Thrombotic Thrombocytopenic Purpura

Genetics and Genetic Testing in Hemolytic Uremic Syndrome/ Thrombotic Thrombocytopenic Purpura Marina Noris, PhD,* and Giuseppe Remuzzi, MD*,† Summary: The hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) are rare diseases that manifest with thrombocytopenia and microangiopathic hemolytic anemia accompanied by renal and neurologic dysfunction. Most childhood cases of HUS are caused by Shiga-toxin–producing bacteria and have a good prognosis. The other form, atypical HUS (aHUS), accounts for 10% of cases. Prognosis of aHUS and TTP has changed over time from fatal disorders to 60% to 80% survival in the plasma therapy era. In the past 10 years the molecular bases of aHUS and TTP have been discovered that mostly lead to uncontrolled activation of the complement system in aHUS and to abnormal von Willebrand factor processing in TTP. Identification of the underlying abnormality in an individual patient can provide prognostically significant information in predicting long-term outcome, response to therapies, and transplant outcome. It also paves the way for the use of specific new therapies in the near future. Semin Nephrol 30:395-408 © 2010 Elsevier Inc. All rights reserved. Keywords: Hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, complement, ADAMTS13

emolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) share a lesion of vessel wall thickening (mainly arterioles or capillaries), intraluminal platelet thrombosis, and partial or complete obstruction of the vessel lumina that is called thrombotic microangiopathy (TMA). Laboratory features of thrombocytopenia and microangiopathic hemolytic anemia are almost invariably present in patients with TMA lesions and reflect consumption and disruption of platelets and erythrocytes in the microvasculature of kidney, brain, and other organs.1

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*Mario Negri Institute for Pharmacological Research, Clinical Research Center for Rare Diseases, Aldo e Cele Daccò, Villa Camozzi, Ranica, Bergamo, Italy. †Department of Nephrology and Dialysis, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Italy. Supported in part by grants from the Fondazione ART La Ricerca Sui Trapianti, Fondazione Aiuto Ricerca Malattie Rare, Istituto Superiore di Sanità, and the Telethon Foundation. Address reprint requests to Dr. Marina Noris, Mario Negri Institute for Pharmacological Research, Clinical Research Center for Rare Diseases, Aldo e Cele Daccò, Via Camozzi, 3–24020 Ranica (Bergamo), Italy. E-mail: [email protected] 0270-9295/ - see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.semnephrol.2010.06.006

Seminars in Nephrology, Vol 30, No 4, July 2010, pp 395-408

Injury to the endothelial cell is the central and likely inciting factor in the sequence of events leading to TMA. In HUS there is primarily glomerular endothelial injury, resulting in acute renal impairment, whereas in TTP there is predominantly brain microvascular endothelial cell injury, resulting in neurologic disturbance.1 Because HUS can involve brain manifestations and TTP can sometimes involve severe renal diseases, the two can be difficult to distinguish on clinical grounds only.2 Newly identified pathophysiologic mechanisms have allowed differentiation of the various clinical syndromes on the basis of the underlying etiogenesis. HEMOLYTIC UREMIC SYNDROME HUS is rare with an overall incidence of 2 cases for 100,000 persons/year, with a peak of 6.1 per 100,000/year in children younger than age 5. In adults ages 50 to 59 years the incidence approaches 0.5 per 100,000/year.3 395

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Most cases of HUS—more than 90% in children—are secondary to infection by certain strains of Escherichia coli (serotypes O157:H7, O111:H8, O103:H2, O123, O26, and others) or Shigella dysenteriae, which produce a powerful exotoxin, the Shiga-like toxin (Stx), and manifest with diarrhea, often bloody.4 Stx-producing E coli closely adhere to the epithelial cells of the gastrointestinal mucosa, causing destruction of brush-border villi. Stx is picked up by polarized gastrointestinal cells via transcellular pathways, translocates across the gut epithelium into the circulation, and is transported to capillary endothelial cells in the kidney and other organs. Through specific receptors, Stx then binds to glomerular endothelial cells, causing cell damage, and promotes a prothrombotic environment in renal microcirculation.5,6 Bacteriologic diagnosis rests on detection of free fecal Stx or isolation of colonies of E coli O157:H7 and other Stx-producing bacteria in sorbitolMacConkey stool cultures. Serologic diagnosis is based on identification by enzyme-linked immunosorbent assay of antibodies against Stx and against O157 and the other Stx-producing E coli serogroups. After exposure to Stx-E coli, 38% to 61% of individuals develop hemorrhagic colitis and 3% to 9% (in sporadic infections) to 20% (in epidemic forms) progress to overt HUS.7,8 Stx-HUS is characterized by prodromal diarrhea followed by acute renal failure. In typical Stx-HUS of children treatment is based on supportive management of anemia, renal failure, hypertension, and electrolyte and water imbalances. StxHUS has a relatively favorable outcome, 75% of patients retain normal renal function long term.9 However, 3% to 5% of patients die during the acute phase. Streptococcus pneumoniae causes a distinctive severe form of HUS accounting for 40% of cases unrelated to Stx-producing bacteria.10 S pneumoniae neuraminidase de-sialates glycocalyx of human cells, exposing the ThomsenFriedenreich antigen that binds preformed IgM antibodies.11 Approximately 10% of cases of HUS are not caused by either Stx-producing bacteria or S pneumoniae and are classified as atypical (aHUS) (incidence, 2 cases per 1,000,000/y).3,12

