Jean-Pierre Girma, INSERM U143, 84 Rue du Général Leclerc, 94 276 Le Kremlin Bicêtre cedex, France. Tel.: +33 1 4959 5633; fax: +33 1 4671 9472; e-mail: email@example.com
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.
Thrombotic thrombocytopenic purpura (TTP) is a life-threatening systemic illness potentially fatal in the absence of treatment by plasma therapy. It is characterized by hemolytic anemia, thrombocytopenia and microvascular thrombosis involving several organs. In most cases, TTP is an acquired disease attacking healthy adults [1–3]. Rarely, TTP is hereditary and it is now clearly established to correspond to Upshaw–Schulman syndrome. These rare forms of TTP usually start at birth with hematological symptoms and jaundice followed by a recurrent course, which may progressively involve kidneys and brain and the requirement for extensive plasmatherapy .
The pathogenesis of TTP is still obscure. However, the microvascular thrombi in TTP are rich in platelets and von Willebrand factor (VWF) but relatively poor in fibrin , suggesting a major role of VWF. Also, studies performed in the last 8 years support that a severe deficiency of the VWF-cleaving metalloprotease (ADAMTS13) activity leads to the accumulation of unusually large VWF multimers in plasma and to the spontaneous formation of platelet aggregates in the microvessels, and thus to be responsible for TTP [6,7]. This functional deficiency, as the various TTP forms, may be either acquired via plasma inhibitors or congenital [8,9].
ADAMTS13 consists of 1427 amino acid residues and contains a metalloprotease domain of the reprolysin/adamalysin type, which activity requires both zinc and calcium ions, a disintegrin domain, a first thrombospondin type 1 (TSP1) repeat, a characteristic cysteine-rich and spacer domain, seven additional TSP1 motifs and two CUB domains (Complement components C1r/C1s, Urinary epidermal growth factor, and Bone morphogenetic protein-1) [10–12]. The ADAMTS13 gene was identified by genomic linkage analysis in families affected by congenital TTP . ADAMTS13 gene is located on chromosome 9q34 and spans 29 exons encompassing 37 kb.
Since the first report of ADAMTS13 mutations in seven families with congenital TTP , several studies have supported the cause–effect relationship between ADAMTS13 mutations and Upshaw–Schulman syndrome [14–22]. In addition, expression studies of mutated recombinant ADAMTS13 have confirmed the deleterious effect of some mutations either on ADAMTS13 activity or on the impairment of its secretion [14,15,21,22].
In previous reports [23–25], we described several French patients with familial congenital TTP related to a severe deficiency of ADAMTS13 activity in plasma. In the present study, we investigated six of them and their family for ADAMTS13 mutations. Our study identified 10 candidate mutations (seven missense, one splice and two deletions).
Subjects, materials and methods
Appropriate consent was obtained from all subjects according to the Declaration of Helsinki. A ‘Research and Clinical Innovation Contract’ for this investigation was obtained from the Institutional Review Board of Assistance Publique, Hôpitaux de Paris (France).
Venous blood was collected into 1 : 10 final volume of 3.8% sodium citrate, before any treatment. Platelet-poor plasma (PPP) was obtained by centrifugation at 2500 × g for 20 min. DNA was isolated from peripheral nucleated blood cells by using DNA Isolation Kit for Mammalian Blood (Roche Applied Science, Meylan, France).
TTP families We investigated six unrelated patients with congenital TTP described previously [23–25]. Their clinical features are summarized in Table 1. Briefly, the diagnosis of congenital TTP was based on a recurrent course of microangiopathic hemolytic anemia and thrombocytopenia associated in most cases with visceral ischemia involving kidneys, brain or other organs. All patients were regularly treated by plasmatherapy. Familial history revealed a parental consanguinity in family 1 and some affected siblings in families 1 and 5. In family 1, one affected sibling died in the neonatal period from severe jaundice and anemia. In family 5, the affected sibling presented neonatal hemolysis and thrombocytopenia requiring exchange transfusion, and after several relapses he died at 5 years old with multivisceral thrombotic microangiopathy confirmed at autopsy.
