Stephen Newhouse

Genome-wide association study identifies genes for biomarkers of cardiovascular disease: serum urate and dyslipidemia.

Many common diseases are accompanied by disturbances in biochemical traits. Identifying the genetic determinants could provide novel insights into disease mechanisms and reveal avenues for developing new therapies. Here, we report a genome-wide association analysis for commonly measured serum and urine biochemical traits. As part of the WTCCC, 500,000 SNPs genome wide were genotyped in 1955 hypertensive individuals characterized for 25 serum and urine biochemical traits. For each trait, we assessed association with individual SNPs, adjusting for age, sex, and BMI. Lipid measurements were further examined in a meta-analysis of genome-wide data from a type 2 diabetes scan. The most promising associations were examined in two epidemiological cohorts. We discovered association between serum urate and SLC2A9, a glucose transporter (p = 2 x 10(-15)) and confirmed this in two independent cohorts, GRAPHIC study (p = 9 x 10(-15)) and TwinsUK (p = 8 x 10(-19)). The odds ratio for hyperuricaemia (defined as urate >0.4 mMol/l) is 1.89 (95% CI = 1.36-2.61) per copy of common allele. We also replicated many genes previously associated with serum lipids and found previously recognized association between LDL levels and SNPs close to genes encoding PSRC1 and CELSR2 (p = 1 x 10(-7)). The common allele was associated with a 6% increase in nonfasting serum LDL. This region showed increased association in the meta-analysis (p = 4 x 10(-14)). This finding provides a potential biological mechanism for the recent association of this same allele of the same SNP with increased risk of coronary disease.

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.

Chromosome 9p21 in sporadic amyotrophic lateral sclerosis in the UK and seven other countries: a genome-wide association study

Summary Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motor neurons that results in progressive weakness and death from respiratory failure, commonly within about 3 years. Previous studies have shown association of a locus on chromosome 9p with ALS and linkage with ALS–frontotemporal dementia. We aimed to test whether this genomic region is also associated with ALS in an independent set of UK samples, and to identify risk factors associated with ALS in a further genome-wide association study that combined data from the independent analysis with those from other countries. Methods We collected samples from patients with sporadic ALS from 20 UK hospitals and obtained UK control samples from the control groups of the Depression Case Control study, the Bipolar Affective Case Control Study, and the British 1958 birth cohort DNA collection. Genotyping of DNA in this independent analysis was done with Illumina HumanHap550 BeadChips. We then undertook a joint genome-wide analysis that combined data from the independent set with published data from the UK, USA, Netherlands, Ireland, Italy, France, Sweden, and Belgium. The threshold for significance was p=0·05 in the independent analysis, because we were interested in replicating a small number of previously reported associations, whereas the Bonferroni-corrected threshold for significance in the joint analysis was p=2·20×10−7 Findings After quality control, samples were available from 599 patients and 4144 control individuals in the independent set. In this analysis, two single nucleotide polymorphisms in a locus on chromosome 9p21.2 were associated with ALS: rs3849942 (p=2·22×10−6; odds ratio [OR] 1·39, 95% CI 1·21–1·59) and rs2814707 (p=3·32×10−6; 1·38, 1·20–1·58). In the joint analysis, which included samples from 4312 patients with ALS and 8425 control individuals, rs3849942 (p=4·64×10−10; OR 1·22, 95% CI 1·15–1·30) and rs2814707 (p=4·72×10−10; 1·22, 1·15–1·30) were associated with ALS. Interpretation We have found strong evidence of a genetic association of two single nucleotide polymorphisms on chromosome 9 with sporadic ALS, in line with findings from previous independent GWAS of ALS and linkage studies of ALS–frontotemporal dementia. Our findings together with these earlier findings suggest that genetic variation at this locus on chromosome 9 causes sporadic ALS and familial ALS–frontotemporal dementia. Resequencing studies and then functional analysis should be done to identify the defective gene. Funding ALS Therapy Alliance, the Angel Fund, the Medical Research Council, the Motor Neurone Disease Association of Great Britain and Northern Ireland, the Wellcome Trust, and the National Institute for Health Research Dementias and Neurodegenerative Diseases Research Network (DeNDRoN).

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.

The C9ORF72 expansion mutation is a common cause of ALS+/-FTD in Europe and has a single founder.

A massive hexanucleotide repeat expansion mutation (HREM) in C9ORF72 has recently been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we describe the frequency, origin and stability of this mutation in ALS+/−FTD from five European cohorts (total n=1347). Single-nucleotide polymorphisms defining the risk haplotype in linked kindreds were genotyped in cases (n=434) and controls (n=856). Haplotypes were analysed using PLINK and aged using DMLE+. In a London clinic cohort, the HREM was the most common mutation in familial ALS+/−FTD: C9ORF72 29/112 (26%), SOD1 27/112 (24%), TARDBP 1/112 (1%) and FUS 4/112 (4%) and detected in 13/216 (6%) of unselected sporadic ALS cases but was rare in controls (3/856, 0.3%). HREM prevalence was high for familial ALS+/−FTD throughout Europe: Belgium 19/22 (86%), Sweden 30/41 (73%), the Netherlands 10/27 (37%) and Italy 4/20 (20%). The HREM did not affect the age at onset or survival of ALS patients. Haplotype analysis identified a common founder in all 137 HREM carriers that arose around 6300 years ago. The haplotype from which the HREM arose is intrinsically unstable with an increased number of repeats (average 8, compared with 2 for controls, P<10−8). We conclude that the HREM has a single founder and is the most common mutation in familial and sporadic ALS in Europe.

