Inherited Thrombotic Thrombocytopenic Purpura in Children Wolf Achim Hassenpflug, MD1 Ulrich Budde, MD2 Reinhard Schneppenheim, MD, PhD1 1 Department of Pediatric Hematology and Oncology, University
Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 Medilys Laborgemeinschaft mbH, Coagulation Laboratory, Hamburg, Germany
Sonja Schneppenheim, MD2
Address for correspondence Reinhard Schneppenheim, MD, PhD, Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany (e-mail: [email protected]).
Semin Thromb Hemost
Abstract
Keywords
► ADAMTS-13 ► Upshaw–Schulman syndrome ► thrombotic thrombocytopenic purpura ► von Willebrand factor ► molecular genetics
Congenital thrombotic thrombocytopenic purpura (TTP) or Upshaw–Schulman syndrome is caused by homozygous or compound heterozygous mutations in the ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) gene. We investigated 30 patients with congenital TTP and analyzed clinical data and underlying ADAMTS-13 mutations. All patients showed virtually no ADAMTS-13 activity in plasma. Individual disease burden ranged from mild courses with rare episodes of mild thrombocytopenia to severe courses with chronic kidney disease and central nervous system (CNS) lesions. Two patients died due to complications of TTP. If initiated in a timely manner, plasma transfusions offer a reliable treatment to prevent organ damage. We identified 30 different causative mutations in the ADAMTS13 gene. Our data do not support the idea of a tight correlation between ADAMTS-13 genotype and severity of disease. The type and magnitude of exogenous triggers for acute bouts of TTP as well as endogenous individual factors participating in the inflammatory response likely represent the foremost determinants of individual clinical courses. Future developments should aim at improving early diagnosis of TTP. To improve feasibility of prophylaxis and treatment of congenital TTP, recombinant ADAMTS-13 therapeutics are highly anticipated.
Thrombotic thrombocytopenic purpura (TTP) is a rare lifethreatening disorder that is caused by congenital or acquired deficiency of the von Willebrand factor-cleaving protease ADAMTS-13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13).1 ADAMTS-13 cleaves von Willebrand factor (VWF) at a specific cleavage site in the A2 domain between residues Thr1605 and Met1606.2 Lack of ADAMTS-13 activity results in the persistence of ultra-large multimers of VWF (ULVWF) in plasma (►Fig. 1). Under conditions of high shear flow ULVWF can extend to long fibers that expose numerous platelet binding sites to which
Issue Theme An Update on the Thrombotic Microangiopathies Hemolytic Uremic Syndrome (HUS) and Thrombotic Thrombocytopenic Purpura (TTP); Guest Editors, Magdalena Riedl, MD, Dorothea Orth-Höller, MD, and Reinhard Würzner, MD, PhD.
platelets readily adhere. Typical triggers of episodic TTP bouts such as infections or pregnancy, increase VWF plasma levels and stimulate agglutination of ULVWF and platelets. As a result, TTP patients suffer from microangiopathic hemolytic anemia, thrombocytopenia, fever, renal failure, and central nervous system (CNS) impairment. These symptoms had come to be known as the classical pentad of TTP. The first description of a TTP case has been ascribed to Eli Moschcowitz.3 In 1924, he reported on a previously healthy 16-year-old girl, who presented with fever, hemolytic anemia, and thrombocytopenia and who
Copyright © by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0034-1376152. ISSN 0094-6176.
Inherited TTP in Children
Hassenpflug et al.
Fig. 1 von Willebrand factor (VWF) multimers in Upshaw–Schulman syndrome (USS)/thrombotic thrombocytopenic purpura (TTP) patients. Plasma samples of USS/TTP patients and normal control plasma (NP) were separated by sodium dodecyl sulfate electrophoresis in 1 and 1.5% agarose gels, respectively, followed by immunoluminescent detection of VWF. As compared with normal controls TTP plasma displays ultra-large VWF multimers (boxed areas). These high-molecular-weight multimers can be detected more easily in low resolution gels (1% agarose, left panel). Additional samples were analyzed using medium resolution gels (1.5% agarose, right panel) which offer a clearer separation of proteolytic sub-bands. Cleavage of VWF by ADAMTS-13 results in VWF fragments represented by subbands which are fainter in TTP patients as compared with normal control plasma (right panel, NP, arrows depict proteolytic sub-bands).
died of multiorgan failure a week after falling ill.3 Histologic specimen obtained at autopsy revealed hyalinic thrombi in the terminal arterioles and capillaries of various organs. Based on these findings, Moschcowitz proposed that the disease was due to a “powerful poison with both agglutinative and hemolytic properties.” Further reports by Schulman and Upshaw recognized that normal plasma contained a factor that was able to correct TTP symptoms.4,5 A role for VWF was first recognized in 1982 when Moake discovered supranormal multimers of VWF in plasma of TTP patients and proposed a defect in VWF processing.6 In 1996, this processing defect was identified as deficiency of a VWF-specific metalloprotease by Furlan et al and Tsai et al.7,8 Finally, the identification of ADAMTS-13 as VWF-cleaving protease in 2001 enabled the definition of TTP at the molecular level.9–12 Mutations in the ADAMTS-13 gene cause congenital TTP11 (syn. Upshaw–Schulman syndrome (USS), OMIM #274150). Autoantibodies against ADAMTS-13 are the cause of acquired TTP. While acquired TTP mostly affects adult patients, congenital TTP or USS is the predominant form of TTP in children. Due to the rarity of USS, only a limited number of patients have been described worldwide. We were able to analyze 30 patients with USS and we summarize our results on molecular genetics and clinical features here.
