Molecular characterization of Iranian patients with type 3 von Willebrand disease.

von Willebrand's disease (VWD) type 3 is a rare but severe autosomal-recessive inherited bleeding disorder with a prevalence higher in certain locations where consanguineous marriages are relatively frequent. The genetic defects causing recessive type 3 VWD in 10 unrelated families from Iran have been investigated and the genetic heterogeneity among these patients was evaluated. All exons and their flanking regions of von Willebrand factor gene were amplified by PCR and sequenced using specific primers. Eight patients were fully characterized at the molecular level. Six different gene alterations were identified. All the mutations caused null alleles, three being nonsense mutations (Q104X, Q793X and E1981X), two possible splice site mutations (2443-1G>C and 1110-1G>A) and one small deletion (3237delA). Three of them have not been described previously. Most patients were born from consanguineous marriages and all were homozygous for their mutations. The results confirm that molecular defects in type 3 VWD are heterogeneous with mutations arising randomly within the entire gene.

Factor VIII genotype and inhibitor development in patients with haemophilia A: highest risk in patients with splice site mutations.

The appearance of inhibitory antibodies against factor VIII (FVIII) is the most severe and costly complication of replacement therapy in patients with haemophilia A (HA). To determine the relationship between FVIII genotype and inhibitor development, baseline FVIII activity, genotype and inhibitor development were reviewed in 1104 patients with HA. In patients with severe HA, splicing errors present the highest frequency of inhibitors, ahead of inversion of intron 1 and of intron 22, nonsense mutations and large deletions. The lowest inhibitor frequency in severe HA is found in patients with missense mutations and small deletions/insertions. Subanalyses indicate that nonsense mutations and small deletions/insertions leading to a frameshift in the light chain are associated with a significant higher risk of inhibitor formation than similar mutations occurring in the heavy chain (27% vs. 14%). These mutation types also have a higher frequency of inhibitors when occurring in exons 23-26, where a second FVIII transcript originates, compared with similar mutations in exons 1-22 (28% vs. 17%). These results suggest that complete absence of FVIII because of null mutations, including splice site mutations, or the absence of a second transcript result in an increased risk of inhibitor development.

Factor VIII (FVIII) gene mutations in 120 patients with hemophilia A: detection of 26 novel mutations and correlation with FVIII inhibitor development.

BACKGROUND: As the publication of the sequence of the factor VIII gene (FVIII) in 1984, a large number of mutations that cause hemophilia A (HA) have been identified. Thanks to the advances in the detection of mutations, it is now possible to identify a putative FVIII sequence alteration in the vast majority of patients with HA. OBJECTIVES: Our main objective was to report on the spectrum of FVIII mutations and their distribution throughout the gene in 120 patients with HA. METHODS: Screening of FVIII mutations was performed using direct sequencing. Newly described missense mutations were further studied by molecular modeling. RESULTS: A total of 47 different HA causative FVIII mutations have been identified, 26 of which are described for the first time. These novel mutations include 14 missense and six nonsense mutations, two small deletions, one large deletion and three splice-site mutations. We further investigated the development of FVIII-specific inhibitors in all patients with HA. We found that four novel mutations (Ser882X, Tyr1786Ser, Ala2218Thr and a splice-site defect in intron 22) were associated with inhibitor development. CONCLUSION: These data extend our insight into the mechanisms by which novel amino acid substitutions may lead to HA, and how HA patient genotypes influence the risk of FVIII inhibitor development.

Ten candidate ADAMTS13 mutations in six French families with congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome).