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aHUS has a poorer prognosis, with a mortality rate of up to 25% in the acute phase. Fifty percent of patients progress to end-stage renal disease (ESRD).3,10 Fewer than 20% of aHUS cases are familial. Reports date back to 1965, when Campbell and Carre13 described hemolytic anemia and azotemia in concordant monozygous twins. Since then, familial HUS has been reported in children and, less frequently, in adults. Although some were in siblings, suggesting autosomal-recessive transmission, others were across 2 to 3 generations, suggesting an autosomal-dominant mode.14 The prognosis is poor (cumulative incidence of death or ESRD, 50%-80%). Sporadic aHUS encompasses cases without family history of the disease. Triggering conditions for sporadic aHUS4 include human immunodeficiency virus infection, anticancer drugs (eg, mitomycin, cisplatin, bleomycin, and gemcitabine), immunotherapeutic agents (eg, cyclosporine, tacrolimus, OKT3, interferon, and quinidine), antiplatelet agents (ticlopidine and clopidogrel), malignancies, transplantation, and pregnancy.15 De novo posttransplant HUS has been reported in patients receiving renal transplants or other organs because of calcineurin inhibitors or humoral rejection. It occurs in 5% to 15% of renal transplant patients who receive cyclosporine and in approximately 1% of those who are given tacrolimus.16-18 Dose reduction or changing one calcineurin inhibitor for another sometimes results in recovery and suggests a causative role. In 10% to 15% of female patients with aHUS the disease manifests during pregnancy or postpartum.3,4 aHUS may present at any time during pregnancy but mostly in the last trimester and about the time of delivery. It is sometimes difficult to distinguish it from pre-eclampsia. The HEmolytic anemia, increased Liver enzymes, and Low Platelets (HELLP syndrome) is a lifethreatening disorder of the last trimester or parturition with severe thrombocytopenia, microangiopathic hemolytic anemia, renal failure, and liver involvement. These forms are always an indication for prompt delivery that is usually followed by complete remission.4 Postpartum HUS manifests within 3 months of delivery in most cases. The outcome is usually poor.

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About 50% of sporadic aHUS cases show no clear trigger (idiopathic HUS).

Complement Abnormalities During the past 10 years a clear link has been shown between genetic abnormalities of proteins of the complement system and familial aHUS, and also sporadic aHUS, mainly idiopathic. Mutations in cases of pregnancy-associated (including the HELLP syndrome), drug-induced, and de novo posttransplant sporadic aHUS19,20 also have been reported. Reduced serum levels of C3 with normal C4 levels were reported since 1974.21 In cases of familial aHUS, serum C3 levels were low even during remission, hinting of genetic defects.22 Low C3 levels reflected complement activation and consumption as documented by high levels of activated products, C3b, C3c, and C3d,23 and by C3 deposits in glomeruli and arterioles of biopsies during acute disease.21,24 The complement system is part of innate immunity and consists of several plasma and membrane-bound proteins that protect against invading organisms.25 Three activation pathways, the classic, lectin, and alternative pathways (APs), produce protease complexes, termed C3 and C5 convertases, that cleave C3 and C5, respectively, eventually leading to the membrane attack complex. The C3 convertases of the classic/lectin pathways are formed by C2 and C4 fragments, whereas the AP convertase requires cleavage of C3 only.25 Thus, low serum C3 levels in aHUS with normal C4 levels indicated selective AP activation.22 A variety of genetic abnormalities in members of the AP of complement have been described in aHUS, which account for about 60% of cases. In 1981 Thompson and Winterborn26 first described deficiency of the plasma regulator of the AP, complement factor H (CFH), in two brothers with aHUS. The parents were first cousins with half-normal CFH levels, indicating an inherited defect. Since then, complete or partial CFH deficiencies have been reported.22,27 It was only in 1998 that the seminal report by Warwicker et al28 showed linkage of aHUS to the chromosome 1q32 locus, containing genes for CFH and other complement regulators and found a heterozygous CFH mutation in cases and obligate