Patients To date, we have investigated a total of 30 patients with USS who were mainly from Germany (n ¼ 20), including four Seminars in Thrombosis & Hemostasis
Turkish families (n ¼ 4). Ten samples were referred to our laboratory from Croatia (n ¼ 1), Denmark (n ¼ 1), Poland (n ¼ 4), Spain (n ¼ 2), Sweden (n ¼ 1), and Australia (n ¼ 1). The diagnosis of USS is based on the presence of clinical symptoms (low platelet count, Coombs negative hemolytic anemia, kidney failure, CNS impairment, and thrombosis) and lack of ADAMTS-13 activity (< 5%) in plasma without inhibitory ADAMTS-13 antibodies. ADAMTS-13 activity was measured using a chromogenic ELISA (TECHNOZYM ADAMTS-13 ELISA, Technoclone, Vienna, Austria) or other commercially available assays as established in the local laboratories of referring centers. Relevant data from patient files were used to establish individual levels of disease severity. The clinical course was designated mild (recurrent isolated thrombocytopenia only), intermediate (additional renal and/or CNS impairment requiring hospitalization at least once), or severe (severe impairment of CNS or renal function and permanent need of prophylactic plasma therapy). All patients or their parents were informed about the nature of this study and their consent was obtained according to the declaration of Helsinki.
Clinical Findings All patients displayed severe ADAMTS-13 deficiency below 5% (►Table 1). Male and female patients are represented equally in our cohort. More than half of the patients showed neonatal onset ( 14 y and adults (including pregnancy onset in 1 patient)
2/30 (6%)
Unknowna
5/30 (17%)
Clinical courseb Mild
10/30 (33%)
Intermediate
12/30 (40%)
Severe (including DOD in two patients)
8/30 (27%)
Treatment On demand
11/30 (37%)
Prophylaxis
16/30 (53%)
Information not available
3/30 (10%)
Mutation Missense/missense
4/30 (13%)
Missense/truncating
15/30 (50%)
Truncating/truncating
11/30 (37%)
Domains
c
MDTS/MDTS
5/30 (17%)
MDTS/TSP-CUB
15/30 (50%)
TSP-CUB/TSP-CUB
10/30 (33%)
Notes: All patients suffer from severe ADAMTS-13 deficiency. Male and female patients are represented equally. More than half of the patients showed first signs of TTP during the first 4 weeks of life. a Due to incomplete patient histories, data on first onset of TTP is missing in 5 cases. b Mild course refers to recurrent episodes of isolated thrombocytopenia only, without any organ involvement, intermediate course refers to additional renal and/or CNS impairment requiring hospitalization at least once, and severe course refers to persistent sequelae of CNS or renal function and permanent need of prophylactic plasma therapy. DOD refers to death of disease. “Truncating” refers to mutations that could potentially result in a truncated gene product and includes nonsense and splice site mutations, frameshift mutations, and large deletions. c MDTS refers to the metalloprotease, disintegrin, TSP1–1 through spacer domains; TSP-CUB refers to the TSP1–2 through CUB2 domains.
4143 (formerly c.4143insA) is now being described as a duplication (c.4143dupA). In all patients we were able to detect homozygous or compound heterozygous mutations in the ADAMTS-13 gene. Mutation screening revealed two larger and three small deletions, including two splice site mutations, two small duplications, 15 missense mutations, and seven nonsense
Fig. 2 Course of platelet counts in a patient with Upshaw–Schulman syndrome with neonatal thrombotic thrombocytopenic purpura (TTP) onset. Platelet count (109/L) in the first 4 weeks of life in a representative case of neonatal onset of TTP. After birth, platelet count is already decreased with further decline on days 2 and 3. This patient received a platelet transfusion at 4 days of age (open arrow) at a platelet count of 27 109/L. Due to hemolytic anemia with hyperbilirubinemia (peak value 24 mg/dL) he received intermittent phototherapy (shaded box) as well as one transfusion of packed red blood cells at a minimal hemoglobin concentration of 6.7 g/dL (shaded arrow).
mutations (►Fig. 4). Mutations were distributed to the metalloprotease domain (MP, n ¼ 5), the disintegrin domain (n ¼ 2), the TSP1 domains (n ¼ 8), and the 2 CUB domains (n ¼ 3). Seven patients had homozygous mutations including four homozygotes for the 4143dupA duplication. The remaining patients had compound heterozygous mutations. The distribution of mutations within the gene and protein structure of ADAMTS-13 is illustrated in ►Fig. 4.
Thrombotic Thrombocytopenic Purpura Genotype We were able to detect homozygous or compound heterozygous mutations in all patients reported here. Forty percent of all mutations are missense mutations. Missense mutations are slightly overrepresented in the N-terminal domains of ADAMTS-13 containing the metalloprotease domain. Other gene defects account for 32% of all mutations. The mutation c.4143dupA is particularly common in our cohort. It has first been identified in four unrelated patients from Germany where it is rather frequent,13 but this mutation is generally also frequently found in other central and Northern European countries. As could be shown by haplotype analysis it might represent a founder mutation most probably derived from a common ancestor.19 The prevalence of c.4143dupA in cohorts from southern and western Europe or other parts of the world is significantly lower.21,22
Thrombotic Thrombocytopenic Purpura Phenotype To prevent organ damage and severe sequelae of acute bouts, an early diagnosis of TTP is mandatory. This can be Seminars in Thrombosis & Hemostasis
Inherited TTP in Children
Hassenpflug et al.
