Abiodun Onipinla

SLC2A9 Is a High-Capacity Urate Transporter in Humans

Background Serum uric acid levels in humans are influenced by diet, cellular breakdown, and renal elimination, and correlate with blood pressure, metabolic syndrome, diabetes, gout, and cardiovascular disease. Recent genome-wide association scans have found common genetic variants of SLC2A9 to be associated with increased serum urate level and gout. The SLC2A9 gene encodes a facilitative glucose transporter, and it has two splice variants that are highly expressed in the proximal nephron, a key site for urate handling in the kidney. We investigated whether SLC2A9 is a functional urate transporter that contributes to the longstanding association between urate and blood pressure in man. Methods and Findings We expressed both SLC2A9 splice variants in Xenopus laevis oocytes and found both isoforms mediate rapid urate fluxes at concentration ranges similar to physiological serum levels (200–500 μM). Because SLC2A9 is a known facilitative glucose transporter, we also tested whether glucose or fructose influenced urate transport. We found that urate is transported by SLC2A9 at rates 45- to 60-fold faster than glucose, and demonstrated that SLC2A9-mediated urate transport is facilitated by glucose and, to a lesser extent, fructose. In addition, transport is inhibited by the uricosuric benzbromarone in a dose-dependent manner (K i = 27 μM). Furthermore, we found urate uptake was at least 2-fold greater in human embryonic kidney (HEK) cells overexpressing SLC2A9 splice variants than nontransfected kidney cells. To confirm that our findings were due to SLC2A9, and not another urate transporter, we showed that urate transport was diminished by SLC2A9-targeted siRNA in a second mammalian cell line. In a cohort of men we showed that genetic variants of SLC2A9 are associated with reduced urinary urate clearance, which fits with common variation at SLC2A9 leading to increased serum urate. We found no evidence of association with hypertension (odds ratio 0.98, 95% confidence interval [CI] 0.9 to 1.05, p > 0.33) by meta-analysis of an SLC2A9 variant in six case–control studies including 11,897 participants. In a separate meta-analysis of four population studies including 11,629 participants we found no association of SLC2A9 with systolic (effect size −0.12 mm Hg, 95% CI −0.68 to 0.43, p = 0.664) or diastolic blood pressure (effect size −0.03 mm Hg, 95% CI −0.39 to 0.31, p = 0.82). Conclusions This study provides evidence that SLC2A9 splice variants act as high-capacity urate transporters and is one of the first functional characterisations of findings from genome-wide association scans. We did not find an association of the SLC2A9 gene with blood pressure in this study. Our findings suggest potential pathogenic mechanisms that could offer a new drug target for gout.

Genome-wide scan identifies CDH13 as a novel susceptibility locus contributing to blood pressure determination in two European populations

Hypertension is a complex disease that affects a large proportion of adult population. Although approximately half of the inter-individual variance in blood pressure (BP) level is heritable, identification of genes responsible for its regulation has remained challenging. Genome-wide association study (GWAS) is a novel approach to search for genetic variants contributing to complex diseases. We conducted GWAS for three BP traits [systolic and diastolic blood pressure (SBP and DBP); hypertension (HYP)] in the Kooperative Gesundheitsforschung in der Region Augsburg (KORA) S3 cohort (n = 1644) recruited from general population in Southern Germany. GWAS with 395 912 single nucleotide polymorphisms (SNPs) identified an association between BP traits and a common variant rs11646213 (T/A) upstream of the CDH13 gene at 16q23.3. The initial associations with HYP and DBP were confirmed in two other European population-based cohorts: KORA S4 (Germans) and HYPEST (Estonians). The associations between rs11646213 and three BP traits were replicated in combined analyses (dominant model: DBP, P = 5.55 × 10–5, effect –1.40 mmHg; SBP, P = 0.007, effect –1.56 mmHg; HYP, P = 5.30 × 10−8, OR = 0.67). Carriers of the minor allele A had a decreased risk of hypertension. A non-significant trend for association was also detected with severe family based hypertension in the BRIGHT sample (British). The novel susceptibility locus, CDH13, encodes for an adhesion glycoprotein T-cadherin, a regulator of vascular wall remodeling and angiogenesis. Its function is compatible with the BP biology and may improve the understanding of the pathogenesis of hypertension.

