An unusual case of haemolytic uraemic syndrome following endoscopic retrograde cholangiopancreatography rapidly improved with eculizumab.

Atypical haemolytic uraemic syndrome (aHUS) is a rare but life-threatening complement system-related disorder, characterized by renal failure, non-immune haemolytic anaemia and thrombo-cytopenia. We report on a young woman who developed a pancreatitis-induced aHUS following a routine procedure of endoscopic retrograde cholangiopancreatography. The patient was successively treated by 2 plasma exchanges with fresh frozen plasma and eculizumab, a monoclonal antibody designed to block terminal complement activation. The last treatment resulted in the immediate improvement of haemolytic parameters and to the definitive suspension of plasma exchanges. This is likely the first description of the use of a complement inhibitor to treat post-pancreatitis aHUS. We discussed treatment options and concluded that eculizumab could be a beneficial alternative to plasma exchanges in the management of such complications.

The role of ADAMTS-13 activity and complement mutational analysis in differentiating acute thrombotic microangiopathies.

UNLABELLED: ESSENTIALS: Molecular diagnostics has improved the differentiation of acute thrombotic microangiopathys (TMAs). Atypical hemolytic uremic syndrome may have features mimicking thrombotic thrombocytopenic purpura. We identified novel complement mutations and a high incidence of CD46, with favorable long term outcomes. Complement mutation analysis in TMA where the diagnosis is unclear and ADAMTS-13 activity is >10%. BACKGROUND: Differentiation of acute thrombotic microangiopathy (TMA) at presentation has historically been dependent on clinical parameters. Confirmation of thrombotic thrombocytopenic purpura (TTP) is increasingly reliant on demonstrating deficient ADAMTS-13 activity. The identification of alternative complement pathway abnormalities in atypical hemolytic uremic syndrome (aHUS), along with the proven efficacy of terminal complement inhibitors in treatment, has increased the need for rapid differentiation of TTP from aHUS. OBJECTIVES: We describe the clinical phenotype and nature of complement mutations in a cohort of aHUS patients referred as acute TMAs. PATIENTS/METHODS: Fourteen consecutive aHUS patients were screened for mutations in C3, CD46, CFH, CFI, and CFB, as well as factor H (FH) antibodies. All aHUS patients had ADAMTS-13 activity > 10%. RESULTS: Of 14 aHUS patients, 11 (79%) had platelet counts < 30 × 10(9) /L during the acute phase. Median presenting creatinine level was 295 µmol L(-1) , while five (36%) of 14 presented with a serum creatinine level < 200 µmol L(-1) . Alternative complement pathway mutations were detected in 9 (64%) of 14 patients, including CD46 mutations in five (36%) of 14 patients. Patients were identified with novel mutations in CFB and C3 that have not been previously reported. CONCLUSIONS: We demonstrate that diagnostic differentiation based on platelet count and renal function is insufficient to predict an underlying complement mutation in some aHUS cases. Specifically, we demonstrate a high frequency of functionally significant CD46 mutations which may mimic TTP. ADAMTS-13 activity > 10% in a patient with a TMA should necessitate genetic screening for complement abnormalities.

A national specialized service in England for atypical haemolytic uraemic syndrome-the first year's experience.

BACKGROUND: In 2013 NHS England commissioned the use of eculizumab for both new patients with atypical haemolytic uraemic syndrome (aHUS) and those undergoing transplantation. This national service is delivered locally but coordinated by an expert centre at the Newcastle upon Tyne Hospitals NHS Foundation Trust. RESULTS: In the first year of service, 43 aHUS patients received eculizumab, 15 children and 28 adults. Twenty-three were new patients and 20 prevalent. Fifteen of the 23 new patients required dialysis before eculizumab was started, 8 of these recovered renal function. Twelve of the 20 prevalent patients who received eculizumab were transplant patients, 8 with prophylactic use and 4 for recurrent disease; the outcome in all was good. Eculizumab was withdrawn in 14 patients, 5 were patients who had not recovered renal function. In 3 of the 14 patients, it was necessary to reintroduce eculizumab because of recurrent disease (2 extra-renal and 1 renal). There were 2 deaths in the 43 patients, and neither was associated with use of eculizumab. There were no episodes of meningococcal disease. CONCLUSIONS: The establishment of this national service has enabled aHUS patients in England to receive eculizumab when they need it for as long as they need it.