Gene-centric Association Signals for Lipids and Apolipoproteins Identified via the HumanCVD BeadChip

Blood lipids are important cardiovascular disease (CVD) risk factors with both genetic and environmental determinants. The Whitehall II study (n=5592) was genotyped with the gene-centric HumanCVD BeadChip (Illumina). We identified 195 SNPs in 16 genes/regions associated with 3 major lipid fractions and 2 apolipoprotein components at p<10(-5), with the associations being broadly concordant with prior genome-wide analysis. SNPs associated with LDL cholesterol and apolipoprotein B were located in LDLR, PCSK9, APOB, CELSR2, HMGCR, CETP, the TOMM40-APOE-C1-C2-C4 cluster, and the APOA5-A4-C3-A1 cluster; SNPs associated with HDL cholesterol and apolipoprotein AI were in CETP, LPL, LIPC, APOA5-A4-C3-A1, and ABCA1; and SNPs associated with triglycerides in GCKR, BAZ1B, MLXIPL, LPL, and APOA5-A4-C3-A1. For 48 SNPs in previously unreported loci that were significant at p<10(-4) in Whitehall II, in silico analysis including the British Womens Heart and Health Study, BRIGHT, ASCOT, and NORDIL studies (total n>12,500) revealed previously unreported associations of SH2B3 (p<2.2x10(-6)), BMPR2 (p<2.3x10(-7)), BCL3/PVRL2 (flanking APOE; p<4.4x10(-8)), and SMARCA4 (flanking LDLR; p<2.5x10(-7)) with LDL cholesterol. Common alleles in these genes explained 6.1%-14.7% of the variance in the five lipid-related traits, and individuals at opposite tails of the additive allele score exhibited substantial differences in trait levels (e.g., >1 mmol/L in LDL cholesterol [approximately 1 SD of the trait distribution]). These data suggest that multiple common alleles of small effect can make important contributions to individual differences in blood lipids potentially relevant to the assessment of CVD risk. These genes provide further insights into lipid metabolism and the likely effects of modifying the encoded targets therapeutically.

Association of WNK1 Gene Polymorphisms and Haplotypes With Ambulatory Blood Pressure in the General Population

Background— Blood pressure (BP) is a heritable trait of major public health concern. The WNK1 and WNK4 genes, which encode proteins in the WNK family of serine-threonine kinases, are involved in renal electrolyte homeostasis. Mutations in the WNK1 and WNK4 genes cause a rare monogenic hypertensive syndrome, pseudohypoaldosteronism type II. We investigated whether polymorphisms in these WNK genes influence BP in the general population. Methods and Results— Associations between 9 single-nucleotide polymorphisms (SNPs) in WNK1 and 1 in WNK4 with ambulatory BP were studied in a population-based sample of 996 subjects from 250 white European families. The heritability estimates of mean 24-hour systolic BP (SBP) and diastolic BP (DBP) were 63.4% and 67.9%, respectively. We found statistically significant (P<0.05) associations of several common SNPs and haplotypes in WNK1 with mean 24-hour SBP and/or DBP. The minor allele (C) of rs880054, with a frequency of 44%, reduced mean 24-hour SBP and DBP by 1.37 (95% confidence interval, −2.45 to −0.23) and 1.14 (95% confidence interval, −1.93 to −0.38) mm Hg, respectively, per copy of the allele. Conclusions— Common variants in WNK1 contribute to BP variation in the general population. This study shows that a gene causing a rare monogenic form of hypertension also plays a significant role in BP regulation in the general population. The findings provide a basis to identify functional variants of WNK1, elucidate any interactions of these variants with dietary intake or with response to antihypertensive drugs, and determine their impact on cardiovascular morbidity and mortality.

Microarray, qPCR, and KCNJ5 Sequencing of Aldosterone-Producing Adenomas Reveal Differences in Genotype and Phenotype between Zona Glomerulosa- and Zona Fasciculata-Like Tumors