A population study of ethnic variations in the angiotensin-converting enzyme I/D polymorphism: relationships with gender, hypertension and impaired glucose metabolism.

BACKGROUND The presence of the deletion allele of the angiotensin-converting enzyme (ACE) I/D polymorphism is associated with an excess risk of vascular disease and diabetic nephropathy. OBJECTIVE To examine the importance of this polymorphism as a determinant of hypertension and impaired glucose metabolism in a population-based study of three ethnic groups and assess the potential modifying effect of gender. DESIGN Population-based cross-sectional study in South London. The population-based sample of 1577 men and women, age 40-59 years, was obtained from stratified random sampling of general practice lists where 25% of the residents were born outside the UK. The ACE I/D polymorphism was determined for 1366 individuals (86.6%): 462 whites, 462 of African descent and 442 of South Asian origin. RESULTS The genotype frequency within each ethnic group was in Hardy-Weinberg equilibrium. The frequencies were similar in whites and those of African descent (II, ID, DD: 18.4%, 49.6%, 32.0% for whites and 18.4%, 50.5%, 30.9% for those of African descent), but there was a much higher frequency of the II genotype in those of South Asian origin (39.8%, 41.8%, 18.3%; chi2 = 77.6; P < 0.0001). There was no association between the I/D polymorphism and impaired glucose metabolism in any ethnic group. There were also no significant associations between the I/D polymorphism and hypertension in whites and in those of South Asian origin. This contrasts with a highly significant association between the D allele and hypertension in women of African descent (OR = 2.54; 95% CI 1.38-4.65; P = 0.003) but not in men of African descent (0.79; 0.36-1.72) (test for differences between sexes P = 0.023). CONCLUSIONS These observations provide estimates of the frequency distribution of the ACE I/D polymorphism in whites, in people of African descent and in people of South Asian origin. Moreover, these results highlight the potential importance of gender-dependent interactions between genetic background and expression of hypertensive phenotype.

Targeting 160 Candidate Genes for Blood Pressure Regulation with a Genome-Wide Genotyping Array

The outcome of Genome-Wide Association Studies (GWAS) has challenged the field of blood pressure (BP) genetics as previous candidate genes have not been among the top loci in these scans. We used Affymetrix 500K genotyping data of KORA S3 cohort (n = 1,644; Southern-Germany) to address (i) SNP coverage in 160 BP candidate genes; (ii) the evidence for associations with BP traits in genome-wide and replication data, and haplotype analysis. In total, 160 gene regions (genic region±10 kb) covered 2,411 SNPs across 11.4 Mb. Marker densities in genes varied from 0 (n = 11) to 0.6 SNPs/kb. On average 52.5% of the HAPMAP SNPs per gene were captured. No evidence for association with BP was obtained for 1,449 tested SNPs. Considerable associations (P<10−3) were detected for the genes, where >50% of HAPMAP SNPs were tagged. In general, genes with higher marker density (>0.2 SNPs/kb) revealed a better chance to reach close to significance associations. Although, none of the detected P-values remained significant after Bonferroni correction (P<0.05/2319, P<2.15×10−5), the strength of some detected associations was close to this level: rs10889553 (LEPR) and systolic BP (SBP) (P = 4.5×10−5) as well as rs10954174 (LEP) and diastolic BP (DBP) (P = 5.20×10−5). In total, 12 markers in 7 genes (ADRA2A, LEP, LEPR, PTGER3, SLC2A1, SLC4A2, SLC8A1) revealed considerable association (P<10−3) either with SBP, DBP, and/or hypertension (HYP). None of these were confirmed in replication samples (KORA S4, HYPEST, BRIGHT). However, supportive evidence for the association of rs10889553 (LEPR) and rs11195419 (ADRA2A) with BP was obtained in meta-analysis across samples stratified either by body mass index, smoking or alcohol consumption. Haplotype analysis highlighted LEPR and PTGER3. In conclusion, the lack of associations in BP candidate genes may be attributed to inadequate marker coverage on the genome-wide arrays, small phenotypic effects of the loci and/or complex interaction with life-style and metabolic parameters.