Thrombotic Microangiopathy as a Cause of Chronic Kidney Transplant Dysfunction: Case Report Demonstrating Successful Treatment with Eculizumab.

Atypical hemolytic uremic syndrome is a rare disease associated with genetic or acquired defects in complement regulation which frequently leads to renal failure. Disease often recurs early after kidney transplantation, leading to a rapid irreversible loss of function. Extrarenal features, such as hemolysis and thrombocytopenia, may not always occur, and diagnosis is made by demonstrating the classic features of thrombotic microangiopathy on renal biopsy. Eculizumab, a terminal complement inhibitor, has been used successfully to treat fulminant early recurrent disease after transplantation. We describe a case of disease recurrence presenting in the second year after transplantation with a gradual decline in function and the first report of eculizumab treatment for chronic thrombotic microangiopathy in a transplanted kidney. The resultant diagnostic challenges and successful response to eculizumab in this setting are discussed.

A novel method for direct measurement of complement convertases activity in human serum.

Complement convertases are enzymatic complexes that play a central role in sustaining and amplification of the complement cascade. Impairment of complement function leads directly or indirectly to pathological conditions, including higher infection rate, kidney diseases, autoimmune- or neurodegenerative diseases and ischaemia-reperfusion injury. An assay for direct measurement of activity of the convertases in patient sera is not available. Existing assays testing convertase function are based on purified complement components and, thus, convertase formation occurs under non-physiological conditions. We designed a new assay, in which C5 blocking compounds enabled separation of the complement cascade into two phases: the first ending at the stage of C5 convertases and the second ending with membrane attack complex formation. The use of rabbit erythrocytes or antibody-sensitized sheep erythrocytes as the platforms for convertase formation enabled easy readout based on measurement of haemolysis. Thus, properties of patient sera could be studied directly regarding convertase activity and membrane attack complex formation. Another advantage of this assay was the possibility to screen for host factors such as C3 nephritic factor and other anti-complement autoantibodies, or gain-of-function mutations, which prolong the half-life of complement convertases. Herein, we present proof of concept, detailed description and validation of this novel assay.

Terminal complement inhibitor eculizumab in atypical hemolytic-uremic syndrome.

BACKGROUND: Atypical hemolytic-uremic syndrome is a genetic, life-threatening, chronic disease of complement-mediated thrombotic microangiopathy. Plasma exchange or infusion may transiently maintain normal levels of hematologic measures but does not treat the underlying systemic disease. METHODS: We conducted two prospective phase 2 trials in which patients with atypical hemolytic-uremic syndrome who were 12 years of age or older received eculizumab for 26 weeks and during long-term extension phases. Patients with low platelet counts and renal damage (in trial 1) and those with renal damage but no decrease in the platelet count of more than 25% for at least 8 weeks during plasma exchange or infusion (in trial 2) were recruited. The primary end points included a change in the platelet count (in trial 1) and thrombotic microangiopathy event-free status (no decrease in the platelet count of >25%, no plasma exchange or infusion, and no initiation of dialysis) (in trial 2). RESULTS: A total of 37 patients (17 in trial 1 and 20 in trial 2) received eculizumab for a median of 64 and 62 weeks, respectively. Eculizumab resulted in increases in the platelet count; in trial 1, the mean increase in the count from baseline to week 26 was 73×10(9) per liter (P<0.001). In trial 2, 80% of the patients had thrombotic microangiopathy event-free status. Eculizumab was associated with significant improvement in all secondary end points, with continuous, time-dependent increases in the estimated glomerular filtration rate (GFR). In trial 1, dialysis was discontinued in 4 of 5 patients. Earlier intervention with eculizumab was associated with significantly greater improvement in the estimated GFR. Eculizumab was also associated with improvement in health-related quality of life. No cumulative toxicity of therapy or serious infection-related adverse events, including meningococcal infections, were observed through the extension period. CONCLUSIONS: Eculizumab inhibited complement-mediated thrombotic microangiopathy and was associated with significant time-dependent improvement in renal function in patients with atypical hemolytic-uremic syndrome. (Funded by Alexion Pharmaceuticals; C08-002 ClinicalTrials.gov numbers, NCT00844545 [adults] and NCT00844844 [adolescents]; C08-003 ClinicalTrials.gov numbers, NCT00838513 [adults] and NCT00844428 [adolescents]).