Fig. 3 Clinical symptoms of TTP observed in 30 patients with Upshaw–Schulman syndrome. (A) Cumulative incidence of TTP-associated symptoms (multiple items allowed). Numbers represent the number of patients with a positive history for a specific symptom at any time during their disease. For only three patients, limited data were available. Thrombocytopenia was defined as platelet count below 100 109 /L. Hemolysis was defined by decline of hemoglobin levels of 1 g/dL or below lower limit of normal and elevation of lactate dehydrogenase or reduced haptoglobin levels. Renal disease was defined by elevation of creatinine above the upper limit of normal or reduction of glomerular filtration rate. The classical pentad of TTP comprises the upper five items (gray shaded boxes). Overt thrombosis was observed in two patients and has been added as an additional item (the light gray shaded box). (B) Relative distribution of the most common combinations of TTP symptoms during initial bouts before TTP was diagnosed in an individual patient. Due to the retrospective nature of our study, sufficient data were available for only 19 patients. Of these patients, 16% showed the full pentad of TTP symptoms, including a girl who died of disease before diagnosis of TTP was made. 17 Almost three-fourths of the patients presented with a combination of thrombocytopenia and hemolytic anemia either with or without fever. CNS, central nervous system; TTP, thrombotic thrombocytopenic purpura.
challenging when patients show only a subset of the diseasedefining symptoms.17 Distinct symptoms of TTP are not necessarily all present at the same time. Our data indicate that oligosymptomatic courses may be very common in USS and that the full pentad of symptoms is restricted to severe and prolonged bouts. Probably the most sensitive indicator of TTP activity is a decline in platelet count (►Fig. 3).18 It has been stressed before that oligosymptomatic TTP must not be mistaken as chronic immune thrombocytopenia, atypical hemolytic uremic syndrome (aHUS), or Evans syndrome.17,18 Indeed, thrombocytopenia and hemolytic anemia with or without concomitant fever is the most common combination of TTP symptoms during the initial presentations (►Fig. 3). Interestingly, half of our patients had a history of neonatal thrombocytopenia or severe hemolytic anemia and jaundice. Clinical observations in other cohorts of TTP patients confirm this phenomenon.21 We propose that low platelet counts and
jaundice after birth are common findings in USS patients that might facilitate early diagnosis. The diagnosis of TTP/USS should be considered in all patients with neonatal thrombocytopenia, especially in patients with concomitant Coombsnegative hemolytic anemia. As of today reliable and less time consuming tests for ADAMTS-13 deficiency are available in many laboratories, therefore the access to adequate diagnostic measures should not be an obstacle anymore. This should lower the hurdles to order such tests which in positive cases might prevent severe organ damage or even death of affected children by simple plasma transfusion.
Genotype–Phenotype Relationship As confirmed by our data, variability of clinical courses among individual patients is high. Within the same family, carriers of identical ADAMTS-13 mutations may or may not experience
Fig. 4 Location and spectrum of mutations in Upshaw–Schulman syndrome. (A) The upper part of the panel shows the ADAMTS-13 gene structure with shaded bars representing exons 1 to 29. Blank circles represent deletions, black shaded circles represent missense mutations, and circles with a mid-vertical line represent insertions. Circles with a cross represent nonsense mutations. Homozygous mutations are linked by open brackets. The lower panel represents the ADAMTS-13 protein with its domains: SP, signal peptide; Pro, propeptide; M, metalloprotease domain; Zn, zinc binding site; D, disintegrin domain; T1, thrombospondin type 1–like domain; following TSP1 domains are marked T2 for TSP1–2, etc.; C, cysteine-rich domain; RGDS, arginine–glycine–aspartate–serine sequence; S, spacer domain; C1, C2, CUB domains 1 and 2. (B) Classification of ADAMTS-13 mutations. Seminars in Thrombosis & Hemostasis
Inherited TTP in Children severe TTP bouts. While well-known exogenous triggers of TTP (e.g., pregnancy) might explain the onset of TTP in some cases, in general no predicting parameters for disease severity exist. Recently, Lotta et al proposed a genotype–phenotype correlation in USS patients.23 In a cohort of 29 TTP/USS patients, mutations in the N-terminal domains of ADAMTS13 were associated with an early onset and more severe clinical course of TTP. Mutations in the N-terminal domains of ADAMTS-13 correlated with lower levels of residual ADAMTS-13 activity in patients’ serum as analyzed by a novel mass spectrometry-based assay.23 In our cohort, we cannot provide any data on residual ADAMTS-13 activities below the lower detection limit of commercial assays in the range of 3 to 5%. However, the high frequency of the c.4143dupA mutation enabled us to analyze the impact of different single mutations on the common background of c.4143dupA on the second allele. There was no phenotypic difference between patients with mutations in the N-terminal or C-terminal domains of ADAMTS-13. Within the group of homozygotes for c.4143dupA, there were one mild, two intermediate, and one severe courses. Obviously, the available data are not sufficient to confirm or dismiss genotype–phenotype considerations: The patient number is low. All clinical data have been analyzed retrospectively. Treatment strategies were not standardized. Some patients already had organ damage before TTP was diagnosed. From the practical perspective, “mild” mutations do not exclude severe bouts. In contrary, even patients with “severe” mutations do not necessarily need primary prophylactic plasma transfusions.