Contrasting associations between aldosterone synthase gene polymorphisms and essential hypertension in blacks and in whites.

Background Genetic variability in the gene for aldosterone synthase – a key enzyme in the production of aldosterone – can affect sodium homeostasis and thereby blood pressure. The possibility of impaired aldosterone production for the development of hypertension is of particular relevance in black Afro-Caribbeans exposed to a high dietary sodium intake. Objectives To compare the frequency of three variants (−344C/T, intron 2 conversion, and the K173R polymorphism) of the aldosterone synthase gene in blacks and whites, and to determine any association of the variants with hypertension. Design and methods We made case–control comparisons of the three gene variants in relation to ethnic background and to essential hypertension in 193 white (51% hypertensive) and 245 black individuals (59% hypertensive) living in south London. Results The frequency of each of the variants was significantly different between the two ethnic groups. The T and the K alleles were more frequent in the black participants (79 compared with 50% for the −344T allele and 81 compared with 50% for K173 allele), whereas the frequency of the intron 2 conversion allele was much lower in that group (8 compared with 38%). None of these variants was associated with essential hypertension in the black participants. In contrast, in the white participants there was a significant and graded association between the intron 2 conversion allele and essential hypertension (odds ratio 1.86, 95% confidence interval 1.16 to 2.98; P = 0.01). Moreover, among the white population, the presence of homozygosity both of the T allele and of the intron 2 conversion alleles was associated with a much greater frequency of hypertension (71 compared with 43%; χ2 P = 0.03). Conclusions The contrasting associations between these variants and essential hypertension do not necessarily exclude the possibility that other, as yet undefined, variants of the aldosterone synthase gene could be linked with hypertension in black people. Nonetheless, the strong association between the intron 2 conversion allele and essential hypertension in the white population reinforces the view that the increased blood pressure may be due, at least in part, to abnormal expression of enzymes involved in the metabolism of adrenal mineralocorticoids.

Polymorphisms in the WNK1 gene are associated with blood pressure variation and urinary potassium excretion.

WNK1 - a serine/threonine kinase involved in electrolyte homeostasis and blood pressure (BP) control - is an excellent candidate gene for essential hypertension (EH). We and others have previously reported association between WNK1 and BP variation. Using tag SNPs (tSNPs) that capture 100% of common WNK1 variation in HapMap, we aimed to replicate our findings with BP and to test for association with phenotypes relating to WNK1 function in the British Genetics of Hypertension (BRIGHT) study case-control resource (1700 hypertensive cases and 1700 normotensive controls). We found multiple variants to be associated with systolic blood pressure, SBP (7/28 tSNPs min-p = 0.0005), diastolic blood pressure, DBP (7/28 tSNPs min-p = 0.002) and 24 hour urinary potassium excretion (10/28 tSNPs min-p = 0.0004). Associations with SBP and urine potassium remained significant after correction for multiple testing (p = 0.02 and p = 0.01 respectively). The major allele (A) of rs765250, located in intron 1, demonstrated the strongest evidence for association with SBP, effect size 3.14 mmHg (95%CI:1.23–4.9), DBP 1.9 mmHg (95%CI:0.7–3.2) and hypertension, odds ratio (OR: 1.3 [95%CI: 1.0–1.7]).We genotyped this variant in six independent populations (n = 14,451) and replicated the association between rs765250 and SBP in a meta-analysis (p = 7×10−3, combined with BRIGHT data-set p = 2×10−4, n = 17,851). The associations of WNK1 with DBP and EH were not confirmed. Haplotype analysis revealed striking associations with hypertension and BP variation (global permutation p<10−7). We identified several common haplotypes to be associated with increased BP and multiple low frequency haplotypes significantly associated with lower BP (>10 mmHg reduction) and risk for hypertension (OR<0.60). Our data indicates that multiple rare and common WNK1 variants contribute to BP variation and hypertension, and provide compelling evidence to initiate further genetic and functional studies to explore the role of WNK1 in BP regulation and EH.

