Examining the Association of Rare Allelic Variants in Urate Transporters SLC22A11, SLC22A13, and SLC17A1 with Hyperuricemia and Gout.

Genetic variations in urate transporters play a significant role in determining human urate levels and have been implicated in developing hyperuricemia or gout. Polymorphism in the key urate transporters, such as ABCG2, URAT1, or GLUT9 was well-documented in the literature. Therefore in this study, our objective was to determine the frequency and effect of rare nonsynonymous allelic variants of SLC22A11, SLC22A13, and SLC17A1 on urate transport. In a cohort of 150 Czech patients with primary hyperuricemia and gout, we examined all coding regions and exon-intron boundaries of SLC22A11, SLC22A13, and SLC17A1 using PCR amplification and Sanger sequencing. For comparison, we used a control group consisting of 115 normouricemic subjects. To examine the effects of the rare allelic nonsynonymous variants on the expression, intracellular processing, and urate transporter protein function, we performed a functional characterization using the HEK293A cell line, immunoblotting, fluorescent microscopy, and site directed mutagenesis for preparing variants in vitro. Variants p.V202M (rs201209258), p.R343L (rs75933978), and p.P519L (rs144573306) were identified in the SLC22A11 gene (OAT4 transporter); variants p.R16H (rs72542450), and p.R102H (rs113229654) in the SLC22A13 gene (OAT10 transporter); and the p.W75C variant in the SLC17A1 gene (NPT1 transporter). All variants minimally affected protein levels and cytoplasmic/plasma membrane localization. The functional in vitro assay revealed that contrary to the native proteins, variants p.P519L in OAT4 (p ≤ 0.05), p.R16H in OAT10 (p ≤ 0.05), and p.W75C in the NPT1 transporter (p ≤ 0.01) significantly limited urate transport activity. Our findings contribute to a better understanding of (1) the risk of urate transporter-related hyperuricemia/gout and (2) uric acid handling in the kidneys.

Alterations in lipidome profiles distinguish early-onset hyperuricemia, gout, and the effect of urate-lowering treatment.

BACKGROUND: Currently, it is not possible to predict whether patients with hyperuricemia (HUA) will develop gout and how this progression may be affected by urate-lowering treatment (ULT). Our study aimed to evaluate differences in plasma lipidome between patients with asymptomatic HUA detected ≤ 40 years (HUA ≤ 40) and > 40 years, gout patients with disease onset ≤ 40 years (Gout ≤ 40) and > 40 years, and normouricemic healthy controls (HC). METHODS: Plasma samples were collected from 94 asymptomatic HUA (77% HUA ≤ 40) subjects, 196 gout patients (59% Gout ≤ 40), and 53 HC. A comprehensive targeted lipidomic analysis was performed to semi-quantify 608 lipids in plasma. Univariate and multivariate statistics and advanced visualizations were applied. RESULTS: Both HUA and gout patients showed alterations in lipid profiles with the most significant upregulation of phosphatidylethanolamines and downregulation of lysophosphatidylcholine plasmalogens/plasmanyls. More profound changes were observed in HUA ≤ 40 and Gout ≤ 40 without ULT. Multivariate statistics differentiated HUA ≤ 40 and Gout ≤ 40 groups from HC with an overall accuracy of > 95%. CONCLUSION: Alterations in the lipidome of HUA and Gout patients show a significant impact on lipid metabolism. The most significant glycerophospholipid dysregulation was found in HUA ≤ 40 and Gout ≤ 40 patients, together with a correction of this imbalance with ULT.

Novel Somatic UBA1 Variant in a Patient With VEXAS Syndrome.

OBJECTIVE: Somatic mutations in UBA1 have recently been causally linked to a severe adult-onset inflammatory condition referred to as VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. Ubiquitin-activating enzyme E1 (UBA-1) is of fundamental importance to the modulation of ubiquitin homeostasis and to the majority of downstream ubiquitylation-dependent cellular processes. Direct sequencing analysis of exon 3 containing the prevalent variants p.Met41Leu, p.Met41Val, and/or p.Met41Thr is usually used to confirm the disease-associated mutations. METHODS: We studied the clinical, biochemical, and molecular genetic characteristics of a 59-year-old man with a 2-year history of arthritis, fever, night sweats, nonspecific skin rash, lymphadenopathy, and myelodysplastic syndrome with multilineage dysplasia. RESULTS: The mutational analysis revealed a previously undescribed sequence variant c.1430G>C in exon 14 (p.Gly477Ala) in the gene UBA1. In vitro enzymatic analyses showed that p.Gly477Ala led to both decreased E1 ubiquitin thioester formation and E2 enzyme charging. CONCLUSION: We report a case of a patient of European ancestry with clinical manifestations of VEXAS syndrome associated with a newly identified dysfunctional UBA-1 enzyme variant. Due to the patient's insufficient response to various immunosuppressive treatments, allogeneic hematopoietic stem cell transplantation was performed, which resulted in significant improvement of clinical and laboratory manifestations of the disease.

