A novel mutation in the SLCO2A1 gene, encoding a prostaglandin transporter, induces chronic enteropathy.

Chronic enteropathy associated with SLCO2A1 gene (CEAS) is caused by loss-of-function mutations in SLCO2A1, which encodes a prostaglandin (PG) transporter. In this study, we report a sibling case of CEAS with a novel pathogenic variant of the SLCO2A1 gene. Compound heterozygous variants in SLCO2A1 were identified in an 8-year-old boy and 12-year-old girl, and multiple chronic nonspecific ulcers were observed in the patients using capsule endoscopy. The splice site mutation (c.940 + 1G>A) of the paternal allele was previously reported to be pathogenic, whereas the missense variant (c.1688T>C) of the maternal allele was novel and had not yet been reported. The affected residue (p.Leu563Pro) is located in the 11th transmembrane domain (helix 11) of SLCO2A1. Because SLCO2A1 mediates the uptake and clearance of PGs, the urinary PG metabolites were measured by liquid chromatography coupled to tandem mass spectrometry. The urinary tetranor-prostaglandin E metabolite levels in the patients were significantly higher than those in unaffected individuals. We established cell lines with doxycycline-inducible expression of wild type SLCO2A1 (WT-SLCO2A1) and the L563P mutant. Immunofluorescence staining showed that WT-SLCO2A1 and the L563P mutant were dominantly expressed on the plasma membranes of these cells. Cells expressing WT-SLCO2A1 exhibited time- and dose-dependent uptake of PGE2, while the mutant did not show any uptake activity. Residue L563 is very close to the putative substrate-binding site in SLCO2A1, R561 in helix 11. However, in a molecular model of SLCO2A1, the side chain of L563 projected outside of helix 11, indicating that L563 is likely not directly involved in substrate binding. Instead, the substitution of Pro may twist the helix and impair the transporter function. In summary, we identified a novel pathogenic variant of SLCO2A1 that caused loss-of-function and induced CEAS.

Introduction

Chronic enteropathy associated with SLCO2A1 gene (CEAS), which was originally called chronic nonspecific multiple ulcers of the small intestine (CNSU), is caused by loss-of-function mutations in the solute carrier organic anion transporter family, member 2A1 (SLCO2A1) gene [1]. Human SLCO2A1 is located on chromosome 3q22.1-q22.2 and consists of 14 exons that encode a cell-surface prostaglandin (PG) transporter comprising 643 amino acids and 12 transmembrane (TM) domains [2]. Prostanoids, which are synthesized from arachidonic acid (AA) via cyclooxygenase (COX)-mediated pathways, are released from cells and bind to their cognate receptors to enact their biological activities [3]. Prostanoid signaling is terminated by cellular uptake of prostanoids, followed by their intracellular oxidation by 15-hydroxyprostaglandin dehydrogenase (15-PGDH) [4, 5], and thus, this uptake is a critical step in prostanoid inactivation [6, 7].

It is generally accepted that chemical or genetic inhibition of SLCO2A1 elevates tissue concentrations of prostaglandin E2 (PGE2), resulting in augmentation of PGE2 signaling through PGE receptors (EPs) [8]. Clinical studies with individuals bearing recessive mutations in SLCO2A1 have demonstrated that SLCO2A1 is a causative gene for primary hypertrophic osteoarthropathy (PHO) and CEAS, in which PGE2 catabolism is defective [1, 9]. CEAS is an autosomal recessive inherited disease [1], and the multiple ulcers that form in the small intestine cause the chronic loss of blood and protein in these patients [10, 11]. Previously, CEAS was diagnosed based on clinical symptoms, including intestinal edema, abdominal pain, and gastrointestinal bleeding, and the identification of distinctive intestinal ulcers using capsule endoscopy or balloon-assisted endoscopy was required to distinguish CEAS from intestinal Crohn’s disease [12, 13]. However, given that CEAS has been shown to be driven by SLCO2A1 loss-of-function [1, 11], the disease is diagnosed by clinical features and a genetic test in the SLCO2A1 gene. In this study, we report a sibling case of CEAS with a novel mutation in the SLCO2A1 gene that resulted in loss-of-function of PGE2 uptake by SLCO2A1.

