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Title:
SOLUTE CARRIER
Document Type and Number:
WIPO Patent Application WO/2009/063200
Kind Code:
A2
Abstract:
We describe genetic polymorphisms within the gene that encodes the solute transporter SCL2A9 and the association of these polymorphisms with disease conditions associated with hyperuhcaemia. We also described screening assays for the identification of agents that are beneficial in the treatment of hyperuhcaemia.

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Inventors:
WRIGHT, Alan (MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, GB)
CAMPBELL, Harry (Community Health Sciences, University of Edinburgh, Edinburgh EH8 9AG, GB)
HASTIE, Nick (MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, GB)
RUDAN, Igor (Croatian Centre for Global Heath, Faculty of MedecineUniversity of Split, Split, 21000, HR)
RUDAN, Pavao (Institute for Anthropological Research, Zagreb, 10000, HR)
Application Number:
GB2008/003821
Publication Date:
May 22, 2009
Filing Date:
November 14, 2008
Export Citation:
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Assignee:
MEDICAL RESEARCH COUNCIL (20 Park Crescent, London W1B 1AL, GB)
WRIGHT, Alan (MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, GB)
CAMPBELL, Harry (Community Health Sciences, University of Edinburgh, Edinburgh EH8 9AG, GB)
HASTIE, Nick (MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, GB)
RUDAN, Igor (Croatian Centre for Global Heath, Faculty of MedecineUniversity of Split, Split, 21000, HR)
RUDAN, Pavao (Institute for Anthropological Research, Zagreb, 10000, HR)
International Classes:
G01N33/50; G01N33/566; G01N33/68
Other References:
LI SIGUANG ET AL: "The GLUT9 gene is associated with serum uric acid levels in Sardinia and Chianti cohorts." PLOS GENETICS NOV 2007, vol. 3, no. 11, 9 November 2007 (2007-11-09), page e194, XP002519771 ISSN: 1553-7404
AUGUSTIN ROBERT ET AL: "Identification and characterization of human glucose transporter-like protein-9 (GLUT9) - Alternative splicing alters trafficking" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 279, no. 16, 16 April 2004 (2004-04-16), pages 16229-16236, XP002519772 ISSN: 0021-9258
VITART VERONIQUE ET AL: "SLC2A9 is a newly identified urate transporter influencing serum urate concentration, urate excretion and gout" NATURE GENETICS, vol. 40, no. 4, April 2008 (2008-04), pages 437-442, XP002519773 ISSN: 1061-4036
DOERING ANGELA ET AL: "SLC2A9 influences uric acid concentrations with pronounced sex-specific effects" NATURE GENETICS, vol. 40, no. 4, April 2008 (2008-04), pages 430-436, XP002519774 ISSN: 1061-4036
MANOLESCU ANDREI R ET AL: "A highly conserved hydrophobic motif in the exofacial vestibule of fructose transporting SLC2A proteins acts as a critical determinant of their substrate selectivity" MOLECULAR MEMBRANE BIOLOGY, TAYLOR AND FRANCIS, GB, vol. 24, no. 5-6, 1 January 2007 (2007-01-01), pages 455-463, XP009113798 ISSN: 0968-7688
Attorney, Agent or Firm:
GILHOLM HARRISON (Marlborough House, Westminster PlaceYork Business Park,Nether Poppleton, York YO26 6RW, GB)
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Claims:
Claims

1. The use of a polypeptide encoded by a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 4b, or a nucleic acid molecule that hybridizes under stringent hybridization conditions to a sequence comprising Figure 4b and which encodes a polypeptide with uric acid transporting activity for the identification of agents that modulate the uric acid transporting activity of said polypeptide.

2. Use according to claim 1 wherein said polypeptide is encoded by a nucleic acid molecule comprising or consisting of a nucleic acid sequence as represented in Figure

4b.

3. Use according to claim 1 or 2 wherein said nucleic acid molecule encodes a polypeptide as represented in Figure 5a or 5b, or a variant polypeptide that varies from a reference amino acid sequence as represented in Figure 5a or 5b by addition, deletion or substitution of at least one amino acid residue and which has uric acid transporting activity.

4. Use according to any of claims 1-3 wherein said agent is an inhibitor of uric acid transport.

5. A screening method for the identification of an agent that has solute transporter modulating activity comprising the steps of: i) providing a polypeptide encoded by a nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 4b; b) a nucleic acid molecule comprising nucleic acid sequences that hybridise to the sequence identified in (a) above under stringent hybridization conditions and which encodes a polypeptide that has solute transporter activity; ii) providing at least one candidate agent to be tested; iii) forming a preparation that is a combination of (i) and (ii) above; and iv) testing the effect of said agent on the activity of said solute transporter.

6. A method according to claim 5 wherein said polypeptide is represented by the amino acid sequence in Figure 5a or 5b.

7. A method according to claim 5 or 6 wherein said polypeptide is expressed by a cell wherein said cell is transformed or transfected with a nucleic acid molecule that encodes a solute transporter polypeptide.

8. A method according to claim 7 wherein said nucleic acid molecule is part of a vector adapted for recombinant expression of said nucleic acid molecule.

9. A method according to any of claims 5-8 wherein said vector is provided with a promoter which enables the expression of said nucleic acid molecule to be regulated.

10. A method according to any of claims 7-9 wherein said cell is a human cell.

11. A method according to claim 10 wherein said human cell is a kidney cell.

12. A method according to claim 10 wherein said human cell is a chondrocyte.

13. A method according to any of claims 7-9 wherein said cell is a Xenopus oocyte.

14. A modelling method to determine the association of an agent with a solute transporter polypeptide comprising the steps of: i) providing computational means to perform a fitting operation between an agent and a polypeptide defined by the amino acid sequence in Figure 5a or 5b; and ii) analysing the results of said fitting operation to quantify the association between the agent and the solute transporter polypeptide.

15. A method for the rational design of at least one mutation in a solute transporter polypeptide comprising the steps of: i) providing a first polypeptide and analyzing the amino acid sequence; ii) providing a second polypeptide wherein said second polypeptide is a modified sequence variant of said first polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue as represented in Figure 5a or 5b;

iii) testing the effect of said modification on the uric acid transporting activity of said second polypeptide when compared to said first polypeptide.

16. A method to diagnose a human subject having a predisposition to hyperuricaemia or a disease condition associated with hyperuricaemia comprising: i) providing an isolated sample to be tested; ii) sequencing at least part of a nucleic acid molecule comprising the nucleic acid sequence as represented in Figure 4a; iii) comparing the sequence with a control nucleic acid sequence corresponding to the nucleic acid sequence obtained in ii); and iv) determining the difference in nucleic acid sequence in said sample with the nucleic acid sequence of said control.

17. A method according to claim 16 wherein hyperuricaemia is primary hyperuricaemia.

18. A method according to claim 16 wherein hyperuricaemia is secondary hyperuricaemia.

19. A method according to any of claims 16-18 wherein said difference is a modification that results in the addition, deletion or substitution of at least one nucleotide base.

20. A method according to claim 19 wherein said modification is the deletion of at least one nucleotide base.

21. A method according to claim 20 wherein said difference is a single nucleotide polymorphism.

22. A method according to any of claims 16-21 wherein said modification is a modification to an intronic nucleotide sequence.

