A pedigree analysis of deep venous thrombosis caused by rare compound heterozygous PROC mutations combined with a heterozygous THBD mutation

Abstract

Venous thromboembolism is a common fatal disease that includes pulmonary embolism (PE) and deep vein thrombosis (DVT), and many genetic risk factors are associated with its pathogenesis. We describe a patient with compound heterozygous mutations in PROC combined with a heterozygous mutation in THBD, who was diagnosed with DVT. Genetic sequencing identified three missense mutations in the proband: PROC c.565 C > T (p.R189W), PROC c.1218G > A (p.M406I), and THBD c.1456G > T (p.D486Y), this genotype not previously documented in association with thrombotic disease. His father carried heterozygous mutations of PROC p.R189W and THBD p.D486Y, while his mother and maternal grandfather were heterozygous for PROC p.M406I. These mutations were not detected in other family members.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12959-025-00824-7.

Introduction

Deep Venous Thrombosis (DVT) is a common disorder characterized by thrombus formation within the deep venous system. The annual incidence of venous thrombosis is approximately 1–2 per1000 individuals, with about 60% of cases presenting as DVT [1, 2]. Acquired risk factors for DVT include surgery, cancer, trauma, advanced age, immobilization, pregnancy, and postpartum conditions. In addition, gene mutation plays a crucial role in thromboembolic propensity, accounting for approximately 50–60% of venous thromboembolism cases [3–5]. In the Chinese population, mutations in PROC, PROS1, and SERPINC1 are common genetic risk factors for venous thrombosis [6–8], whereas THBD mutations are rarely reported. Here, we report the first familial case of severe DVT associated with compound heterozygous PROC mutations combined with a heterozygous THBD mutation, a genotype not previously documented in association with thrombotic disease.

Study subjects and methods

Case presentation

In November and December 2018, a 25-year-old male went to the hospital for treatment due to unknown lower limb pain and swelling. He was diagnosed with hereditary thrombophilia and lower extremity DVT, for which he was hospitalized and received anticoagulant therapy. During hospitalization, his condition worsened, with multiple thromboses and secondary cerebral infarction. Coagulation tests showed elevated D-dimer (10.42 µg/L) and fibrin degradation products (FDP, 41.4 µg/mL). Fibrinogen (FIB) was 5.46 g/L (normal: 2–4 g/L), activated partial thromboplastin time (APTT) was 45.2 s (normal: 23.3–32.5 s), thrombin time (TT) was 19.7 s (normal: 14–21.9 s), prothrombin time (PT) was 14.6 s, and antithrombin activity (AT: A) was 98% (normal: 75–125%). Vascular Doppler ultrasound revealed thrombi in the right superficial femoral vein, popliteal vein, bilateral iliac veins, and right internal jugular vein. Cerebral angiography demonstrated thrombosis in the superior sagittal sinus, right transverse sinus, and sigmoid sinus (Fig. 1). Cranial computed tomography confirmed cerebral infarction (Fig. 1). Symptoms improved after guideline-directed therapy. The patient was discharged on rivaroxaban and followed regularly. With informed consent, peripheral blood was collected from the patient and family members for genetic and laboratory analyses. Additional workup for acquired thrombophilia (lupus anticoagulant, anticardiolipin antibodies, anti-β2 glycoprotein I, homocysteine) was negative.

Fig. 1.

Cerebral angiography imaging and cranial computed tomography of the proband

Pedigree investigation

A three-generation pedigree was constructed based on medical history interviews.

DNA extraction and sequencing

Blood samples (2–3 ml) were drawn from the subjects’ peripheral veins and anticoagulated with sodium citrate (0.109 mol/L). The samples were centrifuged (1500×g, 5 min), and genomic DNA was then extracted from the cell pellet for mutation detection by sequencing. The second-generation sequencing technology based on Illumina platform was used to detect the proband. Through comparative analysis of sequencing data, it was found that the proband carried three different gene mutations: PROC: p.A189T, PROC: p.M406I, and THBD: p.A486T. By designing primers separately to perform Sanger sequencing on the direct relatives of the proband, the genetic mutation carrying status of the proband’s direct relatives can be clarified.

