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Nephrol Dial Transplant (2012) 27: 1035–1041 doi: 10.1093/ndt/gfr419 Advance Access publication 2 August 2011
Two novel homozygous SLC2A9 mutations cause renal hypouricemia type 2 Dganit Dinour1,*, Nicola K. Gray2,*, Liat Ganon1, Andrew J.S. Knox3, Hanna Shalev4, Ben-Ami Sela5, Susan Campbell6, Lindsay Sawyer7, Xinhua Shu6, Evgenia Valsamidou7, Daniel Landau4, Alan F. Wright6 and Eliezer J. Holtzman1 1
Institute of Nephrology and Hypertension, Sheba Medical Center, Tel-Hashomer and the Sackler school of Medicine, Tel Aviv University, Tel Aviv, Israel, 2MRC Centre for Reproductive Health, Queens Medical Research Institute, University of Edinburgh, Edinburgh, UK, 3School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland, 4Pediatric Nephrology, Soroka University Medical Center and Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva, Israel, 5Institute of Chemical Pathology, Sheba Medical Center, Tel-Hashomer and the Sackler school of Medicine, Tel Aviv University, Tel Aviv, Israel, 6MRC Human Genetics Unit, Institute for Genetics and Molecular Medicine, Western General Hospital, Edinburgh, UK and 7Institute of Structural & Molecular Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Correspondence and offprint requests to: Dganit Dinour; E-mail:
[email protected] *These authors contributed equally to this work.
Abstract Background. Elevated serum uric acid (UA) is associated with gout, hypertension, cardiovascular and renal disease. Hereditary renal hypouricemia type 1 (RHUC1) is caused by mutations in the renal tubular UA transporter URAT1 and can be complicated by nephrolithiasis and exercise-induced acute renal failure (EIARF). We have recently shown that loss-of-function homozygous mutations of another UA transporter, GLUT9, cause a severe type of hereditary renal hypouricemia with similar complications (RHUC2). Methods. Two unrelated families with renal hypouricemia were clinically characterized. DNA was extracted and SLC22A12 and SLC2A9 coding for URAT1 and GLUT9, respectively, were sequenced. Transport studies into Xenopus laevis oocytes were utilized to evaluate the function of the GLUT9 mutations found. A molecular modeling study was undertaken to structurally characterize and probe the effects of these mutations. Results. Two novel homozygous GLUT9 missense mutations were identified: R171C and T125M. Mean serum UA level of the four homozygous subjects was 0.15 6 0.06 mg/dL and fractional excretion of UA was 89–150%. None of the affected subjects had nephrolithiasis, EIARF or any other complications. Transport assays revealed that both mutant proteins had a dramatically reduced ability to transport UA. Modeling showed that both R171C and T125M mutations are located within the inner channel that transports UA between the cytoplasmic and extracellular regions.
