Genetic Analysis of a Family With Hereditary Factor V Deficiency Induced by Compound Heterozygous Variants (IVS24+3A>T and p.Asp2222Gly)

ABSTRACT

Objective

Factor V(FV) is a critical cofactor in the coagulation cascade. The diagnosis and investigation of novel variant contribute to improving the clinical treatment and management of patient with Factor V deficiency.

Methods

Coagulation screening, FV activity, FV antigen levels and thrombin generation assay (TGA) were performed on plateletpoor plasma samples collected from eleven family members. All exons and flanks of F5 gene were analysed by Sanger sequencing and novel variant were confirmed by reverse sequencing. Online bioinformatics tools were used to investigate possible adverse and effects on splicing of proband's F5 gene novel variant. Thromboelastography (TEG) and TGA were utilized to detect and evaluate the coagulation function of proband and heterozygous carrier family members.

Results

The proband was a middle‐aged Chinese woman with menorrhagia, presenting as an FV deficiency patient with FV activity and antigen levels of 7% (reference: 86%–114%) and 6% (reference: 70%–140%), respectively. Genetic testing revealed that proband carried compound heterozygous variants in F5 gene: c.6528+3A>T(IVS24+3A>T) and c.6665A>G(p.Asp2222Gly). Multiple online tools indicate that the novel variant represents a genuine splice‐site, leading to the deletion of a fragment and triggering exon skipping. The TGA results indicate decreased thrombin generation capacity in the proband, while TEG showed prolonged clotting initiation time.

Conclusion

The proband with hereditary factor V deficiency carries compound heterozygous variants, IVS24+3A>T and p.Asp2222Gly, which have led to alterations in clinical phenotype, genotype, and function. The IVS24+3A>T variant in the F5 gene is a novel splice‐site variant reported here for the first time.

1. Introduction

Coagulation factor V(FV) is a crucial nonenzymatic protein in the coagulation cascade, which is a large single‐chain glycoprotein(330‐kDa) composed of A1‐A2‐B‐A3‐C1‐C2 domains, primarily synthesized in the liver (approximately 80%), with the remaining 20% produced in megakaryocytes and stored in platelet α‐granules [1, 2]. Upon activation by thrombin or factor Xa, FV is converted to its active form, FVa. FVa consists of a heavy chain (residues 1–709, A1‐A2 domains) and a light chain (residues 1546–2196, A3‐C1‐C2 domains), which associate noncovalently in a calcium‐dependent manner [3].

As an essential component of the prothrombinase complex, FVa accelerates the activation of prothrombin to thrombin by nearly 300,000‐fold, positioning it at a penultimate and critical step of the coagulation cascade [4]. Consequently, FV has been the subject of extensive research.

The mature FV protein consists of 2196 amino acids and is encoded by the F5 gene [3, 5]. Inherited FV deficiency, a rare autosomal recessive disorder, is typically caused by various F5 gene variants and presents with highly variable clinical phenotypes, ranging from mild bleeding to severe haemorrhages [6, 7]. Although traditionally classified by plasma factor V activity (FV:C) levels, a notable genotype–phenotype discrepancy exists, as FV:C alone often fails to predict clinical severity. Diagnosis usually begins with prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT), confirmed by specific FV assays; however, due to the poor correlation between laboratory parameters and bleeding symptoms, global haemostasis assays such as thrombin generation tests (TGA) or thromboelastography (TEG) are increasingly emphasized to provide a more integrated assessment of coagulation potential [8]. Management of FV deficiency is challenging due to the lack of specific concentrates. Treatment is primarily supportive: fresh frozen plasma (FFP) for acute bleeding or surgical prophylaxis, antifibrinolytics for mucosal bleeding, and occasionally platelet transfusions in severe cases [9].

This study describes a Chinese family with inherited FV deficiency, where the proband exhibits menorrhagia and easy bruising. Genetic analysis identified two heterozygous F5 gene variants: a novel splice‐site variant (IVS24+3A>T), unreported in global databases, and a known missense variant (p.Asp2222Gly). Through clinical phenotyping, laboratory assays, and molecular analysis, we characterize the molecular basis of this deficiency, offering insights into genotype–phenotype correlations.

