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 RESULTS: In 19 patients, we detected genetic allelic variants affecting TPMT activity; in 1 case, it was an unpublished heterozygous variant c.85T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 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.

Subtype-specific gout susceptibility loci and enrichment of selection pressure on ABCG2 and ALDH2 identified by subtype genome-wide meta-analyses of clinically defined gout patients.

OBJECTIVES: Genome-wide meta-analyses of clinically defined gout were performed to identify subtype-specific susceptibility loci. Evaluation using selection pressure analysis with these loci was also conducted to investigate genetic risks characteristic of the Japanese population over the last 2000-3000 years. METHODS: Two genome-wide association studies (GWASs) of 3053 clinically defined gout cases and 4554 controls from Japanese males were performed using the Japonica Array and Illumina Array platforms. About 7.2 million single-nucleotide polymorphisms were meta-analysed after imputation. Patients were then divided into four clinical subtypes (the renal underexcretion type, renal overload type, combined type and normal type), and meta-analyses were conducted in the same manner. Selection pressure analyses using singleton density score were also performed on each subtype. RESULTS: In addition to the eight loci we reported previously, two novel loci, PIBF1 and ACSM2B, were identified at a genome-wide significance level (p<5.0×10-8) from a GWAS meta-analysis of all gout patients, and other two novel intergenic loci, CD2-PTGFRN and SLC28A3-NTRK2, from normal type gout patients. Subtype-dependent patterns of Manhattan plots were observed with subtype GWASs of gout patients, indicating that these subtype-specific loci suggest differences in pathophysiology along patients' gout subtypes. Selection pressure analysis revealed significant enrichment of selection pressure on ABCG2 in addition to ALDH2 loci for all subtypes except for normal type gout. CONCLUSIONS: Our findings on subtype GWAS meta-analyses and selection pressure analysis of gout will assist elucidation of the subtype-dependent molecular targets and evolutionary involvement among genotype, phenotype and subtype-specific tailor-made medicine/prevention of gout and hyperuricaemia.

Rapid and reliable HILIC-MS/MS method for monitoring allantoin as a biomarker of oxidative stress.

Allantoin is an excellent biomarker of oxidative stress in humans as the main product of uric acid oxidation by reactive oxygen species. Yet, allantoin determination is still not routinely performed in clinical laboratories. Therefore, we developed a fast, simple, selective, and sensitive UHPLC-MS/MS method for allantoin determination in human serum using an isotopically labeled internal standard. Our analytical protocol provided high sensitivity by mass spectrometry detection and high throughput by HILIC-MS/MS analysis within 4 min, with one-step serum sample preparation approximately within 7 min. Lastly, our protocol was fully validated to demonstrate its reliability in allantoin determination in human serum. The method showed an excellent linear range from 0.05 to 100 µM, with precision ranging from 1.8 to 11.3% (RSD), and with accuracy (relative error %) within ±6.0%. The method was then applied to analyze the concentration of allantoin in serum samples from 71 patients with chronic gout without treatment with xanthine oxidase inhibitors. The median serum allantoin concentration in the cohort was 2.8 µM (n = 71). Overall, our simple analytical protocol has the potential to be easily implemented in clinical routine practice for monitoring allantoin as a key oxidative stress biomarker.

Genome-wide association study revealed novel loci which aggravate asymptomatic hyperuricaemia into gout.

OBJECTIVE: The first ever genome-wide association study (GWAS) of clinically defined gout cases and asymptomatic hyperuricaemia (AHUA) controls was performed to identify novel gout loci that aggravate AHUA into gout. METHODS: We carried out a GWAS of 945 clinically defined gout cases and 1003 AHUA controls followed by 2 replication studies. In total, 2860 gout cases and 3149 AHUA controls (all Japanese men) were analysed. We also compared the ORs for each locus in the present GWAS (gout vs AHUA) with those in the previous GWAS (gout vs normouricaemia). RESULTS: This new approach enabled us to identify two novel gout loci (rs7927466 of CNTN5 and rs9952962 of MIR302F) and one suggestive locus (rs12980365 of ZNF724) at the genome-wide significance level (p<5.0×10-8). The present study also identified the loci of ABCG2, ALDH2 and SLC2A9. One of them, rs671 of ALDH2, was identified as a gout locus by GWAS for the first time. Comparing ORs for each locus in the present versus the previous GWAS revealed three 'gout vs AHUA GWAS'-specific loci (CNTN5, MIR302F and ZNF724) to be clearly associated with mechanisms of gout development which distinctly differ from the known gout risk loci that basically elevate serum uric acid level. CONCLUSIONS: This meta-analysis is the first to reveal the loci associated with crystal-induced inflammation, the last step in gout development that aggravates AHUA into gout. Our findings should help to elucidate the molecular mechanisms of gout development and assist the prevention of gout attacks in high-risk AHUA individuals.