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carriers within one family and another heterozygous mutation in a sporadic case. More than 80 CFH mutations (interactive FH-HUS mutations database, http://www.FH-HUS.org) have been identified in aHUS patients (mutation frequency, 25%-30%).19,29-32 CFH is a plasma-protein containing 20 homologous short consensus repeats. CFH regulates the AP by competing with complement factor B (CFB) for C3b recognition, by acting as a cofactor for complement factor I (CFI), and by enhancing dissociation of C3 convertase.33 These functions are located in the N-terminal region. CFH additionally binds to glycosaminoglycans in basement membranes and endothelium via its C-terminal end.34 Therein, CFH down-regulates the AP activation on structures, like glomerular basement membranes, lacking other regulators, and contributes with membrane-bound regulators to endothelial protection. The vast majority of CFH mutations in aHUS are heterozygous, causing amino acid changes or translation interruption, and mainly cluster in the CFH C-terminus (http://www.FH-HUS. org). Most CFH mutants are secreted and can normally control complement in plasma.35 However, these CFH mutants bind poorly to glycosaminoglycans/endothelial cells and to surface-bound C3b, which results in lower density of mutant CFH on cell surfaces and diminished cofactor activity for C3b degradation.34-36 Less than 15% to 20% of CFH mutations in aHUS are homozygous, leading to quantitative CFH deficiency and very low C330 levels. Besides point mutations, complex abnormalities in the CFH genomic area have been reported. High-degree sequence identity between CFH and the genes CFHR1-5 -encoding fivefactor H–related proteins (CFHR) located in tandem to CFH may predispose to nonallelic recombinations. About 3% to 5% of patients with aHUS carry a heterozygous hybrid gene derived from an anomalous crossing-over between CFH and CFHR1, which contains the first 21 exons of CFH and the last two exons of CFHR1.37 The gene product lacks complement regulatory activity on the endothelial surface. About 6% to 10% of aHUS patients develop anti-CFH autoantibodies.38,39 They bind to CFH

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C-terminus and reduce CFH binding to C3b, and surface cofactor activity,40 without, however, influencing fluid-phase cofactor activity. Lack of complement control on cells despite control in fluid-phase mimics CFH mutations of aHUS. Anti-CFH antibodies seem genetically determined. Indeed, patients usually have homozygous deletion of the 84-kb genomic fragment with loss of CFHR1 and CFHR3.41,42 Another deletion, incorporating CFHR1 and CFHR4, recently has been reported in association with anti-CFH antibodies.43,44 Of note, patients lacking only CFHR3 do not develop anti-CFH antibodies,44 suggesting that generation of these antibodies is related specifically to CFHR1 deficiency. However, the causal link between CFHR1 deletion and the autoantibodies is still elusive. In 2003 two groups45,46 described mutations in MCP, encoding the widely expressed transmembrane regulator, membrane cofactor protein, in affected individuals of four families. MCP serves as a cofactor for CFI to cleave C3b and C4b on cell surfaces. MCP mutations account for about 10% of aHUS cases.19 Most are heterozygous, and about 25% are either homozygous or compound heterozygous (http:// www.FH-HUS.org). The majority cluster in critical extracellular modules for regulation. Expression on blood leukocytes was reduced for about 75% of mutants, causing a quantitative defect. Other mutants instead have low C3bbinding capability and decreased cofactor activity.19,47 CFI is a plasma serine protease that regulates the three complement pathways by cleaving C3b and C4b in the presence of cofactor proteins. CFI mutations affect 4% to 10% of aHUS patients.19,48-50 All mutations are heterozygous, 80% cluster in the serine-protease domain. Approximately 50% of mutations result in low CFI levels. Others disrupt C3b and C4b cleavage.19,48-50 Gain-of-function mutations that affect genes encoding the AP C3 convertase components CFB and C3 have been reported recently in aHUS.51,52 CFB mutations are rare (1%-2%).52 Patients have chronic AP activation with low C3 levels and, usually, normal C4 levels.52 CFB mutants have excess C3b affinity and form a hyper-

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active C3 convertase resistant to dissociation by DAF and CFH,52,53 which results in enhanced C3b deposition on endothelial cells.53 About 4% to 10% of aHUS patients have heterozygous mutations in C3, usually with low C3 levels.51 Most C3 mutations reduce C3b binding to CFH and MCP, severely impairing degradation of mutant C3b.51 Mutations in the gene THBD encoding thrombomodulin, a membrane-bound glycoprotein with anticoagulant properties that modulates complement activation on cell surfaces, have been associated very recently with aHUS.54 About 5% of aHUS patients carry heterozygous THBD mutations. Cells expressing these variants inactivate C3b less efficiently than cells expressing wild-type thrombomodulin.54 These data document a functional link between complement and coagulation, opening new perspectives for candidate gene research in aHUS. Finally, 3% to 5% of aHUS patients have combined mutations, usually in CFH with either MCP or CFI, each inherited from healthy parents.19,55 Few patients with mutations in CFH, CFI, MCP, or C3 also carrying anti-CFH antibodies have been reported.43