Results

Identification of compound heterozygous variants in SLCO2A1 gene

A 12-year-old girl (patient III-1) and 8-year-old boy (patient III-3) were born to healthy non-consanguineous parents (Fig 1A). Initially, the boy exhibited hypoproteinemia and severe anemia, and was referred to a former institution. No remarkable past medical and consanguineous marriage history were found in his parents. Because the fecal occult blood test was positive, esophagogastroduodenoscopy and colonoscopy were performed, but we could not confirm obvious mucosal abnormalities. However, we observed multiple circular and tape-like shallow ulcers in the entire small intestine and predominantly in the lower ileum using intestinal capsule endoscopy, strongly indicating CEAS (Fig 1B). We identified compound heterozygous variants in the SLCO2A1 gene using direct sequencing. The splice site mutation (c.940 + 1G>A) of the paternal allele was previously reported [14], whereas the missense variant (c.1688T>C) of the maternal allele was novel and had not been reported before (Fig 1C). The affected residue (p.Leu563Pro) is located in helix 11 of SLCO2A1 (Fig 1D). Screening of SLCO2A1 orthologs using the NCBI HomoloGene database (http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=homologene&dopt=MultipleAlignment&list_uids=38077) revealed that the Leu563 residue is completely conserved among humans, chimpanzees, monkeys, dogs, cows, mice, rats, chickens, and frogs (Fig 1D), which suggests that the missense mutation is possibly pathogenic. Through genetic analysis of the parents and siblings, we identified the same heterozygous variants in the SLCO2A1 gene of patient III-1, who had past medical history of recurrent abdominal pain, but it had not been scrutinized at medical institutes. After the identification of the genetic mutation, she was found to have hypoproteinemia and anemia similar to patient III-3 by laboratory test. She also exhibited multiple shallow and tape-like ulcers in entire small intestine and predominantly in the lower ileum by capsule endoscopy (Fig 1B). In addition, because SLCO2A1 defects also cause another disease primary hypertrophic osteoarthropathy (PHO)/pachydermoperiostosis (PDP), we consulted two CEAS patients to a dermatologist in our hospital and got the answer that there were no characteristic findings of PHO/PDP.

Fig 1

Pedigree of the chronic enteropathy associated with SLCO2A1 (CEAS) family with a novel mutation of SLCO2A1 gene.

(A) The segregation of the SLCO2A1 mutations c.940 + 1G>A (splice site) and c.1688 T>C (L563P) is indicated. Squares represent male family members, circles represent female family members, and black symbols represent clinically affected family members. (B) Capsule endoscopic findings. Multiple shallow tape-like ulcers (patient III-1) and multiple circular and tape-like shallow ulcers (patient III-3) were observed in the small intestinal mucous, respectively. (C) Trio-based sequence chromatogram of the SLCO2A1 mutations. (D) Schematic diagram representing the position of L563P in the 11th transmembrane helix. L563 in the helix is highly conserved among different species. R561 is the putative substrate-binding site.

The urinary tetranor-prostaglandin metabolites levels

As SLCO2A1 mediates the uptake and clearance of PGs, the urinary PG metabolites of the patients were measured using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Tetranor-prostaglandin E metabolite (t-PGEM) is a major metabolite of PGE2 and is used as a urinary marker of PGE2 biosynthesis (Fig 2A) [15]. The levels of urinary t-PGEM and tetranor-prostaglandin F metabolite (t-PGFM) in the CEAS patients were significantly higher than those in unaffected individuals, whereas tetranor-prostaglandin D metabolite (t-PGDM) levels were relatively comparable (Fig 2B).

Fig 2

Urinary levels of tetranor-prostaglandin metabolites, t-PGEM, t-PGFM, and t-PGDM.

