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. 2018 May;29(5):1525-1535.
doi: 10.1681/ASN.2017080856. Epub 2018 Feb 23.

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

Affiliations

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

Jeremy W Prokop et al. J Am Soc Nephrol. 2018 May.

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.

Keywords: CRISPR/Cas9; GWAS; Genomic Variants; Rare Variants; SHROOM3; TCF7L2.

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Figures

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.
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.
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.
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.
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, 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.
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.

Comment in

  • Using Large Datasets to Understand CKD.
    Drysdale TA. Drysdale TA. J Am Soc Nephrol. 2018 May;29(5):1351-1353. doi: 10.1681/ASN.2018030288. Epub 2018 Apr 11. J Am Soc Nephrol. 2018. PMID: 29643114 Free PMC article. No abstract available.

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