Understanding Congenital FXI Deficiency: Genetic Diagnosis and Correlation of Variant Detection Rate to Factor XI Activity

Abstract

Background

Factor XI (FXI) deficiency is an autosomal bleeding disorder characterized by low FXI levels, resulting in bleeding after trauma or surgery. Genetic variants affecting FXI structure and function often result in bleeding diatheses.

Aim

This study aimed to estimate the variant detection rate (VDR), and assess its correlation with FXI activity (FXI:C) in a large cohort of FXI-deficient patients.

Material and Methods

Genetic defects in the F11 gene were analyzed in 316 index patients (IPs) using Sanger or next-generation sequencing. Multiplex ligation-dependent probe amplification or copy number variation analysis was used to detect duplications and deletions.

Results

Genetic defects were identified in 249 IPs (VDR of 79%). A strong negative correlation (Pearson coefficient: −0.891) was found between FXI:C levels and VDRs: higher FXI:C levels corresponded to a lower likelihood of detecting genetic alterations, with a significant decline in VDR beyond 60 IU/dL. A total of 286 genetic variants were identified in F11 gene: 56% missense, 24% nonsense, 11% small deletions/insertions, and 6% splice-site variants. Large deletions were rare (3%). A total of 48 novel variants were detected. Ashkenazi Jewish founder variants were the most frequent (14.3%). Variants p.Gln134Ter, p.Ile215_Asp216del, and p.Glu315Lys (27% of cases) were recurrent. In four cases, large deletions extended beyond the F11 gene and included the neighboring KLKB1 gene, encoding prekallikrein.

Conclusion

This study demonstrated a significant negative correlation between FXI:C levels and VDRs, underscoring the importance of genetic testing. Findings included combined deficiencies in FXI and prekallikrein due to large deletions affecting both F11 and KLKB genes.

Keywords

FXI deficiency - bleeding - genetic analysis - F11 gene - KLKB1 gene

Introduction

Factor XI (FXI) is a key component of the intrinsic coagulation pathway, playing a critical role in thrombin generation and the pro-inflammatory kallikrein-kinin system.[1] [2] It is essential for the propagation of the coagulation cascade and in downregulating fibrinolysis, underscoring its importance in maintaining hemostasis.[3]

FXI deficiency (MIM*264900) is considered as an autosomal recessive rare bleeding disorder characterized by reduced plasma FXI levels (FXI:C), leading to variable bleeding tendencies[4] with affected individuals being either homozygous or compound heterozygous for pathogenic variants in F11 gene. However, in some cases, the dominant-negative transmission of the disease due to autosomal inheritance patterns has been reported.[5] [6] The autosomal recessive form is most commonly observed among symptomatic patients. Despite its clinical significance, FXI deficiency often remains asymptomatic throughout an individual's life due to the weak correlation between FXI levels and bleeding severity. In many cases, bleeding is unexpected and occurs only during surgery or after trauma.[7] [8] [9]

In healthy individuals, FXI:C levels typically range from 60 to 150 IU/dL. Partial deficiency, typically observed in heterozygotes, is characterized by FXI:C levels ranging from 20 to 60 IU/dL, whereas severe deficiency, often seen in homozygotes or compound heterozygotes, is defined by levels below 20 IU/dL.[10] [11] [12]

The occurrence of FXI deficiency in the general population is exceedingly rare, with an estimated frequency of one per million. However, its prevalence is significantly higher in certain populations, such as Ashkenazi Jews, where it affects 1 in 450 individuals. Recent studies have revealed that FXI deficiency may occur 2 to 20 times more frequently than expected in various ethnic groups.[8] [13] [14] These findings highlight the importance of understanding population-specific genetic risk factors to improve diagnosis and management of FXI deficiency.

The F11 gene, encoding FXI protein spans 15 exons interspersed with 14 introns.[15] [16] Genetic variants disrupting the native structure and function of FXI are the primary cause of FXI deficiency. Over 190 pathogenic variants have been identified to date, including missense, nonsense, and frameshift variants.[17] However, the clinical heterogeneity of FXI-deficient patients results in a weak correlation between genotype and phenotype.[18]

The F11 gene is located adjacent to the KLKB1 gene on chromosome 4q35.2, which encodes plasma prekallikrein (PK). This close proximity raises the possibility of large deletions or structural variations affecting both genes. Both PK and FXI are serine proteases involved in the contact activation pathway of coagulation and share significant structural homology, indicating a common ancestry from a duplication event of the KLKB1 gene during early mammalian evolution.[1] [19]

