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Homozygous SLC2A9 Mutations Cause Severe Renal

ʈ Dganit Dinour,* Nicola K. Gray,†‡§ Susan Campbell,† Xinhua Shu,† Lindsay Sawyer, William Richardson,†‡§ Gideon Rechavi,¶ Ninette Amariglio,¶** Liat Ganon,* Ben-Ami Sela,†† Hilla Bahat,‡‡ Michael Goldman,‡‡ Joshua Weissgarten,§§ Michael R. Millar,§ Alan F. Wright,† and Eliezer J. Holtzman*

*Nephrology and Hypertension Institute, ¶Cancer Research Laboratory, **Institute of Hematology, and ††Institute of Chemical Pathology, Sheba Medical Center, Tel-Hashomer and the Sackler School of Medicine, and Departments of ‡‡Pediatrics and §§Nephrology and Hypertension, Assaf Harofeh Medical Center, Zerifin, and the Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; †MRC Human Genetics Unit, Institute for Genetics and Molecular Medicine ʈ Western General Hospital, Edinburgh, United Kingdom; Institute of Structural and Molecular Biology, School of Biological Sciences, and ‡School of Clinical Sciences and Community Health, University of Edinburgh, Edinburgh, United Kingdom; and §MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, Edinburgh, United Kingdom

ABSTRACT Hereditary hypouricemia may result from mutations in the renal tubular transporter URAT1. Whether mutation of other uric acid transporters produces a similar phenotype is unknown. We studied two families who had severe hereditary hypouricemia and did not have a URAT1 defect. We performed a genome-wide homozygosity screen and linkage analysis and identified the candidate SLC2A9, which encodes the 9 (GLUT9). Both families had homozygous SLC2A9 mutations: A missense mutation (L75R) in six affected members of one family and a 36-kb deletion, resulting in a truncated , in the other. In vitro, the L75R mutation dramatically impaired transport of uric acid. The mean concentration of serum uric acid of seven homozygous individuals was 0.17 Ϯ 0.2 mg/dl, and all had a fractional excretion of uric acid Ͼ150%. Three individuals had nephrolithiasis, and three had a history of exercise-induced acute renal failure. In conclusion, homozygous loss-of-function mutations of GLUT9 cause a total defect of uric acid absorption, leading to severe renal hypouricemia complicated by nephrolithiasis and exercise-induced acute renal failure. In addition to clarifying renal handling of uric acid, our findings may provide a better under- standing of the pathophysiology of acute renal failure, nephrolithiasis, , and .

J Am Soc Nephrol 21: 64–72, 2010. doi: 10.1681/ASN.2009040406

In most mammals, uric acid (UA) is oxidized by antioxidant, and low serum UA levels have been the hepatic enzyme uricase to highly soluble al- linked to several neurologic diseases.2 lantoin. In humans, however, this enzyme is in- Studies of renal handling of UA in humans have active as a result of mutational silencing,1 making UA the end product of purine metabolism. Se- Received April 16, 2009. Accepted September 23, 2009. rum UA concentration depends on both UA pro- Published online ahead of print. Publication date available at duction and UA removal by the kidneys and in- www.jasn.org. testinal tract and is high in humans compared D.D. and N.K.G. contributed equally to this work. with other mammals. Elevation of serum UA lev- Correspondence: Dr. Dganit Dinour, Nephrology and Hyperten- els has been associated with various diseases, in- sion Institute, Sheba Medical Center, Tel-Hashomer, 52621, Is- cluding gout, hypertension, and cardiovascular rael. Phone: 972-3-5302581; Fax: 972-3-5392582; E-mail: and renal disease.2 Conversely, it has been sug- [email protected] gested that UA has a beneficial role as a natural Copyright ᮊ 2010 by the American Society of Nephrology

