Genetic analysis of a pedigree with hereditary coagulation factor XII deficiency

doi:10.21203/rs.3.rs-4978926/v1

Objective: Analyze the clinical phenotype and gene mutations of a family with hereditary FXII deficiency, and preliminarily explore its molecular pathogenic mechanism.

Methods: The routine coagulation indicators and related coagulation factors were measured.. Thromboelastography and thrombin generation tests simulated coagulation and anticoagulation states in vitro and in vivo. PCR direct sequencing was utilized to analyze all exons and flanking sequences of the F12 gene in the proband, confirming suspected mutations through reverse sequencing, and identifying corresponding mutation sites in family members. Using ClustalX-2.1-win to analyze the conservation of the variant, and employing online software to predict the pathogenicity of mutations.

Results: The proband exhibited significantly prolonged APTT (169.1 seconds) and a pronounced decrease in FXII:C to 1.0%. Thromboelastography testing indicated a diminished function of the endogenous coagulation system, while thrombin generation testing revealed a normal ability for thrombin production in the proband. Gene sequencing revealed that the proband harbored a deletion mutation c.303_304delCA in exon 5 and a substitution mutation c.800+1G>A in intron 8. All three bioinformatics software indicated that the mutations were pathogenic and could lead to the production of a terminator, potentially altering the structure and function of the protein.

Conclusion: The deletion mutation c.303_304delCA and substitution mutation c.800+1G>A are associated with a decreased in FXII levels in this family, with the c.800+1G>A mutation being the first reported mutation worldwide.

Coagulation FXII deficiency

Genetic analysis

Bioinformatics

Gene mutation

FXII is a serine protease synthesized in the liver, playing a pivotal role in regulating both the blood coagulation and fibrinolysis systems. FXII participates in fibrinolysis through activation of the kinin-forming and kinin-releasing enzyme systems, with a protein structure closely resembling that of fibrinolysis system proteins^[1]. Therefore, the lack of FXII leads to increased thrombosis rather than increased bleeding. Hereditary FXII deficiency is an autosomal inherited disease caused by mutations in the gene encoding FXII protein. While most patients display autosomal recessive inheritance, some also show dominant inheritance ^[2]. Patients with hereditary FXII deficiency typically do not manifest clinical symptoms associated with other coagulation factor deficiencies, such as spontaneous bleeding or severe post-injury bleeding.^[3] This condition is often identified during preoperative screening.

This study aims to identify the pathogenic gene mutation site and preliminarily explore its molecular pathogenic mechanism by conducting coagulation index and gene mutation detection on a family with hereditary FXII deficiency.

Patients

The proband, a 24-year-old female, is two months pregnant. During routine antenatal check-ups, routine coagulation tests showed significantly prolonged APTT. Further coagulation factor testing revealed a significant decrease in FXII:C, while other coagulation indicators were within the normal range. but there was no clinical evidence of bleeding. A pedigree investigation was conducted on six individuals spanning four generations (Fig. 1). All members had normal liver and kidney function, with no history of spontaneous bleeding or thrombosis.

100 healthy subjects were recruited for this study as healthy controls, comprising 58 males and 42 females, with an average age of 35 years (range: 19–63 years). None of them had a history of abnormal bleeding or thrombosis tendency, and they did not have liver or kidney diseases.

Ethics

Our study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (KY2022-R193). Written informed consent was obtained from all participants prior to their involvement in the study.

Blood Samples

Collect 2.7 mL of peripheral venous blood from the subject, anticoagulated with 0.109 mol/L sodium citrate (ratio 1:9), and centrifuge at 3000 r/min for 15 minutes. The upper layer of platelet depleted plasma is used for coagulation index detection, which is completed within 2 hours. The lower layer of blood cells is used for genomic DNA extraction, PCR amplification, and sequencing.

Coagulation routine testing

PT, APTT, FIB, coagulation factors, and other coagulation indicators were measured using the one-stage coagulation method, while D-D was measured using immunoturbidimetry. All coagulation tests were conducted on the French Stago STA-R fully automatic blood coagulation analyzer with the original manufacturer's reagents.All procedures are strictly conducted in accordance with the reagent instructions.

F12 gene sequencing

Extract genomic DNA from the proband and family members using the NP968-G fully automatic nucleic acid extractor and its accompanying reagents (Xi'an Tianlong Company). Amplify the 12 exons, flanking sequences, 5' and 3' non-coding regions of the F12 gene from the proband using specific primer sequences and amplification conditions. Sanger sequencing was performed on the amplified product using the 3730xl s/n 1524-034 sequencer (ABI, USA). The sequencing results were compared with the F12 gene sequence (NM:000505.3) using Chromas software to identify mutation sites. After confirming the mutation site, amplify the corresponding exons of the family members accordingly.

