Clinical Research

Characterization of Coding/Noncoding Variants for SHROOM3 in Patients with CKD

Jeremy W. Prokop, Nan Cher Yeo, Christian Ottmann, Surya B. Chhetri, Kacie L. Florus, Emily J. Ross, Nadiya Sosonkina, Brian A. Link, Barry I. Freedman, Candice J. Coppola, Chris McDermott-Roe, Seppe Leysen, Lech-Gustav Milroy, Femke A. Meijer, Aron M. Geurts, Frank J. Rauscher, Ryne Ramaker, Michael J. Flister, Howard J. Jacob, Eric M. Mendenhall and Jozef Lazar
JASN May 2018, 29 (5) 1525-1535; DOI: https://doi.org/10.1681/ASN.2017080856

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 mechanisms that likely affect CKD.

The combination of current sequencing and genome wide association studies (GWAS) has linked hundreds of human loci with CKD. Yet the majority of causative variant(s) and mechanism(s) within each locus that confers risk remain largely unknown. Thus, rapidly growing volumes of genetic data without functional validation have dramatically increased the catalog of variants that have not been categorized with respect to function. We and others postulate that the next greatest challenge faced by genetic research is to develop strategies that will rapidly characterize variants and provide insight for personalizing disease interventions.1

Here, we deployed our sequence-to-structure-to-function approach2 that integrates genetic, computational, biochemical, CRISPR/Cas9 editing, molecular, and physiologic data to characterize coding and noncoding genomic variants identified for CKD3–10 risk within SHROOM3. SHROOM3 is an actin-binding protein involved in cell shape, neural tube formation, and epithelial morphogenesis.11,12 A recent study showed that rs17319721 was associated with changes in expression of SHROOM3, and is a leading SNP increasing renal fibrosis in patients with kidney transplantation.13,14 Our group identified missense variants within Shroom3 of the FHH rat that affect normal maintenance of kidney glomerular filtration.15 In mice, genetic deletion of Shroom3 confirms its role in glomerular function and maintenance of proper podocyte morphology, with alterations of apically distributed actin.12 The apical construction role of SHROOM3 was first documented in neurulation.16 This paper lays out mechanistic insights into both noncoding GWAS-associated common variants and rare coding variants of SHROOM3, laying out a workflow for additional GWAS LD block analysis.

Methods

Analysis of SHROOM3 Regulation

The roadmap epigenomics 15 core data17 were viewed for SHROOM3 and sorted on the basis of expression and isoform detection. Start sites predicted for SHROOM3 were identified using SwitchGear. The LD block for the GWAS near SHROOM3 was identified using the SNAP tool18 with a 0.8 correlation. Biotin-conjugated DNA probes were used to perform LightShift Chemiluminescent Electrophoresis Mobility Shift Assays (EMSA) (ThermoFisher) as previously published.19

CRISPR/Cas9 Modification

CRISPR/Cas9 replacement of rs17319721 was performed using gRNAs following previous published conditions20 into HEK293T cells. Cells underwent clonal expansion and variants were confirmed with Sanger sequencing. Real-time qPCR was performed using the RNeasy Plus Mini kit for mRNA isolation, QuantiTect SYBR Green PCR Kit, a QuantStudio 6 Flex Real-Time PCR system, and relative gene expression using the ΔΔ CT method21 with GAPDH as a reference.

Zebrafish Experiments to Test the Short Isoform of SHROOM3

Human SHROOM3 cDNA ORF (NM_020859) was purchased from OriGene Technologies and the ASD2 (∆ASD2) or PDZ (∆PDZ) were removed using Phusion site-directed mutagenesis. Zebrafish coinjections were performed on one- to four-cell–stage zebrafish embryos, and dextran clearance assay performed as previously described.15 A p53 morpholino was coinjected to reduce off-target cell dealth,22 and efficiency of morpholino has been previously published.15

P1244L Analysis

Human coding variants for SHROOM3 were pulled from the gnomAD database23 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. Isothermal 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 after 10 days of incubation at 4°C; and diffraction data collected at the ×06SA/PXI beamline.

