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.
- MeSH
- dna (nemoc) * genetika MeSH
- hyperurikemie * genetika MeSH
- kotransportní proteiny pro sodík a fosfát - typ I * genetika MeSH
- kyselina močová metabolismus MeSH
- lidé MeSH
- přenašeče organických aniontů nezávislé na sodíku * genetika MeSH
- přenašeče organických aniontů * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Outer membrane vesicles (OMVs) carrying virulence factors of enterohemorrhagic Escherichia coli (EHEC) are assumed to play a role in the pathogenesis of life-threatening hemolytic uremic syndrome (HUS). However, it is unknown if and how OMVs, which are produced in the intestinal lumen, cross the intestinal epithelial barrier (IEB) to reach the renal glomerular endothelium, the major target in HUS. We investigated the ability of EHEC O157 OMVs to translocate across the IEB using a model of polarized Caco-2 cells grown on Transwell inserts and characterized important aspects of this process. Using unlabeled or fluorescently labeled OMVs, tests of the intestinal barrier integrity, inhibitors of endocytosis, cell viability assay, and microscopic techniques, we demonstrated that EHEC O157 OMVs translocated across the IEB. OMV translocation involved both paracellular and transcellular pathways and was significantly increased under simulated inflammatory conditions. In addition, translocation was not dependent on OMV-associated virulence factors and did not affect viability of intestinal epithelial cells. Importantly, translocation of EHEC O157 OMVs was confirmed in human colonoids thereby supporting physiological relevance of OMVs in the pathogenesis of HUS.
- Publikační typ
- časopisecké články MeSH
- MeSH
- antibakteriální látky MeSH
- kolaterální senzitivita MeSH
- lidé MeSH
- mikrobiologické techniky * metody MeSH
- odběr biologického vzorku MeSH
- řízení kvality MeSH
- Salmonella enterica izolace a purifikace patogenita MeSH
- Salmonella izolace a purifikace klasifikace patogenita MeSH
- séroskupina MeSH
- Staphylococcus lugdunensis izolace a purifikace patogenita MeSH
- statistika jako téma MeSH
- testování odbornosti laboratoří * metody statistika a číselné údaje MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- zprávy MeSH
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.
- MeSH
- biologický transport MeSH
- dna (nemoc) * genetika metabolismus MeSH
- HEK293 buňky MeSH
- hyperurikemie * genetika MeSH
- kyselina močová metabolismus MeSH
- lidé MeSH
- přenašeče organických aniontů nezávislé na sodíku * genetika MeSH
- protein 1 přenášející organické anionty * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- buněčné klony MeSH
- buňky K562 MeSH
- CRISPR-Cas systémy MeSH
- genový knockout MeSH
- homozygot MeSH
- hyperhomocysteinemie farmakoterapie genetika MeSH
- kultivované buňky MeSH
- kyselina listová terapeutické užití MeSH
- lidé MeSH
- megaloblastová anemie farmakoterapie genetika MeSH
- mladiství MeSH
- posunová mutace MeSH
- recidiva MeSH
- sekvenční delece MeSH
- sekvenování exomu MeSH
- sodíko-vodíkový výměnný transportér 1 nedostatek genetika MeSH
- vitamin B 12 terapeutické užití MeSH
- Check Tag
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- Publikační typ
- dopisy MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
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
- MeSH
- ABC transportér z rodiny G, člen 2 genetika metabolismus MeSH
- běloši genetika MeSH
- biologický transport MeSH
- dítě MeSH
- dna (nemoc) genetika krev metabolismus MeSH
- dospělí MeSH
- genetická predispozice k nemoci MeSH
- HEK293 buňky MeSH
- hyperurikemie genetika krev MeSH
- jednonukleotidový polymorfismus MeSH
- kohortové studie MeSH
- kyselina močová krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- nádorové proteiny MeSH
- předškolní dítě MeSH
- přenašeče organických aniontů MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Geografické názvy
- Česká republika MeSH
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.
- MeSH
- dítě MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- močové kameny genetika MeSH
- mutační analýza DNA MeSH
- přenašeče organických aniontů genetika MeSH
- proteiny přenášející organické kationty genetika MeSH
- proteiny usnadňující transport glukosy genetika MeSH
- vrozené poruchy tubulárního transportu genetika MeSH
- Xenopus MeSH
- zvířata MeSH
- Check Tag
- dítě MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
OBJECTIVE: Using European descent Czech populations, we performed a study of SLC2A9 and SLC22A12 genes previously identified as being associated with serum uric acid concentrations and gout. This is the first study of the impact of non-synonymous allelic variants on the function of GLUT9 except for patients suffering from renal hypouricemia type 2. METHODS: The cohort consisted of 250 individuals (150 controls, 54 nonspecific hyperuricemics and 46 primary gout and/or hyperuricemia subjects). We analyzed 13 exons of SLC2A9 (GLUT9 variant 1 and GLUT9 variant 2) and 10 exons of SLC22A12 by PCR amplification and sequenced directly. Allelic variants were prepared and their urate uptake and subcellular localization were studied by Xenopus oocytes expression system. The functional studies were analyzed using the non-parametric Wilcoxon and Kruskall-Wallis tests; the association study used the Fisher exact test and linear regression approach. RESULTS: We identified a total of 52 sequence variants (12 unpublished). Eight non-synonymous allelic variants were found only in SLC2A9: rs6820230, rs2276961, rs144196049, rs112404957, rs73225891, rs16890979, rs3733591 and rs2280205. None of these variants showed any significant difference in the expression of GLUT9 and in urate transport. In the association study, eight variants showed a possible association with hyperuricemia. However, seven of these were in introns and the one exon located variant, rs7932775, did not show a statistically significant association with serum uric acid concentration. CONCLUSION: Our results did not confirm any effect of SLC22A12 and SLC2A9 variants on serum uric acid concentration. Our complex approach using association analysis together with functional and immunohistochemical characterization of non-synonymous allelic variants did not show any influence on expression, subcellular localization and urate uptake of GLUT9.
- MeSH
- alely MeSH
- běloši MeSH
- biologický transport MeSH
- dna (nemoc) genetika patologie MeSH
- dospělí MeSH
- exprese genu MeSH
- frekvence genu MeSH
- hyperurikemie genetika patologie MeSH
- jednonukleotidový polymorfismus * MeSH
- lidé středního věku MeSH
- lidé MeSH
- přenašeče organických aniontů genetika MeSH
- proteiny přenášející organické kationty genetika MeSH
- proteiny usnadňující transport glukosy genetika MeSH
- senioři MeSH
- studie případů a kontrol MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