(A) Metabolic pathways of prostaglandin E2 and prostaglandin F2α. (B) Urinary t-PGEM, t-PGFM, and t-PGDM were measured using UPLC-MS/MS. Each bar represents the mean ± SEM (n = 3). ***, p < 0.0001; **, p < 0.001; *, p < 0.01 (One-way ANOVA and Tukey’s multiple comparisons test).

Functional analysis of WT-SLCO2A1 and L563P mutants

For functional analysis of the newly identified SLCO2A1 mutation, we established Flp-In T-Rex 293 cell lines with doxycycline-inducible expression of the intact or Leu563Pro (L563P) mutated SLCO2A1. Western blot analysis revealed that the expressions of wild type (WT)-SLCO2A1 and the L563P mutant were comparable (Fig 3A), and immunofluorescence staining showed that WT-SLCO2A1 and the L563P mutant were both dominantly expressed on the plasma membrane (Fig 3B). Cells expressing WT-SLCO2A1 displayed time-dependent uptake of 3H-labeled PGE2 ([11] PGE2), but the mutant did not show any uptake activity at all (Fig 3C). In an assay measuring the transport rate of PGE2, cells expressing WT-SLCO2A1 showed significant [3H]PGE2 uptake in a dose-dependent manner, while the L563P mutation resulted in complete disruption of SLCO2A1 activity at 1, 10, and even 100 nM PGE2 (Fig 3D).

Fig 3

Expression, subcellular localization, and PGE2 uptake activities of SLCO2A1 and the L563P mutant.

(A) Membrane fractions of Flp-In T-REx 293 cells expressing WT-SLCO2A1 and the L563P mutant were subjected to western blotting analysis. (B) Cells were treated with doxycycline (DOX) for 24 hr and fixed. Immunofluorescence staining was performed with anti-SLCO2A1 antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG. (C) Cells were treated with DOX, and then incubated for the indicated periods with 1 nM [3H]PGE2. After extensive washing with HBSS, the cells were lysed with 0.1 N NaOH and the radioactivity was measured. Data represent the mean ± SEM (n = 4). ***, p < 0.0001, ‘ns’, not significant. (One-way ANOVA and Tukey’s multiple comparisons test). (D) Cells were treated with DOX and incubated for 1 min with 1, 10, and 100 nM [3H]PGE2. The radioactivity was measured as described above. The uptake values were normalized by the protein concentrations in the cell lysates. Experiments were repeated three times, with quadruplicates for each sample (n = 4). Each bar represents the mean ± SEM. ***, p < 0.0001 (One-way ANOVA and Tukey’s multiple comparisons test).

The structure model of SLCO2A1

To illustrate the effects of the missense mutation in helix 11 of SLCO2A1, a molecular model of SLCO2A1 was constructed using the crystal structure of a glycerol-3-phosphate transporter (GlpT, PDB ID:1PW4) [16]. The molecular model of SLCO2A1 was constructed with the SWISS-MODEL server and Swiss-PdbViewer from the template PDB, and was analyzed using PyMol. The substrate may occupy a pocket between the transmembrane helixes similar to that of GlpT. The L563 residue is located close to the putative substrate-binding site at R561, and the L563 side chain projects outward from helix 11 (Fig 4). This suggests that while the L563 side chain is not directly involved in substrate binding, its substitution with proline may twist the helix and impair the transporter function.

Fig 4

Molecular model of SLCO2A1.

A molecular model of SLCO2A1 was constructed using the crystal structure of a glycerol-3-phosphate transporter. The Leu563 in the 11th transmembrane helix is shown in red. The mutations reported in the text are displayed in orange.