23. A method according to claim 22 wherein said modification is an intronic single nucleotide polymorphism; preferably said single nucleotide polymorphism is selected from the group consisting of: (1) rs737267 (intron 7), (2) rs1014290 (intron 3), (3) rs6449213 (intron 4), (4) rs13129697 (intron 7), (5) rs733175 (5 1 intergenic), (6) rs13131257 (intron 6), (7) rs4447863 (intron 7) of the SLC2A9 genomic sequence represented in Figure 4a;

24. A method according to any of claims 16-21 wherein said modification is to an exonic nucleotide sequence. 25. A method according to claim 24 wherein said exon modification is a single nucleotide polymorphism selected from the group consisting of: T17A; S22N; G25R (isoform 2 (short) only); T275M; D281H; V282I; T275M; R294H; L350P as represented in the amino acid sequence in Figure 5b.

26. A method according to any of claims 16-21 wherein said nucleic acid molecule encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence varies at amino acid residue leucine 46 of the sequence shown in Figure 5a

27. A method according to any of claims 16-21 wherein said nucleic acid molecule encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence varies at amino acid residue leucine 75 of the sequence shown in Figure 5b. 28. A method according to claim 26 or 27 wherein amino acid residue leucine is substituted with arginine.

29. A method according to any of claims 16-28 wherein said method is a polymerase chain reaction that amplifies at least part of the nucleic acid sequence represented in Figure 4a. 30. A method according to any of claims 16-29 wherein said disease condition is gout.

31. A method according to any of claims 16-29 wherein disease condition is metabolic syndrome.

32. A method according to any of claims 16-29 wherein said disease condition is type Il diabetes.

33. A method according to any of claims 16-29 wherein said disease condition is cardiovascular disease.

34. A method according to claim 33 wherein the cardiovascular disease is hypertension. 35. A method according to claim 34 wherein hypertension is primary hypertension.

36. A method according to any of claims 16-29 wherein said disease condition is urate nephropathy.

37. A method according to any of claims 16-29 wherein said disease condition is cerebrovascular disease. 38. A kit comprising: at least one pair of oligonucleotide primers adapted to anneal and amplify at least part of the nucleic acid sequence presented in Figure 4a.

39. A kit according to claim 38 wherein said kit includes; a plurality of oligonucleotide primers, thermostable DNA polymerase, deoxynucleotide triphosphates and reaction buffer. 40. A kit according to claim 38 or 39 wherein said at least one primer pair is selected from the primer pairs presented in Table 6.

41. A method to diagnose and treat a human subject suffering from or having a predisposition to hyperuricaemia comprising: i) providing an isolated sample to be tested; ii) sequencing at least part of a nucleic acid molecule comprising the nucleic acid sequence as represented in Figure 4a; iii) comparing the sequence with a control nucleic acid sequence corresponding to the nucleic acid sequence obtained in ii); and iv) determining the difference in nucleic acid sequence in said sample with the nucleic acid sequence of said control; and v) determining if the subject will benefit from administration of at least one agent that is effective in the treatment of hyperuricaemia.

42. A method according to claim 41 wherein hyperuricaemia is primary hyperuricaemia.

43. A method according to claim 41 wherein hyperuricaemia is secondary hyperuricaemia.

44. A method according to any of claims 41-43 wherein said agent is a thiazide diuretic.

45. A method according to any of claims 41-43 wherein said agent is a uricosuric agent.

46. A method according to any of claims 41-43 wherein said agent is a glucocorticoid.

47. A method according to any of claims 41-43 wherein said agent is a non-steroidal anti-inflammatory.

48. A method according to any of claims 41-43 wherein said agent is a xanthine oxidase inhibitor.

Description:

Solute Carrier

The invention relates to a screening assay for agents that have beneficial effects in the regulation of hyperuricaemia and a diagnostic test to determine genetic polymorphisms in a solute carrier of the SLC2A family.

Solute carriers (SLC) function to regulate the uptake and efflux of compounds such as sugars, amino acids, nucleotides, inorganic ions and drugs into and out of cells and organelles. Carriers can be divided into active and passive transporters. Passive transporters, also known as facilitated transporters, allow the passage of solutes across membranes down an electrochemical gradient. Active transporters consume energy and create an ion/solute gradient that actively moves compounds across membranes. This latter form of carrier is further characterised as a primary or secondary carrier in accordance with the directness of coupling to ATP hydrolysis. For example, primary ATP-dependent transporters include the ABC transporter family and ion pumps such as ATPases. There are 43 different SLC families that include 318 members (SLC1A1- SLC45A4 see http://www.genecards.org/index.shtml).

Each family is characterised both by the sequence homology between members of a family and by function. For example SLC1 has 7 family members and function as high affinity glutamate and neutral amino acid transporters. SLC25 is the largest family having 27 members and represent a group of mitochondrial transporters. SLC 35 has

17 family members and functions in nucleoside-sugar transport. However it is not possible to derive the function of a transporter merely by sequence analysis and the only confirmed means to determine the function of a carrier is by functional assays.

SLC genes and proteins are known to be associated with pathological conditions. For example SLC10A2 is disclosed in WO02/083944 and describes allelic polymorphisms that respond differentially to cholesterol lowering drugs. W02004/SE/001073 describes expression profiles that include amongst other genes, expression of SLC6A414 and SLC26A2, as a means to distinguish between ulcerative colitis and Crohn's disease. Similarly, expression profiling was undertaken in WO2006/110593 which associates the expression of SLC12A2, SLC27A3 and SLC27A1 in combination with other genes in the diagnosis of cancer, in particular colon, lung, ovarian, prostate, pancreatic and bladder cancers. Furthermore, WO2007/101122 describes the assessment of SLC17A1 expression as a measure of the effectiveness of epidermal growth factor targeted agents

in the treatment of cancer. It is apparent that SLC genes are associated with a number of pathological conditions and represent a group of targets useful in the identification of agents in the treatment of disease.

The SLC2A family (also known as the facilitative glucose carriers or GLUT family) consists of 14 known members (SLC2A1-14), which facilitate the transport of a variety of solutes, including hexose and pentose sugars and myo-inositol. The SLC2A family are part of the major facilitator superfamily (MFS) which is a large family of secondary carrier proteins. The most characteristic structural feature of the MFS is that they are all single polypeptides with 12-transmembrane spanning (TMS) protein topology. The distinctive features of the SLC2A9 family include the characteristic 12 TMS helices with a longer cytoplasmic amino terminal extension (55 amino acids compared with 9-23 amino acids); a large exofacial domain including a potential N-glycosylation site (Asn 90 in humans); 7 conserved glycine residues within the helices; several conserved acidic and basic residues in intracellular regions of the protein; two conserved tryptophan residues; two conserved tyrosine residues 4 . There are three sub-families defined on the basis of sequence similarities and phylogenetic analysis: class I (SLC2A1-4), which are known to be glucose transporters; class Il (SLC2A5, 7, 9, 11), which includes the fructose transporter SLC2A5; and class III (SLC2A6, 8, 10, 12 and the myo-inositol transporter HMIT1) with unknown or varied functions.