Beyond their structural and evolutionary connection, FXI and PK play complementary roles within the contact activation system. FXI primarily amplifies thrombin generation through the intrinsic coagulation pathway, while PK contributes to both coagulation and inflammation via the kallikrein-kinin system. This interplay may influence both hemostatic and inflammatory processes. Emerging evidence suggests that genetic interactions between F11 and KLKB1 may modulate the clinical phenotypes of FXI deficiency, though this area requires further investigation.[2]

This study provides a comprehensive overview of FXI deficiency, encompassing its genetic underpinnings. By elucidating these aspects, we seek to enhance the understanding of FXI deficiency and contribute to the development of more effective diagnostic strategies.

Materials and Methods

Patient Cohort

Over a 15-year period (2009–2023), EDTA blood samples from 334 consecutive index patients (IPs) in Germany were submitted to our laboratory for the identification of genetic defects associated with factor XI (FXI) deficiency. The diagnosis was determined by the treating physician based on a combination of laboratory data (FXI:C), clinical presentation, and, when available, familial members for segregation analysis of variants of uncertain significance (VUS). In 18 IPs, FXI deficiency was excluded due to confounding factors, including associated liver disease, inconsistent laboratory data, or incomplete clinical documentation. Written informed consent for molecular genetic testing was obtained from all participants in accordance with ethical guidelines outlined in the Declaration of Helsinki.

Genetic Analysis

Genetic analyses were conducted at the Department of Molecular Hemostaseology, University Hospital Bonn. Genomic DNA was extracted from peripheral blood collected in EDTA tubes using the Blood Core Kit (Qiagen, Hilden, Germany).

Molecular genetic analyses included sequencing of all coding regions and intron/exon boundaries of the F11 gene (NM_000131) and was performed on ABI Prism 3130 genetic analyzer for Sanger sequencing (Thermo Fisher Scientific, Langenselbold, Germany) and Mini-Seq genome sequencer (Illumina, Santa Clara, CA, USA) for next-generation sequencing (NGS). Of the 316 IPs, 240 (76%) were analyzed by Sanger sequencing, while the remaining 76 (24%) underwent analysis by NGS. Screening for large deletions and duplications was performed in cases with FXI activity <20 IU/dL when only a single genetic variant was identified, as well as for all samples analyzed by NGS, where this analysis was included by default. For NGS, a minimum coverage depth of 30× per base pair was required for variant calling.

Data was evaluated by SeqScape Version 2.7 (Thermo Fisher Scientific) and SeqPilot (JSI Medical Systems, Ettenheim, Germany) software. Primers and conditions are available on request. For the description of sequence variations at the DNA and protein level, the guidelines of the Human Genome Variation Society (HGVS) were used. The genetic variant interpretation and criteria used to establish variant pathogenicity was performed according to the American College of Medical Genetics (ACMG) and Association for Molecular Pathology (AMP) guidelines for the interpretation of sequence variants.[20] The disease causality of genetic variants was compared in Human Gene Mutation Database (HGMD)[21] and ClinGen database.[22]

Large deletions and duplications were analyzed with multiplex ligation-dependent probe amplification analysis (MLPA) or copy number variation (CNV) analysis. MLPA was performed according to the manufacturer's recommendations, using SALSA MLPA Kit P440-A2 (MRC-Holland, Amsterdam, Netherlands). Amplification products were run on an ABI PRISM 3130XL DNA Sequencer (Thermo Fisher Scientific) with the GeneScan 500 ROX size standard (Thermo Fisher Scientific). Dosage analyses were performed by Coffalyser (V5.3) software (MRC-Holland). CNV evaluation was achieved by SeqPilot (JSI Medical Systems GmbH).

Statistical Analysis

Statistical analyses were performed with SPSS software version 19.1. Pearson coefficient of correlation between variant detection rate (VDR) and FXI:C was analyzed. Pearson coefficient correlation thresholds were defined as: <0.3, weak correlation; 0.3–0.5, moderate correlation; and ≥0.5, strong correlation. The Pearson correlation coefficient ranges from −1 to +1 corresponding to positive or negative correlations.