2. Materials and Methods

2.1. Specimen Collection and Family Pedigree

The proband was a 47‐year‐old Chinese female who presented with prolonged PT and APTT during preoperative evaluation for cervical polypectomy. She reported a history of heavy menstrual bleeding and a tendency to develop bruises, with no other significant bleeding symptoms. Further testing revealed markedly decreased levels of both FV:C and Factor V antigen (FV:Ag), leading to a preliminary diagnosis of FV deficiency type I (characterized by simultaneous reduction in both indexes). Family history investigation revealed a history of cerebral haemorrhage in her younger brother, while no other family members exhibiting spontaneous bleeding manifestations. There is no biological relationship between parents. The family pedigree of FV deficiency was investigated across 3 generations encompassing a total of 11 members, as shown in Figure 1.

FIGURE 1.

The family tree of the inherited coagulation FV deficiency.

2.2. Coagulation Index Assays and Genome Sequencing

Peripheral venous blood samples were collected from each subject using a soft‐connected 0.7 mm disposable blood collection needle (Weigao, Weihai, China). For coagulation assays, 2.7 mL of blood was drawn into vacuum tubes containing 0.3 mL of 109 mmol/L sodium citrate (Improve Medical, Guangzhou, China), achieving a final blood‐to‐anticoagulant ratio of 9:1, and gently mixed. A second tube containing the same anticoagulant was collected for TEG. A third tube containing EDTA‐K2 (Improve Medical, Guangzhou, China) was collected for DNA extraction. Platelet‐poor plasma was obtained by centrifuging the first sodium citrate tube at 1500 g for 15 min (Baiyang, Beijing, China). The separated plasma was used for routine coagulation parameter testing and TGA. Whole blood from the second sodium citrate tube was used for TEG analysis within the recommended time frame. Blood samples from the proband's brother and other family members were not collected concurrently with those from the proband.

Genomic DNA was extracted from EDTA‐anticoagulated whole blood using the Tian'anpu Gene Kit (Tian'anpu, Beijing, China) according to the manufacturer's protocol. All exons and exon–intron boundaries of the F5 gene were amplified using an ABI 7500 real‐time quantitative PCR instrument (Thermo Fisher Scientific, USA) under previously described conditions [10]. The amplified products were subsequently sequenced by Sunsoon Biotechnology Co., Ltd. (Shanghai, China) using Sanger sequencing. The obtained sequences were aligned with the human F5 reference sequence (GenBank: AY364535) using Chromas software. All identified variants were confirmed by bidirectional sequencing.

FV:Ag levels were determined using a double‐antibody sandwich ELISA kit (Changfeng, Wenzhou, China). The von Willebrand factor (vWF) was measured via immunoturbidimetry, and fibrinogen (FIB) levels were assessed using the Clauss method. PT, APTT, and the activities of coagulation factors II, V, VII, VIII, and X (expressed as FII:C, FV:C, FVII:C, FVIII:C, FX:C) were measured using the one‐stage clotting assay on an STA‐R‐Max automated analyser (Stago, France). All reagents for these assays were sourced from Siemens Healthcare Diagnostics Products GmbH (Germany). All procedures were performed strictly in accordance with the manufacturers' instructions.

2.3. Online Multiple Bioinformatics Software Analysis

The online informatics tools NetGene2‐2.42 (https://services.healthtech.dtu.dk/services/NetGene2‐2.42/), Ensembl VEP (https://www.ensembl.org/Homo_sapiens/Tools/VEP), and the Rare Disease Database (RDD, https://rddc.tsinghua‐gd.org/zh/tool) were used to comprehensively evaluate the variant's potential impact on splice sites, its pathogenicity, and its effects on splicing patterns.

