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Prepublished online as a Blood First Edition Paper on October 17, 2002; DOI 10.1182/blood-2002-08-2399.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Children's University Hospital,
Hamburg-Eppendorf; Lab Association Prof Arndt and Partners, Coagulation
Laboratory, Hamburg; Children's University Hospital, Magdeburg;
Children's University Hospital, Muenster; Children's University
Hospital, Jena; Children's University Hospital, Ulm; Children's
University Hospital, Munich; Children's University Hospital, Kiel; and
Medical School Hannover, Children's Hospital, Hannover,
Germany.
Thrombotic thrombocytopenic purpura (TTP) is caused by the
persistence of the highly reactive high-molecular-weight multimers of
von Willebrand factor (VWF) due to deficiency of the specific VWF-cleaving protease (VWF-CP) ADAMTS13, resulting in microangiopathic disease. The acquired form is caused by autoantibodies against VWF-CP,
whereas homozygous or compound heterozygous mutations of ADAMTS13 are
responsible for recessively inherited TTP. We investigated 83 children
with hemolytic or thrombocytopenic episodes with or without additional
neurologic symptoms or renal failure. The presumed diagnosis was
chronic idiopathic thrombocytopenic purpura (ITP; n = 50), TTP
(n = 8), hemolytic uremic syndrome (HUS; n = 24), and
Evans syndrome (n = 1). A severe deficiency of VWF-CP ( Thrombotic thrombocytopenic purpura (TTP) is a
life-threatening microangiopathic disorder. Before the introduction of
plasma exchange therapy, the mortality rate was extremely
high.1 It is related to hemolytic uremic syndrome (HUS)
but can be differentiated from HUS by the more general involvement of
many organs compared with the more localized manifestation of HUS in
the kidneys, although some overlap of symptoms may occur. It was first
described by Moschcowitz in 1924,2 who presented the case
of a previously healthy 16-year-old girl with fever, pallor, petechiae,
acute renal failure, and paralysis who died 1 week after falling ill. Postmortem histology revealed hyaline platelet thrombi in almost every
organ. Reports on similar cases with a congenital onset or familial
manifestation, compared with first manifestation in adolescence and
adulthood, indicated the presence of an inherited and an acquired form
of the disease, respectively.3-9 Although the beneficial
effect of plasma infusions suggested a deficiency of an essential
plasma factor in the patients,4,6 the nature of the
disease-causing factor remained obscure until Moake et al detected
unusually large von Willebrand factor (VWF) multimers (ULVWFMs) in the
plasma of 4 patients during their remission from TTP.10
Relapses of TTP were accompanied by a decrease of these ULVWFMs,
suggesting consumption by binding to aggregating platelets. The authors
proposed a defect in the processing of the very large VWF multimers
after synthesis and secretion by endothelial cells, making the patient
susceptible to repeated relapses. This processing defect was identified
as deficiency of a VWF-specific metalloprotease,11,12 which cleaves VWF at a defined position in the VWF A2 domain between Tyr1605 and Met1606.13 A partial amino acid sequence was
determined independently by 3 groups in 2001.14-16 Based
on this information, a VWF-cleaving protease (VWF-CP) was identified as
a member of the ADAMTS family of metalloproteases. The cDNA sequence
was determined,17 and a database search revealed the
structure of the gene on chromosome 9q34. Parallel to the biochemical
approach, the responsible gene locus was mapped to 9q34 by linkage
analysis and identification of specific mutations in
ADAMTS13.18 Review of the literature reveals very similar courses of congenital TTP in different patients over the years. The reported cases display the full spectrum of symptoms, though not always concurrent at the same time. Occasionally, thrombocytopenia can be the only symptom, but the full-blown clinical picture seems to manifest in later childhood or after puberty and to
correlate with certain episodes of infection, stress, alcohol consumption, or pregnancy.3-9 Therefore, especially in
early childhood, oligosymptomatic forms may be more frequent,
complicating the differential diagnosis. To assess the clinical picture
of VWF-CP deficiency in cases of chronic relapsing thrombocytopenia and
hemolytic anemia, we determined VWF-CP activity in children with the
presumed diagnosis of ITP, TTP, HUS, and Evans syndrome and carried out
mutation analysis of the ADAMTS13 gene to establish the
molecular background and the spectrum of mutations in our population.
Patients
All patients or their parents were informed about the nature of this
study and their consent was obtained according to the Declaration of
Helsinki. Approval for this investigation was obtained from the
institutional review board of the Children's University Hospital
Hamburg-Eppendorf.
