Variants of ADRA2A are associated with fasting glucose, blood pressure, body mass index and type 2 diabetes risk: meta-analysis of four prospective studies.

AIMS/HYPOTHESIS: We quantified the effect of ADRA2A (encoding α-2 adrenergic receptor) variants on metabolic traits and type 2 diabetes risk, as reported in four studies. METHODS: Genotype data for ADRA2A single nucleotide polymorphisms (SNPs) rs553668 and rs10885122 were analysed in >17,000 individuals (1,307 type 2 diabetes cases) with regard to metabolic traits and type 2 diabetes risk. Two studies (n = 9,437), genotyped using the Human Cardiovascular Disease BeadChip, provided 12 additional ADRA2A SNPs. RESULTS: Rs553668 was associated with per allele effects on fasting glucose (0.03 mmol/l, p = 0.016) and type 2 diabetes risk (OR 1.17, 95% CI 1.04-1.31; p = 0.01). No significant association was observed with rs10885122. Of the 12 SNPs, several showed associations with metabolic traits. Overall, after variable selection, rs553668 was associated with type 2 diabetes risk (OR 1.38, 95% CI 1.09-1.73; p = 0.007). rs553668 (per allele difference 0.036 mmol/l, 95% CI 0.008-0.065) and rs17186196 (per allele difference 0.066 mmol/l, 95% CI 0.017-0.115) were independently associated with fasting glucose, and rs17186196 with fasting insulin and HOMA of insulin resistance (4.3%, 95% CI 0.6-8.1 and 4.9%, 95% CI 1.0-9.0, respectively, per allele). Per-allele effects of rs491589 on systolic and diastolic blood pressure were 1.19 mmHg (95% CI 0.43-1.95) and 0.61 mmHg (95% CI 0.11-1.10), respectively, and those of rs36022820 on BMI 0.58 kg/m(2) (95% CI 0.15-1.02). CONCLUSIONS/INTERPRETATION: Multiple ADRA2A SNPs are associated with metabolic traits, blood pressure and type 2 diabetes risk. The α-2 adrenergic receptor should be revisited as a therapeutic target for reduction of the adverse consequences of metabolic trait disorders and type 2 diabetes.

Level of ex vivo interleukin 6 expression in human peripheral fat compared with other tissues.

OBJECTIVES: Adipose tissue is able to secrete a variety of active mediators into the circulation. One of these is Interleukin 6 (IL6). IL6 may play a causal role in the development of atherosclerosis. It has therefore been suggested that IL6 may form part of the link between obesity and vascular disease. The aim of this study was to quantify the relative IL6 expression in adipose tissue compared to other tissues. METHODS: Tissue (vein, fat, muscle, blood) was collected from 32 patients undergoing varicose vein surgery. RNA was extracted and mRNA measured using RT-PCR relative quantification. The mean relative IL6 mRNA levels were compared between tissues using the Mann Whitney U test and the independent t-test. Tissue levels were compared for individuals using the Wilcoxon signed rank test. RESULTS: Mean relative IL6 mRNA levels (mean+/-SEM) were significantly greater in adipose tissue 44.8+/-16.1 than in other tissues (leukocytes 1.1+/-0.3, vein 2.0+/-0.8, muscle 0.06+/-0.03: p<0.001). mRNA expression levels were also significantly higher in fat than in all other tissue types in individuals (p<0.001). CONCLUSIONS: IL6 mRNA expression is significantly higher in adipose than in many other tissues known to express IL6.

Angiotensin II type I receptor gene polymorphism: anthropometric and metabolic syndrome traits.