CONTEXT Aldosterone-producing adenomas (APA) are heterogeneous. The recent finding of somatic KCNJ5 mutations suggests a genetic explanation. OBJECTIVES The objectives of this study were the following: 1) to compare transcriptional profiles in APA and adjacent adrenal gland (AAG); 2) to test whether gene expression profile clusters with different cell histology; and 3) to measure the frequency of KCNJ5 mutations and determine the genotype-phenotype relationship. DESIGN/SETTING The design of the study included laboratory analyses of 46 unselected APA. PATIENTS The patients in this study had primary hyperaldosteronism with unilateral APA. INTERVENTIONS The objectives of this study were the following: 1) Illumina beadchip analysis of RNA from eight paired APA-AAG; 2) a blinded review of cell histology for 46 APA; 3) laser capture microdissection of zona glomerulosa (ZG) and zona fasciculata (ZF) cells; and 4) sequencing of KCNJ5 in 46 APA. MAIN OUTCOME MEASURES The main outcome measures of this study were the following: 1) a difference in gene expression profile and a correlation with histological markers of ZF; 2) a frequency of KCNJ5 mutations and phenotypic comparisons of wild type with mutant APA. RESULTS The results of the study were the following: 1) a cluster analysis of microarray data separated APA from AAG. APA at opposite ends of the APA cluster had an approximately 800-fold difference in CYP17A1 mRNA expression, whereas histology showed 0% ZF-like cells in one vs. 100% in the other. A heat map ranking APA by CYP17A1 expression correctly predicted several genes (e.g. KCNK1, SLC24A3) to be enriched in laser capture microdissection samples of ZG; 2) known or novel mutations of KCNJ5 were found in 20 of 46 consecutive APA [43% (95% confidence interval [CI] (29, 58)%)]. The APA with KCNJ5 gene mutations were larger compared with tumors harboring the wild type, 1.63 [95% CI (1.37, 1.88)] vs. 1.14 [0.97, 1.30] cm (P = 0.0013), had predominantly ZF-like cells, and their CYP17A1 (log(2)-fold change) was higher than in wild type: -0.96 [95% CI (-0.07, -1.85)] vs. -2.54 [-1.61, -3.46], (P = 0.017). CONCLUSIONS KCNJ5 mutations are common in APA, particularly those arising from ZF. The long-recognized heterogeneity among APA may have a genetic basis.

An MND/ALS phenotype associated with C9orf72 repeat expansion: Abundant p62-positive, TDP-43-negative inclusions in cerebral cortex, hippocampus and cerebellum but without associated cognitive decline

The transactive response DNA binding protein (TDP‐43) proteinopathies describe a clinico‐pathological spectrum of multi‐system neurodegeneration that spans motor neuron disease/amyotrophic lateral sclerosis (MND/ALS) and frontotemporal lobar degeneration (FTLD). We have identified four male patients who presented with the clinical features of a pure MND/ALS phenotype (without dementia) but who had distinctive cortical and cerebellar pathology that was different from other TDP‐43 proteinopathies. All patients initially presented with weakness of limbs and respiratory muscles and had a family history of MND/ALS. None had clinically identified cognitive decline or dementia during life and they died between 11 and 32 months after symptom onset. Neuropathological investigation revealed lower motor neuron involvement with TDP‐43‐positive inclusions typical of MND/ALS. In contrast, the cerebral pathology was atypical, with abundant star‐shaped p62‐immunoreactive neuronal cytoplasmic inclusions in the cerebral cortex, basal ganglia and hippocampus, while TDP‐43‐positive inclusions were sparse. This pattern was also seen in the cerebellum where p62‐positive, TDP‐43‐negative inclusions were frequent in granular cells. Western blots of cortical lysates, in contrast to those of sporadic MND/ALS and FTLD‐TDP, showed high p62 levels and low TDP‐43 levels with no high molecular weight smearing. MND/ALS‐associated SOD1, FUS and TARDBP gene mutations were excluded; however, further investigations revealed that all four of the cases did show a repeat expansion of C9orf72, the recently reported cause of chromosome 9‐linked MND/ALS and FTLD. We conclude that these chromosome 9‐linked MND/ALS cases represent a pathological sub‐group with abundant p62 pathology in the cerebral cortex, hippocampus and cerebellum but with no significant associated cognitive decline.

Candidate Blood Proteome Markers of Alzheimer's Disease Onset and Progression: A Systematic Review and Replication Study

A blood-based protein biomarker, or set of protein biomarkers, that could predict onset and progression of Alzheimers disease (AD) would have great utility; potentially clinically, but also for clinical trials and especially in the selection of subjects for preventative trials. We reviewed a comprehensive list of 21 published discovery or panel-based (> 100 proteins) blood proteomics studies of AD, which had identified a total of 163 candidate biomarkers. Few putative blood-based protein biomarkers replicate in independent studies but we found that some proteins do appear in multiple studies; for example, four candidate biomarkers are found to associate with AD-related phenotypes in five independent research cohorts in these 21 studies: α-1-antitrypsin, α-2-macroglobulin, apolipoprotein E, and complement C3. Using SomaLogics SOMAscan proteomics technology, we were able to conduct a large-scale replication study for 94 of the 163 candidate biomarkers from these 21 published studies in plasma samples from 677 subjects from the AddNeuroMed (ANM) and the Alzheimers Research UK/Maudsley BRC Dementia Case Registry at Kings Health Partners (ARUK/DCR) research cohorts. Nine of the 94 previously reported candidates were found to associate with AD-related phenotypes (False Discovery Rate (FDR) q-value < 0.1). These proteins show sufficient replication to be considered for further investigation as a biomarker set. Overall, we show that there are some signs of a replicable signal in the range of proteins identified in previous studies and we are able to further replicate some of these. This suggests that AD pathology does affect the blood proteome with some consistency.