the AT:A and AT:Ag of proband (III₂), mother (II₃), and uncle (II₁) showed a simultaneous decrease in AT:A and AT:Ag, with values of 38%, 44 mg/dl, 50%, 48 mg/dl, 40%, and 54 mg/dl, respectively, indicating a type I AT deficiency. Sequencing analysis revealed a heterozygous missense mutation c.851 T > C (p.Met252Thr) in the above three members of pedigree 3. The sequencing diagram is shown in Figure 2. Three corresponding sites in 100 healthy subjects were sequenced and excluded genetic polymorphisms. The FV‐Leiden and prothrombin G20210A of common thrombogenic gene mutations were screened and showed none existing in three probands and their family members.

TABLE 1.

Phenotype results of three pedigrees with inherited AT deficiency

Member	Gender	Age (years)	PC:A (%)	PS:A (%)	AT:A (%)	AT:Ag (mg/dl)
Pedigree 1
I1 (father)	Male	56	88	76	48*	42*
I2 (mother)	Female	54	93	87	99	102
II1 (sister)	Female	30	98	93	120	118
II2 (husband)	Male	28	79	120	107	106
II3 (proband)	Female	27	110	119	49*	52*
II4 (younger brother)	Male	20	108	120	118	99
III1 (daughter)	Female	1	124	130	40*	45*
Pedigree 2
I1 (proband)	Male	74	115	120	32*	97
I2 (wife)	Female	70	90	86	109	102
II1 (eldest daughter)	Female	50	89	98	110	89
II2 (eldest son)	Male	45	109	105	43*	95
II3 (eldest daughter‐in‐law)	Female	44	86	70	109	102
II4 (second daughter)	Female	43	94	85	52*	93
II5 (second son‐in‐law)	Male	45	105	120	117	118
II6 (younger son)	Male	40	127	130	97*	90
III1 (grandson)	Male	22	106	96	106	113
III2 (grandson)	Male	21	99	87	114	109
III3 (granddaughter)	Female	18	87	102	48*	94
Pedigree 3
I2 (grandmother)	Female	86	90	86	109	102
II1 (uncle)	Male	54	89	98	38*	44*
II2 (father)	Male	53	109	105	108	109
II3 (mother)	Female	51	86	70	50*	48*
III1 (husband)	Male	32	106	96	106	113
III2 (proband)	Female	30	99	87	40*	54*
IV1 (daughter)	Female	4	87	102	110	94
Reference range	–		70–130	65–135	98–119	80–120

FIGURE 2.

Sequencing results: (A) c. 1358 T wild type, (B) c. 1358 T > C heterozygous mutation of proband 1; (C) c. 1346 T wild type, (D) c.1346 T > A heterozygous mutation of proband 2; (E) c.851 T wild type, (F) c.851 T > C heterozygous mutation of proband 3

The results of plasma AT protein from Western blot tests are shown in Figure 3. The plasma AT molecular weight of the three probands was consistent with the molecular weight of the normal mixed plasma control, but the plasma AT levels of probands 1 and 3 were significantly lower than those of the normal control. There was no difference between AT content of proband 2 and the normal control.

FIGURE 3.

WB results of Plasma AT protein. The control was human mixed plasma. Results 1, 2, and 3 were proband 1, 2, and 3 respectively

3.2. Bioformatics

The bioinformatics prediction results are shown in Table 2. MutationTaster, PolyPhen‐2, PROVEAN, LRT, and SIFT all predicted the p.Met252Thr, and p.Ile421Thr mutations as “pathogenic and harmful”. For the p. Leu417Gln mutation, MutationTaster, PolyPhen‐2, and LRT suggested that it was harmful; SIFT and PROVEAN software labeled the mutations as “tolerable” and “neutral” respectively. ¹⁸ The ACMG guidelines classified p.Met252Thr and p.Ile421Thr mutations as “pathogenic,” and the p. Leu417Gln mutation as “likely pathogenic.” The conservation analysis results showed that the Met252, Leu417, and Ile421 were highly conserved among homologous species (Figure 4). In the p.Ile421Thr wild type, when Ile421 was replaced by Thr421, the original hydrogen bond was unchanged, but new hydrogen bonds were formed between Thr421 and Ile412, and Thr421 and Ile422, as shown in Figure 5. In the wild‐type AT protein, the main chain of Leu417 formed two hydrogen bonds with the side chain of Glu414 and Ser52. In the wild‐type AT model, there were two hydrogen bonds between the main chain of Met252 residues and Met320. When the Met252 was substituted by Thr, the original hydrogen bond remained, and an additional hydrogen bond was formed with Met320 (as shown in Figure 5).

TABLE 2.