Assay of VWF-CP by the collagen-binding method
Inhibitor assay Detection of inhibitory activity in the sample was carried out by using a screening test, by mixing pooled normal plasma and patient's plasma (1:1), and by incubating for 30 minutes at 37°C. Thereafter the mixture was diluted 1:10 in Tris/urea, pH 8.0, and processed further as the other plasma samples (see "Assay of VWF-CP by the collagen-binding method").Molecular studies DNA was isolated from peripheral nucleated blood cells by standard methods. Polymerase chain reactions (PCRs) were carried out in the T-Gradient and Trio Thermal Cycler, respectively (Biometra, Göttingen, Germany). Direct sequencing was done by means of the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit on an ABI Prism 310 or an ABI 377 (ABI, Foster City, CA) using either PCR primers or additional internal primers (Table 1). Sequencing was carried out on both strands. Mutations were confirmed by an independent method such as the restriction enzyme digest (Table 2). If not otherwise stated, in the case of using kits, we followed the instructions of the provider.
Mutation screening of the VWF-A2 domain Besides VWF-CP deficiency itself, TTP could theoretically also be caused by an intrinsic resistance of VWF to proteolysis. However, naturally occurring respective VWF mutations have not been reported to date. We analyzed the VWF A2 domain of all patients with HUS and of 2 patients with TTP for whom no VWF-CP data were available, for possible VWF cleavage site mutations by PCR amplification of VWF exon 28 and direct sequencing as reported previously.20Mutation screening of ADAMTS13 All coding exons and flanking intron sequences of the ADAMTS13 gene were amplified by PCR using either published primers18 or primers chosen from the published sequence (GenBank accession numbers: AL158826, AC002325; Table 1). All PCR products were sequenced directly using an ABI 310 or an ABI 377 automatic sequencer. In the case of a dinucleotide deletion and a single nucleotide insertion, respectively, PCR products were also sequenced after cloning.
VWF-CP Data obtained from the VWF-CP assay are illustrated in Figure 1. Among the 24 patients with HUS, none had VWF-CP activities below 62% (range, 62%-185%; median, 103%) Among the patients with the presumed diagnosis of ITP, 2 had VWF-CP deficiency with activities less than 2% of normal. However, because both also had mild Coombs-negative hemolytic anemia, the former diagnosis was changed to possible TTP. All other ITP patients had values of 33% or higher (range, 33%-175%; median, 59%) All tested patients with the diagnosis of TTP had VWF-CP values of 5% or less and, except one, even below the limit of detection at 2% at least at one or more occasions (Figure 1).
The VWF collagen-binding assay is relatively easy to perform and is
faster than VWF multimer analysis for determination of VWF-CP activity.
However, the action of VWF-CP on VWF is better visualized by the
specific VWF cleavage pattern. VWF-CP causes loss of large VWF
multimers and generates the typical triplet structure of plasma VWF
oligomers. (Actually, a quintuplet structure with inner subbands is
present that can be visualized by high-resolution gels.)22
This triplet structure is absent in native TTP plasma, in untreated
rVWF, and, accordingly, after incubation of rVWF with VWF-CP-deficient
plasma. The appearance of pronounced triplets after mixing either
VWF-CP-deficient plasma or rVWF with plasma from a patient deficient
in VWF but with normal VWF-CP illustrates the specific action of the
protease on VWF (Figure 2).
Molecular studies VWF A2 domain. In the patients with HUS and in 2 patients with TTP for whom no VWF-CP data were available, we analyzed VWF exon 28 that includes the sequence encoding the VWF A2 domain with the specific VWF cleavage site. No mutations in this region were detected that could possibly provide resistance against the action of VWF-CP. ADAMTS13 mutations.
The complete coding sequence of ADAMTS13 and flanking intron
sequences were analyzed by direct sequencing of PCR products for
mutations that could cause VWF-CP deficiency. Altogether, 8 different
mutations were identified on 14 disease alleles in 7 patients (Table
3; Figure
3). One patient had the
former diagnosis of ITP, another had the diagnosis of Evans syndrome,
and 5 patients had a diagnosis of TTP. No mutations were detected in 4 TTP patients with VWF-CP inhibitors.