Impact of Genetic Testing on Clinical Practice A full analysis for all mutations in the earliermentioned genes and for anti-CFH antibodies is currently time consuming and does not influence the initial treatment of patients, which should be started as soon as possible. Low C3 levels are not a sensitive screening test for identifying complement abnormalities in aHUS because 40% to 70% of patients with mutations have normal C3 levels, with the only exception being patients with C3 mutations (80% with low C3 levels). However, screening for specific aHUS-predisposing defects may have profound implications for prevention of the disease in unaffected carriers and for long-term clinical management of affected patients (Table 1). Mutations in complement genes are predisposing rather than directly causal. aHUS penetrance among CFH or MCP or CFI mutation carriers is 40% to 50%.19 Healthy carriers of CFB, C3, and THBD mutations are being reported too.51,52,54,56 Hence, individuals with

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Table 1. Indication to Specific Therapy and to Transplantation on the Basis of the Specific

Underlying Abnormality in aHUS and TTP Abnormality Complement Anti-CFH antibodies

Plasma exchange combined with steroid or immunosuppressive therapy (remission in 70%-80% of cases)

CFH mutations

Plasma infusion/exchange (effective in 50%-60% of cases)

CFI mutations

Plasma infusion/exchange (remission in 30%-40% of cases)

C3 mutations

Plasma infusion/exchange (remission in 50%-60% of cases)

THBD mutations

Plasma infusion/exchange (effective in 50%-60% of cases)

CFB mutations

Plasma infusion/exchange (remission in 30%-40% of cases)

MCP mutations

Probably no indication to plasma (relatively good outcome with conservative therapy alone)

ADAMTS13 Anti-ADAMTS13 antibodies ADAMTS 13 deficiency

Transplantation (Comments)

Treatment (Comments)

Plasma exchange combined with steroid or immunosuppressive therapy or rituximab (life saving) Plasma infusion/exchange (life saving)

Yes, isolated kidney transplant (only if anti-CFH antibody titer is maintained at a low level by immunosuppressive therapy) No, isolated kidney transplant (80%-90% recurrence) Yes, combined liver-kidney transplant No, isolated kidney transplant (70%-80% recurrence) Yes, combined liver-kidney transplant Isolated kidney transplant risky (40%-50% recurrence) No indication to combined liver-kidney transplant* Probably no indication to isolated kidney transplant (2 post-transplant aHUS reported) No indication to combined liver-kidney transplant Probably no indication to isolated kidney transplant Probably no indication to combined liver-kidney transplant† Yes, isolated kidney transplant (10%-15% recurrence)

N/A

N/A

N/A, not applicable. *Because of substantial extrahepatic synthesis of C3. †These patients have severe vascular diseases that increase the surgical risks.

such mutations cannot be told that they will develop aHUS, but instead can be told that overall they have an approximately 50% chance of developing the disease. Conditions that trigger complement activation directly (bacterial and viral infections), or indirectly by causing

endothelial insult (drugs or pregnancy), precipitated the disease in approximately 60% of patients.3 Identification of unaffected family members with a mutation would allow monitoring such at-risk subjects, particularly during triggering events such as infections and pregnancies.

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In addition, carriers can be advised to avoid known precipitants of disease, such as the oral contraceptive pill. The phenotype of aHUS patients is very variable; although some develop early onset HUS and ESRD, others make complete recovery. The course and outcome of the disease are influenced by the genes involved. About 60% to 70% of patients with CFH, CFI, and C3 mutations and one third of children with anti-CFH autoantibodies lose renal function or die during the presenting episode, or develop ESRD after relapses.19,55,57 CFB mutations are associated with poor renal outcome (renal function loss in 7 of 8 patients).52,57 About 20% of patients with CFH mutations have cardiovascular complications and excess mortality. Indeed, long-term survival is worse in patients with CFH mutations (40%-50% at 10 years) than in those with CFI and C3 mutations or anti-CFH autoantibodies (80%-90% at 10 years).19,55,57 MCP-mutation carriers have a good prognosis (complete remission, 80%-90%). Recurrences are frequent but long-term outcome is good because 80% of patients remain dialysis-free.19,55,57 However, rare patients with MCP mutations had severe disease, immediate ESRD, intractable hypertension, and coma,19 possibly because of concurrent genetic abnormalities.58 Genotyping of patients also could allow optimization of treatment (Table 1). Plasma exchange or plasma infusion have become the first-line management of patients presenting with the features of aHUS. Of note, however, there are no controlled clinical trials of plasma therapy in aHUS and treatment is based on several case reports and retrospective series in the literature.57 Guidelines suggest that plasma therapy (plasma-exchange, 1-2 plasma volumes/d; plasma infusion, 20-30 mL/kg/d) should be started within 24 hours of diagnosis.57 Plasma exchange allows supplying larger amounts of plasma than would be possible with infusion, while avoiding fluid overload. Because CFH is a plasma protein, plasma infusion or exchange provides normal CFH to patients carrying CFH mutations.19,55 In two children with homozygous mutations and complete CFH deficiency, plasma infusion increased