Functional Characterization of Rare Variants in OAT1/SLC22A6 and OAT3/SLC22A8 Urate Transporters Identified in a Gout and Hyperuricemia Cohort.

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.

The Examination of a TPMT Gene Before Administration of Azathioprine in Rheumatology Practice and Identification of a Novel Variant p.W29R.

METHODS: Two-hundred patients were assessed for the presence of genetic allelic variants using PCR amplification and direct sequencing. RESULTS: In 19 patients, we detected genetic allelic variants affecting TPMT activity; in 1 case, it was an unpublished heterozygous variant c.85T>C (p.W29R); of those, 15 patients were switched from AZA to a different medication, and 1 patient was prescribed a reduced dose of AZA. CONCLUSIONS: Our findings show the importance of testing for variants of the TPMT gene before the administration of AZA in clinical rheumatology practice. Patients with documented episodes of leukopenia or elevated liver biochemical tests while on AZA should undergo TPMT genotype testing and/or TPMT enzyme activity testing.

Identification of a dysfunctional exon-skipping splice variant in GLUT9/SLC2A9 causal for renal hypouricemia type 2.

Renal hypouricemia (RHUC) is a pathological condition characterized by extremely low serum urate and overexcretion of urate in the kidney; this inheritable disorder is classified into type 1 and type 2 based on causative genes encoding physiologically-important urate transporters, URAT1 and GLUT9, respectively; however, research on RHUC type 2 is still behind type 1. We herein describe a typical familial case of RHUC type 2 found in a Slovak family with severe hypouricemia and hyperuricosuria. Via clinico-genetic analyses including whole exome sequencing and in vitro functional assays, we identified an intronic GLUT9 variant, c.1419+1G>A, as the causal mutation that could lead the expression of p.Gly431GlufsTer28, a functionally-null variant resulting from exon 11 skipping. The causal relationship was also confirmed in another unrelated Macedonian family with mild hypouricemia. Accordingly, non-coding regions should be also kept in mind during genetic diagnosis for hypouricemia. Our findings provide a better pathogenic understanding of RHUC and pathophysiological importance of GLUT9.

Renal Hypouricemia 1: Rare Disorder as Common Disease in Eastern Slovakia Roma Population.

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 Kosice 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>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>T, and two compound heterozygotes for c.1400C>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.

Identification of Two Dysfunctional Variants in the ABCG2 Urate Transporter Associated with Pediatric-Onset of Familial Hyperuricemia and Early-Onset Gout.

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.

Evaluation of the Influence of Genetic Variants of SLC2A9 (GLUT9) and SLC22A12 (URAT1) on the Development of Hyperuricemia and Gout.

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.

Interaction of the p.Q141K Variant of the ABCG2 Gene with Clinical Data and Cytokine Levels in Primary Hyperuricemia and Gout.

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.

Functional Characterization of Clinically-Relevant Rare Variants in ABCG2 Identified in a Gout and Hyperuricemia Cohort.

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.

The impact of dysfunctional variants of ABCG2 on hyperuricemia and gout in pediatric-onset patients.

BACKGROUND: ABCG2 is a high-capacity urate transporter that plays a crucial role in renal urate overload and extra-renal urate underexcretion. Previous studies have suggested an association between hyperuricemia and gout susceptibility relative to dysfunctional ABCG2 variants, with rs2231142 (Q141K) being the most common. In this study, we analyzed the ABCG2 gene in a hyperuricemia and gout cohort focusing on patients with pediatric-onset, i.e., before 18 years of age. METHOD: The cohort was recruited from the Czech Republic (n = 234) and consisted of 58 primary hyperuricemia and 176 gout patients, with a focus on pediatric-onset patients (n = 31, 17 hyperuricemia/14 gouts); 115 normouricemic controls were used for comparison. We amplified, sequenced, and analyzed 15 ABCG2 exons. The chi-square goodness-of-fit test was used to compare minor allele frequencies (MAF), and the log-rank test was used to compare empirical distribution functions. RESULTS: In the pediatric-onset cohort, two common (p.V12M, p.Q141K) and three very rare (p.K360del, p.T421A, p.T434M) allelic ABCG2 variants were detected. The MAF of p.Q141K was 38.7% compared to adult-onset MAF 21.2% (OR = 2.4, P = 0.005), to the normouricemic controls cohort MAF 8.5% (OR = 6.8, P <  0.0001), and to the European population MAF 9.4% (OR = 5.7, P <  0.0001). The MAF was greatly elevated not only among pediatric-onset gout patients (42.9%) but also among patients with hyperuricemia (35.3%). Most (74%) of the pediatric-onset patients had affected family members (61% were first-degree relatives). CONCLUSION: Our results show that genetic factors affecting ABCG2 function should be routinely considered in a hyperuricemia/gout diagnosis, especially in pediatric-onset patients. Genotyping of ABCG2 is essential for risk estimation of gout/hyperuricemia in patients with very early-onset and/or a family history.

