Fig 2: ITSN1 and ITSN2 are GEFs for Cdc42, regulating NS-related podocyte function, and Itsn2-/- mice develop incompletely penetrant NS upon LPS injection. a Myc-tagged ITSN1-WT increased active Cdc42 levels upon overexpression in HEK293T cells as compared to mock, whereas three out of five ITSN1 mutants from pTSNS patients failed to show any significant increase in active Cdc42 upon overexpression. b Active Cdc42 level was measured in COS7 cells. Myc-tagged ITSN2-WT overexpression increased active Cdc42 levels as compared to Mock. However, all three ITSN2 mutants from pTSNS patients failed to show any significant increase in active Cdc42 upon overexpression. Error bars are defined as the standard error of at least four independent experiments. One-way ANOVA with Dunnet’s post hoc test vs. wild-type (WT) ITSN2 expressing control. c Filopodia induction in cultured human podocytes was quantified by counting the transfected cells with filopodia and showed as a percentage of cells. For each construct, 50 transfected cells from three independent experiments were analyzed. One-way ANOVA with Dunnet’s post hoc test vs. WT ITSN1 expressing control. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. d Filopodia induction in cultured human podocytes was quantified by counting the transfected cells with filopodia and showed as a percentage of cells. For each construct, 50 transfected cells from three independent experiments were analyzed. One-way ANOVA with Dunnet’s post hoc test vs. WT ITSN2 expressing control. Data are presented as the mean ± SEM. * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. e Kidneys of WT and Itsn2 L-/L- mice were stained with periodic acid Schiff. The glomeruli of Itsn2 L-/L- mice showed normal findings, similar to WT. f Urine albumin of Itsn2 L-/L-, Itsn2 +/L-, and WT mice was measured before and after LPS injection. Albuminuria level was increased in Itsn2 L-/L- mice from 12 to 48 h after LPS injection compared with WT and Itsn2 +/L- mice. Two-tailed Student’s t-tests. Data are presented as the mean ± SEM. *P < 0.05. g Representative electron microscopy images. LPS injection-induced foot process (FP) effacement in both WT and Itsn2 L-/L- mice within 24 h. At 48 h after LPS injection, FP effacement was still observed in Itsn2 L-/L- mice, although WT mice recovered from FP effacement

Fig 3: High-throughput sequencing reveals recessive mutations of MAGI2, TNS2, DLC1, CDK20, ITSN1, or ITSN2 as causing NS in humans. a Renal histology of individual A5146-21 with focal segmental glomerulosclerosis (FSGS) and MAGI2 mutation (scale bar = 50 µm). b Exon structure of human MAGI2 cDNA and mutations. Below is the protein domain structure of MAGI2, showing GuK, WW1, WW2, and six different PDZ domains. Two different homozygous truncating mutations of MAGI2 were detected in two families with NS and neurological impairment. c Homozygosity mapping identifies ten recessive candidate loci (red circles) in family A1358 with NS, and WES identifies a homozygous mutation of TNS2 (p.Arg292Gln). Non-parametric lod scores (NPL) were calculated and plotted across the human genome. The TNS2 locus (arrowhead) is positioned within one of the maximum NPL peaks on chromosome 12q. d Exon and protein domain structure of human TNS2. Six different TNS2 mutations were detected in five families with NS. Family numbers and amino acid changes are given (Supplementary Table 1). Arrow heads denote altered nucleotides. Lines and arrows indicate positions of mutations in relation to exons and protein domains. Family numbers with compound heterozygous mutations (het) are highlighted in gray. e Renal histology of individual A4967-21 with FSGS and DLC1 mutations. TEM reveals podocyte foot process effacement (arrow heads, magnification 8000x). f Exon and protein domain structure of human DLC1. The SAM, RhoGAP, and START domains are depicted by colored bars, in relation to encoding exon position. Six different DLC1 mutations were detected in four families with NS. Positions of amino acid changes (Supplementary Table 1) are marked with arrowheads. g Renal histology of individual A5013-21 with membranoproliferative glomerulonephritis (MPGN) and mutation in CDK20. TEM reveals podocyte foot process effacement and thickening of glomerular basement membrane (arrow heads, magnification 7000x). h Homozygosity mapping identifies twelve recessive candidate loci (red circles) in family A5013 with NS, and WES identifies a homozygous mutation of CDK20 (p.Phe204Leu), positioned within one of the maximum NPL peaks on chromosome 9q. i Exon and protein domain structure of human CDK20. The serine threonine kinase (S_TKc) domain is depicted by a colored bar, in relation to encoding exon position. One homozygous mutation in CDK20 was detected in family A5013 with NS. j Homozygosity mapping in family A3706 with NS identifies 17 recessive candidate loci (red circles), and WES identifies a homozygous mutation of ITSN1 (p.Pro180Ser), positioned within one of the maximum NPL peaks on chromosome 21q. k Exon and protein domain structure of human ITSN1. Five different ITSN1 mutations were detected in three families with NS. l Renal histology of Patient-1 with mutations in ITSN2 revealed minimal change disease (scale bar = 50 µm). EM showed foot process effacement (scale bar = 2 µm). m Exon and protein domain structure of human ITSN2. Three different ITSN2 mutations were detected in two families with NS