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Renal hypouricemia (RHUC) is caused by an inherited defect in the main reabsorption system of uric acid, SLC22A12 (URAT1) and SLC2A9 (GLUT9). RHUC is characterized by a decreased serum uric acid concentration and an increase in its excreted fraction. Patients suffer from hypouricemia, hyperuricosuria, urolithiasis, and even acute kidney injury. We report clinical, biochemical, and genetic findings in a cohort recruited from the Košice region of Slovakia consisting of 27 subjects with hypouricemia and relatives from 11 families, 10 of whom were of Roma ethnicity. We amplified, directly sequenced, and analyzed all coding regions and exon–intron boundaries of the SLC22A12 and SLC2A9 genes. Sequence analysis identified dysfunctional variants c.1245_1253del and c.1400C&gt, T in the SLC22A12 gene, but no other causal allelic variants were found. One heterozygote and one homozygote for c.1245_1253del, nine heterozygotes and one homozygote for c.1400C&gt, T, and two compound heterozygotes for c.1400C&gt, T and c.1245_1253del were found in a total of 14 subjects. Our result confirms the prevalence of dysfunctional URAT1 variants in Roma subjects based on analyses in Slovak, Czech, and Spanish cohorts, and for the first time in a Macedonian Roma cohort. Although RHUC1 is a rare inherited disease, the frequency of URAT1-associated variants indicates that this disease is underdiagnosed. Our findings illustrate that there are common dysfunctional URAT1 allelic variants in the general Roma population that should be routinely considered in clinical practice as part of the diagnosis of Roma patients with hypouricemia and hyperuricosuria exhibiting clinical signs such as urolithiasis, nephrolithiasis, and acute kidney injury.

ObjectivesMicroRNAs (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.MethodsTotal 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.ResultsWe 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 (pConclusionFive 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&gt, T (p.M131I) and c.706C&gt, 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.

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&gt, 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&gt, G (SLC2A9) reduce the risk of gout, while p.N82N (SLC22A12) increases the risk.

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.

Career situation of first and presenting author Student for a master or a PhD. Introduction Serum uric acid concentration is significantly influenced by urate transporters, such as ABCG2 (encoded by ABCG2 gene), GLUT9 (SLC2A9 gene) and URAT1 (SLC22A12 gene). The main function of ABCG2 is uric acid secretion, whereas GLU9 and URAT1 also ensure reabsorption. Pathogenic allelic variants in SLC2A9 and SLC22A12 are not only associated with hyperuricemia and gout, but they also lead to rare hereditary renal hypouricemia (type 1 – OMIM #220150 or type 2 – OMIM # 612076). Objectives Previously, we analyzed ABCG2 gene and detected non-synonymous variants that lead to hyperuricemia and early onset of the gout.1 The aim of this study was to find a possible correlation between variants in SLC2A9 and SLC22A12 and hypouricemia, hyperuricemia and gout.2 Methods We recruited a cohort of 232 individuals with primary gout and hyperuricemia. We examined coding regions of SLC2A9 (13 exons) and SLC22A12 (10 exons) by Sanger sequencing. We also analyzed SLC2A9 and SLC22A12 in five patients with suspect hypouricemia. Results In the cohort of 232 individuals, we detected five synonymous variants, 18 intron variants and seven missense variants in SLC2A9: A17T, G25R, T275M, D281H, V282I, R294H, and P350L. In SLC22A12 gene, we found six synonymous variants and seven intron variants. We detected several pathogenic variants in patients with suspect hypouricemia. Intronic variant c.1419+1G>A in SLC2A9 most likely affects the splicing. In SLC22A12, we found rare pathogenic variants T467M and L415_G417del. These variants have according to our previous study high frequency in the Czech and Slovak Roma population.3 Conclusions The uric acid level is determined by a complex mechanism that is not yet fully understood. Disorders of urate transporters can not only lead to hyperuricemia, but in rare cases also to hypouricemia. References Stiburkova B, et al. Rheumatology (Oxford) 2017 November 1;56(11):1982–1992. doi:10.1093/rheumatology/kex295 Hurba O, et al. PLoS One 2014 September 30;9(9):e107902. doi:10.1371/journal.pone.0107902 Gabrikova D, et al. Urolithiasis 2015 October;43(5):441–5. doi:10.1007/s00240-015-0790-4 Acknowledgements This study was supported by the grant from the Czech Republic Ministry of Health AZV 15-26693A. Disclosure of Interest None declared.

