Type 2N Research Articles

Type 2N von Willebrand disease: clinical manifestations, pathophysiology, laboratory diagnosis and molecular biology

Type 2N von Willebrand disease encompasses all patients with factor VIII deficiency caused by a markedly decreased affinity of von Willebrand factor for factor VIII. It is recessively inherited and clinically similar to mild haemophilia. The differential biological diagnosis is of major importance for providing the optimal treatment and relevant genetic counselling. This accurate diagnosis is based on an evaluation of the factor VIII-binding capacity of plasma von Willebrand factor. Furthermore, molecular biology techniques allow the identification of missense mutations in the von Willebrand factor gene. All of these induce the substitution of amino acid residues located in the N terminal part of the mature von Willebrand factor molecule, which contains the factor VIII binding site. Most of them induce a classical type 2N von Willebrand disease phenotype with factor VIII deficiency but a normal level and multimeric pattern of von Willebrand factor.

On Sequences with Zero Autocorrelation and Orthogonal Designs

We propose some methods for multiplying the length and type of sequences with elements on a set of commuting variables which have zero non-periodic autocorrelation function. We use base sequences of lengths n+1, n+1, n, n in order to construct four directed sequences of lengths n+1, n+1, n, n and type (2n+1, 2n+1) with zero NPAF as well as normal sequences of length n in order to construct four directed sequences of length 2n and type (2n, 2n, 2n, 2n) with zero NPAF. We construct two and four directed sequences with zero PAF of length 34 and type (34, 34), and (34, 34, 34, 34), respectively, as well as four directed sequences with zero NPAF of lengths 34, 34, 33, 33 and type (67, 67). We also indicate that from m directed sequences of lengths n1, n2, …, nm which consist of t variables, we obtain k·m directed sequences of lengths n1, n2, …, nm (k sequences will be of lengths ni, i=1, 2, …, m) which consist of k·t variables for k=1, 2, …. The above methods lead to the construction of many new orthogonal designs.

Existence of holey LSSOM of type 2n with application to G7-packings of Kv

In this paper, we show that the set of all integers n⩾4 is the spectrum of two mutually orthogonal holey Latin squares, of type 2n, in which one square is symmetric. As an application, we determine the maximum number of pairwise edge disjoint copies of G7 in Kv for each v, and all possible graphs that arise from the unused edges.

Unbalanced X-chromosome inactivation with a novel FVIII gene mutation resulting in severe hemophilia A in a female

This report is of a 14-month-old girl affected with severe hemophilia A. Both her parents had normal values for factor VIII activity, and von Willebrand disease type 2N was excluded. Karyotype analysis demonstrated no obvious alteration, and BclI Southern blot did not reveal F8 gene inversions. Direct sequencing of F8 gene exons revealed a frameshift-stop mutation (Q565delC/ter566) in the heterozygous state in the proposita only. F8 gene polymorphism analysis indicated that the mutation must have occurred de novo in the paternal germline. Furthermore, analysis of the pattern of X chromosome methylation at the human androgen receptor gene locus demonstrated a skewed inactivation of the derived maternal X chromosome from the lymphocytes of the proband's DNA. Thus, the severe hemophilia A in the proposita results from a de novo F8 gene frameshift-stop mutation on the paternally derived X chromosome, associated with a nonrandom pattern of inactivation of the maternally derived X chromosome. (Blood. 2000;96:4373-4375)

Von Willebrand factor binding to heparin in various types of von Willebrand disease

The purpose was to study von Willebrand factor (vWF) binding to heparin in different types of von Willebrand disease (vWD). Plasma samples from 92 patients were representative of most vWD subtypes as they included 13 type 1, ten type 2N, 27 type 2A, 23 type 2B, and 19 type 2M patients. We selected assay conditions suitable for the screening of plasma vWF concentrations as low as 15 U/dl vWF:Ag. We determined the range of vWF concentrations in plasma where the percentage of (125)I-MAb/vWF complexes bound to heparin-agarose beads was constant. This range of dilution allowed circumvention of potential competition by other plasma heparin-binding proteins. The multimeric composition of vWF had hardly any influence on the ability of vWF to bind to heparin. Results were expressed as the ratio of heparin-binding capacity of patients' plasma to that of normal pool plasma. We found a ratio of 0.99+/-0.004 (mean+/-s.e.m.) for 23 normal individual donors. Furthermore, when comparing the mean values of plasma vWF-heparin binding ratios by ANOVA F-test in the six groups (one normal and five vWD), we found significant differences between them (P<0.0001). Pairwise comparison of multiples by the Scheffe's test indicated that the mean values of ratios in type 2A on the one hand and type 2M on the other, were significantly lower than in normal plasma, type 2N, type 2B and type 1. Our data suggest a relationship between the ability of vWF to bind to heparin and to the platelet GPIb receptor, since type 2B and 2N patients have an increased or normal ability to bind to GPIb whereas type 2A and 2M patients have an impaired interaction with that receptor.

