Recurrent Founder Variant in Inherited FVII Deficiency: Molecular Spectrum Analysis in Southeast China Recurrent Founder Variant in Inherited FVII Deficiency: Molecular Spectrum Analysis in Southeast China

doi:10.21203/rs.3.rs-4674402/v1

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Recurrent Founder Variant in Inherited FVII Deficiency: Molecular Spectrum Analysis in Southeast China Recurrent Founder Variant in Inherited FVII Deficiency: Molecular Spectrum Analysis in Southeast China

https://doi.org/10.21203/rs.3.rs-4674402/v1

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Background

Factor VII (FVII) deficiency is a rare congenital coagulation disorder inherited in an autosomal recessive manner, characterized by reduced plasma FVII levels. Several variants in the F7 gene have been identified across different populations, with higher prevalence in certain regions. However, limited reports exist on predominant variants in Chinese and East Asian populations, highlighting the need for this study to fill the gap and improve understanding of genotype-phenotype correlations.

Result

This study characterizes severe FVII deficiency in 85 unrelated Chinese families, 57 exhibited severe FVII defects. Notably, 31 out of 57 experienced various bleeding manifestations, with the most prevalent being gingival bleeding, epistaxis, and menorrhagia. The Pearson's rank correlation test yielded correlation coefficients of − .496 (P = .000), − .266 (P = .014), and .149 (P = .175) between FVII activity and PT, FVII activity and bleeding scores, and PT and bleeding scores, respectively. Direct sequencing of the exons of the F7 gene and its border regions was conducted on all patients. The five variants with the highest allele frequencies were p.Arg364Gln, p.His408Gln, p.Arg337Cys, p.Cys10Profs16, and p.Cys389Gly. The p.Arg364Gln variant was identified in 11 individuals with homozygotes and 6 with compound heterozygosity, with the highest allele frequency of 30.51%. The variants p.Gln160Leu, p.Leu170*, p.Ser329Pro, and p.Trp344Gly were previously identified and reported by our research group. Additionally, a novel variant, p.Ile421Serfs76, is reported for the first time. This study, representing one of the largest cohorts of FVII variants in China, suggests that the prevalence of FVII deficiency may be underestimated. This conclusion is supported by the high proportion of asymptomatic subjects found to have low FVII levels.

Conclusion

The findings of this study, which represents one of the largest cohorts of FVII variants in China, indicate that the prevalence of FVII deficiencies may be underestimated. This is due to the high proportion of asymptomatic individuals with low FVII:C levels. The significant association between FVII:C levels, prothrombin time, and bleeding symptoms highlights the necessity for continued genetic analysis to enhance our comprehension of genotype-phenotype correlations and to refine diagnostic and management strategies for FVII deficiency in the Chinese population.

Factor VII Deficiency

Inherited bleeding disorders

Variant

Gene frequence

Factor VII (FVII) (OMIM: 227500) is a key factor in the initiation of coagulation. FVII deficiency initially reported by B. Alexander et al. in 1951[1], is a rare congenital bleeding disorder characterized by low or undetectable plasma levels of FVII, affecting approximately 1 in 500,000 people worldwide[2]. It is caused by variants in the F7 gene located in the 13q34 region of the human chromosome. Patients with the disease exhibit reduced FVII activity (FVII:C) in plasma, with clinical bleeding symptoms varying greatly from asymptomatic to severe[3]. The severity of clinical manifestations is closely linked to the number, location, and functional impact of the gene variants[4]. However, there is a close connection between the number of gene variants, their locus, and their impact on function[5, 6]. Variants leading to the complete absence of active plasma FVII are linked to severe phenotypes, such as neonatal death or irreversible organ damage, including intracranial hemorrhage (ICH)[7]. However, even trace amounts of active protein, adequate to initiate coagulation, markedly influence the phenotype[8]. This is particularly true when genetic information is combined with relevant clinical data and modeling analyses, aiding in determining the pathogenicity of a variant[9–11].

The FVII Variant Database (https://www.hgmd.cf.ac.uk/docs/login.html) has cataloged over 200 variants in the F7 gene, primarily identified through small patient studies and individual case reports. This extensive database provides valuable insights into the genetic diversity and pathogenic mechanisms underlying FVII deficiency.