Discussion

In the present study, we report a novel pathogenic variant of SLCO2A1 identified in a sibling case of CEAS. Among the known SLCO2A1 mutations, a splice-site mutation at intron 7 (c.940 + 1G>A; rs765349238) is the most frequently observed among CEAS patients, with 54% of mutant allele frequency. This mutation results in the deletion of the entire exon 7 from SLCO2A1, leading to a frameshift at amino acid position 288 and the creation of a premature stop codon after six amino acid residues (p.R288Gfs*7) [1]. Based on the data from the Human Genetic Variation Database (HGVD) [17], the allele frequency of c.940 + 1G>A in the Japanese population is 0.091% (2/2188). The missense variant (c.1688T>C) identified in this work has not previously been identified, and the affected residue (p.Leu563Pro) is located in the 11th transmembrane domain of SLCO2A1. Several mutations, including G554R, R561C, L571P [14], Q556H [18], F557S [19], and W565G [20], have been reported in the 11th transmembrane domain. SLCO2A1 belongs to the superfamily of 12-transmenbrane organic anion transporting polypeptides (OATPs) [6-8], and R561 and K614 in the 11th and 12th transmembrane domains, respectively, are thought to be involved in ligand binding [8, 21]. The novel variant of L563 is close to R561, and may influence the ligand binding activity of the transporter (Fig 4).

PGE2 is degraded through two main steps. First, prostaglandin transporters (PGTs), including SLCO2A1, SLCO3A1, and SLCO4A1, mediate PGE2 cellular uptake, which allows it to subsequently be oxidized to 15-keto-PGE2 by 15-PGDH [22] and then degraded to t-PGEM [23]. The levels of PGE2 and t-PGEM in the serum and urine of CNSU patients are significantly higher than in control groups [14, 24]. Because SLCO2A1 is highly expressed in the lungs [2, 25] and is localized to vascular endothelial cells, SLCO2A1 expression in lung vascular cells may contribute to PGE2 uptake [26]. Patient III-3 (8-year-old boy, Fig 1A), with hypoproteinemia and severe anemia, showed the highest t-PGEM levels, while patient III-1 (12-year-old girl, Fig 1A), with milder symptoms, showed the second highest t-PGEM level in this family. These results suggest that the t-PGEM level may reflect the severity of the disease in patients. The urinary levels of t-PGFM and t-PGDM were much lower than those of t-PGEM, but the ratio between these metabolites in patients and healthy controls was similar (Fig 2). This can be explained by the fact that SLCO2A1 can transport PGF2α and PGD2, in addition to PGE2 [2], and the degradation pathways of PGF2α and PGD2 are similar to PGE2.

Slco2a1-knockout mice were previously generated and analyzed [26, 27], and the impact of the SLCO2A1 mutation on the plasma t-PGEM levels was different in these organisms was different compared to humans. The urinary t-PGEM levels of Slco2a1-deficient mice and human patients with CEAS were higher than in the control mice and healthy controls, respectively. In contrast, while the plasma t-PGEM levels of Slco2a1-deficient mice were lower than in control mice [27], the plasma t-PGEM levels in CEAS patients were higher than in healthy controls [9]. This might be due to differences in the PGE2 metabolic activities between mice and humans. Further, although recent studies have shown that SLCO2A1 function is lost in patients with CEAS, Slco2a1-deficient mice do not spontaneously develop enteritis [28]. This might be because Slco2a1/SLCO2A1 has different functions in mice and humans. Alternatively, patients with CEAS might have a “second-hit factor,” other than a lack of SLCO2A1, such as other genetic factors or certain pathogens in the intestinal lumen. Considering the clinical impact of these differences, this mouse model may not be the best tool for analyzing CEAS, which mainly affects the small intestine.

PGE2 concentrations in patients with CEAS should theoretically be higher than in those without this mutation because of reduced PGE2 metabolism. However, the t-PGEM concentrations in patients with CEAS have been shown to be higher than those in patients with Crohn’s disease [29]. Measuring t-PGEM is useful for distinguish CEAS from Crohn’s disease, although the roles of PGE2 in enteritis remain uncertain.

An in vitro overexpression experiment showed that the expression of WT-SLCO2A1 and the L563P mutant was comparable (Fig 3A), and that both the WT-SLCO2A1 and the L563P mutant were dominantly expressed on the plasma membrane (Fig 3B). These results suggest that the L563P mutation did not affect the stability and localization of the protein, but instead impaired the transport function of SLCO2A1. In previous reports, cells transiently expressing the transporter were used for PGE2 uptake assays; however, these studies did not address the expression and the cellular localization of the transporter [1, 20]. The transporter expression on the plasma membrane requires multiple steps, including protein folding and membrane sorting. Our strategy will be useful for analyzing the molecular functions of mutant SLCO2A1 transporters with missense mutations.