Bioinformatics analysis

We performed online prediction analysis on the mutations using MutationTaster (https://www.mutationtaster.org) and the Franklin database (https://franklin.genoox.com).

Thromboela-stogram

Extract 2 mL of peripheral venous blood, add kaolin, mix well, and let it stand; After activation,insert the sample cup into the TCA6000 thromboela-stography instrument to automatically record TEG images and parameters, mainly including coagulation reaction time (R), coagulation formation time (K), alpha angle (α angle, Angle), and maximum thrombus amplitude (MA). R refers to the latency period from the start of the coagulation system to the formation of fibrin clots, K refers to the time from the start of coagulation to the amplitude of 20 mm in the recording image, and Angle refers to the time from the start of coagulation to the amplitude of 20 mm in the recording image. The formation rate of fibrin clots, MA refers to the final strength of fibrin clots. ^[4]

Thrombin generation test

According to reference ^[5], a calibrated automatic thrombin generation method (CAT) is used to assess thrombin generation. We conducted experiments using the reagent kit and analytical tools provided by Thrombinoscope BV (Rijswijk, Netherlands) with the Fluoroskan Ascent FL reader (Thermo Electron and Fisher Scientific, Waltham, Massachusetts, USA). The measured parameters included delay time (lag time), thrombin potential (ETP), peak (peak), and time to peak (time to peak) to assess the total amount of thrombin produced by the subjects.

Coagulation routine test results

The proband's APTT was extended to 169.1 seconds (reference range: 29.0–43.0 seconds), and FXII:C decreased to 1% (reference range: 72–113%). Meanwhile, the APTT of the proband's grandfather, father, mother, and daughter all showed slight prolongation, with FXII: C levels of 45%, 41%, 51%, and 24%, respectively. All other indicators of the family members were within normal ranges. (Table)

Thromboela-stogram and Thrombin generation test

The thromboelastogram results indicated a notable increase in the R value, with all other parameters within the normal reference range. The thrombin production test results showed that the proband's production curve was generally consistent with the reference, with all parameters closely aligned. (Fig. 2)

Genetic analysis

Gene sequence analysis revealed that the proband harbored a compound heterozygous mutation: a deletion mutation c.303_304delCA in exon 5 and a substitution mutation c.800 + 1G > A in intron 8. The proband's father carries a heterozygous deletion mutation c.303_304delCA, while the proband's grandfather, mother, and daughter carry a heterozygous substitution mutation c.800 + 1G > A. These test results indicate that the proband inherited the two mutations from the father and mother, respectively. (Fig. 3)

Bioinformatics analysis

MutationTaster and the Franklin database predict that both mutations are deleterious. Two RNA splicing prediction models for this mutation were established on the RDDC platform developed by the Guangzhou Rare Disease Gene Therapy Alliance. (Fig. 4)

In early coagulation theory, FXII was considered one of the components in contact with the coagulation system, activating FXII, which in turn activates FXI, FX, FIX, and FXI through the endogenous coagulation pathway to enter the common coagulation pathway and exert coagulation effects. Therefore, defects in FXII can lead to prolonged APTT ^[6–7]. However, since coagulation reactions in the body primarily occur through the extrinsic pathway, FXII deficiency alone does not lead to bleeding phenomena. On the contrary, due to the significant role FXII plays in activating the fibrinolytic system, defects or activation disorders may decrease fibrinolytic activity in the body, potentially leading to thrombotic diseases. In recent years, studies have shown that FXII plays an important role in thrombosis and is not essential in endogenous coagulation processes ^[8].

In our study, the total R value of the thrombelastogram result of the proband significantly increased, indicating that the patient's comprehensive coagulation state is in a hypocoagulable state, with overall weak activity of the endogenous coagulation system’s coagulation factors, which is consistent with the prolonged APTT and the extremely low FXII activity. However, the above experiments were conducted in vitro to simulate the activation of the endogenous coagulation pathway and may not fully reflect the actual coagulation process in the body ^[9]. Therefore, we conducted thrombin generation test to simulate the dynamic changes in thrombin production under the combined influence of the body’s coagulation and anticoagulation systems. ^[10]The results showed that the proband's thrombin generation ability was essentially consistent with that of the normal control, which also explains why the proband did not experience any bleeding or coagulation abnormalities on a daily basis or during pregnancy clinical manifestations.

