Dysfunctional missense variant of OAT10/SLC22A13 decreases gout risk and serum uric acid levels

Organic anion transporter 10 (OAT10), also known as SLC22A13, has hitherto been identified as a urate transporter by in vitro analyses.1 Despite the reported expression of OAT10 on the apical membrane of the renal proximal tubular cells,1 the physiological impact of OAT10 on urate handling in humans remains to be elucidated. Accumulating evidence suggests that functional variants of already-characterised, physiologically important urate transporters—URAT1/SLC22A12, GLUT9/SLC2A9, BCRP/ABCG2 and NPT1/SLC17A1—affect serum uric acid (SUA) levels and susceptibility of gout,2–6 the most common form of inflammatory arthritis. However, there are no reports on the association between OAT10 gene and either hyperuricaemia or gout. Here, for the first time, we reveal that a dysfunctional variant of OAT10 decreases both gout risk and SUA levels, suggesting OAT10 to be physiologically involved in urate reabsorption in the human kidney, as described below.

To explore exonic variants in OAT10 potentially associated with gout susceptibility, we sequenced all exons of OAT10 in 480 gout cases and 480 controls of Japanese male6 and conducted an association analysis (see online supplementary tables S1 and S2), followed by a replication study on 924 gout cases and 2113 controls (see online supplementary figure S1). In two identified OAT10 variants with minor allele frequency (MAF) >0.5%, only rs117371763 (c.1129C>T; p.Arg377Cys [R377C]) was significantly associated with gout susceptibility after Bonferroni correction (p=0.014). The significant association between rs117371763 and gout susceptibility was replicated, and our meta-analysis showed a significant protective effect of rs117371763 on gout susceptibility (OR=0.67; 95% CI 0.53 to 0.85; pmeta …

fragmented and adapter-ligated using a SureSelect QXT Library Prep Kit (Agilent Technologies, Santa Clara, CA, USA). The fragmented libraries with distinct indexed adapters were pooled at equimolar amounts. Target enrichment was then conducted using the SeqCap EZ Choice System (Roche Diagnostics, Tokyo, Japan). A DNA probe set complementary to OAT10 was selected using NimbleDesign (https://design.nimblegen.com). The libraries were sequenced on an Illumina HiSeq 2500 platform with a 2×100-bp paired-end module (Illumina, San Diego, CA, USA). As the quality control step, the reads containing the Illumina adapter sequences and low quality sequences were trimmed using Trimmomatic 6 . The sequence reads were aligned to human reference genome (hg19) using BWA 7 . The aligned reads were processed for removal of PCR duplicates using Picard tools (https://broadinstitute.github.io/picard/), and for local realignment and base quality recalibration by G AT K 8 . Single nucleotide variants (SNVs) and insertions and deletions were detected using GATK's HaplotypeCaller 8 . Functional annotation of the identified variants was implemented by ANNOVAR 9 .
After variant calling and annotation, we selected missense and nonsense SNVs as well as insertions and deletions in the exons of OAT10 based on the DNA reference sequence NM_004256; we excluded synonymous SNVs in OAT10 exons as well as variants in introns or untranslated regions for the downstream association analysis. In the replication phase of the association analysis, rs117371763 (1129C>T: p. Arg377Cys [R377C]) of OAT10 was genotyped using a TaqMan method (ThermoFisher Scientific Inc., MA, USA) with a LightCycler 480 (Roche Diagnostics, Mannheim, Germany) as previously described 10 . In a quantitative trait locus (QTL) analysis of SUA, we used genotyping data of normouricemic controls obtained in both discovery and replication phases of the association analysis.
Additionally, we genotyped rs117371763 of hyperuricemic controls with the TaqMan method 10 . To confirm rs117371763 genotypes, >100 samples were subjected to direct sequencing. Direct sequencing was performed with the following primers: forward 5′-TGGTGTGTGTTGGCAGAG-3′ and reverse 5′-GGTCCCATCCACTGGAAC-3′. DNA sequence analysis was performed with a 3130xl Genetic Analyzer (ThermoFisher Scientific) 10 . Statistical analyses were performed using SPSS v.22.0J (IBM Japan Inc., Tokyo, Japan) and the software R (version 3.1.1) with meta package. The χ2 test was used for association analyses; the univariate linear regression analysis was used for the QTL analysis. In the discovery phase, we chose nonsynonymous OAT10 variants with minor allele frequency (MAF) greater than 0.5% either in cases or controls and analyzed their association with gout susceptibility. After the replication phase, we also performed a meta-analysis using the DerSimonian and Laird random-effects model. Cochran's Q test and I 2 were used for the evaluation of heterogeneity between two phases of the association analysis. A p value less than 0.05 was considered statistically significant. In addition, this statistical threshold was adjusted by Bonferroni correction accordingly.