2.4. TGA Test

TGA is determined using the calibrated automated thrombin generation assay, with plasma thrombin generation process recorded via the Fluoroskan Ascent FL reader. By measuring four parameters: lag time, peak height (Peak), endogenous thrombin potential (ETP) and time to peak(ttPeak)—the test reflects the overall level of thrombin generation within the subject's body to evaluate coagulation function. All experiments were conducted following the manufacturers' protocols for the respective kits and instruments.

2.5. TEG Test

After thorough mixing of whole blood with kaolinite and a standing period for activation, the sample was loaded into the TCA6000 thromboelastography analyser (Shengyu Medical Technology Co., Ltd., China) to record the TEG curve and parameters. The measured parameters include the clotting initiation time (R‐value), clotting kinetics (K‐value), clot formation angle (Angle), and maximum amplitude (MA). Specifically, the R‐value reflects the activation rate of coagulation factors. The K‐value and Angle assess fibrinogen function and the speed of clot formation; a shortened K‐value or increased Angle indicates enhanced fibrinogen activity and reflects a faster rate of fibrin clot formation. The MA represents the maximum strength of the fibrin clot.

3. Results

3.1. Phenotype and Genotype Test Results of Pedigree

The proband exhibited significantly prolonged PT (25.6 s; reference: 12.5–14.5 s) and APTT (58.3 s; reference: 29.0–43.0 s), along with markedly reduced factor V activity (FV:C, 7%; reference: 86%–114%) and antigen levels (FV:Ag, 6%; reference: 70%–140%). Her younger brother also showed severely decreased FV:C and FV:Ag levels (3% and 5%, respectively). Among the remaining family members, only her husband and sister‐in‐law had normal coagulation parameters without significant abnormalities. The FV:C and FV:Ag levels in other family members were approximately half of the reference ranges, as detailed in Table 1.

TABLE 1.

Phenotypes and genotypes of pedigree.

Family members	Age	PT (s)	APTT (s)	FIB (g/l)	vWF (%)	FII: C (%)	FVII:C (%)	FVIII:C(%)	FX:C (%)	FV:C (%)	FV:Ag (%)	Nucleotide changes
Mother (I2)	73	15.6	49.6	2.15	113	95	89	93	88	63	58	IVS24+3A>T heterozygote
Eldest sister (II1)	52	15.9	46.8	2.28	121	90	100	99	89	65	66	IVS24+3A>T heterozygote
Second sister (II2)	50	16.3	45.3	2.06	124	89	102	103	91	61	59	p. Asp2222Gly heterozygote
Proband (II3)	47	25.6	58.3	2.26	98	101	95	130	92	7	6	compound heterozygote
Husband (II4)	48	13.8	34.2	2.36	85	90	105	112	114	91	89	Normal
Younger brother (II5)	45	22.7	55.7	2.53	105	89	96	96	80	3	5	compound heterozygote
Sister‐in‐law (II6)	43	12.9	32.5	2.99	116	103	101	109	97	104	99	Normal
Daughter (III1)	20	16.3	48.6	2.36	93	105	91	98	89	63	65	IVS24+3A>T heterozygote
Son (III2)	17	15.9	50.3	2.06	89	112	93	102	105	58	62	p. Asp2222Gly heterozygote
Niece (III3)	16	17.1	49.6	2.48	101	108	98	100	103	53	60	IVS24+3A>T heterozygote
Niece (III4)	13	15.6	48.3	2.24	112	107	96	95	103	57	66	p. Asp2222Gly heterozygote
Reference range	—	12.5–14.5	29.0–43.0	2.00–4.00	75–125	86–116	86–120	78–130	80–120	86–114	70–140	—

Abbreviations: APTT, activated partial thromboplastin time; FIB, fibrinogen; FII:C, factor II activity; FV:Ag, factor V antigen; FV:C, factor V activity; FVII:C, factor VII activity; FVIII:C, factor VIII activity; FX:C, factor X activity; PT, prothrombin time; vWF, von Willebrand factor.