In this study, additional genetic profiles can enhance our understanding of genotype-phenotype correlations in this rare coagulation disorder. We conducted a comprehensive analysis of variants and clinical presentations in 85 probands diagnosed with FVII deficiency, along with their respective relatives from the Chinese population. The data were collected over a fourteen-year period from 2010 to the present at the First Affiliated Hospital of Wenzhou Medical University. Genetic counseling was offered to other relatives upon identification of one or more variants in a family.

The objective of this study was to investigate the correlation between genotype and phenotype and to summarize the specific variant spectrum of the F7 gene in 85 patients with FVII deficiency from the Chinese population.

2.1 Participants

The retrospective cohort study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China. A total of 85 unrelated probands (FVII:C < 72% ) from 2010 to 2024 in our study provides a reasonable sample size to investigate FVII deficiency. The parents of probands numbered 1, 3, 6, 7, 8, 12–15, 24, and 25 are consanguineous (Table 1). The patient's demographic details, including age, sex, bleeding history, and consanguinity, were duly recorded. Exclusion criteria included a history of hepatic and renal disease, as well as acquired FVII defects. Among the population with detected variants, 66.1% exhibited symptoms indicative of bleeding, while 33.9% did not demonstrate any such symptoms. Other platelet or coagulation factor defects were also excluded. Bleeding scores of 85 patients were calculated according to ISTH-BAT, a bleeding assessment tool that Rodeghiero introduced[12]. The normal ranges for bleeding scores are 0–3 for adult males and 0–5 for adult females. The FⅦ levels were measured before transfusion. Other platelet or coagulation defects were excluded. Details of patients with detected variants are presented in Table 1, whilst information of patients without detected variants is provided in Supplementary Table 1. The reference sequences table is included in Supplementary Table 2 (both supplementary tables are available as separate files)

