Volume 95, Issue 1, Pages 57-61 (January 2019) Recessive mutation in CD2AP causes focal segmental glomerulosclerosis in humans and mice  Tomoko Takano, Eric Bareke, Naoki Takeda, Lamine Aoudjit, Cindy Baldwin, Philip Pisano, Jun Matsuda, Jasmine El Andalousi, Lina Muhtadie, Chantal Bernard, Jacek Majewski, Toru Miyazaki, Ken-ichi Yamamura, Indra R. Gupta  Kidney International  Volume 95, Issue 1, Pages 57-61 (January 2019) DOI: 10.1016/j.kint.2018.08.014 Copyright © 2018 International Society of Nephrology Terms and Conditions

Figure 1 Whole exome sequencing (WES) identified a homozygous frameshift mutation in the CD2AP gene. (a) Pedigree of the affected family. Three siblings (2 males and 1 female) born to consanguineous parents of Lebanese origin (first cousins) developed focal segmental glomerulosclerosis (FSGS) in their teens and had progression to end-stage renal disease by ∼20 years of age. (b) Kidney biopsy of brother 2 (left) and sister (right) showing FSGS. Top left: Masson’s trichrome staining shows a glomerulus with early segmental lesion (arrow) and a normal glomerulus (asterisk). Bar = 100 nm. Bottom left: Periodic acid-Schiff (PAS) staining shows a hypertrophic (asterisk) as well as a globally sclerosed (arrow) glomerulus with a few adjacent atrophic tubules (arrow). Bar = 80 nm. Top right: Silver staining shows an enlarged glomerulus with a well-developed segmental sclerosing lesion with a capsular adhesion (asterisk). The adjacent glomeruli show some thickening of the Bowman’s capsule (arrows). Bar = 70 nm. Bottom right: PAS staining shows 2 well-preserved glomeruli, except for early capsular adhesion (arrow). Bar = 90 nm. (c) WES identified an insertion of 4 nucleotides in the CD2AP gene that causes a frameshift at S198, resulting in a stop codon (p.S198fs). Sanger sequencing confirmed that all 3 siblings were homozygous for the mutation while the unaffected father was heterozygous. (d) Expected protein structures of the wild type (WT) and S198fs. CC, coiled-coil domain; SH3, Src homology 3 domain. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2019 95, 57-61DOI: (10.1016/j.kint.2018.08.014) Copyright © 2018 International Society of Nephrology Terms and Conditions

Figure 2 Cd2ap knock-in mice for S198fs develop heavy proteinuria and die of kidney failure. p.Ser198fs mutation was introduced into the mouse embryonic stem cells by using CRISPR/Cas9 gene editing, and knock-in mice were generated (C57BL/6). When heterozygous mice carrying the insertion were bred, wild-type (WT), heterozygous (Het), and homozygous (Homo) mice were born at the expected Mendelian frequency. (a) Urine gel electrophoresis and Coomassie Blue staining of 2-week-old mice. Homo mice showed heavy albuminuria. (b) By 4 weeks, elevated blood urea nitrogen (BUN), serum creatinine (sCr), and hypoalbuminemia (sAlb) levels were observed. These changes were not seen in heterozygotes or WT. Homo mice died at ∼6–8 weeks of kidney failure. n = 3–5 mice each. ∗P < 0.05, **P < 0.01 vs. WT and Het. (c) Histological changes in the kidney of knock-in mice. PAS staining of the kidneys. By 2–3 weeks, Homo mice showed mild glomerulosclerosis, tubular atrophy, and interstitial leukocyte infiltration. By 4–6 weeks, histological changes worsened. These changes were not seen in heterozygotes or WT. See Supplementary Figure S1 for quantification. BSA, bovine serum albumin. Bar = 50 μm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2019 95, 57-61DOI: (10.1016/j.kint.2018.08.014) Copyright © 2018 International Society of Nephrology Terms and Conditions