Results

Transcript Characterization of SHROOM3 Isoforms in Human and Mouse Tissues

Although the SHROOM3 locus has been implicated in CKD,3–10 the molecular mechanisms that affect CKD through SHROOM3 are unknown. Data from the human Roadmap Epigenomics Project17 were scanned for transcriptional regulation surrounding SHROOM3 (Figure 1). Analysis of the core 15-state model25 suggests that there is active SHROOM3 transcription (green) in most tissue types. However, the transcriptional state (green, Figure 1) is of various sizes and can be binned into three isoforms on the basis of different transcriptional start sites (TSSs: TSS1 at chr4:77356253, TSS2 at chr4:77507377, and TSS3 at chr4:77610545). Neuronal cells contain active histone marks suggestive of a full-length isoform with a promoter (red, Figure 1) annotated at TSS1 (blue box, Figure 1). Tissues such as fetal kidney (red box, Figure 1) and fetal adrenal gland (green box, Figure 1) have a shorter isoform (isoform 2) with transcription starting between exons two and three (of long isoform 1) with TSS2 and TSS3 identified as promoter state (red, Figure 1). Analysis of frequency of reads from RNAseq for the Human Roadmap Epigenomics Program (Supplemental Figure 1), the Human Protein Atlas (Supplemental Table 2), and 1830 FANTOM CAGE datasets (Supplemental Figure 2) shows strong correlation with data from the core 15-state model for a full-length isoform in neuronal cells (isoform 1) and a shorter isoform found in most other tissues including kidney (isoform 2/3). Sixty RNAseq datasets generated from different cell types (Supplemental Figure 3) were analyzed for reads that map to SHROOM3, showing peaks of transcription start at TSS2/3 in kidney and TSS1 in brain. Analysis of RNAseq from isolated mouse glomeruli and 20 single cell RNAseq runs from podocyte show a similar short isoform (NM_001077595) that is not present in cells from tubule. HEK293 cells, used for variant analysis, show a similar isoform to podocytes but with lower levels of transcripts. Breaking down individual glomeruli and podocyte datasets with SHROOM3 expression, a fourth novel mouse isoform was mapped that results in a 45.9 kD protein (Supplemental Figure 4) that was confirmed in addition to isoform 2/3 protein in immunoprecipitations of primary podocytes (Supplemental Figure 5). The CKD-associated LD block was identified (zoomed in bottom, Figure 1) after TSS1 and is located approximately 100 kb upstream of TSS2/3. The implication of the shorter isoform in kidney is relevant to CKD, because early papers claimed the associated SNP was intronic,13 so changing how one interprets the biologic role of SNPs.

Figure 1.
Figure 1.

Analysis of SHROOM3 data from Roadmap Epigenetics. Core 15-state model for multiple human tissue types for SHROOM3 gene. Colors indicate the predicted states: red=active TSS, orange red=flanking active TSS, green=transcript, yellow=enhancer, gray=repressed polycomb, white=quiescent. All 15 colors for each state can be found at http://egg2.wustl.edu/roadmap/web_portal/chr_state_learning.html#core_15state. Three active TSSs (labeled on top), resulting in three isoforms for the SHROOM3 gene. Neural tissue is boxed in blue, fetal kidney in red, and adrenal gland in green. The bottom of the figure is the zoomed in view of the CKD-associated LD block of SNPs associated in GWAS showing a breakdown of the 15-state model, 25-state model, DNase hypersensitivity, H3kme1, H3kme3, vertebrate conservation, human GWAS lead SNPs, and the HapMap CEU Utah LD analysis. The red intensity shows the correlation of any two points (red is highest correlation) on the chromosome for coinheritance of genetic variants, such that the point of the triangle is the correlation of the two edges of the base. CEU, Utah Residents with Northern and Western European Ancestry; LD, linkage disequilibrium; SNP, single nucleotide polymorphism.

