Edwin Garcia

Genes and Hypertension

The combination of investigation of rare Mendelian forms of hypertension, candidate gene studies, comparative mapping and genome-wide screening in both animal models and man has led to significant progress in determining new mechanisms of blood pressure control. In this review, the newly discovered blood pressure/cardiovascular genes, WNK kinases and angiotensin converting enzyme 2 and the development of a new anti-hypertensive agent PST2238 are discussed. Major genes causing essential hypertension have yet to be discovered, however, there are now over 20 published genome-wide screens for blood pressure controlling genes. Several regions demonstrate suggestive linkage to the trait and there is some overlap of regions between the different studies. It is hoped that new blood pressure genes will ultimately be discovered using this method. Pharmacogenetic studies in hypertension have only been initiated recently, some are described in this paper. Small studies upon single candidate genes, suggest that the contribution of genetics to the inter-individual variation in blood pressure response to anti-hypertensive therapy, is small, approximately 3-5%. Recently micro-arrays with multiple polymorphisms in multiple genes have been used. After accounting for the additive affects of multiple blood pressure loci, an individuals genetic profile appeared to explain up to 50% of the variation in blood pressure response to therapy. Knowledge of the genetic variants that cause hypertension and influence response to anti-hypertensive therapy will ultimately provide a greater understanding of the molecular mechanisms underlying blood pressure control.

A mutation and expression analysis of the oncogene BRAF in pituitary adenomas

Objective  BRAF is an oncogene that is commonly mutated in both melanomas and papillary thyroid carcinomas, usually at position V600E that leads to constitutive activity in the Ras–mitogen‐activated protein kinase (MAPK) pathway. We speculated that this same gene may be either mutated at this site, or overexpressed, in pituitary adenomas.

Genetic studies on the ghrelin, growth hormone secretagogue receptor (GHSR) and ghrelin O-acyl transferase (GOAT) genes

Ghrelin is a 28 amino acid peptide hormone that is produced both centrally and peripherally. Regulated by the ghrelin O-acyl transferase enzyme, ghrelin exerts its action through the growth hormone secretagogue receptor, and is implicated in a diverse range of physiological processes. These implications have placed the ghrelin signaling pathway at the center of a large number of candidate gene and genome-wide studies which aim to identify the genetic basis of human heterogeneity. In this review we summarize the available data on the genetic variability of ghrelin, its receptor and its regulatory enzyme, and their association with obesity, stature, type 2 diabetes, cardiovascular disease, eating disorders, and reward seeking behavior.

The role of ghrelin and ghrelin-receptor gene variants and promoter activity in type 2 diabetes

BACKGROUND Ghrelin and its receptor play an important role in glucose metabolism and energy homeostasis, and therefore they are functional candidates for genes carrying susceptibility alleles for type 2 diabetes. METHODS We assessed common genetic variation of the ghrelin (GHRL; five single nucleotide polymorphisms (SNP)) and the ghrelin-receptor (GHSR) genes (four SNPs) in 610 Caucasian patients with type 2 diabetes and 820 controls. In addition, promoter reporter assays were conducted to model the regulatory regions of both genes. RESULTS Neither GHRL nor GHSR gene SNPs were associated with type 2 diabetes. One of the ghrelin haplotypes showed a marginal protective role in type 2 diabetes. We observed profound differences in the regulation of the GHRL gene according to promoter sequence variants. There are three different GHRL promoter haplotypes represented in the studied cohort causing up to 45% difference in the level of gene expression, while the promoter region of GHSR gene is primarily represented by a single haplotype. CONCLUSION The GHRL and GHSR gene variants are not associated with type 2 diabetes, although GHRL promoter variants have significantly different activities.

Combined blockade of signalling pathways shows marked anti-tumour potential in phaeochromocytoma cell lines

Currently, there is no completely effective therapy available for metastatic phaeochromocytomas (PCCs) and paragangliomas. In this study, we explore new molecular targeted therapies for these tumours, using one more benign (mouse phaeochromocytoma cell (MPC)) and one more malignant (mouse tumour tissue (MTT)) mouse PCC cell line - both generated from heterozygous neurofibromin 1 knockout mice. Several PCC-promoting gene mutations have been associated with aberrant activation of PI3K/AKT, mTORC1 and RAS/RAF/ERK signalling. We therefore investigated different agents that interfere specifically with these pathways, including antagonism of the IGF1 receptor by NVP-AEW541. We found that NVP-AEW541 significantly reduced MPC and MTT cell viability at relatively high doses but led to a compensatory up-regulation of ERK and mTORC1 signalling at suboptimal doses while PI3K/AKT inhibition remained stable. We subsequently investigated the effect of the dual PI3K/mTORC1/2 inhibitor NVP-BEZ235, which led to a significant decrease of MPC and MTT cell viability at doses below 50 nM but again increased ERK signalling. Accordingly, we next examined the combination of NVP-BEZ235 with the established agent lovastatin, as this has been described to inhibit ERK signalling. Lovastatin alone significantly reduced MPC and MTT cell viability at therapeutically relevant doses and inhibited both ERK and AKT signalling, but increased mTORC1/p70S6K signalling. Combination treatment with NVP-BEZ235 and lovastatin showed a significant additive effect in MPC and MTT cells and resulted in inhibition of both AKT and mTORC1/p70S6K signalling without ERK up-regulation. Simultaneous inhibition of PI3K/AKT, mTORC1/2 and ERK signalling suggests a novel therapeutic approach for malignant PCCs.