64 ISSN : 1046-6673/2101-64 J Am Soc Nephrol 21: 64–72, 2010 www.jasn.org BASIC RESEARCH provided evidence for a historical model of urinary UA excretion, been shown to be expressed in renal tubular cells and to trans- which consists of four components: Free glomerular filtration, port UA in vitro. Recently, heterozygous mutations of GLUT9 tubular absorption, secretion, and postsecretion reabsorption. were shown to cause renal hypouricemia.16 The location and molecular physiology of the three tubular trans- In this report, we show that homozygous mutations of port components, however, have not been completely clarified.3 GLUT9 cause severe hereditary hypouricemia complicated by The first renal UA transporter, URAT1, was identified in 2002 nephrolithiasis and EIARF. Our findings provide further evi- by Enomoto et al.4 The significance of URAT1 in the handling of dence for the key role played by GLUT9 in renal UA handling. UA was demonstrated by genetic analysis of Japanese patients with hereditary renal hypouricemia.4,5 These patients were char- acterized by very low levels of serum UA, high fractional excretion RESULTS of UA, and attenuated response of urinary urate excretion to pyr- azinamide and probenecid.5 Most of these patients were asymp- Clinical Characteristics tomatic, but some had nephrolithiasis or were predisposed to ex- Family 1. ercise-induced acute renal failure (EIARF). The Japanese patients The index patient (IV6; Figure 1A) was a previously healthy were found to possess homozygous or compound heterozygous 18-yr-old man, who presented with ARF after physical exer- loss-of-function mutations in the gene SLC22A12 coding for hu- tion and required hemodialysis for 3 wk. One month after man URAT1; most of them carry at least one allele with the trun- hospital discharge, serum urea and creatinine were normal and cation mutation W258X.4–6 serum UA level was 0.1 mg/dl. The clinical course of the patient Mutations in SLC22A12 seem to be very rare outside Japan. A was described in detail in our previous report.17 mutation analysis of renal hypouricemia in Korea showed that This patient is a member of a highly consanguineous Israeli- three of four patients with URAT1 mutations carried the W258X Arab family (Figure 1A). We evaluated all available family mutation.7 We previously described hereditary hypouricemia as a members and found five additional individuals with extremely result of a homozygous SLC22A12 missense mutation (R496C) in low hypouricemia and fractional excretion of UA Ͼ150%; two three Israeli families of Iraqi origin.8 Although serum UA level and had a history of EIARF (III9 and IV5), and two reported renal fractional excretion of UA were similar to those of the Japanese stones (II5 and III10). Clinical features and data related to UA patients, none of our patients developed EIARF. handling of all individuals are shown in Table 1. A recent meta-analysis of 14 genome-wide association scans in Europe demonstrated significant association of serum UA Family 2. concentration with several other , including SLC22A11 The index (III2) is a 69-yr-old Ashkenazi-Jewish man whose coding for organic anion transporter 4 (OAT4), SLC17A1 cod- parents are first cousins (Figure 1B). He was found to have ing for NPT4, the ATP-binding cassette transporter ABCG2, extremely low serum UA levels in routine repeated examina- and SLC2A9 coding for the glucose-facilitated transporter tions in the past 5 yr (0.0 to 0.1 mg/dl). He had one episode of GLUT9.9 OAT4,10 NPT1,11 ABCG2,12 and GLUT913–16 have renal colic and confirmed nephrolithiasis approximately 30 yr

Figure 1. Pedigrees of two unrelated consanguineous families with severe renal hypouricemia and SLC2A9 mutations. (A) Pedigree of family 1. (B) Pedigree of family 2. Solid symbols denote affected family members, open symbols denote unaffected family members, half-solid denote heterozygous family members, and dotted symbols denote family members who were not available for examination. Circles represent female family members, squares represent male family members, and crosses represent dead family members. Arrows indicate index patients. #Low serum UA (1.5 mg/dl), not available for genetic evaluation.