Analysis of LD for rs17319721 identified 11 noncoding SNPs with 0.8 correlation in the Utah Residents with Northern and Western European Ancestry population, four of which have been associated with CKD. Using RegulomeDB26 to prioritize these 11 SNPs, only rs17319721 is predicted to have high effect with high conservation, DNase hypersensitivity (bottom Figure 1), and prediction to alter TCF7L2 or FOXO1 binding. Utilizing publicly available HiChIP data,27 we show that in a cell line, GM12878 (with no detectable levels of SHROOM3), chromatin contacts exist between areas near rs17319721 and TSS2 (Supplemental Figure 6), suggesting a looping structure potentially involved in gene repression. Analyses of current ENCODE ChIP-Seq data for transcription factor (TF) binding near rs17319721 and TSS2 reveal eight factors shared between both sites (Supplemental Figure 7), including association of TCF7L2 to TSS2 and association of linker protein CTCF at both sites. A network analysis of HEK293 cells for TFs near TSS2 shows association of TCF7L2 and also multiple TFs for gene repression such as TRIM28/KAP1 (Supplemental Figure 8). This data suggests that rs17319721 is potentially the functional SNP for CKD as a regulatory site for TSS2 within the shorter isoform of SHROOM3.

EMSA to Map Functional Noncoding Variants

To confirm the potential role of the noncoding rs17319721, we performed biochemical TF interaction tests using EMSA, identifying only two of the LD SNPs to have potential altered binding (Supplemental Figure 9). Taking nuclear extract from multiple cell lines with variable expression of SHROOM3, we show a strong shifted band for rs17319721 that can be cold-outcompeted (Supplemental Figure 9). Nuclear extracts were taken from three primary kidney cell types (glomerular endothelial, tubule, and podocyte) and HEK293 cell line. These extracts were mixed with rs17319721 DNA probes containing either major (G) or minor (A) alleles, and EMSA was performed in three independent experiments (Figure 2A). Podocytes for rs17319721 had the largest and most significant shift between major and minor alleles, with no changes in binding observed in HEK293 or glomerular endothelial extracts, and some relative loss of binding from tubule nuclear extracts.

Figure 2.
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 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 and FOXO1 compared with the shifting seen by the podocyte nuclear lysate. The bands for TCF7L2 are identified in blue and those for FOXO1 in magenta with the quantification of three separate experiments shown below the representative EMSA. An additional scrambled probe was used for the EMSA where eight bases where mutated in the middle of the probe. (C) Models for both TCF7L2 (blue) and FOXO1 (magenta) interacting with the DNA near variant rs17319721. DNA is shown in gray with the variant location colored in red. Nuc, nuclear.

Because rs17319721 falls between FOXO1 and TCF7L213 binding motifs (Figure 2C), we evaluated ability of recombinant protein for each to compete with shifts induced by podocyte nuclear extracts. TCF7L2 protein shifts the probe to the position of the lower band (Figure 2A) whereas FOXO1 shifts to the higher band, both of which were present in podocyte nuclear lysate shifts (Figure 2B). The lower band shift by TCF7L2 is altered by the minor allele to the same level as an 8-bp scramble of the loci, whereas FOXO1 minor allele has less alteration than the scramble element. Of note, lack of complete loss of binding by TCF7L2 is not a surprise because it is a minor groove binding protein, with high nonspecific DNA interaction. This suggests that within podocytes rs17319721 changes the binding of TCF7L2, a factor with known human CKD effect28 and suggested podocyte function.29