Table 1. Clinical features of the six probands with congenital thrombotic thrombocytopenic purpura
Sex/age at diagnosis of
Onset of the disease hemolysis
Neonatal hemolysis and thrombocytopenia
Neonatal hemolysis and thrombocytopenia
Neonatal hemolysis and thrombocytopenia
Neonatal hemolysis and thrombocytopenia
Neonatal hemolysis and thrombocytopenia
Neonatal hemolysis and thrombocytopenia
Chronic hemolysis (Hb 7 g dL−1) and thrombocytopenia (Plt 43 × 109 L−1)
Chronic hemolysis (Hb 10 g dL−1) and thrombocytopenia (Plt 20 × 109 L−1)
Chronic hemolysis (Hb 7 g dL−1) and thrombocytopenia (Plt 45 × 109 L−1)
Chronic hemolysis (Hb 8 g dL−1) and thrombocytopenia (Plt 27 × 109 L−1)
Hb 12 g dL−1 Plt 200 × 109 L−1 between relapses
Chronic hemolysis (Hb 9 g dL−1) and thrombocytopenia (Plt 26 × 109 L−1)
End-stage renal failure and transplantation at 15 years. Recurrence and return to hemodialysis
Transient diplopia at 11 years. Middle cerebral artery thrombosis at 14 years. Transient amaurosis, retinal ischemia, seizures, coma at 15 years
Coma at 3 and 6 years
Lobster claw hand
Diarrhea, melena and hematemesis
In four families, both parents and the living siblings were investigated. In two cases, only the propositus was tested. Unfortunately, neither plasma nor DNA from the dead siblings had been stored.
Controls Fifty healthy volunteers were used as controls for protease activity and mutation screening of ADAMTS13.
Assay of ADAMTS13 activity by immunoradiometric assay
Measurement of ADAMTS13 activity in plasma was performed as previously described . Briefly, the method relies on the hydrolysis of a constant amount of wild-type recombinant VWF used as substrate by serial dilutions of tested plasma used as ADAMTS13 provider. Plasma samples treated with Pefabloc (2 mm final) for 10 min were serially diluted from 1 : 10 to 1 : 640 in 5 mm Tris–HCl pH 8, 1.5 m urea. Aliquots of 90 µL were preincubated with 10 µL of 100 mm BaCl2 for 5 min at room temperature. This mixture (60 µL) was added into wells of microtitration plates to 40 µL of wild-type recombinant VWF (0.05 IU) previously dialyzed against 5 mm Tris–HCl pH 8, 1.5 m urea. Incubation was performed for 48 h at 37 °C. The proteolysis was stopped by addition of 5 µL of 200 mm EDTA in water. The residual VWF antigen contained in the hydrolysate was then estimated by a two-site immunoradiometric assay (IRMA) as previously described  using the anti-C-ter-VWF MoAb 453 for coating and a pool of 125I-anti-N-ter-VWF MoAbs for staining. A normal pooled plasma (NPP) was arbitrarily defined as containing 100% of ADAMTS13 activity and used as an internal control.
Inhibitor for ADAMTS13 was assayed by measuring the residual ADAMTS13 activity in mixtures of TTP patient plasma and NPP at 1 : 1, 2 : 1 and 3 : 1 v/v ratios after a 1-h preincubation at room temperature.
Mutation screening of ADAMTS13
All 29 exons of the ADAMTS13 gene, including the intron–exon boundaries, were amplified by polymerase chain reaction (PCR) with corresponding intron primers using either Taq polymerase (Invitrogen, Cergy Pontoise, France), the GC-RICH kit (Roche Diagnostics, Meylan, France) for exons 19, 21 and 25 or the FastStart TaqDNA polymerase (Roche Diagnostics) for exons 7, 8 and 12. Sequences of primers used for the amplification of ADAMTS13 exons and intron–exon boundaries are available on request. PCR products were sequenced in both direction with PCR primers and a BigDye Terminator Kit (Applied Biosystems, Les Ulis, France) on a 310 genetic analyzer (Applied Biosystems).
ADAMTS13 activity in plasma
Results of ADAMTS13 activity have already been reported for propositi . Briefly, all six TTP patients demonstrated a constant and severe deficiency of ADAMTS13 activity in plasma (levels <5%) with no inhibitor (Fig. 1). Surprisingly, ADAMTS13 activity was also < 5% in the propositus' sister (17 years old) in family 3 (Fig. 1). She never had obvious manifestations of thrombotic microangiopathy except a neonatal hemolysis/thrombocytopenia treated by exchange transfusions and an isolated mild thrombocytopenia (around 80 × 109 L−1). In tested families, ADAMTS13 activity was > 50% in the healthy siblings as well as in the parents.
ADAMTS13 activity was within the normal range (50–178%) in the 50 controls .
Mutations in ADAMTS13 gene
All 29 exons of ADAMTS13 gene of the six propositi were analyzed by sequencing.