Background:The level of uric acid is largely determined by the functions of urate transporters, which are located in the kidney and intestine. The ABCG2 protein is the major excretor of uric acid and its dysfunction may lead to the development of hyperuricemia and gout.Objectives:The aim of our study was to detect the occurrence and frequency of allelic variants in the ABCG2 gene that can lead to impaired function of the ABCG2 protein and to the development of hyperuricemia and gout.Methods:We examined allelic variants of ABCG2 using PCR amplification and Sanger sequencing of all coding regions and exon-intron boundaries in 359 patients with primary hyperuricemia and gout.Results:We found a rare in-frame deletion p.K360del and 15 missense variants, two of which were common (p.V12M, p.Q141K) and 13 were very rare (p.M71V, p.G74D, p.M131I, p.R147W, p.T153M, p.I242T, p.R236X, p.F373C, p.T421A, p.T434M, p.S476P, p.S572R, p.D620N). The p.R236X variant leads to a premature stop codon. The p.V12M variant probably has a protective effect against gout (minor allele frequency – MAF – in our cohort = 0,025 / MAF in the European population = 0,061), while the p.Q141K variant increases the risk of gout (MAF in our cohort = 0,213 / MAF in the European population = 0,094) (1). As for the rare variants, the p.R147W, p.T153M, p.F373C, p.T434M, p.S476P and p.S572R according to functional analyzes reduce the function of the ABCG2 protein (2). Based on in silico prediction, the impact on reduced function is expected for variants p.M71V, p.G74D, p.M131I, p.R147W, p.I242T, p.F373C, p.T434M, p.S476P and p.S572R.Conclusion:Our data suggest that the common variant p.Q141K and most of the rare variants in the ABCG2 gene affect the function of the ABCG2 urate transporter and are a genetic risk factor for hyperuricemia and gout.References:[1]Stiburkova B, et al. Functional non-synonymous variants of ABCG2 and gout risk. Rheumatology (Oxford). 2017 Nov 1; 56(11):1982-1992.[2]Toyoda Y, et al. Functional characterization of clinically-relevant rare variants in ABCG2 identified in a gout and hyperuricemia cohort. Cells. 2019 Apr 18;8(4).Acknowledgements:This study was supported by the project for conceptual development of research organization 00023728 (Institute of Rheumatology) and RVO VFN64165.Disclosure of Interests:None declared

Background: Gout and diabetes mellitus type 2 (DM) are frequently co-existing. Metformin is the first choice of treatment for patients with DM type 2, and might – based on previous studies - have beneficial clinical effects on gout through a putative anti-inflammatory as well as serum uric acid (SUA) lowering effect. 1,2,3 Objectives: To investigate the anti-inflammatory and SUA lowering effect of metformin in patients with gout starting uric acid lowering treatment (ULT). Methods: Patients with clinical diagnosis of gout, a first outpatient visit between January 2010 and March 2018 and a follow-up of at least 6 months were included in a retrospective cohort study, conducted in two rheumatology centres in the Netherlands. From this cohort patients with DM starting ULT were selected. Patients with metformin use were compared to patients using other or no antidiabetic medication (control group). Metformin use was defined as use ≥80% of the time during the first six months after initiation of ULT. To evaluate the anti-inflammatory effect, the differences in incidence density (ID) of gout flares in the first six months after starting ULT was measured and analysed using Poisson regression. To evaluate the SUA lowering effect, the difference in baseline SUA, proportion of patients reaching target serum uric acid (SUA Results: Of 2108 gout patients, 309 patients who started ULT also had DM, with 155 in the metformin group and 154 in the control group (table 1). ID of flares was 2.8 and 3.3 per patient year in the control and metformin group respectively, resulting in an incidence rate ratio of 0.93 (95% CI 0.75 – 1.15). SUA levels at baseline were similar, between the two groups (Table 1, p=0.31). At six months 46.1% and 57.4% reached target SUA in the control and metformin group respectively, odds ratio of 1.30 (95%CI 0.80 – 2.08). No difference was found in allopurinol dose at time of reaching target SUA between the groups (1.14 mg, 95% CI -38.89 – 41.19). Conclusion: In contrast to a previous report1, in this study in patients with DM and gout and starting ULT, metformin does not have a clinically relevant added anti-inflammatory or urate lowering effect. References: [1]Vazirpanah N et al. Ann Rheum Dis. 2019;78(5):663-71. [2]Simao AN et al. Expert opinion on therapeutic targets. 2012;16(12):1175-87. [3]Barskova VG et al. Klin Med (Mosk). 2009;87(7):41-6. Disclosure of Interests: : Frouwke Veenstra: None declared, L.M. Verhoef: None declared, Merel Opdam: None declared, Alfons den Broeder: None declared, Wing-Yee Kwok: None declared, Inger Meek: None declared, Frank van den Hoogen: None declared, Marcel Flendrie Grant/research support from: M. Flendrie has received grants from Menarini and Grunenthal. , Consultant of: M. Flendrie has received consultancy fees from Menarini and Grunenthal., Noortje van Herwaarden: None declared