Table 1

Laboratory Findings and Clinical Phenotypes of Patients with Mutated FVII Deficiency. **Note**: The meaning of all abbreviations has been previously indicated.
Sr.no	Gender/Age	PT(s)	FⅦ:C(%)	Bleeding manifestation	Bleeding score	Nucleotide change	Mature protein alteration	Predicted pathogenicity	Consanguinity	carriers/families	Zygosity
1	F/23	30.9	3	SB	4	c.1224T > G	His408Gln	Damaging	Yes	3/6	Homo
2	M/7	32.8	3	PB/EP/SB	3	c.27_28delCT	Cys10Profs*16	Damaging	No	2/5	Homo
3	M/41	35.1	3	N	0	c.1090C > T	Arg364Trp	Damaging	Yes	2/4	Homo
4	F/72	26.5	2	N	0	c.27_28delCT	Cys10Profs*16	Damaging	No	N	Homo
5	M/53	34	2	GB/PB	3	c.1090C > T	Arg364Trp	Damaging	No	N	Homo
6	M/38	37.8	2	EB/EP/HU	7	c.1030T > G	Trp344Gly	Damaging	Yes	5/7	Homo
7	F/27	31.1	2	EP/MN	4	c.1090C > T	Arg364Trp	Damaging	Yes	N	Homo
8	F/55	36.2	3	GB/SB	3	c.1091G > A	Arg364Gln	Tolerable	Yes	N	Homo
9	F/23	20.3	3	N	0	c.1009C > T	Arg337Cys	Damaging	No	3/4	Homo
10	F/70	38.7	3	N	0	c.1091G > A	Arg364Gln	Tolerable	No	N	Homo
11	F/67	30.8	3	N	0	c.1091G > A	Arg364Gln	Tolerable	No	N	Homo
12	M/36	32.5	3	N	0	c.1091G > A	Arg364Gln	Tolerable	Yes	N	Homo
13	F/25	42.1	3	GB/PB	3	c.1091G > A	Arg364Gln	Tolerable	Yes	N	Homo
14	M/48	30.9	4	EP	1	c.1224T > G	His408Gln	Damaging	Yes	3/5	Homo
15	M/57	33.8	4	N	0	c.1091G > A	Arg364Gln	Tolerable	Yes	N	Homo
16	F/48	39.3	2	N	0	c.1165T > G	Cys389Gly	Damaging	No	3/6	Homo
17	M/56	30.2	3	ICB	7	c.722C > A	Thr241Asn	Damaging	No	3/4	Homo
18	F/75	39.1	2	N	0	c.1224T > G	His408Gln	Damaging	No	N	Homo
19	M/75	36.8	2	N	0	c.1165T > A	Cys389Gly	Damaging	No	N	Homo
20	F/53	53.8	4	N	0	c.1009C > T	Arg337Cys	Damaging	No	N	Homo
21	F/54	34.2	6	N	0	c.1091G > A	Arg364Gln	Tolerable	No	3/5	Homo
22	M/75	27.2	3	N	0	c.27_28delCT	Cys10Profs*16	Damaging	No	N	Homo
23	M/35	36.1	2	N	0	c.1091G > A	Arg364Gln	Tolerable	No	N	Homo
24	M/32	26.7	6	N	0	c.910G > A	Ala304Thr	Damaging	Yes	2/4	Homo
25	F/31	20.5	8	GB	2	c.1009C > T	Arg337Cys	Damaging	Yes	N	Homo
26	m/28	28.3	3	MN	1	c.1009C > T	Arg337Cys	Damaging	No	N	Homo
27	m/54	32.2	3	GI-MA	3	c.1091G > A	Arg364Gln	Tolerable	No	N	Homo
28	F/56	37.9	2	GB	4	/ c.1165T > G	IVS6 + 1G > T Cys389Gly	/ Damaging	No	N	Compound het
29	F/47	25.3	4	N	0	N	IVS1a + 5G > A IVS6 + 1G > T	/ /	No	N	Compound het
30	F/57	31.2	2	GB	1	c.1165T > G c.1224T > G	Cys389Gly His408Gln	Damaging Damaging	No	3/5	Compound het
31	M/67	24.5	8	ICB	4	c.27_28delCT c.1238G > C	Cys10Profs*16 Arg413Pro	Damaging Damaging	No	4/6	Compound het
32	F/52	19.9	15	N	0	c.752C > T c.1165T > G	Ala251Val Cys389Gly	Damaging Damaging	No	5/7	Compound het
33	M/59	32.8	3	EP	1	c.1165T > G c.1224T > G	Cys389Gly His408Gln	Damaging Damaging	No	N	Compound het
34	F/40	22.7	4	N	0	c.1009C > T c.1224T > G	Arg337Cys His408Gln	Damaging Damaging	No	N	Compound het
35	F/43	27.1	4	N	0	c.1009C > T c.27_28delCT	Arg337Cys Cys10Profs*16	Damaging Damaging	No	2/4	Compound het
36	M/55	39.6	2	N	0	c.27_28delCT c.1091G > A	Cys10Profs*16 Arg364Gln	Damaging Tolerable	No	4/6	Compound het
37	F/52	19.4	16	MN	1	c.691-693delTTG c.1224T > G	Leu170* His408Gln	Damaging Damaging	No	3/4	Compound het
38	F/21	36.3	2	EP/GB/PPH	7	c.1201A > G /	Lys401Glu IVS5-1G > A	Tolerable /	No	N	Compound het
39	F/43	35.4	2	MN	1	c.1224T > G c.1238G > A	His408Gln Arg413Gln	Damaging Damaging	No	N	Compound het
40	F/66	21.8	13	GI-MA	1	c.1238G > A /	Arg413Gln IVS6 + 1 G > T	Damaging /	No	N	Compound het
41	F/34	33.1	2	N	0	c.1091G > A c.1238G > A	Arg364Gln Arg413Gln	Tolerable	No	3/5	Compound het
42	F/47	36.1	2	MN	1	/ c.1238G > A	IVS6 + 1G > T Arg413Gln	/ Damaging	No	N	Compound het
43	F/30	35.7	2	SB/GB/PB	5	c.1091G > A c.1224T > G	Arg364Gln His408Gln	Tolerable	No	N	Compound het
44	F/61	38.8	3	SDH	6	c.1224T > G /	His408Gln IVS5 + 1G > A	Damaging /	No	N	Compound het
45	F/60	34.8	3	GI-MA/GB/EP	5	c.1224T > G c.698-162insT	His408Gln /	Damaging /	No	3/6	Compound het
46	M/25	25.2	3	PPH	3	c.1009C > T c.698-162insT	Arg337Cys /	Damaging /	No	N	Coupound het
47	F/42	34.8	2	GB/SB	2	c.1201A > G c.27_28delCT	Lys401Glu Cys10Profs*16	Tolerable Damaging	No	4/6	Compound het
48	M/25	39	2	N	0	c.1224T > G c.1091G > A	His408Gln Arg364Gln	Damaging Tolerable	No	N	Compound het
49	M/28	76.4	4	EB/SB/GB	5	c.1009C > T c.1270G > T	Arg337Cys Val384Phe	Damaging Damaging	No	N	Compound het
50	F/37	28.2	2	MN	2	c.698-162insT c.1009C > T	/ Arg337Cys	/ Damaging	No	N	Compound het
51	F/25	33.7	2	EP/SB	3	c.479A > T c.1224T > G	Gln160Leu His408Gln	Damaging Damaging	No	3/5	Compound het
52	M/56	36.3	5	N	0	c.985T > C c.1165T > G	Ser329Pro Cys389Gly	Damaging Damaging	No	2/4	Compound het
53	F/46	35.6	2	MN/PPH/PB	5	c.1090C > T c.1165T > G	Arg364Trp Cys389Gly	Damaging Damaging	Yes	N	Compound het
54	M/48	34	2	N	0	g.11895C > T c.1317G > T	c.805 + 120C > T Arg439Ser	/ Damaging	No	N	Compound het
55	M/30	35.7	4	GB/EB/PB	4	c.1009C > T c.27_28delCT	Arg337Cys Cys10Profs*16	Damaging Damaging	No	N	Compound het
56	F/14	19.5	6	N	0	c.1201A > G c.910G > A	Lys401Glu Ala304Thr	Tolerable	No	N	Compound het
57	F/20	13.9	7	GI-MA	3	c.1091G > A /	Arg364Gln Cys10Profs*16	Tolerable	No	N	Compound het
58	F/26	21.1	8	N	0	c.1165T > G c.1224T > G	Cys389Gly His408Gln	Damaging Damaging	No	N	Compound het
59	F/46	33.1	2	MN	1	c.722C > A c.1261_1261delA	Thr241Asn Ile421Ser*fs76	Damaging Damaging	No	3/5	Compound het
Reference range	11.3-14.5s	72%-133%	N	N	N	N	N	N	N	N	N

