Defining the genetics of thrombotic microangiopathies.

The spectrum of the thrombotic microangiopathies (TMA) encompasses a heterogeneous group of disorders with hereditary and acquired forms. Endothelial cell injury in the microvasculature is common to all TMAs, whatever the pathophysiological process. In this review we describe genetic mutations characteristic of certain TMAs and review their contributions to disease. Recent identification of novel pathologic mutations has been enabled by exome studies. The monogenic forms of TMA are more frequently caused by recessive alterations in von Willebrand factor cleaving protease ADAMST13, leading to congenital thrombotic thrombocytopenic purpura, or cobalamine C and DGKE genes, leading to an atypical hemolytic-uremic syndrome (aHUS)-like TMA. aHUS, whether idiopathic or linked to a known complement amplifying condition, is a TMA that primarily affects kidney function. It often results from a combination of an underlying genetic susceptibility with environmental factors activating the alternative complement pathway. Pathogenic variants in at least five complement genes coding for complement factor H (CFH) complement factor I (CFI), MCP (CD46), C3 and complement factor B (CFB) have been demonstrated to increase the risk of developing aHUS, but several more genes have been implicated. A new challenge is to separate disease-associated genetic variants from the broader background of variants or polymorphisms present in all human genomes that are rare, potentially functional, but may or may not be pathogenic.

A prevalent C3 mutation in aHUS patients causes a direct C3 convertase gain of function.

Atypical hemolytic uremic syndrome (aHUS) is a rare renal thrombotic microangiopathy commonly associated with rare genetic variants in complement system genes, unique to each patient/family. Here, we report 14 sporadic aHUS patients carrying the same mutation, R139W, in the complement C3 gene. The clinical presentation was with a rapid progression to end-stage renal disease (6 of 14) and an unusually high frequency of cardiac (8 of 14) and/or neurologic (5 of 14) events. Although resting glomerular endothelial cells (GEnCs) remained unaffected by R139W-C3 sera, the incubation of those sera with GEnC preactivated with pro-inflammatory stimuli led to increased C3 deposition, C5a release, and procoagulant tissue-factor expression. This functional consequence of R139W-C3 resulted from the formation of a hyperactive C3 convertase. Mutant C3 showed an increased affinity for factor B and a reduced binding to membrane cofactor protein (MCP; CD46), but a normal regulation by factor H (FH). In addition, the frequency of at-risk FH and MCP haplotypes was significantly higher in the R139W-aHUS patients, compared with normal donors or to healthy carriers. These genetic background differences could explain the R139W-aHUS incomplete penetrance. These results demonstrate that this C3 mutation, especially when associated with an at-risk FH and/or MCP haplotypes, becomes pathogenic following an inflammatory endothelium-damaging event.

Sheep Erythrocyte Preparation for Hemolytic Tests Exploring Complement Functional Activities.

Sheep erythrocytes (SE) are commonly used in complement functional tests. Non sensitized SE are useful to study the FH activity of cell protection. Indeed, as the cell surface of sheep erythrocytes is rich in sialic acids, Factor H (FH) is able to bind on it and therefore they represent a model of nonactivating surface. Because of their high capacity of complement regulation SE need to be modified to explore other functionality of the complement pathways, like the Complement hemolytic 50 (CH50) or the AP C3 convertase decay assays. For these tests, SE are sensitized with an anti-sheep red blood cell stroma antibody. In presence of serum or plasma complement components, sensitized SE may initiate complement cascade activation via the classic pathway explored in the CH50 assay. Sensitized SE may also be used to prepare C3b-coated SE that, with the use of buffers favoring AP, are suitable for the C3 Nef hemolytic assay and for the hemolytic assay studying the AP decay activity of FH. In this chapter we describe how to prepare SE for these different hemolytic tests.

Analytical validation of an alternative method to quantify specific antibodies in 3 applications.

The detection and the quantification of specific antibodies represent essential tools for the diagnosis and for the biological monitoring of immune humoral response in many clinical situations in particular in autoimmune diseases or in the context of immunotherapy using monoclonal antibodies. This article focuses on the development of a specific antibody measuring method (Patent n°PCT/IB2014/064437). The principle of this method is based on the combined use of a monoclonal antibody as standard and the protein G as immunoglobulins detecting agent. We performed a complete analytical validation of this method for the quantification of antibodies in three different applications: autoantibodies, alloantibodies and therapeutic monoclonal antibody. The results showed good performances compatible with the use of these assays as diagnostic tools. This method allows avoiding the use of products from human origin as reagent that causes ethical and infectious concerns but also storage and long term stock management problems. Moreover, this approach is particularly useful when no commercial reagent is available, especially in the case of rare diseases.

Clinical and Genetic Spectrum of a Large Cohort With Total and Sub-total Complement Deficiencies.

