Analysis of 363 Genetic Variants in F5 via an Interactive Web Database Reveals New Insights into FV Deficiency and FV Leiden

Abstract

The inherited bleeding disorder Factor V (FV) deficiency and clotting risk factor FV Leiden are associated with genetic variants in the F5 gene. FV deficiency occurs with mild, moderate, severe, or asymptomatic phenotypes, and either dysfunctional or reduced amounts of plasma FV protein. Here we present an interactive web database containing 363 unique F5 variants derived from 801 patient records, with 199 FV deficiency-associated variants from 245 patient records. Their occurrence is rationalized based on the 2,224 residue sequence and new FV protein structures. The 199 FV deficiency variants correspond to 26 (13%) mild, 22 (11%) moderate, 49 (25%) severe, 35 (18%) asymptomatic, and 67 (34%) unreported phenotypes. Their variant distributions in the FV domains A1, A2, A3, B, C1 and C2 were 28 (14%), 32 (16%), 34 (17%), 42 (21%), 16 (8%), and 19 variants (10%), respectively, showing that these six regions contain similar proportions of variants. Variants associated with FV deficiency do not cluster near known protein-partner binding sites, thus the molecular mechanism leading to the phenotypes cannot be explained. However, the widespread distribution of FV variants in combination with a high proportion of buried variant residues indicated that FV is susceptible to disruption by small perturbations in its globular structure. Variants located in the disordered B domain also appear to disrupt the FV structure. We discuss how the interactive database provides an online resource that clarifies the clinical understanding of FV deficiency.

Keywords: coagulation; coagulation factors; gene mutations; protein structure.

Fig. 1

Distribution of the 363 unique variants identified within the F5 gene and FV protein. The FV protein is comprised of the A1-A2-a2-B-a3-A3-C1-C2 domains and linkers in that order, and is shown as gray boxes that are not drawn to scale. N and C represent the N- and C-termini, respectively. Residue numbering marks the first and last amino acids which frame each domain, reported in HGVS format (starting with 1 at the signal peptide). The number of variants in each domain is shown above each protein domain. The number of variants associated with FV deficiency ( red ), thrombosis or FV Leiden ( blue ), or have an unknown association ( gray ) are indicated. Intronic variants (19 in total), undefined or not applicable variants (nine), and variants affecting multiple domains (two) are not shown. Below the protein domains, the gene arrangement of 25 exons is shown as alternating light gray and maroon boxes drawn to scale. The FV protein domain to which each exon codes for are indicated. FV, Factor V.

Fig. 2

Distribution of the 199 unique FV deficiency variants found in the F5 gene. The panels ( a–c ) indicate breakdowns of the 199 FV deficiency variants into variant type, effect, and location within the F5 gene sequence. ( a ) The relative frequency of six distinct types of unique variants in the F5 gene. ( b ) Effect of the 147 point variants found in the F5 gene sequence. ( c ) Distribution of the 199 FV variants across the F5 gene and FV protein domains, although six undefined or not applicable variants are not shown. FV, Factor V.

Fig. 3

Structural and schematic views of FV deficiency variants within the FV domains. ( a ) The full FV structure is shown in ribbon format from the AlphaFold prediction. The structure is shown in rainbow colors, with blue corresponding to the N-terminal region and red corresponding to the C-terminal region. The N-terminus and C-terminus are denoted by N and C, respectively. ( b ) The FV structure from ( a ) is shown schematically in cartoon form in the same orientation and colors. The globular A1, A2, A3, C1, and C2 domains are denoted by filled circles. The disordered B domain is schematically represented by a green line. ( c ) The 100 missense variants are mapped to the ribbon diagram, where the phenotype classifications of mild, moderate, and severe effects are denoted as the traffic light colors of green, yellow, and red, respectively. Missense variants with unreported phenotype are shown in gray and asymptomatic cases are shown in purple. ( d ) The 25 most commonly reported variants are shown as spheres in the ribbon structure of FV shown in ( a ). Blue spheres denote the fifth to twenty-fifth more common variants, and the four magenta spheres denote the four most common variants in FV ( Supplementary Fig. S3 below). FV, Factor V.

Fig. 4

The six individual FV domain structures and their 100 missense variants. Each domain is shown as a ribbon diagram in rainbow colors from the N-terminus ( blue ) to the C-terminus ( red ) for clarity. The structurally similar A1, A2, and A3 domains are shown with their secondary structure ribbons depicted in the same orientations, and likewise the structurally similar C1 and C2 domains. The blue spheres denote the missense mutations associated with FV deficiency in each domain: A1, 22 variants; A2, 20 variants; A3, 22 variants; B, nine variants; C1, eight variants; C2, 17 variants. These variants total 98, where the remaining two variants (not shown) occur in the signal peptide (one variant) and the a2 linker (one variant). FV, Factor V.

Fig. 5

Distribution of FV deficiency variants in the six FV domains . ( a ) The number of missense variants in each of the six FV domains is shown above the red bars. If the number of 98 missense variants are normalized in proportion to amino acid residues present in the sequence of each domain, the outcome is shown as blue bars. The two missense variants that occur in the linker region and the signal peptide are not shown. ( b ) The distribution of 42 variants in the B domain ( green ) is compared against the five types of 157 genetic variants that occur in FV ( orange ). FV, Factor V.

Fig. 6

Substitution analysis of 100 FV deficiency missense variants in the F5 gene. The four substitution analyses predict the damaging effects of substitution variants on the protein structures. ( a ) Analysis of the variants determined by their PolyPhen-2 scores. ( b ) Analysis of variants determined by their SIFT scores. ( c ) Analysis of variants determined by their PROVEAN scores. The PROVEAN threshold used was −2.5. ( d ) Analysis of the variants determined by their Grantham scores. FV, Factor V.

Fig. 7

Accessibility analyses of 100 missense variants associated with FV deficiency in the FV protein structure . ( a ) Accessibilities are presented for the full protein. FV variants in the intact cryo-EM structure (PDB ID: 7KVE) and the full-length AlphaFold structure ( Supplementary Fig. S1 ) were grouped by their phenotypic classification (bottom row). The variants were further subdivided according to the native residue accessibility (accessibilities of 0–1, 2–3 and 4+) of the intact protein. The accessibility was determined using DSSP ( Supplementary Fig. S1 ). Accessibilities of 0 or 1 indicate side chain burial and values of >1 indicate side chain exposure to solvent. ( b ) Accessibilities are presented for the six separated domains. FV variants were again grouped by phenotypic classification and accessibility. Here, accessibility refers to the change in residue accessibility when the cryo-EM FV structure (PDB ID: 7KVE) or the AlphaFold structure ( Supplementary Fig. S1 ) was separated into the six A1, A2, A3, B, C1, and C2 domains. Interdomain linkers are not included in this comparison. FV, Factor V.