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Table 1. Clinical data and SLC2A9 mutations of the patients with renal hypouricemia and their family members Serum Urine Fractional Age Serum UA Urine UA Patient Gender History SLC2A9 Mutation Creatinine Creatinine excretion (yr) (mg/dl) (mg/dl) (mg/dl) (mg/dl) of UA (%) F1 II5 M 67 Nephrolithiasis, CKD L75R/L75R 0.67 67.9 1.53 88.2 Ͼ150.0 II6 F 64 L75R/WT 4.50 47.2 0.89 172.9 5.4 III1 F 31 WT 4.00 93.5 0.74 186.6 9.3 III2 M 46 WT 7.90 NA 1.23 NA NA III3 F 46 WT 4.30 69.9 0.93 119.5 12.6 III4 F 28 L75R/WT 2.00 80.4 0.78 114.3 21.7 III5 F 35 WT 3.40 30.7 0.70 56.4 11.2 II6 F 38 L75R/WT 2.20 69.3 0.85 136.8 19.6 III7 M 44 WT 5.90 78.6 0.93 214.4 5.8 III8 F 48 L75R/WT 3.40 23.1 0.70 63.6 7.5 III9 M 46 EIARF L75R/L75R 0.20 80.3 0.88 174.8 Ͼ150.0 III10 M 36 Nephrolithiasis L75R/L75R 0.04 19.0 0.79 74.1 Ͼ150.0 III11 F 40 L75R/WT 3.70 50.5 0.65 118.7 7.4 III12 F 44 L75R/WT 3.10 55.6 0.84 121.4 12.4 IV1 M 5 L75R/WT 2.60 NA NA NA NA IV3 F 15 L75R/WT 2.40 NA NA NA NA IV4 F 10 L75R/L75R 0.01 34.8 0.51 74.4 Ͼ150.0 IV5 M 24 EIARF L75R/L75R 0.20 45.8 0.93 114.9 Ͼ150.0 IV6 M 19 EIARF L75R/L75R 0.10 92.1 0.93 281.7 Ͼ150.0 IV7 M 16 L75R/WT 2.00 41.0 0.60 71.6 17.0 IV8 F 19 WT 6.40 83.9 0.70 287.3 3.2 F2 III2 M 69 Nephrolithiasis delExon7/delExon7 0.10 11.4 1.14 49.5 Ͼ150.0 CKD, chronic kidney disease. ago but no history of renal failure. One of his daughters had DNA sequencing of SLC2A9 in the index patient of fam- hypouricemia (serum UA of 1.5 mg/dl). She and the other ily 1 (Figure 2A) identified a novel homozygous missense family members were unavailable for genetic studies. mutation, GLUT9L-L75R/GLUT9S-L46R. The L75R/L46R mutation creates an Age1 restriction site. Restriction en- Molecular Analysis zyme analysis using Age1 of all family members detected six We first excluded mutations in the URAT1-encoding gene, patients bearing homozygous L75R/L46R SLC2A9 muta- SLC22A12, in the index patients of families 117 and 2. Family 1 tion, nine heterozygous carriers, and six members with the consists of three generations of patients with hereditary hy- wild type (WT) SLC2A9 gene (Figure 2B). The presence of pouricemia (Table 1, Figure 1A). This pattern could suggest an homozygous or heterozygous L75R/L46R mutation was also autosomal dominant inheritance; however, because both fam- confirmed in all affected members by direct sequencing. ilies described here are consanguineous and some family mem- This mutation was absent in a control group of 150 unre- bers have intermediate serum UA levels (Table 1), compatible lated normal control subjects (300 alleles), including 100 with heterozygosity, the disease was more consistent with an Israeli-Arabs. autosomal recessive mode of inheritance. Therefore, we chose Whereas the Leu75 (or Leu46) residue is conserved in all to perform a genome-wide screening of family 1 under a hy- known GLUT9 orthologs, it is present only in seven of 14 SLC2 pothesis of homozygosity by descent for an ancestral mutation. family paralogues (SLC2A1 through 14; data not shown). The genome-wide search identified five genomic intervals DNA sequencing of SLC2A9 in patient III2 (family 2) iden- of homozygosity of Ͼ4 Mb on 4, 5, 8, 11, and 17. tified only two common polymorphisms: G25R and P350L. To limit the candidate genomic regions, we performed genetic However, exon 7 was not detected in the genomic DNA. We analysis of additional family members as well as patient III2 of therefore sequenced SLC2A9 transcript and demonstrated family 2, using microsatellite markers located in these regions skipping of the entire exon 7 and a direct transition from exon (see the Supplemental Appendix for a complete list of micro- 6 to 8 causing a translational frameshift introducing a prema- satellite markers used in this study). By this method, we were ture termination codon after 14 amino acids, resulting in a able to exclude all but one interval on 4 between truncated protein of 231 amino acids instead of 540. PCR of markers D4S403 and D4S419 (20.1 Mb), which contains the genomic DNA showed a rearrangement with a deletion of ap- gene SLC2A9. SLC2A9 encodes for two variants of GLUT9: proximately 36 kb including parts of intron 6, exon 7, and part Short (GLUT9S) and long (GLUT9L).13 of intron 7 (Figure 2C).