2.2 Blood Samples

From 2010 to 2024, we collected peripheral venous blood samples in standard 129 mmol/L trisodium citrate tubes from 85 individuals suspected of having inherited FⅦ deficiency, along with 100 healthy controls. After centrifugation at 3000 rpm for 15 minutes at 8°C, Platelet-Poor Plasma and blood cells were obtained. PPP was utilized for coagulation tests, and human genomic DNA was extracted from leukocytes using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China), following the manufacturer's instructions.

2.3 Coagulation assays

The prothrombin time (PT), activated partial thromboplastin time (APTT), FⅦ:C, coagulation factor Ⅱ activity (FⅡ:C), coagulation factor Ⅴ activity (FⅤ:C), coagulation factor Ⅹ activity (FⅩ:C) and Lupus anticoagulant (LA) were determined by a coagulation assay on the STAR analyzer (STAGO, France) using matched commercially available kits. All of them were determined by one-step clotting method. The reference ranges of these indexes in our laboratory were established with 100 healthy subjects.

2.4 Genetic Analysis

The F7 gene, including all exons and adjacent intronic regions near splice sites, was amplified on an ABI Thermocycler 2720r (Applied Biosystems, California, USA), following procedures and materials as previously described [13]. The amplification products were identified and sequenced by Saiheng Bio-Technology Corporation (Shanghai, China). Sequencing results were compared with the F7 gene sequence published in the NCBI database (GenBank: AK298404.1) using Chromas software. PyMOL software was utilized to analyze and predict the effect of amino acid changes on the three-dimensional spatial structure of the FVII protein. Crystal structure data for the FVII protein can be accessed from the Protein Data Bank at https://www.rcsb.org/ (PDB ID: 1F7E).

