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.

von Willebrand disease (vWD) type IIB is characterized by an increased reactivity of von Willebrand factor (vWF) with platelets and a lack of large multimers. Exon 28 of the vWF gene encodes for functional domains involved in the binding of vWF to GPIb, and it is presumed that the defects in type IIB vWD lie within or adjacent to these functional domains. We screened overlapping DNA fragments generated by the polymerase chain reaction (PCR) that spanned the 1,379 bp of exon 28 of a type IIB vWD patient using denaturing gradient gel electrophoresis (DGGE). To increase the power of DGGE to detect base changes, we used the PCR to attach a G + C-rich sequence. In the type IIB patient, a DNA fragment at the 5′ end of exon 28 demonstrated homoduplex and heteroduplex complexes after DGGE, a pattern characteristic of heterozygous genes after melting and reannealing during the PCR. Sequencing of the cloned insert from the patient showed a duplication of an ATG in one gene coding for a Met at amino acids 540 to 541 in the mature vWF subunit. This duplication leads to three consecutive methionines in the patient's sequence. The duplicated Met resides within a disulfide bond loop proposed to be important in the function of the GPIb binding domain of vWF. The patient's nephew, who also has type IIB vWD, showed the same duplicated codon, linking the defect to the abnormal phenotype in this family. These nucleotide changes were not found in 100 chromosomes analyzed either by DGGE or hybridization with an allele specific oligonucleotide containing the duplicated ATG codon. In addition, the same oligonucleotide hybridized only to DNA from type IIB vWD individuals and not to DNA from normal members of the family. Therefore, we conclude that this duplicated Met modifies the GPIb binding domain of vWF and causes type IIB vWD in this family.

No abstract

No abstract