Ten distinct mutations distributed over the gene were discovered (Table 2). Seven missense mutations (237C→G, 607T→C, 803G→C, 1520G→A, 1787C→T, 2272T→C and 2723G→C) were identified in exons 3, 6, 7, 13, 16, 19 and 21, respectively. The nucleotides are numbered from the A of the initiation Met codon. These mutations lead to amino-acid substitutions Ile79Met, Ser203Pro, Arg268Pro, Arg507Gln, Ala596Val, Cys758Arg and Cys908Ser (Table 2). All these mutations are novel except the mutation Arg268Pro, which was previously found in a TTP patient . We identified one mutation G→A at the acceptor splice of exon 12. In addition, two deletions were detected. The first one deleted nucleotides CT at position 3252–3253 in exon 25 leading to a frameshift with a stop codon at amino acid residue 1096. The second one deleted 29 nucleotides at the intron–exon 8 junction (Table 2). No other abnormalities were found in the ADAMTS13 gene of the six propositi. Figure 1 shows pedigrees of the patient families. All the mutations were detected at the compound heterozygous state in the propositi except for family 5, in which the propositus is homozygote for the mutation Ala596Val. When analyzed, parents of the propositi exhibited a heterozygous state for one of the mutations. Interestingly, in family 3 the propositus' sister exhibited an identical genotype as her brother, associated with a severe deficiency of ADAMTS13 activity in plasma. However, the expression of her disease is limited to a mild thrombocytopenia.
Table 2. ADAMTS13 gene mutations identified in six French families with congenital thrombotic thrombocytopenic purpura
Del 29 bp
Acc splice G→A
The presence of mutations was searched for in the gene of 50 healthy controls. Only the substitution Ile79Met was found at the heterozygous state in eight controls.
Figure 2 localizes the position of the distinct mutations along the sequence of ADAMTS13. Missense mutations Ile79Met, Ser203Pro and Arg268Pro are within the metalloprotease domain. Arg507Gln is in the Cys-rich domain and Ala596Val in the spacer domain. Mutations Cys758Arg and Cys908Ser are located in the TSP1 repeats 3 and 5, respectively. The dinucleotide deletion within the exon 25 exchanges Arg1096 for a stop codon in the TSP1-8 repeat and can lead to a truncated protein. The effects of the truncating mutations, G→A of the acceptor splice of exon 12 and deletion of 29 bp in the intron/exon 8 boundary, on the structure of the protein cannot be predicted.
Four single nucleotide polymorphisms associated with an amino acid substitution, Arg7Trp, Gln448Glu, Val900Ala and Ala1033Thr, were identified in our TTP families. These polymorphisms were present in control alleles with variable frequency and were previously reported by other authors [13,14].
In the present study, the specific involvement of ADAMTS13 in the etiology of congenital TTP is further supported by the finding of 10 novel mutations in six unrelated TTP families from France, Haiti and Mauritius.
In accordance with all previous reports [13–22], ADAMTS13 gene mutations found in the probands were identified in both alleles. All but one patient were compound heterozygotes. None of the patients exhibited two null alleles of ADAMTS13 gene, which further suggests that a total deletion of the gene leading to a complete deficiency of ADAMTS13 may be lethal. In all our families, the parents were clinically unaffected, and exhibited ADAMTS13 activity levels in plasma > 50% concordant with their status of heterozygous carriers of one mutant ADAMTS13 allele and confirming the recessive inheritance of congenital TTP. Except in family 1, where the parents are first cousins but are probably not carriers of the same mutation, no consanguinity was reported. However, in the Haitian family 5, the parents were born in the same area and then may have a common ancestor as suggested by the homozygous status of the propositus.
As previously reported [13,14,18,19], a potential correlation between phenotype and genotype also remains very difficult to establish in our patients. First, the various genotypes of the probands were all associated with ADAMTS13 activity levels in plasma < 5%. Second, in spite of a common onset of the disease in the neonatal period (Table 1), the six probands exhibited a large variety of clinical symptoms of TTP, ranging from isolated hematological involvement (patient 2), sometimes associated with a transient proteinuria (patients 3, 4 and 6) to a multivisceral disease involving kidneys and brain and affecting several siblings (families 1 and 5). No relationship between the features of ADAMTS13 mutations (missense or truncating mutations, location on the gene) and severity of the disease can be derived from a comparative analysis of the six families presented here. Furthermore, as illustrated by the siblings of family 3, an identical ADAMTS13 genotype may lead to quite different phenotypes: indeed, although the 14-year-old proband exhibited a chronic hemolysis and thrombocytopenia requiring extensive plasmatherapy, the expression of the disease in his 18-year-old sister has been so far limited to a neonatal hemolysis and a fluctuating mild thrombocytopenia (80 × 109 L−1) requiring no treatment. This symptom variability, even in the same family, further emphasizes a major contribution of other still unidentified genetic and/or environmental factors.