Hyperuricaemia is associated with gout 14 15 , in which hyperuricaemia is well established as the major risk factor, leading to deposition of crystals of monosodium urate, particularly within tissues such as joints and kidney, causing tissue damage and inflammatory reactions 14 . Hyperuricaemia is also associated with cardiovascular 16 17 and cerebrovascular diseases 18"20 but cause and effect relationships are less clear 21 .

Dietary purines, endogenous synthesis and re-utilisation of purines are the main sources of urate formation in mammals. In humans and higher primates, uric acid is the main end product of purine catabolism, since these species lack uricase activity, the enzyme that oxidises uric acid to the more soluble allantoin in other mammals 10 . As a result, serum uric acid concentrations in humans (-300 μM) are substantially higher than in most other mammals. The human diet contains very little urate so that serum uric acid reflects the balance between synthesis, which mainly occurs in the liver, and to a lesser extent in small intestine, and excretion by the kidneys, where 70% of daily production is eliminated 11 . More than 96% of serum uric acid is not bound to protein. Uric acid in

extracellular fluids at pH 7.4 exists mainly as urate ion, predominantly as the relatively insoluble mono-sodium urate, because of the high sodium concentration in extracellular fluids. If urate concentrations exceed the solubility of mono-sodium urate (~6.8 mg/dl), there is a risk of crystal formation and deposition in tissues.

Uric acid is freely filtered from the blood by the glomerulus but 90% of the filtered load is reabsorbed by the proximal tubules. Defects in tubular reabsorption by URAT1 cause hypouricaemia while defects in the renal excretion of uric acid are associated with hyperuricaemia. Renal uric acid transport is complex and only partially understood 12 . Four members of the organic ion transporter (OAT) family (OAT1 , 3, 4, URAT1), which is also a member of the major facilitator superfamily have been shown to transport urate 12 . Renal urate transport also seems to involve two sodium-coupled monocarboxylate transporters (SLC5A8, SLC5A12), which may be involved in a multi-molecular complex , called a "urate transportsome" 13 . Transport of monocarboxylate anions from the renal tubular lumen into the proximal tubule cells is thought to be driven by a coupled sodium gradient, which in turn creates an outwards anion gradient to drive inwards entry of urate into the cell 13 .

This disclosure relates to SLC2A9 and homologues thereof which is a uric acid transporter. Two major isoforms are found in human tissues, a full length (FL) form consisting of 540 amino acids and an amino-terminally shortened form (GLUT9δN) form, with 511 amino acids, which are expressed in the basolateral and apical plasma membrane respectively of MDCK cells 5 . The inventors also disclose a correlation between SLC2A9 polymorphisms and a predisposition to develop hyperuricaemia which is a known determining factor in gout and related pathological conditions.

According to an aspect of the invention there is provided the use of a polypeptide encoded by a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 4b, or a nucleic acid molecule that hybridizes under stringent hybridization conditions to a sequence comprising Figure 4b and which encodes a polypeptide with uric acid transporting activity for the identification of agents that modulate the uric acid transporting activity of said polypeptide.

Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic

acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The T m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:

Very High Stringency (allows sequences that share at least 90% identity to hybridize) Hybridization: 5x SSC at 65°C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65°C for 20 minutes each

High Stringency (allows seguences that share at least 80% identity to hybridize) Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: 1 x SSC at 55°C-70°C for 30 minutes each

Low Stringency (allows seguences that share at least 50% identity to hybridize) Hybridization: 6x SSC at RT to 55°C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.

In a preferred embodiment of the invention said polypeptide is encoded by a nucleic acid molecule comprising or consisting of a nucleic acid sequence as represented in Figure 4b.

In a preferred embodiment of the invention said nucleic acid molecule encodes a polypeptide as represented in Figure 5a or 5b, or variant polypeptides that varying from a reference amino acid sequence as represented in Figure 5a or 5b by addition, deletion or substitution of at least one amino acid residue and which has uric acid transporting activity.

In a preferred embodiment of the invention said polypeptide varies at amino acid residue leucine 46 of the amino acid sequence shown in Figure 5a.

In a preferred embodiment of the invention said polypeptide varies at amino acid leucine 75 of the amino acid sequence shown in Figure 5b.

A variant, i.e. a fragment polypeptide and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations which may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like character. The following non-limiting list of amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and asparatic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalaine, tyrosine and tryptophan. Most highly preferred are variants which retain the same biological function and activity as the reference polypeptide from which it varies. Alternatively said amino acid substitutions are non-conservative and alter the function of the polypeptide.

A functionally equivalent polypeptide of Figure 5a or 5b is a variant in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues, or one in which one or more amino acid residues includes a substituent group. Conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, VaI, Leu and lie; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and GIu; substitution between amide residues Asn and GIn; exchange of the basic residues Lys and Arg; and replacements among aromatic residues Phe and Tyr.

In addition, the invention features polypeptide sequences having at least 75% identity with the polypeptide sequences illustrated in Figures 5a or 5b, or fragments and functionally equivalent polypeptides thereof. In one embodiment, the polypeptides have at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, still more preferably at least 97% identity, and most preferably at least 99% identity with the amino acid sequences illustrated in Figure 5a or 5b.

In a preferred embodiment of the invention said agent is an inhibitor of uric acid transport.

According to an aspect of the invention there is provided a screening method for the identification of an agent that has solute transporter modulating activity comprising the steps of: i) providing a polypeptide encoded by a nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 4b; b) a nucleic acid molecule comprising nucleic acid sequences that hybridise to the sequence identified in (a) above under stringent hybridization conditions and which encodes a polypeptide that has solute transporter activity; ii) providing at least one candidate agent to be tested; iii) forming a preparation that is a combination of (i) and (ii) above; and iv) testing the effect of said agent on the activity of said solute transporter.

In a further preferred method of the invention said polypeptide is represented by the amino acid sequence in Figure 5a or 5b.

In a preferred method of the invention said polypeptide is expressed by a cell wherein said cell is transformed or transfected with a nucleic acid molecule that encodes a solute transporter polypeptide. Preferably said nucleic acid molecule is part of a vector adapted for recombinant expression of said nucleic acid molecule. Preferably said vector is provided with a promoter which enables the expression of said nucleic acid molecule to be regulated.

In a preferred method of the invention said cell is a human cell; preferably a kidney cell (e.g. MDCK cells, African Green Monkey epithelial cells, human embryonic kidney cells e.g. HEK 293 cell-line, human kidney cell-line e.g. HT1080) or a chondrocyte.

In an alternative preferred method of the invention said cell is an amphibian cell; for example a Xenopus oocyte.

According to a further aspect of the invention there is provided a modelling method to determine the association of an agent with a solute transporter polypeptide comprising the steps of:

i) providing computational means to perform a fitting operation between an agent and a polypeptide defined by the amino acid sequence in Figure 5a or 5b; and ii) analysing the results of said fitting operation to quantify the association between the agent and the solute transporter polypeptide.