BACKGROUND: The renin angiotensin system is important in the regulation of vascular tone and fluid and electrolyte balance. The angiotensin converting enzyme gene (ACE) genotype has been shown to affect exercise response and glucose load response dependent on birth weight. Angiotensin II type I receptor (AGTR1) A1166C has previously been associated with the development of hypertension and coronary disease, but its metabolic effects have not been investigated. METHOD: AGTR1 A1166C was genotyped by allele specific PCR in 378 individuals from Hertfordshire, UK, who had been characterised for metabolic syndrome traits. RESULTS: Genotype counts were: AA, 183; AC, 170; CC, 25, consistent with Hardy-Weinberg equilibrium. The CC genotype was associated with significantly lower body mass index (by 1.7 units) in men (p = 0.03), and the same magnitude effect in women with significant lower weight in both genders (p = 0.01), also lower waist circumference and waist-hip ratio (p = 0.01) in men, with a trend for lower waist circumference in women also. Additionally, the CC genotype and/or C allele was associated with lower fasting glucose and insulin, and 30 and 120 min glucose in men (respectively, p = 0.08, 0.04, 0.01, 0.06). Lower means of systolic blood pressure, pulse pressure, cholesterol, and fasting triglyceride were also observed for the CC genotype in both genders though these were not statistically significant. CONCLUSIONS: The AGTR1 1166 CC genotype appears to predispose to favourable anthropometric and metabolic traits, relative to cardiovascular risk.

An efficient procedure for genotyping single nucleotide polymorphisms.

Analysis of single nucleotide polymorphisms (SNPs) has been and will be increasingly utilized in various genetic disciplines, particularly in studying genetic determinants of complex diseases. Such studies will be facilitated by rapid, simple, low cost and high throughput methodologies for SNP genotyping. One such method is reported here, named tetra-primer ARMS-PCR, which employs two primer pairs to amplify, respectively, the two different alleles of a SNP in a single PCR reaction. A computer program for designing primers was developed. Tetra-primer ARMS-PCR was combined with microplate array diagonal gel electrophoresis, gaining the advantage of high throughput for gel-based resolution of tetra-primer ARMS-PCR products. The technique was applied to analyse a number of SNPs and the results were completely consistent with those from an independent method, restriction fragment length polymorphism analysis.

Characterisation of an epitope specific to the neuron-specific isoform of human enolase recognised by a monoclonal antibody raised against a synthetic peptide corresponding to the C-terminus of beta/A4-protein.

Antibodies to synthetic peptides corresponding to different regions of beta/A4-protein recognize deposits of amyloid in the brains of patients with Alzheimer's disease. Down's syndrome cases and in the normal ageing brain. We have prepared a monoclonal antibody, mAb 22.212, raised against a synthetic C-terminal peptide of beta/A4 protein (residues 28-40) which labelled senile plaques in Alzheimer's disease after proteolytic treatment of tissue sections. In addition to recognising synthetic beta/A4-peptides that include the C-terminal residues 28-42, the mAb 22.212 was found to cross-react with a soluble, 47 kDa protein found in brain homogenates. This protein was shown, by amino acid sequence analysis and immunoassay, to be neuron-specific enolase (NSE). The mAb 22.212 did not recognize the non-neuronal enolase (NNE) or muscle-specific enolase (MSE) isoforms and its epitope was mapped to a short stretch of amino-acids unique to NSE, near the C-terminus. The cross-reactive NSE epitope is sited between residues 402-423 in NSE and shows no common sequence with beta/A4, perhaps suggesting that it is a conformational epitope. The significance and applications of these findings are discussed.

Microplate-array diagonal-gel electrophoresis (MADGE) and melt-MADGE: tools for molecular-genetic epidemiology.

Microplate-array diagonal-gel electrophoresis (MADGE) was invented for molecular-genetic epidemiological studies. It combines direct compatibility with microplates, convenient polyacrylamide-gel electrophoresis and economy of time and reagents at minimal capital cost, and enables one user to run up to several-thousand gel lanes per day for the direct assay of single-base variations. Melt-MADGE adds temporal-thermal-ramp apparatus to achieve similar throughput for de novo mutation scanning.

Microarray analysis of nicotine-induced changes in gene expression in endothelial cells.

Cigarette smoking causes vascular endothelial dysfunction and is a major risk factor for cardiovascular diseases. Nicotine, a major constituent of cigarette smoke, has been shown to alter gene expression in endothelial cells; however, the regulatory pathways involved remain to be defined. We hypothesized that there might be distinct pathways that could be identified by systematic transcriptome analysis. Using the cDNA microarray approach, we ascertained the expression of over 4,000 genes in human coronary artery endothelial cells and identified a number of nicotine-modulated genes encoding a protein involving in signal transduction or transcriptional regulation. Among these were phosphatidylinositol phosphate kinase and diacylglycerol kinase, which are regulators of the inositol phospholipid pathway. Changes were also detected for transcription factors cAMP response element binding protein and nuclear factor-kappaB, of which the activities of both have been previously shown to be altered in nicotine-stimulated cells. The data from this study are relevant to understanding the mechanisms underlying the pathophysiological effect of nicotine and smoking, particularly on endothelial function and pathogenesis of atherosclerosis.