Conclusions. This is the second report of renal hypouricemia caused by homozygous GLUT9 mutations. Our findings confirm the pivotal role of GLUT9 in UA transport and highlight the similarities and differences between RHUC1 and RHUC2. Keywords: GLUT9; hereditary renal hypouricemia; SLC2A9; URAT1; uric acid
Introduction In most mammals, uric acid (UA) is oxidized by the hepatic enzyme uricase to highly soluble allantoin. In humans, however, this enzyme is inactive due to mutational silencing [1] making UA the end product of purine metabolism. Serum UA concentration depends on both UA production and UA removal by the kidneys and intestinal tract and is high in humans compared to other mammals. Elevation of serum UA levels has been associated with various diseases, including gout, hypertension, cardiovascular and renal disease [2, 3]. On the other hand, it has been suggested that UA has a beneficial role as a natural antioxidant, and low serum UA levels have been linked to several neurological diseases [2, 3]. Studies of renal handling of UA in humans have provided evidence for the historical model of urinary UA excretion, which consists of four components: free glomerular filtration, tubular absorption, secretion and post-secretion
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reabsorption. However, the location and molecular physiology of the three tubular transport components have not been completely clarified [4]. The first identified renal UA transporter, URAT1 (SLC22A12 gene), was described in 2002 by Enomoto et al. [5]. URAT1 is a urate/anion exchanger located in the apical membrane of the proximal tubule epithelial cells. The significance of URAT1 for the handling of UA was demonstrated by genetic analysis of Japanese patients with hereditary renal hypouricemia [5–7]. These patients were characterized by low levels of serum UA, high fractional excretion of UA (FEUA) and attenuated response of urinary urate excretion to pyrazinamide and probenecid [6]. Most of these patients were asymptomatic, but some had nephrolithiasis or were predisposed to exercise-induced acute renal failure (EIARF). The Japanese patients were found to possess homozygous, heterozygous or compound heterozygous loss-of-function mutations in the gene SLC22A12 coding for human URAT1 (hereditary renal hypouricamia type 1, RHUC1, OMIM #220150; http://www.ncbi.nlm.nih.gov/omim/220150). Most of them carry at least one ancestral allele with the truncation mutation W258X [5–7]. Several cases of familial hypouricemia and hyperuricosuria in Iraqi-Jews have been previously reported [8, 9], however, the molecular basis of hypouricemia in these cases was unknown. We have recently described hereditary hypouricemia due to loss-of-function mutations of URAT1 in three Israeli families of Iraqi-Jewish origin [10]. As the missense mutation R496C was detected in affected subjects of all three families, it is likely to be the common Iraqi mutation. Although serum UA levels and FEUA were similar to those of the Japanese patients, none of our patients developed EIARF. Recent reports revealed a second type of hereditary renal hypouricemia caused by mutations in another UA transporter, GLUT9 [hereditary renal hypouricamia type 2 (RHUC2, OMIM #612076; http://www.ncbi.nlm.nih.gov/ omim/612076)]. GLUT9, a member of the facilitated glucose transporters, encoded by SLC2A9, was associated with serum UA levels and gout in several population studies [11–13]. SLC2A9 encodes two variants of GLUT9—short (GLUT9S) and long (GLUT9L)–localized to the apical and basolateral tubular membrane, respectively [14]. Both variants were shown to be potent UA transporters in vitro [11, 15, 16]. The clinical importance of GLUT9 in humans was confirmed by the detection of SLC2A9 mutations in individuals with renal hypouricemia. Anzai et al. [15] reported a heterozygous GLUT9 mutation (GLUT9L-P412R) in one man with serum UA of 2.4 mg/dL. Matsuo et al. [16] identified two different missense heterozygous GLUT9 mutations (GLUT9L-R380M and R198C) in three subjects with renal hypouricemia. In a previous study, we described two families with ‘homozygous’ SLC2A9 mutations: a missense mutation (GLUT9L-L75R) in one family and a 36-kb deletion, resulting in a truncated protein, in the other. In vitro, the L75R mutation dramatically impaired transport of UA. The mean concentration of serum UA of homozygous individuals was 0.17 0.2 mg/dL, and all had a FEUA >150%. Three of six individuals had nephrolithiasis and three had a history of EIARF [17].
