Rheumatology key message Dysfunctional variants of urate transporter ABCG2 are an important genetic factor in hyperuricaemia and gout. Sir, Gout is caused by hyperuricaemia. Several genes involved in renal urate transport, such as SLC2A9, ABCG2 and SLC22A12, have been identified as risk factors for hyperuricaemia and gout [1–5]. More recently, a genome-wide association study of gout uncovered that single-nucleotide polymorphisms (SNPs) of SLC2A9 and ABCG2 are associated with two types of gout, renal underexcretion and renal overload, respectively [6]. The genome-wide association study, however, has limitations in establishing causality of disease-associated SNPs. Therefore, experimental validations are essential to determine if interesting variants are indeed responsible for clinical symptoms. As reported in our previous study of common SLC2A9 allelic variants, we detected variants with no evidence of an association with hyperuricaemia and gout [7]. In this letter we present a biochemical, molecular genetic and functional case study suggesting a causal link between a hitherto undescribed sequence variant in the ABCG2 gene and a severe gouty phenotype. The patient, a participant of our study on common SLC2A9 allelic variants [7], was a 47-year-old male who started having gout attacks at the age of 29 years. His medical history was unremarkable and no close relative was known to have gout or hyperuricaemia. Unfortunately, none of his close relatives agreed to an analysis of their biological material. When first seen in our clinic, he already suffered from chronic debilitating polyarthritis with widespread bulky tophi located mainly on his hands and feet and around the elbows and knees, with intermittent discharge of a chalky substance (supplementary Fig. S1, available at Rheumatology Online). Monosodium urate crystals were microscopically verified from the SF aspirated from his knee. Without treatment his serum uric acid level was 647 μmol/l. To explore the cause of gout, we performed a metabolic investigation. The results suggested that his hyperuricaemia was mainly due to a defect in the urate excretion system and did not result from an excess production of urate associated with purine metabolic disorders and/or with uromodulin-associated disorders (normal excretion of uromodulin, xanthine and hypoxanthine). Based on two separate measurements of the patient’s fractional excretion of urate (FEUA) and urinary urate excretion (UUE) (FEUA 4.21%, UUE 14.8 mg/h/1.73m2; and FEUA 1.67%, UUE 31 mg/h/1.73 m2), his hyperuricaemia could be classified as either renal underexcretion or a combined renal underexcretion and renal overload type, which corresponds to an ABCG2 dysfunction [8]. Next we performed a sequencing analysis focusing on physiologically important urate transporters for further investigation. The analysis of SLC22A12 revealed two sequence variants in intron regions and two synonymous variants in exon regions. The analysis of SLC2A9 revealed eight variants in intron regions, three synonymous variants in exon regions and three heterozygous non-synonymous variants: rs2276961 (p.G25R), rs3733591 (p.R294H) and rs2280205 (p.P350L). Our previously reported approach, which used association analysis together with functional and immunohistochemical characterization of these variants in SLC2A9, did not show any influence on expression, subcellular localization or urate uptake of GLUT9 [7]. The analysis of ABCG2 revealed eight variants in intron regions, an unpublished heterozygous intron variant c.689+1G>A and two exon variants, synonymous rs35622453 and heterozygous non-synonymous rs22231137 (p.V12M), which had little effect on the expression and urate transport activity of ABCG2 [6]. Two abnormal ABCG2 splicing variants were identified: r.[532_689del] deletion of exon 6 (ΔE6), and r.