Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls.

Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed approximately 19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated approximately 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with disease-IRGM for Crohn's disease, HLA for Crohn's disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetes-although in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases.

Characterization of the molecular genetic pathology in patients with 11ß-hydroxylase deficiency.

OBJECTIVE: Steroid 11ß-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia. Nonclassic or mild 11OHD appears to be a rare condition. Our study assessed the residual CYP11B1 function of detected mutations, adding to the spectrum of mild 11OHD, and illustrates the variability of the clinical presentation of 11OHD. PATIENTS AND METHODS: Five patients presented with mild to moderate 11OHD. Two women presented with mild hirsutism and in one case with secondary amenorrhoea. Two men presented with precocious pseudopuberty, gynaecomastia and elevated blood pressure. One 46,XX female patient was diagnosed with virilization of the external genitalia 2 years after birth. Direct DNA sequencing was carried out to perform CYP11B1 mutation analysis. The CYP11B1 mutations were functionally characterized using an in vitro expression system. RESULTS: CYP11B1-inactivating mutations were detected in all patients. Two novel missense mutations (p.P42L and p.A297V) and the previously characterized p.R143W mutation had residual CYP11B1 activities between 10% and 27%. A novel p.L382R and the previously uncharacterized p.G444D mutation both caused complete loss of CYP11B1 enzymatic activity. CONCLUSION: Mutations causing partial impairment of 11ß-hydroxylase activity (residual activity of 10% or above) are associated with a less severe clinical presentation of 11OHD, which can be classified as a nonclassic form. Our data demonstrate that patients with nonclassic 11OHD can present with androgen excess, precocious pseudopuberty and increased blood pressure. Timely diagnosis of nonclassic 11OHD and consequently initiation of personalized treatment is essential to prevent co-morbidities caused by androgen excess and hypertension.

Quantitative variation in plasma angiotensin-I converting enzyme activity shows allelic heterogeneity in the ABO blood group locus.

Angiotensin-I converting enzyme (ACE) occupies a pivotal role in cardiovascular homeostasis. Major loci for plasma ACE have been identified at ACE on Chromosome 17 and at ABO on Chromosome 9. We sought to characterise the genetic architecture of plasma ACE at finer resolution in two populations. We carried out a GWAS in 1810 individuals of Japanese ethnicity; this identified signals at ACE and ABO that together accounted for nearly half of the population variability of the trait. We conducted measured haplotype analysis at the ABO locus in 1425 members of 248 British families using haplotypes of three SNPs, which together tagged the alleles responsible for the principal blood group antigens A1, A2, B and O. Type O alleles were associated with intermediate plasma ACE activity compared to Type A1 alleles (in whom plasma ACE activity was ∼36% lower) and Type B alleles (in whom plasma ACE activity was ∼36% higher). We demonstrated heterogeneity among A alleles: A2 alleles were associated with plasma ACE activity that was very similar to the O alleles. Variation at ACE accounted for 35% of the trait variance, and variation at ABO accounted for 15%. A further 10% could be ascribed to polygenic effects.

Urinary sodium excretion is the main determinant of mineralocorticoid excretion rates in patients with chronic kidney disease.

BACKGROUND: Blockade of the mineralocorticoid receptor (MR) in patients with chronic kidney disease (CKD) improves surrogate cardiovascular outcomes, such as left ventricular mass. Animal models of renal disease support a pathological role of mineralocorticoids, in the context of a high sodium intake. We aimed to assess the regulation of mineralocorticoid biosynthesis in patients with CKD. METHODS: Seventy patients with CKD stages 3/4 and 30 patients with essential hypertension (EH) were recruited. Patients underwent detailed clinical phenotyping, drug history and biochemical assessment. Patients completed a 24-h urine collection for measurement of urinary tetrahydroaldosterone (THALDO) and tetrahydrocorticosterone (THDOC) excretion rates (measured using gas chromatography-mass spectrometry) and urinary electrolytes. The factors which correlated significantly with THALDO and THDOC excretion were entered into linear regression models. RESULTS: Patients with EH and CKD were well matched with no significant differences in gender, age or weight. The mean estimated glomerular filtration rate (eGFR) in CKD patients was 38.6/min/1.73 m(2). The mean urinary excretion rates of THALDO, THDOC and 24-h urinary sodium (24-h USod) were not significantly different between CKD and EH patients. The level of renal function did not correlate with THALDO or THDOC excretion. In patients with CKD, 24-h USodium (r = 0.614, P < 0.001) and 24-h UPotassium (r = 0.538, P < 0.001) were positively correlated with THALDO excretion. On multivariate linear regression analysis, 24-h USod was the strongest independent predictor (P = 0.004) of THALDO and THDOC excretion in CKD. In patients with EH, no relationship was seen between mineralocorticoid excretion and 24-h urinary sodium excretion. CONCLUSIONS: In patients with CKD, 24-h urinary sodium excretion is the strongest positive predictor of urinary mineralocorticoid excretion. The nature of this relationship is unexpected, novel, not seen in patients with EH and may explain the association seen between high urinary sodium excretion, mineralocorticoids and poor outcomes in patients with CKD.