Modification of LD Block and rs17319721 Using CRISPR/Cas9

To test rs17319721 in a cellular model for SHROOM3 regulation, we used CRISPR/Cas9 to generate the homozygous A allele in HEK293T cells (Figure 3A). HEK293T cells were used as a model for kidney because this cell line expresses the short isoform of SHROOM3, and as a stable cell line facilitated gene editing. HEK293T cells homozygous for the A allele at rs17319721 were confirmed by Sanger sequencing (Figure 3C). The A allele variant resulted in an elevated SHROOM3 expression of the short isoform relative to GAPDH, without changing expression of two flanking genes, SEPT11 and FAM47E (Figure 3D), and still did not produce any detectable long isoform (Figure 3B). To confirm the role of the LD block in TSS2 regulation, we removed the entire LD block from HEK293 cells, which also resulted in an elevation of short and not long isoform of SHROOM3. The number of modified alleles in these HEK293 cells correlates to expression change in SHROOM3 (Supplemental Figure 10). This study demonstrates that the minor allele (A variant) can significantly increase expression of a short isoform of SHROOM3.

Figure 3.
Figure 3.

Demonstrating expression changes in SHROOM3 by allelic specific changes in rs17319721. (A) Transcripts of the SHROOM3 gene with primer combinations designed to identify isoform 1 using primers on exons 2 and 4 (E2+E4) or all isoforms (exons 8+9, E8+E9). (B) RT-PCR of exon primer pairs showing expression of full-length isoform (E2E4) only in RPMI8226 and not HEK293T cells, whereas all other exon pairs can be seen in all samples. (C) Using a donor sequence after CRISPR/Cas9 editing, the wild-type G allele (WT) at rs17319721 converted into a homozygous A allele (Mut) in HEK293T cells confirmed by Sanger sequencing. (D) Expression of short (E8E9) forms of SHROOM3 were seen (larger change of shorter isoforms) to elevate after the generation of the A allele (Mut) of rs17319721. Expression of the genes that flank SHROOM3, SEPT11, and FAM47E were not changed. Error bars for all represent the SEM with significance (*) determined as P<0.05. SNP, single nucleotide polymorphism.

Functionality of the Shorter SHROOM3 Isoform in Kidney

With strong evidence that the shorter SHROOM3 isoform in kidney is altered by the minor allele of rs17319721 in the CKD GWAS LD block, we evaluated the resulting protein. The three isoforms of human transcripts result in two SHROOM3 proteins, with isoform 1 being the longest and isoform 2/3 sharing the same initiator methionine found as an MM sequence at amino acid 177 of full-length isoform 1 (Figure 4A). A fourth isoform resulting from an unknown TSS or splicing has also been identified that results in a 45.9 kD protein (Supplemental Figures 4 and 5); however, little is currently known about the functionality of this isoform 4 besides the fact that it lacks both the PDZ and ASD1 regions. Isoform 1 is the longest protein, containing PDZ, ASD1, and ASD2 domains. The second protein contains all of the same domains except the PDZ (Figure 4A), similar to previously identified mouse shrmS.11 Our sequence-to-structure-to-function approach2 was used to assess conservation in multiple sequences for full-length SHROOM3 protein (Supplemental Figures 11 and 12). To validate our prediction of the potential functional regions of SHROOM3, we ran the same analysis for the highly homologous SHROOM2 (Figure 4B). The PDZ domain of SHROOM3 is highly conserved and has been suggested to be a critical functional region in neural biology,11,30 but it is not known what effect the lack of the PDZ domain in isoform 2/3 has on functional protein within kidney.

Figure 4.
Figure 4.

Kidney functionality of the shortened SHROOM3 protein. (A) Schematic of the full-length human SHROOM3, PDZ, and ASD2 deletion constructs. The resulting proteins from isoform 2/3 relative to isoform 1 result in the removal of the PDZ domain. A control construct removing the ASD2 domain was also designed. Isoform 2/3 starts protein production at the conserved MM site (amino acid 177 of full-length SHROOM3). (B) Evolutionary analysis of SHROOM3 and the highly homologous gene SHROOM2 identifying functional domains and linear motifs. Previously unpublished motifs are seen conserved in SHROOM2 and SHROOM3 (magenta). (C) Representative images of zebrafish dorsal aorta at 1, 24, and 48 hours after injection of 70 kD FITC dextran. (D) Coinjection of shroom3+tp53 MO with SHROOM3∆PDZ but not SHROOM3∆ASD2 mRNA rescued dextran leakage induced by the MO (n=15, 9, 9, and 9, respectively; *P<0.05 versus uninjected). hpi, hours post injection; MM, double methionine start site; MO, morpholino.

