Next-generation sequencing for new gene identification and for diagnosis in steroid-resistant nephrotic syndrome

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Nephrotic syndrome is clinically characterized by massive proteinuria, and hypoalbuminemia. It represents a heterogeneous group of glomerular disorders characterized by distinct causes and histopathologic lesions. The present thesis evaluates the contribution of genetics in conditions commonly associated with NS: the immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN), the C3 glomerulopathy (C3G) and the podocytopathies, usually characterized by steroid-resistant nephrotic syndrome (SRNS). The first part of the thesis focused on IC-MPGN and C3G. We found likely pathogenic (LP) variants in complement alternative pathway genes in 18% of patients and describe, for the first time, thrombomodulin rare pathogenic variants in C3G patients. Interestingly, mutations alone did not increase the risk of developing IC-MPGN or C3G, but they did so when combined with common susceptibility variants. The prevalence of alternative pathway abnormalities (mutations and/or C3 Nephritic Factors, C3NeFs) was similar in IC-MPGN (53-56%) and C3G (64-65%). To investigate for more homogeneous subgroups within IC-MPGN/C3G, an operator-independent approach was applied using genetic, biochemical, histological and clinical data. Four different groups emerged showing distinct pathogenesis, and histological and clinical features. In the second part, the genetic causes of podocytopathies were investigated. LP variants in podocytopathy-associated genes are found in 23% of SRNS patients, prevalently in COL4A3-5 genes. Moreover, 8% of patients carried LP variants in Congenital Anomalies of the Kidney and the Urinary Tract (CAKUT)-associated genes, which were not previously associated with podocytopathies. LP variants in CAKUT-associated and podocytopathy-associated genes frequently combined together. Finally, possibly pathogenic variants in EPB41L45, a candidate gene for podocytopathies, were identified in 3 unrelated patients. In conclusion, this thesis contributes to understand the complex genetic basis of both IC-MPGN/C3G and podocytopathies. It introduces new players in the pathogenesis of these diseases. Finally, it provides evidence of the presence of subgroups within IC-MPGN/C3G with distinct underlying mechanisms, and clinical and histopathologic features.