Online analysis tools predict that both mutations are deleterious. The c.303_104delCA mutation causes a frameshift, substituting serine at position 101 with proline (His101Pro), and generating a premature termination codon in exon 6, leading to the production of a truncated protein. It is speculated that this site may have serious effects on coagulation and fibrinolysis functions, as has been reported ^[11]. The c.800 + 1G > A mutation occurs at the first position downstream of intron 8, precisely at the canonical splice site GT-AG domain, and is regarded as a pathogenic variant. We established an RNA splicing prediction model for this mutation using the RDDC platform developed by Guangzhou Rare Disease Gene Therapy Alliance. The results showed that the mutation disrupts the original donor splice site, affecting the normal mRNA splicing process. There may be two splicing models: one that deletes 166 bp causing exon skipping, and another that inserts 85 bp causing intron retention. Both ultimately result in frameshift mutations and premature termination. The protein translated after the mutation is rapidly degraded due to its high instability. It is also possible that abnormally cleaved mRNA cannot be transported into the cytoplasm, leading to a lack of protein translation ^[12]. The c.800 + 1G > A mutation has not been reported in Query human gene polymorphism database(https://www.ncbi.nlm.nih.gov/snp/) and the Human Gene Mutation Database(http://www.hgmd.cf.ac.uk/ac/index.php).

In summary, we identified a family with coagulation factor XII deficiency, where the proband carried compound heterozygous mutations: c.303_304 delCA inherited from her father and c.800 + 1G > A inherited from her mother. Both mutations can impact the gene structure by introducing different termination signals, thereby affecting the normal function of the protein. This results in varying degrees of decreased FXII activity among family members. The c.800 + 1G > A mutation is reported here for the first time. However, its precise molecular pathogenesis requires further research. The thrombelastogram and thrombin generation test explain why the proband had low FXII activity levels despite showing no clinical manifestations.

FXII：Coagulation factor XII

PCR: polymerase chain reaction

FXII:C：FXII activity

APTT：activated partial thromboplastin time

PT：prothrombin time

FIB：Fibrinogen

D-D：D-Dimer

TEG：thromboela-stogram

Acknowledgements

We appreciate the patient and her family members for their cooperation. All authors declared no competing interests that could be perceived as influencing the findings or conclusions.We are grateful to the patient and her family members for their cooperation. All authors stated that they had no interests which might be perceived as posing a conflict or bias.

Disclaimer statements

Disclosure of interest: The authors have no relevant financial or nonfinancial interests to disclose.

Contributors: All authors have made significant contributions to the study's conception and design, data acquisition, analysis and interpretation, as well as to the drafting and critical revision of the manuscript for important intellectual content. Furthermore, all authors have provided their final approval for the submitted version of the article.

Funding

This work was supported by Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province(2022E10022) and the Wenzhou Science and Technology Bureau Project (Y2023508).

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Table. Phenotypes andgenotypes of the inheritedcoagulation factor XII deficiencyfamily

Patient	PT (s)	APTT (s)	FIB (g/L)	D-D (g/L)	LAC	FⅧ:C (%)	FⅨ:C (%)	FⅪ:C (%)	FⅫ:C (%)	Genetic mutations
Proband	14.8	169.1	2.43	0.48	1.05	93	76	87	1	c.303_304 del CA c.800+1G>A
Grandfather	12.9	44.5	3.86	0.41	1.09	128	86	104	45	c.800+1G>A
Father	13.4	48.2	2.67	0.26	1.01	106	95	89	41	c.303_304 del CA
Mother	13.9	43.56	2.54	0.32	1.04	112	87	96	51	c.800+1G>A
Husband	13.5	35.6	2.58	0.27	1.08	120	90	105	96	Wild-type
Daughter	14.2	44.1	2.24	0.35	1.02	120	88	100	24	c.800+1G>A
Normal range	12.6-14.4	29.0-43.0	2.00-4.00	<0.50	0.80-1.20	78~128	75~128	82~118	72~113	Wild-type

No competing interests reported.

Genetic analysis of a pedigree with hereditary coagulation factor XII deficiency

Status:

Version 1

Abstract

Figures

Introduction

Material and methods

Patients

Ethics

Blood Samples

Coagulation routine testing

F12 gene sequencing

Bioinformatics analysis

Thromboela-stogram

Thrombin generation test

Results

Coagulation routine test results

Thromboela-stogram and Thrombin generation test

Genetic analysis

Bioinformatics analysis

Discussion

Abbreviations

Declarations

References

Table

Additional Declarations

Status:

Version 1