Hassenpflug et al.
very rare disease and oligosymptomatic courses may further obscure the diagnosis. Therefore, atypical cases of chronic ITP, Coombs negative (atypical) Evans syndrome, atypical HUS should be further evaluated for ADAMTS-13 deficiency. Derived from our experience, in particular the neonatal period offers a window of opportunity for early diagnosis of USS: The diagnosis should be considered in all patients with neonatal thrombocytopenia, especially in patients with concomitant Coombs-negative hemolytic anemia, but also in suspected neonatal alloimmune thrombocytopenia (NAIT) and neonatal infection with thrombocytopenia.
Acknowledgments We thank the following colleagues for contributing patient samples and clinical data (alphabetical order): W. Brockhaus (Nuremburg, Germany), J. M. Estella (Barcelona, Spain), J. Ingerslev (Aarhus, Denmark), A. Heitger (Vienna, Austria), D. Karpman Lund (Sweden), A. Klukowska (Warsaw, Poland), K. Kentouche (Jena, Germany), E. Kohne (Ulm, Germany), B. Korczowski (Rzeszow, Poland), K. Kurnik (Munich, Germany), H. Oldigs (Flensburg, Germany), B. Perez de Mendiguren (Burgos, Spain), J. E. Pimanda (Cambridge, UK), A. Reiter (Giessen, Germany), and L. Stapenhorst (Cologne, Germany).
References 1 Moake JL. Thrombotic microangiopathies. N Engl J Med 2002;
347:589–600 2 Dent JA, Berkowitz SD, Ware J, Kasper CK, Ruggeri ZM. Identifica-
Treatment In our experience, transfusion of 15 to 20 mL/kg body weight of fresh–frozen plasma on one to three consecutive days is sufficient to resolve most uncomplicated bouts of TTP. Such a regimen can be applied on demand, which was the treatment strategy in about one-third of the patients investigated here. Fifty percent received prophylactic plasma transfusions. Due to the long half-life of ADAMTS-13 (3 to 4 days) prophylactic transfusions with an interval of were sufficient in most of the patients in our cohort. Plasma prophylaxis might prevent severe sequelae of TTP bouts. In our study, all patients who suffered from chronic kidney disease or CNS lesions were either diagnosed after several bouts of TTP or had catastrophic first events.
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Concluding Remarks Within the given methodical limits our data do not support the idea of an overarching genotype–phenotype correlation. Probably, the inevitably variant nature and strength of exogenous triggers, as well as additional endogenous factors participating in an inflammatory response, which varies between individuals, have a significant impact on clinical courses of TTP. One of the most important issues is not to miss the diagnosis of TTP/USS. This can be difficult since USS is a
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tion of a cleavage site directing the immunochemical detection of molecular abnormalities in type IIA von Willebrand factor. Proc Natl Acad Sci U S A 1990;87:6306–6310 Moschcowitz E. Hyaline thrombosis of the terminal arterioles and capillaries: a hitherto undescribed disease. Proc N Y Pathol Soc 1924;24:21–24 Schulman I, Pierce M, Lukens A, Currinbhoy Z. Studies on thrombopoiesis. I. A factor in normal human plasma required for platelet production; chronic thrombocytopenia due to its deficiency. Blood 1960;16:943–957 Upshaw JD Jr. Congenital deficiency of a factor in normal plasma that reverses microangiopathic hemolysis and thrombocytopenia. N Engl J Med 1978;298:1350–1352 Moake JL, Rudy CK, Troll JH, et al. Unusually large plasma factor VIII: von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. N Engl J Med 1982;307:1432–1435 Furlan M, Robles R, Lamie B. Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. Blood 1996; 87:4223–4234 Tsai HM. Physiologic cleavage of von Willebrand factor by a plasma protease is dependent on its conformation and requires calcium ion. Blood 1996;87:4235–4244 Fujikawa K, Suzuki H, McMullen B, Chung D. Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family. Blood 2001; 98:1662–1666 Gerritsen HE, Robles R, Lammle B, Furlan M. Partial amino acid sequence of purified von Willebrand factor-cleaving protease. Blood 2001;98:1654–1661 Seminars in Thrombosis & Hemostasis
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11 Levy GG, Nichols WC, Lian EC, et al. Mutations in a member of the
17 Schneppenheim R, Budde U, Oyen F, et al. von Willebrand factor
ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 2001;413:488–494 Soejima K, Mimura N, Hirashima M, et al. A novel human metalloprotease synthesized in the liver and secreted into the blood: possibly, the von Willebrand factor-cleaving protease? J Biochem 2001;130:475–480 Hassenpflug WA, Angerhaus D, Budde U, Obser T, Schneppenheim R. Thrombotic thrombocytopenic purpura in childhood. Hamostaseologie 2004;24:71–76 Haberle J, Kehrel B, Ritter J, et al. New strategies in diagnosis and treatment of thrombotic thrombocytopenic purpura: case report and review. Eur J Pediatr 1999;158:883–887 Kentouche K, Budde U, Furlan M, et al. Remission of thrombotic thrombocytopenic purpura in a patient with compound heterozygous deficiency of von Willebrand factor-cleaving protease by infusion of solvent/detergent plasma. Acta Paediatr 2002; 91:1056–1059 Licht C, Stapenhorst L, Simon T, et al. Two novel ADAMTS13 gene mutations in thrombotic thrombocytopenic purpura/hemolyticuremic syndrome (TTP/HUS). Kidney Int 2004;66:955–958
cleaving protease and ADAMTS13 mutations in childhood TTP. Blood 2003;101:1845–1850 Schneppenheim R, Budde U, Hassenpflug W, Obser T. Severe ADAMTS-13 deficiency in childhood. Semin Hematol 2004; 41:83–89 Schneppenheim R, Kremer Hovinga JA, Becker T, et al. A common origin of the 4143insA ADAMTS13 mutation. Thromb Haemost 2006;96:3–6 den Dunnen JT, Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 2000;15:7–12 Fujimura Y, Matsumoto M, Isonishi A, et al. Natural history of Upshaw-Schulman syndrome based on ADAMTS13 gene analysis in Japan. J Thromb Haemost 2011;9(Suppl 1):283–301 Lotta LA, Garagiola I, Palla R, Cairo A, Peyvandi F. ADAMTS13 mutations and polymorphisms in congenital thrombotic thrombocytopenic purpura. Hum Mutat 2010;31:11–19 Lotta LA, Wu HM, Mackie IJ, et al. Residual plasmatic activity of ADAMTS13 is correlated with phenotype severity in congenital thrombotic thrombocytopenic purpura. Blood 2012;120:440–448
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Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 Medilys Laborgemeinschaft mbH, Coagulation Laboratory, Hamburg, Germany
Sonja Schneppenheim, MD2
Address for correspondence Reinhard Schneppenheim, MD, PhD, Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany (e-mail: [email protected]).