Both the proband and his younger brother carry a heterozygous c.6528+3A>T(IVS24+3A>T) variant in intron 24 and a heterozygous missense c.6665A>G(p.Asp2222Gly) variant in exon 25 of the F5 gene. Proband's mother, eldest‐sister, daughter, and eldest niece were heterozygous for IVS24+3A>T, while the second‐sister, son, and youngest‐niece were heterozygous for p.Asp2222Gly (Figure 2A, B). The IVS24+3A>T variant has been confirmed by reverse sequencing and represents the first reported instance of this splice‐site variant. Genetic screening of 100 healthy individuals excluded the possibility of this variant being a common polymorphism. Furthermore, its absence from public databases such as the Human Gene Mutation Database (HGMD), PubMed, and Ensembl confirmed it as a novel variant.

FIGURE 2.

The F5 Gene Sequencing Results and Predicted Splicing Pattern Diagram. Note: 2A: IVS24+3A>T heterozygous variant; a represents forward sequencing, b represents reverse sequencing, c represents wild‐type; 2B: p.Asp2222Gly heterozygous variant; a represents forward sequencing, b represents wild‐type. The arrow indicates the variant site; 2C: IVS24+3A>T splicing results: Solid lines indicate normal splicing patterns, while dashed lines represent abnormal splicing patterns caused by variant.

3.2. Bioinformatics Software Analysis Results

NetGene2 2.42 software predicts a splicing donor score of “0.93” (score range 0–1) for this variant site. A higher score indicates a greater likelihood that the site is a genuine splice‐site. The current score suggests this variant is highly likely to cause abnormal splicing and has a high predictive confidence. Ensembl VEP software prediction indicates that IVS24+3A>T variant is primarily located in the splice donor region (45%) and intron region (45%). The RDD predicts that the IVS24+3A>T variant will cause a 183 bp deletion near exon 25, leading to an abnormal splicing mode that involving exon skipping (Figure 2C).

3.3. TGA and TEG Results

The Figure 3A displays thrombin generation curves for the proband and their daughter and son. Table 2 further quantifies thrombin generation parameters for each sample, including lag time, ETP, Peak and ttPeak. Results showed that the proband exhibited prolonged Lag time to 4.38 min (reference: 1.7 min), a reduced Peak of 184.2Nm (reference: 374.3Nm), an ETP of 929.5 nM·min(reference:1175.7nM·min) and the ttPeak was 7.05 min (reference: 3.37 min), all indicating reduced thrombin generation capacity. The daughter's ttPeak (6.71 min) was prolonged compared to healthy controls, while the son's Peak(255.8Nm) and ETP (1080.8 nM·min) were similarly lower than the control, suggesting mild coagulation abnormalities in both individuals.

FIGURE 3.

TGA and TEG results of Proband. 3A: TGA, thrombin generation assay. 3B: TEG, Dotted lines represent the reference range, while solid lines represent the results. Blue represents the R‐value; purple represents the K‐value; orange represents the Angle; green represents the MA.

TABLE 2.

TGA results of the proband and offspring with hereditary FV deficiency.

	Proband	Daughter	Son	Reference range
Lag time (min)	4.38	4.21	1.67	1.70
ETP (nM·min)	929.5	1161.9	1080.8	1175.7
Peak (nM)	184.2	283.4	255.8	374.3
ttPeak (min)	7.05	6.71	4.38	3.37

Abbreviations: ETP, endogenous thrombin potential; Lag time, delay time; Peak, peak height; TGA, thrombin generation assay; ttPeak, time to peak.

The TEG (Figure 3B) results showed that the proband's R‐value was 8.19min (reference: 5.0–8.0 min), indicating prolonged initiation of the coagulation process, which reflects impaired coagulation function. The K‐value (result: 2.39 min, reference: 1.0–3.0 min) and Angle (result: 64.0°, reference: 50.0–72.0°) remain essentially normal, suggesting that subsequent stages of coagulation were not significantly impaired. The MA was 60.8mm (reference: 50.0–68.0 mm) suggests that the formed thrombus possesses a certain degree of strength and stability.