Insulin-like growth factor II (IGF-II).

Insulin-like growth factor II (IGF-II) plays a key role in mammalian growth, influencing foetal cell division and differentiation and possibly metabolic regulation. The mature 67 amino acid peptide shares sequence homology with both insulin and IGF-I. The liver is the main endocrine source of IGFs, but autocrine/paracrine activity is found in most tissues. The type 1 receptor mediates most of the biological effects of IGF-I and IGF-II; the type 2 receptor is involved with IGF-II degradation. Binding proteins may both localise IGFs to the receptors and regulate their activities. The IGF2 gene is maternally imprinted in mouse and human. Relaxation of IGF2 imprinting occurs in the Beckwith-Wiedemann syndrome of somatic overgrowth, sporadic Wilms' tumour and a number of other cancers. In the general adult population, the IGF2-INS gene cluster may also influence body weight, in which case IGF-II function could become a target for therapeutic intervention in obesity.

Polymorphisms in matrix metalloproteinase-1, -3, -9, and -12 genes in relation to subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Intracranial aneurysm, which underlies the vast majority of subarachnoid hemorrhage incidences, has a multifactorial etiology, and the importance of genetic factors is increasingly recognized. Development and rupture of intracranial aneurysms involve degradation and remodeling of the vascular wall matrix in which the matrix metalloproteinases (MMPs) play an important role. The possible impact of MMP gene polymorphisms on susceptibility to intracranial aneurysms is still controversial, with conflicting data from different reported studies. METHODS: In this study we analyzed 5 different functional promoter polymorphisms in the MMP-1, MMP-3, MMP-9, and MMP-12 genes in a sample of 92 patients with aneurysmal subarachnoid hemorrhage and 158 healthy control subjects, all from southern England. RESULTS: No significant difference was detected between the patient and control groups in genotype distribution of any of the polymorphisms studied. CONCLUSIONS: The data do not support the hypothesis that MMP gene variations influence the development of intracranial aneurysms in the population studied.

Polymorphism in the growth hormone gene, weight in infancy, and adult bone mass.

Epidemiological studies point to the importance of gene-environment interactions during early life as determinants of later osteoporosis and fracture. We examined associations between common single nucleotide polymorphisms in the human GH (GH1) gene and weight in infancy, adult bone mass and bone loss rates, and circulating GH profiles. Two hundred and five men and 132 women, aged 61-73 yr, in the Hertfordshire Cohort Study were included; bone mineral density was measured by dual energy x-ray absorptiometry over 4 yr. Twenty-four-hour circulating GH profiles were constructed in a subset of 71 men and women. Genomic DNA was examined for two single nucleotide polymorphisms in the GH gene (one in the promoter region and one in intron 4). Homozygotes at loci GH1 A5157G and T6331A displayed low baseline bone density and accelerated bone loss; there was also a significant (P = 0.04) interaction among weight at 1 yr, GH1 genotype, and bone loss rate. There was a graded association between alleles and circulating GH concentration among men. This study suggests that common diversity in the GH1 region predisposes to osteoporosis via effects on the level of GH expression. The interaction with infant weight suggests that early environment may influence the effect of GH1 genotype on bone loss.

Associations of IGF2 ApaI RFLP and INS VNTR class I allele size with obesity.

Body mass index (BMI) is an established epidemiological predictor of coronary disease, diabetes and hypertension. In a previous study of 2560 healthy British Caucasoid males aged 50-61 years (Northwick Park Heart Study II; NPHSII), we showed that IGF2 ApaI AA homozygotes display a mean body weight 3.3 kg lower than GG homozygotes (P = 0.0002) independent of height. Two RFLPs in the insulin (INS) gene, +1127/PstI shown previously and -23/HphI in this study, both of which are in strong linkage disequilibrium with class I/III alleles of the INS 5' variable number tandem repeat (VNTR), are not associated with weight or BMI. The IGF2 ApaI polymorphism therefore appears to mark an effect independent of INS VNTR class I vs class III. We now show by regression that there is a positive correlation of BMI with INS VNTR class I allele size, with an average 0.33% (95% CI = 0.13%, 0.50%) increase in BMI per extra tandem repeat (P < 0.0001) representing variation of 4.8% over the allele size range. However, an alternative interpretation is of 'step' rather than 'slope', the small class I subclass allele group (mode 669 bp) being lighter than the large subclass group (mode 814 bp). This small effect would not be evident as an association between INS VNTR class I/I1 genotype and BMI. The IGF2 ApaI association and INS VNTR class I subclass regression association account for at least 1.1% of population BMI variance. Neither, both, or a third site may be aetiological.