ADAMTS13, the specific von Willebrand factor (VWF)-cleaving metalloprotease, prevents the spontaneous formation of platelet thrombi in the microcirculation by degrading the highly adhesive ultralarge VWF multimers into smaller forms. ADAMTS13 severe enzymatic deficiency and mutations have been described in the congenital thrombotic thrombocytopenic purpura (TTP or Upshaw-Schulman syndrome), a rare and severe disease related to multivisceral microvascular thrombosis. We investigated six French families with congenital TTP for ADAMTS13 enzymatic activity and gene mutations. Six probands with congenital TTP and their family were tested for ADAMTS13 activity in plasma using a two-site immunoradiometric assay and for ADAMTS13 gene mutations using polymerase chain reaction and sequencing. ADAMTS13 activity was severely deficient (< 5%) in the six probands and one mildly symptomatic sibling but normal (> 50%) in all the parents and the asymptomatic siblings. Ten novel candidate ADAMTS13 mutations were identified in all families, showing either a compound heterozygous or a homozygous status in all probands plus the previous sibling and a heterozygous status in the parents. The mutations were spread all over the gene, involving the metalloprotease domain (I79M, S203P, R268P), the disintegrin domain (29 bp deletion in intron/exon 8), the cystein-rich domain (acceptor splice exon 12, R507Q), the spacer domain (A596V), the 3rd TSP1 repeat (C758R), the 5th TSP1 repeat (C908S) and the 8th TSP1 repeat (R1096stop). This study emphasizes the role of ADAMTS13 mutations in the pathogenesis of congenital TTP and suggests that several structural domains of this metalloprotease are involved in both its biogenesis and its substrate recognition process.

Type 2 von Willebrand disease causing defective von Willebrand factor-dependent platelet function.

Type 2 von Willebrand disease causing defective von Willebrand factor-dependent platelet function comprises mainly subtypes 2A, 2B and 2M. The diagnosis of type 2 von Willebrand disease may be guided by the observation of a disproportionately low level of ristocetin cofactor activity or collagen-binding activity relative to the von Willebrand factor antigen level. The decreased platelet-dependent function is often associated with an absence of high molecular weight multimers (types 2A and 2B), but the high molecular weight multimers may also be present (type 2M and some type 2B), and supranormal multimers may exist (as in the Vicenza variant). Today, the identification of mutations in particular domains of the pre-provon Willebrand factor is helpful to classify these variants and to provide further insight into the structure-function relationship and the biosynthesis of von Willebrand factor. Thus, mutations in the D2 domain, involved in the multimerization process, are found in patients with type 2A, formerly named IIC von Willebrand disease. Mutations in the D3 domain characterize the Vicenza variant, or type IIE patients. Mutations in the A1 domain may modify the binding of von Willebrand factor multimers to platelets, either increasing (type 2B) or decreasing (types 2M and 2A/2M) the affinity of von Willebrand factor for platelets. In type 2A disease, molecular abnormalities identified in the A2 domain, which contains a specific proteolytic site, are associated with alterations in folding that impair the secretion of von Willebrand factor or increase its susceptibility to proteolysis. Finally, a mutation localized in the C terminus cysteine knot domain, which is crucial for the dimerization of von Willebrand factor subunit, has been identified in a rare subtype 2A, formerly named IID.

Ser968Thr mutation within the A3 domain of von Willebrand factor (VWF) in two related patients leads to a defective binding of VWF to collagen.

We report the identification of a new mutation of von Willebrand Factor (VWF) gene within exon 30 occurring in two related patients (mother and daughter) with a hemorrhagic syndrome. A T-->A transvertion at nucleotide 5441 was found changing the serine 968 to threonine of the mature VWF subunit (S1731T of the preproVWF). The Ser968Thr mutation is located within the VWF A3 domain which interacts with type I and III collagens. Both patients were found to be heterozygous for the mutation. The propositus (daughter) exhibited a slightly prolonged bleeding time, levels of VWF:Ag and VWF:RCo at the lower limit of normal, contrasting with normal levels of VIII:C. Her mother exhibited borderline bleeding time and moderately decreased levels of VWF and VIII:C. In both patients multimeric structure of VWF and ristocetin- as well as botrocetin-induced binding of VWF to GPIb were normal; however both patients repeatedly showed decreased binding of VWF to collagen. The Ser968Thr substitution was reproduced by site-directed mutagenesis on the full-length cDNA of VWF. The mutated recombinant VWF (rVWF), T968rVWF, and the hybrid S/T968rVWF were transiently expressed by COS-7 cells. Both rVWF exhibited normal multimeric pattern and normal ristocetin- as well as botrocetin-induced binding to GPIb. T968rVWF showed significantly decreased binding to collagen while the hybrid S/T968rVWF bound to collagen in a similar way to that of the patients' plasma VWF. Thus, our data demonstrate that the Ser968Thr mutation of the VWF A3 domain is clearly responsible for the abnormal binding of VWF to collagen observed in both patients. The Ser968Thr substitution of the VWF is the first mutation identified in two patients leading to a decreased affinity of VWF for collagen and a normal multimeric structure.