Conformational changes in the D′ domain of von Willebrand factor induced by CYS 25 and CYS 95 mutations lead to factor VIII binding defect and multimeric impairment

We report 2 new mutations identified in 3 patients and characterized by the markedly decreased affinity of von Willebrand factor (vWF) for factor VIII (FVIII). Patients 2 and 3, who have a typical type 2N phenotype, were found to be compound heterozygous for Arg91Gln and Cys25Tyr or Cys95Phe, respectively. Patient 1, who is the first cousin of patient 2, had an FVIII binding defect of vWF, low levels of vWF, and multimeric impairment. She was found to be compound heterozygous for the mutations Cys25Tyr and a stop codon (D93ter) in exon 4. Transient expression of recombinant vWF (rvWF) containing either Cys25Tyr or Cys95Phe mutations resulted in mutated rvWF with markedly reduced FVIII binding ability, multimeric structure impairment, and a significant decrease in the vWF expression level. Moreover, the use of anti-vWF monoclonal antibodies that inhibit the FVIII binding showed that these 2 mutations likely induce a conformational change in the D′ domain. These results show that the native conformation of the D′ domain of vWF is not only required for FVIII binding but also for normal multimerization and optimal secretion.

Conformational changes in the D′ domain of von Willebrand factor induced by CYS 25 and CYS 95 mutations lead to factor VIII binding defect and multimeric impairment

AbstractWe report 2 new mutations identified in 3 patients and characterized by the markedly decreased affinity of von Willebrand factor (vWF) for factor VIII (FVIII). Patients 2 and 3, who have a typical type 2N phenotype, were found to be compound heterozygous for Arg91Gln and Cys25Tyr or Cys95Phe, respectively. Patient 1, who is the first cousin of patient 2, had an FVIII binding defect of vWF, low levels of vWF, and multimeric impairment. She was found to be compound heterozygous for the mutations Cys25Tyr and a stop codon (D93ter) in exon 4. Transient expression of recombinant vWF (rvWF) containing either Cys25Tyr or Cys95Phe mutations resulted in mutated rvWF with markedly reduced FVIII binding ability, multimeric structure impairment, and a significant decrease in the vWF expression level. Moreover, the use of anti-vWF monoclonal antibodies that inhibit the FVIII binding showed that these 2 mutations likely induce a conformational change in the D′ domain. These results show that the native conformation of the D′ domain of vWF is not only required for FVIII binding but also for normal multimerization and optimal secretion.

Two novel type 2N von Willebrand disease–causing mutations that result in defective factor VIII binding, multimerization, and secretion of von Willebrand factor

AbstractTwo novel mutations, a T-to-C transition at nucleotide 2612 and a T-to-G transversion at nucleotide 3923 of the von Willebrand factor (vWF) complementary DNA, were detected by analysis of the vWF gene in DNA from members of 2 families with atypical von Willebrand disease. The T2612C transition predicts substitution of cysteine by arginine at amino acid position 788 (C788R), and the T3923G transversion predicts substitution of cysteine by glycine at position 1225 (C1225G) of pre-pro-vWF. The patients homozygous for the C788R and C1225G mutations both had a partial vWF deficiency (0.18 IU/mL and 0.07 IU/mL vWF antigen, respectively); vWF in plasma from patients homozygous for either the C788R or the C1225G mutation failed to bind factor VIII and lacked high molecular weight multimers. Recombinant (r) vWF molecules having the C788R or C1225G mutation were expressed in COS-7 cells. Both rvWF C788R and rvWF C1225G exhibited significantly impaired secretion and failed to bind factor VIII. Recombinant vWF C788R in COS-7 culture medium showed a severe reduction in high molecular weight multimers, whereas rvWF C1225G showed a very mild reduction in high molecular weight multimers when compared with wild-type rvWF.