Postpartum aHUS secondary to a genetic abnormality in factor H acquired through liver transplantation.

We report here a young female who underwent a successful deceased donor liver transplant for hepatic vein thrombosis. Five years after transplantation she developed postpartum atypical hemolytic uremic syndrome (aHUS). She did not recover renal function. Mutation screening of complement genes in her DNA did not show any abnormality. Mutation screening of DNA available from the donor showed a nonsense CFH mutation leading to factor H deficiency. Genotyping of the patient showed that she was homozygous for an aHUS CD46 at-risk haplotype. In this individual, the development of aHUS has been facilitated by the combination of a trigger (pregnancy), an acquired rare genetic variant (CFH mutation) and a common susceptibility factor (CD46 haplotype).

Complement polymorphisms: geographical distribution and relevance to disease.

The evolution of man has been characterised by recurrent episodes of migration and settlement with infectious disease a constant threat. This long history of demographic change, together with the action of evolutionary forces such as natural selection and genetic drift, has shaped human genetic diversity. In particular, the interaction between humans, pathogens and the environment has played a crucial role in generating patterns of human genetic variation. The complement system plays a crucial role in the early protective immune response after exposure to a pathogen. Pathogens, over time, have developed mechanisms to circumvent the effects of complement which in turn has led to development of a more complex complement system. During the evolution of the complement system genes coding complement proteins have evolved polymorphisms, some of which have a functional effect, and this may reflect human-pathogen interaction and geographical origin. An example is the polymorphism Ile62Val (rs800292 (A>G)) in the complement regulator Factor H gene which alters the susceptibility to age-related macular degeneration (AMD), with the Ile62 polymorphism protecting against AMD. When sub-Saharan African and European populations are compared, the frequency of this polymorphism shows a very marked geographical distribution. Polymorphisms in other complement genes such as complement factor B show similar trends. This paper describes the geographical variation present in complement genes and discusses the implications of these observations. The analysis of genetic variation in complement genes is a promising tool to unravel mechanisms of host-pathogen interaction and can provide new insights into the evolution of the human immune system.

Successful isolated liver transplantation in a child with atypical hemolytic uremic syndrome and a mutation in complement factor H.

A male infant was diagnosed with atypical hemolytic uremic syndrome (aHUS) at the age of 5.5 months. Sequencing of the gene (CFH) encoding complement factor H revealed a heterozygous mutation (c.3644G>A, p.Arg1215Gln). Despite maintenance plasmapheresis he developed recurrent episodes of aHUS and vascular access complications while maintaining stable renal function. At the age of 5 years he received an isolated split liver graft following a previously established protocol using pretransplant plasma exchange (PE) and intratransplant plasma infusion. Graft function, renal function and disease remission are preserved 2 years after transplantation. Preemptive liver transplantation prior to the development of end stage renal disease is a valuable option in the management of aHUS associated with CFH mutations.

Impact of compound heterozygous complement factor H mutations on development of atypical hemolytic uremic syndrome-A pedigree revisited.

Atypical hemolytic uremic syndrome (aHUS) is associated with mutations in the gene CFH encoding the complement regulator factor H (CFH). We previously reported a family, in which three individuals had partial CFH deficiency but only one was affected by aHUS. We have investigated this family further to show that the partial CFH deficiency is associated with a heterozygous CFH mutation (c.2768T>G, p.Tyr899Asp). We used the polymorphic CFH variant p.His402Tyr to track expression of p.Tyr899Asp, and found that this mutant was expressed in minimal quantities in serum. In the one affected individual we found a second CFH mutation (c.3581G>A, p.Gly1194Asp) on the other allele which was expressed normally. We showed that this mutant, which has been described previously in aHUS, has impaired regulation of cell surface complement activation. The affected individual in this family is therefore a compound heterozygote for two functionally significant CFH mutations. Two individuals (mother and male sib) in the pedigree carried only c.2768T>G, p.Tyr899Asp and one (father) carried only c.3581G>A, p.Gly1194Asp, and all three were asymptomatic. Thus, further investigation of this family has enabled us to clarify the genotype-phenotype correlation.