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.

A lifetime of aldosterone excess: long-term consequences of altered regulation of aldosterone production for cardiovascular function.

Up to 15% of patients with essential hypertension have inappropriate regulation of aldosterone; although only a minority have distinct adrenal tumors, recent evidence shows that mineralocorticoid receptor activation contributes to the age-related blood pressure rise and illustrates the importance of aldosterone in determining cardiovascular risk. Aldosterone also has a major role in progression and outcome of ischemic heart disease. These data highlight the need to understand better the regulation of aldosterone synthesis and its action. Aldosterone effects are mediated mainly through classical nuclear receptors that alter gene transcription. In classic epithelial target tissues, signaling mechanisms are relatively well defined. However, aldosterone has major effects in nonepithelial tissues that include increased synthesis of proinflammatory molecules and reactive oxygen species; it remains unclear how these effects are controlled and how receptor specificity is maintained. Variation in aldosterone production reflects interaction of genetic and environmental factors. Although the environmental factors are well understood, the genetic control of aldosterone synthesis is still the subject of debate. Aldosterone synthase (encoded by the CYP11B2 gene) controls conversion of deoxycorticosterone to aldosterone. Polymorphic variation in CYP11B2 is associated with increased risk of hypertension, but the molecular mechanism that accounts for this is not known. Altered 11beta-hydroxylase efficiency (conversion of deoxycortisol to cortisol) as a consequence of variation in the neighboring gene (CYP11B1) may be important in contributing to altered control of aldosterone synthesis, so that the risk of hypertension may reflect a digenic effect, a concept that is discussed further. There is evidence that a long-term increase in aldosterone production from early life is determined by an interaction of genetic and environmental factors, leading to the eventual phenotypes of aldosterone-associated hypertension and cardiovascular damage in middle age and beyond. The importance of aldosterone has generated interest in its therapeutic modulation. Disadvantages associated with spironolactone (altered libido, gynecomastia) have led to a search for alternative mineralocorticoid receptor antagonists. Of these, eplerenone has been shown to reduce cardiovascular risk after myocardial infarction. The benefits and disadvantages of this therapeutic approach are discussed.

Urinary corticosteroid excretion predicts left ventricular mass and proteinuria in chronic kidney disease.

Blockade of the MR (mineralocorticoid receptor) in CKD (chronic kidney disease) reduces LVMI [LV (left ventricular) mass index] and proteinuria. The MR can be activated by aldosterone, cortisol and DOC (deoxycorticosterone). The aim of the present study was to explore the influence of mineralocorticoids on LVMI and proteinuria in patients with CKD. A total of 70 patients with CKD and 30 patients with EH (essential hypertension) were recruited. Patients underwent clinical phenotyping; biochemical assessment and 24 h urinary collection for THAldo (tetrahydroaldosterone), THDOC (tetrahydrodeoxycorticosterone), cortisol metabolites (measured using GC-MS), and urinary electrolytes and protein [QP (proteinuira quantification)]. LVMI was measured using CMRI (cardiac magnetic resonance imaging). Factors that correlated significantly with LVMI and proteinuria were entered into linear regression models. In patients with CKD, significant predictors of LVMI were male gender, SBP (systolic blood pressure), QP, and THAldo and THDOC excretion. Significant independent predictors on multivariate analysis were THDOC excretion, SBP and male gender. In EH, no association was seen between THAldo or THDOC and LVMI; plasma aldosterone concentration was the only significant independent predictor. Significant univariate determinants of proteinuria in patients with CKD were THAldo, THDOC, USod (urinary sodium) and SBP. Only THAldo excretion and SBP were significant multivariate determinants. Using CMRI to determine LVMI we have demonstrated that THDOC is a novel independent predictor of LVMI in patients with CKD, differing from patients with EH. Twenty-four hour THAldo excretion is an independent determinant of proteinuria in patients with CKD. These findings emphasize the importance of MR activation in the pathogenesis of the adverse clinical phenotype in CKD.