Except for the mutation Arg268Pro already identified by another group , none of the other nine ADAMTS13 mutations identified in the present study has been reported so far. Seven missense and three truncating mutations were found. Interestingly, the combination of mutated ADAMTS13 alleles in the compound heterozygous probands exhibits an association of either two missense mutations (probands 2 and 4) or one missense and one truncating mutation (probands 1, 3 and 6) (Fig. 1). The only homozygote proband (patient 5) carried a missense mutation (Ala596Val) on both alleles (Fig. 1). This heterogeneous panel renders speculation difficult about the final effect of ADAMTS13 gene mutations on the protein in these TTP patients. Mutations may induce an impaired secretion of a potentially active ADAMTS13. This was documented for the mutant R268P-ADAMTS13 by Kokame et al. , by Matsumoto et al.  for mutants R193W-, I673F-, C908Y- and R1123C-ADAMTS13, and by Pimanda et al.  for the truncated protease due to a frameshift mutation after S1381. Also, mutations may produce a normally secreted but non-functional ADAMTS13 as described so far for most of the studied mutants (mutants P475S and Q449stop  as well as mutants H96D, R102C, T196I, R398H, R692C, C951G, C1213Y, R1219W ). However, a cause–effect relationship between ADAMTS13 mutations identified in our patients and their congenital TTP is suggested by several arguments: first, except for mutation Ile79Met, these mutations are not present in 100 control alleles; second, one of them (Ala596Val) is associated with a homozygous status and was also present in two unrelated families; third, almost all of them involve ADAMTS13 domains previously described to contain mutations in TTP patients. Finally mutation Ile79Met also identified at the heterozygous state in eight controls may appear as a polymorphism. However, sequencing the 29 exons of the propositus of family 4 did not allow the detection of any additional mutation but the Arg268Pro mutation on the other allele. Thus, the mutation Ile79Met appears a candidate in participating in the deleterious effect on ADAMTS13 activity in this patient. Such a prevalence of a deleterious mutation in normal individuals has already been observed for Pro475Ser substitution . However, definitive clarification of the causative role of the Ile79Met mutation will require the expression and characterization of the corresponding mutated recombinant ADAMTS13.
As reported in previous studies [13–22], the 10 mutations identified in our TTP patients are not clustered in one region but distributed along the ADAMTS13 gene. In our series, seven mutations are N-terminal to or within the spacer domain. This observation further emphasizes the role of these domains in VWF cleavage by ADAMTS13 that was recently demonstrated by the characterization of carboxy-terminal truncated ADAMTS13 mutants [27,28]. Indeed, ADAMTS13 truncated after the metalloprotease domain, the desintegrin domain, the first TSP1 repeat or the cystein-rich domain, is unable to cleave VWF, whereas addition of the spacer domain completely restores ADAMTS13 activity. These data not only demonstrate that the spacer region is necessary for normal ADAMTS13 activity toward VWF, but also suggest that the more C-terminal TSP1 motifs and CUB domains are dispensable in vitro[27,28]. However, in vivo as well as in vitro, the role of these latter domains on the biological activity of ADAMTS13 was clearly supported by the effect of mutations identified in the C-terminal TSP1 repeats ([13,16,17–19] and the present report) and in the CUB domains [13,16–19,22] of TTP patients.
Beyond the crucial role of ADAMTS13 mutations in the pathogenesis of congenital TTP, this study highlights that VWF cleavage by ADAMTS13 is probably not limited to the role of the ADAMTS13 metalloprotease domain. The absence of mutational hotspots in ADAMTS13 underlines the function of other structural domains in both ADAMTS13 biogenesis and its substrate recognition process . Together, all these studies converge to show that ADAMTS13 mutations found in congenital TTP share several features. Patients with compound heterozygosity are much more frequent than homozygous patients, and no patient with two null alleles has been described suggesting that a complete deficiency of ADAMTS13 may be lethal. Even though missense, nonsense, frameshift or splice mutations have been identified, missense mutations are the most frequent.
The authors thank the following physicians for providing plasma samples as well as clinical and biological data: H. Nivet, S. Cloarec, G. Deschênes (CHU de Tours); E. Fressinaud, F. Mechinaud, M. Hamidou (CHU de Nantes); M. Foulard (CHU de Lille); P. Delattre (Centre Hospitalier de Cayenne); E. Haddad, V. Baudoin, A.L. Lapeyraque, M.A. Macher (Hôpital Robert Debré, Paris); M. Lakhadari (CHU de Gonesse). We are grateful to A. Houllier and B. Gouritin for expert technical assistance.