The rational design of binding entities for proteins is known in the art and there are a large number of computer programs that can be utilised in the modelling of 3- dimensional protein structures to determine the binding of chemical entities to functional regions of proteins and also to determine the effects of mutation on protein structure. This may be applied to binding entities and also to the binding sites for such entities. The computational design of proteins and/or protein ligands demands various computational analyses which are necessary to determine whether a molecule is sufficiently similar to the target protein or polypeptide. Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., Waltham, Mass.) version 3.3, and as described in the accompanying User's Guide, Volume 3 pages. 134-135. The Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e. moving structures). When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure.

The person skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with a target. The screening process may begin by visual inspection of the target on the computer screen, generated from a machine-readable storage medium. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within the binding pocket.

Useful programs to aid the person skilled in the art in connecting the individual chemical entities or fragments include: CAVEAT (P. A. Bartlett et al, "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules". In Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc, 78, pp. 182-196 (1989)). CAVEAT is available from the University of California, Berkeley, California. 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, California). This is reviewed in Y. C. Martin, "3D Database Searching in Drug

Design", J. Med. Chem., 35, pp. 2145-2154 (1992); and HOOK (available from Molecular Simulations, Burlington, Mass.). These citations are incorporated by reference.

Once the agent has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. The computational analysis and design of molecules, as well as software and computer systems are described in US Patent No 5,978,740 which is included herein by reference.

According to a further aspect of the invention there is provided a method for the rational design of at least one mutation in a solute transporter polypeptide comprising the steps of: i) providing a first polypeptide and analyzing the amino acid sequence; ii) providing a second polypeptide wherein said second polypeptide is a modified sequence variant of said first polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue as represented in Figure 5a or 5b; iii) testing the effect of said modification on the uric acid transporting activity of said second polypeptide when compared to said first polypeptide.

According to a further aspect of the invention there is provided a method to diagnose a human subject having a predisposition to hyperuricaemia or a disease condition associated with hyperuricaemia comprising: i) providing an isolated sample to be tested; ii) sequencing at least part of a nucleic acid molecule comprising the nucleic acid sequence as represented in Figure 4a; iii) comparing the sequence with a control nucleic acid sequence corresponding to the nucleic acid sequence obtained in ii); and iv) determining the difference in nucleic acid sequence in said sample with the nucleic acid sequence of said control.

In a preferred method of the invention hyperuricaemia is primary hyperuricaemia.

In an alternative preferred method of the invention hyperuricaemia is secondary hyperuricaemia.

In a preferred method of the invention said difference is a modification that results in the addition, deletion or substitution of at least one nucleotide base; preferably said modification is the deletion of at least one nucleotide base.

In a preferred method of the invention said difference is a single nucleotide polymorphism.

Single nucleotide polymorphisms (SNPs) arise due to the substitution, deletion or insertion of a nucleotide residue to create a polymorphic site. Such variations are referred to as SNPs. SNPs may occur in protein coding regions, in which case different polymorphic forms of the sequence may give rise to variant protein sequences. Other SNPs may occur in non-coding regions, for example promoter regions. In either case, SNPs may result in defective proteins or regulation of genes, thus resulting in disease or predisposing the subject to a pathological condition. Other SNPs may have no direct phenotypic effects, but may show linkage to disease states, thus serving as markers for disease. In a preferred method of the invention said modification is a modification to an intronic nucleotide sequence.

In a preferred method of the invention said modification is an intronic single nucleotide polymorphism; preferably said single nucleotide polymorphism is selected from the group consisting of: (1) rs737267 (intron 7), (2) rs1014290 (intron 3), (3) rs6449213 (intron 4), (4) rs13129697 (intron 7), (5) rs733175 (5' intergenic), (6) rs13131257 (intron 6), (7) rs4447863 (intron 7) of the SLC2A9 genomic sequence represented in Figure 4a;

In an alternative preferred method of the invention said modification is to an exonic nucleotide sequence.

In a preferred method of the invention said exon modification is single nucleotide polymorphism selected from the group consisting of: T17A; S22N; G25R (isoform 2 (short) only); T275M; D281 H; V282I; T275M; R294H; L350P, as represented in the amino acid sequence in Figure 5b (numbering for isoform 1 unless indicated otherwise).

In a preferred embodiment of the invention said method is a polymerase chain reaction that amplifies at least part of the nucleic acid sequence represented in Figure 4a.

In an alternative preferred method of the invention said nucleic acid molecule encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence varies at amino acid residue leucine 46 of the sequence shown in Figure 5a. Preferably, amino acid residue leucine is substituted with arginine.

In an alternative preferred method of the invention said nucleic acid molecule encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence varies at amino acid residue leucine 75 of the sequence shown in Figure 5b. Preferably, amino acid residue leucine is substituted with arginine.

In a preferred method of the invention said disease condition is gout.

In an alternative preferred method of the invention said disease condition is metabolic syndrome.

In a further alternative preferred method of the invention said disease condition is type Il diabetes.

In a further preferred method of the invention said disease condition is cardiovascular disease; for example hypertension. Preferably hypertension is primary hypertension.

In a preferred method of the invention said disease condition is cerebrovascular disease. In a preferred method of the invention said disease condition is urate nephropathy. According to a further aspect of the invention there is provided a kit comprising: at least one pair of oligonucleotide primers adapted to anneal and amplify at least part of the nucleic acid sequence presented in Figure 4a.

In a preferred embodiment of the invention said kit includes; a plurality of oligonucleotide primers, thermostable DNA polymerase, deoxynucleotide triphosphates and buffer. In a preferred embodiment of the invention said at least one primer pair is selected from the primer pairs presented in Table 6.

According to a further aspect of the invention there is provided a method to diagnose and treat a human subject suffering from or having a predisposition to hyperuricaemia comprising: i) providing an isolated sample to be tested;

ii) sequencing at least part of a nucleic acid molecule comprising the nucleic acid sequence as represented in Figure 4a; iii) comparing the sequence with a control nucleic acid sequence corresponding to the nucleic acid sequence obtained in ii); and iv) determining the difference in nucleic acid sequence in said sample with the nucleic acid sequence of said control; and v) determining if the subject will benefit from administration of at least one agent that is effective in the treatment of hyperuricaemia or a disease condition associated with hyperuricaemia.

In a preferred method of the invention hyperuricaemia is primary hyperuricaemia.

In an alternative preferred method of the invention hyperuricaemia is secondary hyperuricaemia.

In a preferred method of the invention said agent is a thiazide diuretic, for example thiazides derived from benzothiadiazine, e.g. chlorothiazide, hydrochlorothiazide or bendroflumethiazide.

In a preferred method of the invention said agent is a uricosuric agent, for example probenecid.

In an alternative preferred method of the invention said agent is a glucocorticoid, for example prednisone.

In a further alternative preferred method of the invention said agent is a non-steroidal anti-inflammatory.