2.5 Statistical Analysis and Allelic Frequencies

In this study, both Pearson correlation and Chi-squared tests were used to assess the relationships and associations between variables. All statistical analyses were performed using Python, and significance was determined at the 0.05 level. Allele frequencies were calculated based on genotype data obtained from the sample population. Exon proportions were determined by analysing the sequence data.

3.1 Clinical Phenotype of 85 Patients

Eighty-five probands (47 females and 38 males) aged between 7 and 79 years, were enrolled in the study. All probands exhibited significantly reduced plasma FVII levels; specifically, 57 patients had FVII:C < 5%, while the remainder had FVII:C levels below 72%. Levels of FII:C, FV:C, FX:C, and LA were within normal ranges for all patients. The patients had a mean age of 46 years, an average FVII level of 12.13%, and a mean PT level of 25.0 seconds.

Thirty-nine parobands presented with various bleeding symptoms. The most common manifestations were gingival bleeding (16%), epistaxis (14%), subcutaneous bleeding (12%), and menorrhagia (12%). Additional symptoms included postoperative bleeding (8%), postpartum hemorrhage (6%), gastrointestinal melena (4%), easy bruising (4%), and hematuria (2%). Bleeding scores in these patients ranged from 0 to 7, as listed in Table 1. In total, five probands were administered fresh frozen plasma or recombinant activated coagulation factor VIIa (rFVIIa) as a treatment option. Among them, probands 1, 18, 31, and 38 showed improvement in bleeding symptoms. Unfortunately, proband 17, who had intracranial bleeding (ICB), died despite receiving treatment. The remaining 46 probands showed no signs of abnormal bleeding.

3.2 Genotypes of 85 patients

In this study, a total of 25 variants were identified, with four variants (p.Trp344Gly, p.Leu170*, p.Gln160Leu, and p.Ser329Pro) being first reported by our team. Additionally, the variant p.Ile421Serfs*76 was reported globally for the first time. Goodeve, Reitsma, and McVey[14] numbered all residues using the Human Genome Variation Society (HGVS) nomenclature. The starting methionine of the reference protein sequence (NP_000122.1) was numbered as + 1, which may differ from the conventional numbering of + 60, leading to the formation of disulfide-bonded double-stranded molecules. Variants were found throughout the structural domain of the protein encoded by the F7 gene (Fig. 1), with introns and splice sites are not depicted in Fig. 1. Nineteen missense variants were identified, distributed across the Peptide S1, Signal, and Propep structural domains. Furthermore, the exon distribution of the variants was quantified, as illustrated in Fig. 2. Out of the 59 probands with variants, 14 unrelated families had the p.Arg364Gln variant, 14 had the p.His408Gln variant, 10 had the p.Arg337Cys variant, 9 had p.Cys389Gly variant, and 9 had the p.Cys10Profs*16 variant. These five variants are prevalent variant sites in the Chinese population (Data source: https://www.ensembl.org/index.html). Additionally, four families had the p.Arg413Gln variant, three unrelated families had the IVS6 + 1G > T, c.698-162insT, and p.Arg364Trp variants, and two unrelated families had the p.Thr241Asn variant. The remaining 13 variants were each found only once. All variants occur at amino acid positions within a signal peptide.

3.3 Analysis of Protein Structure and ACMG

The four missense variants and a nonsense variant identified by our reserch group were analyzed for protein modeling using PyMOL (Fig. 3). The analysis revealed that all four missense variants and one nonsense variant induced conformational changes in the FVII protein. Specifically, two of the missense variants (p.Gln160Leu, p.Ser329Pro) were found to reduce hydrogen bonding.