Thiopurine-induced toxicity is associated with dysfunction variant of the human molybdenum cofactor sulfurase gene (xanthinuria type II).

BACKGROUND: The aim of our study was to identify the genetic background of thiopurine-induced toxicity in a patient with a wild-type thiopurine methyltransferase genotype and activity. A 38-year-old Caucasian woman presented with cutaneous necrotizing vasculitis pancytopenia one month after starting azathioprine therapy. METHODS: During a routine biochemical follow-up of the patient, undetectable serum uric acid (<10 µl) was observed. A high performance liquid chromatography analysis of urinary purines revealed increased levels of xanthine (137 mmol/mol creatinine). The suspected diagnosis of hereditary xanthinuria, a rare autosomal recessive disorder of the last two steps of purine metabolism, was confirmed by sequence analysis. RESULTS: An analysis of XDH/XO and AOX1 revealed common polymorphisms, while analysis of the MOCOS gene identified a rare homozygous variant c.362C > T. Dysfunction of this variant was confirmed by significantly decreased xanthine dehydrogenase/oxidase activity in the patient's plasma (<2% of control mean activity). CONCLUSIONS: We present a biochemical, enzymatic, and molecular genetic case study suggesting an important association between a hitherto undescribed dysfunction variant in the MOCOS gene and thiopurine-induced toxicity. The identified variant c.362C > T results in slower thiopurine metabolism caused by inhibition of 6-mercaptopurine oxidation (catabolism) to 6-thioxanthine and 6-thiouric acid, which increases the formation of the nucleotide 6-thioguanine, which is toxic. This is the first clinical case to identify the crucial role of the MOCOS gene in thiopurine intolerance and confirm the impact of genetic variability of purine enzymes on different therapeutic outcomes in patients undergoing thiopurine treatment.

Functional non-synonymous variants of ABCG2 and gout risk.

Objectives: Common dysfunctional variants of ATP binding cassette subfamily G member 2 (Junior blood group) (ABCG2), a high-capacity urate transporter gene, that result in decreased urate excretion are major causes of hyperuricemia and gout. In the present study, our objective was to determine the frequency and effect on gout of common and rare non-synonymous and other functional allelic variants in the ABCG2 gene. Methods: The main cohort recruited from the Czech Republic consisted of 145 gout patients; 115 normouricaemic controls were used for comparison. We amplified, directly sequenced and analysed 15 ABCG2 exons. The associations between genetic variants and clinical phenotype were analysed using the t-test, Fisher's exact test and a logistic and linear regression approach. Data from a New Zealand Polynesian sample set and the UK Biobank were included for the p.V12M analysis. Results: In the ABCG2 gene, 18 intronic (one dysfunctional splicing) and 11 exonic variants were detected: 9 were non-synonymous (2 common, 7 rare including 1 novel), namely p.V12M, p.Q141K, p.R147W, p.T153M, p.F373C, p.T434M, p.S476P, p.D620N and p.K360del. The p.Q141K (rs2231142) variant had a significantly higher minor allele frequency (0.23) in the gout patients compared with the European-origin population (0.09) and was significantly more common among gout patients than among normouricaemic controls (odds ratio = 3.26, P < 0.0001). Patients with non-synonymous allelic variants had an earlier onset of gout (42 vs 48 years, P = 0.0143) and a greater likelihood of a familial history of gout (41% vs 27%, odds ratio = 1.96, P = 0.053). In a meta-analysis p.V12M exerted a protective effect from gout (P < 0.0001). Conclusion: Genetic variants of ABCG2, common and rare, increased the risk of gout. Non-synonymous allelic variants of ABCG2 had a significant effect on earlier onset of gout and the presence of a familial gout history. ABCG2 should thus be considered a common and significant risk factor for gout.

Genetic background of uric acid metabolism in a patient with severe chronic tophaceous gout.

Hyperuricemia depends on the balance of endogenous production and renal excretion of uric acid. Transporters for urate are located in the proximal tubule where uric acid is secreted and extensively reabsorbed: secretion is principally ensured by the highly variable ABCG2 gene. Enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) plays a central role in purine metabolism and its deficiency is an X-linked inherited metabolic disorder associated with clinical manifestations of purine overproduction. Here we report the case of a middle-aged man with severe chronic tophaceous gout with a poor response to allopurinol and requiring repeated surgical intervention. We identified the causal mutations in the HPRT1 gene, variant c.481G>T (p.A161S), and in the crucial urate transporter ABCG2, a heterozygous variant c.421C>A (p.Q141K). This case shows the value of an analysis of the genetic background of serum uric acid.