Semin Thromb Hemost
Abstract
Keywords
► ADAMTS-13 ► Upshaw–Schulman syndrome ► thrombotic thrombocytopenic purpura ► von Willebrand factor ► molecular genetics
Congenital thrombotic thrombocytopenic purpura (TTP) or Upshaw–Schulman syndrome is caused by homozygous or compound heterozygous mutations in the ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) gene. We investigated 30 patients with congenital TTP and analyzed clinical data and underlying ADAMTS-13 mutations. All patients showed virtually no ADAMTS-13 activity in plasma. Individual disease burden ranged from mild courses with rare episodes of mild thrombocytopenia to severe courses with chronic kidney disease and central nervous system (CNS) lesions. Two patients died due to complications of TTP. If initiated in a timely manner, plasma transfusions offer a reliable treatment to prevent organ damage. We identified 30 different causative mutations in the ADAMTS13 gene. Our data do not support the idea of a tight correlation between ADAMTS-13 genotype and severity of disease. The type and magnitude of exogenous triggers for acute bouts of TTP as well as endogenous individual factors participating in the inflammatory response likely represent the foremost determinants of individual clinical courses. Future developments should aim at improving early diagnosis of TTP. To improve feasibility of prophylaxis and treatment of congenital TTP, recombinant ADAMTS-13 therapeutics are highly anticipated.
Thrombotic thrombocytopenic purpura (TTP) is a rare lifethreatening disorder that is caused by congenital or acquired deficiency of the von Willebrand factor-cleaving protease ADAMTS-13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13).1 ADAMTS-13 cleaves von Willebrand factor (VWF) at a specific cleavage site in the A2 domain between residues Thr1605 and Met1606.2 Lack of ADAMTS-13 activity results in the persistence of ultra-large multimers of VWF (ULVWF) in plasma (►Fig. 1). Under conditions of high shear flow ULVWF can extend to long fibers that expose numerous platelet binding sites to which
Issue Theme An Update on the Thrombotic Microangiopathies Hemolytic Uremic Syndrome (HUS) and Thrombotic Thrombocytopenic Purpura (TTP); Guest Editors, Magdalena Riedl, MD, Dorothea Orth-Höller, MD, and Reinhard Würzner, MD, PhD.
platelets readily adhere. Typical triggers of episodic TTP bouts such as infections or pregnancy, increase VWF plasma levels and stimulate agglutination of ULVWF and platelets. As a result, TTP patients suffer from microangiopathic hemolytic anemia, thrombocytopenia, fever, renal failure, and central nervous system (CNS) impairment. These symptoms had come to be known as the classical pentad of TTP. The first description of a TTP case has been ascribed to Eli Moschcowitz.3 In 1924, he reported on a previously healthy 16-year-old girl, who presented with fever, hemolytic anemia, and thrombocytopenia and who
Copyright © by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0034-1376152. ISSN 0094-6176.
Inherited TTP in Children
Hassenpflug et al.
Fig. 1 von Willebrand factor (VWF) multimers in Upshaw–Schulman syndrome (USS)/thrombotic thrombocytopenic purpura (TTP) patients. Plasma samples of USS/TTP patients and normal control plasma (NP) were separated by sodium dodecyl sulfate electrophoresis in 1 and 1.5% agarose gels, respectively, followed by immunoluminescent detection of VWF. As compared with normal controls TTP plasma displays ultra-large VWF multimers (boxed areas). These high-molecular-weight multimers can be detected more easily in low resolution gels (1% agarose, left panel). Additional samples were analyzed using medium resolution gels (1.5% agarose, right panel) which offer a clearer separation of proteolytic sub-bands. Cleavage of VWF by ADAMTS-13 results in VWF fragments represented by subbands which are fainter in TTP patients as compared with normal control plasma (right panel, NP, arrows depict proteolytic sub-bands).