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In this study, we analyzed two novel loss-of-function GLUT9 mutations, which cause hereditary renal hypouricemia with extremely low serum UA levels but no complications. Materials and methods Clinical analysis Subjects were evaluated for clinical history of exercise-induced acute kidney injury, renal stones or other renal diseases. Blood and spot urine samples were collected for measurement of UA and creatinine levels and for genetic analysis. The study was approved by the institutional and Ministry of Health Review Boards for human experimentation. All participants gave written informed consent. Parental consent for children under 18-year-old was obtained. Molecular analysis DNA extraction and sequencing. Genomic DNA was isolated from peripheral blood cells applying the ArchivePure DNA Blood Kit (5 PRIME, Gaithersburg, USA) according to the manufacturer’s instructions. The coding areas and splice sites of SLC22A12 and SLC2A9 were amplified by polymerase chain reaction (PCR), using intronic primers as previously described [10, 17]. All PCR products were sequenced directly (ABI Prism 3100; Applied Biosystems, Foster City, CA). Restriction enzyme analysis. We used restriction enzyme analysis to screen normal controls for the GLUT9L-T125M/GLUT9S-T96M. Exon 4 of SLC2A9 was amplified using flanking intronic primers [17] and digested with the restriction enzyme FaqI, which cleaves the wild-type (WT) sequence but not the mutant. The digested fragments were detected using gel electrophoresis. Molecular/functional studies Plasmids. pLuc-MS2 and pSLC2A9_S have previously been described [11, 18]. For oocyte transport studies, pSLC2A9_ST96M and pSLC2A9_SR142C were created by site-directed mutagenesis using a Stratagene kit according to the manufacturer’s instructions and were confirmed by sequencing. Transport studies. Following linearization with BglII (luc-MS2) or XbaI (pSLC2A9 and derivates), messenger RNAs (mRNAs) were transcribed, adenylated and purified as described [11]. Five or 0.5 ng of mRNA was injected into defolliculated Stage VI oocytes and uptake assays were performed 2 days after injection as described [17]. Three or four pools of five oocytes were collected per experimental point in each experiment. Modeling Homology modeling. A homology model of GLUT9 was constructed based on the X-ray structure of GlpT from Escherichia coli (PDB ID. 1PW4) [19]. Models were built based on multiple-threading alignments by LOMETS and iterative TASSER [20–22] simulations using the iTASSER server. iTASSER has recently been ranked as the top server for protein structure prediction in recent CASP7/8 experiments. The topranking model was subsequently passed through a 500 ps minimization routine using YASARA (http://www.yasara.org/) to refine the model [23]. The lowest energy model generated was validated using Procheck [24] and ProSA-Web [25]. Molecular dynamics simulation. The molecular dynamics simulations were performed using the YASARA dynamics module in a membrane environment of lipid composition, 100% phosphatidylethanolamine with the AMBER03 force field [26]. The protein was firstly scanned for hydrophobic amino acids and embedded in the membrane and an initial 250 ps restrained simulation was run to allow lipids to surround the protein and fill gaps. The cell was subsequently filled with water and the AMBER03 electrostatic potential was evaluated for all water molecules. Those water molecules with the lowest potential were changed to K1 ions and the highest potential were changed to Cl ions to produce a neutralized cell. A 2 ns simulation was executed at 298 K and 0.9% NaCl and trajectories were captured every 25 ps. The average model was output and used in the final analysis.
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Results Clinical characteristics Family 1. The index patient (Figure 1A; Subject II1) was a 7.5-year-old otherwise healthy girl who was evaluated for short stature (third percentile). The only abnormal finding in her blood tests was a UA level of 0.1 mg%. Urine UA level was 44 mg% and FEUA was 138%. This patient is a member of a consanguineous Israeli-Arab family (Figure 1A): the parent’s grandmothers were sisters. Her father and mother had normal serum UA levels, but her 5.5-year-old brother and her 2.3-year-old sister had also very low serum UA levels and a high FEUA (Table 1). Their stature was normal for their age (25th and 10th percentile, respectively). All family members were asymptomatic and had no history of renal stones or acute renal failure. The index patient had a blood pressure of 100/65. The blood pressures of her brother and sister were 101/67 and 90/40, respectively. The blood pressures