In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a disease characterized by microvascular thrombosis, often associated with deficiency of the vonWillebrand 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.

Genetics of HUS: the impact of MCP, CFH, and IF mutations on clinical presentation, response to treatment, and outcome.

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. MCP-mutated 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.

Complement factor H mutation in familial thrombotic thrombocytopenic purpura with ADAMTS13 deficiency and renal involvement.

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.

Hemolytic uremic syndrome: a fatal outcome after kidney and liver transplantation performed to correct factor h gene mutation.

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.

Complement factor H mutations and gene polymorphisms in haemolytic uraemic syndrome: the C-257T, the A2089G and the G2881T polymorphisms are strongly associated with the disease.

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.

Single Strand Conformation Polymorphism (SSCP) as a quick and reliable method to genotype M235T polymorphism of angiotensinogen gene.

OBJECTIVES: Conflicting results on the relationship between M235T polymorphism of angiotensinogen (AGT) gene and diabetic nephropathy are reported in the literature, probably due to the small number of subjects, to different inclusion criteria and the different genotype analysis methods used. The aim of the present study was to set up a fast, cheap and reliable method to allow the genotyping of M235T polymorphism in a large number of subjects. DESIGN AND METHODS: We developed in our laboratory a new specifically designed PCR-SSCP method for M235T genotyping whose specificity was compared with that of Allele Specific PCR (ASPCR) and Mutagenically Separated PCR (MS-PCR). The exact M235T genotype was estabilished by direct sequencing. The new PCR-SSCP method was then used to genotype a population of 1171 hypertensive, normoalbuminuric type II diabetes mellitus patients. The patients were also genotyped for ACE I/D polymorphism. For comparison a group of hypertensive non diabetic patients (n = 88) were also screened. RESULTS: The PCR-SSCP method identified the M235T polymorphism with no misinterpretation at variance with ASPCR and MS-PCR methods that showed a preferential amplification of the T allele. The rare Y248C polymorphism of the AGT gene was also detected by PCR-SSCP. In diabetic hypertensive patients the prevalence of TT genotype was higher than in normotensive healthy controls and equivalent to that found in hypertensive non diabetic patients. CONCLUSIONS: The PCR-SSCP method for detection of M235T polymorphism is a powerful and sensitive tool for rapid, cheap and efficient screening of a large number of samples. The results obtained with this method demonstrate an association of the TT genotype of AGT gene with hypertension, both in diabetic and non diabetic patients.