To test functionality, we created a human SHROOM3 mutant allele lacking either ASD2 (∆ASD2) or PDZ (∆PDZ, podocyte isoform 2) (Figure 4A) and tested these alleles using a zebrafish assay for renal function. Coinjection of a shroom3+tp53 morpholino with SHROOM3∆PDZ mRNA successfully prevented the dextran leakage phenotype resulted by injection of the morpholino alone similar to isoform 1; whereas, SHROOM3∆ASD2 mRNA did not (Figure 4, C and D). This suggests that isoforms 1 and 2/3 both have functional capacity in kidney. Therefore, the shorter isoform 2/3 identified in podocytes is sufficient for kidney function.

Rare Variants in Human Populations for SHROOM3

Given the evidence that commonly inherited variants within regulatory regions of SHROOM3 may be functional in GWAS, we set out to look for rare coding variants that also could contribute to CKD but are present at lower frequencies than needed for genome-wide significance. This analysis serves a two-fold importance for this paper: (1) identification of rare missense variants within a gene near GWAS can serve as a secondary independent confirmation of disease linkage, thus allowing for the breakdown of complex CKD GWAS LD blocks; and (2) discovery of novel SHROOM3 mechanisms through rare disease-associated variants in unknown regions of the SHROOM3 protein. We queried all of the human SHROOM3 variants identified in the gnomAD database23 (covering an average of 112,849 individual whole genomes or exomes) and identified 2237 variants in SHROOM3 nucleotides (Supplemental Table 3). Of these variants, 1043 were missense (Figure 5A). Each variant was run through PolyPhen2 with 518 of 1043 (49.66%) identified as benign, 195 of 1043 (18.70%) as possibly damaging, and 330 of 1043 (31.64%) as “probably damaging.” To prioritize the “probably damaging” variants, we created an effect score by multiplying the allele counts, codon selection score, and 21-codon sliding window score for each variant, identifying 35 variants with an effect score of at least 100 (red box, Figure 5A; details provided in Supplemental Table 4). It should be noted that our evolutionary metrics used in this paper add codon selection, detection of conserved linear motifs, and allele frequencies to outputs of PolyPhen2 prediction status.

Figure 5.
Figure 5.

Biochemistry of the CKD-associated P1244L SHROOM3 variant. (A) Within gnomAD are 1043 missense variants, with 35 having an effect score >100 (red box). The variant our group previously identified in association with CKD,15 P1244L, (OR 7.95; 95% CI 1.53 to 41.46, P=0.01) is found in East Asian individuals within gnomAD (blue box). (B) Evolutionary analysis of the P1244 location (red) showing conserved sites for 14–3-3 binding (gray) and LATS1/2 kinase recognition (yellow). The site is also conserved in the SHROOM2 protein. (C) LATS2 kinase assay on peptides of SHROOM3 containing multiple mutations including the P1244L CKD variant. Wild-type SHROOM3 (black) and SHROOM3 P1244L (red) were both phosphorylated. Removal of the Histidine (SHROOM3 H123A), no peptide, and scrambled (SHROOM3 SS1241AA) all failed to phosphorylate. (D) Crystal structure of 14–3-3 (gray) interacting with either SHROOM3 WT (cyan) or P1244L (red). (E) Final electron density map (2Fo-Fc, contoured at 1σ) of the WT (top) or P1244L variant showing detailed changes to binding pocket particularly around the P to L change with no modification for the binding of the phosphorylated Ser. (F) Water coordination is increased in the crystal structure for P1244L relative to WT. (G) Molecular dynamic simulations were performed for 125 nanoseconds on both the WT and P1244L SHROOM3–14–3-3 structures using the AMBER03 force field. All amino acids interacted the same except for the one at position 1244. L, leucine; P, proline; WT, wild type.