of the parents were 115/70 (father) and 100/70 (mother). Family 2. An 84-year-old male was found to have very low serum UA levels in repeated routine blood test. His medical history was remarkable for congestive heart failure and paroxysmal atrial fibrillation treated with Warfarin. Transitional cell carcinoma was diagnosed 6 years previously and since then he was regularly followed in the urology clinic. Blood pressure at the time of the study was 147/73. His serum creatinine was 0.9 mg%, sodium 142 mEq/L, potassium 3.8 mEq/L, calcium 9.3 mg% and phosphate 3.3 mg%. UA level was 0.2 mg% and FEUA was 151% (Table 1). The patient had no history of nephrolithiasis and had never had an episode of EIARF despite a very active lifestyle over the years. He was born in Israel to parents who were not known to be related, but both were of Sephardi-Jewish origin living in Israel for eight generations. Molecular analysis DNA sequencing of SLC22A12 was normal in both families. Sequencing of SLC2A9 identified two novel missense mutations of GLUT9: the index patient of Family 1 and her two siblings had a homozygous GLUT9L-R171C/GLUT9SR142C mutation, while both parents were heterozygous carriers [Figure 1B (1)]. The mutation was absent in a control group of 53 unrelated Israeli-Arabs (106 alleles), as shown by direct sequencing of SLC2A9 Exon 5. The patient of Family 2 was found to have a homozygous GLUT9L-T125M/ GLUT9S-T96M mutation [Figure 1B (2)]. The mutation was absent in a control group of 50 unrelated Israeli-Jews (100 alleles), as confirmed by restriction enzyme analysis. Both mutations replace amino acids, which are highly conserved in various species [Figure 1C (1)], but only R171 is also conserved in other GLUT proteins [Figure 1C (2)]. Functional studies in oocytes To determine the extent to which the identified mutations compromised the capacity of GLUT9 to transport UA, [8-14C]-UA transport was measured in oocytes injected
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with control, WT or mutant mRNAs as described [11, 17]. Both isoforms of GLUT9 efficiently transported UA compared to the control (luciferase) mRNA which was similar in activity to uninjected oocytes (data not shown). Importantly, the transport activity of GLUT9S-T96M and GLUT9S-R142C was severely reduced, retaining only 19.8% (0.005 0.002 pmol/oocyte/min) and 16% (0.004 0.002 pmol/oocyte/min) of the activity of the WT short isoform (0.025 0.002 pmol/oocyte/min), respectively (Figure 2). A similar loss of activity was seen when the mutations were analyzed in the context of the long isoform (T125M and R171C) or when mRNA concentrations were varied 10-fold (data not shown). GLUT9 model and mutations To establish a structural model to probe the mutational effects on the GLUT9 transporter, we have developed a homology model with the E.coli glycerol phosphate transporter (GlpT) [19]. Both the initially generated model (iTASSER) [20–22] and refined model (YASARA dynamics) were subjected to statistical analysis with Procheck [24] and ProSA-web [25]. Ramachandran plots indicated that the initial model had an overall G-factor (dihedrals and covalent) of 0.65 with residues distributed as follows—75.4% core, 18.2% allowed, 4.8% generously allowed and 1.5% in disallowed regions. After model refinement, the following was obtained—the overall G-factor was 0.09 and residues were distributed; 87.3% core, 10.1% allowed, 1.5% generously allowed, 1.1% disallowed. G-factors provide a measure of how normal or unusual a stereochemical property is, whereby negative G-factors indicate that the property corresponds to a low probability conformation. Pal et al. [27] previously noted that residues in disallowed regions of a ramachandran plot are usually located at the surface of a molecule in short loops and also at the interface between two secondary structure elements. A good quality model of ~2Å would be expected to have >90% of residues located in favored regions. Based on G-factors and ramachandran analysis our homology model appears to meet these criteria. Final validation with ProSAweb indicated that the overall quality of our model was excellent (score calculated of 4.45) and was distributed among those scores of all experimentally determined protein chains currently available in the Protein Data Bank (http://www.rcsb.org). Figure 3A (1–3) shows the characteristic MFS fold helical topology of GLUT9 with the mutations described in this study highlighted. It can be seen that those missense mutations are located within the inner channel that expels UA from the cytoplasmic to extracellular regions. These findings provide an explanation for the mechanism by which these two mutations block UA transport in proximal tubular cells.