Analysis of the 5'-AAUAAA motif and its flanking sequence in human RNA: relevance to cDNA library sorting.

The motif, N-8..N-1AAUAAAN1..N8 (where N is A, C, G or U), and its flanking sequence in human mRNA were examined by database analysis. Approximately 20% of 5'-AAUAAA in 3'-noncoding regions appear not to direct mRNA cleavage-polyadenylation. In coding regions, Asn-Lys, Ile-Lys and Ile-Asn are proven not to be unfavourable, and AAUAAA is not an unfavourable choice of coding sequence, occurring in 16% of mRNAs. Neither immediate flanking sequence nor associated motifs bear sufficient information content to account for the cleavage specificity observed. The unusual distribution and properties of the motif, AATAAA, in cDNA invite novel strategies for sorting cDNA libraries.

Molecular cloning of cDNA coding for human PGP 9.5 protein. A novel cytoplasmic marker for neurones and neuroendocrine cells.

The co-ordinate sequencing of the human neuronal and neuroendocrine marker protein PGP 9.5 and its cDNA is described. The cDNA encodes the complete protein (212 amino acids), and the 340 nucleotide 3'-noncoding region including the polyadenylation signal, indicating an mRNA slightly larger than 1 kb in size. Protein sequencing of 50% of PGP 9.5 confirms the deduced protein sequence.

Sequence conservation in the 3'-untranslated regions of neurone-specific enolase, lymphokine and protooncogene mRNAs.

The C-terminal protein-coding and the entire 3'-untranslated regions of a cDNA corresponding to human neurone-specific enolase mRNA have been sequenced. The 3'-untranslated region is 892 bases long and shows a high degree of homology with the 3'-untranslated region of rat neurone-specific enolase mRNA. This sequence conservation is not seen in non-neuronal enolase mRNAs. Features of the conserved sequence include an A-rich region approx. 250 bases from the stop codon at a point corresponding to the polyadenylation signal site in non-neuronal enolase mRNA, and a repeating ATTT sequence. This unusual motif in eukaryotic mRNAs has previously been reported in the 3'-untranslated regions of lymphokine and protooncogene mRNAs.

Patterns of DNA methylation of the parathyroid hormone-related protein gene in human lung carcinoma.

Humoral hypercalcaemia of malignancy often results from production of parathyroid hormone-related protein (PTHrP) by the tumour. We have investigated whether malignancy is associated with epigenetic changes in the PTHrP gene in lung. In normal and tumour tissue, there was a general background of nonmethylation in the PTHrP gene. In the 5' region, there appeared to be increased methylation of sites upstream of the promoter, P2. The extent of methylation increased from germ line to normal tissue to tumour tissue to tumour cell line, indicating that new methylation events in this region mark neoplastic change in lung cells.

SNP genotyping by combination of 192-well MADGE, ARMS and computerized gel image analysis.

A new modification of the microplate array diagonal gel electrophoresis (MADGE) system accommodates the dual amplification refractory mutation system (ARMS) products of 96 samples on one 192-well gel. Simultaneous electrophoresis of a number of horizontal ARMS-MADGE gels achieves high throughput. Gels are imaged digitally, here using the FluorImager 595 fluorescent scanning system. Customized software by Phoretix enables rapid computerized calling of band patterns in ARMS-MADGE arrays, in which the two wells receiving a pair of allele-specific assays for a single template are juxtaposed to form one virtual track, with genotype data exported directly into Microsoft Excel for statistical analysis. An ARMS assay of the A/T base change at the -23/HphI RFLP in the insulin gene promoter, which initiates from 2.5 ng template DNA, was used here to demonstrate this improved general approach for population SNP analyses.