Insulin rapidly stimulates L-arginine transport in human aortic endothelial cells via Akt.

Insulin stimulates endothelial NO synthesis, at least in part mediated by phosphorylation and activation of endothelial NO synthase at Ser1177 and Ser615 by Akt. We have previously demonstrated that insulin-stimulated NO synthesis is inhibited under high culture glucose conditions, without altering Ca(2+)-stimulated NO synthesis or insulin-stimulated phosphorylation of eNOS. This indicates that stimulation of endothelial NO synthase phosphorylation may be required, yet not sufficient, for insulin-stimulated nitric oxide synthesis. In the current study we investigated the role of supply of the eNOS substrate, L-arginine as a candidate parallel mechanism underlying insulin-stimulated NO synthesis in cultured human aortic endothelial cells. Insulin rapidly stimulated L-arginine transport, an effect abrogated by incubation with inhibitors of phosphatidylinositol-3'-kinase or infection with adenoviruses expressing a dominant negative mutant Akt. Furthermore, supplementation of endothelial cells with extracellular L-arginine enhanced insulin-stimulated NO synthesis, an effect reversed by co-incubation with the L-arginine transport inhibitor, L-lysine. Basal L-arginine transport was significantly increased under high glucose culture conditions, yet insulin-stimulated L-arginine transport remained unaltered. The increase in L-arginine transport elicited by high glucose was independent of the expression of the cationic amino acid transporters, hCAT1 and hCAT2 and not associated with any changes in the activity of ERK1/2, Akt or protein kinase C (PKC). We propose that rapid stimulation of L-arginine transport contributes to insulin-stimulated NO synthesis in human endothelial cells, yet attenuation of this is unlikely to underlie the inhibition of insulin-stimulated NO synthesis under high glucose conditions.

Insulin-stimulated phosphorylation of endothelial nitric oxide synthase at serine-615 contributes to nitric oxide synthesis.

Insulin stimulates endothelial NO (nitric oxide) synthesis via PKB (protein kinase B)/Akt-mediated phosphorylation and activation of eNOS (endothelial NO synthase) at Ser-1177. In previous studies, we have demonstrated that stimulation of eNOS phosphorylation at Ser-1177 may be required, yet is not sufficient for insulin-stimulated NO synthesis. We therefore investigated the role of phosphorylation of eNOS at alternative sites to Ser-1177 as candidate parallel mechanisms contributing to insulin-stimulated NO synthesis. Stimulation of human aortic endothelial cells with insulin rapidly stimulated phosphorylation of both Ser-615 and Ser-1177 on eNOS, whereas phosphorylation of Ser-114, Thr-495 and Ser-633 was unaffected. Insulin-stimulated Ser-615 phosphorylation was abrogated by incubation with the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, infection with adenoviruses expressing a dominant-negative mutant PKB/Akt or pre-incubation with TNFalpha (tumour necrosis factor alpha), but was unaffected by high culture glucose concentrations. Mutation of Ser-615 to alanine reduced insulin-stimulated NO synthesis, whereas mutation of Ser-615 to aspartic acid increased NO production by NOS in which Ser-1177 had been mutated to an aspartic acid residue. We propose that the rapid PKB-mediated stimulation of phosphorylation of Ser-615 contributes to insulin-stimulated NO synthesis.

Altered corticosteroid biosynthesis in essential hypertension: A digenic phenomenon.

Aldosterone plays an important role in electrolyte and blood pressure homeostasis. Our studies have focused on the role of aldosterone in essential hypertension. We have shown that plasma aldosterone and ARR are heritable characteristics and that aldosterone concentrations in older subjects are inversely correlated with birthweight and positively correlated with blood pressure. Aldosterone levels are also associated with polymorphic variation in the CYP11B2 gene, which encodes aldosterone synthase, the enzyme responsible for aldosterone production. Interestingly, CYP11B2 polymorphisms are also associated with less efficient activity of 11beta-hydroxylase, encoded by the neighbouring, highly homologous CYP11B1 gene. We propose that a digenic effect leads to increased aldosterone production, with inefficient 11beta-hydroxylation causing a long-term increase in ACTH drive to the adrenal gland and enhanced expression of CYP11B2, thereby resulting in chronically raised aldosterone secretion in response to factors such as angiotensin II and potassium. In susceptible subjects this is likely, over many years, to result in hypertension with relative aldosterone excess.