Discussion In this study, we evaluated a consanguineous Israeli-Arab family in which all three siblings had extremely low serum UA levels and an elevated FEUA (Table 1, F1A). The affected children were found to be homozygous for a novel
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Fig. 1. Pedigree and SLC2A9 mutations found in two unrelated families with hereditary hypouricemia. (A) Pedigree of Family 1. Circles represent female subjects; squares represent male subjects. The double lines indicate consanguinity: the father’s grandmother and the mother’s grand-grandmother were sisters. Solid symbols denote homozygous and half-solid denote heterozygous family members. The arrow indicates index patient. [B (1)] Missense mutation (R171C) found in the three affected children of Family 1 as compared with the sequences in the heterozygous father and a healthy control (WT). (2) Missense mutation (T125M) found in the affected subjects of Family 2 as compared with a healthy control (WT). (C) Alignment of the GLU9 amino acids mutated in our families with (1) orthologs (2) paralogs. Table 1. Clinical data and SLC2A9 mutationsa SLC2A9 mutations Patient
Gender
Age (years)
SUA (mg%)
UUA (mg%)
SCR (mg%)
UCR (mg%)
FEUA (%)
Nucleotideb
Aminoc acid
F1 I1 I2 II1 II2 II3d F2
Male Female Female Male Female Male
37 24 7.5 5.5 2.3 84
4.9 3.8 0.1 0.1 0.2 0.2
73 33 44 68 71 14.6
0.81 0.57 0.44 0.38 0.27 0.81
185 153 140 134 108 38.6
6.5 3.2 138 157 88.8 151
C511>T/WT C511>T/WT C511>T/C511>T C511>T/C511>T C511>T/C511>T C375>T/C375>T
R171C/WT R171C/WT R171C/R171C R171C/R171C R171C/R171C T125M/T125M
a
SUA, serum UA; UUA, urine UA; SCR, serum creatinine; UCR, urine creatinine. According to coding sequence of GLUT9L. c According to GLUT9L. d Indicates the index patient of family 1. b
SLC2A9 missense mutation, GLUT9L-R171C/GLUT9SR142C. UA levels in these subjects were similar to those reported in homozygous patients with RHUC2 [17] and much lower than UA levels in homozygous patients with RHUC1 (OMIM 220150). The index patient presented with short stature, most probably unrelated to the hypouricemia, as this disorder has not been described in RHUC and was absent in her two siblings. To date, all three affected siblings are asymptomatic, but all are still very young.
In addition, we described an 84-year-old male (Table 1, F2) with a serum UA near zero, who had never had any symptoms related to hypouricemia despite his age and active lifestyle. In this patient, we identified another novel SLC2A9 missense mutation, GLUT9L-T125M/GLUT9ST96M. Both mutations showed a markedly reduced ability to transport UA (Figure 2), comparable to previously described GLUT9 mutations [15–17], confirming their loss-
Hereditary hypouricemia type 2
of-function nature. It should be noted that none of the reported GLUT9 mutations showed a complete loss of UA transport in oocyte transport studies, as might have been expected from the severe hypouricemia found in affected subjects. This discrepancy may perhaps be explained by the combined effects of reduced UA transport activity and impaired trafficking and membrane targeting of the mutant GLUT9 in humans. Figure 3B clearly illustrates the reasons for the observed loss-of-function occurring because of the missense mutations, T125M and R171C. It can be seen that mutation of
Fig. 2. Severely reduced [8-14C]-UA transport activity in oocytes injected with mutant compared to WT SLC2A9 mRNAs. Oocytes injected with control, SLC2A9_S WT or mutant mRNAs encoding T96M or R142C and subjected to transport assays for 1 h at room temperature. Control (unrelated mRNA) counts were subtracted and WT was set to 100%. The average of three experiments is shown and error bars represent standard error of measurement.