In summary, we identified a novel mutation in SLCO2A1 (L563P) that affects the function of this transporter in PGE2 transport and causes CEAS.

Material and methods

Ethics statement

Written informed consent for the studies was obtained from each individual. Written statement that formal consent was obtained from the parent. The study was approved by the Juntendo University Research Ethics Committee.

Genetic analysis

Genomic DNA was extracted from the peripheral leukocytes of the patients and their parents using a standard procedure with QIAamp DNA Blood Mini Kit (QIA). All 14 exons and exon–intron splice junction regions of the SLCO2A1 gene were amplified by PCR (Ampli Taq Gold ABI, USA and Thermal Cycler C1000 Touch Bio-Rad). PCR products were purified and cycling sequencing was performed using Big Dye terminator Mix V1.1 (ABI) for direct DNA sequence analysis (Genetic Analyzer 3500, ABI). Sequences were analyzed using Seq Scape software V.2.5 (ABI). The reference sequence data is based on GenBank NG_031964.2. Primers for mutation analysis of the SLCO2A1 gene are shown in S1 Table.

Urinary prostaglandin metabolites analysis

The measurements of t-PGEM, t-PGFM, and t-PGDM were performed as previously described [30]. An aliquot of urine (20 μL) was diluted with 100 μL of methanol and incubated for 1 hr at -20°C. After the addition of 1 mL of water, stable isotope-labeled internal standards, t-PGEMd6, and t-PGDMd6, the solution was loaded onto Oasis HLB cartridges (Waters Corporation, Milford, MA, USA). The column was sequentially washed with water containing 0.1% formic acid (Solvent A), 5% methanol containing 0.1% formic acid, and petroleum ether containing 0.1% formic acid, and the samples were eluted with 200 μL of methanol containing 0.1% formic acid. For reverse-phase ultra-high performance liquid chromatography coupled to tandem mass spectrometry (RP-UPLC-MS/MS), we used the ACQUITY UPLC H-Class system (Waters Corporation) and a TSQ Quantum Ultra triple quadrupole mass spectrometer, equipped with an electrospray ionization ion source (Thermo Fisher Scientific, Walford, MA, USA). An aliquot of each sample (20 μL) was diluted with 30 μL of Solvent A and analyzed by RP-UPLC-MS/MS using an ACQUITY UPLC HSS C18 column (2.1 × 100 mm, 1.8 μm particles) (Waters Corporation). The column temperature was set at 41°C, and the samples were separated at a flow rate of 250 μL/min with a linear gradient from 5% to 30% of Solvent B (acetonitrile) in three minutes, and then 100% in 15 minutes. The samples were then kept at 100% for two minutes before re-equilibration for two minutes. The settings for the mass spectrometer were as follows: ESI voltage of 2500 V, tube lens of 110 V, collision energy of 13 V, capillary temperature 225°C, and vaporizer temperature of 250°C. Mass spectrometric analysis was performed in the negative ion mode using selective reaction monitoring, and the transitions that were monitored were: 327.2 > 309.2 m/z (-H2O) for t-PGEM and t-PGDM, 333.2 > 315.2 m/z (-H2O) for t-PGEM-d6 and t-PGDM-d6, respectively, and 329.1 > 311.1 m/z (-H2O) for t-PGFM. Automated peak detection, calibrations, and calculations were carried out using the Xcalibur 2.2 software package (Thermo Fisher Scientific).

The absorbance of urinary creatinine was measured using a Creatinine (urinary) Colorimetric Assay Kit (Cayman Chemical, Ann Arbor, Michigan). The values for t-PGEM, t-PGDM, and t-PGFM are presented as ng/mg creatinine.

