Maris Laan

Genome-wide association study of blood pressure extremes identifies variant near UMOD associated with hypertension

Hypertension is a heritable and major contributor to the global burden of disease. The sum of rare and common genetic variants robustly identified so far explain only 1%–2% of the population variation in BP and hypertension. This suggests the existence of more undiscovered common variants. We conducted a genome-wide association study in 1,621 hypertensive cases and 1,699 controls and follow-up validation analyses in 19,845 cases and 16,541 controls using an extreme case-control design. We identified a locus on chromosome 16 in the 5′ region of Uromodulin (UMOD; rs13333226, combined P value of 3.6×10−11). The minor G allele is associated with a lower risk of hypertension (OR [95%CI]: 0.87 [0.84–0.91]), reduced urinary uromodulin excretion, better renal function; and each copy of the G allele is associated with a 7.7% reduction in risk of CVD events after adjusting for age, sex, BMI, and smoking status (H.R. = 0.923, 95% CI 0.860–0.991; p = 0.027). In a subset of 13,446 individuals with estimated glomerular filtration rate (eGFR) measurements, we show that rs13333226 is independently associated with hypertension (unadjusted for eGFR: 0.89 [0.83–0.96], p = 0.004; after eGFR adjustment: 0.89 [0.83–0.96], p = 0.003). In clinical functional studies, we also consistently show the minor G allele is associated with lower urinary uromodulin excretion. The exclusive expression of uromodulin in the thick portion of the ascending limb of Henle suggests a putative role of this variant in hypertension through an effect on sodium homeostasis. The newly discovered UMOD locus for hypertension has the potential to give new insights into the role of uromodulin in BP regulation and to identify novel drugable targets for reducing cardiovascular risk.

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.

Genomewide association study using a high-density single nucleotide polymorphism array and case-control design identifies a novel essential hypertension susceptibility locus in the promoter region of endothelial NO synthase

Essential hypertension is a multifactorial disorder and is the main risk factor for renal and cardiovascular complications. The research on the genetics of hypertension has been frustrated by the small predictive value of the discovered genetic variants. The HYPERGENES Project investigated associations between genetic variants and essential hypertension pursuing a 2-stage study by recruiting cases and controls from extensively characterized cohorts recruited over many years in different European regions. The discovery phase consisted of 1865 cases and 1750 controls genotyped with 1M Illumina array. Best hits were followed up in a validation panel of 1385 cases and 1246 controls that were genotyped with a custom array of 14 055 markers. We identified a new hypertension susceptibility locus (rs3918226) in the promoter region of the endothelial NO synthase gene (odds ratio: 1.54 [95% CI: 1.37–1.73]; combined P=2.58 · 10−13). A meta-analysis, using other in silico/de novo genotyping data for a total of 21 714 subjects, resulted in an overall odds ratio of 1.34 (95% CI: 1.25–1.44; P=1.032 · 10−14). The quantitative analysis on a population-based sample revealed an effect size of 1.91 (95% CI: 0.16–3.66) for systolic and 1.40 (95% CI: 0.25–2.55) for diastolic blood pressure. We identified in silico a potential binding site for ETS transcription factors directly next to rs3918226, suggesting a potential modulation of endothelial NO synthase expression. Biological evidence links endothelial NO synthase with hypertension, because it is a critical mediator of cardiovascular homeostasis and blood pressure control via vascular tone regulation. This finding supports the hypothesis that there may be a causal genetic variation at this locus.

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.

Extensive Linkage Disequilibrium in Small Human Populations in Eurasia

The extent of linkage disequilibrium (LD) was studied in two small food-gathering populations-Evenki and Saami-and two larger food-producing populations-Finns and Swedes-in northern Eurasia. In total, 50 single-nucleotide polymorphisms (SNPs) from five genes were genotyped using real-time pyrophosphate DNA sequencing, whereas 14 microsatellites were genotyped in two X-chromosomal regions. In addition, hypervariable region I of the mtDNA was sequenced to shed light on the demographic history of the populations. The SNP data, as well as the microsatellite data, reveal extensive levels of LD in Evenki and Saami when compared to Finns and Swedes. mtDNA-sequence variation is compatible with constant population size over time in Evenki and Saami but indicates population expansion in Finns and Swedes. Furthermore, the similarity between Finns and Swedes in SNP allele- and haplotype-frequency distributions indicate that these two populations may share a recent common origin. These findings suggest that populations such as the Evenki and the Saami, rather than the Finns, may be particularly suited for the initial coarse mapping of common complex diseases.

Genetics of Recurrent Miscarriage: Challenges, Current Knowledge, Future Directions

Recurrent miscarriage (RM) occurs in 1–3% of couples aiming at childbirth. Due to multifactorial etiology the clinical diagnosis of RM varies. The design of genetic/“omics” studies to identify genes and biological mechanisms involved in pathogenesis of RM has challenges as there are several options in defining the study subjects (female patient and/or couple with miscarriages, fetus/placenta) and controls. An ideal study would attempt a trio-design focusing on both partners as well as pregnancies of the couple. Application of genetic association studies focusing on pre-selected candidate genes with potential pathological effect in RM show limitations. Polymorphisms in ∼100 genes have been investigated and association with RM is often inconclusive or negative. Also, implication of prognostic molecular diagnostic tests in clinical practice exhibits uncertainties. Future directions in investigating biomolecular risk factors for RM rely on integrating alternative approaches (SNPs, copy number variations, gene/protein expression, epigenetic regulation) in studies of single genes as well as whole-genome analysis. This would be enhanced by collaborative network between research centers and RM clinics.