4. Discussion

Coagulation factor V is proteolytically cleaved to remove the B domain, thereby converting into FVa. Thrombin acts as an amplifier in the coagulation cascade, and any of a central coagulation factor can disrupt the clotting process [11]. Thrombin activity is largely dependent on FVa levels. Decreased FVa levels may lead to a potential risk of bleeding. Animal studies indicate that complete absence of FVa in mice leads to mid‐embryonic lethality or perinatal fatal haemorrhage [12]. However, bleeding manifestations in patients with FV deficiency show poor correlation with the FV: C. Common clinical presentations include mucosal bleeding (such as gingival bleeding and menorrhagia), postoperative or post‐traumatic bleeding tendency, and subcutaneous and soft tissue haemorrhage [6, 7]. FV deficiency can be broadly classified into two types: Type I is a quantitative deficiency, characterized by decreased plasma FV:Ag levels leading to a corresponding reduction in FV:C, manifested as a synchronous and proportional decrease in both; Type II is a functional deficiency, characterized by the presence of functionally abnormal FV in plasma, presenting as decreased FV:C with normal FV:Ag levels [13].

Preoperative screening of the proband revealed prolonged PT and APTT, with concurrent decreases in FV:C and FV:Ag to only 7% and 6%, respectively, leading to a diagnosis of type I FV deficiency. The test results for coagulation‐related indicators, including FIB, vWF, and the FII:C, FVII:C, FVIII:C, FX:C were all within normal levels, which largely rules out the possibility of combined deficiencies with other common coagulation factors. Genetic testing of this family demonstrated that the proband and his younger brother carried compound heterozygous variants in the F5 gene: IVS24+3A>T and c.6665A>G. The proband's mother, eldest‐sister, daughter, and eldest‐niece carried only the IVS24+3A>T heterozygous variant, while the second sister, son, and youngest‐niece carried only the p.Asp2222Gly heterozygous variant. Family segregation analysis indicated that the IVS24+3A>T variant originated from the proband's mother. The proband's heterozygous IVS24+3A>T and p.Asp2222Gly variants were inherited by his daughter and son. The compound heterozygous variants in the younger brother were also inherited to his daughters. These findings indicate that the familial hereditary FV deficiency in this family follows an autosomal recessive inheritance pattern.

The TGA comprehensively reflects the actual coagulation state by continuously monitoring thrombin concentration, provides a more accurate assessment of the procoagulant activity of factor V in plasma [14]. In this study, TGA results from the proband demonstrated a markedly delayed thrombin generation (4.38 min vs. reference 1.7 min) and a reduced peak thrombin level (184.2 nM vs. reference 374.3 nM), with alterations in other parameters indicating a significant impairment in thrombin generation capacity. The TEG reflecting the entire coagulation process from initiation to clot lysis, showed a prolonged coagulation initiation time in the proband (8.19 min vs. reference range 5.0–8.0 min), suggesting coagulation dysfunction. The proband's primary symptoms of menorrhagia and easy bruising are consistent with these laboratory findings. Both the proband and his younger brother exhibit bleeding tendencies, while other family members are currently asymptomatic. We speculate that the absence of symptoms in other members may be related to their relatively young age, though the actual situation requires long‐term follow‐up of this family.

