Background/Purpose: Variation in SLC2A9, which encodes the urate transporter GLUT9, is the major single genetic determinant of serum uric acid (SUA); however, the causal variant(s) within SLC2A9 have not been identified.  Two distinct N-terminal isoforms, GLUT9a, (540 residues) and GLUT9b (511 residues), are generated by alternative 5′ ends.  Our aim was to characterize the 5′ end of SLC2A9to further understanding of how variation in this gene affects SUA.

Methods: 5′ untranslated region (UTR) exons, alternative splicing, and transcriptional start sites were identified by database mining, 5′-RACE PCR, and RT-PCR.  GLUT9 cDNA expression constructs were characterized by ¹⁴C-urate uptakes in Xenopusoocytes.  Promoter analysis utilized the dual luciferase system.

Results: Seven novel 5′ UTR exons were identified, with substantial alternative splicing; in GLUT9b only exon 2, containing the start codon, is spared from alternative splicing. Alternative splicing that deletes coding exons 3 +/- 4 was also identified, in both GLUT9a and GLUT9b transcripts.  Exon 3 is a cassette exon encoding most of transmembrane domain 1 (TM1) and part of the first, glycosylated extracellular loop.  Surprisingly, GLUT9b constructs with deletion of exon 3 were functional (GLUT9b-delta3), generating urate uptakes that were 10-fold higher than that of control Xenopusoocytes.   Western blotting indicated that GLUT9b-delta3 protein is not glycosylated, presumably due to altered topology of the first extracellular, glycosylated loop.

A single GLUT9a transcriptional start site was identified, flanking exon 1a.  Luciferase constructs for this promoter were highly active in multiple cell lines, with a minimal promoter construct of ~200 bp.  Four GLUT9b start sites were identified, including one ~35 kb 5′ of exon 1b flanking a novel 5′ UTR exon.  Only 2 out of 4 GLUT9b promoters were functional, with substantially lower activity than the GLUT9a promoter.

Using the publically available Atherosclersis Risk in Communities Study dataset a major urate-increasing haplotype (freq=0.50) was identified spanning the promoter elements, in addition to two related urate-lowering haplotypes (freq=0.21) with the remaining haplotypes not significantly associated with urate levels.

Conclusion: There is substantial transcriptional heterogeneity in SLC2A9, with seven new 5′ UTR exons and at least four transcriptional initiation sites.  Alternative splicing that removes exon 3 generates functional GLUT9 urate transporters, despite removal of TM1.  The single GLUT9a promoter is strongly active in luciferase assays, whereas only 2 out of 4 GLUT9b promoters are weakly active. Relating these effects to genetic control of urate will increase understanding of the molecular basis of renal uric acid secretion.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/transcriptional-heterogeneity-of-the-slc2a9-gene-encoding-the-glut9-urate-transporter/