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PLoS One
2014 Jan 01;99:e107902. doi: 10.1371/journal.pone.0107902.
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Complex analysis of urate transporters SLC2A9, SLC22A12 and functional characterization of non-synonymous allelic variants of GLUT9 in the Czech population: no evidence of effect on hyperuricemia and gout.
Hurba O
,
Mancikova A
,
Krylov V
,
Pavlikova M
,
Pavelka K
,
Stibůrková B
.
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OBJECTIVE: Using European descent Czech populations, we performed a study of SLC2A9 and SLC22A12 genes previously identified as being associated with serum uric acid concentrations and gout. This is the first study of the impact of non-synonymous allelic variants on the function of GLUT9 except for patients suffering from renal hypouricemia type 2.
METHODS: The cohort consisted of 250 individuals (150 controls, 54 nonspecific hyperuricemics and 46 primary gout and/or hyperuricemia subjects). We analyzed 13 exons of SLC2A9 (GLUT9 variant 1 and GLUT9 variant 2) and 10 exons of SLC22A12 by PCR amplification and sequenced directly. Allelic variants were prepared and their urate uptake and subcellular localization were studied by Xenopus oocytes expression system. The functional studies were analyzed using the non-parametric Wilcoxon and Kruskall-Wallis tests; the association study used the Fisher exact test and linear regression approach.
RESULTS: We identified a total of 52 sequence variants (12 unpublished). Eight non-synonymous allelic variants were found only in SLC2A9: rs6820230, rs2276961, rs144196049, rs112404957, rs73225891, rs16890979, rs3733591 and rs2280205. None of these variants showed any significant difference in the expression of GLUT9 and in urate transport. In the association study, eight variants showed a possible association with hyperuricemia. However, seven of these were in introns and the one exon located variant, rs7932775, did not show a statistically significant association with serum uric acid concentration.
CONCLUSION: Our results did not confirm any effect of SLC22A12 and SLC2A9 variants on serum uric acid concentration. Our complex approach using association analysis together with functional and immunohistochemical characterization of non-synonymous allelic variants did not show any influence on expression, subcellular localization and urate uptake of GLUT9.
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25268603
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Figure 2. Immunocytochemical analysis of Xenopus oocytes.Immunocytochemical analysis of Xenopus oocytes injected with 50 ng of cRNA encoding the wild-type (wt) or mutant GLUT9 performed with rabbit anti-SLC2A9 polyclonal antibody. The signal of protein is green while autofluorescent granules in cytoplasm of oocytes give a blue signal. (a) Non-injected oocytes, (b) oocytes injected with wt cRNA GLUT9 variant 1, (c) oocytes injected with wt cRNA GLUT9 variant 2, (d) GLUT9 variant 2 A17T, (e) GLUT9 variant 1 G25R, (f) GLUT9 variant 1 V169M, (g) GLUT9 variant 2 V140M, (h) GLUT9 variant 2 T246M, (i) GLUT9 variant 2 D252H, (j) GLUT9 variant 1 V282I, (k) GLUT9 variant 2 V253I, (l) GLUT9 variant 1 R294H, (m) GLUT9 variant 2 R265H, (n) GLUT9 variant 1 P350L, (o) GLUT9 variant 2 P321L. Scale bar represents 50 µm.
Figure 3. Plot of -log10(p-value) for the analysis of association of studied allele variants with hyperuricemia/primary gout and serum uric acid concentration.The dependent variable is the -log10(p-value) of either the Fisher exact test for genotype distribution comparison (top and middle figure) or the -log10(p-value) of the goodness-of-fit test in the linear regression model with and without the respective variant (bottom figure).
Figure 4. SLC2A9 allelic variants.Alignment of the GLUT9 amino acids in the studied allelic variants with chimpanzee, horse, dog, mouse, rat and Xenopus paralogs.
Figure 1. Urate uptake in oocytes.Transport activity is expressed as % urate uptake in oocytes injected with 50 ng of cRNA encoding the wild-type or the particular analyzed allelic variants. The uptake of 100 µM [14C]urate in oocytes was measured after 30 minutes incubation. Our experiments showed similar urate transport activity of GLUT9 variant 1 wt and GLUT9 variant 2 wt. GLUT9 wt variants were set to 100%. The average of six measurements each with five oocytes in group is shown and error bars represent standard error. (A) GLUT9 variant 1 (B) GLUT9 variant 2.
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