Bioinformatics prediction results of mutations

Software	p.Ile421Thr (Score)	p.Met252Thr (Score)	p.Leu417Gln (Score)
MutationTaster	Disease causing (1.000)	Disease causing (1.000)	Disease causing (1.000)
PolyPhen‐2	Probably damaging (1.000)	Probably damaging (1.000)	Probably damaging (1.000)
PROVEAN	Damaging (−5.480)	Damaging (−3.150)	Neutral (−2.030)
LRT	Deleterious (0.000)	Deleterious (0.000)	Deleterious (0.000)
SIFT	Damaging (0.001)	Damaging (0.001)	Tolerated (0.060)

Note: Meanings of scores are as follows: Mutation Taster: a probability close to 1 indicates a high predictive value; PolyPhen‐2: scores are evaluated as 0.000 (most probably benign) to 1.000 (most probably damaging); PROVEAN: the predefined threshold of score is 2.5, the score 2.5 or less (deleterious), greater than 2.5 (neutral); LRT: scores 0.01 or less (deleterious), greater than 0.01 (benign); SIFT: scores range from 0 to 1, 0.05 or less (damaging), greater than 0.05 (tolerated).

FIGURE 4.

Conservation analysis of homologous species of Met252, Leu417, and Ile421

FIGURE 5.

Mutation model analysis (A) p.Ile421Thr wild type, (B) p.Ile421Thr mutant type, (C) p.Met252Thr wild type, (D) p.Met252Thr mutant type. Hydrogen bonds are indicated by green dotted lines.

3.3. Transfection and expression

The AT‐M252T, AT‐I421T, and AT‐L417Q mutant plasmids were successfully constructed by site‐directed mutation without other mutation sites verified by sequencing. The wild type and each mutant plasmid were observed under normal field and fluorescence field after 48 h transfection. The results are shown in Figures S1–S4. The wild‐type and each mutant plasmid were successfully transfected. The efficiency was approximately 85%.

The mRNA expression level of each group was detected by the fluorescence quantitative RT‐PCR method; the results are shown in Figure S2. AT gene expression of the AT‐WT plasmid was 100%, AT‐M252T, AT‐I421T, and AT‐L417Q mutant were 94.65%, 94.62%, and 107.6%, respectively. There was no significant difference between these and AT‐WT in mRNA expression. The nontransfected control and samples transfected with empty vector did not express AT mRNA.

The AT:Ag of the wild‐type and each mutant plasmid group were measured by ELISA. The cell culture supernatant and lysate of the wild‐type group were defined as 100%, and the calculated results were statistically analyzed by GraphPad 8.0 software as shown in Figure S3. The AT expression levels of AT‐M252T and AT‐I421T mutants in the cell culture supernatant were lower than those of wild‐type WT, while the expression levels of AT‐M252T and AT‐I421T mutants in the cell lysate were higher than that of wild‐type WT. The expression of AT of AT‐L417Q mutant in the cell culture supernatant and lysate was equivalent to that of the wild type, and there was no statistical difference.

Semi‐quantitative analyses of AT proteins were performed by Western blot in the cell lysate and culture supernatant. The results are shown in Figure S4, respectively. AT protein of AT‐M252T and AT‐I421T mutants expressed significantly more than the wild‐type in the cell lysate. AT protein expressions of AT‐M252T and AT‐I421T mutant were significantly reduced, compared with the wild type. AT protein expression of AT‐L417Q mutant levels were not much different from wild‐type cells in lysate and culture supernatant. The Western blot results of the three mutants were consistent with the ELISA results.

4. DISCUSSION

Thrombophilia refers to a high thromboembolic tendency caused by certain risk factors, including hereditary and acquired factors. Genetic factors mainly include congenital defects of anticoagulant proteins such as AT, PC, and PS, mutations of prothrombin G20210A, and coagulation factor V Leiden. ¹⁹ Acquired risk factors include, but are not limited to, oral contraceptives, trauma, immobilization, and surgery, and other factors. ²⁰ In this article, we presented 3 families with inherited AT deficiency and found 3 heterozygous missense variants, namely c.1358 T > C (p.Ile421Thr), c.1346 T > A (p.Leu417Gln), and c.851 T > C (p.Met252Thr). However, these three variants were not found in 100 healthy controls, thus excluding genetic polymorphisms.