Successful renal transplantation in factor H autoantibody associated HUS with CFHR1 and 3 deficiency and CFH variant G2850T.

Factor H (CFH) autoantibodies are associated with atypical hemolytic uremic syndrome (aHUS). Peritransplantation plasma exchange therapy and intensification of immunosuppression, with adjuvant use of anti-CD20 monoclonal antibodies has recently been advocated for cases of CFH-autoantibody associated aHUS. In this report, we describe successful deceased donor renal transplantation in a case of CFH-autoantibody associated aHUS with combined CFHR1 and 3 deficiency in addition to the CFH sequence variant, (cG2850T, pGln950His). CFH-autoantibodies were detected 2 weeks prior to transplantation. Disease recurrence was not observed using basiliximab, an IL2-receptor antagonist and high-dose corticosteroids with mycophenolate mofetil. Adjuvant therapies such as Rituximab nor intensification of plasma therapy were employed. Consequently, careful consideration needs to be given to the use of additional immunosuppression in certain cases of CFH-autoantibody associated aHUS. Serial measurement of CFH-autoantibodies is required in the immediate pre- and posttransplantation period to further clarify their role as a factor in the recurrence of aHUS posttransplantation. Furthermore, delineation of the functional significance of CFH-autoantibodies is warranted in individual cases.

The decay accelerating factor mutation I197V found in hemolytic uraemic syndrome does not impair complement regulation.

Hemolytic uremic syndrome is the clinical triad of thrombocytopenia, microangiopathic hemolytic anaemia and acute renal failure. Cases not associated with a preceding Shiga-like toxin producing Escherichia coli are described as atypical HUS (aHUS). Approximately 50% of patients with aHUS have mutations in one of three complement regulatory proteins, Factor H (CFH), membrane cofactor protein (MCP;CD46) or factor I (IF). A common feature of these three proteins is that they regulate complement by cofactor activity. Decay accelerating factor (DAF; CD55) regulates the complement system by disassociating the alternative and classical pathway convertases. Like CFH and MCP, the gene for DAF lies within the regulators of complement activation (RCA) gene cluster at 1q32. In 1998, we described linkage to this region in families with aHUS which led to the discovery of mutations in CFH and MCP. We therefore genotyped DAF in a panel of 46 aHUS patients including families with linkage to the RCA cluster. A mutation, I197V, was identified in one patient with familial HUS which was not found in 100 healthy controls. Molecular modelling of this mutation shows that the I197V mutation does not reside in an area which would be predicted to be important in decay accelerating activity. The expression of I197V on EBV-transformed B lymphocytes was equivalent to that of wild type controls. There was no significant decrease in decay acceleration activity of the recombinantly produced I197V mutant compared with wild type, as measured by a complement-mediated lytic assay. In conclusion, this study, identifies only one mutation in DAF in 46 patients with aHUS. This mutation, I197V, does not impair complement regulation and cannot be implicated in the pathogenesis of aHUS in this patient. This suggests that the complement regulatory abnormality in aHUS is principally one of deficient cofactor activity rather than of decay acceleration activity.

Favorable long-term outcome after liver-kidney transplant for recurrent hemolytic uremic syndrome associated with a factor H mutation.

A male child initially presented with atypical hemolytic uremic syndrome (HUS) at the age of 4 months and progressed within weeks to end stage renal disease (ESRD). At the age of 2 years he received a live-related kidney transplant from his mother, which, despite initial good function, was lost to recurrent disease after 2 weeks. Complement factor H analysis showed low serum levels and the presence of two mutations on different alleles (c.2918G > A, Cys973Tyr and c.3590T > C, Val1197Ala). His survival on dialysis was at risk because of access failure and recurrent bacteremic episodes. Therefore, at the age of 5 years he received a combined liver-kidney transplant with pre-operative plasma exchange. Initial function of both grafts was excellent and this has been maintained for over 2 years. This report suggests that despite setbacks in previous experience, combined liver-kidney transplantation offers the prospect of a favorable long-term outcome for patients with HUS associated with complement factor H mutations.

