ATP-binding cassette subfamily G member 2 (ABCG2) is a physiologically important urate transporter. Accumulating evidence demonstrates that congenital dysfunction of ABCG2 is an important genetic risk factor in gout and hyperuricemia; recent studies suggest the clinical significance of both common and rare variants of ABCG2. However, the effects of rare variants of ABCG2 on the risk of such diseases are not fully understood. Here, using a cohort of 250 Czech individuals of European descent (68 primary hyperuricemia patients and 182 primary gout patients), we examined exonic non-synonymous variants of ABCG2. Based on the results of direct sequencing and database information, we experimentally characterized nine rare variants of ABCG2: R147W (rs372192400), T153M (rs753759474), F373C (rs752626614), T421A (rs199854112), T434M (rs769734146), S476P (not annotated), S572R (rs200894058), D620N (rs34783571), and a three-base deletion K360del (rs750972998). Functional analyses of these rare variants revealed a deficiency in the plasma membrane localization of R147W and S572R, lower levels of cellular proteins of T153M and F373C, and null urate uptake function of T434M and S476P. Accordingly, we newly identified six rare variants of ABCG2 that showed lower or null function. Our findings contribute to deepening the understanding of ABCG2-related gout/hyperuricemia risk and the biochemical characteristics of the ABCG2 protein.

Urate transporters, which are located in the kidneys, significantly affect the level of uric acid in the body. We looked at genetic variants of genes encoding the major reabsorption proteins GLUT9 (SLC2A9) and URAT1 (SLC22A12) and their association with hyperuricemia and gout. In a cohort of 250 individuals with primary hyperuricemia and gout, we used direct sequencing to examine the SLC22A12 and SLC2A9 genes. Identified variants were evaluated in relation to clinical data, biochemical parameters, metabolic syndrome criteria, and our previous analysis of the major secretory urate transporter ABCG2. We detected seven nonsynonymous variants of SLC2A9. There were no nonsynonymous variants of SLC22A12. Eleven variants of SLC2A9 and two variants of SLC22A12 were significantly more common in our cohort than in the European population (p = 0), while variants p.V282I and c.1002+78A>G had a low frequency in our cohort (p = 0). Since the association between variants and the level of uric acid was not demonstrated, the influence of variants on the development of hyperuricemia and gout should be evaluated with caution. However, consistent with the findings of other studies, our data suggest that p.V282I and c.1002+78A>G (SLC2A9) reduce the risk of gout, while p.N82N (SLC22A12) increases the risk.

Objectives: MicroRNAs (miRNAs) are short single-stranded RNAs that play a role in the post-transcriptional regulation of gene expression. Their deregulation can be associated with various diseases, such as cancer, neurodegenerative, and immune-related diseases. The aim of our study was to compare miRNA levels in plasma that could potentially influence the progression of hyperuricemia to gout, since the mechanism of progression is still unclear.Methods: Total RNA, including miRNA, was isolated from the plasma of 45 patients with asymptomatic hyperuricemia, 131 patients with primary gout (including 16 patients having a gout attack), and 130 normouricemic controls. The expression of 18 selected miRNAs (cel-miR-39 and cel-miR-54 as spike-in controls, hsa-miR-16-5p and hsa-miR-25-3p as endogenous controls, hsa-miR-17-5p, hsa-miR-18a-5p, hsa-miR-30a-3p, hsa-miR-30c-5p, hsa-miR-126-3p, hsa-miR-133a-3p, hsa-miR-142-3p, hsa-miR-143-3p, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-miR-222-3p, hsa-miR-223-3p, hsa-miR-488-3p and hsa-miR-920) was measured using qPCR.Results: We found that hsa-miR-17-5p, hsa-miR-18a-5p, hsa-miR-30c-5p, hsa-miR-142-3p, and hsa-miR-223-3p were significantly upregulated (p< 0.001) in the plasma of hyperuricemia and gout patients compared to normouricemic individuals. As part of the follow-up of our previous study, we found a negative correlation between hsa-miR-17-5p, hsa-miR-30c-5p, hsa-miR-126-3p, hsa-miR-142-3p, and hsa-miR-223-3p with plasma levels of chemokine MCP-1. Additionally, we found a positive correlation between CRP and plasma levels of hsa-miR-17-5p, hsa-miR-18a-5p, hsa-miR-30c-5p, hsa-miR-126-3p, hsa-miR-142-3p, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-miR-222-3p, and hsa-miR-223-3p. Five of those miRNAs (hsa-miR-126-3p, hsa-miR-142-3p, hsa-miR-146a-5p, hsa-miR-155-5p, and hsa-miR-222-3p) also had a positive correlation with serum creatinine and therefore a negative correlation with eGFR.Conclusion: Five miRNAs were significantly upregulated in the plasma of patients with hyperuricemia and gout (and those during a gout attack) compared to normouricemic controls. We also found a correlation between the plasma levels of several miRNA and plasma levels of MCP-1, CRP, serum creatinine, and eGFR.

