Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy with manifestations of hemolytic anemia, thrombocytopenia, and renal impairment. Genetic studies have shown that mutations in complement regulatory proteins predispose to non-Shiga toxin-associated HUS (non-Stx-HUS). We undertook genetic analysis on membrane cofactor protein (MCP), complement factor H (CFH), and factor I (IF) in 156 patients with non-Stx-HUS. Fourteen, 11, and 5 new mutational events were found in MCP, CFH, and IF, respectively. Mutation frequencies were 12.8%, 30.1%, and 4.5% for MCP, CFH, and IF, respectively. MCP mutations resulted in either reduced protein expression or impaired C3b binding capability. MCPmutated patients had a better prognosis than CFH-mutated and nonmutated patients. In MCP-mutated patients, plasma treatment did not impact the outcome significantly: remission was achieved in around 90% of both plasma-treated and plasma-untreated acute episodes. Kidney transplantation outcome was favorable in patients with MCP mutations, whereas the outcome was poor in patients with CFH and IF mutations due to disease recurrence. This study documents that the presentation, the response to therapy, and the outcome of the disease are influenced by the genotype. Hopefully this will translate into improved management and therapy of patients and will provide the way to design tailored treatments. (Blood.

Mutations in complement factor H (HF1) gene have been reported in non-Shiga toxin-associated and diarrhoea-negative haemolytic uraemic syndrome (D-HUS). We analysed the complete HF1 in 101 patients with HUS, in 32 with thrombotic thrombocytopenic purpura (TTP) and in 106 controls to evaluate the frequency of HF1 mutations, the clinical outcome in mutation and non-mutation carriers and the role of HF1 polymorphisms in the predisposition to HUS. We found 17 HF1 mutations (16 heterozygous, one homozygous) in 33 HUS patients. Thirteen mutations were located in exons XXII and XXIII. No TTP patient carried HF1 mutations. The disease manifested earlier and the mortality rate was higher in mutation carriers than in non-carriers. Kidney transplants invariably failed for disease recurrences in patients with HF1 mutations, while in non-mutated patients half of the grafts were functioning after 1 year. Three HF1 polymorphic variants were strongly associated with D-HUS: -257T (promoter region), 2089G (exonXIV, silent) and 2881T (963Asp, SCR16). The association was stronger in patients without HF1 mutations. Two or three disease-associated variants led to a higher risk of HUS than a single one. Analysis of available relatives of mutated patients revealed a penetrance of 50%. In 5/9 families the proband inherited the mutation from one parent and two disease-associated variants from the other, while unaffected carriers inherited the protective variants. In conclusion HF1 mutations are frequent in patients with D-HUS (24%). Common polymorphisms of HF1 may contribute to D-HUS manifestation in subjects with and without HF1 mutations.

Thrombotic thrombocytopenic purpura is a rare disorder of small vessels that is associated with deficiency of the von Willebrand factor-cleaving protease ADAMTS13, which favors platelet adhesion and aggregation in the microcirculation. The disease manifests mainly with central nervous system symptoms, but cases of renal insufficiency have been reported. Presented are findings of the genetic basis of phenotype heterogeneity in thrombotic thrombocytopenic purpura in two sisters within one family. The patients had ADAMTS13 deficiency as a result of two heterozygous mutations (causing V88M and G1239V changes). In addition, a heterozygous mutation (causing an S890I change) in factor H of complement was found in the patient who developed chronic renal failure but not in her sister, who presented with exclusive neurologic symptoms. 16: 117716: -118316: , 200516: . doi: 10.1681 T hrombotic thrombocytopenic purpura (TTP) is a disease of small vessels characterized by anemia that is caused by erythrocyte fragmentation in the microcirculation and thrombocytopenia that is caused by intravascular thrombi of aggregated platelets (1). Recent studies provided substantial evidence that 70 to 80% of cases of TTP are triggered by a deficiency of ADAMTS13 (2-4), a plasma metalloprotease that cleaves von Willebrand factor multimers soon after their secretion by endothelial cells (1,5-7). ADAMTS13 deficiency can be constitutive, as a result of homozygous or double heterozygous mutations in the corresponding gene (8 -13), or acquired, as a result of the presence of circulating inhibitory antibodies (1,3,4,14 -20). J Am Soc NephrolTTP manifests mainly with central nervous system symptoms, but cases of renal insufficiency have been reported (1). In rare cases, renal involvement is severe enough to cause endstage renal failure (1,21-25). Those patients' clinical manifestations are difficult to distinguish from those of hemolytic uremic syndrome (HUS), a form of thrombotic microangiopathy characterized by predominant renal involvement, often with renal failure (1,20,26). This difficulty has given rise to a heated debate on whether a severe deficiency of ADAMTS13 activity is enough to distinguish TTP from HUS (27,28).Here we present findings of the genetic basis of phenotype heterogeneity in patients with congenital ADAMTS13 deficiency. We studied a family with two affected sisters, one who presented with exclusive neurologic symptoms and the other one with severe renal involvement that required chronic dialysis. These diverse clinical manifestations suggested to us that the genetic background could be different. Materials and Methods PatientsA woman, now 60 yr old (F48), and her younger sister (F45, died in 2002 at the age of 55 yr) were referred to our International Registry of Recurrent and Familial HUS/TTP in 1996 because of history of recurrent and familial thrombotic microangiopathy. The youngest brother died at the age of 15 yr of leukemia. The other four siblings (three male and one female) all seem to be healthy and have no sig...

