Steroid-sensitive nephrotic syndrome (SSNS) is a rare, mostly childhood kidney disease defined by selective proteinuria, hypoalbuminaemia and minimal histological changes without Ig deposits. It has been postulated that SSNS is immune mediated and involves T-cell dysregulation. Association studies with HLA class-2 alleles are small or limited to an exome array on a South Asian population. Building on our GWAS in membranous nephropathy (MN), (Stanescu et al, NEJM 2011, 364: 616) and a first set of data obtained by GWAS and NGS in 425 and 96 SSNS patients, respectively, our objectives are to identify additional gene variants by GWAS, to sequence the GWAS-tagged loci by NGS, and to investigate the functional significance of the relevant gene variants. This project is very innovative because it combines multi-ethnic and family studies, full sequencing of GWAS-tagged loci, functional studies, and clinical correlations in deeply phenotyped cohorts, as well as a comparison with risk variants in MN. This study comprises 5 workpackages. WP 1: GWAS discovery of new SNPs associated with SSNS We have tripled the number of enrolled patients (non-Chinese available for the current study: 375 patients; Chinese: 500 patients; grand total, 1,400 patients including already studied patients) to discover new SNPs, particularly in non-HLA-D associated loci. WP 2: Deep genotyping of relevant loci by NGS: HLA-D and beyond Our first aim is to provide a fine mapping of loci tagged by GWAS within HLA-D locus and outside of it. Studies will include logistic regression analysis of top SNPs, and identification of ethnicity-specific SNPs which may explain part of the geographic heterogeneity of SSNS, as well as common, trans-ethnic SNPs, which may be associated with key pathways affecting the immune system or the podocyte. A second objective is to compare these data with those in MN (220 patients sequenced, unpublished results) because of shared characteristics with SSNS. WP 3: Family studies Our objective is to discover variants associated with 22 families of SSNS, which may reveal a strong effect of a rare variant of a gene involved as a common variant in sporadic cases. We aim to identify coding variants (mutations) by whole exome studies (WES) as well as non-coding and coding variants within HLA-D locus and outside of it, by targeted sequencing of candidate genes and loci. WP 4: Functional studies of relevant gene variants They will depend on the findings of genetic studies, which may provide clues to permeability factor(s) or to altered signaling pathways. Two lines of research can already be envisaged: • Characterization of regulatory and coding SNPs: We expect to find disease-associated non-coding SNPs within regulatory sequences as well as SNPs in the coding regions. Computational approaches will first guide the design of functional assays. We will use 41 glomerular transcriptomes from SSNS patients to correlate tag and regulatory SNPs with actual transcriptional changes in the glomerulus. • Identification of HLA-class 2 molecules: the road toward identification of T-cell epitopes: We will characterize risk allelotypes, haplotypes and amino acids on HLA class-2 molecules, and we will use the relevant anti-allelotype antibodies to immunoprecipitate the circulating HLA class-2 peptide complexes followed by mass spectometry analysis. WP 5: Clinical correlations Correlations will be established between clinical presentation (demography) and outcome, and genotype (alleles of top SNPs, risk allelotypes and amino acids). With our deeply phenotyped, multi-ethnic cohort also including adult patients (30% of the Spanish and 60% of the Chinese cohorts), we have the unique opportunity to establish correlations with response to treatment depending on age and ethnicity. These studies will contribute to SSNS pathogenesis and hopefully provide new disease biomarkers and drug targets.