Method: The coding polymorphism rs16890979 was selected as a haplotype tagging marker known to be in strong linkage disequilibrium with intronic SNPs identified from genome wide association studies of serum urate (Kolz et al, PLoS Genetics 2009). TaqMan genotyping probes recognising either allele of rs16890979 were obtained from Applied Biosystems. RNA was extracted from whole blood or surplus tissue obtained from nephrectomy or joint replacement surgery using standard techniques. For the promoter-reporter assays 2kb fragments upstream of exon 1 of both short and long splice variants of SLC2A9 were cloned as well as a 2kb region immediately upstream of exon2 which is predicted to have regulatory potential. Promotor constructs were transfected into HEK293 cells using a pGL3 basic vector with expression levels determined by measurement of firefly luciferase.
Result: In samples from patients heterozygous for the rs16890979 polymorphism consistent overexpression of SLC2A9 was observed with the minor ‘A’ allele relative to the major ‘G’ allele. Approximately 6 fold enhanced expression was observed in both peripheral blood and renal tissue. Similar levels of expression were observed in synovial tissue as were seen in renal tissue. Minimal promoter activity was observed in both putative promoter regions upstream of short and long splice variants but approximately 2 fold enhanced expression was seen within a 2kb region upstream of exon2.
Conclusion: Marked differential expression of SLC2A9 is associated with alternative alleles of the rs16890979 polymorphism suggesting that this marker is in linkage disequilibrium with a mutation influencing gene regulation. Modest promoter activity is identified in a region upstream of exon 2 of SLC2A9. Further work will be required to identify variants within this promoter region, as well as to look for further regulatory elements within SLC2A9. SLC2A9 is known to be expressed in the proximal tubule where it is expressed at both apical and basolateral membranes. Better understanding of the role of SLC2A9 in regulating serum urate and also better understanding of the regulation of urate transport in synovium may in time lead to novel strategies for the management of gout.
Disclosure: P. L. Riches, None; S. Gray, None; O. Albagha, None; S. H. Ralston, None.