Serum uric acid increases in patients with systemic autoimmune rheumatic diseases after 3 months of treatment with TNF inhibitors.

In patients with gout, the serum uric acid (SUA) is usually lower during acute gouty attacks than during intercritical periods. It has been suggested that systemic inflammatory response can cause this phenomenon. The objective is to determine whether therapy with TNF inhibitors (TNFis) affects SUA levels in patients with systemic autoimmune rheumatic diseases (SARDs) and whether SUA changes correlate with pro-inflammatory cytokines or with the oxidative stress marker allantoin. In this study, SUA, CRP, creatinine, MCP-1, IFN-α2, IFN-γ, Il-1ß, IL-6, IL-8, IL-10, IL-12, IL-17a, IL-18, IL-23, IL-33, TNF-α, and allantoin levels were measured prior to and after 3 months of TNFis treatment in patients with SARDs. The values obtained in the biochemical assays were then tested for associations with the patients' demographic and disease-related data. A total of 128 patients (rheumatoid arthritis, n = 44; ankylosing spondylitis, n = 45; psoriatic arthritis, n = 23; and adults with juvenile idiopathic arthritis, n = 16) participated in this study. Among the entire patient population, SUA levels significantly increased 3 months after starting treatment with TNFis (279.5 [84.0] vs. 299.0 [102.0] µmol/l, p < 0.0001), while the levels of CRP, IL-6, IL-8, and MCP-1 significantly decreased. Male sex was the most powerful baseline predictor of ΔSUA in univariate and multivariate models. None of the measured laboratory-based parameters had statistically significant effects on the magnitude of ΔSUA. 3 months of anti-TNF therapy increased the levels of SUA in patients with SARDs, but neither the measured pro-inflammatory cytokines nor the oxidation to allantoin appeared responsible for this effect.

Clinical manifestations and molecular aspects of phosphoribosylpyrophosphate synthetase superactivity in females.

Objectives: Phosphoribosylpyrophosphate synthetase (PRPS1) superactivity is an X-linked disorder characterized by urate overproduction Online Mendelian Inheritance in Man (OMIM) gene reference 300661. This condition is thought to rarely affect women, and when it does, the clinical presentation is mild. We describe a 16-year-old African American female who developed progressive tophi, nephrolithiasis and acute kidney failure due to urate overproduction. Family history included a mother with tophaceous gout who developed end-stage kidney disease due to nephrolithiasis and an affected sister with polyarticular gout. The main aim of this study was to describe the clinical manifestations of PRPS1 superactivity in women. Methods: Whole exome sequencing was performed in affected females and their fathers. Results: Mutational analysis revealed a new c.520 G > A (p.G174R) mutation in the PRPS1 gene. The mutation resulted in decreased PRPS1 inhibition by ADP. Conclusion: Clinical findings in previously reported females with PRPS1 superactivity showed a high clinical penetrance of this disorder with a mean serum urate level of 8.5 (4.1) mg/dl [506 (247) µmol/l] and a high prevalence of gout. These findings indicate that all women in families with PRPS1 superactivity should be genetically screened for a mutation (for clinical management and genetic counselling). In addition, women with tophaceous gout, gout presenting in childhood, or a strong family history of severe gout should be considered for PRPS1 mutational analysis.

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.

A heterozygous variant in the SLC22A12 gene in a Sri Lanka family associated with mild renal hypouricemia.