Coagulation assays

Collected peripheral blood was processed as above. Plasma samples were analyzed within 2 h for APTT, PT, TT, FIB, FDP, D-dimer, AT: A, protein C activity (PC: A), protein C antigen (PC: Ag), protein S activity (PS: A), thrombomodulin antigen (TM: Ag) and calibrated automated thrombin generation (CAT). PC: A, PS: A, APTT, PT, TT and FIB were measured by coagulation method; AT: A by chromogenic substrate assay; TM: Ag by chemiluminescence and CAT by fluorescence. PC: Ag was quantified using a commercial ELISA kit (ELK Biotechnology). These tests were conducted 4–5 days after the patient stopped DOAC treatment. In order to avoid false positives in test results and obtain accurate results.

Results

Pedigree and clinical history

The proband (III-1) and eight family members across three generations were studied. The proband’s grandmother (I-2, deceased) was suspected of having thrombosis; no other members had thrombotic events.

Genetic findings

Genetic sequencing identified that the proband carried three missense mutations: PROC c.565 C > T (p.R189W), PROC c.1218G > A (p.M406I), and THBD c.1456G > T (p.D486Y) (Fig. 2). Familial segregation showed that the PROC p.R189W and THBD p.D486Y mutations were inherited from the father (II-1), while the PROC p.M406I mutation came from the mother (II-2) and maternal grandfather (I-3). The proband’s brother (III-2) carried none of these variants (Fig. 3).

Fig. 2.

Genetic analysis of the patient and his pedigree (A. pedigree analysis of the patient. B. DNA sequencing of the PROC and THBD gene mutation; the position of mutational base is indicated with an arrow)

Fig. 3.

Results of calibrated automated thrombin generation in the proband and his family members

Laboratory findings

Coagulation parameters are summarized in Table 1. The proband’s TT, AT: A, and FIB were normal, APTT (54.40 s) and PT (17.10 s) were prolonged, likely influenced by his DOACs therapy. Notably, D-dimer (2.78 µg/L) and FDP (12.29 µg/mL) were markedly elevated, consistent with active thrombosis/hypercoagulability. The routine laboratory coagulation test results of other family members are shown in Table 1.

Table 1.

Laboratory tests of the proband and their family members

Members	I-3	I-4	II-1	II-2	III-1a	III-2	normal range
APTT	42.80	38.60	47.30	41.40	54.40	47.30	20.00 ~ 43.10(s)
PT	14.90	13.70	15.30	15.10	17.10	15.50	10.00 ~ 16.50(s)
TT	20.30	19.30	19.10	18.70	18.10	20.20	10.00 ~ 16.50(s)
AT-Ⅲ:A	96.00	105.00	95.00	108.00	87.00	96.00	80.00 ~ 120.00(%)
D-dimer	0.20	0.28	<0.01	0.20	2.78	0.20	0.00 ~ 0.50(ug/L)
FIB	1.71	1.65	2.08	1.68	2.04	1.46	2.00 ~ 4.00(g/L)
FDP	3.36	1.23	2.34	1.61	12.29	2.75	0.10 ~ 5.00(ug/Ml)
PC: A	49.00	117.00	62.00	53.00	10.00	87.00	70.00 ~ 130.00(%)
PC: Ag	2.43	4.36	3.67	2.14	1.25	4.47	3.00 ~ 6.00(mg/L)
PS: A	86.80	75.90	81.70	74.40	139.90	72.50	55.00 ~ 140.00(%)
TM	9.75	8.93	4.75	7.86	3.22	8.73	3.82–13.35(TU/mL)

a: the proband

PC: A and PC: Ag were markedly reduced in the proband (10% and 1.25 mg/L, respectively), consistent with type I protein C deficiency. His mother and grandfather (carriers of PROC p.M406I) also showed reduced PC: A and PC: Ag, while his father (carrier of PROC p.R189W) had reduced PC: A but normal PC: Ag. PS: A was normal in all, while TM: Ag was slightly lower in the proband (Table 1).

CAT test was used to analyze the whole process of coagulation in patients and his families. The results showed that due to the administration of rivaroxaban, the thrombin generation time of the proband was prolonged, the amount of thrombin produced was significantly reduced, and the inactivation time of thrombin was significantly prolonged (Fig. 3). CAT analysis (excluding the proband on rivaroxaban) revealed higher thrombin generation potential in mutation carriers, especially those with PROC p.M406I. However, different from the proband, the inactivation time of thrombin basically at 20 minutes in other members (Fig. 3).