ACMG analysis yielded the following conclusions: p.Trp344Gly was classified as possibly pathogenic (PS1 + PM2 + PP3); p.Leu170* was identified as pathogenic (PVS1 + PM2); p.Gln160Leu was classified as possibly pathogenic (PS1 + PM2 + PP3); p.Ser329Pro was deemed possibly pathogenic (PM1 + PM2 + PP3); and p.Ile421Serfs*76 was determined to be pathogenic (PVS1 + PM2). The meanings of the relevant abbreviations are explained in the Abbreviations section.

3.4 Statistical Analysis and Allelic Frequencies

A chi-square test was conducted to evaluate the association between FVII:C levels (below or above 30%) and the occurrence of bleeding manifestations. The test yielded a Chi-square statistic (χ²), which is defined as follows: The test yielded a Chi-square statistic (χ²) of 5.302, with a p-value of 0.0213 and degrees of freedom (dof) of 1. The p-value is less than the conventional threshold of 0.05, indicating a statistically significant association. Pearson's rank correlation test indicated a correlation coefficient of -0.496 (P = 0.000) between FVII activity and PT, -0.266 (P = 0.014) between FVII activity and haemorrhage score, and 0.149 (P = 0.175) between PT and haemorrhage score. The correlations between the first two variables were statistically significant, particularly the relationship between PT and FVII:C, which was highly significant. Conversely, the correlation between PT and haemorrhage score was not statistically significant, suggesting that the observed relationship may have been due to chance.

The p.Arg364Gln and p.His408Gln variants were the most prevalent, collectively representing 18.37% of the total number of alleles. The Cys10Profs*16 and Arg337Cys variants were also relatively common, collectively accounting for 14.29% of the total number of alleles. The remaining variants exhibited intermediate- and low-frequency distributions(Table 2).

Table 2

Statistics on the Number of Occurrences and Frequencies of FVII Mutant Alleles in the Study Population
Mutaion	Count	Frequency
Ala251Val	1	0.010204082
Ala304Thr	1	0.010204082
Arg337Cys	14	0.142857143
Arg364Gln	18	0.183673469
Arg364Trp	3	0.030612245
Arg413Pro	1	0.010204082
Arg439Ser	1	0.010204082
Cys10Profs*16	14	0.142857143
Cys389Gly	10	0.102040816
Gln160Leu	1	0.010204082
His408Gln	18	0.183673469
IVS1a + 5G > A	1	0.010204082
IVS5-1G > A	2	0.020408163
IVS6 + 1G > T	3	0.030612245
Ile421Ser*fs76	1	0.010204082
Leu170*	1	0.010204082
Lys401Glu	3	0.030612245
Ser329Pro	1	0.010204082
Thr241Asn	1	0.010204082
Trp344Gly	1	0.010204082
Val384Phe	1	0.010204082
c.805 + 120C > T	1	0.010204082

As of now, the HGMD variant database (https://www.hgmd.cf.ac.uk/ac/validate.php) has identified 397 variants in the F7 gene, including 261 missense/nonsense variants, which account for 65.7% of the total. Through direct sequencing, we identified pathogenic variants in 39 registered symptomatic cases. However, no variants were identified in 6 symptomatic cases and 20 asymptomatic cases. While direct sequencing offers benefits such as low cost, high accuracy, and a short detection period, it may miss cryptic variants, including heterozygous large deletions or insertions, deep intronic variants, or variants in the promoter region. Furthermore, pathogenic variants were identified in 46 asymptomatic registered cases suggesting that a significant proportion of FVII-deficient patients may remain undetected due to methodological limitations and variability in symptoms. Therefore, the actual number of patients with FVII deficiency disorders may be considerably higher than currently recognized.

In this study, 25 types of variants were identified in 85 patients with FVII deficiency, with 73.9% occurring in the Peptide S1 region, the main variant-prone area of the F7 gene, and the remaining 26.1% scattered in the Signal peptide, Propeptide, and EGF like-2 regions. The variants included 27 homozygous and 32 compound heterozygous variants, with 14 missense variants accounting for approximately 60.9% of the total, findings that align with the variant database. Approximately 45.45% of the variants were identified in exon 8, indicating that this exon is a major hotspot for variants. This indicates that Exon 8 plays a pivotal role in the gene's functionality, and variants within this region may significantly impact gene functionality. In the context of genetic testing, it is of paramount importance to focus on regions with a high prevalence of variants, with particular emphasis on Exon 8. Prioritising these areas will enhance the accuracy of identifying gene-related variants, thereby improving the sensitivity and specificity of the tests. This approach also provides a robust basis for subsequent functional studies.