Construction of expression vectors

Expression vectors for pcDNA3.1(+)-WT-SLCO2A1 (NM_005630.2) and the L563P mutant were constructed by VectorBuilder (Chicago, IL). The DNA fragments containing a Kozak sequence and full-length coding sequence were excised by HindIII/ApaI-digestion, and subcloned into pcDNA5/FRT/TO (Invitrogen-Thermo Fisher Scientific, Waltham, MS). Obtained expression vectors were used to establish doxycycline-inducible stable cell lines.

Construction of stable cell lines

Doxycycline-inducible stable cell lines of WT-SLCO2A1 and the L563P mutant using Flp-In T-REx 293 cells were constructed and maintained as previously described [31]. Empty vector pcDNA5/FRT/TO was used to generate control cell lines (Mock). Obtained clones were tested for the expression of SLCO2A1 protein in crude membrane fractions by western blot analysis. Briefly, cells were seeded in 10 cm dish (4×106 cells/dish) containing DMEM high glucose w/o phenol red (Wako Pure Chemicals, Osaka, Japan), supplemented with 10% (v/v) heat-inactivated FBS (Gibco Life Technologies, Grand Island, NY), 100 units/mL penicillin (Nacalai Tesque, Kyoto, Japan), 100 μg/mL streptomycin (Nacalai Tesque, Kyoto, Japan), 150 μg/mL hygromycin B (Wako Pure Chemicals, Osaka, Japan), and 5 μg/mL Blasticidin (InvivoGen, San Diego, CA). 24 hr after the passage, doxycycline (Clontech, Mountain View, CA) was added to the medium to a final concentration of 1 μg/mL, and the cells were further incubated for 24 hr. The cells were collected on ice in ice-cold PBS and lysed by sonication in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and protease inhibitor cocktail (Roche Diagnostics GmBH, Mannheim, Germany). Whole cell lysate was centrifuged at 1,000 × g for 5 min, and the supernatant was further centrifuged at 541,000 × g for 1 hr to obtain crude membrane fraction. After solubilization with the lysis buffer containing 10% (v/v) glycerol and 1% (v/v) NP-40, samples were mixed with Laemmli buffer. SDS-PAGE followed by western blot analysis was conducted as previously described [32]. Primary antibodies used were: anti-SLCO2A1 (HPA013742, Sigma-Aldrich, St. Louis, MO), and anti-Na+/K+ ATPase α1 (sc-21712, Santa Cruz Biotechnology, Santa Cruz, CA). The immunoreactive band intensity of SLCO2A1 was quantified by densitometry using Image J software (National Institutes of Health, Bethesda, MD). Clones expressing similar levels of WT and the L563P mutant were selected for PGE2 uptake assays and immunofluorescence staining.

Immunofluorescence staining

Cells were seeded in 3.5 cm dishes (1×106 cells/dish) containing collagen-I-coated coverslips, and were treated with doxycycline as described above. Immunofluorescence staining and image acquisition was performed as described previously [33]. Antibodies used were: anti-SLCO2A1 (HPA013742, Sigma-Aldrich, St. Louis, MO) as the primary antibody, and Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11034, Molecular probes-Thermo Fisher Scientific, Waltham, MS) as the secondary antibody.

PGE2 uptake assay

Cells were seeded on poly-d-lysine coated 24-well plates (1×105 cells/well), and treated with doxycycline as described above. After preincubation for 5 min in Hanks’ balanced salt solution (HBSS: 125 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.3 mM CaCl2, 5.6 mM d-glucose, and 25 mM HEPES [pH 7.4]), cells were incubated for the indicated periods with 1 nM [3H]PGE2 ([5,6,8,11,12,14,15-3H(N)]-PGE2, 1.85 MBq/mmol, PerkinElmer, Waltham, MA) to perform a time course analysis. To test the concentration dependence of uptake, cells were incubated for 1 min with 1, 10, and 100 nM [3H]PGE2. After extensive washing with HBSS, the cells were lysed with 0.1 N NaOH and radioactivity was measured by liquid scintillation counting as previously described [34]. The uptake values were normalized by protein concentration of cell lysates determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MS). Experiments were repeated three times, with quadruplicates for each sample (n = 4).