66 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 64–72, 2010 www.jasn.org BASIC RESEARCH

level (0.67 mg/dl) was found in a 67-yr-old man with chronic kidney disease (serum creatinine 1.53 mg/dl). Mean serum UA level in heterozygous individuals was 2.88 Ϯ 0.87 mg/dl com- pared with 5.10 Ϯ 1.88 mg/dl in family members with a WT SLC2A9 gene. Fractional excretion of UA was Ͼ150% in all homozygous individuals, compared with 13.00 Ϯ 6.74% in heterozygous and 8.40 Ϯ 3.88% in nonaffected members of the family.

Functional and Expression Studies in Oocytes To determine whether the mutated SLC2A9 gene encodes a compromised UA transporter, we measured [8-14C]UA trans- port in oocytes injected with WT or mutant SLC2A9 compared with a control mRNA. As described previously,14 GLUT9S ef- ficiently transported urate compared with oocytes injected with the control mRNA, averaging 0.041 Ϯ 0.005 (SEM) pmol oocyte/min. The GLUT9S-L46R mutant-injected oocytes showed reduced uptake compared with WT GLUT9S (19.4% of WT uptake; Figure 3B, B). The GLUT9L-L75R mutation also showed reduced uptake (37.8% of WT GLUT9L uptake; Figure 3B, A), although the difference was less than with GLUT9S. The reduced UA trans- port by the mutant transporter cannot be explained by failure to reach the plasma membrane, because both the WT and mu- tant are similarly expressed at the oocyte plasma membrane (Figure 3C). GLUT9S-positive staining was scored at the plasma membrane, and expression of the WT and L46R mutant at the plasma membrane was shown to be identical. The data were derived from a minimum of 40 oocytes per experimental point from three different animals.

DISCUSSION

Hereditary hypouricemia complicated by nephrolithiasis and EIARF has been reported so far only in patients with loss-of-function URAT1 mutations. Most of the patients were of Japanese origin and carried the truncation mutation W258X.4–6,18,19 This study shows that a similar but not Figure 2. SLC2A9 mutations, found in two families with severe hereditary hypouricemia. (A) Missense mutation (c.T224G, p.L75R) identical syndrome is caused by homozygous loss-of-func- found in the index patient of family 1 as compared with the se- tion mutations in the SLC2A9 gene encoding GLUT9. quences in a heterozygous family member and a healthy control We detected a homozygous SLC2A9 missense mutation, subject (WT). (B) Restriction enzyme analysis of family 1, showing L75R, in six members of an Israeli-Arab consanguineous fam- individuals with WT, homozygote, and heterozygote SLC2A9. The ily with severe renal hypouricemia (family 1). Expression stud- L75R mutation creates an AgeI recognition site that does not exist in ies in oocytes demonstrated that the GLUT9L-L75R/GLUT9S- the WT sequence. Digestion fragments of 269 and 83 bp are seen in L46R mutation markedly reduced the UA transport activity of mutated alleles. (C) Schematic presentation of the 36-kb deletion both GLUT9 variants (Figure 3B). These findings confirm the identified in the index patient of family 2 (top) and cDNA sequence loss-of-function nature of the GLUT9-L75R/L46R mutation. of the transition from exon 6 to exon 8 (bottom). The results of oocyte transport studies did not show a complete loss of UA transport as might have been expected from the Genotype–Phenotype Correlations severe clinical findings. This discrepancy may perhaps be ex- In family 1 we detected six individuals with a homozygous plained by the combined effects of reduced UA transport and SLC2A9-L75R mutation, nine with heterozygous mutation, impaired trafficking and membrane targeting of the mutant and six normal individuals. Mean serum UA level in homozy- GLUT9 in humans that cannot be demonstrated in oocytes gous individuals was 0.17 Ϯ 0.20 mg/dl. The highest serum UA (Figure 3).