Proband 1 had p.Ile421Thr mutation and was diagnosed as a type I Inherited AT deficiency. The results of conservation analysis showed that the Ile421 site was highly conserved in homologous species, suggesting that this site plays an important role in the structure or function of AT protein. When le421 was replaced by Thr421, intermolecular hydrophobic force and hydrogen bonds changed, and the protein structure might have been affected, which was in accordance with the results of bioinformatics analysis. The p.Ile421Thr mutation has only been reported since 1994 by overseas researchers. ²⁰ , ²¹ The overseas case presented recurrent venous thrombosis with a family history of thrombosis, and developed DVT and PE after the first pregnancy at the age of 27 years, with similar symptoms after the second and third pregnancies. Similar to our study, proband 1 was 27 years old and had venous thrombosis during puerperium, indicating that the p.Ile421Thr mutation has a higher risk of thrombosis, and when combined with acquired risk factors such as pregnancy and surgery, the p.Ile421Thr mutation has early onset characteristics. The p.Leu417Gln mutation was found in all four members in the second family (proband 2, his son, daughter, and granddaughter) who carried this heterozygous mutation; the details of which had been reported by our group. ¹⁸ All members in the third family including the proband 3, her mother, and uncle carried the p.Met252Thr heterozygous mutation. Western blot results showed that proband 3's plasma AT level was lower than the normal controls, in accordance with the phenotypic results, and diagnosed as a type I hereditary AT deficiency. ²² The bioinformatics software predicted that it would be pathogenic and that the protein structure may be affected. The hydrophobic interaction force and the number of hydrogen bonds between Thr252 and surrounding amino acids had been changed, which had an influence on the structural stability. In family 3, all three members carrying the heterozygous mutation of p.Met252Thr had a history of venous thrombosis; the wild‐type members were asymptomatic, indicating that the mutation had a high correlation with thrombosis. Patients in a South Korean study with p.Met252Arg mutation both had decreased AT activity, antigen level, and venous thrombosis. ⁹ We speculated that Met252 might be a hot spot mutation of the AT gene and carries a higher risk of thrombosis.

In order to further investigate the reasons for the reduction in AT levels caused by these three mutations, we ligated the coding sequence (CDS) of the AT gene into the eukaryotic expression vector to construct the AT‐WT plasmid. RT‐PCR results showed that the expression of mutant AT mRNA was not significantly different from that of the wild type, indicating that these three mutations had no significant effect on AT transcription. Western blot results showed that levels of AT protein of AT‐M252T and AT‐I421T in the cell lysates were significantly higher than that of AT‐WT, but the AT levels of both in the culture supernatant were significantly lower than that of AT‐WT, showing that the mutations of p.Met252Thr and p.Ile421Thr affected the normal secretion of AT protein from the inside to the outside of the cell, resulting in AT being retained in the cell, and causing the AT level to decrease. ELISA results showed the same findings, and further verified the retention of AT protein in the cell. Mutations such as AT‐Trp225Cys, AT‐Ala404Asp ^,23 and AT‐Ala404Thr ²⁴ had previously been reported. In vitro expression experiments showed that these mutations also led to impaired secretion of AT protein, type I inherited AT deficiency. For the p.Leu417Gln mutation, both ELISA and Western blot showed that the AT level expressed by the AT‐L417Q mutant was not significantly different from that of the wild type, which was consistent with the phenotypic test result of proband 2. The AT activity level of the patient was reduced, indicating that dysfunctional AT molecules may be generated by the mutation, thus impairing or damaging anticoagulant activity. ²⁵

In this article, we confirmed that pathogenic molecular mechanisms of p.Met252Thr, p.Ile421Thr, and p.Leu417Gln mutations led to inherited AT defects in three families. However, the genotype is not completely consistent with the clinical phenotype. Among the 25 people in the above‐mentioned three families, ten people carry AT mutations, but only five people displayed clinical manifestations; three probands had venous thrombosis, but only family 3 had a family history of hemorrhagic thrombosis. Family members in Family 1 and Family 2 who carried the same mutations as the proband had no thrombosis or related clinical manifestations, suggesting that patients with simple AT deficiency might not always develop thrombus. Therefore, it is necessary to carry out routine and specific antithrombotic prevention and treatment for such patients.

5. CONCLUSION

We reported three heterozygous missense mutations in the SERPINC1 gene, namely c.1358 T > C (p.Ile421Thr), c.1346 T > A (p.Leu417Gln), and c.851 T > C (p.Met252Thr). The pathogenic mechanisms of the three mutations were preliminarily explored through bioinformatics, protein modeling, and in vitro expression analysis, which enriched the AT genetic mutation database.

AUTHOR CONTRIBUTIONS

MSW and ML designed experiments, analyzed data, wrote the article, and supervised the project. SQL and STJ performed molecular modeling. SQL and YHJ designed experiments, conducted genetic analysis, and coagulation assays. All authors approved the final version of the article.

CONFLICT OF INTEREST

The authors declare no conflicts of interests.

ETHICS STATEMENT

Our study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (China).

Supporting information

Figures S1–S4

Li M, Jiang S, Liu S, Jin Y, Wang M. Analysis of phenotype and gene mutation in three pedigrees with inherited antithrombin deficiency. J Clin Lab Anal. 2022;36:e24732. doi: 10.1002/jcla.24732

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.