When we evaluated the distribution of these 35 variants within specific population cohorts (Supplemental Figure 13, Supplemental Table 5), a specific variant, G186R, within South Asian individuals (allele frequency of 5.09%) had the second highest effect score (14,902). Within the East Asian population P1244L was identified, with an allele frequency of 0.27% (50 of 18,674 alleles) in East Asian individuals (blue box, Figure 5A). We have shown that P1244L is associated with CKD (P=0.01, OR=7.95) and that the mutation is unable to recover the SHROOM3 dextran zebrafish kidney assays, suggesting a loss of function in animal studies.15 However, no mention of cellular mechanisms has been put forth for this variant’s loss of function.

Biophysical Insights into SHROOM3 P1244L CKD-Associated Variant

A deeper analysis of P1244 revealed it to be highly conserved in 125 species that have SHROOM3, and it is also conserved in 128 species within a homologous region of SHROOM2 (Figure 5B). The site is within a predicted linear motif for 14–3-3 interaction (HVRSRSSP), which must be phosphorylated for binding. However, this site within SHROOM3 has not been previously described despite its high conservation. This motif has a highly-conserved histidine, which we predict is a substrate for LATS1/2 kinase–mediated phosphorylation, thus regulating the 14–3-3 binding motif. To test this hypothesis, we conducted an in vitro LATS2 phosphorylation assay. Both the WT and P1244L variant could be phosphorylated by LATS2 in a concentration-dependent mode as expected on the basis of the variant location (Figure 5C), whereas controls were not. Given the fact that P1244L can still be phosphorylated, the 14–3-3 interaction kinetics are the next question to address. We solved the crystal structures (PDB: 6FBB/6FCP, Table 1), to incredibly high resolution allowing for coordination of water to be visualized, of complexes of WT or P1244L mutant with 14–3-3 (Figure 5D). A magnified view of these structures showed proline at 1244 to be well packed with 14–3-3, whereas leucine at 1244 lacks multiple contacts (Figure 5E). The decrease in interaction with 14–3-3 resulting from P1244L also results in an increase in water coordination (Figure 5F), strongly suggesting there is reduced binding to 14–3-3. Molecular dynamic simulations of these two crystal structures confirmed P1244L destabilizes contacts with 14–3-3 (Figure 5G). Further validation using isothermal titration calorimetry and affinity capture showed that P1244L variant decreases binding to 14–3-3 (Supplemental Figure 14). Therefore, we have identified a previously unpublished conserved site within SHROOM3 for LATS1/2 phosphorylation and 14–3-3 interaction that contains a CKD-associated variant that alters the interaction between SHROOM3 and 14–3-3. In affinity capture experiments in vitro for 14–3-3 β, SHROOM3 was one of many binding proteins,31 further supporting an in vitro/vivo interaction of the two proteins. Given the role of LATS1/2 and 14–3-3 in renal disease,32,33 these results create a better understanding of how SHROOM3 might contribute to CKD.

View this table:
Table 1.

Statistics for crystal structure

Discussion

As sequencing expands for clinical diagnosis and to guide treatment, the list of variants will expand for genes already associated with disease, as well as genes not previously connected to pathogenicity. We applied our sequence-to-structure-to-function approach for variants of SHROOM3 in both protein coding and noncoding regions to assign preliminary function to these changes. In this process, we gained considerable insight into SHROOM3 and its role in CKD. This work is not just limited to SHROOM3, but the strategies can be applied to nearly any GWAS LD block, allowing for the breakdown of more complex multigene loci using a combination of common GWAS and rare missense variants within each gene.

