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We previously reported that Indian paediatric patients with atypical haemolytic–uraemic syndrome (aHUS) showed high frequencies of anti‐complement factor H (FH) autoantibodies that are correlated with homozygous deletion of the genes for FH‐related proteins 1 and 3 (FHR1 and FHR3) (FHR1/3 (–/–)). We now report that Indian paediatric aHUS patients without anti‐FH autoantibodies also showed modestly higher frequencies of the FHR1/3 (–/–) genotype. Further, when we characterized epitope specificities and binding avidities of anti‐FH autoantibodies in aHUS patients, most anti‐FH autoantibodies were directed towards the FH cell‐surface anchoring polyanionic binding site‐containing C‐terminal short conservative regions (SCRs) 17–20 with higher binding avidities than for native FH. FH SCR17–20‐binding anti‐FH autoantibodies also bound the other cell‐surface anchoring polyanionic binding site‐containing region FH SCR5–8, at lower binding avidities. Anti‐FH autoantibody avidities correlated with antibody titres. These anti‐FH autoantibody characteristics did not differ between aHUS patients with or without the FHR1/3 (–/–) genotype. Our data suggest a complex matrix of interactions between FHR1‐FHR3 deletion, immunomodulation and anti‐FH autoantibodies in the aetiopathogenesis of aHUS.

Background Mutations in the CD46 gene account for an important proportion of patients with atypical hemolytic uremic syndrome (aHUS) who characteristically show multiple relapses, no response to plasma exchange and low recurrence risk in allograft. We screened for mutations in CD46 in patients with and without circulating anti-factor H (FH) antibodies-associated aHUS. Methods We estimated CD46 surface expression by flow cytometry and sequenced the CD46 gene in 23 and 56 patients with and without circulating anti-FH antibodies, respectively. Human Splicing Finder and PolyPhen2 were used for in silico prediction of pathogenicity. Results Two novel and three known (c.286 +2T > G, c.104G > A and c.565T > G) mutations in CD46 were found in nine (11.4%) patients; one patient had a variant of unknown significance and two patients presented during the first year of life. Novel intronic (c.1127 + 46C > G) and exonic (c.911C > T) mutations are proposed to activate cryptic splicing sites or alter protein conformation. Markedly reduced CD46 surface expression was found in homozygous states in five patients. Conclusion Patients with mutations in CD46 present at all ages, including the first year of life. Mutations in intron 2, (c.286 +2T > G) may be a potential hot spot in Indian children. Flow cytometry for CD46 expression is a satisfactory screening tool enabling early diagnosis.

Background: Atypical hemolytic uremic syndrome (aHUS), an important cause of acute kidney injury (AKI), is characterized by dysregulation of the alternative complement pathway. Autoantibodies to factor H (FH), a chief regulator of this pathway, account for a distinct subgroup. While high anti-FH titers predict relapse, they do not correlate well with disease activity and their functional characterization is required.Methods: Of 781 patients

A homozygous 83-kb deletion encompassing the genes for complement factor-H-related proteins 1 and 3 (FHR 1, FHR3) is known as a risk factor for some immune inflammatory disorders. However, the functional relevance of this FHR1/3 deletion is relatively unexplored. Globally, healthy populations of all ethnic groups tested show an 8-10% prevalence of homozygosity for this deletion polymorphism. We have begun to compare the peripheral leucocyte phenotype and functionality between FHR1/3-/- and FHR1/3+/+ healthy adult individuals. We report that the two groups show significant differences in their peripheral blood innate leucocyte subset composition, although the adaptive immune subsets are similar between them. Specifically, FHR1/3-/- individuals show higher frequencies of patrolling monocytes and lower frequencies of classical monocytes than FHR1/3+/+ individuals. Similarly, FHR1/3-/- individuals show higher frequencies of plasmacytoid dendritic cells (pDCs) and lower frequencies of myeloid DCs (mDCs) than FHR1/3+/+ individuals. Notably, classical monocytes specifically showed cell-surface-associated factor H (FH), and cells from the FHR1/3-/- group had somewhat higher surface-associated FH levels than those from FHR1/3+/+ individuals. FHR1/3-/- monocytes also showed elevated secretion of TNF-α, IL-1β, and IL-10 in response to TLR7/8 or TLR4 ligands. Similarly, FHR1/3-/- mDCs and pDCs showed modest but evident hyper-responsiveness to TLR ligands. Our findings, that the FHR1/3-/- genotype is associated with significant alterations of both the relative prominence and the functioning of monocyte and DC subsets, may be relevant in understanding the mechanism underlying the association of the genotype with immune inflammatory disorders.

Rituximab, a monoclonal antibody targeting B lymphocytes, effectively sustains remission in steroid-dependent nephrotic syndrome (SDNS). We studied its effects on lymphocyte subsets and urinary CD80 excretion (uCD80) in patients with SDNS. Blood and urine samples were collected from 18 SDNS patients before rituximab, and after 1 month and 1 year or at first relapse. T and B lymphocytes and uCD80 were determined by flow cytometry and ELISA, respectively. Treatment was associated with reduction in counts of Th17, Th2, and memory T cells, and increased T-regulatory (Treg) cells. The Th17/Treg ratio declined from baseline (median 0.6) to 1 month (0.2, P = 0.006) and increased during relapse (0.3, P = 0.016). Ratios of Th1/Th2 cells at baseline, 1 month after rituximab, and during relapse were 7.7, 14.0 (P = 0.0102), and 8.7, respectively. uCD80 decreased 1 month following rituximab (45.5 vs. 23.0 ng/g creatinine; P = 0.0039). B lymphocytes recovered earlier in relapsers (60.0 vs.183.0 days; P

Genetic susceptibility to atypical hemolytic uremic syndrome (aHUS) may lie within genes regulating or activating the alternate complement and related pathways converging on endothelial cell activation. We tested 32 Indian patients of aHUS negative for antibodies to complement factor H for genetic variations in a panel of 15 genes, i.e., CFH, CFHR1-5, CFI, CFB, C3, CD46, MASP2, DGKE, ADAMTS13, THBD and PLG using next-generation DNA sequencing and for copy number variation in CFHR1-3. Despite absence of a public database of exome variations in the Indian population and limited functional studies, we could establish a genetic diagnosis in 6 (18.8%) patients using a stringent scheme of prioritization. One patient carried a likely pathogenic variation. The number of patients carrying possibly pathogenic variation was as follows: 1 variation: 5 patients, 2 variations: 9 patients, 3 variations: 5 patients, 4 variations: 9 patients, 5 variations: 2 patients and 6 variations: 2 patients. Homozygous deletion of CFHR1-3 was present in five patients; none of these carried a diagnostic genetic variation. Patients with or without diagnostic variation did not differ significantly in terms of enrichment of genetic variations that were rare/novel or predicted deleterious, or for possible environmental triggers. We conclude that genetic testing for multiple genes in patients with aHUS negative for anti-FH antibodies reveals multiple candidate variations that require prioritization. Population data on variation frequency of the Indian population and supportive functional studies are likely to improve diagnostic yield.