Five different molecular defects were truncating mutations, suggesting a causative nature, 3 were candidate missense mutations. Only 1 patient was homozygous, whereas the remaining 6 patients were compound heterozygotes for 2 mutations each. One mutation (4143insA) is particularly frequent and was found in 4 unrelated German patients. One missense mutation (1058C>T; Pro353Leu) and one nonsense mutation, (2728C>T; Arg910Xaa), respectively, were identified in 2 patients each, whereas 6 mutations were confined to single families. The 695T>A in a Turkish patient (05 I-1) is located in exon 7 and predicts the exchange of leucine 232 to glutamine (Leu232Gln) in the metalloproteinase domain. Homozygosity for the mutation and an intragenic haplotype suggests consanguinity. The clinical course of this patient must be considered as severe due to renal infarction and to neurologic sequelae following stroke, and the need for continuous replacement therapy by fresh-frozen plasma (FFP). The 788C>G is located in exon 7 and predicts the exchange of serine 263 to cysteine (Ser263Cys) in the metalloproteinase domain. It is found in patient 01 II-1 in combination with 4143insA, a truncating single base insertion in exon 29. He inherited Ser263Cys from his father and 4143insA from his mother (Table 3). To date the only symptoms were mild hemolysis and thrombocytopenia. The 1058C>T is located in exon 9 and predicts the exchange of proline 353 to leucine (Pro353Leu) in the disintegrin domain. This mutation was found in 2 unrelated patients, both living in the same region in Northern Germany at the Denmark border. Patient 03 I-1 was also compound heterozygous for the nonsense mutation 2728C>T (Arg910Xaa). She was misdiagnosed with Evans syndrome and died from her disease at the age of 3 years. The correct diagnosis was made from archived DNA, 8 years after her death. Patient 10 II-1, with a clinically benign course and VWF-CP residual activity between less than 2% and 6% of normal, had 4143insA as second mutation, which he inherited from his father (Table 3). The 1169G>A in exon 10 (Trp390Xaa) and an additional truncating mutation, a dinucleotide deletion, in exon 20 (2549-2550delAT) were identified in patient 08 II-1. Trp390Xaa was inherited from the father and 2549-2550delAT from the patient's mother (Table 3). The patient had a severe clinical course with stroke episodes and she is mentally retarded. She is on continuous replacement therapy with FFP. VWF-CP values were not available. The 3100A>T in exon 24 (Arg1034Xaa) and 4143insA were detected in patient 04 II-1. She had severe clinical symptoms including neurologic and renal involvement. Her VWF-CP was less than 2% of normal. She is on continuous replacement therapy with a virus-inactivated commercial plasma preparation. 4143insA was inherited from the father, and Arg1034Xaa from the patient's mother (Table 3). The patient's unaffected brother is heterozygous for Arg1034Xaa. In all investigated cases, asymptomatic family members who were heterozygous for one of the mutations displayed VWF-CP values corresponding to the heterozygous state and were clinically not affected (Table 3). By screening of 100 normal alleles none of the mutations was detected in the controls.
Although reliable incidence data are not available, TTP is a rare disease in childhood. Our study, however, points to the possibility of underdiagnosis in the past. In our cohort, 3 patients were misdiagnosed as having Evans syndrome and ITP, respectively. It is of interest that the child with TTP reported by Schulman also had a previous diagnosis of ITP.4 These cases illustrate that oligosymptomatic forms may occur with the possibility of misdiagnosis. However, most probably even these patients may eventually suffer from severe TTP episodes when precipitating factors trigger the pathophysiologic cascade that has recently been elucidated. To identify such patients and to differentiate between the acquired and the hereditary form is of crucial importance for immediate care and adequate therapy in acute phases of the disease. Today this can be reliably done by assessing the VWF-CP activity, by screening for VWF-CP inhibitors, and recently, by performing mutation analysis of the ADAMTS13 gene. All mutations reported to date were confined to single families and most of them were missense mutations.18 None of the ADAMTS13 gene mutations that we identified in our patients has been described before. In contrast to the first report,18 we found a higher rate of truncating mutations. One mutation (4143insA) is particularly prevalent in our population and was found in 4 compound heterozygous patients. Only 3 mutations were missense mutations that can only be regarded as candidates, because, to date, expression studies are pending. However, one of them (Leu232Gln) was found in homozygous form, and another one (Pro353Leu) was found in 2 unrelated patients. Furthermore, none of the 8 different mutations was detected among 100 alleles of healthy controls. A correlation between phenotype and genotype of congenital TTP is hard to establish. The patient's history suggests that precipitating factors such as infections have more impact on the severity of TTP episodes than the genotype. Due to the complex mutation spectrum in congenital TTP, proof of a genotype-phenotype correlation would require a large number of patients. Although the final events in TTP and childhood HUS are comparable, the initiating triggers are different. Not