Functional mapping of anti-factor IX inhibitors developed in patients with severe hemophilia B.

Development of inhibitory antibodies is a serious complication of treatment with repeated factor IX infusions in a minority of patients with hemophilia B. Such antibodies detected in 8 patients have been characterized. Typing studies revealed that patients' immune response toward factor IX is highly heterogeneous and involves immunoglobulin G (IgG) antibodies, preferentially IgG1 and IgG4. The preservation of the sequence and the 3-dimensional orientation of the amino acids constituting one epitope are highly important for the assembly of an antibody-antigen complex. To localize the epitopes on the factor IX molecule, an original approach was designed using a set of factor X chimeras carrying regions of factor IX. Results showed that some patients' antibodies were directed against both the domain containing the gamma-carboxy glutamic acid residues (Gla domain) and the protease domain of factor IX. In contrast, no binding was observed to the epidermal growth factor-like domains or to the activation peptide. Functional characterization showed that the purified IgG from patients' serum inhibited the factor VIIIa-dependent activation of factor X. Moreover, patients' IgG directed against the Gla domain inhibited the binding of factor IX to phospholipids as well as the binding of factor VIII light chain to factor IXa. These data demonstrate that inhibitors appearing in patients with severe hemophilia B display specificity against restricted functional domains of factor IX.

Mild haemophilia A discovered in a previously multi-operated 73-year-old man: characterization of a new mutation.

A mild haemophilia A (factor VIII [FVIII] 10%) was discovered in a 73-year-old man during the preoperative haemostasis tests before a total knee arthroplasty for treatment of degenerative senile arthropathy. No history of previous abnormal bleeding was noted in spite of several previous challenging surgical procedures. Acquired haemophilia was ruled out and no other cases of haemophilia were found in the family. The surgery was successfully performed under recombinant FVIII aimed at achieving an FVIII level above 60%. This therapy induced the transient appearance of low-titre FVIII antibodies. A still unpublished mutation within the exon 3 (Gly73Ala) was found with molecular studies. This case report underlines the importance of haemostasis tests before high-risk bleeding surgical procedures even in elderly patients without a past history of haemorrhagic tendency.

Somatic mosaicism in hemophilia A: a fairly common event.

Mutations in the large gene of clotting factor VIII (FVIII) are the most common events leading to severe human bleeding disorder. The high proportion of de novo mutations observed in this gene raises the possibility that a significant proportion of such mutations does not derive from a single germ cell but instead should be attributed to a germline or somatic mosaic originating from a mutation during early embryogenesis. The present study explores this hypothesis by using allele-specific PCR to analyze 61 families that included members who had sporadic severe hemophilia A and known FVIII gene defects. The presence of somatic mosaicisms of varying degrees (0.2%-25%) could be shown in 8 (13%) of the 61 families and has been confirmed by a mutation-enrichment procedure. All mosaics were found in families with point mutations (8 [25%] of 32 families). In the subgroup of 8 families with CpG transitions, the percentage with mosaicism increased to 50% (4 of 8 families). In contrast, no mosaics were observed in 13 families with small deletions/insertions or in 16 families with intron 22 inversions. Our data suggest that mosaicism may represent a fairly common event in hemophilia A. As a consequence, risk assessment in genetic counseling should include consideration of the possibility of somatic mosaicism in families with apparently de novo mutations, especially families with the subtype of point mutations.

The arginine-552-cysteine (R1315C) mutation within the A1 loop of von Willebrand factor induces an abnormal folding with a loss of function resulting in type 2A-like phenotype of von Willebrand disease: study of 10 patients and mutated recombinant von Willebrand factor.