Production and cytogenetics of intergeneric hybrids between the three cultivated Brassica diploids and Orychophragmusviolaceus

It has been proposed that both complete and partial separation of the parental genomes during mitosis and meiosis occurs in the intergeneric hybrids between Orychophragmus violaceus (2n=24) and the three cultivated Brassica tetraploids (B. napus, B. carinata and B. juncea). The hypothesis has been that this and the variations in chromosome numbers of these hybrids and their progenies result from the different roles of the A, B and C genomes originating from Brassica. To test this hypothesis, we produced hybrids between O. violaceus and the cultivated Brassica diploids. The hybrids with B. oleracea (2n=18, CC) had an intermediate morphology, but their petals were purple like those of O. violaceus. They were sterile and had the expected chromosome number (2n=21) in their mitotic and meiotic cells. The hybrid with B. campestris (2n=20, AA) was morphologically intermediate, except for its partial fertility and its yellow petals, which were similar to those of B. campestris. It was mixoploid (2n=23-42), and cells with 2n=34 were most frequent. Partial separation of parental genomes during mitosis, leading to the addition of O. violaceus chromosomes to the B. campestris complement, was proposed to explain the findings in the mitotic and meiotic cells of the hybrid and its progeny. In crosses with B. nigra (2n=16, BB), the majority of the F(1) plants were of the maternal type (2n=16), a small fraction had B. nigra morphology but were mixoploids (2n=16-18), predominantly with 2n=16 cells and three plants, each with a specific morphology, were mixoploids consisting of cells with varying ranges of chromosome numbers (2n=17-26, 11-17 and 14-17). The origin of these different types of plants was inferred to be a result of the complete and partial separation of parental genomes and the loss of O. violaceus chromosomes. Our findings in the three crosses suggest that the A genome was more influential than the C genome with respect to complete genome separation during mitosis and meiosis of the hybrids with B. napus. Possible complete and partial genome separation during mitotic divisions of the hybrids with B. carinata was mainly attributed to the role of the B genome. The combined roles of the A and B genomes would thus contribute to the most variable chromosome numbers of mitotic and meiotic cells in the hybrids with B. juncea and their progenies. The possible cytological mechanisms pertaining to these hybrids and the potential of genome separation in the production of Brassica aneuploids and homozygous plants are discussed.

A Common Splice Site Mutation Is Shared by Two Families with Different Type 2N von Willebrand Disease Mutations

Using an ELISA-based method to detect type 2N von Willebrand disease (VWD), we found two individuals with absent FVIII binding. Direct sequencing of the FVIII binding region of the von Willebrand factor (VWF) gene showed that one individual had an R854Q substitution whilst the other had a T791M substitution. The very low FVIII binding and the VWF:Ag levels in both individuals suggested a second defect on the other VWF allele. Conformation sensitive gel electrophoresis of polymerase chain reaction amplified DNA was used to detect an additional change in the VWF gene of each patient. Direct sequencing confirmed a previously unreported G to A transition in the donor splice site in intron 25 of both individuals which should result in a null allele. This was confirmed by mRNA analysis. These two individuals therefore have compound heterozygous VWD in which the only expressed allele has a type 2N mutation. In our population, such compound heterozygosity appears to be a significant cause of type 2N VWD.

New Assay for Measuring Binding of Platelet Glycoprotein IIb/IIIa to Unpurified von Willebrand Factor

Among the numerous variants of vWD, no patient with an abnormal vWF binding to GPIIb/IIIa has been described to date. To search for such potential variants, we developed a two-site assay for measuring the binding of purified GPIIb/IIIa to vWF in biological fluids and we used it to study a large series of plasmas from various types of von Willebrand disease (vWD) and recombinant vWF (rvWF). vWF in plasma or rvWF in culture medium was immobilized onto anti-vWF monoclonal antibodies (MoAb)-coated wells of microtiter plates. After incubation with either unlabeled GPIIb/IIIa and a 125I-anti-GPIIb/IIIa MoAb or 125I-GPIIb/IIIa, binding curves and binding isotherms were respectively established. Normal pool plasma and wild-type rvWF were used as reference samples. We tested plasmas from 85 normal subjects, 115 patients with different types of vWD (64 type 1, 2 type 3, 9 type 2A, 4 type 2M, 16 type 2B, 15 type 2N, 3 type IID and 2 acquired forms) and 50 patients with various bleeding disorders. Four mutated rvWF with 2A (Glu875Lys and Pro885Ser) or 2B (Dupl.Met540 and Val551Phe) substitutions and one rvWF mutated in the RGD domain of the C-terminal part of vWF-subunit (Asp1746Gly) were also studied. Among the various samples tested, only rvWF Asp1746Gly had no affinity for GPIIb/IIIa. In contrast, GPIIb/IIIa similarly bound to the other vWF, independently of the proteic environment, the factor VIII level, the degree of multimerization or the mutation of vWF. Our results indicate that subjects with an abnormal vWF binding to GPIIb/IIIa are probably rare and difficult to target for a specific screening.