In a yet further preferred method of the invention said agent is a xanthine oxidase inhibitor, for example allopurinol.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following tables and figures:

Table 1 Association of the most significant SLC2A9 SNPs from the genome-wide scan with SUA in Croatian and Orcadian samples accounting for a polygenic effect, stratification (in Croatia), sex, age-by-sex and BMI. The significance of the fixed effects was tested using WaId statistics. The fitted SNP effect was additive. Individuals on diuretics and uricosuric drugs were excluded. SUA in Croatians was 151-519μM (range, less 5% extremes), similar to values in other European populations, while in Orkney, SUA concentrations were similar, 153-480 μM (range, less 5% extremes). Statistically significant results are shown in bold;

Table 2 Association of the most significant SLC2A9 SNPs with low fractional excretion of uric acid (FEUA) and/or gout in German and Scottish cohorts, (i) Results from a German cohort with low FEUA (<6.6%) (N=349) compared with normal controls (FEUA >7.4%) (N=255) matched for ethnicity from an urban German population, (ii) Results from a Scottish gout cohort in which 484 cases and 9659 controls were recruited from central Scotland. The odds ratios, 95% confidence intervals and P-values are shown. The results were obtained by logistic regression analyses, with age, gender ,and BMI as covariates. A sex- by-SNP interaction was not significant in these analyses. Odds ratios correspond to the effect of the major allele under an additive SNP model. Statistically significant results are shown in bold;

Supplemental Table 1 : Association of SLC2A9 SNPs and gout in German and Croatian samples. 57 unrelated gout cases (37 males, 20 females) and 440 controls (157 males, 283 females) were analysed in the Croatian dataset and 148 cases (128 males, 20 females) and 231 controls (79 males, 152 females) in the German dataset. The results were obtained by logistic regression analyses using an additive SNP model, with gender (for the combined gender data sets), age (Croatia) and BMI as covariates. Gender-by- SNP interactions were not statistically significant. Results are shown for analyses of both genders combined and separated. SNPs that show statistically significant association (P<0.05) are shown in bold;

Supplemental Table 2: Association of SLC2A9 variants with metabolic syndrome. In the Croatian dataset, effect sizes, P-values and odds ratios for association with metabolic syndrome are shown.. The Odds Ratio (heterozygote) is the odds ratio for the heterozygote against the low risk homozygote. The Odds Ratio (homozygote) is the odds ratio for the high risk homozygote against the low risk homozygote. An additive model was used for both sexes with age as a covariate. Statistically significant results (P<0.05) are shown in bold;

Supplemental Table 3: Association of SLC2A9 SNPs with fasting plasma glucose in the Croatian and Orkney datasets. The results were analysed using a linear mixed model with age, sex, age by sex (Orkney), and population structure (Croatia) as covariates and performed with or without BMI as additional covariate. P-values for the effect of SLC2A9

SNPs on plasma glucose are shown. Individuals on glucose lowering medication (insulin or oral hypoglycaemics) were removed and phenotypes ranked and quantile normalised Statistically significant results (P≤0.05) are shown in bold;

Supplemental Table 4: Association of SLC2A9 SNPs with metabolic syndrome in the Scottish Tayside cohort, including 1678 controls and 417 cases. The data were analysed by logistic regression with an additive model and age as a covariate;

Supplemental Table 5: Association of SLC2A9 SNPs with type 2 diabetics (age 40-60 yrs) from the Tayside cohort. The sample contains 1 ,388 cases and 2,495 controls and was analysed by logistic regression using an additive model for both sexes, with age and BMI as covariates. SNPs that show statistically significant association (P<0.05) are shown in bold;

Supplemental Table 6: Oligonucleotide primers used for polymerase chain reaction amplification and sequencing of SLC2A9.

Figure 1 illustrates the association of SLC2A9 SNPs with SUA (-Iog10 P-values) following a genome wide association scan of 986 Croatians using 317,503 SNPs. The location of associated and flanking SNPs are shown across a 700 kb region on human chromosome 4p16.1 (9,260-9,960 kb) containing the SLC2A9 gene. The genomic location of exons in the long and short SLC2A9 isoforms are shown. SLC2A9 is expressed from right to left as shown in the figure. The nearest flanking genes are DRD5 (dopamine receptor D5) and WDR1 (WD repeat-containing protein 1), both of which show weak associations with SUA. The stringent Bonferroni corrected significance threshold and a "suggestive significance" threshold are represented by horizontal lines. Below are shown the linkage disequilibrium patterns of the Hapmap dataset (Genome build 35) within 240kb centred on the top SNP, using Haploview (http://www.broad.mit.edu/mpg/haploview; standard setting, The subset of 32 SNPs from this region used in the genome scan are marked by vertical lines. The most significantly associated SNPs are numbered as follows: (1) rs737267 (intron 7), (2) rs1014290 (intron 3), (3) rs6449213 (intron 4), (4) rs13129697 (intron 7), (5) rs733175 (5' intergenic), (6) rs13131257 (intron 6), (7) rs4447863 (intron 7);

Figure 2 illustrates meta-analysis of data from gout cases and controls from Croatian, German and UK populations, using the Mantel-Haenszel method to estimate pooled allelic effects under a fixed effect model. Each SLC2A9 SNP showed highly significant associations with gout;

Figure 3 illustrates SLC2A9 (short isoform) displays saturable 14C-uhc acid transport activity in Xenopus laevis oocytes. A, transport of 14C-uric acid by oocytes expressing SLC2A9_S (isoform 2) compared with negative control oocytes, injected with an unrelated mRNA, and positive control oocytes, injected with URAT1 mRNA. B, 14C- urate transport activity (V, pmol 14C-uric acid oocyte-1 min-1) shows saturation kinetics with increasing urate concentrations (S, μM). Inset is an Eadie-Hofstee plot of activity (V) against V/S. The Km is 890μM and Vmax is 5.33 pmol oocyte-1 min-1 , indicating a high capacity low affinity transporter;

Figure 4a is the genomic DNA sequence of SLC2A9; Figure 4b is the cDNA sequence of SLC2A9; and

Figure 5a is the amino acid sequence of short form SLC2A9; Figure 5b is the amino acid sequence of long form SLC2A9.

Materials and Methods Study samples

All studies received appropriate ethical approval and participants gave informed consent. Unselected Croatians (N=986), aged 18-93 years, were recruited into the study from the villages of Vis and Komiza on the Dalmatian island of Vis and were phenotyped for >50 disease-related quantitative traits 5 ' 26 . Orcadian individuals (N=706), aged 16-100 years, were recruited as part of a similar population-based study in the Northern islands of Orkney, UK. The German dataset included 148 cases of gout (128 men, 20 women), individuals with low FEUA (N=349) or normal FEUA (N=255), identified by the Department of Internal Medicine, Carl Gustav Carus Medical School, Dresden, Germany. The low FEUA group had mean SUA of 414.5±79.5μM (±SD) and the normal FEUA group had mean SUA of 290.5±76.7μM. The Scottish Tayside Go-DARTS study consists of 4,789 people with type 2 diabetes and 5,295 non-diabetic controls 27 . All cases were of white European origin with physician-diagnosed type 2 diabetes recruited at primary or secondary care diabetes clinics, or invited to participate from primary care registers from throughout the Tayside region of Scotland. The controls were invited to participate through primary care physicians or through their workplace occupational health departments. None of the controls had a previous diagnosis of diabetes. Individuals were linked to the DARTS/MEMO (Medicines Monitoring Unit), which includes validated prescribing, biochemistry and phenotypic historical data from 1992 to the present 28 . All encashed prescriptions for allopurinol in the population of Tayside (N=387,000) were available between January 1992 and December 2006, and 425 patients who encashed at least two allopurinol prescriptions were identified, which was taken to imply a diagnosis of primary or secondary gout. Allopurinol inhibits xanthine oxidase, the final enzyme in uric acid synthesis, and is widely used to treat gout. An additional group of 59 white European-origin patients from central Scotland had a physician-based diagnosis of gout using American College of Rheumatology criteria 29 , of whom 85% were also taking allopurinol. Controls were 9,659 individuals from Go- DARTS who had not encashed allopurinol prescriptions.

