Florence Mathonnet

Activated protein C sensitivity ratio in pregnant women at delivery

We studied activated protein C sensitivity ratio (APC‐SR), factors V and VIII activity and von Willebrand antigen in control women, women using oral contraceptives, and pregnant women at delivery. The mean APC‐SR of 2.4 in pregnant women was significantly lower than the mean APC‐SR value of 3.5 for both the other groups and 45% of pregnant women had a ratio below the 5th percentile of the control group. None of them carried the R506→Q mutation. This decreased ratio at delivery appeared to be connected, at least in part, with increased VIII activity. Thus, APC‐SR at delivery should not be used to detect APC resistance.

Identification of five novel mutations in the factor XI gene (F11) of patients with factor XI deficiency.

Factor XI (FXI) deficiency is an inherited autosomal recessive disorder associated with bleeding of variable severity. However, many cases of dominant disease transmission have been recently described. This disorder is rare in the general population, whereas it is commonly found in individuals of Ashkenazi Jewish ancestry. This study reports the molecular genetic analysis of FXI deficiencies in 11 unrelated families of different origin. Five novel mutations have been identified. Severe FXI deficiency of two unrelated patients resulted from two novel mutations: one deletion (960–961delGT) in exon 9 predicting a frameshift, and a Ser-4Leu mutation located in the signal peptide. In addition, three novel missense mutations associated with partial FXI deficiency have been identified: Cys122Tyr, Glu297Lys and Glu579Lys.

Three new cases of dysfibrinogenemia: Poissy III, Saint-Germain I and Tahiti.

In order to identify unknown mutations, the FAMA method was used to rapidly screen the fibrinogen chain genes in individuals with dysfibrinogenemias. Chemical cleavage at mismatches on heteroduplexes DNA end-labeled with strand-specific fluorescent dyes reliably detects sequence changes in DNA fragments of up to 1.5 kb and locates them precisely. This method was successfully used for the detection of three new dysfibrinogenemias: Poissy III, Tahiti (heterozygous Aalpha Arg16His) and Saint-Germain I (heterozygous AalphaGly12Val). The mutations were confirmed by dideoxy sequencing.

Role of factor VIII on activated protein C resistance ratio in inflammatory diseases

Activated protein C resistance ratio (APC‐Rr), factor VIIIC (FVIIIC) and plasma fibrinogen levels were studied in patients with inflammatory disease. The patient mean APC‐Rr was significantly lower than in the control group. This decreased ratio in inflammatory diseases appeared to be connected with increased FVIIIC. Moreover, supplementation of plasmas with purified factor VIII decreased the APC‐Rr in plasma from both groups, and suppressed the difference between groups. These data suggest that factor VIIIa and factor Va compete for protein C‐catalysed cleavage. Ratios were identical in both groups when FVIIIC level was lowered by dilution in factor V deficient plasma.

A case of afibrinogenemia associated with A-alpha chain gene compound heterozygosity (HUMFIBRA c.[4110delA]+[3200+1G>T]).

The clinical features and molecular biology data of a case of afibrinogenemia are reported. The propositus is a 14-year-old girl who suffered several bleeding manifestations that were successfully treated with fibrinogen infusion. The afibrinogenemia results from compound heterozygosity for two mutations on the Aα chain gene (c.[4110delA]+[3200+1G>T]). The first mutation is a novel frameshift mutation inherited from her father. The second is a previously described Aα chain gene splice junction mutation inherited from her mother. Neither of the parents fulfills the criteria for hypofibrinogenemia.

Fibrinogen Poissy I: a new case of the A alpha Arg 16His fibrinogen variant.

A fibrinogen variant was identified in a patient with disseminated intravascular coagulation and in one member of her family. Coagulation studies showed marked prolongation of both the thrombin and reptilase times and discrepancy was noted between the levels of plasma fibrinogen, determined by a kinetic vs immunological determination or light scattering assay. Studies on purified fibrinogen revealed an impaired release of fibrinopeptides by thrombin. DNA sequencing revealed a heterozygous A to G point mutation in exon 2 of the A alpha chain, which substituted Arg for His at position 16. This mutation creates a Nla III cleavage site which was used to confirm the mutation.

