Forty-one consecutive unrelated patients (14 males and 27 females) were examined (Table 1). The ages ranged from 9 to 86 years, with a median age of 48.5 years. The laboratory tests revealed that both the APTT and FXI: C were outside the reference range. The median APTT was 82.6 s, while the median FXI: C was 3%. Of the patients studied, 39 had severe FXI defects, which was significantly greater compared to those with partial defects. The Spearman’s rank correlation test revealed a negative correlation between APTT levels and FXI: C levels (coefficient=-0.584, P < .001). The majority of patients were referred for preoperative screening (n = 22, 53.66%) and check-ups (n = 14, 34.15%). In addition, five women were diagnosed with severe FXI defects, including three during antenatal examination and two due to hemostatic difficulties. Of the total cases, 32 patients did not exhibit any bleeding symptoms. Among the cases where abnormal bleeding occurred (n = 9), four cases resulted from bleeding after surgery or trauma. Additionally, three cases involved menorrhagia while nosebleed, gingival bleeding, and urinary tract bleeding each presented once. The median ISTH-BAT bleeding score for these nine patients was 2, and all bleeding was mild. One of only two patients with partial FXI defects (Patient 2, P2) exhibited postoperative bleeding. Furthermore, while P3 did not experience any abnormal bleeding, his brother who was partial defect (FXI: C level of 48%) did experience postoperative bleeding. Four patients (P3, P23, P25, P30) received preoperative supplementation with fresh frozen plasma (FFP), and none of them experienced abnormal postoperative bleeding.

Genetic analysis revealed that a total of 37 patients carried the variant, with a genetic defect rate of 90%. A total of seventeen candidate variants were identified in the F11 gene, of which eleven were missense variants, four were nonsense variants, and two were small deletions (Table 2). As shown in Fig. 1A, variants were distributed throughout FXI, affecting the Ap1 (1), Ap2 (2), Ap3 (3), Ap4 (3), and SP (6) domains. Additionally, the deletion variant c.1136-4delGTTG occurred in intron 10. In our study cohort, the percentage of missense variants was significantly consistent compared to those documented in HGMD, but nonsense variants and small deletions were higher than in the database, and no other types of variants were identified (Fig. 1B). Of these 17 different potential variants, 6 (p.Leu78Pro, p.Thr179Met, p.Cys230Phe, p.Ser313Ile, p.Tyr521Cys, p.Leu442Cysfs*8) were reported for the first time in our laboratory.

The mutational distribution and model in the cohort. (A) The distribution of 17 disease-causing mutations on FXI protein. The signal peptide, apple domain, and peptidase S1 are represented by the green rectangle, red rectangle, and blue rectangle, respectively. (B) Comparison of the mutation model in the study with the mutation model presented in the Mutation Database. Different types of mutations are indicated by different colors, respectively

Two, sixteen, and nineteen patients carried heterozygous, homozygous, and compound heterozygous variants, respectively (Table 1). As expected, patients who were homozygotes or compound heterozygotes presented severe FXI defects, while patients who were heterozygotes presented FXI: C values approximately half of the reference range. Of the nine patients exhibiting a hemorrhagic phenotype, one was a heterozygote, one was a homozygote, and the remaining seven were compound heterozygotes.

Most of the identified potential variants were single amino acid substitutions, nine of which occurred at completely conserved positions corresponding to those of the F11 gene in other mammalian species (excluding p.T179M and p.S313I), demonstrating these variants may play a vital role in biological evolution. Three software programs all predicted that p.H145R and p.T179M were nonpathogenic, while all the other variants were pathogenic (Table 2). Three out of the 11 missense variants were identified as CRM+, seven were identified as CRM-, and the remaining one was uncharacterized. In addition, five variants were previously characterized through in vitro expression, including two CRM + variants (p.H145R and p.V516M) [16, 17]; two CRM- variants (p.T179M and p.G368E) exhibited a defect in the formation of FXI dimers that are not secreted outside the cell [17, 18]; and one CRM- variants (p.G418V) resulted in a reduction in plasma FXI levels via the dominant negative effect (DNE) [18]. This negative effect of the abnormal allele on the normal allele is known as the DNE.