For the metabolic syndrome analysis, a subset of 2,095 non-diabetic control individuals from the Go-DARTS study with complete data on the components of the metabolic

syndrome were used to identify 425 people with metabolic syndrome at recruitment (>3 individual components from the NCEP ATP III definition of metabolic syndrome 14 ).

Genotyping A genome-wide association scan using 317,503 SNPs (Human Hap300, lllumina, San Diego, USA) was initially carried out in 986 Croatian individuals and scored using the Bead studio software v.3 (lllumina). The genotypes in the follow-up studies were generated using Taqman probes (Applied Biosystems, Foster City). 8,915 SNPs had less than 90% genotyping call rates and 393 had a minor allele frequency under 1%, so were removed, leaving 308,195 SNPs in the analyses.

Statistical analysis

Genome-wide association

Summary statistics and population stratification were tested using the genome-wide association tool set PLINK 30 . Seventeen individuals were removed due to low call rates (<90%) and 3 individuals due to non-European ethnicity, leaving 966 individuals (of whom 58% were members of 122 families). The clustering of the Croatian dataset into 2 sub-groups (roughly corresponding to the two villages) was accounted for in the association analysis. Individuals on diuretics and uricosuric drugs were excluded in the SUA analysis, leaving 794 individuals in this analysis.

To take into account the relatedness of many participants, the GRAMMAR (Genome- wide Rapid Association using Mixed Model and Regression) 6 method was used. This involves three steps: first, the residuals were obtained from a mixed linear model, which adjusted the quantitative trait for a random polygenic effect, covariates and fixed effects, using ASReml 31 . SUA was square-root transformed to reduce kurtosis of the residual distribution. Sex, age nested within sex, body mass index (BMI) and population stratification were all significant effects. The residuals were then analysed for association with each SNP using a least squares regression under an additive model for the SNP effect (genotypes coded 0, 1 or 2 corresponding to the number of minor alleles). Data for candidate associated SNPs were then analysed using a full linear mixed model, i.e. including the candidate SNP as an additional fixed effect and estimating SNP effects using ASReml software 31 . The most appropriate genetic model was investigated including SNP-by-sex interactions and allowing for non-additivity of alleles. For SUA, a SNP-by-sex interaction was uncovered (P<0.05). Finally, imputation of SNPs using HapMap data within a 1Mb area surrounding the top candidate SNP rs737267 did not

reveal better candidate causal SNPs.

Replication and analysis of related traits

For continuous outcomes, general linear models were fitted as in the final step of the genome-wide association analysis. In the case of binary traits, with the exception of metabolic syndrome in the Croatian dataset, analyses were performed by standard logistic regression using SPSS software (v.12) (Woking, Surrey, UK).

In the case of the Croatian dataset, in order to use data for all individuals and hence retain maximum power for the analyses, the data on binary ATP III metabolic syndrome phenotypes were analysed assuming an underlying quantitative trait "liability" 32 . This facilitated fitting of a family random effect, modelled through a relationship (A) matrix. Stratification, age and sex were fitted as fixed effects, and a sex-by-SNP interaction was also tested. Analyses were carried out on approximately 923 individuals (varying with SNP due to missing data), of which 559 were unaffected, and 364 were affected. There were 387 men and 536 women. The sex-by-SNP interaction for metabolic syndrome was not significant and thus not retained. The meta-analysis of gout data was performed using the metabin option from the meta package of the R software. Since we did not find significant heterogeneity (I 2 = 0%, P Het > 0.74 for the three SNPs) between studies, we used the Mantel-Haenszel method to estimate pooled allelic effects under a fixed effect model and used the odds ratio as the summary measure.

Re-sequencing of SLC2A9

SLC2A9 exons and their exon-intron boundaries were amplified and sequenced using the primers shown in Supplementary Table 6. After initial denaturation at 96°C for 6 min, DNA was amplified with 35 cycles of 94°C for 30 sec, 55 0 C for 30 sec and 72°C for 1 min, and a final extension at 72°C for 10 min. All PCR products were sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit's and run out on a 3730 genetic analyser (Applied Biosystems) before checking for variants with Mutation Surveyor (www.softgenetics.com) and/or Sequencher v4.7 (www.sequencher.com) software.

14 C-uric acid transport in Xenopus oocytes

Plasmid pLuc-MS2 has been previously described 33 . SLC2A9_S and URAT3 clones were created using Gateway compatible entry clones T3097 (SLC2A9_S or GLUT9δN; accession number BC018897) and T4563 (URAT1 or SLC22A12; accession number AB071863) provided by GeneCopoeia (Germantown, MD, USA). These were transferred into a Gateway compatible version of pcDNA3.1 (Invitrogen) using standard LR clonase reactions. Plasmids were linearised with BgI Il (Luc-MS2) or Xba I (pURATI and pSLC2A9) prior to transcription, which was performed as previously described 33 except that an adenylation step was included (PoIy(A) tailing kit, Ambion) before purification. 25- 50 ng of mRNA was injected into the cytoplasm at the midline of defolliculated stage Vl Xenopus laevis oocytes treated with collagenase (2.5mg/ml). Two days after injection, uptake assays were performed at room temperature with either 25μM or 50μM 14 C-uric acid and varying amounts (25-1525 μM) of cold uric acid in ND-96 buffer 34 for 60 minutes. Following four washes in ND-96 containing 100 μM cold urate, pools of five oocytes were collected, lysed in 200 μl 10% SDS and subject to scintillation counting. At least three pools of five oocytes were collected per experimental point. Statistical analyses including linear regressions were performed using Microsoft Excel software (Microsoft, UK).

Examples

A genome-wide association scan using 317,503 single nucleotide polymorphisms (SNPs) was carried out in 986 individuals sampled as part of a larger study of >50 disease-related quantitative traits (QTs) in a Croatian isolate population 5 . The analysis was performed using a pedigree-based genome-wide quantitative trait locus association method 6 . Serum uric acid (SUA) was found to be strongly associated with seven SNPs, one located just 5' to SLC2A9 (solute carrier family 2, member 9) in chromosomal region 4p16.1 , the others within introns 3-7 of this gene , three of which (rs737267, rs13129697, rs6449213) reached genome-wide significance after Bonferroni correction (P≤1.6*10 7 ) (Figure 1). In the follow-up analysis, a strong SNP allele-by-sex interaction was found for SUA, so that, in women, the effects were large, additive and highly significant, while in men, they were smaller (Tablei ). The rs737267 polymorphism explained 5.3% of the total unadjusted variance in SUA in women and 1.7% of the variance in men.

The result was replicated in a UK population sample from Orkney (N=706). In both populations, SLC2A9 SNPs showed the same allelic associations with SUA 1 the same sex-specificity, magnitude and direction of effects (Table 1).