Fibrinogen Saint-Germain I: a case of the heterozygous Aα GLY 12 → VAL fibrinogen variant

A fibrinogen variant was suspected based on the results of routine coagulation tests in a 2-year-old asymptomatic child. Coagulation studies showed marked prolongation of both the thrombin and reptilase times, and discrepancy was noted between the level of plasma fibrinogen as measured by a kinetic versus immunological determination. Family studies revealed that the father beared the same abnormality. Studies of purified fibrinogen revealed an impaired release of both fibrinopeptides by thrombin. Fibrin monomer polymerization and fibrin stabilization were normal. DNA sequencing revealed a heterozygous G → T point mutation in exon 2 of the gene coding for the Aα chain, which substituted a Gly for Val at position 12. Although the mutation is the same as in fibrinogen Rouen, fibrinogen Saint-Germain I shows a different fibrinopeptide release pattern and a mild factor V deficiency.

Characterization of combined factor VII and factor XI deficiencies.

Constitutive combined coagulation factor deficiencies are rare genetic bleeding disorders. Most of the reported cases comprise combined deficiency of vitamin K-dependent clotting factors and combined deficiency of factor V and factor VIII. These disorders are related to post-translational modifications defects as in gamma carboxylase or vitamin K epoxide reductase mutations, and to defective intracellular trafficking by disruption of LMAN1 and MCFD2 genes [1]. Only one family with a combined factor VII and factor XI deficiency has been previously reported [2]. It is believed to result from by chance inheritance of two distinct defects [1]. We report two new cases of combined FXI and FVII deficiencies. These two factors are not highly homologous, they are both synthesized in the liver, FXI is not vitamin K-dependent, the genes are located on distinct chromosomes (chromosome 4 and 13 for FXI and FVII, respectively), and there is no obvious post-translational shared modification, nor a gene regulatory element that could result in a combined defect limited to factors XI and VII. However, as FXI sequencing of the first individual DNA did not show any mutation, it was initially wondered if a common mechanism could explain the combined defects. Complete sequencing of both genes was undertaken to rule out a coincidence. Routine coagulation studies were performed by established procedures on an automated analyzer STAR (Diagnostica Stago, Asnières France). FXI antigen level (FXI:Ag, normal range 70–150 U dL) was assayed by ELISA based on goat anti-human FXI polyclonal antibodies and a peroxidase-conjugated IgG (Cedarlane Laboratories Ltd, Ontario, Canada). For the propositus, the purified DNA was used for amplification by polymerase chain reaction (PCR) of all the exons of the FVII and FXI genes with primers encompassing the intron-exon boundaries. For other affected members, when available, only the relevant exons were analyzed. Dideoxy sequencing was performed with the DYEnamic ET Terminator Cycle sequencing kit (Amersham biosciences, Piscataway, NJ, USA). The first propositus is a 40-year-old male with schizophrenia. He has no bleeding symptomatology, but minor bleeding of untreated haemorrhoids. Routine coagulation tests found a prolonged activated partial thromboplastin time and PT (Prothrombin Time), and factor assays found a partial combined FVII and FXI defects. He has no circulating anticoagulant. Antigen assays were available for both FVII and FXI (see Table 1). His brother has a partial FVII defect (40 U dL) and a normal FXI activity, but plasma and DNA were not available for further antigen and gene analysis and clinical description is not reliable. The second individual is a 32-year-old woman. At her first pregnancy she exhibited bleeding complications after delivery. At that time an isolated partial FVII deficiency was found. She underwent several uncomplicated dental extractions and a second delivery with FVII concentrate prophylaxis. She had no other personal or familial bleeding history. She was referred to our hospital a few years later for coagulation investigation. Coagulation tests results are given in Table 1. A combined partial FXI and FVII defect was found in the propositus and the two children. Comparison of antigen and activity values (90 U dL and 30 U dL respectively) indicated that the FXI variant is secreted but not active, whereas the FVII variant was not secreted (FVII Correspondence: Philippe de Mazancourt, Laboratoire de biologie, CHI Poissy-St-Germain, F78303 Poissy France. Tel.: +33 1 39 27 47 42; fax: +33 1 47 10 79 23; e-mail: philippe.de-mazancourt@rpc.aphp.fr