The complement system is crucial for defense against pathogens and the removal of dying cells or immune complexes. Thus, clinical indications for possible complete complement deficiencies include, among others, recurrent mild or serious bacterial infections as well as autoimmune diseases (AID). The diagnostic approach includes functional activity measurements of the classical (CH50) and alternative pathway (AP50) and the determination of the C3 and C4 levels, followed by the quantitative analysis of individual components or regulators. When biochemical analysis reveals the causal abnormality of the complement deficiency (CD), molecular mechanisms remains frequently undetermined. Here, using direct sequencing analysis of the coding region we report the pathogenic variants spectrum that underlie the total or subtotal complement deficiency in 212 patients. We identified 107 different hemizygous, homozygous, or compound heterozygous pathogenic variants in 14 complement genes [C1Qß (n = 1), C1r (n = 3), C1s (n = 2), C2 (n = 12), C3 (n = 5), C5 (n = 12), C6 (n = 9), C7 (n = 17), C8 ß (n = 7), C9 (n = 3), CFH (n = 7), CFI (n = 18), CFP (n = 10), CFD (n = 2)]. Molecular analysis identified 17 recurrent pathogenic variants in 6 genes (C2, CFH, C5, C6, C7, and C8). More than half of the pathogenic variants identified in unrelated patients were also found in healthy controls from the same geographic area. Our study confirms the strong association of meningococcal infections with terminal pathway deficiency and highlights the risk of pneumococcal and auto-immune diseases in the classical and alternative pathways. Results from this large genetic investigation provide evidence of a restricted number of molecular mechanisms leading to complement deficiency and describe the clinical potential adverse events of anti-complement therapy.

Anti-factor H autoantibodies assay.

Non-Shiga-toxin-associated hemolytic uremic syndrome (atypical HUS) is a rare form of thrombotic microangiopathy which associates hemolytic anemia, thrombocytopenia, and acute renal failure. In 10 % of cases the disease is linked to presence of autoantibodies directed against Factor H (FH), the main plasmatic alternative complement pathway regulatory protein. Their presence induces an acquired functional FH deficiency. The anti-FH autoantibodies screening must be performed at the very onset of the disease in all cases of HUS, in order, first, to make the proper diagnosis as early as possible, and second to support an appropriate therapy including early plasma exchanges and immunosuppressive treatments. Thus, anti-CFH IgG represents a diagnostic marker and the titer determination is useful for assessing disease evolution, because changes precede clinical symptoms, and for monitoring of treatment. Presence of anti-FH IgG has been recently reported to be associated with other clinical context such as C3 glomerulopathies, but their pathogenicity in these conditions remains to be assessed. Here we describe the ELISA assay allowing the detection of these autoantibodies and report the analysis which can be performed concomitantly to improve the diagnosis.

Standardisation of the factor H autoantibody assay.

The screening of all atypical haemolytic uraemic syndrome (aHUS) patients for factor H autoantibodies is best practice. However, there is no consensus assay for the reporting of factor H autoantibody titres. In this study, three European complement laboratories with expertise in the field of autoantibody testing address this by systematically evaluating several ELISA methods used for the detection of factor H autoantibodies. All methods tested adequately detect high titre samples. However, this study recommends the Paris method for the detection and reporting of factor H autoantibodies to be used when setting up a factor H autoantibody screen. The importance of individual sample background subtraction in these ELISA tests was established. The use of a relative or arbitrary unit index with a common positive and negative serum allowed for consistent comparison of findings from different test centres. Therefore, it is recommended that a standard arbitrary unit scale based on a titration curve from a common positive anti-serum be adopted to allow future establishment of the relative importance of particular titres of factor H autoantibodies in aHUS. Systematic assay for the presence of factor H autoantibodies in patients using the Paris method will provide the longitudinal analysis needed to fully establish the importance of factor H autoantibodies in disease. This will feed into additional research to clarify whether additional factors have a bearing on the phenotype/outcome of autoimmune aHUS.

[Complement deficiencies and human diseases]. / Déficits en protéines du complément et pathologies humaines.

The complement system is a complex system involving serum and membrane proteins interacting in a regulated manner. The complement system plays a major role in antibacterial immunity, in inflammation, and in immune complex processing. Therefore, deficiencies in complement proteins are associated with increased susceptibility to bacterial infections and autoimmune diseases. These deficiencies can be inherited or acquired. Most of them can be screened by simple laboratory tests but require a diagnosis in a specialized laboratory. All sequences of complement genes are known, and the discovery of a deficiency must lead to genetic testing. The discovery of a congenital deficiency requires a familial study and a prophylaxis. In this article, we review the complement cascade, the laboratory tests to explore it, and the main diseases associated with complement deficiencies.