CLINICAL RESEARCH www.jasn.org Characterization of Coding/Noncoding Variants for SHROOM3 in Patients with CKD Jeremy W. Prokop,1,2 Nan Cher Yeo,3 Christian Ottmann,4,5 Surya B. Chhetri,1,6 Kacie L. Florus,1 Emily J. Ross,1,7 Nadiya Sosonkina,1 Brian A. Link,8 Barry I. Freedman,9 Candice J. Coppola,6 Chris McDermott-Roe,10 Seppe Leysen,4 Lech-Gustav Milroy,4 Femke A. Meijer,4 Aron M. Geurts,10 Frank J. Rauscher III,11 Ryne Ramaker,1 Michael J. Flister,10 Howard J. Jacob,1 Eric M. Mendenhall,1,6 and Jozef Lazar1 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Interpreting genetic variants is one of the greatest challenges impeding analysis of rapidly increasing volumes of genomic data from patients. For example, SHROOM3 is an associated risk gene for CKD, yet causative mechanism(s) of SHROOM3 allele(s) are unknown. Methods We used our analytic pipeline that integrates genetic, computational, biochemical, CRISPR/Cas9 editing, molecular, and physiologic data to characterize coding and noncoding variants to study the human SHROOM3 risk locus for CKD. Results We identified a novel SHROOM3 transcriptional start site, which results in a shorter isoform lacking the PDZ domain and is regulated by a common noncoding sequence variant associated with CKD (rs17319721, allele frequency: 0.35). This variant disrupted allele binding to the transcription factor TCF7L2 in podocyte cell nuclear extracts and altered transcription levels of SHROOM3 in cultured cells, potentially through the loss of repressive looping between rs17319721 and the novel start site. Although common variant mechanisms are of high utility, sequencing is beginning to identify rare variants involved in disease; therefore, we used our biophysical tools to analyze an average of 112,849 individual human genome sequences for rare SHROOM3 missense variants, revealing 35 high-effect variants. The high-effect alleles include a coding variant (P1244L) previously associated with CKD (P=0.01, odds ratio=7.95; 95% CI, 1.53 to 41.46) that we find to be present in East Asian individuals at an allele frequency of 0.0027. We determined that P1244L attenuates the interaction of SHROOM3 with 14–3-3, suggesting alterations to the Hippo pathway, a known mediator of CKD. Conclusions These data demonstrate multiple new SHROOM3-dependent genetic/molecular mecha- nisms that likely affect CKD. J Am Soc Nephrol 29: 1525–1535, 2018. doi: https://doi.org/10.1681/ASN.2017080856 The combination of current sequencing and genome wideassociationstudies(GWAS)haslinkedhundredsof Significance Statement human loci with CKD. Yet the majority of causative Although the genetics of CKD are beginning to be deciphered, interpretation of how variants result in Received August 8, 2017. Accepted January 19, 2018. disease remains a challenge that is increasing as more and more genomes are being sequenced. In this paper, Published online ahead of print. Publication date available at we use our workflow designed to assess variants to www.jasn.org. develop mechanistic insights into CKD variants, high- Correspondence: Dr. Jeremy W. Prokop or Dr. Jozef Lazar, lighting new knowledge of both common noncoding HudsonAlpha Institute for Biotechnology, 601 Genome Way and rare coding variants within SHROOM3. The de- Northwest, Huntsville, AL 35806. E-mail: [email protected] tailed knowledge gleaned for function of SHROOM3 in or [email protected] podocytes advances novel pathways and mechanisms for CKD. Copyright © 2018 by the American Society of Nephrology J Am Soc Nephrol 29: 1525–1535, 2018 ISSN : 1046-6673/2905-1525 1525 CLINICAL RESEARCH www.jasn.org faced by genetic research is to develop strate- gies that will rapidly characterize variants and provide insight for personalizing disease in- terventions.1 Here, we deployed our sequence-to-struc- ture-to-function approach2 that integrates ge- netic, computational, biochemical, CRISPR/ Cas9 editing, molecular, and physiologic data to characterize coding and noncoding geno- mic variants identified for CKD3–10 risk within SHROOM3. SHROOM3 is an actin- binding protein involved in cell shape, neural tube formation, and epithelial morphogene- sis.11,12 A recent study showed that rs17319721 was associated with changes in expression of SHROOM3,andisaleadingSNPincreasing renal fibrosis in patients with kidney trans- plantation.13,14 Our group identified missense variants within Shroom3 of the FHH rat that affect normal maintenance of kidney glomer- ular filtration.15 In mice, genetic deletion of Shroom3 confirms its role in glomerular func- tion and maintenance of proper podocyte morphology, with alterations of apically dis- tributed actin.12 The apical construction role of SHROOM3 was first documented in neu- rulation.16 This paper lays