BACKGROUND: Renal hypouricemia is a rare heterogeneous inherited disorder characterized by impaired tubular uric acid transport, reabsorption insufficiency and /or acceleration of secretion. The affected individuals are predisposed to nephrolithiasis and recurrent episodes of exercise-induced acute kidney injury. Type 1 is caused by dysfunctional variants in the SLC22A12 gene (URAT1), while type 2 is caused by defects in the SLC2A9 gene (GLUT9). To date, more than 150 patients with the loss-of-function mutations for the SLC22A12 gene have been found (compound heterozygotes and/or homozygotes), most of whom are Japanese and Koreans. CASE PRESENTATION: Herein, we report a nine year old Sri Lankan boy with renal hypouricemia (serum uric acid 97 µmol/L, fractional excretion of uric acid 33%).The sequencing analysis of SLC22A12 revealed a potentially deleterious missense variant c.1400C > T (p.T467 M, rs200104135) in heterozygous state. This variant has been previously identified in homozygous and/or compound heterozygous state with other causative SLC22A12 variant c.1245_1253del (p.L415_G417del) in Roma population. CONCLUSIONS: This is the first identification of a family with mild renal hypouricemia1 associated to the p.T467 M variant. Detailed investigations of urate blood and urine concentrations in patients with unexplained hypouricemia are needed and renal hypouricemia should also be considered in patients other than those from Japan and/or Korea. Our finding confirms an uneven geographical and ethnic distribution of Romany prevalent SLC22A12 variant that need to be considered in Asian patients (population data Genome Aggregation Database: allele frequency in South Asia 0.007055, in East Asia 0.001330).

GWAS of clinically defined gout and subtypes identifies multiple susceptibility loci that include urate transporter genes.

OBJECTIVE: A genome-wide association study (GWAS) of gout and its subtypes was performed to identify novel gout loci, including those that are subtype-specific. METHODS: Putative causal association signals from a GWAS of 945 clinically defined gout cases and 1213 controls from Japanese males were replicated with 1396 cases and 1268 controls using a custom chip of 1961 single nucleotide polymorphisms (SNPs). We also first conducted GWASs of gout subtypes. Replication with Caucasian and New Zealand Polynesian samples was done to further validate the loci identified in this study. RESULTS: In addition to the five loci we reported previously, further susceptibility loci were identified at a genome-wide significance level (p<5.0×10-8): urate transporter genes (SLC22A12 and SLC17A1) and HIST1H2BF-HIST1H4E for all gout cases, and NIPAL1 and FAM35A for the renal underexcretion gout subtype. While NIPAL1 encodes a magnesium transporter, functional analysis did not detect urate transport via NIPAL1, suggesting an indirect association with urate handling. Localisation analysis in the human kidney revealed expression of NIPAL1 and FAM35A mainly in the distal tubules, which suggests the involvement of the distal nephron in urate handling in humans. Clinically ascertained male patients with gout and controls of Caucasian and Polynesian ancestries were also genotyped, and FAM35A was associated with gout in all cases. A meta-analysis of the three populations revealed FAM35A to be associated with gout at a genome-wide level of significance (p meta =3.58×10-8). CONCLUSIONS: Our findings including novel gout risk loci provide further understanding of the molecular pathogenesis of gout and lead to a novel concept for the therapeutic target of gout/hyperuricaemia.

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.

Functional analysis of novel allelic variants in URAT1 and GLUT9 causing renal hypouricemia type 1 and 2.

BACKGROUND: Renal hypouricemia is a rare heterogeneous inherited disorder characterized by impaired tubular uric acid transport with severe complications, such as acute kidney injury and nephrolithiasis. Type 1 is caused by a loss-of-function mutation in the SLC22A12 gene (URAT1), while type 2 is caused by defects in the SLC2A9 gene (GLUT9). METHODS AND RESULTS: In this article we present clinical, biochemical and molecular genetics of two Czech patients. The serum uric acid in the probands was 57 and 98 µmol/l and expressed as an increase in the fractional excretion of uric acid (40 and 18 %). The sequencing analysis of SLC22A12 and SLC2A9 revealed novel variants p.R92C and p.R203C in URAT1 and p.G72D in GLUT9. Functional studies were performed for these novel variants and for previously reported variants p.I118HfsX27, p.G216R and p.N333S in GLUT9 responsible for renal hypouricemia in three probands from Czech Republic and United Kingdom. Functional studies showed significantly decreased urate uptake for all variants. However, urate uptake of GLUT9 variants prepared for both isoforms were not significantly different. CONCLUSIONS: This is the first complex function characterization of non-synonymous allelic variants in patients with renal hypouricemia regarding both GLUT9 isoforms. Our finding of defects in the SLC2A9 and SLC22A12 genes show the following: renal hypouricemia is not restricted to East Asia populations; urate uptake of GLUT9 variants prepared for both isoforms were not significantly different; renal hypouricemia type 2 has more wide clinical variability than type 1; the phenotypic severity of renal hypouricemia is not correlated with results of functional characterizations of URAT1 and GLUT9 variants.

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.