Statistics

One-way ANOVA and Tukey’s multiple comparisons test was used to determine p values. P values ≤ 0.05 were considered statistically significant. The statistical analyses were performed with GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA).

Primers for mutation analysis of the SLCO2A1 gene.

(DOCX)

Click here for additional data file.

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

22 Sep 2020

PONE-D-20-25461

A novel mutation in the SLCO2A1 gene, encoding a prostaglandin transporter, induces chronic enteropathy

PLOS ONE

Dear Dr. Okuno,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Nov 06 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Sylvie Mazoyer, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Jimbo et al. reported a sibling case with chronic enteropathy associated with SLCO2A1 gene (CEAS) and found a new missense variant (c.1688T>C) in the SLCO2A1 gene. They also confirmed that this mutation causes loss of transporter function by the PGE2 uptake experiment.

Major point

1. Clinical features about the patients are unclear. For example, there is no description of patient III-1 and the location of the small intestinal lesions of patient III-3 is unclear. They should describe in detail the gastrointestinal and extraintestinal features of the patients. If possible, they should discuss the difference between siblings.

Minor point

1. P6L74. The authors wrote, "the disease can now be genetically diagnosed...". but CEAS is diagnosed by clinical features and a genetic test and genetic test just help in diagnosing. Please revise this sentence.

2. P6L81. The word "consanguineous" should be wrong. Please make sure.

3. P9L120. The word "CNSU" should be changed to CEAS.

4. P10L150. There is no explanation about the representation of the black circle and so on in the Fig 3c.

Reviewer #2: This is a new case report for the sibling of CEAS with compound heterozygous variants (c.940+1G>A and c.1688T>C). Jibo et al found a novel SLCO2A1 mutation (c.1688T>C) and confirmed that the SLCO2A1 variant (L563P) had no PGE2 transport activity when it was expressed in Flp-In T-Rex 293 cells. The structure model of SLCO2A1 suggests the substitution of Leu with Pro impairs the transport function. This paper is well-described, and molecular mechanism supports their clinical observation. Information on clinically associated mutations of SLCO2A1 is worthwhile to be shared for efficient genetical diagnosis. The following minor concerns are raised.

1. Figure 2A shows the metabolic pathway of both PGE2 and PGF2a; however, they are basically the same. They should combine them and show only essential parts of the pathway.

2. SLCO2A1 defects also cause PHO/PDP. Have the authors observed any clinical features of PHO/PDP in these patients? It would be appreciated to describe or mention clinical manifestations of PHO/PDP.

3. In Figure 4, the authors suggest that L563 side chain is not directly involved in substrate biding because the side chain is outward (line 168). Does this mean that the side of the leucine outward from the helix does not affect substrate binding? This description is a little bit hard to understand. It would be nicer If the authors clarified this molecular basis more clearly.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Junji Umeno

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

14 Oct 2020

Point-by-point Responses

Reviewer #1: Jimbo et al. reported a sibling case with chronic enteropathy associated with SLCO2A1 gene (CEAS) and found a new missense variant (c.1688T>C) in the SLCO2A1 gene. They also confirmed that this mutation causes loss of transporter function by the PGE2 uptake experiment.

Major point

1. Clinical features about the patients are unclear. For example, there is no description of patient III-1 and the location of the small intestinal lesions of patient III-3 is unclear. They should describe in detail the gastrointestinal and extraintestinal features of the patients. If possible, they should discuss the difference between siblings.

Response: Thank you very much for your critical comments. We described clinical features about the patients as follows:

P6L86: However, we observed multiple circular and tape-like shallow ulcers in the entire small intestine and predominantly in the lower ileum using intestinal capsule endoscopy, strongly indicating CEAS (Fig. 1B).

P7L98: Through genetic analysis of the parents and siblings, we identified the same heterozygous variants in the SLCO2A1 gene of patient III-1, who had past medical history of recurrent abdominal pain, but it had not been scrutinized at medical institutes. After the identification of the genetic mutation, she was found to have hypoproteinemia and anemia similar to patient III-3 by laboratory test. She also exhibited multiple shallow and tape-like ulcers in entire small intestine and predominantly in the lower ileum by capsule endoscopy (Fig. 1B).