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arginine to cysteine would result in a loss of stability and most likely folding propensity of GLUT9 through removal of the hydrogen bond contacts made by Arg171 (Helix 4) to Thr113 (TM Domain: Helix 2) and Asn74 (TM Domain: Helix 1). Figure 3C (1–2) depicts the steric hindrance resulting from the T125M mutation (Helix 2) and loss of hydrogen-bonding potential that would challenge passage of UA through the central channel. Interestingly, the two heterozygous subjects for the GLUT9L-R171C mutation had serum UA levels and FEUA values within the normal range (Table 1, F1I1 and F1I2). The finding of normal UA is conflicting since previous reports have shown low serum UA in subjects with other heterozygous GLUT9 mutations. Anzai et al. [15] found a serum UA level of 2.4 mg/dL in a man with the heterozygous GLUT9-P412R mutation. Matsuo et al. [16] described a mother and her son who had serum UA levels of 2.7 and 1.5 mg/dL, respectively, and a FEUA of ~15% as well as an unrelated subject with a serum UA of 2.1 mg/dL, all of them were heterozygous for loss-of-function GLUT9 mutations. In our previous report [17], we identified nine individuals with a heterozygous loss-of-function GLUT9 mutation (L75R). Their serum UA levels ranged between 2.0 and 4.5 mg/dL and the range of FEUA values was 21.7 to 5.4%. The variability of serum UA levels in heterozygous individuals is also supported by the data on rare GLUT9 variants reported by Vitart et al. [11]. By sequencing 95 samples from a Croatian dataset, they identified two rare heterozygous variants of SLC2A9: a missense mutation (T125R) was associated with a serum UA of 2.2 mg/dL and a
Fig. 3. [A (1)] Side view depicting relative positions of helices of GLUT9. Mutations R171C and T125M (green) located within the inner channel are represented with van der Waals surface. Figure drawn using Yasara (http://www.yasara.org). (2) View of GLUT9 homology model from the extracellular side showing the configuration of 12 transmembrane helices. Figure drawn using Pymol (http://pymol.sourceforge.net). (3) Cytoplasmic view of GLUT9 homology model from the intracellular side. (B) Location of and interactions made by R171. Three key hydrogen-bonding interactions are made between the guanidinium moiety of R171 and N74, T113 stabilizing Helices 1, 2 and 4. Figure drawn using Yasara (http://www.yasara.org). [C (1)] Intracellular view of GLUT9 (surface rendered) with Thr125 highlighted. Figure drawn using Pymol (http://pymol.sourceforge.net). (2) Intracellular view of GLUT9 (surface rendered) with mutation T125M highlighted. Figure drawn using Pymol (http://pymol.sourceforge.net).
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Table 2. Characterizations of renal hereditary hypouricemia (RHUC) types
Mutated gene Mutated protein Reported homozygous mutations Mutation’s effect Serum UA FEUA UA transport defect Complications
RHUC type 1
RHUC type 2
SLC22A12 URAT1 W258X; E298D, T217N, R90H, R406C; G444R; Loss-of-function 0.5–1.0 mg/dL 40–90% Pre-secretory absorption defect Nephrolithiasis and EIARF
SLC2A9 GLUT9 L75R; delExon7; R171C; T125M Loss-of-function 0–0.2 mg/dL 88 to >150% Total reabsorption defect Nephrolithiasis and EIARF
heterozygous deletion (77delC), predicting to result in a truncated protein, was associated with serum UA values of 3.1–5.8 mg/dL in four individuals. Of note, a similar spectrum of serum UA levels has been reported in heterozygous carriers of URAT1 mutations [7, 10]. Based on the available data, we conclude that heterozygous loss-of-function GLUT9 mutations cause mild hypouricemia, most probably through haploinsufficiency. However, in contrast to individuals with homozygous loss-of-function GLUT9 mutations, the renal tubular cells of heterozygous subjects retain the ability to transport UA. Therefore, the degree of UA absorption may vary depending on other genetic or non genetic variables such as sex, age, diet, medications and volume status. Our study highlights the similarity and discrepancy