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CFH from undetectable to 20% normal levels and stopped hemolysis.59,60 Plasma was given chronically every second week (estimated CFH half-life, 6 d) because HUS recurred rapidly after plasma tapering-off.60 Long-term treatment, however, may fail owing to development of plasma resistance. In a third child with CFH homozygous mutation, chronic plasma infusions maintained remission for 4 years, but thereafter the child had a relapse and developed ESRD.61 Heterozygous CFH mutation carriers usually have normal levels of CFH, but half molecules are dysfunctional. The beneficial effect of plasma is strongly dependent on amount, frequency, and modality of administration, with plasma exchange being superior to plasma infusion for remission and prevention of recurrencies.62,63 In respect to infusion, plasma exchange has the theoretical benefit of removing mutant CFH that could antagonize the normal protein. Among three sisters with a heterozygous CFH mutation and childhood-onset HUS, the oldest one not receiving plasma developed immediate ESRD. The two siblings receiving daily plasma exchange underwent remission. One sibling thereafter interrupted plasma therapy and had three HUS relapses and developed ESRD. The other sibling received prophylactic plasma exchanges and maintained normal renal function for 6 years.62 Overall published data19,55,57 in patients with CFH mutations show either complete or partial (hematologic normalization with renal sequelae) remission of 60% of plasma-treated episodes. Plasma exchange removes anti-CFH antibodies,38,55 however, the effect usually is transient. Immunosuppressants (corticosteroids and azathioprine or mycophenolate-mofetil) and rituximab, an anti-CD20 antibody, combined with plasma exchange allowed long-term, dialysisfree survival in 60% to 70% of patients.38-40,64 Measurement of anti-CFH antibodies in patients with aHUS could help identify those patients who potentially would benefit from combining plasma exchange with immunosuppressive therapy. Patients with CFI mutations show only a partial response to plasma and remission was achieved in about 30% to 40% treated episodes.19,55,57,65 Forty percent of patients with CFB mutations and 50% of those with C3 muta-

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tions responded to plasma infusion or exchange.51,52,57 It is possible that these patients need abundant and frequent plasma exchanges to clear the hyperfunctional mutant CFB and C3.57 Because MCP is a cell-associated protein, a beneficial effect of plasma therapy is unlikely to occur in patients with a mutation in MCP. Indeed, 80% to 90% of patients undergo remission of an acute episode, independently of plasma treatment.19,55,57,65 Of note, few reported patients with MCP mutations who developed severe forms of aHUS responded poorly to plasma therapy.19,66 The highest utility of genetic testing is in patients who are on renal replacement therapy and wish to be considered for a renal transplant (Table 1). Renal transplantation in aHUS is associated with an overall 50% rate of recurrence in the allograft.67,68 The outcome, however, varies according to the underlying genetic abnormality. Mutations in CFH, CFI, CFB, and C3 all lead to abnormalities in circulating proteins, mainly of liver source. These abnormalities persist after kidney transplantation. aHUS recurred in 80% to 90% of transplanted patients with CFH or CFI mutations.3,48,55,68 Intensive chronic plasma prophylaxis prevented recurrence and preserved renal graft function for 1 year in a patient with a CFH mutation69 but failed in another case.70 Patients with CFB and C3 mutations appear at risk of posttransplant recurrence as well.51,52 Data about the outcome of renal transplantation in patients with anti-CFH autoantibodies are limited but they suggest that there is a significant risk of recurrence. However, there is evidence that minimization of antibody titer by the use of plasma exchange in combination with immunosuppressants or rituximab enables a positive outcome of the graft.64 Live-related donation is contraindicated by high risk of recurrences71,72 and may expose the donor to the risk of de novo disease because the donor could carry the same disease-predisposing abnormality of the affected relative. An adult male with a heterozygous CFH mutation developed de novo HUS after donating a kidney to his child.72 If live donation is considered, it is essential that both the recipient and the donor undergo genotyping for CFH, MCP, CFI, C3,

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CFB, and THBD given the incomplete penetrance reported for mutations. Simultaneous kidney and liver transplant was performed in two children with aHUS and CFH mutations with the aim to correct the genetic defect and prevent recurrences.73,74 However, both cases were complicated by premature liver graft failure.73,74 It was reasoned that the surgical stress with ischemia/reperfusion induced complement activation in liver that could not be regulated because of transient CFH deficiency. A modified approach to the combined transplant was applied to 8 cases,75,76 including extensive plasma exchange before surgery to provide normal CFH until the liver graft recovered synthetic functions. This procedure was successful in 7 patients. However, another child76 developed severe hepatic thrombosis and fatal encephalopathy. The risks of kidney and liver transplantation require a careful assessment of benefits for candidate patients. Screening for mutations in complement genes should allow patients and clinicians to make informed decisions regarding listing transplantation based on risk of recurrence. A position paper76 has defined the groups of patients in whom isolated kidney transplantation is extremely risky, although a combined kidney-liver transplant is recommended (namely patients with CFH or CFI mutations), and those eligible for an isolated kidney transplantation (patients with MCP mutation; Table 1). THROMBOTIC THROMBOCYTOPENIC PURPURA TTP is a rare disease with an incidence of approximately 2 to 4 cases per million persons/ year.77 It is more common in women (female to male ratio, 3:2 to 5:2). Although the peak incidence is in the third and fourth decades of life, TTP can affect any age group.77,78 TTP classically presents with the pentad of thrombocytopenia, microangiopathic hemolytic anemia, fever, neurologic dysfunction, and renal dysfunction.79 Neurologic symptoms can be seen in more than 90% of patients during the entire course of the disease. Central nervous system involvement mainly represents thrombo-occlusive disease of the grey matter, but also can include headache, cranial nerve palsies, confusion, stupor, and coma. Renal