Diminished fractional excretion of uric acid (FEUA) is the major risk factor for hyperuricaemia, and is present in >90% of subjects with gout 7 . Data from a sample of 349 German subjects with low FEUA (<6.6%) and 255 controls matched for ethnicity with normal FEUA (>7.4%) were analysed by logistic regression for association with SLC2A9 SNPs (Table 2). When both sexes were combined, low FEUA was significantly associated with intragenic SLC2A9 SNPs, with odds ratios of 1.53-1.67 .(Table 2).

We then sought associations between SLC2A9 variants and gout. No interaction between SNP and sex was statistically significant in these analyses. The German FEUA cohort included 148 cases of gout (128 men, 20 women), reflecting the known male excess in gout 2 and 231 controls (79 men, 152 women). The Croatian sample included 57 unrelated people with gout (37 men, 20 women) . Both small samples showed borderline significant associations with SLC2A9 SNPs when sexes were analysed separately (Supplementary Table 1).

The association of SLC2A9 SNPs with gout was investigated further in a Scottish sample of 484 individuals with gout, ascertained either from allopurinol prescription data or by their attendance at rheumatology outpatient clinics, compared with 9,659 controls who had never received allopurinol. The data were analysed by logistic regression and showed highly significant associations between gout and SLC2A9 SNPs (P-values 0.0001-0.003) with odds ratios of 1.32-1.40 (Table 2).

A meta-analysis was performed using data from gout cases and controls from Croatian, German and UK populations, using the Mantel-Haenszel method to estimate pooled allelic effects under an additive fixed effect model with both sexes combined. Each SLC2A9 SNP showed highly significant associations with gout (rs1014290 T allele, odds ratio (OR)=1.38, P=1.57*10 '5 ; rs6449213 T allele, OR=1.34, P=3.77 χ 10 "4 ; rs737267, C allele, OR=1.34, P=6.9*10 "5 ) (Figure 2).

Hyperuricaemia is also associated with metabolic syndrome 8"10 and cardiovascular disease 11 12 , although cause and effect relationships are unclear 11 . Metabolic syndrome is a state of insulin resistance associated with elevated blood pressure, plasma glucose

and triglyceride, decreased high density lipoprotein cholesterol and abdominal obesity 13 . In order to clarify the relationship between SUA and metabolic syndrome, we examined SLC2A9 variants in people with and without metabolic syndrome from Croatian and two UK population samples. The NCEP Adult Treatment Panel III (ATP III) definition of metabolic syndrome was used 14 . The analysis was initially performed on 923 Croatians (387 men, 536 women), of whom 364 had metabolic syndrome and 559 did not. Six SLC2A9 SNPs showed significant associations with metabolic syndrome (P=O.006- 0.047) (Supplementary Table 2). The SNP-by-sex effect was non-significant in this trait. Individual components of the metabolic syndrome were evaluated separately for association but only plasma glucose was significantly associated with SLC2A9 SNPs, and only in the Croatian dataset (P=0.036-0.005; Supplementary Table 3). The same SNP alleles and direction of effect as with SUA were associated with plasma glucose and metabolic syndrome in the Croatian dataset.

Two UK cohorts with metabolic syndrome were also analysed by logistic regression for association with SLC2A9 SNPs. The first was a Scottish (Tayside) population sample of 417 people with metabolic syndrome and 1 ,678 without metabolic syndrome. The second was a population-based cohort of 166 people with metabolic syndrome and 540 people without metabolic syndrome from the Scottish islands of Orkney. The results showed no evidence of association with metabolic syndrome or plasma glucose in either cohort (Supplementary Tables 2-4). Similarly, only a single SNP was associated (P=0.025) with type 2 diabetes (aged 40-60 yrs) in the Tayside cohort (1 ,388 people with diabetes and 2,495 without diabetes), which was non-significant after correction for multiple testing (Supplementary Table 5). The reported association of SUA and metabolic syndrome 8 ' 9 was clearly evident in the Tayside cohort, since 78% of gout cases had metabolic syndrome (P=8.9*10 '33 ; OR=3.8). The issue of cause and effect relationships between SUA and metabolic syndrome remains unresolved and probably requires much larger cohorts.

The SLC2A9 gene contains 1 non-coding and 13 coding exons and spans 214 kb. It encodes two major transcripts, a long isoform with 540 amino acids and a short (S) isoform (SLC2A9_S) with 511 amino acids. SLC2A9 is a member of the SLC2 (GLUT2) family of facilitated hexose and polyol transporters. Ten coding SNPs have been reported, five of which give rise to non-synonymous amino acid substitutions (T17A, G25R, V282I, R294H, L350P). The latter SNPs showed less significant associations with SUA than intronic SNPs, so that causal variant(s) remain to be identified. A

recombination hot spot (10cM/Mb) is located in intron 8 in the HapMap dataset, consistent with the low, background levels of association to SUA shown by more distal SNPs (Figure 1). In order to identify additional candidate SNPs, exons 1-8 and adjacent introns were re-sequenced either in the entire Croatian dataset (exon 8) or in 95 Croatian samples from each extreme of the SUA distribution (exons 1-7). This identified four rare polymorphisms (S22N, T275M, D281 H, R300H) and two rare variants, one of which (77delC, allele frequency 0.2%) is predicted to result in a reading frameshift which truncates the protein after 38 amino acids, consistent with a null allele. This allele was associated with low SUA (187-216μM) in three female heterozygotes and with average SUA (348μM) in a male, but interpretation was complicated by drugs effects. The other rare variant (T125R, allele frequency 0.1 %) was also associated with low SUA (133μM; Z-score -1.7) and occurs at a site that is conserved in mammals. Loss-of-function alleles may therefore reduce SUA levels, similar to the uric acid transporter URAT1 15 , although the effect may be sex limited and remains to be confirmed experimentally.

The entire SLC2A9 coding and exon junctions were sequenced in 48 unrelated UK individuals with early onset or familial hyperuricaemia of unknown cause. The results showed one individual with a heterozygous non-conservative G216R substitution within a residue that is conserved in mammals and birds and is absent from 250 UK controls. No clear association with familial hyperuricaemia was evident.

SLC2A9 contains the characteristic 12 transmembrane helices, sugar transporter motifs and other signatures of facilitated sugar transporters, although it lacks the features of a high affinity glucose transporter 16 , suggesting alternative substrates. SLC2A9 shows greatest sequence similarity to the fructose transporter SLC2A5, a class Il transporter 17 and has recently been confirmed as both a fructose transporter (K n , 0.42 mM) and a low affinity glucose transporter (K n , 0.61 mM) 4 . The long isoform of SLC2A9 is expressed in basolateral membranes of proximal renal tubular cells, liver and placenta, while the short isoform is expressed only in the apical membranes of proximal renal tubular cells and placenta 18 ' 19 . SLC2A9 is also expressed in chondrocytes from human articular cartilage, a major site of uric acid deposition in gout 20 .