a single patient among our 24 with HUS displayed VWF-CP values below 62% of normal, not even those with "atypical" HUS. This agrees with the reports of others,23,24 although contradictory observations were also published.25 However, in conclusion, it seems that severe VWF-CP deficiency with values below 5% of normal is indicative of TTP rather than HUS. Although theoretically possible, we could not detect mutations of VWF that would provide resistance against the proteolytic action of VWF-CP in the patients with HUS. VWF-CP resistance does not seem to contribute significantly to the pathomechanism of HUS. Two different forms of childhood TTP were observed among our patients. Four patients had the acquired form, most probably caused by autoantibodies against the protease. The inconsistent detection of VWF-CP inhibitors even during longer periods of severe VWF-CP deficiency in these patients complicates the correct classification, which is crucial for the adequate treatment. Possibly, the Bethesda method for the identification of VWF-CP antibodies is not sensitive enough to detect low titer, though effective, inhibitors. Patients with an inhibitor need a more aggressive therapy, including plasma exchange and immunosuppression, whereas patients with the hereditary form can be treated by VWF-CP replacement therapy alone. Due to the long half-life of VWF-CP of 2 to 3 days26 and the clinical efficacy even at low VWF-CP values, plasma infusion every 2 weeks seems sufficient. This is also confirmed by the clinical response to such prophylaxis in some of our patients.27,28 In all of our patients without an inhibitor we identified ADAMTS13 gene mutations on both alleles, which is in accordance with the autosomal recessive inheritance of inborn VWF-CP deficiency, whereas no mutation was found in patients with a significant inhibitor detected at least at one time point. Although exact data are lacking, it seems that childhood TTP is more often caused by hereditary VWF-CP deficiency, which would agree with the lower incidence of autoimmune disease in children compared with adults. Given the impact of the correct classification of TTP with respect to therapy and genetic counseling, we propose molecular genetic testing in children with severe VWF-CP deficiency.
A second report on ADAMTS13 gene mutations has been published during revision of this manuscript.29
Submitted August 6, 2002; accepted October 2, 2002.
Prepublished online as Blood First Edition Paper, October 17, 2002; DOI 10.1182/blood-2002-08-2399.
R.S. and U.B. contributed equally to this study.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Reinhard Schneppenheim, University Hospital Hamburg-Eppendorf, Department of Pediatric Hematology and Oncology, Martinistr 52, D-20246 Hamburg, Germany; e-mail: schneppenheim{at}uke.uni-hamburg.de.
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J. E. Pimanda, A. Maekawa, T. Wind, J. Paxton, C. N. Chesterman, and P. J. Hogg Congenital thrombotic thrombocytopenic purpura in association with a mutation in the second CUB domain of ADAMTS13 Blood, January 15, 2004; 103(2): 627 - 629. [Abstract] [Full Text] [PDF]
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 J. Caprioli, F. Castelletti, S. Bucchioni, P. Bettinaglio, E. Bresin, G. Pianetti, S. Gamba, S. Brioschi, E. Daina, G. Remuzzi, et al. Complement factor H mutations and gene polymorphisms in haemolytic uraemic syndrome: the C-257T, the A2089G and the G2881T polymorphisms are strongly associated with the disease Hum. Mol. Genet., December 15, 2003; 12(24): 3385 - 3395. [Abstract] [Full Text] [PDF]
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K. Soejima, M. Matsumoto, K. Kokame, H. Yagi, H. Ishizashi, H. Maeda, C. Nozaki, T. Miyata, Y. Fujimura, and T. Nakagaki ADAMTS-13 cysteine-rich/spacer domains are functionally essential for von Willebrand factor cleavage Blood, November 1, 2003; 102(9): 3232 - 3237. [Abstract] [Full Text] [PDF]
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X. Zheng, K. Nishio, E. M. Majerus, and J. E. Sadler Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13 J. Biol. Chem., August 8, 2003; 278(32): 30136 - 30141. [Abstract] [Full Text] [PDF]
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| Copyright © 2003 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
5%) was
found in all investigated patients with TTP and in none of those with
HUS. Additionally, 2 of 50 patients with a prior diagnosis of ITP were
deficient for VWF-CP. Antibodies against VWF-CP were found in 4 children. Mutation analysis of the ADAMTS13 gene in the
patients deficient in VWF-CP by direct sequencing of all 29 exons
identified 8 different mutations, suggesting the hereditary form of TTP
in 1 patient with ITP, in the patient with Evans syndrome, and in 5 of
the 8 patients with TTP. The phenotype of TTP in childhood can
be rather variable. Besides the classical clinical picture,
oligosymptomatic forms may occur that can delay the identification of
patients at risk.
(Blood. 2003;101:1845-1850)
80°C. After thawing it was equilibrated with
1.5 M urea and used as the substrate for the VWF-CP. Plasma of a
patient with severe von Willebrand disease (VWD) type 3 and normal
VWF-CP served as a control. Digestion of rVWF (final concentrations,
0.6-0.9 U/mL) by VWF-CP and the collagen-binding assay of the digested
samples was performed as described.
an alternative assay for detection of von Willebrand factor multimers.
Thromb Haemost.
1988;60:133-136