Minor point

1. P6L74. The authors wrote, "the disease can now be genetically diagnosed...". but CEAS is diagnosed by clinical features and a genetic test and genetic test just help in diagnosing. Please revise this sentence.

Response: Thank you for your critical comment. We revised our manuscript as follow.

P6L74: the disease is diagnosed by clinical features and a genetic test in the SLCO2A1 gene.

2. P6L81. The word "consanguineous" should be wrong. Please make sure.

Response: Thank you for your useful comment. We modified our manuscript as follow.

P6L81: to healthy non-consanguineous parents (Fig. 1A).

3. P9L120. The word "CNSU" should be changed to CEAS.

Response: Thank you for your useful comment. We modified our manuscript as follow.

P9L127: in the CEAS patients were significantly higher

4. P10L150. There is no explanation about the representation of the black circle and so on in the Fig 3c.

Response: Thank you for your useful comment. We added the representations of the black circle, open circle, and open triangle in the Fig 3c.

We thank Reviewer #1 for the constructive and insightful comments, which helped us to substantially improve our manuscript.

Reviewer #2: This is a new case report for the sibling of CEAS with compound heterozygous variants (c.940+1G>A and c.1688T>C). Jibo et al found a novel SLCO2A1 mutation (c.1688T>C) and confirmed that the SLCO2A1 variant (L563P) had no PGE2 transport activity when it was expressed in Flp-In T-Rex 293 cells. The structure model of SLCO2A1 suggests the substitution of Leu with Pro impairs the transport function. This paper is well-described, and molecular mechanism supports their clinical observation. Information on clinically associated mutations of SLCO2A1 is worthwhile to be shared for efficient genetical diagnosis. The following minor concerns are raised.

1. Figure 2A shows the metabolic pathway of both PGE2 and PGF2a; however, they are basically the same. They should combine them and show only essential parts of the pathway.

Response: Thank you for useful comments. We have combined the metabolic pathway of PGE2 and PGF2a in Figure 2A.

2. SLCO2A1 defects also cause PHO/PDP. Have the authors observed any clinical features of PHO/PDP in these patients? It would be appreciated to describe or mention clinical manifestations of PHO/PDP.

Response: Thank you for critical comments. We consulted two CEAS patients to a dermatologist in our hospital and got the answer that there were no characteristic findings of PHO/PDP. Therefore, we modified our manuscript as follows.

P7L104: (Fig. 1B). In addition, because SLCO2A1 defects also cause another disease primary hypertrophic osteoarthropathy (PHO)/pachydermoperiostosis (PDP), we consulted two CEAS patients to a dermatologist in our hospital and got the answer that there were no characteristic findings of PHO/PDP.

3. In Figure 4, the authors suggest that L563 side chain is not directly involved in substrate biding because the side chain is outward (line 168). Does this mean that the side of the leucine outward from the helix does not affect substrate binding? This description is a little bit hard to understand. It would be nicer If the authors clarified this molecular basis more clearly.

Response: Thank you for useful comments. We consider that the side chain of the leucine outward from the helix is not involved in substrate binding. We modified the manuscript as follows:

P12L172: The substrate may occupy a pocket between the transmembrane helixes similar to that of GlpT.

We thank Reviewer #2 for the constructive and insightful comments, which have helped us to substantially improve our manuscript.

Submitted filename: Response_to_ Reviewers.docx

Click here for additional data file.

22 Oct 2020

A novel mutation in the SLCO2A1 gene, encoding a prostaglandin transporter, induces chronic enteropathy

PONE-D-20-25461R1

Dear Dr. Okuno,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Sylvie Mazoyer, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Additional information about clinical features in these CEAS patients is highly appreciated and useful.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Takeo Nakanishi

29 Oct 2020

PONE-D-20-25461R1

A novel mutation in the SLCO2A1 gene, encoding a prostaglandin transporter, induces chronic enteropathy

Dear Dr. Okuno:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr Sylvie Mazoyer

Academic Editor

PLOS ONE