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impairment has been reported in around 25% of patients.78 In the microvasculature of patients with TTP, systemic platelet thrombi are developed, mainly formed by platelets and von Willebrand factor (VWF). This protein plays a major role in primary hemostasis, forming platelet plugs at sites of vascular injury under high shear stress. VWF is a large glycoprotein synthesized in vascular endothelial cells and megakaryocytes. The multimeric VWF is stored in the Weibel-Palade bodies of endothelial cells and ␣-granules of platelets. Upon stimulation, VWF is secreted by endothelial cells as ultra large multimers that form string-like structures attached to the endothelial cells, possibly through interaction with P-selectin. Under fluid shear stress, the ultra large VWF strings are cleaved to generate the range of VWF multimer sizes that normally circulate in the blood, from approximately 500 kd to 20 million d.80 The proteolytic cleavage of VWF multimers appears to be critical to prevent thrombosis in the microvasculature. Furlan et al81 and Tsai and Lian82 partially purified and characterized a plasma metalloprotease that physiologically reduces ultra large VWF multimers by cleaving VWF at the peptide bond between 842Y and 843M residues in the A2 domain of the subunit. The activity of this protease was found deficient in the majority of patients with TTP, leading to accumulation of ultra large VWF multimers that are highly reactive with platelets. In 2001 the protease was identified as ADAMTS13, the 13th member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin type 1 domains) family.83 This metalloprotease is encoded by the homonymous gene located on chromosome 9q34. ADAMTS13 is expressed predominantly in liver and consists of a N-terminal signal peptide, a propeptide, a reprolysin-like metalloprotease domain, a disintegrinlike domain, a first thrombospondin type-1 motif (TSP1), a cysteine-rich domain, a spacer domain, seven additional TSP1 repeats, and two CUB domains.

ADAMTS13 Defects in TTP Two mechanisms for deficiency of the ADAMTS13 activity have been identified in patients with TTP, an acquired deficiency re-

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sulting from the formation of anti-ADAMTS13 autoantibodies (acquired TTP), and a genetic deficiency resulting from homozygous or compound heterozygous mutations in ADAMTS13 gene (congenital TTP) (Table 1). Acquired TTP is an immune-mediated, nonfamilial form of TTP that most likely accounts for the majority of cases (from 60% to 90%) reported to date as acute idiopathic or sporadic TTP. The disease is characterized by a severe deficiency of ADAMTS13 whose activity is inhibited by specific autoantibodies that develop transiently and tend to disappear during remission.77,82,84,85 These inhibitory anti-ADAMTS13 antibodies are mainly IgG,82,84,86 although IgM and IgA anti-ADAMTS13 antibodies also have been described.86 Patients with TTP secondary to hematopoietic stem cell transplantation, malignancies, or human immunodeficiency virus infection rarely have severe ADAMTS13 deficiency and inhibitory IgG antibodies.87 TTP associated with ticlopidine and clopidogrel (thienopyridine drugs that inhibit platelet aggregation) represent interesting exceptions of secondary TTP consistent with a drug-induced autoimmune disorder. Severe ADAMTS13 deficiency and ADAMTS13 inhibitory antibodies were detected in 80% to 90% of patients with ticlopidine-associated TTP87 and in few patients with clopidogrel-induced TTP.87 The deficiency resolved after the drugs were discontinued. The ADAMTS13 epitopes recognized by autoantibodies have been mapped and so far all plasmas contain at least some antibodies directed against the Cys-rich/spacer domain.88,89 In some cases the antibodies were directed only against these epitopes, but in the majority of patients combinations of antibodies were found, including antibodies against the CUB domains in approximately 64%, against the first TSP1 repeat in 56%, against TSP1 repeats 2 to 8 in 28%, and against the ADAMTS13 propeptide in 20% of patients.89 When cloned and prepared as monoclonal antibodies, many of these antibodies inhibit ADAMTS13 activity in vitro and in vivo in mice.87 Evidence of the pathogenetic role of TTP-associated anti-ADAMTS13 autoantibodies is derived by finding that they usually disappear from the circulation when remission is