Discussion

Genetic factors are the important underlying factors of venous thrombosis, and there are ethnic differences in the inherited traits of venous thrombosis. PROC, PROS1, and SERPINC1 gene mutations are common risk factors for thrombophilia in Chinese population [8], especially the PROC gene mutations. This report describes a patient with compound heterozygous PROC mutations (p.R189W and p.M406I) plus a heterozygous THBD p.D486Y mutation, resulting in severe thrombotic manifestations. The co-inheritance of compound heterozygous PROC mutations and a THBD variant in a single individual is rare and has not been previously reported. This combination likely leads to synergistic impairment of the protein C pathway, where severe PC deficiency diminishes activated protein C (APC) generation, while the THBD p.D486Y variant may further compromise APC generation efficiency by reducing TM’s cofactor activity. This dual defect in both the quantity and activation efficiency of PC likely explains the severe and early-onset thrombosis in the proband.

The proband’s prolonged APTT and PT are most likely attributable to his therapeutic anticoagulation with rivaroxaban, a direct factor Xa inhibitor known to prolong these global coagulation assays. Despite this anticoagulant effect, the significantly elevated D-dimer and FDP levels indicate a persistent hypercoagulable state and ongoing fibrin turnover. CAT plays an important role in assessing the risk of bleeding or thrombosis. Although the proband produced very little thrombin due to the medication, compared with the substantial thrombin generation observed in other family members—which was largely inactivated within approximately 20 min—the proband’s thrombin inactivation time was significantly prolonged. This suggests a clear defect in the proband’s anticoagulant system.

Protein C (PC) is a vitamin K-dependent zymogen that circulates in an inactive form [9, 10]. Upon activation, activated PC (APC) inhibits thrombin generation by cleaving activated coagulation factors V and VIII. Variants in the PROC gene can impair the structure and function of PC, leading to inherited PC deficiency and thrombotic risk. The pathogenicity of mutations within the same gene varies considerably across different sites. Over 500 PROC mutations have been recorded in the Human Gene Mutation Database (HGMD). In Chinese populations, PROC p.R189W and PROC p.K193del are hotspot mutations [11]. The prevalence of R189W is 0.9% in the general population and 5.21% in thrombotic patients [11–13]. This mutation alters the EGF-2 domain of the PC light chain, reducing PC: A while minimally affecting secretion, thus leaving PC: Ag relatively normal [14, 15]. In this study, the father carried the PROC p.R189W mutation and exhibited reduced PC: A with normal PC: Ag, consistent with previous reports. The proband carried two PROC mutations and presented with severely decreased PC: A and PC: Ag. The marked reduction in PC: Ag (< 50%) likely reflects the combined impact of the p.M406I mutation—which impairs protein secretion and stability—and the p.R189W mutation, which may further destabilize the protein or shorten its half-life, leading to a severe type I deficiency phenotype.

The PROC c.1218G > A (p.M406I) mutation identified in the proband is a known pathogenic variant. It impairs PC secretion and stability, reducing both PC: A and PC: Ag [16–19]. In a Korean population, M406I was reported as the second most common mutation in patients with venous thrombosis [20]. Compared with other mutations, M406I significantly increases the risk of venous thrombosis [21, 22]. Accordingly, reduced PC: A and PC: Ag were observed in the mother and maternal grandfather, who carried the PROC p.M406I mutation. Their thrombin generation potential was also significantly elevated compared with other family members. However, neither has developed thrombosis, likely due to the heterozygous state. Notably, the mother exhibited increased thrombin generation and thrombin generation potential in CAT assays but experienced no thrombosis during pregnancy or delivery.

The clinical manifestations of a single heterozygous mutation in PROC gene are highly heterogeneous. Homozygotes and compound heterozygotes of PROC gene mutations are less common [23]. However, patients with homozygous or compound heterozygous mutations had a significantly increased risk of thrombosis [24, 25]. The proband did not have a fulminant onset in his neonatal period despite carrying compound heterozygous mutations. The variable clinical expressivity within this family is noteworthy. The proband’s father (II-1), who carries both PROC p.R189W and THBD p.D486Y, remains asymptomatic despite laboratory evidence of reduced PC activity and relatively low TM antigen. This highlights the incomplete penetrance and multifactorial nature of inherited thrombophilia. Environmental factors (e.g., mobility, diet, absence of triggering events), epigenetic modifications, or the influence of other genetic modifiers may attenuate the thrombotic risk in carriers. This underscores the importance of considering gene-gene and gene-environment interactions in the clinical evaluation of thrombophilia families.