Results

Patient Data

A total of 316 unrelated IPs with FXI deficiency were included in our study. The mean FXI:C level was 37 IU/dL, with a range spanning from 1 to 60 IU/dL. The cohort comprised 58.6% females (185 IPs) and 41.4% males (131 IPs). Of these, 75 IPs (24%) were classified as severe FXI deficiency (FXI:C <20 IU/dL), while the remaining 241 IPs (76%) exhibited moderate to mild deficiency. Within the latter group, the majority of patients (143 IPs, 59%) presented FXI:C levels of between 30 and 50 IU/dL. The mean age of the cohort was 26.6 years, ranging from 1 to 87 years. Notably, the severe form of FXI deficiency tended to be diagnosed at a younger age, with the majority of patients (67%) being under 30 years of age at the time of diagnosis.

Genetic Landscape

Comprehensive Analysis of Genetic Variants and Variant Detection Rate

All 316 IPs underwent comprehensive genetic analysis through direct sequencing of all 15 exons of the F11 gene, including the exon–intron boundaries, to identify pathogenic variants. Additionally, the samples were screened for large deletions or duplications. Genetic defects were successfully identified in 249 IPs, resulting in a high VDR of 79%, confirming the utility of genetic testing in the diagnosis of FXI deficiency. Among these 249 patients, the majority (179 IPs; 72%) harbored variants in a heterozygous state, while the remaining 70 patients (28%) had genetic alterations evenly distributed between homozygous and compound heterozygous forms, with 35 cases in each group.

The identified variants were broadly distributed across the F11 gene, affecting all functional domains, including Apple (Ap)1, Ap2, Ap3, Ap4, and the Serine Protease (SP) domain ([Fig. 1]). Although the genetic defects demonstrated significant variability and were dispersed throughout the gene, no substantial clustering in specific exons was observed, apart from recurrent variants.

Fig. 1 Structure of FXI protein and F11 gene with the domain specificity of detected genetic variants and their distribution throughout F11 gene. Novel variants are marked in bold. Ap, Apple domain; E, exon; SP, Serine Protease domain.

The profile of genetic defects was heterogeneous, encompassing all types of genetic alterations (missense, nonsense, small deletion/insertion, splice-site, and large deletions). A total of 286 genetic variants were identified, with missense variants being the most common (160; 56%), followed by nonsense variants (69; 24%). Small deletions or insertions accounted for 11% (32 variants), while splice-site variants constituted 6% (17 variants). Large deletions were rare, identified in only seven individuals (3% of variants), and no large duplications were detected ([Fig. 2A]).

Fig. 2 Overall variant spectrum and variant detection rate (VDR). (A) Molecular genetic profile in 316 patients with FXI deficiency. (B) VDR in patients with FXI deficiency. IU/dL, International Units/deciliter.

From all 286 genetic variants, 129 (45%) unique variants were detected, of which 81 (63%) were classified as pathogenic based on database records and literature, while 48 (37%) variants were categorized as VUS. A total of 15 variants were reclassified as likely pathogenic due to the type of the defect (e.g., nonsense variants, small deletions, or splice-site variants affecting positions ± 1) that were predicted to impair FXI protein function. Based on segregation analyses in family members with and without FXI deficiency, 16 variants were reclassified as likely pathogenic. The remaining 17 variants were retained as VUS, requiring cautious interpretation and further investigation ([Table 1]).