The study identified 10 patients from 6 families with prolonged bleeding time, decreased von Willebrand factor (vWF) ristocetin cofactor activity (RCoF) to vWF:Ag (antigen) ratio, and reduced ristocetin-induced platelet agglutination as well as ristocetin- or botrocetin-induced binding of plasma vWF to platelet glycoprotein Ib (GpIb). In addition, all patients showed a decrease of intermediate-molecular-weight (intermediate-MW) and high-molecular-weight (HMW) multimers of vWF. In the heterozygous state, a cysteine-to-threonine (C --> T) transversion was detected at nucleotide 4193 of the VWF gene of all patients and lead to the arginine (R)522C substitution in the A1 loop of vWF mature subunit (R1315C in the preprovWF). By in vitro mutagenesis of full-length complementary DNA (cDNA) of vWF and transient expression in COS-7 cells, the mutated C552 recombinant vWF (C552rvWF) was found to exhibit decreased expression, abnormal folding, and lack of intermediate-MW and HMW multimers. In addition, direct binding of botrocetin to C552rvWF, as well as ristocetin- and botrocetin-induced binding of C552rvWF to GpIb, was markedly decreased. Although being localized in an area of the A1 loop of vWF where most of the type 2B mutations that induce a gain-of-function have been identified, the R552C mutation induces a 2A-like phenotype with a decrease of intermediate-MW and HMW multimers as well as a loss-of-function of vWF in the presence of either ristocetin or botrocetin. (Blood. 2001;97:952-959)

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)

Somatic mosaicism and compound heterozygosity in female hemophilia B.

Sequencing the complete factor IX gene of 2 sisters with hemophilia B with different phenotypes and no family history of hemorrhagic diathesis revealed a common 5' splice site mutation in intron 3 (T6704C) in both and an additional missense mutation (I344T) in one. The presence of dysfunctional antigen in the latter strongly suggested that these mutations are in trans. Neither mutation was found in leukocyte DNA from the asymptomatic parents, but the mother was in somatic mosaicism for the shared splice site mutation. This case illustrates the importance of defining the phenotype and considering somatic mosaicism in sporadic cases. It underlines the limitations of complete gene sequencing for the detection of mosaicism and has implication for genetic counseling. (Blood. 2000;96:1585-1587)

A novel cause of mild/moderate hemophilia A: mutations scattered in the factor VIII C1 domain reduce factor VIII binding to von Willebrand factor.

The mechanisms responsible for the low factor VIII (fVIII) activity in the plasma of patients with mild/moderate hemophilia A are poorly understood. In such patients, we have identified a series of fVIII mutations (Ile2098Ser, Ser2119Tyr, Asn2129Ser, Arg2150His, and Pro2153Gln) clustered in the C1 domain and associated with reduced binding of fVIII to von Willebrand factor (vWf). For each patient plasma, the specific activity of mutated fVIII was close to that of normal fVIII. Scatchard analysis showed that the affinity for vWf of recombinant Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII mutants was reduced 8-fold, 80-fold, and 3-fold, respectively, when compared with normal fVIII. Given the importance of vWf for the stability of fVIII in plasma, these findings suggested that the reduction of fVIII binding to vWf resulting from the above-mentioned mutations could contribute to patients' low fVIII plasma levels. We, therefore, analyzed the effect of vWf on fVIII production by Chinese hamster ovary (CHO) cells transfected with expression vectors for recombinant B domain-deleted normal, Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII. These 3 mutations impaired the vWf-dependent accumulation of functional fVIII in culture medium. Analysis of fVIII production by transiently transfected CHO cells indicated that, in addition to the impaired stabilization by vWf, the secretion of functional Ile2098Ser and Arg2150His fVIII was reduced about 2-fold and 6-fold, respectively, by comparison to Ser2119Tyr and normal fVIII. These findings indicate that C1-domain mutations resulting in reduced fVIII binding to vWf are an important cause of mild/moderate hemophilia A.

Fast and efficient mutation detection method using multiplex PCR and cycle sequencing--application to haemophilia B.

A method using multiplex PCR followed by cycle-sequencing has been developed to detect mutations in the FIX gene. The procedure was evaluated in 45 severe or mild haemophilia B patients from 45 unrelated families. At least one deleterious mutation was identified in every haemophiliac demonstrating the efficiency of the method. Furthermore the described procedure offers many advantages compared to other screening detection methods: it is fast (less than 48 h), simple (partly automated) and of relatively low cost (it requires only one PCR).