Factor H genotype-phenotype correlations: lessons from aHUS, MPGN II, and AMD.

Missense mutations in the C-terminal region of Factor H are associated with atypical hemolytic uremic syndrome, whereas homozygous Factor H deficiency is more frequently associated with membranoproliferative glomerulonephritis type II (MPGN II). The report of Licht et al. of a mutation in the complement-regulatory N-terminal region of Factor H in MPGN II provides additional insight into the pathogenesis of this condition.

The development of atypical haemolytic-uraemic syndrome is influenced by susceptibility factors in factor H and membrane cofactor protein: evidence from two independent cohorts.

BACKGROUND: In both familial and sporadic atypical haemolytic-uraemic syndrome (aHUS), mutations have been reported in regulators of the alternative complement pathway including factor H (CFH), membrane cofactor protein (MCP), and the serine protease factor I (IF). A characteristic feature of both MCP and CFH associated HUS is reduced penetrance and variable inheritance; one possible explanation for this is that functional changes in complement proteins act as modifiers. OBJECTIVE: To examine single nucleotide polymorphisms in both CFH and MCP genes in two large cohorts of HUS patients (Newcastle and Paris). RESULTS: In both cohorts there was an association with HUS for both CFH and MCP alleles. CFH and MCP haplotypes were also significantly different in HUS patients compared with controls. CONCLUSIONS: This study suggests that there are naturally occurring susceptibility factors in CFH and MCP for the development of atypical HUS.

Inherited dysregulation of the complement system.

In recent years there has been a substantial increase in the understanding of the genetics and pathogenesis of HUS. Mutations in factor H, a fluid-phase regulator of the alternative complement pathway, have been identified in 15-30% of patients with both familial and sporadic (D-) HUS. The mutations mainly cluster in the C terminal part of factor H, a region that is important for both binding to c3b and also polyanionic structures on cell surfaces. This leads to loss of protection against complement mediated endothelial injury. Mutations in the membrane bound complement regulator, membrane cofactor protein (MCP; CD46) have also been described in three families. These result in an impairment of inactivation of surface bound c3b. Finally mutations in the serine protease, factor I that lead to deficiency of the protein have been reported in two HUS patients. There is therefore now overwhelming evidence that dysregulation of the alternative complement pathway predisposes to the development of a thrombotic microangiopathy.

Effect of insulin upon protein degradation in cultured human myocytes.

BACKGROUND: The anabolic effects of insulin are well recognized but the mechanism by which insulin decreases muscle protein degradation in human is unclear. However, in a variety of catabolic conditions it is believed to be changes in the activity of the ATP-dependent ubiquitin proteolytic pathway that are responsible for changes in protein degradation in skeletal muscle. The aim of this study was to test the hypothesis that insulin regulates the ATP-dependent ubiquitin proteolytic pathway in human muscle. MATERIAL AND METHODS: The effects of insulin and acidosis on protein degradation were measured in human myocytes using L-[14C]phenylalanine. The effect of insulin on the activity of the ATP-dependent ubiquitin pathway was assessed from the mRNA expression of ubiquitin and the ubiquitin-conjugating enzyme E214k in human myocytes. RESULTS AND CONCLUSIONS: Coincubation of human myocytes with 100 nM of insulin was associated with a significant reduction in protein degradation. Metabolic acidosis is known to increase skeletal muscle protein degradation rates, and in our experiments protein degradation at a pH of 7.0 was significantly higher than pH 7.35. Eight-hour exposure to 100 nM of insulin resulted in a significant reduction in the expression of E214k but no change in the expression of ubiquitin. CONCLUSIONS: In human muscle we have demonstrated regulation by insulin of the ATP-dependent ubiquitin pathway at the level of ubiquitin conjugation.