died of multiorgan failure a week after falling ill.3 Histologic specimen obtained at autopsy revealed hyalinic thrombi in the terminal arterioles and capillaries of various organs. Based on these findings, Moschcowitz proposed that the disease was due to a “powerful poison with both agglutinative and hemolytic properties.” Further reports by Schulman and Upshaw recognized that normal plasma contained a factor that was able to correct TTP symptoms.4,5 A role for VWF was first recognized in 1982 when Moake discovered supranormal multimers of VWF in plasma of TTP patients and proposed a defect in VWF processing.6 In 1996, this processing defect was identified as deficiency of a VWF-specific metalloprotease by Furlan et al and Tsai et al.7,8 Finally, the identification of ADAMTS-13 as VWF-cleaving protease in 2001 enabled the definition of TTP at the molecular level.9–12 Mutations in the ADAMTS-13 gene cause congenital TTP11 (syn. Upshaw–Schulman syndrome (USS), OMIM #274150). Autoantibodies against ADAMTS-13 are the cause of acquired TTP. While acquired TTP mostly affects adult patients, congenital TTP or USS is the predominant form of TTP in children. Due to the rarity of USS, only a limited number of patients have been described worldwide. We were able to analyze 30 patients with USS and we summarize our results on molecular genetics and clinical features here.
Patients To date, we have investigated a total of 30 patients with USS who were mainly from Germany (n ¼ 20), including four Seminars in Thrombosis & Hemostasis
Turkish families (n ¼ 4). Ten samples were referred to our laboratory from Croatia (n ¼ 1), Denmark (n ¼ 1), Poland (n ¼ 4), Spain (n ¼ 2), Sweden (n ¼ 1), and Australia (n ¼ 1). The diagnosis of USS is based on the presence of clinical symptoms (low platelet count, Coombs negative hemolytic anemia, kidney failure, CNS impairment, and thrombosis) and lack of ADAMTS-13 activity (< 5%) in plasma without inhibitory ADAMTS-13 antibodies. ADAMTS-13 activity was measured using a chromogenic ELISA (TECHNOZYM ADAMTS-13 ELISA, Technoclone, Vienna, Austria) or other commercially available assays as established in the local laboratories of referring centers. Relevant data from patient files were used to establish individual levels of disease severity. The clinical course was designated mild (recurrent isolated thrombocytopenia only), intermediate (additional renal and/or CNS impairment requiring hospitalization at least once), or severe (severe impairment of CNS or renal function and permanent need of prophylactic plasma therapy). All patients or their parents were informed about the nature of this study and their consent was obtained according to the declaration of Helsinki.
Clinical Findings All patients displayed severe ADAMTS-13 deficiency below 5% (►Table 1). Male and female patients are represented equally in our cohort. More than half of the patients showed neonatal onset ( 14 y and adults (including pregnancy onset in 1 patient)
2/30 (6%)
Unknowna
5/30 (17%)
Clinical courseb Mild
10/30 (33%)
Intermediate
12/30 (40%)
Severe (including DOD in two patients)
8/30 (27%)
Treatment On demand
11/30 (37%)
Prophylaxis
16/30 (53%)
Information not available
3/30 (10%)
Mutation Missense/missense
4/30 (13%)
Missense/truncating
15/30 (50%)
Truncating/truncating
11/30 (37%)
Domains
c
MDTS/MDTS
5/30 (17%)
MDTS/TSP-CUB
15/30 (50%)
TSP-CUB/TSP-CUB
10/30 (33%)
Notes: All patients suffer from severe ADAMTS-13 deficiency. Male and female patients are represented equally. More than half of the patients showed first signs of TTP during the first 4 weeks of life. a Due to incomplete patient histories, data on first onset of TTP is missing in 5 cases. b Mild course refers to recurrent episodes of isolated thrombocytopenia only, without any organ involvement, intermediate course refers to additional renal and/or CNS impairment requiring hospitalization at least once, and severe course refers to persistent sequelae of CNS or renal function and permanent need of prophylactic plasma therapy. DOD refers to death of disease. “Truncating” refers to mutations that could potentially result in a truncated gene product and includes nonsense and splice site mutations, frameshift mutations, and large deletions. c MDTS refers to the metalloprotease, disintegrin, TSP1–1 through spacer domains; TSP-CUB refers to the TSP1–2 through CUB2 domains.
4143 (formerly c.4143insA) is now being described as a duplication (c.4143dupA). In all patients we were able to detect homozygous or compound heterozygous mutations in the ADAMTS-13 gene. Mutation screening revealed two larger and three small deletions, including two splice site mutations, two small duplications, 15 missense mutations, and seven nonsense
Fig. 2 Course of platelet counts in a patient with Upshaw–Schulman syndrome with neonatal thrombotic thrombocytopenic purpura (TTP) onset. Platelet count (109/L) in the first 4 weeks of life in a representative case of neonatal onset of TTP. After birth, platelet count is already decreased with further decline on days 2 and 3. This patient received a platelet transfusion at 4 days of age (open arrow) at a platelet count of 27 109/L. Due to hemolytic anemia with hyperbilirubinemia (peak value 24 mg/dL) he received intermittent phototherapy (shaded box) as well as one transfusion of packed red blood cells at a minimal hemoglobin concentration of 6.7 g/dL (shaded arrow).
mutations (►Fig. 4). Mutations were distributed to the metalloprotease domain (MP, n ¼ 5), the disintegrin domain (n ¼ 2), the TSP1 domains (n ¼ 8), and the 2 CUB domains (n ¼ 3). Seven patients had homozygous mutations including four homozygotes for the 4143dupA duplication. The remaining patients had compound heterozygous mutations. The distribution of mutations within the gene and protein structure of ADAMTS-13 is illustrated in ►Fig. 4.