We examined the function of SLC2A9 by expression in Xenopus laevis oocytes and assayed transport activity with 14 C-uric acid as substrate. The results showed 31 -fold higher urate uptake by SLC2A9 than control oocytes and 7-fold higher activity than

URAT1 -injected oocytes, at a urate concentration of 150μM (Figure 3). Urate uptake

was saturable and partially inhibited by the uricosuric agent benzbromarone (50μM; 41% inhibition). Kinetic analysis showed it to be a high capacity low affinity transporter with V max of 5.33 pmol oocyte "1 min "1 and K n , of 890μM (Figure 3). The organic anion transporters URAT1 and OAT4 are the only other apically expressed urate transporters potentially involved in urate reabsorption from glomerular filtrate by proximal tubules 15 ' 21 ' 22 . These proteins are either low capacity intermediate affinity transporters (URAT1 , V max 0.4 pmol oocyte '1 min "1 , K n , 371 μM) 15 or low capacity low affinity transporters (OAT4, <50% of URAT1 activity, K n , 925μM) 21 . URAT1 is proposed to account for 50% of urate reabsorption by proximal tubules 1523 so SLC2A9 may account for much of the remainder.

It is interesting that SLC2A9 appears to be both a fructose and urate transporter. Animal data suggest a causal relationship between fructose intake, SUA and metabolic syndrome 10 . Fructose is the only sugar known to increase SUA 24 and increased dietary intake over the past 30 years has been implicated in the rising SUA levels 10 and increased global prevalence of metabolic syndrome 8 ' 9 . The failure to confirm an association between SLC2A9 variants and metabolic syndrome may result from lack of power so leaves this important question open. The rapid uptake and metabolism of fructose to lactate 25 in SLC2A9-expressing cells such as proximal renal tubules and liver may trans-stimulate urate transport and promote hyperuricaemia. Further investigation of SLC2A9 should help to clarify the complex relationship between genotype and phenotype in gout, hyperuricaemia and metabolic syndrome, which affects over 25% of adults in westernised countries. The identification of SLC2A9 as a novel urate transporter may also lead to new drugs for lowering uric acid levels in a broad range of conditions associated with hyperuricaemia.

Hypertension

Hypertension affects about 25% of industrialised populations in contrast to aboriginal populations and other mammals, in whom it is uncommon. Raised serum urate has long been known to be associated with hypertension but cause and effect relationships are unclear. About one-half of untreated primary hypertensives have hyperuricaemia.

However there is growing evidence that raised urate has a causal effect in early primary hypertension and that lowering of urate levels in this same group is beneficial. This is supported by animal and human data. Animal experiments in which serum urate levels are increased by a uricase inhibitor lead to hypertension which is reversed by lowering of urate.

In clinical studies, hyperuricaemia is a feature of primary but not secondary hypertension, indicating possible specificity. All except one out of 16 clinical studies found that hyperuricaemia predicts the development of hypertension over the next 5-10 years, independent of other risk factors. The only exception was a study of late-onset hypertensives (>60 yrs).

A recent double-blind placebo controlled crossover trail in humans using the xanthine oxidase inhibitor allopurinol resulted in 86% of patients in whom serum urate was successfully lowered achieving normal blood pressure, compared with 3% of controls. Large scale trials are now recommended. The use of allopurinol was not recommended for widespread use due to its toxicity (fatal hypersensitivity reactions can occur) but there are limitation to alternative uricosuric drugs, possibly due to their failure to achieve low intracellular levels. The proposed mechanisms of urate-induced hypertension involves effects on renal afferent arterioles (endothelium and smooth muscle cells) leading to activation of the renin-angiotensin system and eventually a salt-sensitive hypertension. At later stages, structural changes in the kidney maintain the hypertension and the sensitivity to urate lowering drugs may decline, although this remains to be investigated.

Our study sought an association between hypertension and serum urate but failed to identify one in over 5,000 cases compared with controls, as did the recent study of Caulfield et a/., including four population cohorts (>11 ,000 people) and 6 case-controls series (5,249 cases). While this argues against a causal role for urate in hypertension, the relationships with age-of-onset, stratification by urate level and potential dietary factors such as fructose remain to be studied.

Elevated serum urate predicts the development of hypertension but the relationship is complex. There is increasing clinical and animal data suggesting a causal role for urate in the early stages of hypertension before structural renal changes become irreversible and in those with abnormally high serum urate levels (>390 μM in females, >420 μM in males).

Urate nephropathy

There is increasing evidence that raised serum urate can damage the kidneys independent of crystal deposition although it is unclear to what extent this is independent of associated disorders such as hypertension. Essentially all untreated gout patients

show characteristic renal lesion, including glomerulosclerosis, interstitial fibrosis and arteriosclerosis. However, many of these changes are similar to those found in hypertensive nephropathy. Similar renal lesions are found in hyperuricosaemic rats, together with activation of the renin-angiotensin system. However, three studies have showed that lowering of serum uric acid appears to slow progression of renal disease.

Renal hypouricaemia and exercise-induced acute renal failure

Autosomal recessive mutations in the URAT1 gene, encoding a known proximal renal tubular urate transporter, are not uncommon in the Japanese population (0.1-0.7%), where a founder mutation has a frequency of 2%. Some affected individuals show nephrolithiasis (urate stones) and about 10% develop exercise-induced acute renal failure. Average serum urate levels were 55 μM (normal range ~240-350 μM) and fractional excretion of uric acid was reduced. The mechanism is unclear but two have been suggested. First, a direct urate nephropathy may result from increased serum urate after strenuous exercise (urate crystals are rarely seen in affected kidneys). Second, low urate reduces the antioxidant capacity of blood and causes renal vasoconstriction due to exercise-induced reactive oxygen species formation.

Recently, we have shown that two families containing individuals with homozygous mutations in SLC2A9 have very low or unmeasurable serum urate concentrations, some of whom have developed severe renal disease (E. Holzman et a/., unpublished). In one consanguineous Arab family, a homozygous Leu75Arg mutation in SLC2A9 was found to be associated with very low serum uric acid concentrations, substantially raised fractional excretion of uric acid and acute renal failure in an affected individual. We then showed that the Leu75Arg mutation allele only retains 18±5% of wildtype urate transport activity in Xenopus oocyte assays (unpublished data). Molecular modelling suggested that the Leu75 residue is on helix 2 of SLC2A9, pointing into the centre of the vestibule in a hydrophobic pocket. The introduction of a strong positive charge in this position would be expected to compromise urate transport. These findings are consistent with a model in which SLC2A9 has a role in urate reabsorption in the proximal renal tubules, influencing both apical (lumen to tubular cell) and basolateral (tubular cell to interstitium and blood) transport. The data suggest a model in which raised uric acid can be toxic in tissues such as the kidney, in this case due to apical transport of urate into tubular cells (via URAT1 and residual SLC2A9) but failure to transport it further, either into the urine or the blood, leading to raised intracellular urate concentrations and renal failure.

Renal disease may result from direct effects of raised urate anion, causing arteriolar damage and interstitial fibrosis, but this may be compounded by renal microvascular disease due to hypertension. The discovery of families with mutations in SLC2A9 associated with hypouricaemia, hyperuricuria and renal failure suggests failure of urate transport out of tubular cells and direct toxicity. Identification of an inhibitor specifically targeting the apical but not the basal tubular transporter would therefore be desirable.

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