Table 1 Novel genetic variants identified in the cohort of 316 index patients

No.	Exon/Intron	HGVS nucleotide position	HGVS protein position	Type of variant	FXI activity (IU/dL)	Zygosity	Classification
1	2	c.39_43delTACTT	p.Thr14Serfs	Small deletion	45	Heterozygous	Likely pathogenic
2	3	c.89T > G	p.Phe30Cys	Missense	40	Heterozygous	VUS
3	3	c.138C > A	p.Cys46Ter	Nonsense	36	Heterozygous	Likely pathogenic
4	3	c.166T > C	p.Cys56Arg	Missense	37	Heterozygous	VUS
5	3	c.206A > G	p.Asp70Gly	Missense	1	Compound heterozygous(with c.599G > A, p.Cys200Tyr)	Likely pathogenic (segregation)
6	4	c.328G > A	p.Ala109Thr	Missense	2	Compound heterozygous (with c.908delG, p.Gly303Glufs)	VUS
7	5	c.329G > C	p.Cys110Ser	Missense	50	Heterozygous	Likely pathogenic (segregation)
8	5	c.333C > A	p.Asn111Lys	Missense	20	Homozygous	Likely pathogenic (segregation)
9	6	c.518G > C	p.Gly173Ala	Missense	58	Heterozygous	VUS
10	6	c.523C > A	p.Pro175Thr	Missense	25	Heterozygous	VUS
11	6	c.539A > G	p.Lys180Arg	Missense	33	Heterozygous	Likely pathogenic (segregation)
12	6	c.608A > G	p.Asp185Gly	Missense	35	Heterozygous	VUS
13	6	c.560G > T	p.Gly187Val	Missense	38	Heterozygous	Likely pathogenic (segregation)
14	6	c.559G > T	p.Gly187Ter	Nonsense	50	Heterozygous	Likely pathogenic
15	6	c.561delA	p.Ser189Hisfs	Small deletion	56	Heterozygous	Likely pathogenic
16	7	c.644_649delTCGACA	p.Ile197_Asp198del	Small deletion	17	Heterozygous	Likely pathogenic
17	7	c.623C > T	p.Thr208Met	Missense	9	Compound heterozygous (with c.644_649delTCGACA, p.Ile215_Asp216del)	Likely pathogenic (segregation)
18	7	c.704G > A	p.Gly235Asp	Missense	55	Heterozygous	Likely pathogenic (segregation)
19	7	c.712T > C	p.Phe238Leu	Missense	47	Heterozygous	VUS
20	8	c.770T > C	p.Leu257Pro	Missense	45	Heterozygous	Likely pathogenic (segregation)
21	8	c.787G > T	p.Gly263Ter	Nonsense	8	Compound heterozygous (with c.1327C > G, p.Arg443Gly)	Likely pathogenic
22	8	c.865G > A	p.Val289Met	Missense	51	Heterozygous	VUS
23	9	c.919G > A	p.Asp307Asn	Missense	49	Heterozygous	Likely pathogenic (segregation)
24	9	c.961_962delTG	p.Cys321Hisfs	Small deletion	9	Compound heterozygous (with c.400C > T, p.Gln134Ter)	Likely pathogenic
25	9	c.1028 + 1G > T	p.?	Splice site	5	Compound heterozygous (with c.1079InsCGGG, p.Leu360Glyfs)	Likely pathogenic
26	10	c.1075A > G	p.Ile359Val	Missense	37	Heterozygous	VUS
27	10	c.1076InsCGGG	p.Leu360Glyfs	Small insertion	5	Compound heterozygous (with c.1028 + 1G > T)	Likely pathogenic
28	11	c.1187G > T	p.Arg396Leu	Missense	18	Compound heterozygous (with c.328T > G, p.Cys110Gly)	VUS
29	11	c.1291C > T	p.His431Tyr	Missense	5	Homozygous	Likely pathogenic (segregation)
30	12	c.1327C > G	p.Arg443Gly	Missense	8	Compound heterozygous (with c.787G > T, p.Gly263Ter)	VUS
31	12	c.1373_1375insCA	p.Ser459Hisfs	Small insertion	13	Compound heterozygous (with c.1371C > G, p.Asp457Glu)	Likely pathogenic
32	12	c.1391A > G	p.Gln464Arg	Missense	41	Compound heterozygous (with c.1247G > A, p.Cys416Tyr)	VUS
33	12	c.1411C > T	p.Gln471Ter	Nonsense	58	Heterozygous	Likely pathogenic
34	12	c.1437T > A	p.Tyr479Ter	Nonsense	36	Heterozygous	Likely pathogenic
35	12	c.1442T > A	p.Ile 481Asn	Missense	44	Heterozygous	VUS
36	12	c.1481–1G > A	p.?	Splice site	7	Compound heterozygous (with c.1613C > T, p.Pro538Leu)	Likely pathogenic
37	13	c.1497A > G	p.Ile499Met	Missense	50	Heterozygous	Likely pathogenic (segregation)
38	13	c.1505C > T	p.Pro502Leu	Missense	55	Heterozygous	Likely pathogenic (segregation)
39	13	c.1543T > A	p.Trp515Arg	Missense	51	Heterozygous	VUS
40	14	c.1667A > G	p.His556Arg	Missense	65	Heterozygous	VUS
41	14	c.1627G > A	p.Gly570Lys	Missense	4	Compound heterozygous (with c.901T > C, p.Phe301Leu)	Likely pathogenic (segregation)
42	14	c.1712G > A	p.Cys571Tyr	Missense	2	Compound heterozygous (with c.325 + 1G > A)	Likely pathogenic (segregation)
43	15	c.1727G > A	p.Gly576Glu	Missense	38	Heterozygous	VUS
44	15	c.1732C > T	p.Pro578Ser	Missense	44	Heterozygous	Likely pathogenic (segregation)
45	15	c.1760G > A	p.Trp587Ter	Nonsense	5	Compound heterozygous (with c.1817G > A, p.Gly606Asp)	Likely pathogenic
46	15	c.1768delG	p.Val590Ter	Nonsense	51	Heterozygous	Likely pathogenic
47	15	c.1817G > A	p.Gly606Asp	Missense	5	Compound heterozygous (with c.1760G > A, p.Trp587Ter)	VUS
48	15	c.1817G > C	p.Gly606Ala	Missense	47	Heterozygous	Likely pathogenic (segregation)

Abbreviations: HGVS, Human Genome Variation Society; NO., number; VUS, variant of uncertain significance.