The novel variant site IVS24+3A>T is located at position +3 of the splice donor site within intron 24 of the F5 gene, situated within the conserved splice signal region. Bioinformatics analyses using both NETGENE2 and Ensembl VEP strongly support the classification of this variant as a bona fide splice‐site variant. Predictions from the Rare Disease Database further suggest that the novel variant may cause a 183‐base pair deletion, leading to exon skipping and resulting in a predicted loss of function or protein truncation (Figure 2C). Cross‐validation of the results confirmed that the variant at this site causes alterations in protein synthesis. To date, over 150 variants in the F5 gene have been reported, with splicing variants accounting for approximately 16% [15]. However, only a limited number of splicing site variants in the F5 gene have been well documented worldwide. For instance, Claudia Dall‘Osso et al. [16]. reported three variants affecting F5 gene splicing: IVS8+6T>C, IVS21+1G>A and IVS24+1_+4delGTAG. Functional studies demonstrated that abnormal transcripts were detected after transfection of mutant constructs into COS‐1 cells. The IVS21+1G>A and IVS24+1_+4delGTAG variants activated a cryptic donor splice site, while the IVS8+6T>C mutation caused exon 8 skipping. Additionally, Ilaria Guella et al. [17]. showed through in vitro experiments that the IVS12+5G>A variant leads to exon 12 skipping, resulting in deletion of the C‐terminal region of the A2 domain. This likely compromises the stability and secretion of the mutant FV protein while also partially activating a cryptic donor splice site. Notably, the novel IVS24+3A>T variant identified in this study resides within the same donor splice site region as the previously reported IVS24+1_+4delGTAG. Therefore, it is speculated that this variant may also induce abnormal splicing events by interfering with donor splice‐site recognition.

The other familial variant, p.Asp2222Gly, is a previously reported functional site associated with the R2 haplotype, located within the C2 domain of the FV protein. This domain mediates the binding of FVa to phospholipid membranes—a critical step for the functional expression of FV activity. The Asp2222 residue is highly conserved across species, and its substitution leads to localized changes in charge and conformational stability, thereby weakening its affinity for phospholipid membranes [4]. Literature reports indicate that plasma FV levels decrease by approximately 20% in individuals carrying the heterozygous p.Asp2222Gly variant, while in vitro experiments show a reduction of about 34% in recombinant FV levels [18]. These findings suggest that p.Asp2222Gly may impair FV activity by disrupting its interaction with phospholipid membranes. Other variants located within the C2 domain (such as p.Met2120Thr and p.Arg2074Cys) exhibit similar pathogenic mechanisms [19, 20]. The findings above are consistent with the coagulation phenotypes observed in family members II2, III2, and III4, who carry only the heterozygous p.Asp2222Gly variant.

Recent studies have successfully established viable FV‐deficient mouse models through gene editing technology, providing more precise tools for disease research. De Pablo‐Moreno et al. [21] utilized CRISPR/Cas9 technology to introduce a missense mutation (Thr1898Met), associated with a mild human phenotype, into the mouse genome. They successfully obtained a heritable, non‐embryonic lethal FV deficiency model with a mild phenotype, exhibiting approximately 25% of normal FV activity. More importantly, Miguel‐Batuecas et al. [22] further explored the impact of truncating mutations on embryogenesis, revealing that the integrity of the FV protein is not only crucial for coagulation function but also closely related to embryonic development through its direct influence on thrombin generation. This provides a novel perspective for understanding the mechanisms underlying the clinical phenotypic heterogeneity resulting from different FV mutations. The existence of these new animal models facilitates pathophysiological research and the development of novel therapies for FV deficiency.

In summary, this study performed phenotypic, genetic, and functional analyses on a family with hereditary factor V deficiency. We identified a novel splice‐site variant (IVS24+3A>T) and a heterozygous missense variant (p.Asp2222Gly) in the F5 gene. The compound heterozygous effects of these variants led to markedly reduced FV activity and antigen levels in the proband, aligning with the genetic and molecular basis of FV deficiency. This work preliminarily elucidates the pathogenic mechanism of a novel splicing variant in a familial context, expands the mutational spectrum of the F5 gene and provides new molecular markers for the diagnosis and management of hereditary factor V deficiency.

Author Contributions

Ben Huang and Yan Zhang: data analysis and interpretation, draft preparation. Litao Zhang: sample collection, revision of key academic content. Li Wang: data acquisition, data analysis and interpretation. Kankan Su: research design, final version review and submission. All authors have made substantial contributions to the article or revising it critically for important intellectual content, and have given their final approval for the version to be submitted.

Funding

This work was supported by National Key Clinical Department of Laboratory Medicine of China in Nanjing (ZDXK202239).

Ethics Statement

This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University, China (Approval No. 2026‐SR‐125). All subjects provided written informed consent prior to participation.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

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