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erozygous GLUT9 mutations and 40 to 90% in patients with homozygous loss of URAT1.5,6,8 The human SLC2A9 gene, cloned in 2000,20 encodes two isoforms of GLUT9—long and short (Figure 3A)—through the use of alternative pro- moters. In the kidney, GLUT9L is localized to the basolateral membrane of proximal tubular epi- thelial cells, whereas GLUT9S is localized to the apical membrane of these cells.21 On the basis of sequence similarity to other members of the facil- itative sugar transporter family, GLUT9 was first predicted to be a sugar transporter. Uptake stud- ies in Xenopus oocytes demonstrated that GLUT9 exhibits glucose transport activity.21 Surprising, recent population studies showed significant as- sociation between SLC2A9 genotype and serum UA concentrations.9,13,22 Moreover, two rare variants, including one null allele, were found to be associated with low serum UA.13 Analysis of GLUT9 expression in Xenopus oocytes provided strong evidence for its role as a UA transport- er.13,15 UA uptake for GLUT9-expressing oocytes was 31-fold higher versus control and seven-fold higher versus URAT1-expressing oocytes.13 In- creased UA uptake by overexpression of GLUT9 was also confirmed in transfected human and mouse cells.14 The significance of GLUT9 func- Figure 3. (A) Schematic representation of the variants GLUT9L and GLUT9S tion for human UA handling was further sup- showing the location of the mutation (L75R, L45R) found in family 1. (B) Reduced ported by the recent report of Matsuo et al.,16 who [8-14C]UA transport activity in oocytes injected with L75R mutant (MUT; A) or described three individuals, including a mother L46R mutant (MUT; B) compared with WT SLC2A9 mRNA. Oocytes were in- and a son, with hypouricemia caused by loss-of- jected with control, WT, or mutant mRNA, and transport assays were performed function heterozygous GLUT9 mutations. Their at room temperature for 1 h, 2 d after injection. Activity is expressed as counts 14 report led to the definition of hereditary renal hy- per minute of [8- C]UA uptake, as a percentage of WT. The average of either pouricemia type 2 (RHUC2; OMIM 612076; three or four experiments is shown, and error bars represent SEs. (C) The L46R http://www.ncbi.nlm.nih.gov/entrez/dispomim. mutation does not significantly impair transport of GLUT9S to the plasma ϭ membrane. Immunodetection with an anti-GLUT9S antibody shows that the WT cgi?id 612076) in addition to hereditary renal and L46R proteins are expressed at the plasma membrane, whereas fluores- hypouricemia type 1, caused by URAT1 muta- cence levels were undetectable in control oocytes or in the absence of GLUT9S tions (RHUC1; OMIM 220150; http://www.ncbi. antibody (data not shown). nlm.nih.gov/entrez/dispomim.cgi?idϭ220150). Interestingly, mutations in canine SLC2A9 were In one patient of a consanguineous Ashkenazi-Jewish family recently found to affect UA handling by the kidney and the (family 2), who exhibited clinical characteristics very similar to liver of Dalmatian , which exhibit hyperuricosuria and those of family 1, we found an approximately 36-kb deletion in relative hyperuricemia.23 the SLC2A9 gene. Because the deletion results in a truncated 231– Our findings provide definitive proof of the pivotal role amino acid protein, we assume that it leads to loss of function. played by GLUT9 in UA renal absorption in human. Ichida et Although patients with homozygous GLUT9 mutations can al.5 concluded their survey of renal hypouricemia in Japan with present with clinical features resembling hereditary hypouri- the statement that the ability of a UA transporter other than cemia as a result of URAT1 mutations, we note an important URAT1 to regulate serum UA levels should not be greater than difference: Patients with loss of function of GLUT9 (as a result that of URAT1. We show that the impact of GLUT9 deficiency of either the L46/75R mutation or the truncating deletion mu- on UA renal excretion and serum UA levels exceeds that of tation) have much lower serum UA levels (near 0) compared URAT1. with patients with loss of URAT1 function (0.5 to 1.0 mg/dl).5,6 On the basis of our and others’ findings, we speculate that Furthermore, renal excretion of UA in these patients is mark- UA efflux is mediated solely by basolateral GLUT9L. In con- edly higher: Fractional excretion of UA in all homozygous trast, UA absorption from the tubular lumen is carried out not members was Ͼ150%, compared with 13% in those with het- only by URAT1 but also by GLUT9S and possibly other apical