Combined Effects of the Variants FSHB −211G>T and FSHR 2039A>G on Male Reproductive Parameters

CONTEXT A polymorphism in the FSHB promoter (-211G>T, rs10835638) was shown to influence male serum FSH levels, whereas a polymorphism in the FSH receptor gene (FSHR; 2039A>G, rs6166) was previously shown to be associated with FSH levels in women only. OBJECTIVE The objective of the study was to analyze the effects of both FSHB -211G>T and FSHR 2039A>G on male reproductive parameters. DESIGN AND SETTING A total of 1213 German men attending an infertility clinic were genotyped by TaqMan assay. PATIENTS Patients included male partners in infertile couples without known causes for male infertility. MAIN OUTCOME MEASURES An association analysis of single and combined single-nucleotide polymorphism genotypes with clinical parameters was performed. RESULTS The FSHB -211G>T T-allele showed significant dosage effects for FSH (-0.51 U/liter per T-allele), LH (0.28 U/liter), and bitesticular volume (-3.2 ml). Statistical significance was enhanced severalfold after a meta-analysis comprising 3017 men. TT carriers were significantly more prevalent among men with lower sperm counts. The FSHR 2039A>G G-allele exhibited nonsignificant trends for associations with higher FSH and reduced testicular volumes. However, in the combined model, FSHR 2039A>G significantly modulated the more dominant effect of FSHB -211G>T on serum FSH and testicular volume among the T-allele carriers. CONCLUSIONS By analyzing both single-nucleotide polymorphisms for the first time, we convincingly show that indeed FSHR 2039A>G has an effect also in males. In the proposed model of the combined effects, FSHB -211G>T acts strongly on male reproductive parameters, whereas the FSHR 2039A>G effects were approximately 2-3 times smaller. Clinically this is of importance because oligozoospermic patients carrying unfavorable variants affecting FSH action may benefit from FSH treatment.

Natural antisense transcript of natriuretic peptide precursor A (NPPA): structural organization and modulation of NPPA expression.

BackgroundMammalian transcriptome contains a large proportion of diverse and structurally complex noncoding RNAs. One class of such RNAs, natural antisense transcripts (NATs), are derived from the opposite strand of many protein-coding genes. Although the exact structure and functional relevance of most of the NATs is unknown, their emerging role as gene expression regulators raises the hypothesis that NATs might contribute to development of complex human disorders. The goal of our study was to investigate the involvement of NATs in regulation of candidate genes for blood pressure.ResultsFirst we analysed blood pressure candidate genes for the presence of natural antisense transcripts. In silico analysis revealed that seven genes (ADD3, NPPA, ATP1A1, NPR2, CYP17A1, ACSM3, SLC14A2) have an antisense partner transcribed from the opposite strand. We characterized NPPA and its antisense transcript (NPPA-AS) in more detail. We found that NPPA-AS is expressed in a number of human tissues as a collection of alternatively spliced isoforms and that NPPA-AS and NPPA can form RNA duplexes in vivo. We also demonstrated that a specific NPPA-AS isoform is capable of down-regulating the intron-retained NPPA mRNA variant. We studied the evolutionary conservation of NPPA-AS and were able to detect the presence of Nppa-as transcript in mouse.ConclusionOur results demonstrate functional interaction of NPPA-AS with NPPA at the RNA level and suggest that antisense transcription might be an important post-transcriptional mechanism modulating NPPA expression.

FSHB promoter polymorphism within evolutionary conserved element is associated with serum FSH level in men

BACKGROUND No polymorphisms affecting serum FSH levels have been described in the human FSHB gene. We have identified a potential regulatory single nucleotide polymorphism (SNP, rs10835638; G/T) 211 bp upstream from the FSHB mRNA transcription start-site, located within a highly conserved region among placental mammals. We aimed to determine the correlation of carrier status of rs10835638 alternative alleles with serum FSH level in men, and testicular and hormonal parameters. METHODS A quantitative genetic association study using a cohort of healthy men (n = 554; age 19.2 ± 1.7 years) visiting the Centre of Andrology, Tartu University Hospital, Estonia. RESULTS Rs10835638 (allele frequencies: G 87.6%, T 12.4%) was significantly associated with serum FSH level (analysis of variance: F = 13.0, P = 0.0016, df = 1; regression testing for a linear trend: P = 0.0003). Subjects with the GG genotype exhibited higher FSH levels (3.37 ± 1.79 IU/l, n = 423) compared with heterozygotes (2.84 ± 1.54 IU/l, n = 125) (P = 0.0005), the group of T-allele carriers (GT+TT, 2.78 ± 1.51 IU/l, n = 131) (P = 0.0005) and TT-homozygotes (2.02 ± 0.81 IU/L, n = 6) (P = 0.031). Rs10835638 was also associated with significant (P < 0.05) reduction in free testosterone index and testes volume, but increased semen volume, sex hormone-binding globulin, serum testosterone and estradiol. LH and inhibin-B levels did not differ significantly between groups. CONCLUSIONS The identification of a regulatory SNP in FSHB promoter paves the way to study the effect of constitutively low FSH on male health and fertility. As FSH contributes to follicular development and sex steroid production in women, the role of this FSHB variant in female reproductive success is still to be addressed.