Recurrent Variants

Although genetic variants were dispersed across F11 gene, certain exons exhibited a higher frequency of alterations. Approximately 45% of all identified genetic variants were localized within exons 5, 9, and 7. In exon 5, the most common genetic variants were associated with p.Gln134Ter (detected 12 times) and p.Glu135Ter (detected 30 times). The variant p.Glu135Ter is a founder variant in the Ashkenazi Jewish population predicted to result in premature termination of the protein. Both variants were observed in a homozygous or compound heterozygous state, accounting for nearly half of the cases, linked to severe FXI deficiency (FXI:C levels ranging between 2 and 10 IU/dL).

Exon 7 featured a recurrent 6-bp deletion, resulting in the loss of amino acid residues Ile215 and Asp216, identified in 12 cases. Similarly, exon 9 demonstrated an increased prevalence of two highly recurrent genetic variants: p.L p.Phe301Leu, commonly referred to as the type III variant, identified almost solely in Ashkenazi Jews (11 cases), and p.Glu315Lys (13 cases).

These recurrent variants significantly contributed to the variant burden observed in the cohort, underlining their critical role in the pathogenesis of FXI deficiency. Additionally, variants p.Gln134Ter, p.Ile215_Asp216del, and p.Glu315Lys represented 27% of cases, making them rather common in our cohort.

Correlation between Variant Detection Rate and FXI Activity

The FXI:C levels in patients with detectable variants ranged from 1 to 60IU/dL. To explore the relationship between VDR and FXI:C, the entire cohort was stratified into sub-groups based on FXI:C levels ([Fig. 2B]).

The first group consisted of 75 IPs (24%) with severe FXI deficiency (FXI:C <20 IU/dL). Genetic defects were identified in 72 individuals, yielding a remarkably high VDR of 96%. Nearly all genetic defects, except for one case, were found in either a homozygous or compound heterozygous state, both strongly associated with severe FXI deficiency.

The second group consisted of 20 individuals (6%) with FXI activity levels between 21 and 30 IU/dL. Genetic alterations were identified in 16 of these cases, resulting in a VDR of 80%. For groups with FXI:C levels of up to 60 IU/dL, the VDR remained high (70–92%), indicating a consistent association between genetic alterations and moderate to mild FXI deficiency. However, beyond 60 IU/dL, the VDR sharply declined to 14%, suggesting a significantly lower likelihood of identifying causative genetic variants in individuals with near-normal FXI activity levels.

In partial FXI-deficient cases (FXI:C between 20 and 60 IU/dL), the majority of defects were found in a heterozygous state. This pattern reflects the milder clinical impact of heterozygous variants on FXI:C levels and highlights the differential genetic architecture underlying varying degrees of FXI deficiency.

A Pearson correlation coefficient of −0.891 demonstrated a significant negative dependence between FXI:C levels and VDRs. Among patients without identifiable genetic defects, FXI:C levels ranged from 3 to 60 IU/dL, including three patients with severe FXI deficiency.

Large Deletions in F11 and KLKB1 Genes

Large deletions were identified in seven IPs (2%). In two patients, the deletions were partial, involving exons 9 to 14 and exons 1 to 15, respectively. In the remaining five cases, the large deletions encompassed the entire F11 gene, resulting in a complete loss of its coding sequence. In four of these cases, the deletion extended to the neighboring KLKB1 gene.

All large deletions, whether partial or complete, were identified in the heterozygous state. FXI:C levels in these patients ranged from 35 to 56 IU/dL, consistent with the heterozygous genetic alterations. Interestingly, all patients with large deletions affecting both the F11 and KLKB1 genes also carried a second genetic defect in the KLKB1 gene, reducing their PK activity to nearly undetectable levels (<1 IU/dL). Importantly, no additional bleeding diathesis was observed in these patients, despite the near-complete loss of PK activity, suggesting the presence of compensatory mechanisms.

Discussion

Since its discovery in 1953 in three families presenting with a mild hemorrhagic phenotype, research into congenital FXI deficiency has steadily advanced, leading to significant progress in understanding its pathogenesis and identifying the genetic variants responsible for the disorder.[23] [24]

Diagnosing FXI deficiency is challenging due to the poor correlation between FXI plasma levels and bleeding phenotype. Severe deficiency may not cause bleeding, while heterozygous individuals can exhibit symptoms. Global hemostasis tests such as activated partial thromboplastin time (aPTT) are limited in their capacity to detect partial FXI defects in over one-third of patients, particularly when FXI:C levels are above 30%.[12] [25] Molecular analysis may be useful as a definitive diagnostic tool, especially where FXI:C levels are inconclusive. The identification of pathogenic variants in the F11 gene not only confirms the diagnosis but also provides insight into the molecular mechanisms of the disease, which is crucial given the variability in FXI:C and its overlapping clinical manifestations with other bleeding disorders. Importantly, a confirmed genetic diagnosis supports clinical decision-making as it may assist in predicting bleeding risk, guides perioperative management strategies, enables genetic counseling in affected families, and helps distinguish FXI deficiency from other inherited bleeding disorders with similar laboratory features. Thus, molecular diagnostics serve not only as a confirmatory tool but also as a foundation for personalized clinical management.

Our analysis of 316 IPs revealed a high VDR of 79%, with the majority of pathogenic or likely pathogenic variants identified predominantly in individuals with severe and moderate FXI deficiency. A significant negative correlation between FXI:C levels and VDR underscores the genetic basis of the disorder. Notably, as FXI:C levels exceeded 60 IU/dL, the likelihood of identifying causative genetic variants decreased sharply (14%), highlighting the diagnostic utility of genetic testing, especially in these cases where clinical and biochemical parameters alone may be insufficient to establish a definitive diagnosis.

This study also highlights the molecular heterogeneity of the F11 gene, encompassing both rare and recurrent variants associated with FXI deficiency. Although FXI deficiency occurs across all ethnicities, certain variants display notable population-specific distributions due to founder effects. Several population-specific variants have been described including the p.Cys56Arg in the Basque region, p.Gln106Ter in Nantes, and p.Cys146Ter in British Caucasians.[11] [26] [27] East Asians also show unique variants, such as p.Glu281Ter and p.Leu442Cysfs[*]8.[28] In our cohort, Ashkenazi Jewish founder variants[14] were most frequent (14.3%), primarily linked to severe FXI deficiency (FXI:C <20 IU/dL) in homozygous or compound heterozygous states and moderate deficiency (30–60 IU/dL) in heterozygotes. Additionally, variants such as p.Gln134Ter, p.Ile215_Asp216del, and p.Glu315Lys accounted for 27% of cases and appeared to be unique to the German population, as they are not commonly reported in other ethnicities.

Our study uncovered a broad spectrum of rare variants, identifying 129 unique sequencing alterations. Missense variants predominated, followed by nonsense, small deletions/insertions, and splice-site variants, all uniformly distributed across the five functional domains and linker regions of the F11 gene. Large deletions were rare, with approximately 5% prevalence typically observed in other genes.[8] [15] [17] [29] [30] Notably, we identified 48 novel genetic changes, 31 of which were reclassified as likely pathogenic based on family segregation analysis and the type of the genetic defect. The remaining variants were classified as VUS.

It is worth noting that while 70 of 249 IPs (28%) with identified genetic variants were homozygous or compound heterozygous, 75 of the entire cohort (24% of 316 IPs) exhibited severe FXI deficiency (FXI:C <20 IU/dL). This suggests that our focus on exonic and flanking regions of the F11 gene may have missed pathogenic variations in non-coding regions, such as introns or promoter sequences, or even in other putative genes implicated in FXI deficiency. These observations reinforce the clinical and genetic heterogeneity of FXI deficiency and highlight the need for extended genomic analysis in unresolved cases.

Additionally, in three patients with compound heterozygosity, FXI:C levels ranged between 50 and 55 IU/dL. Family analyses confirmed that both variants were located on the same allele, aligning with the observed FXI activity levels.

Furthermore, our analysis identified several noteworthy cases that underscore the complexity and variability of FXI deficiency. These unique instances reveal potential gaps in current diagnostic approaches and highlight the need for comprehensive genetic screening. One case presents a patient initially genotyped as homozygous for a missense defect, corresponding to FXI:C levels of 5 IU/dL. Subsequent analysis revealed a large deletion encompassing the entire F11 gene, resulting in a pseudo-homozygous sequencing result. This highlights the importance of screening for large deletions, even in cases presenting as homozygous variants, and underscores the implications for genetic counseling and routine diagnostic workflows.

Furthermore, in four patients with large deletions of the complete F11 gene, the deletion also extended into the KLKB1 gene. Although specific reports of large deletions encompassing both F11 and KLKB1 are limited, the close genomic proximity of these genes on chromosome 4q35.2 suggests that such deletions, simultaneously affecting both genes, could occur, potentially leading to combined deficiencies in FXI and PK, presenting complex clinical phenotypes, potentially affecting both coagulation and inflammatory pathways.[1] [30] Interestingly, despite near-complete loss of PK activity, these patients showed no bleeding diathesis, suggesting the presence of compensatory mechanisms. This finding emphasizes the need for further research to understand the physiological and clinical implications of combined deficiencies.

The advent of NGS offers numerous advantages over traditional methods like Sanger sequencing. NGS facilitates comprehensive analysis of the F11 gene, including all 15 exons and exon–intron boundaries. This approach is particularly beneficial in FXI deficiency, where genetic defects span the entire gene and include diverse types, such as missense, nonsense, small insertions/deletions, splice-site variations, and large deletions that may extend into neighboring genes like KLKB1. Interestingly, despite near-complete loss of PK activity, these patients showed no bleeding diathesis. This is consistent with previous reports indicating that PK deficiency alone does not lead to a bleeding tendency, likely due to compensatory mechanisms such as thrombin-mediated activation of FXI independent of the contact system.[31]

In summary, the genetic landscape of FXI deficiency in our cohort revealed significant heterogeneity, with variants distributed throughout the entire F11 gene. Combined with the low bleeding risk observed in many FXI-deficient patients, the wide spectrum of F11 variants identified across different populations, and the high incidence of FXI deficiency reported in various studies, including ours, these findings suggest that FXI deficiency may be underdiagnosed.

Genetic testing, particularly through NGS, is essential for improved diagnosis of FXI deficiency. Beyond diagnosis, genetic testing plays a crucial role in guiding patient management, facilitating family counseling, and supporting future research to unravel the molecular and clinical intricacies of FXI deficiency.

Conflict of Interests

The authors declare that they have no conflict of interest.

Authors' Contributions

B.Pez. and A.P.: conceptualization; A.B. and A.P.: data analysis; U.S., B.Z., and J.O.: investigation; B.Prei. A.P., and B.Pez.: data curation; A.P., A.B., and B.Pez.: writing—original draft preparation; all authors: writing—review and editing; B.Pez. and A.P.: visualization. All authors have read and agreed to the published version of the manuscript.

^* Contributed equally to this work as senior authors.

• References

• 1 Ponczek MB, Shamanaev A, LaPlace A. et al. The evolution of factor XI and the kallikrein-kinin system. Blood Adv 2020; 4 (24) 6135-6147
• 2 Wheeler AP, Gailani D. Why factor XI deficiency is a clinical concern. Expert Rev Hematol 2016; 9 (07) 629-637
• 3 Puy C, Rigg RA, McCarty OJT. The hemostatic role of factor XI. Thromb Res 2016; 141 (Suppl. 02) S8-S11
• 4 Duga S, Salomon O. Factor XI deficiency. Semin Thromb Hemost 2009; 35 (04) 416-425
• 5 Dai L, Rangarajan S, Mitchell M. Three dominant-negative mutations in factor XI-deficient patients. Haemophilia 2011; 17 (05) e919-e922
• 6 Franchini M, Veneri D, Lippi G. Inherited factor XI deficiency: a concise review. Hematology 2006; 11 (05) 307-309
• 7 Palla R, Peyvandi F, Shapiro AD. Rare bleeding disorders: diagnosis and treatment. Blood 2015; 125 (13) 2052-2061
• 8 Asselta R, Paraboschi EM, Rimoldi V. et al. Exploring the global landscape of genetic variation in coagulation factor XI deficiency. Blood 2017; 130 (04) e1-e6
• 9 Peyvandi F, Palla R, Menegatti M. et al; European Network of Rare Bleeding Disorders Group. Coagulation factor activity and clinical bleeding severity in rare bleeding disorders: results from the European Network of Rare Bleeding Disorders. J Thromb Haemost 2012; 10 (04) 615-621
• 10 Berber E. Molecular characterization of FXI deficiency. Clin Appl Thromb Hemost 2011; 17 (01) 27-32
• 11 Bolton-Maggs PHB, Peretz H, Butler R. et al. A common ancestral mutation (C128X) occurring in 11 non-Jewish families from the UK with factor XI deficiency. J Thromb Haemost 2004; 2 (06) 918-924
• 12 Lewandowska MD, Connors JM. Factor XI deficiency. Hematol Oncol Clin North Am 2021; 35 (06) 1157-1169
• 13 Martín-Salces M, Jimenez-Yuste V, Alvarez MT, Quintana M, Hernández-Navarro F. Review: Factor XI deficiency: review and management in pregnant women. Clin Appl Thromb Hemost 2010; 16 (02) 209-213
• 14 Shpilberg O, Peretz H, Zivelin A. et al. One of the two common mutations causing factor XI deficiency in Ashkenazi Jews (type II) is also prevalent in Iraqi Jews, who represent the ancient gene pool of Jews. [see comments] Blood 1995; 85 (02) 429-432
• 15 Rimoldi V, Paraboschi EM, Menegatti M. et al. Molecular investigation of 41 patients affected by coagulation factor XI deficiency. Haemophilia 2018; 24 (02) e50-e55
• 16 Mohammed BM, Matafonov A, Ivanov I. et al. An update on factor XI structure and function. Thromb Res 2018; 161: 94-105
• 17 Harris VA, Lin W, Perkins SJ. Analysis of 272 genetic variants in the upgraded interactive FXI web database reveals new insights into FXI deficiency. TH Open 2021; 5 (04) e543-e556
• 18 Santoro C, Di Mauro R, Baldacci E. et al. Bleeding phenotype and correlation with factor XI (FXI) activity in congenital FXI deficiency: results of a retrospective study from a single centre. Haemophilia 2015; 21 (04) 496-501
• 19 Ponczek MB, Gailani D, Doolittle RF. Evolution of the contact phase of vertebrate blood coagulation. J Thromb Haemost 2008; 6 (11) 1876-1883
• 20 Richards S, Aziz N, Bale S. et al; ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015; 17 (05) 405-424
• 21 Stenson PD, Mort M, Ball EV, Shaw K, Phillips A, Cooper DN. The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine. Hum Genet 2014; 133 (01) 1-9
• 22 Rehm HL, Berg JS, Brooks LD. et al; ClinGen. ClinGen—the clinical genome resource. N Engl J Med 2015; 372 (23) 2235-2242
• 23 Rosenthal RL, Dreskin OH, Rosenthal N. New hemophilia-like disease caused by deficiency of a third plasma thromboplastin factor. Proc Soc Exp Biol Med 1953; 82 (01) 171-174
• 24 Dorgalaleh A, Bahraini M, Shams M. et al. Molecular basis of rare congenital bleeding disorders. Blood Rev 2023; 59: 101029
• 25 Esteban J, de la Morena-Barrio ME, Salloum-Asfar S. et al. High incidence of FXI deficiency in a Spanish town caused by 11 different mutations and the first duplication of F11: results from the Yecla study. Haemophilia 2017; 23 (06) e488-e496
• 26 Zivelin A, Bauduer F, Ducout L. et al. Factor XI deficiency in French Basques is caused predominantly by an ancestral Cys38Arg mutation in the factor XI gene. Blood 2002; 99 (07) 2448-2454
• 27 Quélin F, Trossaërt M, Sigaud M, Mazancourt PDE, Fressinaud E. Molecular basis of severe factor XI deficiency in seven families from the west of France. Seven novel mutations, including an ancient Q88X mutation. J Thromb Haemost 2004; 2 (01) 71-76
• 28 Zhang K, Qin L, Xu F. et al. The spectrum of factor XI deficiency in Southeast China: four recurrent variants can explain most of the deficiencies. Orphanet J Rare Dis 2024; 19 (01) 224
• 29 Mitchell M, Mountford R, Butler R. et al. Spectrum of factor XI (F11) mutations in the UK population—116 index cases and 140 mutations. Hum Mutat 2006; 27 (08) 829
• 30 Guéguen P, Chauvin A, Quémener-Redon S. et al. Revisiting the molecular epidemiology of factor XI deficiency: nine new mutations and an original large 4qTer deletion in western Brittany (France). Thromb Haemost 2012; 107 (01) 44-50
• 31 Riano I, Prasongdee K. A rare cause of isolated prolonged activated partial thromboplastin time: an overview of prekallikrein deficiency and the contact system. J Investig Med High Impact Case Rep 2021 9. ;9:23247096211012187

Publication History

Received: 25 February 2025

Accepted: 14 July 2025

Article published online:
15 October 2025