Five prevalent variants (p.His408Gln, p.Arg364Gln, p.Cys389Gly, p.Cys10Profs*16, and p.Arg337Cys) were identified in unrelated Chinese families, with the p.His408Gln variant, found in approximately 16% of patients when blood relatives were not present. Current data suggest that p.His408Gln induced low FVII does not prevent the development of VTE[15], although clinical manifestations varied considerably among individuals. The p.Arg364Gln variant, located in a CpG dinucleotide hotspot, affects FVII binding to TF, thereby impacting FX activation[16]. The p.Cys389Gly variant disrupts the disulfide bond between p.Cys370 and p.Cys389, affecting protein stability and function, with cases showing a range of presentations from asymptomatic to severe[17]. While primarily associated with non-severe bleeding, rare cases with additional coagulation factor deficiencies (e.g., FX) can lead to lifelong severe bleeding[18]. The p.Cys10Profs16 variant results in a frameshift and premature stop codon, likely resulting in a nonfunctional protein, which may be associated with severe bleeding symptoms. The p.Arg337Cys variant affects the protein's structure and function, and cases with this variant may also exhibit a wide range of clinical manifestations, from mild to severe bleeding.

We also identified five missense/nonsense variants, including one novel variant and four previously reported by our group. The p.Ser329Pro variant was associated with hemoptysis and resolved with treatment. The p.Gln160Leu variant was linked to recurrent epistaxis, while the p.Leu170* variant was associated with prolonged menstrual symptoms. In both cases, the bleeding symptoms resolved spontaneously without the need for haemostatic treatment. One patient had severe bleeding with a heterozygous p.Trp344Gly variant, while another patient experienced hematuria for over two weeks with a homozygous p.Trp344Gly variant. Further genetic analysis indicated that clinical phenotypes cannot be solely attributed to these variants, suggesting potential interactions with other genetic or environmental factors. ACMG analyses classified these variants as possibly pathogenic or pathogenic.

The bleeding tendency linked with FVII deficiency can manifest in various ways, ranging from more severe than hemophilia to very mild or even asymptomatic cases. In the study, only 39 out of 85 patients with FVII deficiency experienced bleeding, with excessive menstrual bleeding being the most prevalent symptom among the women. Studies on FVII deficiency have suggested that the reduction in FVII:C may not be associated with bleeding manifestations[15]. However, a statistical analysis of the study cohort yielded markedly different results.

The chi-square test demonstrated that there is a notable increase in the likelihood of bleeding when FVII:C levels reach a very low point. Pearson's test revealed a statistically significant correlation between FVII:C and both PT and bleeding score. Specifically, there was a statistically significant inverse relationship between PT and FVII:C, with PT demonstrating a decrease as FVII:C increased. Furthermore, a higher FVII:C was associated with a lower bleeding score, although this relationship was weaker and still statistically significant. Conversely, no statistically significant correlation was observed between PT and bleeding score, suggesting that any observed relationship between these two variables may be coincidental. These findings underscore the importance of FVII:C levels in influencing PT and haemorrhage risk, while highlighting the need for further investigation into the factors affecting bleeding scores.

A review of the allele frequencies revealed that the Arg364Gln and His408Gln variants were the most prevalent in the study population. This indicates a significant prevalence of these variants in the study population, which suggests that they may be of high genetic importance or health relevance in this population. Further investigation is required to elucidate the significance of these high-frequency variants. The high frequency of these variants may be associated with specific molecular mechanisms that may have important implications for the pathogenesis of the disease. Future studies should concentrate on investigating the specific functions of these variants in molecular mechanisms, examining their association with related diseases, and exploring their potential for therapeutic applications.