The evaluation of factor VIII binding activity of von Willebrand factor by means of an ELISA method: significance and practical implications.

One of the functions of von Willebrand factor (vWF) is to serve as a carrier of clotting factor VIII (FVIII). Deficiency of this function results in the von Willebrand disease (vWD) variant type 2N, which resembles hemophilia A. We describe a new sandwich enzyme-linked immunosorbent assay (ELISA) to study the ability of vWF to bind exogenous recombinant FVIII (rFVIII), in which anti-vWF-coated plates are incubated with plasma vWF, followed by exogenous FVIII and a peroxidase-coupled anti-FVIII antibody. Dose-response curves obtained using normal plasma vWF and purified normal vWF revealed a hyperbolic relationship between the optical density and the vWF concentration. The assay allows the quantification of FVIII binding with values expressed in U/dL; 100 U/dL was the amount present in normal plasma. The sensitivity and specificity of the method are demonstrated by its ability to measure binding levels as low as 1 to 2 U/dL and the fact that no FVIII binding was observed using plasma known to contain less than 1 U/dL vWF. To verify the accuracy of the assay, three patients with type 2N vWD with characterized vWF gene mutations were studied using an existing chromogenic assay and our ELISA. A patient who was homozygous for the R53W mutation and had no FVIII binding capacity according to the chromogenic method showed undetectable FVIII binding by ELISA. The remaining two patients, one who was homozygous for the R91Q mutation and one with compound heterozygosity for the R91Q and R53W mutations, showed markedly decreased FVIII binding by the chromogenic method and yielded ELISA values ranging from 4 to 8 U/dL. Therefore, although the two methods produce qualitatively similar results, the ELISA method offers the advantage of allowing quantification of the FVIII binding function. FVIII binding was also analyzed in 20 patients with type 1 vWD; we found a decrease of FVIII binding that was proportionate to the decrease in vWF levels, showing a normal FVIII binding activity/vWF molecule ratio. We define the binding activity measured by this assay as vWF:FVIII binding activity and propose its use in the functional analysis of vWF.

Type 2N von Willebrand Disease: Rapid Genetic Diagnosis of G2811A (R854Q), C2696T (R816W), T2701A (H817Q) and G2823T (C858F) – Detection of a Novel Candidate Type 2N Mutation: C2810T (R854W)

The majority of patients with type 2N von Willebrand disease (VWD type 2N) have mutations in the region of the von Willebrand factor (VWF) gene encoding the factor VIII binding domain of VWF. Two mutations predominate among VWD type 2N patients: G2811A and C2696T, which respectively bring about the amino acid substitutions R854Q and R816W in VWF. Several other mutations have been found in VWD type 2N, including T2701A (H817Q) and G2823T (C858F). We have developed a genetic test which permits rapid screening for these four mutations in a single polymerase chain reaction (PCR). The test employs induced heteroduplex formation using two universal heteroduplex generators, one of which detects G2811A (R854Q) and G2823T (C858F), the other detects C2696T (R816W) and T2701A (H817Q). The allele frequency of the common G2811A (R854Q) mutation was investigated in the local (S. Wales) population by examination of 216 VWF genes (108 individuals) and was found to be 0.01. The heteroduplex-based test additionally detected a novel candidate type 2N mutation, C2810T (R854W) and a previously described polymorphism, G2805A (R852Q). The polymorphism showed allele frequencies of 0.92 (G nucleotide) and 0.08 (A nucleotide) in the population study.

Re-examination of the chromosome homology between two subspecies of Japanese raccoon dogs (Nyctereutes procyonoides albus and N.p. viverrinus)

SUMMARY- We investigated the chromosomal relationships between subspecies of Japanese raccoon dogs from Hokkaido (Nyctereutes procyonoides albus; NP A) and all of the other major Japanese Islands (N. p. viverrinus; NPV). Chromosomes of wild-caught NPA were analyzed by conventional stain, G-banding and C-banding techniques. The basic karyotype of this subspecies is composed of met acentric (M) and acrocentric (A) chromosomes plus a variable number of B chromosomes (2n = 38 ( = 26M + 10A + XY or XX) + Bs) and is the same as that of NPV. This study of NP A suggests that the Island type (2n = 38) only occurs in Japanese islands including Hokkaido, whereas all animals from mainland Asia and eastern Europe that have been examined have the Continental type (2n = 54).