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achieved by effective treatment and this occurs in parallel with the normalization of ADAMTS13 activity. In patients with acquired ADAMTS13 deficiency a relapse risk as high as 50% has been reported,78 and undetectable ADAMTS13 activity and persistence of antiADAMTS13 inhibitors during remission are predictor of recurrences. Congenital TTP is a rare form associated with a genetic defect of ADAMTS13 and accounts for about 5% of all of cases of TTP.84 TTP associated with congenital ADAMTS13 deficiency presented either in families or in patients with no familial history of the disease.83-85 In both cases the disease is inherited as a recessive trait, as documented by the fact that ADAMTS13 levels in unaffected relatives of patients fell into a bimodal distribution with a group with halfnormal levels, consistent with carriers, and the other with normal values. In 2001, Levy et al,83 by means of linkage analysis performed in 4 pedigrees with congenital TTP, showed that mutations in the ADAMTS13 gene, resulting in severely reduced ADAMTS13 activity in plasma, are responsible for the autosomal-recessive inheritance of the disease. To date more than 80 ADAMTS13 mutations have been identified in patients with TTP.90 Approximately 60% of these mutations are missense, causing single amino acid substitutions, and the remaining are nonsense, deletions or insertions causing frameshifts, or splice site mutants leading to a truncated protein. Although the mutations that result in a truncated protein are distributed equally throughout the molecule, more than 75% of the missense mutations cluster in the first half of the protein (from the metalloprotease through the Tsp-1 to -2 domains).90 Most patients are carriers of compound heterozygous mutations; only 15 mutations have been observed in homozygous form. Studies on secretion and activity of the mutated forms of the protease showed that most of these mutations led to an impaired secretion from the cells, and, when the mutated protein is secreted, the proteolytic activity is greatly reduced.91

Clinical Impact of ADAMTS13 Testing in TTP In the first two large studies of ADAMTS13 activity in patients with thrombotic microan-

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giopathies,82,84 severe deficiency of ADAMTS13 activity (⬍10% activity) was found in the large majority of patients with a diagnosis of TTP but not in those with HUS. This observation generated the paradigm that a deficiency of ADAMTS13 activity may point to a specific diagnosis of TTP. Clinical limitations of ADAMTS13 measurements became apparent when testing became more widely available. Greater experience revealed that some patients with characteristic clinical features of TTP did not have severely deficient ADAMTS13 activity and that patients with other disorders, such as sepsis and cirrhosis, did have severe ADAMTS13 defects.92,93 Emerging data indicate that patients with a clinical diagnosis of HUS85,94 also may have a complete lack of ADAMTS13 activity, albeit less frequently. Thus, on clinical grounds a possible defect of ADAMTS13 cannot be excluded only on the basis of predominant renal localization of disease manifestation, and measurement of ADAMTS13 activity should be performed in HUS patients as well. However, severe ADAMTS13 deficiency remains the most important risk factor for TTP and documentation of ADAMTS13 deficiency can be important to confirm diagnosis. If ADAMTS13 activity is found to be less than 10%, detection of antiADAMTS13 antibodies should be undertaken. Search for mutations in the ADAMTS13 gene should be performed in patients with less than 10% activity and no antibodies. ADAMTS13 measurements can provide important information for prognosis. Patients with anti-ADAMTS13 antibodies experience a more severe manifestation of the disease and have a higher mortality rate than patients without antibodies.92 High titers of anti-ADAMTS13 autoantibodies are correlated with relapsing disease and poor prognosis. Approximately 60% of patients with congenital deficiency of ADAMTS13 experience their first acute episode of disease in the neonatal period or during infancy, but a second group (10%-20%) manifests the disease after the third decade of life. TTP recurrences are common but their frequency varies widely. Emerging data suggest that the type and location of ADAMTS13 mutations may influence the age at

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onset of TTP and the penetrance of the disease in mutation carriers.90 One of the most frequently reported ADAMTS13 mutations, the 4143-4144insA in the second CUB domain, leading to a frameshift and loss of the last 49 amino acids of the protein, is associated with neonatalchildhood onset, indeed only 1 of 16 reported carriers, either homozygous or compound heterozygous with other ADAMTS13 mutations, reached adult age without developing TTP.90 In vitro expression studies revealed that the mutation causes a severe impairment of protein secretion combined with a strongly reduced specific protease activity. On the other hand, mutations in the sixth and the seventh TSP190 appear to lead to an adult onset and a milder course of TTP. Expression studies revealed that these mutations result in severe defects in secretion of the metalloprotease, although a small fraction of the mutant protein is released in the supernatant, but the mutants maintain normal specific protease activity.90,95 It is possible that in carriers of these mutations some ADAMTS13 activity is present in the circulation, which is enough to prevent onset of the disease in childhood or even in adulthood. The latter possibility is supported by descriptions of asymptomatic carriers of such mutations who never developed TTP.90,91 Plasma manipulation is a cornerstone in the therapy of the acute episode of all forms of TTP1 and measurement of ADAMTS13 activity is not essential for the critical initial management decision. However, identification of severely deficient ADAMTS13 activity combined or not with autoantibodies can provide important information for decisions regarding adjunctive treatment (Table 1). For example, in patients with anti-ADAMTS13 antibodies, as compared with infusion, exchange may offer the advantage of also rapidly removing anti-ADAMTS13 antibodies. This, however, needs to be proven in controlled trials. Corticosteroids might be of benefit in autoimmune forms of TTP by inhibiting the synthesis of anti-ADAMTS13 autoantibodies. In a series of 35 patients with undetectable ADAMTS-13 activity and anti-ADAMTS13 antibodies, combined treatment with plasma exchange and prednisone was associated with disease remission in around 90% of cases.86 The

