Jean-Maurice Lavergne

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.

Mutations in the catalytic domain of human coagulation factor IX: Rapid characterization by direct genomic sequencing of DNA fragments displaying an altered melting behavior

Deficiency in coagulation factor IX, a plasma glycoprotein constituent of the clotting cascade, results in hemophilia B, an inherited recessive X-linked bleeding disorder. Some affected individuals, referred to as antigen positive or CRM+, express an inactive factor IX gene product at normal levels and are expected to have natural mutations altering domains of the molecule that are critical for its correct function. The serine protease catalytic domain of activated factor IX, encoded by exons VII and VIII of the gene, is a possible target for such mutations. We designed a strategy allowing rapid analysis of this region through enzymatic amplification of genomic DNA, analysis of the amplification products by denaturing gradient gel electrophoresis, and direct sequencing of the fragments displaying an altered melting behavior. This procedure permitted us to characterize two previously undescribed mutations. Factor IX Angers is a G-to-A substitution generating an Arg in place of a Gly at amino acid 396 of the mature factor IX protein. Factor IX Bordeaux is an A-to-T substitution introducing a nonsense codon in place of the normal codon for Lys at position 411. Moreover, the already described factor IX Vancouver defect was found in three apparently independent families. These results provide further insight into the molecular heterogeneity of hemophilia B. In addition, we demonstrate the usefulness of this rapid screening procedure, which has broad applications in human genetics and can be used as an alternative to RFLP analysis in carrier detection or prenatal diagnosis studies.

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

Summary.  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.

Cross-reacting material in congenital factor VIII deficiencies (Haemophilia A and von Willebrand's disease)

Abstract Using a heterologous specific and precipitating anti human Factor VIII antiserum, the presence of cross-reacting material (C.R.M.) was detected in cryoprecipitate as well as in plasma of 83 patients with Haemophilia A (49 without and 34 with a circulating Factor VIII antibody). The ratio of Factor VIII activity to antigen was lower in known carriers of Haemophilia than in normal women. In 15 patients with von Willebrands disease, C.R.M. was absent or seemed only to be proportional to the amount of biologically active Factor VIII.

Heterogeneity of von Willebrand's disease: Study of 40 Iranian Cases

Summary. Forty Iranian patients with von Willebrands disease were tested for bleeding time, platelet retention to glass beads, ristocetin‐induced platelet aggregation, and assay of factor VIII procoagulant activity (VIII:C), Willebrand factor activity (VIIIRrWF), and factor VIH‐related antigen (VIIIR:AG) by two methods (Laurell and immunoradiometric assay). In 22 cases from 11 families, levels of VIII:C, VIIIR:WF and VIIIR:AG (Laurell) were below 5% and the immunoradiometric assay showed total lack of VIIIR:AG in all cases (sensitivity of the method 0.01%). In 10 of these families, the parents were related, raising the possibility that these patients are homozygous. The occurrence of precipitating antibodies to factor VIII was demonstrated in one of these severe patients. In seven cases from five families the anomaly was less severe, with results of VIII:C between 5 and 17%. In 11 cases from six families VIII:C was normal or moderately decreased, contrasting with lower levels of VIIIR:WF and VIHR:AG. The presence of an abnormal factor VHI/von Willebrand factor protein was assessed by double‐cross immunoelectro‐phoresis and gel filtration.

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.

Precipitating antibodies in von Willebrand's disease

WE report the presence of precipitating antibodies in three patients with von Willebrands disease (VWD), an inherited deficiency of factor VIII (anti-haemophilic factor, AHF). To our knowledge this is the first description of patients with complete biological and immunological deficiency of a plasma-clotting protein responding to replacement therapy by the production of antibodies with properties indistinguishable from those elicited in animals. AHF is a high molecular weight (> 106) glycoprotein complex1 associated with two biological activities; one necessary for normal clotting (VIII: C) and the other for normal platelet function (Willebrand factor, VIIIR: WF). Specific antigenic determinants (VIIIR: AG) can be reliably quantified using heterologous antisera2,3. Any concept of the structure or the molecular genetics of AHF needs to incorporate the clinical and immunological findings in the two congenital disorders associated with AHF deficiency: haemophilia A and VWD. The former is a sex-linked deficiency of VIII: C alone whereas the latter represents a deficiency of all three parameters of the AHF complex and is inherited as an autosomal trait. Non-precipitating antibodies to AHF, acquired after replacement therapy, are common in haemophilia A (ref. 4) but only one case has been reported in VWD5,6.

