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.

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.

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.

Solution structures of complement components by X-ray and neutron scattering and analytical ultracentrifugation.

The short consensus/complement repeat (SCR) domain (also known as the complement control protein domain) is the most abundant domain type in the complement system. Crystal and NMR structures for proteins that contain single and multiple SCR domains have now been published. These contain inter-SCR linkers of between three and eight residues, and the structures show much variability in inter-SCR orientations. X-ray and neutron scattering, combined with analytical ultracentrifugation and constrained modelling based on known subunit structures will yield a medium-resolution structure for the protein of interest. The fewer parameters that are associated with the structure of interest, the more defined the structure of interest becomes. These solution studies have been applied to several SCR-containing proteins in the complement system, most notably Factor H with 20 SCR domains, a complement receptor type 2 fragment with two SCR domains, and rat complement receptor-related protein (Crry) which contains five SCR domains. The results show great conformational variability in the inter-SCR orientation, and these will be reviewed. Even though the rotational orientation cannot be modelled, it is nonetheless possible to measure the degree of extension of the multi-SCR proteins and, from this, to obtain functionally useful results.

Is decreased treatment time in hemodialysis patients harmful if solute clearance is maintained?

An association between decreased duration of hemodialysis and increased morbidity and mortality in patients has been suggested. Whether this is due only to decreased solute clearance is unclear. In this prospective randomised study the effect of reducing treatment time whilst maintaining constant solute clearance was examined in fourteen patients. The study lasted for a period of 36 weeks (3x12 week study periods) and used a crossover design. The patients dialysis prescription (KW) was not changed on entering the study and was maintained during short (150 minutes) and long dialysis (240 minutes) by varying blood flow, dialysate flow and dialyzer surface area. The delivered KW was kinetically assessed. Fractional urea clearance was also measured during each treatment period by measurement of urea concentration in spent dialysate and total body water using 3H2O. At the end of each treatment period a full biochemical and hematological profile, nutritional intake and status, 24 h ambulatory blood pressure, nerve conduction studies, and quality of life questionnaire were performed. Within patients the delivered single pool KW was uniform throughout the 3 treatment periods and fractional urea clearance did not vary. However, Kt/W assessed using equilibrated models (Daugardis and Smye) was significantly lower in the short dialysis period. No differences between short and long dialysis sessions were noted in any of the measured variables. Thus, over a 36 week period there is no evidence to suggest that hemodialysis patients are adversely affected by decreased duration of treatment provided that solute clearance is maintained.

Sodium bicarbonate treatment and ubiquitin gene expression in acidotic human subjects with chronic renal failure.

BACKGROUND: In chronic renal failure, metabolic acidosis is associated with increased whole body protein degradation. In rats this effect of acidosis occurs in skeletal muscle and is associated with increased ubiquitin mRNA expression. This has not been demonstrated in humans. MATERIALS AND METHODS: Six patients with chronic renal failure and acidosis underwent muscle biopsy before and after 1 month's treatment with sodium bicarbonate. RNA was extracted from the biopsy, and the expression of the genes for ubiquitin and the proteasome component, C2, were measured by Northern blotting. RESULTS AND CONCLUSIONS: There was no significant difference in the expression of ubiquitin or C2 after bicarbonate treatment. This is contrast with results from animal models of acidosis and some other catabolic conditions in humans. This may reflect the complexity of the ubiquitin-dependent pathway, and it may be that changes in ubiquitin expression are only seen with more severe and/or acute changes in pH.

The voltage-dependent Cl(-) channel ClC-5 and plasma membrane Cl(-) conductances of mouse renal collecting duct cells (mIMCD-3).

1. We have tested the hypothesis that the voltage-dependent Cl(-) channel, ClC-5 functions as a plasma membrane Cl(-) conductance in renal inner medullary collecting duct cells. 2. Full-length mouse kidney ClC-5 (mClC-5) was cloned and transiently expressed in CHO-K1 cells. Fast whole-cell patch-clamp recordings confirmed that mClC-5 expression produces a voltage-dependent, strongly outwardly rectifying Cl(-) conductance that was unaffected by external DIDS. 3. Slow whole-cell recordings, using nystatin-perforated patches from transfected CHO-K1 cells, also produced voltage-dependent Cl(-) currents consistent with ClC-5 expression. However, under this recording configuration an endogenous DIDS-sensitive Ca(2+)-activated Cl(-) conductance was also evident, which appeared to be activated by green fluorescent protein (GFP) transfection. 4. A mClC-5-GFP fusion protein was transiently expressed in CHO-K1 cells; confocal laser scanning microscopy (CLSM) showed localization at the plasma membrane, consistent with patch-clamp experiments. 5. Endogenous expression of mClC-5 was demonstrated in mouse renal collecting duct cells (mIMCD-3) by RT-PCR and by immunocytochemistry. 6. Using slow whole-cell current recordings, mIMCD-3 cells displayed three biophysically distinct Cl(-)-selective currents, which were all inhibited by DIDS. However, no cells exhibited whole-cell currents that had mClC-5 characteristics. 7. Transient transfection of mIMCD-3 cells with antisense mClC-5 had no effect on the endogenous Cl(-) conductances. Transient transfection with sense mClC-5 failed to induce the Cl(-) conductance seen in CHO-K1 cells but stimulated levels of the endogenous Ca(2+)-activated Cl(-) conductance 24 h post-transfection. 8. Confocal laser scanning microscopy of mIMCD-3 cells transfected with mClC-5-GFP showed that the protein was absent from the plasma membrane and was instead localized to acidic endosomal compartments. 9. These data discount a major role for ClC-5 as a plasma membrane Cl(-) conductance in mIMCD-3 cells but suggest a role in endosomal function.