out mechanistic in- sights into both noncoding GWAS-associated common variants and rare coding variants of SHROOM3,layingoutaworkflow for addi- tional GWAS LD block analysis. Figure 1. Analysis of SHROOM3 data from Roadmap Epigenetics. Core 15-state METHODS model for multiple human tissue types for SHROOM3 gene. Colors indicate the predicted states: red=active TSS, orange red=flanking active TSS, green=transcript, Analysis of SHROOM3 Regulation yellow=enhancer, gray=repressed polycomb, white=quiescent. All 15 colors for each 17 state can be found at http://egg2.wustl.edu/roadmap/web_portal/chr_state_learning. The roadmap epigenomics 15 core data html#core_15state. Three active TSSs (labeled on top), resulting in three isoforms for were viewed for SHROOM3 and sorted on the SHROOM3 gene. Neural tissue is boxed in blue, fetal kidney in red, and adrenal the basis of expression and isoform detec- gland in green. The bottom of the figure is the zoomed in view of the CKD-associated tion. Start sites predicted for SHROOM3 LD block of SNPs associated in GWAS showing a breakdown of the 15-state model, were identified using SwitchGear. The LD 25-state model, DNase hypersensitivity, H3kme1, H3kme3, vertebrate conservation, block for the GWAS near SHROOM3 was human GWAS lead SNPs, and the HapMap CEU Utah LD analysis. The red intensity identified using the SNAP tool18 with a 0.8 shows the correlation of any two points (red is highest correlation) on the chromo- correlation. Biotin-conjugated DNA probes some for coinheritance of genetic variants, such that the point of the triangle is the were used to perform LightShift Chemilumi- correlation of the two edges of the base. CEU, Utah Residents with Northern and nescent Electrophoresis Mobility Shift As- Western European Ancestry; LD, linkage disequilibrium; SNP, single nucleotide polymorphism. says (EMSA) (ThermoFisher) as previously published.19 CRISPR/Cas9 Modification variant(s) and mechanism(s) within each locus that confers risk CRISPR/Cas9 replacement of rs17319721 was performed remainlargelyunknown.Thus,rapidlygrowingvolumesofgenetic using gRNAs following previous published conditions20 data without functional validation have dramatically increased the into HEK293T cells. Cells underwent clonal expansion catalog of variants that have not been categorized with respect to and variants were confirmed with Sanger sequencing. function. We and others postulate that the next greatest challenge Real-time qPCR was performed using the RNeasy Plus 1526 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1525–1535, 2018 www.jasn.org CLINICAL RESEARCH Zebrafish Experiments to Test the Short Isoform of SHROOM3 Human SHROOM3 cDNA ORF (NM_020859) was purchased from OriGene Technolo- gies and the ASD2 (ΔASD2) or PDZ (ΔPDZ) were removed using Phusion site-directed mutagenesis. Zebrafish coin- jections were performed on one- to four- cell–stage zebrafish embryos, and dextran clearance assay performed as previously described.15 A p53 morpholino was coin- jected to reduce off-target cell dealth,22 and efficiency of morpholino has been previ- ously published.15 P1244L Analysis Human coding variants for SHROOM3 were pulled from the gnomAD data- base23 and potential functional variants were assessed using our sequence-to- structure-to-function tools.2 LATS2 phosphorylation was analyzed using multiple custom peptides, and ADP-Glo Kinase Assay. For determining Kd and solving the crystal structure of 14–3-3 with SHROOM3 (either WT or P1244L) the peptides were synthesized24 and combined with purified 14–3-3. Isother- mal titration calorimetry measurements were performed with Malvern MicroCal iTC200. Crystals were set up using 14–3-3 and SHROOM3 peptide dissolved in crystallization buffer and mixed in a 1:1 stoichiometry, set up for sitting-drop crystallization; crystals were captured af- ter 10 days of incubation at 4°C; and dif- fraction data collected at the 306SA/PXI beamline. Figure 2. Nuclear interactions with the SHROOM3 CKD noncoding associated SNPs. (A) EMSA using probes with minor (A) or major (G) alleles of rs17319721 and nuclear lysates from HEK293 and the three primary kidney cell nuclear extracts (endothelial, tubule, and RESULTS podocyte). Shown to the side of the representative EMSA is the quantification of free probe and shifted probe from three separate replicates. The stars are sites significantly different from the control. (B) EMSA assays of rs17319721 using recombinant TCF7L2 Transcript Characterization of and FOXO1 compared with the shifting seen by the podocyte nuclear lysate. The bands SHROOM3 Isoforms in Human and for TCF7L2 are identified in blue and those for FOXO1 in magenta with the quantification Mouse Tissues of three separate experiments