Background The urate transporters are one of the main genetic determinants of serum uric acid concentrations. Objectives In this study we investigated the effects of non-synonymous allelic variants of urate transporters in a cohort of patients with primary gout and/or asymptomatic hyperuricemia. Methods The cohort consisted of 165 gout patients (151 men, 14 women); 58 hyperuricemic individuals (39 men/19 women); 115 normouricemic controls were used for comparison. Gouty arthritis was diagnosed according to the 1977 preliminary criteria of the American College of Rheumatology. Coding regions of ABCG2, SLC2A9, SLC22A11, SLC22A8, SLC17A3, and SLC17A1 genes were amplified and sequenced directly. To estimate the functions of the identified non-synonymous allelic variants, we used the protein prediction algorithms. Results In ABCG2 gene, we detected nine non-synonymous variants (two common, seven rare including one novel): p.V12M, p.Q141K, p.R147W, p.T153M, p.K360del, p.F373C, p.T434M, p.S476P and p.D620N. The p.Q141K (rs2231142) variant had a significantly higher minor allele frequency (0.23) in the gout patients compared to the European-origin population (0.09) and was significantly more common among gout patients than among normouricemic controls (OR=3.15, p A which is associated with two abnormal splicing variants, leading to premature introduction of the stop codon, mislocalized ABCG2 signal on plasma membrane and no urate uptake activity2. In SLC2A9 gene, seven missense variants were identified (six common, one rare): p.A17T, p.G25R, p.T275M, p.D281H, p.V282I, p.R294H, and p.P350L. In SLC17A3, the analysis revealed common allelic variants p.A100T and p.G279R. Rare non-synonymous variants p.V202M and p.R343L were found in SLC22A11. Only one common missense variant p.T269I was identified in SLC17A1. In SLC22A8 sequencing revealed three rare variants: p.R149C, p.V448I and p.R513G. The precise impact of SLC22A11, SLC22A8, SLC17A3, and SLC17A1 in the context of hyperuricemia and gout in our cohort is unclear and will be further studied include functional characterisation of selected dysfunctional variants. Conclusions Genetic variants of ABCG2, common and rare, increased the risk of gout and had a significant effect on earlier onset of gout and the presence of a familial gout history. Genotyping the rare variants of ABCG2 along with its common variants is essential for evaluating the individual risk for gout. References [1] Stiburkova B, et al. Functional non-synonymous variants of ABCG2 and gout risk. Rheumatology (Oxford)2017Nov 1;56(11):1982–1992. [2] Stiburkova B, et al. Novel dysfunctional variant in ABCG2 as a cause of severe tophaceous gout: biochemical, molecular genetics and functional analysis. Rheumatology (Oxford)2016Jan;55(1):191–4. Acknowledgements This study was supported by the grant from the Czech Republic Ministry of Health AZV 15–26693A. Disclosure of Interest None declared

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.

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.