Factor VIII and von Willebrand Factor

IntroducationFactor VIII and von Willebrand factor are plasma glycoproteins whose deficiency or structural defects cause hemophilia A and von Willebrand disease, respectively (1). These diseases are the most common inherited bleeding disorders of man. Factor VIII and vWF are synthesized by different cell types and circulate in plasma as a tightly bound complex. Factor VIII is synthesized in the liver (2), and functions as a cofactor for activated factor IX in the intrinsic activation of factor X on a membrane surface (3). vWF is synthesized in endothelial cells (4, 5) and megakaryocytes (6). vWF has a dual role in hemostasis: it promotes platelet adhesion to subendothelium after vessel injury (7, 8) and it acts as a carrier protein of factor VIII (1).The distinction between factor VIII and vWF was unclear for many years, because severe Von Willebrand disease is associated with factor VIII deficiency and because early preparations of factor VIII concentrates contained vWF and were therefore effective in correcting the platelet adhesion defects in patients with von Willebrand disease (9). Since factor VIII and vWF form a tightly bound non-covalent complex in plasma, both proteins are copurified when isolated from plasma, unless special measures are taken (1). The stoichiometry of factor VIII and vWF in plasma is approximately 1:50 and factor VIII and monomeric vWF have similar molecular weights of approximately 240 kDa. Therefore, vWF represents 98% of the molecular mass of the factor VIII-vWF complex (10) and almost all the antibodies raised against the complex react to vWF. In the 1980’s, factor VIII and vWF have each been purified to homogeneity and the genes for these proteins have been cloned. This set the stage for studies with purified proteins which have elucidated structure-function relationships for both proteins. Also, the interaction between both proteins could be studied using proteolytic fragments, small peptides, and monoclonal antibodies. In the last few years, the construction of recombinant mutants and fragments of both factor VIII (11-13) and vWF (14-16) has proven to be a powerful tool in the elucidation of the structure and function of both proteins.Binding of factor VIII to vWF is essential for the survival of factor VIII in vivo (17, 18). The underlying mechanism is probably that factor VIII bound to vWF is protected from phospholipid dependent proteolysis by activated protein C and factor Xa (19, 20). The binding site for factor VIII has been located at the amino terminus of vWF (21, 22). A tryptic fragment containing this binding site was not sufficient to protect factor VIII against activated protein C-mediated degradation according to some groups (23, 24). In contrast, a recent study using comparable vWF fragments showed protection of factor VIII equivalent to mature vWF (16).In 1989, a new variant of von Willebrand disease was discerned (type Normandy or 2N), distinct from the more than 20 subtypes known, characterized by a mutant vWF that is structurally and functionally normal, except that it does not bind to and stabilize factor VIII (25, 26). Since then, several mutations in the factor VIII binding site on vWF have been found (27). A number of reports have shown that factor VIII binds vWF via a high affinity binding site on its light chain (28-30). Two recent studies suggest that this binding site consists of two separate binding sites (31, 32).This review summarizes current knowledge on the interaction between factor VIII and vWF. Emphasis will be laid on the biological importance of, and the domains involved in binding, and on the stoichiometry and kinetics of complex formation.

Single stranded conformational polymorphism screening for von Willebrand disease type 2N mutations

Single stranded conformational polymorphism screening for von Willebrand disease type 2N mutations

Estimation of 2n Megagametophyte Heterozygosity in a Diploid Blueberry (Vaccinium darrowi Camp) Clone Using RAPDs

To determine the mode(s) of 2n gamete formation in a Vaccinium darrowi Camp (2n = 2x = 24) clone Fla4B, the transmission of parental heterozygosity was estimated in 2n eggs through randomly amplified polymorphic DNA (RAPD) analysis of 41 progeny from crosses of Fla4B to tetraploid V. corymbosum (2n = 4x = 48). The level of estimated heterozygosity transmitted ranged from 5.3-100%, with an overall mean heterozygosity of 72.7%, which suggests that first division restitution (FDR) type 2n gamete forming mechanisms are operating in this clone. However, there was evidence that 17% of the 2n eggs had less than 50% estimated heterozygosity, which supports second division restitution (SDR) mechanisms operating as well. Both SDR and FDR modes of 2n egg formation are also supported by a nonnormal, bimodal frequency distribution of estimated heterozygosity transmission. Recombination also appears to be prevalent. The predominance of 2n gametes genetically equivalent to FDR is generally considered desirable for breeding autotetraploid crops.