Materials for all based molecular analyses
All chemicals used were commercially available and were of analytical grade. Critical materials used are summarized in Supplementary Table S5.

Construction of OAT10-containing expression vector
As a sub-cloning source, an I.M.A.G.E Full length cDNA clone (GenBank: BC035973.1) was purchased from K.K. DNAFORM (Kanagawa, Japan). We corrected some mutations in the clone using a

Preparation of protein lysates and immunoblotting
Whole cell lysates were prepared with cell lysis buffer A containing 50 mM Tris/HCl (pH 7.4), 1 mM dithiothreitol, 1% (w/v) Triton X-100, and a protease inhibitor cOmplete, EDTA free (Roche, Basel, Switzerland), followed by treatment with Peptide N-glycosidase F (PNGase F) (New England Biolabs, Inc., Ipswich, MA, USA) as described previously 11 . The protein concentration was determined using the Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific) with BSA as a standard according to the manufacturer's protocol.
Immunoblotting was performed according to our previous study 3 . Blots were probed with appropriate antibodies (Supplementary Table S5), and then the signals were visualized by a chemiluminescent method. All antibodies were used at 1:1000 (first antibody) or 1:2000 (second antibody) dilution. After washing with Tris-buffered saline containing 0.05% Tween 20 for 1 hour at

Confocal microscopic observation
Forty-eight hours after transfection, 293A cells were fixed with ice cold methanol and subjected to TO-PRO-3 Iodide (Molecular Probes, Eugene, OR, USA) staining as described previously 13 . After the visualization of nuclei, the cells mounted in VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA) were observed using an FV10i Confocal Laser Scanning Microscope (Olympus, Tokyo, Japan). The obtained data were analyzed using an Imaris software (Carl Zeiss Japan, Tokyo, Japan).

Cell-based transport assay using OAT10-expressing 293A cells
The urate uptake assay using O AT 1 0 -expressing 293A cells was conducted according to a previous study 14 with some modifications. In brief, 48 hours after the plasmid transfection, the cells were washed twice with a transport buffer (TP-Buffer: 130 mM NaCl, 4 mM KCl, 1 mM Na 2 HPO 4 , 1 mM MgSO 4 , 1 mM CaCl 2 , 20 mM HEPES, 18 mM D-glucose, and pH 7.4) and pre-incubated in TP-Buffer for 15 minutes at 37°C. Then, the buffer was exchanged with a pre-warmed fresh TP-Buffer containing 10 μM

Quantification and statistical analysis for cell-based molecular analyses
All statistical analyses were performed using Excel 2013 (Microsoft Corp., Redmond, WA, USA) with Statcel3 add-in software (OMS publishing Inc., Saitama, Japan). Different statistical tests were used for different experiments as described in the figure legends. Briefly, when analyzing multiple groups, the similarity of variance between groups was compared using Bartlett's test. When passing the test for homogeneity of variance, a parametric Dunnett's test was used. In the case of a single pair of quantitative data, after comparing the variances of a set of data by F-test, unpaired Student's t-test was performed. Statistical significance was defined in terms of p values less than 0.05 or 0.01.