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rationale of combined treatment is that plasma exchange will have only a temporary effect on the presumed autoimmune basis of the disease and additional immunosuppressive treatment may cause a more durable response. Recent prospective studies successfully and safely have used rituximab in patients who had failed to respond to standard daily plasma exchange and methylprednisolone and in patients with relapsed acute TTP who previously had shown antibodies to ADAMTS13.96 Treatment was associated with clinical remission in all patients, disappearance of anti-ADAMTS13 antibodies, and with an increase of ADAMTS13 activity to levels greater than 10%. Rituximab also has been used electively to prevent relapses in patients with autoantibodies and recurrent disease.77,96,97 In one study, 5 patients with persistent undetectable ADAMTS13 activity and high titers of autoantibody were treated with rituximab as pre-emptive therapy during remission. ADAMTS13 activity ranged from 15% to 75% and disappearance of inhibitors was achieved after 3 months in all patients, and activity was still greater than 20% at 6 months. Three patients maintained a disease-free status after 29, 24, and 6 months, respectively.90,97 Relapses were documented at 13 and 51 months in the remaining 2 patients during follow-up evaluation. Longitudinal evaluation of ADAMTS13 activity and autoantibody levels may help monitor patient response to treatment. Re-treatment with rituximab should be considered when ADAMTS13 activity decreases and inhibitors reappear into the circulation, to avoid a relapse. In contrast, in patients with congenital ADAMTS13 deficiency plasma infusion may be enough to induce remission of the disease by replacing defective protease activity (Table 1). Actually, providing just a 5% normal enzymatic activity is sufficient to degrade large VWF multimers, which may be relevant to induce remission of the microangiopathic process, and this effect is sustained over time because of the relatively long half-life (2-4 d) of the protease. Although some patients with congenital ADAMTS13 deficiency depend on frequent chronic plasma infusions to prevent recurrences, many patients who achieved clinical remission after plasma treatment remain in a disease-free status for

Genetics and genetic testing in HUS and TTP

long periods of time after plasma discontinuation, despite the absence of protease activity.90 No information is available on whether the type and position of ADAMTS13 mutations may affect the amount and the frequency of plasma therapy needed to maintain remission. CONCLUSIONS Multiple and complex genetic and acquired abnormalities that are associated with aHUS and TTP have been identified. Hopefully, this will translate into an improvement in the management of these diseases and pave the way for tailored treatments such as complement inhibitors and plasma-purified or recombinant CFH for aHUS, or recombinant ADAMTS13 for TTP. Currently, a number of complement inhibitors are under preclinical and clinical development. Phase III clinical trials showed efficacy and tolerance of the humanized anti-C5 monoclonal antibody eculizumab in paroxysmal nocturnal hemoglobinuria. That C5 blockade could be effective to induce remission of acute episodes of aHUS or to treat recurrence in the graft is supported by recent case reports.98-102 Results from single cases should be taken cautiously and eculizumab efficacy in aHUS needs confirmation in controlled trials. Because eculizumab leaves upstream complement activation intact, high levels of C3-activated products still might trigger inflammation,103 sustaining microangiopathic injury. Open-label controlled trials of eculizumab in patients with aHUS are ongoing (www.ClinicalTrials.gov). REFERENCES 1. Ruggenenti P, Noris M, Remuzzi G. Thrombotic microangiopathies. In: Wilcox C, editor. Therapy in nephrology & hypertension a companion to Brenner & Rector’s the kidney. 3rd ed. Philadelphia: Saunders Elsevier; 2008. p. 294-312. 2. Remuzzi G. HUS and TTP: variable expression of a single entity. Kidney Int. 1987;32:292-308. 3. Noris M, Remuzzi G. Atypical hemolytic-uremic syndrome. N Engl J Med. 2009;361:1676-87. 4. Besbas N, Karpman D, Landau D, et al. A classification of hemolytic uremic syndrome and thrombotic thrombocytopenic purpura and related disorders. Kidney Int. 2006;70:423-31. 5. Muthing J, Schweppe CH, Karch H, Friedrich AW. Shiga toxins, glycosphingolipid diversity, and endothelial cell injury. Thromb Haemost. 2009;101:252-64.

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