[532_689del; 944_949del] deletion of exon 6 and deletion of the first six base pairs of exon 9 (ΔE6-ΔE9′). Predictions for these variants showed an extra stop codon resulting from the frame shift. To examine the effect of ΔE6 and ΔE6-ΔE9′ variants on the expression and function of the ABCG2 protein, we performed in vitro experiments. Fig. 1A shows the structure inserted in pcDNA3.1(+). Fig. 1B shows the immunoblot analysis. ABCG2 WT (wild-type) was estimated to be ∼70 kDa (glycosylated form) and 65 kDa (non-glycosylated form), whereas the ΔE6 and ΔE6-ΔE9′ variants were ∼18 kDa. In addition, protein expression levels of ΔE6 and ΔE6-ΔE9′ variants were lower than that of the WT version, suggesting that the mutated ABCG2 protein is not stable. Fig. 1C shows the immunofluorescence images of HEK293A cells expressing the ABCG2 protein. Strong ABCG2 signals were observed at the plasma membrane in cells expressing ABCG2 WT; however, the ΔE6 and ΔE6-ΔE9′ variant proteins were only detected within the intracellular compartment. These results indicate that the ΔE6 and ΔE6-ΔE9′ variants could not reach the plasma membrane. Finally, to assess the effect of ΔE6 and ΔE6-ΔE9’ mutations on ABCG2 transport activity, ABCG2-mediated urate transport experiments were performed using plasma membrane vesicles prepared from ABCG2-expressing HEK293A cells. As shown in Fig. 1D, the ΔE6 and ΔE6-ΔE9′ variants of ABCG2 had no urate transport activity. Dysfunction of ABCG2 ΔE6 and ΔE6-ΔE9’ variants as urate transporters (A) Schematic illustration of myc-tagged ABCG2 ORF and translational region. Shaded box shows the translational region. (B) Expression of ΔE6 and ΔE6-ΔE9′ variants as a truncated form of the ABCG2 protein. Forty-eight hours after the transfection of each expression vector with polyethylenimine MAX, HEK293A cells were subjected to immunoblot analysis with myc-specific antibody for detecting myc-tagged ABCG2 proteins. (C) Intracellular localization of ΔE6 and ΔE6-ΔE9′ variants in HEK293A cells. ABCG2 proteins were probed with anti-myc antibody and then labelled with green fluorescence dye (Alexa Fluor 488 goat anti-rabbit IgG); the nuclei were labelled with TOPRO3 (blue). (D) No transport activity in the ΔE6 and ΔE6-ΔE9′ variants. Bars are mean (s.d.); n = 3. **P < 0.01 compared with WT by Dunnett’s post hoc test. AA: amino acids; bp: base pair; ΔE6 and ΔE6-ΔE9′: mutant variants (see main text); Mt: mutant variants; Mw: molecular weight; Na+/K+-ATPase: a control for equal loading; ORF: open reading frame; WT: wild-type. In conclusion, our findings suggest a causal relationship between the hitherto undescribed dysfunctional mutation of the ABCG2 gene and a severe gouty phenotype, and strongly support the pathophysiological significance of ABCG2 as a risk factor for gout. The authors would like to thank Petr Vyletal, PhD for uromodulin analysis (Institute of Inherited Metabolic Disorders, Prague, Czech Republic). In addition, the authors’ thanks go to Dr Hirotaka Matsuo (National Defense Medical College, Saitama, Japan) and Dr Kimiyoshi Ichida (Jikei University School of Medicine, Tokyo, Japan) for engaging in a fruitful discussion on gout risk. T.T. was supported by Japan Society for the Promotion of Science (JSPS) Kakenhi Grant Number 23689008, Kobayashi International Scholarship Foundation, the Mochida Memorial Foundation for Medical and Pharmaceutical Research and the Gout Research Foundation of Japan. Y.T. was a JSPS research fellow. All the authors were involved in drafting the article or revising it critically for important intellectual content and all the authors approved the final version for publication. Study conception and design: B.S., H.M., Y.T., T.T., H.S.; clinical observation: J.Z., M.T.; acquisition of data: B.S., H.M.; analysis and interpretation of data: B.S., H.M., G.S., Y.T., T.T., K.P. Funding: This study was supported by the grants from the Czech Republic Ministry of Health (RVO 023728 Institute of Rheumatology, RVO VFN64165, AZV 15-26693A). Disclosure statement: The authors have declared no conflicts of interest.
No takes yet. Share an insight, caveat, or question.
Stibůrková et al. (2015) studied this question.
Synapse has enriched 5 closely related papers on similar clinical questions. Consider them for comparative context: