Genotype-phenotype correlations of low-frequency variants in the complement system in renal disease and age-related macular degeneration.

Genetic alterations in the complement system have been linked to a variety of diseases, including atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G), and age-related macular degeneration (AMD). We performed sequence analysis of the complement genes complement factor H (CFH), complement factor I (CFI), and complement C3 (C3) in 866 aHUS/C3G and 697 AMD patients. In total, we identified 505 low-frequency alleles, representing 121 unique variants, of which 51 are novel. CFH contained the largest number of unique low-frequency variants (n = 64; 53%), followed by C3 (n = 32; 26%) and CFI (n = 25; 21%). A substantial number of variants were found in both patients groups (n = 48; 40%), while 41 (34%) variants were found only in aHUS/C3G and 32 (26%) variants were AMD specific. Genotype-phenotype correlations between the disease groups identified a higher frequency of protein altering alleles in short consensus repeat 20 (SCR20) of factor H (FH), and in the serine protease domain of factor I (FI) in aHUS/C3G patients. In AMD, a higher frequency of protein-altering alleles was observed in SCR3, SCR5, and SCR7 of FH, the SRCR domain of FI, and in the MG3 domain of C3. In conclusion, we observed a substantial overlap of variants between aHUS/C3G and AMD; however, there is a distinct clustering of variants within specific domains.

Complement activation patterns in atypical haemolytic uraemic syndrome during acute phase and in remission.

Atypical haemolytic uraemic syndrome (aHUS) is associated with (genetic) alterations in alternative complement pathway. Nevertheless, comprehensive evidence that the complement system in aHUS patients is more prone to activation is still lacking. Therefore, we performed a thorough analysis of complement activation in acute phase and in remission of this disease. Complement activation patterns of the aHUS patients in acute phase and in remission were compared to those of healthy controls. Background levels of complement activation products C3b/c, C3bBbP and terminal complement complex (TCC) were measured using enzyme-linked immunosorbent assay (ELISA) in ethylenediamine tetraacetic acid (EDTA) plasma. In vitro-triggered complement activation in serum samples was studied using zymosan-coating and pathway-specific assay. Furthermore, efficiencies of the C3b/c, C3bBbP and TCC generation in fluid phase during spontaneous activation were analysed. Patients with acute aHUS showed elevated levels of C3b/c (P < 0·01), C3bBbP (P < 0·0001) and TCC (P < 0·0001) in EDTA plasma, while values of patients in remission were normal, compared to those of healthy controls. Using data from a single aHUS patient with complement factor B mutation we illustrated normalization of complement activation during aHUS recovery. Serum samples from patients in remission showed normal in vitro patterns of complement activation and demonstrated normal kinetics of complement activation in the fluid phase. Our data indicate that while aHUS patients have clearly activated complement in acute phase of the disease, this is not the case in remission of aHUS. This knowledge provides important insight into complement regulation in aHUS and may have an impact on monitoring of these patients, particularly when using complement inhibition therapy.

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.

A new era in the diagnosis and treatment of atypical haemolytic uraemic syndrome.

The haemolytic uraemic syndrome (HUS) is characterised by haemolytic anaemia, thrombocytopenia and acute renal failure. The majority of cases are seen in childhood and are preceded by an infection with Shiga-like toxin producing Escherichia coli (STEC-HUS; so-called typical HUS). Non-STEC or atypical HUS (aHUS) is seen in 5 to 10% of all cases and occurs at all ages. These patients have a poorer outcome and prognosis than patients with STEC-HUS. New insights into the pathogenesis of aHUS were revealed by the identification of mutations in genes encoding proteins of the alternative pathway of the complement system in aHUS patients. Specific information of the causative mutation is important for individualised patient care with respect to choice and efficacy of therapy, the outcome of renal transplantation, and the selection of living donors. This new knowledge about the aetiology of the disease has stimulated the development of more specific treatment modalities. Until now, plasma therapy was used with limited success in aHUS, but recent clinical trials have demonstrated that patients with aHUS can be effectively treated with complement inhibitors, such as the monoclonal anti-C5 inhibitor eculizumab.

Tissue transglutaminase mRNA expression in apoptotic cell death.

INTRODUCTION: Tissue transglutaminase (t.TG) is an enzyme that catalyzes the cross-linking of intracellular proteins, thus assembling a protein scaffold that prevents leakage of intracellular components. t.TG is activated during the apoptotic cell death cascade and plays a key role in the formation of apoptotic bodies. The aim of this study was to determine to what amount t.TG-mRNA becomes expressed during apoptosis and whether the t.TG-mRNA expression level could be used as trace marker of recent apoptosis and in individual cases for quantification of apoptosis. METHODS: Expression of t.TG-mRNA was determined using TaqMan based, real-time RT-PCR, a semi-quantitative RT-PCR technique. The t.TG-mRNA expression was measured in cultured cells (MCF-7, human endothelial cells) and in peripheral blood mononuclear cells (PBMCs) before and after induction of apoptosis in vitro. RESULTS: The TaqMan RT-PCR of t.TG proved to be reliable, reproducible (CV's inter and intraassay precisions of 0.8-2.8%, measured at two levels), and specific for apoptotic cell death. t.TG-mRNA expression increases in response to apoptosis induction and is not expressed during the process of necrotic cell death. The expression during apoptotic cell death changes in the dose dependent manner in cultured cells as well as in the PBMCs, treated in vitro. The increase t.TG-mRNA expression level was up to 20 times, depending on the intensity of the apoptosis induction treatment and incubation time afterwards. PBMCs of patients with myelodysplasia showed spontaneous expression of t.TG-mRNA in agreement with their increased apoptotic cell death in vivo. CONCLUSION: t.TG-mRNA expression increases significantly in response to apoptosis inducing treatment. The observed changes are dose and time dependent. This leads to the conclusion that t.TG expression can be used as a trace marker for detection and quantification of apoptosis.