There are few reports of THBD mutations in Chinese patients with hereditary thrombophilia. Although numerous variants in THBD are recorded in population databases such as gnomAD, only a limited number have been established as pathogenic for thrombosis in mutation databases such as HGMD. The THBD p. D486Y mutation, first identified and reported by AK Ohlin in 1995 [26], results in the substitution of tyrosine with aspartic acid and conformational changes in key regions. Thus, the anticoagulant activity of TM was affected and TM antigen was reduced. This mutation has also been identified in patients with Atypical Hemolytic Uremic Syndrome (aHUS) [27, 28]. The proband and his father with THBD p. D486Y present lower TM: Ag than the others, indicating p. D486Y variant has a certain effect of TM. However, in ClinVar statistics, reports of THBD p.D486Y mutation are mostly benign mutations, and there is insufficient evidence for the pathogenesis of thrombotic events. This may be related to the rare coexistence of this mutation with other pathogenic anticoagulant protein mutations. It is noteworthy that while both the proband and his father carried the THBD p.D486Y variant, only the proband exhibited subnormal TM: Ag levels. This may reflect the compounded impact of concurrent severe protein C deficiency, which could further dysregulate endothelial protein C receptor (EPCR)-TM complex stability or expression. Additionally, biological variability in TM antigen measurement and the modest functional effect of the THBD p.D486Y variant alone may explain why the father—despite carrying the mutation—maintained TM: Ag within the normal range, albeit at the lower end compared to non-carrier family members.

In summary, we identified three different heterozygous missense mutations of PROC and THBD gene in a proband and his family members. There is obvious clinical heterogeneity of PROC mutations, and the compound mutations in proband obviously hits to the PC: A and PC: Ag. THBD gene mutation may also impair TM, resulting in serious damage to the PC system, and greatly increase the risk of thrombosis. The coexistence of PROC and THBD mutations in this family highlights the potential for multi-gene interactions in thrombotic risk. While the proband presented with severe thrombosis, his father—who carries both PROC p.R189W and THBD p.D486Y—remained asymptomatic, illustrating the variable penetrance and expressivity commonly observed in inherited thrombophilia. Environmental factors, epigenetic modifications, or other genetic modifiers may influence clinical outcomes. The detection of the mutation gene in family members also reminded them to pay attention to the acquired risk factors of thrombosis in life and early prevention to reduce the incidence of thrombosis.

Limitations

This study has several limitations. First, the functional impact of the THBD p.D486Y variant on TM cofactor activity was not experimentally validated in vitro; thus, its pathogenic contribution remains inferential. Second, the sample size is small, confined to a single family, which limits the generalizability of the findings. Third, although we conducted a thorough coagulation analysis, the impact of DOACs on certain tests such as thrombin production in patients has made the interpretation of baseline pre thrombotic potential complex.Future studies with larger cohorts and functional assays are needed to confirm the synergistic effect of combined PROC and THBD mutations and to identify additional modifiers influencing penetrance.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Y. Xiao was mainly responsible for project design and the acquisition of financial support. Y. Feng and Y. Xiao contributed to conception of the study. Data analysis and writing were completed by R. Tuo and LX. Chen. QL. Mo and XX. Xiao prepared figures. CL. Chen. and QL. Mo collected clinical data. All authors reviewed the manuscript.

Funding

The work was supported by the Major Science and Technology projects of Shenzhen Municipal Nanshan District Health System (No. NSZD2023054). Shenzhen Clinical Research Center for hematologic disease (No. LCYSSQ20220823091401002).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The gene testing and clinical examination was performed in accordance with the ethical committee of Shenzhen Qianhai Shekou Pilot Free Trade Zone Hospital. We obtained peripheral blood samples from the patient and his family for laboratory examinations and genetic detection after obtaining consent.

Consent for publication

We have ensured obtaining the consent of patients and their families for publication.

Competing interests

The authors declare no competing interests.