High-throughput genotyping using horizontal polyacrylamide gels with wells arranged for microplate array diagonal gel electrophoresis (MADGE).

Genotyping (typing of genetic variation) typically involves PCR followed by an allele-specific oligonucleotide-binding assay, restriction enzyme digest or direct check of the outcome of a PCR designed to distinguish genotype. Electrophoresis can resolve "bound" from "free" oligonucleotide, as well as resolve PCR fragments and digests, but it is traditionally regarded as cumbersome and laborious in comparison with solution assays. Here we describe simple horizontal polyacrylamide gels which can receive a 96-well array of samples directly, which can be stacked in tanks and which are bound to a robust support of glass. The line of electrophoresis is on a 71.6 degree diagonal relative to the columns of the array (microplate array diagonal gel electrophoresis [MADGE]). Several thousand reactions can conveniently be analyzed in a shoebox-sized apparatus in a couple of hours. High resolution is achieved in the range of 20-1000 bp, information processing is simplified and automation is possible.

A World Wide Web site for low-density lipoprotein receptor gene mutations in familial hypercholesterolemia: sequence-based, tabular, and direct submission data handling.

Familial hypercholesterolemia is an autosomal dominant inherited condition characterized by a mutation in the low-density lipoprotein receptor (LDLR) gene. A database has been set up on the World Wide Web for mutations in the LDLR gene.

Analysis of the association of a heat shock protein70-1 gene promoter polymorphism with myocardial infarction and coronary risk traits.

Heat shock proteins (HSP) are induced during coronary ischaemia, and abnormal expression of one HSP gene may cause hypertension in rats. We examined association of a promoter polymorphism in the major stress-inducible hsp70 gene (hsp70-1 or HSP70A1) on chromosome 6 (p21.3) with coronary disease traits. This C-->A base substitution (AAACCCC) is at nucleotide position-110 in the heat shock transcription factor binding site (heat shock element, HSE). The first study sample (ECTIM), recruited from Belfast and three centers in France, consisted of 578 myocardial infarction cases and 698 age-matched controls. The frequency of the A-110 allele was 0.381 (95% CI = 0.35-0.41) and 0.384 (95% CI = 0.36-0.41) in cases and controls respectively. Homozygotes for the rarer A-110 allele had a higher BMI (27.3 kg/m2 +/- 3.9) compared with homozygotes for the common C-110 allele (26.3 kg/m2 +/- 3.3). The rarer homozygotes were shorter and heavier than the common homozygotes. A follow-up study involved 1431 healthy, middle aged men from the UK (NPHS II group). The frequency of the A-110 allele was 0.385 (95% CI = 0.37-0.40), and there was no association of genotype with BMI. Thus there appears to be no strong association of the Hsp70-1 promoter polymorphism with risk of myocardial infarction, BMI or any coronary disease traits analysed here.

A mitochondrial DNA sequence variant associated with schizophrenia and oxidative stress.

We have previously reported a changed mitochondrial (mt) gene expression in brain from patients with schizophrenia [Schizophr. Res. 14 (1995) 203]; now, we describe the distribution in the mtDNA from lymphocytes of a heteroplasmic sequence variation that was originally found in the mtDNA from the postmortem brain of a patient with schizophrenia. The variant is m.12027T>C and results in the change from isoleucine to threonine at position 423 of the ND4 subunit of NADH-ubiquinone reductase. Using a PCR-RFLP method, we have determined the heteroplasmy as the ratio of variant to total (variant ratio) at m.12027 in 184 controls and 181 patients with schizophrenia as well as 24 postmortem brain samples. The distribution of variants is bimodal having peaks at variant ratios of 0.262 and 0.732. The variant-rich fraction is very significantly associated with schizophrenia in males (47%), while there is only 18% in control males. There are significantly more variant-rich control females (36%) than control males (18%), suggesting that the female population is less sensitive to the presence of a variant in terms of liability to schizophrenia. In variant-rich samples from postmortem brain originating from both sexes, there is an increased superoxide production, suggesting that the variation contributes to oxidative stress. Antioxidant glycosides, such as quercetin rutoside, quench the superoxide production without (in contrast to neuroleptic drugs) interfering with the electron transfer activity of the reductase.