between RHUC1 and RHUC2 (Table 2): both disorders impair UA transport in the proximal tubular cells, causing hypouricemia and increased UA excretion. However, the extent of impairment is markedly different. Patients with homozygous loss-of-function mutations of GLUT9 have lower serum UA levels (0–0.2 mg/dL) compared to patients with loss of URAT1 function (0.5–1.0 mg/dL) [6, 7]. Furthermore, renal excretion of UA in patients with lossof-function of GLUT9 is higher than in patients with loss of URAT1 function: FEUA in previously described subjects with homozygous GLUT9 mutations was >150% [17], compared to 13% in those with heterozygous GLUT9 mutations and 40–90% in patients with homozygous loss of URAT1 [6, 7, 10]. In the present study, we found a FEUA of 138–151% in three of four homozygous subjects. In one very young child (Table 1, F1II3) with a homozygous GLUT9L-R171C/GLUT9S-R142C mutation, serum UA was 0.2 mg% and FEUA was 88.8%. The relatively low FEUA could result from an inaccurate measurement of serum UA, which is possible in such low values. As the type of RHUC may be predicted from clinical parameters and confirmed by molecular analysis, the traditional pyrazinamide/probenecid test seems no longer to be required. Similar to patients with URAT1 mutations, patients with loss-of-function GLUT9 mutations may develop renal stones or EIARF [17] but may also be asymptomatic, despite extremely low serum UA levels. At this time, it is impossible to compare the rate of complications between RHUC1 and RHUC2 because of the small number of RHUC2 patients described to date. The present findings also support previous speculation about the role of GLUT9 in UA handling by the human kidney. Based on our and on others’ findings, it was suggested that UA efflux is mediated solely by basolateral
GLUT9L, whereas UA absorption from the tubular lumen is carried out by URAT1 as well as other apical UA transporters, including apical GLUT9S [3, 17, 28]. Thus, in RHUC1, the loss of URAT1 function produces a partial UA absorption defect, while the loss-of-function of GLUT9 in RHUC2 precludes UA absorption by all the apical transporters (including URAT1) through complete blocking of UA efflux, resulting in a total UA reabsorption defect. The finding of a FEUA of >100% in most cases of RHUC2 confirms the existence of a functioning UA excretion pathway, yet to be defined. A strong candidate is the ABCG2 gene identified in genome-wide association studies to be associated which serum UA concentrations and gout. Recent studies have shown that ABCG2 is a high-capacity UA secretion transporter and that non-functional variants of ABCG2 block gut and renal urate excretion and cause gout [29, 30]. The role of GLUT9 in renal handling of UA was also revealed by genome-wide studies looking for association with serum UA and gout. It is tempting to assume that gain-of-function mutations in GLUT9 will cause renal hyperuricemia and gout as a mirror image of renal hypouricemia. However, so far, no GLUT9 mutation causing hyperuricemia has been identified. In fact, coding SNPs showed less significant association with serum UA than intronic SNPs [11]. Thus, the association of hyperuricemia and gout with GLUT9 may be explained by non-coding variants or combination of variants that affect recycling or degradation of GLUT9 protein, resulting in increased expression on tubular membranes. This is the second report of renal hypouricemia caused by homozygous loss-of-function GLUT9 mutations. It contributes to the characterization of RHUC type 2 as opposed to RHUC type 1 and confirms the pivotal role played by GLUT9 in renal UA transport. A better understanding of UA handling by the kidney may also improve our understanding of more common clinical problems such as hyperuricemia, nephrolithiasis and gout. Acknowledgements. N.K.G. is funded by MRC core, an MRC Senior NonClinical Fellowship and by Welcome Trust project grant funding.
Conflict of interest statement. None declared.
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