Thrombotic Thrombocytopenic Purpura Genotype We were able to detect homozygous or compound heterozygous mutations in all patients reported here. Forty percent of all mutations are missense mutations. Missense mutations are slightly overrepresented in the N-terminal domains of ADAMTS-13 containing the metalloprotease domain. Other gene defects account for 32% of all mutations. The mutation c.4143dupA is particularly common in our cohort. It has first been identified in four unrelated patients from Germany where it is rather frequent,13 but this mutation is generally also frequently found in other central and Northern European countries. As could be shown by haplotype analysis it might represent a founder mutation most probably derived from a common ancestor.19 The prevalence of c.4143dupA in cohorts from southern and western Europe or other parts of the world is significantly lower.21,22
Thrombotic Thrombocytopenic Purpura Phenotype To prevent organ damage and severe sequelae of acute bouts, an early diagnosis of TTP is mandatory. This can be Seminars in Thrombosis & Hemostasis
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Fig. 3 Clinical symptoms of TTP observed in 30 patients with Upshaw–Schulman syndrome. (A) Cumulative incidence of TTP-associated symptoms (multiple items allowed). Numbers represent the number of patients with a positive history for a specific symptom at any time during their disease. For only three patients, limited data were available. Thrombocytopenia was defined as platelet count below 100 109 /L. Hemolysis was defined by decline of hemoglobin levels of 1 g/dL or below lower limit of normal and elevation of lactate dehydrogenase or reduced haptoglobin levels. Renal disease was defined by elevation of creatinine above the upper limit of normal or reduction of glomerular filtration rate. The classical pentad of TTP comprises the upper five items (gray shaded boxes). Overt thrombosis was observed in two patients and has been added as an additional item (the light gray shaded box). (B) Relative distribution of the most common combinations of TTP symptoms during initial bouts before TTP was diagnosed in an individual patient. Due to the retrospective nature of our study, sufficient data were available for only 19 patients. Of these patients, 16% showed the full pentad of TTP symptoms, including a girl who died of disease before diagnosis of TTP was made. 17 Almost three-fourths of the patients presented with a combination of thrombocytopenia and hemolytic anemia either with or without fever. CNS, central nervous system; TTP, thrombotic thrombocytopenic purpura.
challenging when patients show only a subset of the diseasedefining symptoms.17 Distinct symptoms of TTP are not necessarily all present at the same time. Our data indicate that oligosymptomatic courses may be very common in USS and that the full pentad of symptoms is restricted to severe and prolonged bouts. Probably the most sensitive indicator of TTP activity is a decline in platelet count (►Fig. 3).18 It has been stressed before that oligosymptomatic TTP must not be mistaken as chronic immune thrombocytopenia, atypical hemolytic uremic syndrome (aHUS), or Evans syndrome.17,18 Indeed, thrombocytopenia and hemolytic anemia with or without concomitant fever is the most common combination of TTP symptoms during the initial presentations (►Fig. 3). Interestingly, half of our patients had a history of neonatal thrombocytopenia or severe hemolytic anemia and jaundice. Clinical observations in other cohorts of TTP patients confirm this phenomenon.21 We propose that low platelet counts and
jaundice after birth are common findings in USS patients that might facilitate early diagnosis. The diagnosis of TTP/USS should be considered in all patients with neonatal thrombocytopenia, especially in patients with concomitant Coombsnegative hemolytic anemia. As of today reliable and less time consuming tests for ADAMTS-13 deficiency are available in many laboratories, therefore the access to adequate diagnostic measures should not be an obstacle anymore. This should lower the hurdles to order such tests which in positive cases might prevent severe organ damage or even death of affected children by simple plasma transfusion.
Genotype–Phenotype Relationship As confirmed by our data, variability of clinical courses among individual patients is high. Within the same family, carriers of identical ADAMTS-13 mutations may or may not experience
Fig. 4 Location and spectrum of mutations in Upshaw–Schulman syndrome. (A) The upper part of the panel shows the ADAMTS-13 gene structure with shaded bars representing exons 1 to 29. Blank circles represent deletions, black shaded circles represent missense mutations, and circles with a mid-vertical line represent insertions. Circles with a cross represent nonsense mutations. Homozygous mutations are linked by open brackets. The lower panel represents the ADAMTS-13 protein with its domains: SP, signal peptide; Pro, propeptide; M, metalloprotease domain; Zn, zinc binding site; D, disintegrin domain; T1, thrombospondin type 1–like domain; following TSP1 domains are marked T2 for TSP1–2, etc.; C, cysteine-rich domain; RGDS, arginine–glycine–aspartate–serine sequence; S, spacer domain; C1, C2, CUB domains 1 and 2. (B) Classification of ADAMTS-13 mutations. Seminars in Thrombosis & Hemostasis
Inherited TTP in Children severe TTP bouts. While well-known exogenous triggers of TTP (e.g., pregnancy) might explain the onset of TTP in some cases, in general no predicting parameters for disease severity exist. Recently, Lotta et al proposed a genotype–phenotype correlation in USS patients.23 In a cohort of 29 TTP/USS patients, mutations in the N-terminal domains of ADAMTS13 were associated with an early onset and more severe clinical course of TTP. Mutations in the N-terminal domains of ADAMTS-13 correlated with lower levels of residual ADAMTS-13 activity in patients’ serum as analyzed by a novel mass spectrometry-based assay.23 In our cohort, we cannot provide any data on residual ADAMTS-13 activities below the lower detection limit of commercial assays in the range of 3 to 5%. However, the high frequency of the c.4143dupA mutation enabled us to analyze the impact of different single mutations on the common background of c.4143dupA on the second allele. There was no phenotypic difference between patients with mutations in the N-terminal or C-terminal domains of ADAMTS-13. Within the group of homozygotes for c.4143dupA, there were one mild, two intermediate, and one severe courses. Obviously, the available data are not sufficient to confirm or dismiss genotype–phenotype considerations: The patient number is low. All clinical data have been analyzed retrospectively. Treatment strategies were not standardized. Some patients already had organ damage before TTP was diagnosed. From the practical perspective, “mild” mutations do not exclude severe bouts. In contrary, even patients with “severe” mutations do not necessarily need primary prophylactic plasma transfusions.
