The genetic diagnosis of several rare diseases is rather complex: these diseases are characterized by a high genetic heterogeneity –i.e. in different patients the same disease may be caused by mutations in different genes-. Traditional sequencing methods, such as Sanger sequencing, are not suitable for genetic diagnosis, since they require long analytical time and are expensive. In our laboratory we have developed diagnostic panels that are specific for the genes associated with some rare diseases such as the atypical hemolytic syndrome (aHUS), C3 glomerulopathy (C3G), Immune Complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) and steroid resistant nephrotic syndrome (SRNS). These panels coupled with the next generation sequencing platform allow the analysis of several genes together in shorter time and with reduced costs than Sanger sequencing. We designed this project with the goal to ameliorate the diagnostic resources of rare disease and to improve at the same time our knowledge of the genetic causes of these diseases through the inclusion in the panels of new candidate genes.

The evaluation of the functional consequences of gene mutations associated with rare diseases and the investigation of how these abnormalities influence the synthesis and/or the function of the encoded proteins, have a crucial impact in the comprehension of the molecular mechanisms responsible for the onset of a given disease. The effects of gene mutations may be very diverse depending on the type of the mutation and on its localization in the protein. Indeed, at cellular level, different mutations may have different impact on the synthesis, the cellular transport, the localization and the function of the mutant protein. To study the functional consequences of the mutations in known disease-associated genes as well as in new genes that we identified in our patients with aHUS, C3G, IC-MPGN or SRNS, we generate the recombinant mutant and wild-type proteins and we induce their expression in mammalian and insect stable cell lines. Thereafter, we evaluate the effects of the mutations on protein synthesis and secretion and on protein function by specific biochemical and molecular tests.

During the last 10 years several types of autoantibodies have been identified that are responsible of the activation of the alternative pathway of complement, resulting in tissue and organ injury in patients with aHUS, IC-MPGN and C3G. In aHUS the most common antibodies target a complement regulatory protein called factor H, whereas about half of patients with IC-MPGN or C3G carry an heterogeneous group of antibodies collectively called C3NeFs. C3NeFs stabilize the C3 convertase, a protease complex that is crucial for the activation of the alternative pathway of complement. Through the purification of the antibodies from the serum of patients with either aHUS or IC-MPGN/C3G and after setting up specific functional tests, we could document that antibodies isolated from different patients have different molecular targets and different functional effects in the complement cascade. More importantly we found that the abnormalities associated with the specific autoantibodies correlate with clinical parameters. Further studies are ongoing in our laboratory to characterize the specific autoantibodies and to identify the causes underlying their formation in the circulation of patients. Several drugs are under clinical development, which inhibit the complement cascade at various levels. The results of our studies will hopefully help designing personalized therapies, targeted to counteract the effect of the specific autoantibodies identified in each patient.