Increased Support for Linkage of a Novel Locus on Chromosome 5q13 for Essential Hypertension in the British Genetics of Hypertension Study

Human hypertension arises from a combination of genetic factors and lifestyle influences. With cardiovascular disease set to become the number 1 cause of death worldwide, it is important to understand the etiologic mechanisms for hypertension, because these might provide new routes to improved treatment. The British Genetics of Hypertension Study has recently published a primary genome screen that identified 4 chromosomal regions of interest. We have now genotyped additional markers to confirm the most promising regions for follow-up studies. Thirty-four additional microsatellites were genotyped in our severely hypertensive affected sibling pair resource (now 1635 families with 2142 affected sibling pairs), leading to a substantial increase in information content in the regions of interest. We found increased support for linkage of chromosome 5q13 to human hypertension (multipoint logarithm of odds=2.50) with 3 adjacent markers yielding single point logarithm of odds scores of 3.22, 2.84, and 2.51. The placement of additional markers on 2q, 6q, and 9q diminished support for linkage in these regions. However, the addition of new data and families identified novel regions of interest on chromosomes 1q and 11q. The 3 positive markers in the chromosome 5 region were also genotyped in 712 distinct parent–offspring trios with the same severe phenotype to replicate linkage and association. Borderline support for replication was found (P=0.07). We found increased evidence for linkage and borderline-significant evidence for association for a hypertension susceptibility locus on chromosome 5q13 that is worthy of detailed fine mapping and assessment of candidate genes.

Differences between disease-associated endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms in cellular expression, interactions with tumour necrosis factor receptor 1 (TNF-R1) and regulation by cytokines.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) processes peptides for major histocompatibility complex (MHC) class I presentation and promotes cytokine receptor ectodomain shedding. These known functions of ERAP1 may explain its genetic association with several autoimmune inflammatory diseases. In this study, we identified four novel alternatively spliced variants of ERAP1 mRNA, designated as ΔExon‐11, ΔExon‐13, ΔExon‐14 and ΔExon‐15. We also observed a rapid and differential modulation of ERAP1 mRNA levels and spliced variants in different cell types pretreated with lipopolysaccharide (LPS). We have studied three full‐length allelic forms of ERAP1 (R127‐K528, P127‐K528, P127‐R528) and one spliced variant (ΔExon‐11) and assessed their interactions with tumour necrosis factor receptor 1 (TNF‐R1) in transfected cells. We observed variation in cellular expression of different ERAP1 isoforms, with R127‐K528 being expressed at a much lower level. Furthermore, the cellular expression of full‐length P127‐K528 and ΔExon‐11 spliced variant was enhanced significantly when co‐transfected with TNF‐R1. Isoforms P127‐K528, P127‐R528 and ΔExon‐11 spliced variant associated with TNF‐R1, and this interaction occurred in a region within the first 10 exons of ERAP1. Supernatant‐derived vesicles from transfected cells contained the full‐length and ectodomain form of soluble TNF‐R1, as well as carrying the full‐length ERAP1 isoforms. We observed marginal differences between TNF‐R1 ectodomain levels when co‐expressed with individual ERAP1 isoforms, and treatment of transfected cells with tumour necrosis factor (TNF), interleukin (IL)‐1β and IL‐10 exerted variable effects on TNF‐R1 ectodomain cleavage. Our data suggest that ERAP1 isoforms may exhibit differential biological properties and inflammatory mediators could play critical roles in modulating ERAP1 expression, leading to altered functional activities of this enzyme.

Recent advances in the identification of genes for human hypertension

It is a well-established fact that genes are involved in the etiology of hypertension. However, identification of the gene variants still remains a challenge. Over the years, different approaches and technologies, including genome-wide scans, case-control association studies, experiments on inbred rodent models and expression profiling, have been utilized to elucidate hypertension susceptibility genes, but so far the results have been equivocal. During the last year, further chromosomal regions harboring blood pressure loci have been identified, and transcriptomics has been applied to aid the identification of disease genes. There are great expectations for the future with regards to further advancements in transcriptomics and proteomics. This review reports primarily on work that has been carried out in the last 12 months in the field, and considers its contribution towards a better understanding of the genetic mechanisms involved in blood pressure regulation and hypertension.