The ABCG2 gene is a well-established hyperuricemia/gout risk locus encoding a urate transporter that plays a crucial role in renal and intestinal urate excretion. Hitherto, p.Q141K—a common variant of ABCG2 exhibiting approximately one half the cellular function compared to the wild-type—has been reportedly associated with early-onset gout in some populations. However, compared with adult-onset gout, little clinical information is available regarding the association of other uricemia-associated genetic variations with early-onset gout; the latent involvement of ABCG2 in the development of this disease requires further evidence. We describe a representative case of familial pediatric-onset hyperuricemia and early-onset gout associated with a dysfunctional ABCG2, i.e., a clinical history of three generations of one Czech family with biochemical and molecular genetic findings. Hyperuricemia was defined as serum uric acid (SUA) concentrations 420 μmol/L for men or 360 μmol/L for women and children under 15 years on two measurements, performed at least four weeks apart. The proband was a 12-year-old girl of Roma ethnicity, whose SUA concentrations were 397–405 µmol/L. Sequencing analyses focusing on the coding region of ABCG2 identified two rare mutations—c.393G>T (p.M131I) and c.706C>T (p.R236X). Segregation analysis revealed a plausible link between these mutations and hyperuricemia and the gout phenotype in family relatives. Functional studies revealed that p.M131I and p.R236X were functionally deficient and null, respectively. Our findings illustrate why genetic factors affecting ABCG2 function should be routinely considered in clinical practice as part of a hyperuricemia/gout diagnosis, especially in pediatric-onset patients with a strong family history.

The OAT1 (SLC22A6) and OAT3 (SLC22A8) urate transporters are located on the basolateral membrane of the proximal renal tubules, where they ensure the uptake of uric acid from the urine back into the body. In a cohort of 150 Czech patients with primary hyperuricemia and gout, we examined the coding regions of both genes using PCR amplification and Sanger sequencing. Variants p.P104L (rs11568627) and p.A190T (rs146282438) were identified in the gene for solute carrier family 22 member 6 (SLC22A6) and variants p.R149C (rs45566039), p.V448I (rs11568486) and p.R513Q (rs145474422) in the gene solute carrier family 22 member 8 (SLC22A8). We performed a functional study of these rare non-synonymous variants using the HEK293T cell line. We found that only p.R149C significantly reduced uric acid transport in vitro. Our results could deepen the understanding of uric acid handling in the kidneys and the molecular mechanism of uric acid transport by the OAT family of organic ion transporters.

Gout is an inflammatory arthritis influenced by environmental risk factors and genetic variants. The common dysfunctional p.Q141K allele of the ABCG2 gene affects gout development. We sought after the possible association between the p.Q141K variant and gout risk factors, biochemical, and clinical determinants in hyperuricemic, gouty, and acute gouty arthritis cohorts. Further, we studied the correlation of p.Q141K allele and levels of pro-/anti-inflammatory cytokines. Coding regions of the ABCG2 gene were analyzed in 70 primary hyperuricemic, 182 gout patients, and 132 normouricemic individuals. Their genotypes were compared with demographic and clinical parameters. Plasma levels of 27 cytokines were determined using a human multiplex cytokine assay. The p.Q141K variant was observed in younger hyperuricemic/gout individuals (p = 0.0003), which was associated with earlier disease onset (p = 0.004), trend toward lower BMI (p = 0.056), and C-reactive protein (CRP, p = 0.007) but a higher glomerular filtration rate (GFR, p = 0.035). Levels of 19 cytokines were higher, mainly in patients with acute gouty arthritis (p < 0.001), irrespective of the presence of the p.Q141K variant. The p.Q141K variant influences the age of onset of primary hyperuricemia or gout and other disease-linked risk factors and symptoms. There was no association with cytokine levels in the circulation.