68 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 64–72, 2010 www.jasn.org BASIC RESEARCH transporters (Figure 4A). Thus, in RHUC1, the loss of URAT1 tion defect (Figure 4C). The finding of a fractional excretion of function produces a partial UA absorption defect (fractional UA of Ͼ100% (Ͼ150% in our patients) confirms the existence excretion of UA of 40 to 90%; Figure 4B). In contrast, the loss of a functioning UA excretion pathway, yet to be defined. of function of GLUT9 in RHUC2 precludes UA absorption by Three of seven patients with homozygous SLC2A9 muta- all of the apical transporters (including URAT1) through com- tions had experienced one or more episodes of renal colic. plete blocking of UA efflux, resulting in a total UA reabsorp- Urolithiasis is a known complication of hereditary hypourice- mia as a result of URAT1 mutations with a prevalence of 8.5%, compared with 2.0 to 3.0% in the general population.6 The high prevalence of urolithiasis is attributed to increased urine UA concentration. Because the renal UA absorption defect is more severe in SLC2A9-associated hypouricemia than in URAT1-associated disease, a higher prevalence of urolithiasis might be expected. The most severe complication of hereditary renal hypouri- cemia is EIARF. This disorder was previously described in pa- tients bearing the Japanese URAT1 mutation (W258X).4–6,24 We show here that EIARF occurs also in hereditary renal hy- pouricemia as a result of homozygous SLC2A9 mutations. Thus, predisposition to EIARF seems to be related to a reduced renal UA absorption, regardless of the genetic or environmen- tal background. Two mechanisms have been previously proposed to explain the mechanism of EIARF. The first is acute urate nephropathy, caused by increased production of UA during physical exercise and leading to increased urinary UA excretion and resulting in renal UA precipitation.3 Acute urate nephropathy occurs in conditions of rapid increase in serum UA levels such as the tumor lysis syndrome. It is characterized by typical histologic findings in kidney biopsy25 that have not been detected in af- fected patients with hereditary hypouricemia, making this ex- planation very unlikely. The second explanation is ischemic kidney injury secondary to vasoconstriction of renal vessels mediated by an exercise- induced increase in oxygen free radicals.3 UA is the most abun- dant aqueous antioxidant in humans and has been shown to preserve endothelial dilation in the face of oxidative stress.26 It is thus possible that the lack of UA in plasma of patients with hereditary hypouricemia predisposes them to ischemic acute renal failure. This hypothesis is supported by reported imaging results27 as well as biopsy results of patients with EIARF show- ing mainly acute tubular necrosis28; however, EIARF has never been described in patients with xanthinuria, whose serum UA levels are as low as those of patients with renal hypouricemia. We propose a third mechanism for EIARF: Reduced clearance of urate-coupled anions by URAT1 as a result of loss-of-func- tion mutations of either URAT1 or GLUT9 may exert toxic effects on renal proximal tubules, leading to toxic acute tubular necrosis. We show here that loss-of-function mutations of GLUT9 cause severe hypouricemia in homozygous individuals and moderately low serum UA levels in heterozygous carriers. In- Figure 4. A simplified model of UA handling by the proximal terestingly, several polymorphisms in the same gene were renal tubular cell, modified after Anzai et al.15 and Matsuo et al.16 found to be associated with hyperuricemia and gout in large (A) Normal physiology. (B) Loss-of-function of URAT1. (C) Loss-of- population studies.13,22,29,30 This may suggest that the patho- function of GLUT9 as a result of homozygous mutations. genesis of hyperuricemia and gout involves increased tubular

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UA reabsorption as a result of a gain of function of GLUT9, Microarray Studies. rather than reduced UA secretion, as postulated previously.31 DNA of three individuals with severe renal hypouricemia (II5, III9, Similar transport-related mirror-image diseases caused by dif- and III10; Figure 1A), assumed to be carrying a homozygous muta- ferent mutations in the same molecules were described in the tion in the candidate gene, were analyzed using GeneChip 500 K Map- epithelial sodium channel in which a loss of function mutation ping Arrays (Affymetrix, Santa Clara, CA) according to the manufac- causes pseudohypoaldosteronism with hypotension type 1A, turer’s protocol. For detection of regions that are homozygous in all and a gain-of-function mutation causes Liddle syndrome with three individuals, the GeneChip genotyping analysis software was hypertension.32 used to run dynamic model mapping analysis. In summary, this study describes the clinical and molecular characteristics of a severe type of hereditary renal hypourice- Haplotype Analysis. mia (RHUC2), caused by homozygous loss-of-function muta- Haplotype analysis for identifying homozygous regions in all mem- tions in the SLC2A9 gene, coding for the UA transporter bers of families 1 and 2 was used to narrow the regions of homozy- GLUT9. Our clinical and molecular findings may contribute to gosity detected by GeneChip analysis. Genotyping was performed at the understanding of the physiology of renal UA handling and the Center for Genomic Technologies at the Hebrew University of the pathogenesis of common diseases such as ARF, nephroli- Jerusalem, Israel, using microsatellite markers from the Genethon thiasis, hyperuricemia, and gout. human linkage map (Applied Biosystems). PCR product electro- phoresis and detection were performed using the 3700 Automated DNA Analyzer (Applied Biosystems). Sizing and genotyping were CONCISE METHODS performed using GENESCAN and GENOTYPER software (Applied Biosystems). Haplotypes were constructed and compared between family members. Clinical Analysis Individuals were evaluated for clinical history of exercise-induced acute renal injury, renal stones, or other renal diseases. Blood and Molecular/Functional Studies spot urine samples were collected for measurement of UA and Plasmids. Plasmid pLuc-MS2 has been previously described.33 For oocyte trans- creatinine levels and for genetic analysis. The study was approved port and immunohistochemistry studies, plasmids pSLC2A9_S and by the institutional and Ministry of Health review boards for hu- pSLC2A9_L were created as follows. Human SLC2A9 (long form L man experimentation. All participants gave written informed con- and short form S) cDNAs were PCR-amplified with forward primer sent. Parental consent for children who were younger than 18 yr 5Ј-GATGGCAAGGAAACAAAATAGG-3Ј (SLC2A9_L) or 5Ј-GAT- was obtained. GAAGCTCAGTAAAAAGGAC-3Ј (SLC2A9_S) and with the reverse primer 5Ј-GTTAAGGCCTTCCATTTATCTTACC-3Ј (both forms). Molecular Analysis The PCR products were ligated into pGEM-Teasy vector (Promega). DNA and RNA extraction and sequencing. The mutant form of L75R in SLC2A9_L and of L46R in SLC2A9_S Genomic DNA was isolated from peripheral blood cells using the were generated (CTG to CGG) by site-directed mutagenesis following ArchivePure DNA Blood Kit (5 PRIME, USA) according to the man- the manufacturer’s protocol (Stratagene). All constructs were verified ufacturer’s instructions. Total RNA was extracted from whole blood by sequencing. with TRIzol Reagent (Life Technologies-BRL, Paisley, UK) according to the manufacturer’s instructions. Antibody Production. The coding areas and splice-sites of SLC22A12 and SLC2A9 were The GLUT9S antibody was made by expression of amino acids 1 amplified by PCR, using intronic primers (see the Supplemental Ap- through 22 from GLUT9S (NP_001001290; MKLSKKDRGEDEESD- pendix for primer sequences). All PCR products were sequenced di- SAKKKLD) in rabbits using Genomic Antibody Technology. rectly (ABI Prism 3100; Applied Biosystems, Foster City, CA). RNA from blood leukocytes of patient III2, family 2, was reverse-tran- Immunohistochemistry. scribed using the Reverse-iT 1st strand Synthesis Kit (ABgene, Surrey, Two days after injection, oocytes were fixed in Dents (80%/methanol, UK). cDNA of exons 6 through 8 of SLC2A9 was sequenced using 20% DMSO), paraffin-embedded, and sectioned at 5 ␮m. Sections coding area primers. For better characterization of the deletion of were antigen-retrieved in a pressure cooker using 0.01 M citrate (pH exon 7, primer pairs were designed to amplify short amplicons (ap- 6) for 5 min followed by a 20-min stand in hot buffer. Slides were proximately 200 bp) on each side of the exon in a stepwise manner. An incubated in 3% H2O2 in methanol for 30 min at room temperature. appropriate WT control was used in every reaction. For primer se- After a 30-min incubation in 20% normal goat serum in Tris-buffered quences, see Supplemental Appendix Table 1D. saline (0.05 M [pH 7.4] and 0.85% NaCl), sections were incubated with a GLUT9S antibody (1:5000) at 4°C overnight, washed twice in Restriction Enzyme Analysis (Family 1). TBS, and incubated in goat anti-rabbit peroxidase Fab secondary an- Exon 3 of SLC2A9 was amplified using flanking intronic primers and tibody (1:500). After two additional washes, sections were incubated digested with the restriction enzyme AgeI. The digested fragments with Tyramide Cy5 (NEN) for 10 min. Sections had two final washes were detected using gel electrophoresis. in TBS before coverslipping. The sections were visualized under a

70 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 64–72, 2010 www.jasn.org BASIC RESEARCH

Zeiss LSM 510 confocal laser scanning microscope, using a HeNe 633 Salomaa V, Samani NJ, Schlessinger D, Uda M, Volker U, Waeber G, laser and dichroics and a long-pass 650 emission filter. Waterworth D, Wang-Sattler R, Wright AF, Adamski J, Whitfield JB, Gyllensten U, Wilson JF, Rudan I, Pramstaller P, Watkins H, Doering A, Wichmann HE, Spector TD, Peltonen L, Volzke H, Nagaraja R, Vollen- Transport Studies. weider P, Caulfield M, Illig T, Gieger C: Meta-analysis of 28,141 Plasmids were linearized with BglII (pLuc-MS2) or XbaI (pSLC2A9_S/ individuals identifies common variants within five new loci that influ- pSLC2A9_S_L46R and pSLC2A9_L/pSLC2A9_L_L75R), and mRNAs ence uric acid concentrations. PLoS Genet 5: e1000504, 2009 were transcribed, adenylated, and purified as described previously.13 Ten 10. Hagos Y, Stein D, Ugele B, Burckhardt G, Bahn A: Human renal or 40 ng of mRNA was injected, as stated, into defolliculated stage VI organic anion transporter 4 operates as an asymmetric urate trans- 14 porter. J Am Soc Nephrol 18: 430–439, 2007 Xenopus laevis oocytes, and 2 d after injection, [8- C]UA uptake assays 11. Uchino H, Tamai I, Yamashita K, Minemoto Y, Sai Y, Yabuuchi H, were carried out as described previously13 for 60 min using 25 ␮M Miyamoto K, Takeda E, Tsuji A: p-Aminohippuric acid transport at [8-14C]UA (American Radiolabeled Chemicals). Three or four pools of renal apical membrane mediated by human inorganic phosphate five oocytes were collected per experimental point, and statistical analyses transporter NPT1. Biochem Biophys Res Commun 270: 254–259, were done using Microsoft Excel software. 2000 12. Woodward OM, Kottgen A, Coresh J, Boerwinkle E, Guggino WB, Kottgen M: Identification of a urate transporter, ABCG2, with a com- mon functional polymorphism causing gout. Proc Natl Acad Sci U S A ACKNOWLEDGMENTS 106: 10338–10342, 2009 13. Vitart V, Rudan I, Hayward C, Gray NK, Floyd J, Palmer CN, Knott SA, Kolcic I, Polasek O, Graessler J, Wilson JF, Marinaki A, Riches PL, Shu We thank Evgenia Valsamidou for help with the homology modeling, X, Janicijevic B, Smolej-Narancic N, Gorgoni B, Morgan J, Campbell S, Rotem Ron for technical assistance, and Eithan Friedman for provid- Biloglav Z, Barac-Lauc L, Pericic M, Klaric IM, Zgaga L, Skaric-Juric T, ing DNA samples of Israeli-Arabs. Wild SH, Richardson WA, Hohenstein P, Kimber CH, Tenesa A, Don- nelly LA, Fairbanks LD, Aringer M, McKeigue PM, Ralston SH, Morris AD, Rudan P, Hastie ND, Campbell H, Wright AF: SLC2A9 is a newly identified urate transporter influencing serum urate concentration, DISCLOSURES urate excretion and gout. 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