Immunoradiometric Assay of Factor VIII: Coagulant Antigen using Four Human Antibodies. Study of 27 Cases of Haemophilia A

Summary. Immunoradiometric assay (IRMA) of VIII:C antigen was performed using either IgG or monovalent Fab fragments from four antibodies arisen in polytransfused haemophilia A patients (titre between 100 and 1500 U/ml). Using IgG isolated by a solid or a liquid phase system, only the high titre (1000 U/ml) antibodies could be used for IRMA, with a sensitivity of 0.2% VIII:CAg. Using Fab fragments isolated by liquid phase, high and low (150 U/ml) titre antibodies could be used and the IRMA was significantly improved with a 10‐fold higher sensitivity. The affinity of the antibodies for VIII:CAg, studied by displacement curves using a modification of the IRMA, was found not to depend upon the titre of the antibody. Comparative levels of VIII:C and VIII:CAg in 27 cases of haemophilia A emphasize the heterogeneity of this disorder, two types of severe and three types of mild haemophilia being observed.

Defects in type IIA von Willebrand disease: a cysteine 509 to arginine substitution in the mature von Willebrand factor disrupts a disulphide loop involved in the interaction with platelet glycoprotein Ib‐IX

Summary Type IIA von Willebrand disease (vWD) is characterized by the loss of high and intermediate weight multimers of von Willebrand factor (vWF) from plasma. The 3’end of exon 28 in the vWF gene from four type IIA vWD patients was amplified by the polymerase chain reaction, cloned and sequenced. Sequencing identified two potential missense mutations resulting in the amino acid substitutions Arg 834Gln and Glu 875Lys in the mature vWF subunit within an area of vWF where mutations in type IIA vWD have been reported. Neither of these amino acid substitutions was found in over 100 normal alleles tested by allele specific oligonucleotide hybridization. A polymorphism (Val 802Leu) was identified in another patient. Other areas of exon 28 were analysed by denaturing gradient gel electro‐phoresis (DGGE) and DNA from one patient demonstrated an irregular DGGE pattern on the 5’end of the exon. Sequencing demonstrated an amino acid substitution of an arginine for cysteine at position 509 adjacent to an area of vWF where defects associated with type IIB vWD have been found. This substitution was not found in 100 normal chromosomes tested by restriction enzyme digestion. The Cys 509Arg substitution eliminates an intramolecular disulphide bridge formed by Cys 509 and Cys 695 which is important to maintain the configuration of vWF functional domains that interact with platelet glycoprotein Ib‐IX.

Variants of von Willebrand's disease. Demonstration of a decreased antigenic reactivity by immunoradiometric assay.

Abstract Factor VIII related antigen (VIIIR:AG) was studied by immunoradiometric assay (IRMA) in fourteen cases of atypical von Willebrands disease (vWd) using two different antibodies (rabbit and goat). The dose response curves showed two types of abnormalities. The decrease in maximal bound radioactivity (plateau) was consistent in all cases whereas the lack of parallelism of the linear portion of the curves only occurred in five of them. These findings were more evident with the rabbit than with the goat antibodies. However with both antibodies, the level of VIIIR:AG measured by IRMA was always lower than that measured by Laurell. These data indicate a reduced and/or abnormal antigenic reactivity in “variants” of vWd. Since the same abnormalities of IRMA are found in the supernatant of cryoprecipitate, this technique may reflect the degree of polymerization of the Factor VIII/von Willebrand Factor protein.