FVII plays a crucial role in the exogenous coagulation pathway, and managing congenital FVII deficiency presents a challenge in accurately assessing bleeding risk. Therefore, FVII preparations are currently the preferred treatment option due to their effectiveness and low risk of blood-borne infections[19]. Recombinant technology offers an effective method for large-scale production of human FVII, accompanied by complex post-translational modifications. Various recombinant expression systems have been established in recent years[20], ensuring proper protein folding, accurate post-translational modifications, and correct glycosylation sites. These factors are crucial for maintaining the stability of recombinant proteins, particularly those containing complex disulfide bonds or post-translational modifications. It is hoped that the study of FVII will serve as a reference for the future development in recombinant human FVII using a recombinant expression system[21].

This study presents the variant spectrum of FVII deficiency in a southeastern Chinese population, highlighting significant correlations between FVII:C levels and clinical outcomes. The highest allele frequencies were observed in p.Arg364Gln, p.His408Gln, p.Arg337Cys, p.Cys10Profs*16, and p.Cys389Gly, with p.Arg364Gln being the most predominant, suggesting a potential founder effect. The presence of asymptomatic individuals with low FVII:C levels indicates that the prevalence of FVII deficiency may be underestimated. Therefore, comprehensive genetic screening is essential to accurately understand genotype-phenotype correlations. Such screening will improve diagnostic accuracy and inform better management strategies for FVII deficiency in the Chinese population.

Declarations

Ethics approval and consent to participate

The study received approval from the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (KY2022-R193). All participants provided written informed consent prior to their involvement in the study.

Consent for publication

Not applicable.

Competing interests

All the authors declare no conflicts of interest.

Funding

We would like to acknowledge the support provided by the Wenzhou Basic Medical and Health Science and Technology Project (Y20220125) and the Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province (2022E10022) for this work.

Author Contributions

LY analyzed data and wrote the manuscript. LQ, SW, YJ, and LY collected the raw data. JP and LY provided advices on interpretation of the results. MW and YX critically reviewed and revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors express their gratitude to the patients and their family members for their cooperation. They declare no interests that could be perceived as posing a conflict or bias.

Availability of data and material

All data generated or analyzed during this study are included in this published article. Additional data used in the current study are not publicly available due to privacy restrictions but can be obtained from the corresponding authors upon reasonable request. Please contact Yaosheng Xie (email: [email protected]) or Mingshan Wang (email: [email protected]) for access to the datasets.

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Running title: Spectrum of Variants in the F7 Gene

Abbreviations

FVII Coagulation factor VII

FVII:C Coagulation factor VII activity

ICH Including intracranial hemorrhage

ELISA Enzyme-linked immunosorbent assay

PCR Polymerase chain reaction

PT Prothrombin time

APTT Activated partial thromboplastin time

FⅡ:C Coagulation factor Ⅱ activity

FⅤ:C Coagulation factor Ⅴ activity

FⅩ:C Coagulation factor Ⅹ activity

LA Lupus anticoagulant

GB Gingival bleeding

EP Epistaxis

SB Subcutaneous bleeding

MN Menorrhagia

PB Postoperative bleeding

PPH Postpartum Hemorrhage

GI-MA Gastrointestinal Melena

EB Easy to bruise

HU Hematuria

SDH Subdural haematoma

N No bleeding

F Female

M Man

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Recurrent Founder Variant in Inherited FVII Deficiency: Molecular Spectrum Analysis in Southeast China Recurrent Founder Variant in Inherited FVII Deficiency: Molecular Spectrum Analysis in Southeast China

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Version 1

Abstract

Background

Result

Conclusion

Figures

1 INTRODUCTION

2 MATERIALS AND METHODS

2.1 Participants

2.2 Blood Samples

2.3 Coagulation assays

2.4 Genetic Analysis

2.5 Statistical Analysis and Allelic Frequencies

3 RESULTS

3.1 Clinical Phenotype of 85 Patients

3.2 Genotypes of 85 patients

3.3 Analysis of Protein Structure and ACMG

3.4 Statistical Analysis and Allelic Frequencies

4 DISCUSSION

5 CONCLUSION

Declarations

Declarations

Funding

Author Contributions

Acknowledgements

Availability of data and material

References

Unsectioned Paragraphs

Supplementary Files

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Version 1