Next-Generation Sequencing for New Gene Identification and for Diagnosis in Steroid-Resistant Nephrotic Syndrome

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

Unraveling the Molecular Mechanisms Underlying Complement Dysregulation by Nephritic Factors in C3G and IC-MPGN

Membranoproliferative glomerulonephritis (MPGN) was recently classified as C3 glomerulopathies (C3G), and immune-complex (IC) mediated MPGN. Dysregulation of the complement alternative pathway, driven by acquired and/or genetic defects, plays a pathogenetic role in C3G. However, alternative pathway abnormalities were also found in IC-MPGN. The most common acquired drivers are the C3 nephritic factors (C3NeFs), heterogeneous autoantibodies that stabilize the C3 convertase, C3bBb. C3NeFs are traditionally detected by hemolytic assays based on sheep erythrocyte lysis, which however do not provide a direct molecular estimation of C3bBb formation and decay. We set up a microplate/western blot assay that specifically detects and quantifies C3bBb, and its precursor, the C3 proconvertase C3bB, to investigate the complex mechanistic effects of C3NeFs from patients with primary IC-MPGN (n = 13) and C3G (n = 13). In the absence of properdin, 9/26 patients had C3NeF IgGs stabilizing C3bBb against spontaneous and FH-accelerated decay. In the presence of properdin the IgGs of all but one patient had C3bBb-stabilizing activity. Properdin-independent C3NeFs were identified mostly in DDD patients, while properdin-dependent C3NeFs associated with either C3GN or IC-MPGN and with higher incidence of nephrotic syndrome. When we grouped patients based on our recent cluster analysis, patients in cluster 3, with highly electron-dense intramembranous deposits, low C3, and mostly normal sC5b-9 levels, had a higher prevalence of properdin-independent C3NeFs than patients in clusters 1 and 2. Conversely, about 70% of cluster 1 and 2 patients, with subendothelial, subepithelial, and mesangial deposits, low C3 levels and high sC5b-9 levels, had properdin-dependent C3NeFs. The flexibility of the assay allowed us to get deep insights into C3NeF mechanisms of action, showing that: (1) most C3NeFs bind strongly and irreversibly to C3 convertase; (2) C3NeFs and FH recognize different epitopes in C3 convertase; (3) C3NeFs bind rapidly to C3 convertase and antagonize the decay accelerating activity of FH on newly formed complexes; (4) C3NeFs do not affect formation and stability of the C3 proconvertase. Thus, our study provides a molecular approach to detecting and characterizing C3NeFs. The results highlight different mechanisms of complement dysregulation resulting in different complement profiles and patterns of glomerular injury, and this may have therapeutic implications

CFH and CFHR Copy Number Variations in C3 Glomerulopathy and Immune Complex-Mediated Membranoproliferative Glomerulonephritis

C3 Glomerulopathy (C3G) and Immune Complex-Mediated Membranoproliferative glomerulonephritis (IC-MPGN) are rare diseases characterized by glomerular deposition of C3 caused by dysregulation of the alternative pathway (AP) of complement. In approximately 20% of affected patients, dysregulation is driven by pathogenic variants in the two components of the AP C3 convertase, complement C3 (C3) and Factor B (CFB), or in complement Factor H (CFH) and Factor I (CFI), two genes that encode complement regulators. Copy number variations (CNVs) involving the CFH-related genes (CFHRs) that give rise to hybrid FHR proteins also have been described in a few C3G patients but not in IC-MPGN patients. In this study, we used multiplex ligation-dependent probe amplification (MLPA) to study the genomic architecture of the CFH-CFHR region and characterize CNVs in a large cohort of patients with C3G (n = 103) and IC-MPGN (n = 96) compared to healthy controls (n = 100). We identified new/rare CNVs resulting in structural variants (SVs) in 5 C3G and 2 IC-MPGN patients. Using long-read single molecule real-time sequencing (SMRT), we detected the breakpoints of three SVs. The identified SVs included: 1) a deletion of the entire CFH in one patient with IC-MPGN; 2) an increased number of CFHR4 copies in one IC-MPGN and three C3G patients; 3) a deletion from CFHR3-intron 3 to CFHR3-3′UTR (CFHR34–6Δ) that results in a FHR3-FHR1 hybrid protein in a C3G patient; and 4) a CFHR31–5-CFHR410 hybrid gene in a C3G patient. This work highlights the contribution of CFH-CFHR CNVs to the pathogenesis of both C3G and IC-MPGN

Rare Functional Variants in Complement Genes and Anti-FH Autoantibodies-Associated aHUS

Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and renal failure. It is caused by genetic or acquired defects of the complement alternative pathway. Factor H autoantibodies (anti-FHs) have been reported in 10% of aHUS patients and are associated with the deficiency of factor H-related 1 (FHR1). However, FHR1 deficiency is not enough to cause aHUS, since it is also present in about 5% of Caucasian healthy subjects. In this study we evaluated the prevalence of genetic variants in CFH, CD46, CFI, CFB, C3, and THBD in aHUS patients with anti-FHs, using healthy subjects with FHR1 deficiency, here defined “supercontrols,” as a reference group. “Supercontrols” are more informative than general population because they share at least one risk factor (FHR1 deficiency) with aHUS patients. We analyzed anti-FHs in 305 patients and 30 were positive. The large majority were children (median age: 7.7 [IQR, 6.6–9.9] years) and 83% lacked FHR1 (n = 25, cases) due to the homozygous CFHR3-CFHR1 deletion (n = 20), or the compound heterozygous CFHR3-CFHR1 and CFHR1-CFHR4 deletions (n = 4), or the heterozygous CFHR3-CFHR1 deletion combined with a frameshift mutation in CFHR1 that generates a premature stop codon (n = 1). Of the 960 healthy adult subjects 48 had the FHR1 deficiency (“supercontrols”). Rare likely pathogenetic variants in CFH, THBD, and C3 were found in 24% of cases (n = 6) compared to 2.1% of the “supercontrols” (P-value = 0.005). We also found that the CFH H3 and the CD46GGAAC haplotypes are not associated with anti-FHs aHUS, whereas these haplotypes are enriched in aHUS patients without anti-FHs, which highlights the differences in the genetic basis of the two forms of the disease. Finally, we confirm that common infections are environmental factors that contribute to the development of anti-FHs aHUS in genetically predisposed individuals, which fits with the sharp peak of incidence during scholar-age. Further studies are needed to fully elucidate the complex genetic and environmental factors underlying anti-FHs aHUS and to establish whether the combination of anti-FHs with likely pathogenetic variants or other risk factors influences disease outcome and response to therapies

C4 nephritic factor in patients with immune-complex-mediated membranoproliferative glomerulonephritis and C3-glomerulopathy

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Direct Reprogramming of Human Bone Marrow Stromal Cells into Functional Renal Cells Using Cell-free Extracts

The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs), also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes—formation of “domes” and tubule-like structures—and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy

Disruption of PTPRO Causes Childhood-Onset Nephrotic Syndrome

Idiopathic nephrotic syndrome (INS) is a genetically heterogeneous group of disorders characterized by proteinuria, hypoalbuminemia, and edema. Because it typically results in end-stage kidney disease, the steroid-resistant subtype (SRNS) of INS is especially important when it occurs in children. The present study included 29 affected and 22 normal individuals from 17 SRNS families; genome-wide analysis was performed with Affymetrix 250K SNP arrays followed by homozygosity mapping. A large homozygous stretch on chromosomal region 12p12 was identified in one consanguineous family with two affected siblings. Direct sequencing of protein tyrosine phosphatase receptor type O (PTPRO; also known as glomerular epithelial protein-1 [GLEPP1]) showed homozygous c.2627+1G>T donor splice-site mutation. This mutation causes skipping of the evolutionarily conserved exon 16 (p.Glu854_Trp876del) at the RNA level. Immunohistochemistry with GLEPP1 antibody showed a similar staining pattern in the podocytes of the diseased and control kidney tissues. We used a highly polymorphic intragenic DNA marker—D12S1303—to search for homozygosity in 120 Turkish and 13 non-Turkish individuals in the PodoNet registry. This analysis yielded 17 candidate families, and a distinct homozygous c.2745+1G>A donor splice-site mutation in PTPRO was further identified via DNA sequencing in a second Turkish family. This mutation causes skipping of exon 19, and this introduces a premature stop codon at the very beginning of exon 20 (p.Asn888Lysfs∗3) and causes degradation of mRNA via nonsense-mediated decay. Immunohistochemical analysis showed complete absence of immunoreactive PTPRO. Ultrastructural alterations, such as diffuse foot process fusion and extensive microvillus transformation of podocytes, were observed via electron microscopy in both families. The present study introduces mutations in PTPRO as another cause of autosomal-recessive nephrotic syndrome

Myo1E Mutations And Childhood Familial Focal Segmental Glomerulosclerosis

BACKGROUND Focal segmental glomerulosclerosis is a kidney disease that is manifested as the nephrotic syndrome. It is often resistant to glucocorticoid therapy and progresses to end-stage renal disease in 50 to 70% of patients. Genetic studies have shown that familial focal segmental glomerulosclerosis is a disease of the podocytes, which are major components of the glomerular filtration barrier. However, the molecular cause in over half the cases of primary focal segmental glomerulosclerosis is unknown, and effective treatments have been elusive. METHODS We performed whole-genome linkage analysis followed by high-throughput sequencing of the positive-linkage area in a family with autosomal recessive focal segmental glomerulosclerosis (index family) and sequenced a newly discovered gene in 52 unrelated patients with focal segmental glomerulosclerosis. Immunohistochemical studies were performed on human kidney-biopsy specimens and cultured podocytes. Expression studies in vitro were performed to characterize the functional consequences of the mutations identified. RESULTS We identified two mutations (A159P and Y695X) in MYO1E, which encodes a nonmuscle class I myosin, myosin 1E (Myo1E). The mutations in MYO1E segregated with focal segmental glomerulosclerosis in two independent pedigrees (the index family and Family 2). Patients were homozygous for the mutations and did not have a response to glucocorticoid therapy. Electron microscopy showed thickening and disorganization of the glomerular basement membrane. Normal expression of Myo1E was documented in control human kidney-biopsy specimens in vivo and in glomerular podocytes in vitro. Transfection studies revealed abnormal subcellular localization and function of the A159P-Myo1E mutant. The Y695X mutation causes loss of calmodulin binding and of the tail domains of Myo1E. CONCLUSIONS MYO1E mutations are associated with childhood-onset, glucocorticoid-resistant focal segmental glomerulosclerosis. Our data provide evidence of a role of Myo1E in podocyte function and the consequent integrity of the glomerular filtration barrier.Wo