Broadly, the network of factors found in this study suggests an enrichment of apoptotic signaling pathways and Hippo signaling for SHROOM3 (including FOXO1, TCF7L2, LATS1/2, and 14–3-3 proteins). The Hippo pathway has previously been associated with kidney function34–37 and is a known repressor of the Wnt/β-catenin signaling system,38 of which regulation for the noncoding variant rs17319721 by TCF7L2 is Wnt/β-catenin dependant.13 In evaluating noncoding variants of SHROOM3, we determined that there are multiple isoforms predicted to encode three proteins, two that lack the PDZ domain. Public databases revealed this shorter isoform is expressed in kidney and more specifically podocytes. Isoforms of Shroom in Drosophila melanogaster are known to change cellular localization and thus actomyosin networks and cellular morphology.39 In mouse, Shroom3 isoforms lacking the PDZ domain were originally identified by gene trap, resulting in the same sized protein as TSS2-derived human SHROOM3 protein.11 We show using ENCODE datasets that the rs17319721 region loops back on TSS2 in cells that do not express SHROOM3. More importantly, both sites have CTCF association, a factor known to serve a looping roll in insulating transcription.40 The HEK293 TF binding suggests factors such as TCF7L2 serve a repression role of TSS2, with association of factors such as HDAC241 and TRIM28/KAP142 bound near TSS2 known to associate with transcriptional repression. The rs17319721 A allele decreases TCF7L2 binding and thus possibly results in the loss of looping with TSS2 that results in TSS2 transcriptional activation (Figure 6). To our knowledge this is the first time a single noncoding SNP has been integrated into a genome using CRISPR/Cas9 and showed changed gene regulation with effect on CKD, highlighting the future potential of precision genome editing in deciphering complex disease mechanisms. In conclusion, we show for the first time that variants in SHROOM3 that alter gene expression and protein function have the ability to affect CKD, laying a foundation for future CKD and disease mechanistic insights.

Figure 6.
Figure 6.

Schematic of potential mechanism for SHROOM3 rs17319721 TSS2 regulation proposed in this study for individuals (A) without or (B) with rs17319721. LD, linkage disequilibrium.

Disclosures

None.

Acknowledgments

National Institutes of Health (NIH)–K01ES025435 (to J.W.P.), NIH-R01HL069321 (to H.J.J.), NIH-R01EY014167 (to B.A.L.), the Collaborative Research Centre 1093 through the Deutsche Forschungsgemeinschaft (to C.O.), and HudsonAlpha Institute for Biotechnology.

J.W.P., N.C.Y., B.I.F., F.J.R., M.J.F., H.J.J., E.M.M., and J.L. designed the studies; J.W.P., N.C.Y., C.O., B.A.L., S.B.C., C.M.-R., A.M.G., K.L.F., E.J.R., N.S., S.L., L.-G.M., F.A.M., C.J.C., and R.R. carried out the experiments and analyzed the data; J.W.P. and N.C.Y. made the figures; J.W.P., N.C.Y., H.J.J., E.M.M., and J.L. drafted and revised the manuscript; all authors approved the final version of the manuscript.

Footnotes

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Characterization of Coding/Noncoding Variants for SHROOM3 in Patients with CKD
Jeremy W. Prokop, Nan Cher Yeo, Christian Ottmann, Surya B. Chhetri, Kacie L. Florus, Emily J. Ross, Nadiya Sosonkina, Brian A. Link, Barry I. Freedman, Candice J. Coppola, Chris McDermott-Roe, Seppe Leysen, Lech-Gustav Milroy, Femke A. Meijer, Aron M. Geurts, Frank J. Rauscher, Ryne Ramaker, Michael J. Flister, Howard J. Jacob, Eric M. Mendenhall, Jozef Lazar
JASN May 2018, 29 (5) 1525-1535; DOI: 10.1681/ASN.2017080856
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