Genetic Factors Associated with Anti-Factor H Autoantibodies in Atypical Hemolytic Uremic Syndrome (aHUS)

Atypical hemolytic uremic syndrome (aHUS) is a rare form of thrombotic microangiopathy characterized by renal failure and determined by genetic and acquired defects of alternative pathway (AP) of the complement system. Autoantibodies against factor H (anti-FHs), a regulator of the AP, were reported in 10% of patients, and are associated with the deficiency of factor H related 1 (FHR1), a FH homologous protein. The aim of this thesis was to evaluate the contribution of genetics to the development of anti-FHs in aHUS. Thirty patients affected by aHUS resulted positive for anti-FHs (9.8%) and FHR1 deficiency was present in 83.3% of them. A healthy control also showed anti-FHs in concomitance with FHR1 deficiency documenting that the lack of FHR1 strongly predisposed to anti-FH development also in healthy subjects although this condition was not sufficient for the disease manifestation. The presence of infectious prodromal signs and an age at the disease onset around 8 years indicated that common infections may trigger the development of autoantibodies in subjects with at risk genetic background. Likely pathogenetic variants in complement genes were observed in 37% of our patients with anti-FHs. At variance, common variants in complement genes did not seem to contribute to the disease, as documented by comparing patients with super controls, unaffected subjects carrying FHR1 deficiency. Finally, I report that the HLA-DRB1*11:04 allele could be a predisposing genetic variant for anti-FH associated aHUS. Further works will be necessary to confirm this finding and to explore the presence of other genetic susceptibility factors that, in combination with the HLA-DRB1*11:04 allele and the FHR1 deficiency, could increase the risk for anti-FHs

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

Rat intestinal precision-cut slices as an in vitro model to study xenobiotic interaction with transporters

ATP-binding cassette (ABC) proteins play key role in tissue defence by transporting metabolic waste and toxic chemicals out of the cells. Consequently, intact cell systems are required to study xenobiotic interactions with ATP-dependent transporters. The aim of the present study was to set up an intestinal precision-cut slice technique to study the interactions of ABC transporters with xenobiotics. Rat intestinal slices were incubated with verapamil, indomethacin and glibenclamide, and the ability of the above-mentioned drugs to inhibit the multidrug resistance glycoprotein (MDR) and/or multidrug-resistance-associated protein (MRP) was assessed by measuring the intracellular conversion of calcein-AM to fluorescent calcein. The ABC transporters' inhibitors caused a time-dependent florescence increase which reached the maximum value at 30 min. Verapamil and glibenclamide promoted a concentration-dependent intracellular accumulation of calcein (IC(50) 8.1×10(-6) M, 1.9×10(-4) M, respectively). The effect of glibenclamide was fully reversed by washing the slices, suggesting the reversible nature of calcein accumulation. These data suggest that the precision-cut intestinal slices are a reliable, simple and fast system to evaluate xenobiotic interactions with ABC transporters in rat and, hopefully, in human intestine

Association of CFHR1 homozygous deletion with acute myelogenous leukemia in the European population

Our data demonstrate an association between the germinal homozygous deletion of the CFHR1 gene and acute myeloid leukemia (AML) in the Caucasian population. The frequency of this deletion is 4 % in the healthy reference and the medulloblastoma populations, while it is 7 % in AML patients. The study may define a novel subtype of AML, with a peculiar etio-pathogenesis related to alterations of the complement factor cascade