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very rare disease and oligosymptomatic courses may further obscure the diagnosis. Therefore, atypical cases of chronic ITP, Coombs negative (atypical) Evans syndrome, atypical HUS should be further evaluated for ADAMTS-13 deficiency. Derived from our experience, in particular the neonatal period offers a window of opportunity for early diagnosis of USS: The diagnosis should be considered in all patients with neonatal thrombocytopenia, especially in patients with concomitant Coombs-negative hemolytic anemia, but also in suspected neonatal alloimmune thrombocytopenia (NAIT) and neonatal infection with thrombocytopenia.
Acknowledgments We thank the following colleagues for contributing patient samples and clinical data (alphabetical order): W. Brockhaus (Nuremburg, Germany), J. M. Estella (Barcelona, Spain), J. Ingerslev (Aarhus, Denmark), A. Heitger (Vienna, Austria), D. Karpman Lund (Sweden), A. Klukowska (Warsaw, Poland), K. Kentouche (Jena, Germany), E. Kohne (Ulm, Germany), B. Korczowski (Rzeszow, Poland), K. Kurnik (Munich, Germany), H. Oldigs (Flensburg, Germany), B. Perez de Mendiguren (Burgos, Spain), J. E. Pimanda (Cambridge, UK), A. Reiter (Giessen, Germany), and L. Stapenhorst (Cologne, Germany).
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347:589–600 2 Dent JA, Berkowitz SD, Ware J, Kasper CK, Ruggeri ZM. Identifica-
Treatment In our experience, transfusion of 15 to 20 mL/kg body weight of fresh–frozen plasma on one to three consecutive days is sufficient to resolve most uncomplicated bouts of TTP. Such a regimen can be applied on demand, which was the treatment strategy in about one-third of the patients investigated here. Fifty percent received prophylactic plasma transfusions. Due to the long half-life of ADAMTS-13 (3 to 4 days) prophylactic transfusions with an interval of were sufficient in most of the patients in our cohort. Plasma prophylaxis might prevent severe sequelae of TTP bouts. In our study, all patients who suffered from chronic kidney disease or CNS lesions were either diagnosed after several bouts of TTP or had catastrophic first events.
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Concluding Remarks Within the given methodical limits our data do not support the idea of an overarching genotype–phenotype correlation. Probably, the inevitably variant nature and strength of exogenous triggers, as well as additional endogenous factors participating in an inflammatory response, which varies between individuals, have a significant impact on clinical courses of TTP. One of the most important issues is not to miss the diagnosis of TTP/USS. This can be difficult since USS is a
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ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 2001;413:488–494 Soejima K, Mimura N, Hirashima M, et al. A novel human metalloprotease synthesized in the liver and secreted into the blood: possibly, the von Willebrand factor-cleaving protease? J Biochem 2001;130:475–480 Hassenpflug WA, Angerhaus D, Budde U, Obser T, Schneppenheim R. Thrombotic thrombocytopenic purpura in childhood. Hamostaseologie 2004;24:71–76 Haberle J, Kehrel B, Ritter J, et al. New strategies in diagnosis and treatment of thrombotic thrombocytopenic purpura: case report and review. Eur J Pediatr 1999;158:883–887 Kentouche K, Budde U, Furlan M, et al. Remission of thrombotic thrombocytopenic purpura in a patient with compound heterozygous deficiency of von Willebrand factor-cleaving protease by infusion of solvent/detergent plasma. Acta Paediatr 2002; 91:1056–1059 Licht C, Stapenhorst L, Simon T, et al. Two novel ADAMTS13 gene mutations in thrombotic thrombocytopenic purpura/hemolyticuremic syndrome (TTP/HUS). Kidney Int 2004;66:955–958
cleaving protease and ADAMTS13 mutations in childhood TTP. Blood 2003;101:1845–1850 Schneppenheim R, Budde U, Hassenpflug W, Obser T. Severe ADAMTS-13 deficiency in childhood. Semin Hematol 2004; 41:83–89 Schneppenheim R, Kremer Hovinga JA, Becker T, et al. A common origin of the 4143insA ADAMTS13 mutation. Thromb Haemost 2006;96:3–6 den Dunnen JT, Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 2000;15:7–12 Fujimura Y, Matsumoto M, Isonishi A, et al. Natural history of Upshaw-Schulman syndrome based on ADAMTS13 gene analysis in Japan. J Thromb Haemost 2011;9(Suppl 1):283–301 Lotta LA, Garagiola I, Palla R, Cairo A, Peyvandi F. ADAMTS13 mutations and polymorphisms in congenital thrombotic thrombocytopenic purpura. Hum Mutat 2010;31:11–19 Lotta LA, Wu HM, Mackie IJ, et al. Residual plasmatic activity of ADAMTS13 is correlated with phenotype severity in congenital thrombotic thrombocytopenic purpura. Blood 2012;120:440–448
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