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To identify gene loci associated with steroid-resistant nephrotic syndrome (SRNS), we utilized homozygosity mapping and exome sequencing in a consanguineous pedigree with three affected siblings. High-density genotyping identified three segments of homozygosity spanning 33.6 Mb on chromosomes 5, 10, and 15 containing 296 candidate genes. Exome sequencing identified two homozygous missense variants within the chromosome 15 segment; an A159P substitution in myosin 1E (MYO1E), encoding a podocyte cytoskeletal protein; and an E181K substitution in nei endonuclease VIII-like 1 (NEIL1), encoding a base-excision DNA repair enzyme. Both variants disrupt highly conserved protein sequences and were absent in public databases, 247 healthy controls, and 286 patients with nephrotic syndrome. The MYO1E A159P variant is noteworthy, as it is expected to impair ligand binding and actin interaction in the MYO1E motor domain. The predicted loss of function is consistent with the previous demonstration that Myo1e inactivation produces nephrotic syndrome in mice. Screening 71 additional patients with SRNS, however, did not identify independent NEIL1 or MYO1E mutations, suggesting larger sequencing efforts are needed to uncover which mutation is responsible for the phenotype. Our findings demonstrate the utility of exome sequencing for rapidly identifying candidate genes for human SRNS.

Background: Steroid-resistant nephrotic syndrome (SRNS) with focal segmental glomerulosclerosis has emerged as a leading cause of end-stage renal failure (ESRF) in children and adults. In the past decade, immunosuppressive drugs such as cyclosporine (CsA) and cyclophosphamide have been introduced for the treatment of SRNS, but data on long-term clinical outcome (over years) are lacking., Objective: The current study considered the clinical outcome of patients with SRNS who had been treated with CsA for >2 years. The primary objective was to evaluate renal function after years of treatment compared with nontreated or CsA-resistant patients. A secondary objective was to identify renal effects related to the use of CsA, with a major emphasis on renal fibrosis., Methods: In this open-label, nonrandomized, retrospective study, the outcomes of patients of all ages with sporadic SRNS who had been followed up for >2 years (between 1970 and 2002) at 4 Italian clinical institutions were evaluated. Preliminary molecular screenings for genes encoding proteins of the slit-diaphragm (eg, podocin, nephrin, alpha-actinin) were performed to exclude inherited forms of sporadic SRNS., Results: A total of 157 patients were studied; mutations were found in 18 patients (11%). Of the remaining 139 patients (84 men, 55 women; median [interquartile range (IQR)] age at onset of proteinuria, 12 [4-32] years), 84 (60%) were nontreated and 55 (40%) were treated with CsA. Of these 55 treated patients, 35 (64%) were found to be resistant (ie, persistence of proteinuria after 2 months) or intolerant (ie, malignant hypertension or worsening of renal function), and CsA was withdrawn. The median (IQR) durations of follow-up for CsA-resistant and nontreated patients were 41 (23-92) and 48 (28-106) months, respectively. Twenty patients (36%) were responsive to CsA and were followed up for a median (IQR) of 81 (47-115) months. Progression Lo ESRF occurred in 10% of CsA-responsive patients versus 60% of CsA-resistant patients and 62% of nontreated patients (P = 0.002). No sign of renal fibrosis related to drug toxicity was observed in renal biopsies performed at 5-year intervals., Conclusions: This retrospective analysis of SRNS documented a persistent antiproteinuric effect of long-term CsA (>2 years) in the absence of renal fibrosis. Although sensitivity to CsA was associated with normal renal function, resistance or intolerance was associated with progression to ESRF These data suggest that CsA may have a role in the treatment of patients with SRNS.

A total of 179 children with sporadic nephrotic syndrome were screened for podocin mutations: 120 with steroid resistance, and 59 with steroid dependence/frequent relapses. Fourteen steroid-resistant patients presented homozygous mutations that were associated with early onset of proteinuria and variable renal lesions, including one case with mesangial C3 deposition. Single mutations of podocin were found in four steroid-resistant and in four steroid-dependent; five patients had the same mutation (P20L). Among these, two had steroid/cyclosporin resistance, two had steroid dependence, and one responded to cyclosporin. The common variant R229Q of podocin, recently associated with late-onset focal segmental glomerulosclerosis, had an overall allelic frequency of 4.2% versus 2.5% in controls. To further define the implication of R229Q, a familial case was characterized with two nephrotic siblings presenting the association of the R229Q with A297V mutation that were inherited from healthy mother and father, respectively. Immunohistochemistry with anti-podocin antibodies revealed markedly decreased expression of the protein in their kidneys. All carriers of heterozygous coding podocin mutation or R229Q were screened for nephrin mutation that was found in heterozygosity associated with R229Q in one patient. Finally, podocin loss of heterozygosity was excluded in one heterozygous child by characterizing cDNA from dissected glomeruli. These data outline the clinical features of sporadic nephrotic syndrome due to podocin mutations (homozygous and heterozygous) in a representative population with broad phenotype, including patients with good response to drugs. The pathogenetic implication of single podocin defects per se in proteinuria must be further investigated in view of the possibility that detection of a second mutation could have been missed. A suggested alternative is the involvement of other gene(s) or factor(s).

Background: Hyperlipemia characterizes nephrotic syndrome (NS) and contributes to the progression of the underlying nephropathy. The data in the literature support an implication of apolipoprotein E (apoE) in both hyperlipemia and focal segmental glomerulosclerosis (FSGS), a malignant condition associated with NS., Methods: The apoE genotype was determined in 209 nephrotic patients, who were classified according to age and their response to steroids as resistant children (N = 96) and adults (43), and steroid dependent (33) and steroid responder (37) children. A total of 123 presented the histological features of FSGS. In a subgroup of 28 patients, serum and urinary levels of apoE and renal deposits were evaluated by immunofluorescence., Results: The allelic frequencies of the three major haplotypes epsilon2, epsilon3, and epsilon4 were the same in nephrotic patients versus controls, and homozygosity for epsilon3epsilon3 was comparably the most frequent genotype (70 vs. 71%) followed by epsilon3epsilon4, epsilon2epsilon3, epsilon2epsilon4, epsilon4epsilon4. Serum levels of apoE were fivefold higher in NS and in FSGS patients than in controls, with a direct correlation with hypercholesterolemia and proteinuria. ApoE genotypes did not influence serum levels. Urinary levels were 1/10,000 of serum with an increment in nephrotic urines. Finally, immunofluorescence demonstrated the absence of apoE in sclerotic glomeruli, while comparably nephrotic patients with membranous nephropathy had an increased glomerular expression of apoE., Conclusions: ApoE is dysregulated in NS with a marked increment in serum, which is a part of the complex lipid metabolism. Down-regulation of glomerular apoE instead is a peculiarity of FSGS and may contribute to the pathogenesis of the disease. The normal distribution of apoE genotypes in nephrotic patients with FSGS excludes a pathogenetic role of genetic variants.