Gene defects in 150 unrelated French cases with type 2 von Willebrand disease: from the patient to the gene. INSERM Network on Molecular Abnormalities in von Willebrand Disease.

Type 2 vWD is defined by qualitative defects of vWF and is subdivided into four subtypes: 2N, 2B, 2A and 2M. The characterization of 150 unrelated French cases with type 2 vWD emphasizes the heterogeneity of this group. In 51 cases of type 2N vWD, new mutations were found not only in the D' domain (Cys25Tyr and Cys95Phe) but also in the D3 domain (Asp116Asn and Cys297Arg). In 42 cases of type 2B vWD, no new mutation was detected. In 45 cases with type 2A phenotype, three new candidate mutations were found in the A2 domain: Gln793Arg, Val841Phe and Leu876Pro. In addition, four new candidate mutations were detected in the A1 domain: Cys509Gly, Arg545His, Arg552Cys and Cys695Tyr. Finally, five new candidate mutations were identified in 12 patients with 2M (or unclassified) phenotype: Leu513Pro, Gly561A1a, Glu596Lys, Arg611Leu and IIe662Phe. For all candidate mutations, expression studies are in progress. This study of a large number of French variants of vWD brings further insight into the relationship between phenotype and genotype.

Occurrence of myeloproliferative disorder in patients with Noonan syndrome.

We report four cases of Noonan syndrome associated with chronic myelomonocytic leukemia in childhood. These children shared some hematologic features: thrombocytopenia, splenomegaly in the first months of life, occurrence of chronic myelomonocytic leukemia without abnormalities of the initial bone marrow karyotype, and, in three cases, improvement of the hematologic disease. A common pathophysiologic process in such patients is suggested.

A patient with type 2N von Willebrand disease is heterozygous for a new mutation: Gly22Glu. Demonstration of a defective expression of the second allele by the use of monoclonal antibodies.

We report the case of a Chinese patient who has subnormal von Willebrand factor (vWF) level and normal vWF multimeric pattern, but a lack of vWF capacity to bind factor VIII (FVIII). Exons 18 to 20 of the patient's vWF gene were analyzed by DGGE and a G2354 --> A substitution which changes the encoded amino acid sequence from Gly22 to Glu was identified. The patient is heterozygous for this substitution, creating a unique Sac I restriction site. Recombinant vWF (rvWF) containing the candidate mutation was transiently expressed in COS-7 cells. It was processed and secreted normally but failed to bind FVIII. FVIII binding ability of hybrid rvWF, obtained by cotransfection of normal and mutated expression vectors and corresponding to a heterozygous genotype, was moderately decreased. To explain this functional discrepancy between patient's plasma vWF and hybrid rvWF, we used anti-vWF monoclonal antibodies (MoAbs) as capture in an enzyme-linked immunosorbent assay test. MoAb 32B12 recognized both wild-type and mutated rvWFs whereas MoAb 418 did not recognize mutated rvWF. Because MoAb 418 also failed to capture the plasma vWF from propositus, it means that his second nonmutated allele is not expressed or expressed at a very low level.

Detection by denaturing gradient gel electrophoresis of an Arg1689Cys mutation in a Chinese patient with mild hemophilia A.

OBJECTIVE: To detect gene defects of factor VIII (F VIII) in Chinese hemophilia A patients. METHODS: 3' end of exon 14 of F VIII gene from a mild hemophilia A patient of Chinese origin was amplified by polymerase chain reaction (PCR) and identified mutations by denaturing gradient gel electrophoresis (DGGE) combining with direct sequencing. RESULTS: An upward shift band was detected by DGGE in W381. Direct sequencing demonstrated a C to T transition resulting in substitution of Arg1689Cys within a thrombin activation site of mature F VIII protein, which created a unique a thrombin activation site of mature F VIII protein, which created a unique PstI site in amplified fragment of F VIII. CONCLUSIONS: The association of PCR and DGGE can detect a single base substitution; the Arg1689Cys mutation that inhibited activation of F VIII by thrombin is a molecular defect associated with hemophilia A in W381.

Abnormal proteolytic processing of von Willebrand factor Arg611 Cys and Arg611His.

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.