Understanding von Willebrand's disease from gene defects to the patients

von Willebrand's disease (vWD) is caused by qualitative (type 2) and quantitative (types 1 and 3) abnormalities of von Willebrand factor (vWF). vWD type 3, a severe form of the disease with nearly complete deficiency of the protein in plasma, are found to be homozygous or compound heterozygous for null mutations in the vWF gene. Null mutations in both alleles of the vWF gene completely disrupt the protein synthesis resulting in a nearly complete deficiency of the vWF in the type 3 patients. The vWD type 1 patients (mild form with partial deficiency of the protein) could be heterozygous for null mutations or compound heterozygous for the mutations (null mutation + missense mutation) in the gene. The vWD type 2, divided into four variants: types 2A, 2B, 2M and 2N, are caused exclusively by missense mutations within three different domains of the protein (gain or loss of function). The majority of type 2A mutations are located in the A2 domain and the types 2B and 2M mutations are in the A1 domain, while the type 2N mutation is in the FVIII binding domain.

Abnormal Proteolytic Processing of von Willebrand Factor Arg611 Cys and Arg 611 His

The structural and functional properties of plasma and platelet vWF were studied in 8 patients (5 unrelated families) with vWD demonstrating a mutation at position 611 (R611C or R611H). Following reduction, electrophoresis and immunoblotting with a polyclonal anti-reduced vWF antibody, abnormal proteolysis of vWF was demonstrated in plasma and to a lesser extent in platelets from all patients, leading to the formation of a unique 209 kDa fragment undetectable in control as well as in type 2A, 2B or 2N vWF. Immunoblotting with MoAbs to reduced vWF showed that the C-terminal end of the 209 kDa fragment was located beyond residue 1744 of the subunit and that its N-terminus was between residues 523 and 1114. Multimeric analysis of patients vWF showed an abnormal pattern in both plasma and platelets, with a moderate decrease of the HMW multimers together with a significant increase of the lowest MW forms. The specific sensitivity of vWF R611C and vWF R611H to proteolysis was further evidenced using V-8 protease. In all patient's samples the enzyme produced a unique monomeric 80 kDa fragment, absent in V-8 digested normal vWF, which overlapped the N-terminal part of the subunit. The functional analysis of vWF showed a markedly decreased affinity of mutated plasma vWF for platelet GPIb in the presence of ristocetin. Infusion of DDAVP in two of these patients did not lead to significant platelet count change. It induced a limited increase of the HMW multimers in plasma together with a poor correction of the vWF binding to platelet GPIb. In conclusion, our data demonstrate that in addition to a normal proteolysis, vWF mutated at position 611 undergoes a specific cleavage in plasma and platelets. In contrast to the increased proteolysis observed in type 2A and 2B patients' plasma, this additional cleavage produced a unique 209 kDa species but maintained a HMW multimer-like structure of vWF R611C and R611H.

Moderation of Hemophilia A Phenotype by the Factor V R506Q Mutation

Although many examples of unrelated hemophilia A patients carrying identical point mutations in the factor VIII (FVIII) gene have been reported, the clinical phenotype is not always the same among patients sharing the same molecular defect. Possible explanations for this discrepancy include undetected additional mutations in the FVIII gene or coinheritance of mutations at other genetic loci that modulate FVIII function. We report molecular genetic analysis of potential modifying genes in two sets of unrelated patients carrying common FVIII missense mutations but exhibiting different levels of clinical severity. Both mutations (FVIII R1689C and R2209Q) are associated with severe hemophilia A in some patients and mild/moderate disease in others. The common von Willebrand disease type 2N mutation (R91Q) was excluded as a modifying factor in these groups of patients. However, analysis of the recently described factor V (FV) R506Q mutation (leading to activated protein C resistance) identified a correlation of inheritance of this defect with reduced hemophilia A severity. Two moderately affected hemophilia A patients, each with either of two FVIII gene mutations, were heterozygous for FV R506Q, whereas two severely affected patients and two moderately affected patients were homozygous normal at the FV locus. Our results suggest that coinheritance of the FV R506Q mutation may be an important determinant of clinical phenotype in hemophilia A and that modification of the protein C pathway may offer a new strategy for the treatment of FVIII deficiency.