Protein modeling analysis was performed for the remaining five missense variants (Fig. 2). Variants substituting leucine with proline at position 78 and cysteine with phenylalanine at position 230 resulted in pronounced changes in the side chains while retaining the identical hydrogen bonds, potentially causing a disturbance in the local structural stability and intrachain disulfide bond between Cys230 and Cys236. When p.S313 was replaced with p.I313, the two hydrogen bonds that had formed with p.E315 vanished, leading to a decrease in local stability. Likewise, substituting tryptophan with a serine or a cysteine at position 519 eliminated the hydrogen bonds created by the side chain with both p.L449 and p.N530. Additionally, pedigree investigations indicated that these variants were responsible for the decreased plasma FXI.

Genotype proportions, allele frequencies, and geographic origins of nine recurrent mutations. The x-axis displays the count of individuals with distinct genotypes

All six null variants presented CRM- (Table 2). Two variants were studied with recombinant proteins, and they decreased plasma FXI levels by triggering nonsense-mediated mRNA decay (NMD) [17]. The c.1136-4delGTTG variant, located in intron 10, has been confirmed to be pathogenic through reverse transcription-PCR (RT-PCR) analysis due to translational retention in the intron, leading to premature stop codon generation [19].

As shown in Fig. 3, a total of nine variants (3 missense and 6 null) appeared two or more times. The nonsense variant c.841 C > T (p.Gln281*) in 11 patients, with one heterozygote, five homozygotes, and five compound heterozygotes. Similarly, the nonsense variant c.738G > A (p.Trp246*) was detected in 9 patients, one of whom was heterozygous, five of whom were homozygous, and three of whom were compound heterozygous. These two variants had the highest allele frequencies in our cohort (21.25% and 17.50%, respectively) and have been reported in Chinese, Japanese, and Korean populations. Additionally, patients with the p.Gln281* variant exhibited a range of symptoms, from asymptomatic (n = 6) to mildly hemorrhagic (n = 5). Of the five patients with hemorrhagic symptoms, one was heterozygous, and four were compound heterozygous. Both homozygous and compound heterozygous states of the p.Trp246* variant manifested mild hemorrhagic phenotypes in one patient.

The FXI protein modeling analysis with the PyMOL (PDB: 5EOK). The five mutated amino acid residues are illustrated as hot pink spheres and sticks. The orange dashed lines represent the hydrogen bonds. (A) The secondary structures of the separate domains of the FXI proteins are identified through varying colors on the Cartoon, respectively. (B) The p.Leu78Pro variant; (C) The p.Cys230Phe variant; (D) The p.Ser313Ile variant; (E) The p.Trp519Ser and p.Trp519Cys variant

The two variants, the nonsense c.1107 C > A (p.Tyr369*) and the frameshift c.1325delT (p.Leu442Cysfs*8), had an identical allele frequency of 12.50%, which were in the homozygous or compound heterozygous states. Only one patient (P33) with p.Tyr369* and p.Leu442Cysfs*8 experienced mild bleeding. Interestingly, both of these variants have been found only in the Chinese population [20,21,22].

The c.1556G > A (p.Trp519*) was identified as being in a compound heterozygous state. Its allele frequency was only 6.25%, despite being reported in four different countries, specifically China, Japan, Korea, and Turkey. Only one out of five patients developed minor bleeding symptoms.

The missense variants c.1562 A > G (p.Tyr521Cys), c.1546G > A (p.Val516Met), and c.434 A > G (p.His145Arg) and the deletion variant c.1136-4delGTTG were present in only two patients. The allele frequency of the former was 3.75%, while that of the remaining three was 2.50%. Patients who carried the c.1136-4delGTTG and p.Gln281* compound heterozygous variants (P27, 28) presented abnormal post-traumatic bleeding. Furthermore, it has been determined that the c.1136-4delGTTG is exclusive to Chinese individuals. Conversely, the remaining three variants have been found in Lebanese, Korean, and Indonesian populations, in addition to Chinese populations.