Factor H-associated hemolytic uremic syndrome (HUS) is a genetic form of thrombotic microangiopathy characterized by deficient factor H (HF-1) levels/activity and uncontrolled complement activation. The disorder mostly leads to end-stage renal disease and often recurs after kidney transplantation. We previously demonstrated that in a child with HF-1-associated HUS a simultaneous kidney and liver transplantation restored the defective HF-1 with no recurrence of the disease in the transplanted kidney.Here we describe a second childhood case of HF-1-associated HUS treated by combined kidney and liver transplant and complicated by a fatal, primary non-function of the liver graft. Graft hypoperfusion during surgery triggered ischemia/reperfusion changes and complement activation. Conceivably, as a result of defective complement regulatory potential, massive shedding of vascular heparan sulfates was documented in the transplanted liver. This might have impaired the physiological thromboresistance of vascular endothelium ending with widespread microvascular thrombosis and infarction. This case indicates that more fundamental research is needed before combined liver and kidney transplant is considered an option for children with HF-1-associated HUS.

SummaryThrombotic thrombocytopenic purpura (TTP) is a disease characterized by microvascular thrombosis, often associated with deficiency of the von Willebrand factor (VWF) cleaving protease ADAMTS13.We investigated the spectrum of ADAMTS13 gene mutations in patients with TTP and congenital ADAMTS13 deficiency to establish the consequences on ADAMTS13 processing and activity. We describe five missense (V88M, G1239V, R1060W, R1123C and R1219W), 1 nonsense (W1016Stop) and 1 insertion (82_83insT) mutations. In two patients no mutation was identified despite undetectable protease activity. Expression in HEK293 mammalian cells (V88M, G1239V, R1123C and R1219W) documented that three missense mutants were not secreted, whereas theV88M was secreted at low levels and with reduced activity. We also provide evidence that impaired secretion of ADAMTS13 mutants observed in vitro translates into severely reduced ADAMTS13 antigen levels in patients in vivo. To evaluate whether the small amounts of mutant protease present in the circulation of patients had VWF cleaving activity, WT and mutant rADAMTS13 were stably expressed in Drosophila S2 cells under the influence of the Drosophila BiP protein signal sequence, which allows protein secretion. Drosophila expression system showed a 40–60% protease activity in the mutants. Several single nucleotide polymorphisms (SNPs) within exons and intron boundaries were found in patients, suggesting that the interplay of SNPs could at least in part account for ADAMTS13 functional abnormalities in patients without mutations. In conclusion, defective secretion and impaired activity of the mutants concur to determine an almost complete deficiency of ADAMTS13 activity in patients with a homozygous or two heterozygous ADAMTS13 mutations.

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Thrombotic thrombocytopenic purpura (TTP) is a rare disorder of small vessels that is associated with deficiency of the VWF cleaving protease ADAMTS13, causing life-threatening disseminated microvascular thrombosis. Here we report four missense mutations (V88M, G1239V, R1219W, R1123C) in three patients with congenital ADAMTS13 deficiency. The three subjects carrying the mutations had less than 10% normal ADAMTS13 plasma antigen levels (measured by ELISA), which is consistent with previously reported data showing that most ADAMTS13 mutations in TTP patients result in impaired secretion of the protein. To evaluate whether the small amount of the mutant protease present in patients’ plasma had proteolytic activity, we cloned the ADAMTS13 cDNA in the pMT/Bip/His Drosophila expression vector and introduced the respective mutations by directed mutagenesis technique using wild-type cDNA as a template. These wild-type and mutant constructs were stably transfected into S2 Drosophila cells under the influence of the Drosophila BiP protein signal sequence, which allows the protein to be secreted into the medium and overcomes impaired secretion caused by the mutations. The induction of the histidine-tagged ADAMTS13 recombinants following the addition of copper was analyzed by Western blotting using an anti-hexahistidine monoclonal antibody. Equal amounts of recombinant ADAMTS13 proteins were used to evaluate proteolytic activity of recombinant proteins by cleavage of the rVWF A1-A2-A3 substrate. The proteolytic carboxyl terminal product of about 30 kDa was visualized by Western blot with a mouse monoclonal antibody directed against an epitope contained within the A3 domain of VWF. All the four missense mutations exhibited reduced activity: V88M:40%, G1239V: 66%, R1219W: 62% and R1123C: 64% of wild type activity . The results were also confirmed by collagen binding assay. For the the V88M mutation (located in the metalloprotease domain), decreased activity was confirmed by kinetic studies. In conclusion, mutant ADAMTS13 proteins found in patients with TTP, besides defective secretion, also have reduced protease activity. The mechanisms of the deficiency will be the matter of further studies.