Ghrelin Receptor Gene Polymorphisms and Body Size in Children and Adults

BACKGROUND The GH secretagogue receptor type 1a gene (GHSR) encodes the cognate receptor of ghrelin, a gut hormone that regulates food intake and pituitary GH secretion. Previous studies in U.S. families and a German population suggested GHSR to be a candidate quantitative locus for association with human obesity and growth. AIM The aim of the study was to test common genetic variation in GHSR for association with body size in children and adults. METHODS Sequencing was performed to systematically identify novel single nucleotide polymorphisms (SNPs) in GHSR. A set of three haplotype-tagging SNPs that captured all the genetic variation in GHSR was identified. These three haplotype-tagging SNPs were then genotyped in three large population-based U.K. cohort studies (two adult and one childhood cohort) comprising 5807 adults and 843 children. RESULTS No significant genotype or haplotype associations were found with adult or childhood height, weight, or body mass index. CONCLUSION Common variation in GHSR is not associated with body size in U.K. adults or children.

Haplotypes of the β2-adrenergic receptor gene are associated with essential hypertension in a Singaporean Chinese population

Objective To investigate the relation between the gene encoding the β2-adrenergic receptor (B2AR) and essential hypertension in a Singaporean Chinese cohort. Methods Three single nucleotide polymorphisms (SNPs) were genotyped in 190 cases and 323 controls, and eight haplotypes were determined and tested for association using the likelihood test statistic. Results We observed a significant difference in haplotype frequency distributions between the cases and the controls (P < 0.00001). A logistic regression model fitted to the data supported this finding. Conclusion The results suggest that variants at the B2AR locus may play a role in the pathophysiology of hypertension in this population.

Haplotypes of the beta-2 adrenergic receptor associate with high diastolic blood pressure in the Caerphilly prospective study.

Objectives Current evidence demonstrates that both genetic and environmental factors influence blood pressure. The sympathetic nervous system is a key player in blood pressure control and functional genetic variants of the beta-2 adrenergic receptor (B2AR) have been identified and implicated in the pathogenesis of hypertension. The present study aimed to determine the effects of common haplotypes of the B2AR gene upon blood pressure in the Caerphilly Prospective Study. Design Two thousand five hundred and twelve men (aged 45–59 years) participated in the study. We selected individuals in the upper (n = 347) and lower (n = 279) quintiles of the diastolic blood pressure distribution fixed at two time points [phase 2 (1984–88) or phase 3 (1989–93)] as cases and controls. Methods We analysed two functional polymorphisms (Arg16Gly and Gln27Glu) of B2AR and their haplotypes. Results We found a higher risk of hypertension in individuals homozygous for the Gln27 compared to those individuals homozygous for Glu27 [odds ratio (OR) = 1.94; 95% confidence interval (CI) = 1.34–2.81; P = 0.001]. Three haplotypes (Gly16Gln27, Gly16Glu27 and Arg16Gln27) were present in both quintile groups. Logistic regression analysis showed that haplotypes with a Gln27 allele (Gly16Gln27 and Arg16Gln27) conferred a significantly higher risk for hypertension than the Gly16Glu27 haplotype (OR = 1.55; 95% CI = 1.11–2.17, OR = 1.37; 95% CI = 1.04–1.81; P = 0.009 and P = 0.027, respectively). However, there was no evidence to support a statistically significant difference in odds ratios for the Gly16Gln27 and Arg16Gln27 haplotypes (P = 0.477), suggesting that it is the Gln27 allele alone, rather than any haplotype, which best explains the association. Conclusions In a prospectively studied Caucasian male cohort, high diastolic blood pressure was associated with B2AR haplotypes containing the pro-downregulatory Gln27 variant.

Characterization of SNARE Proteins in Human Pituitary Adenomas: Targeted Secretion Inhibitors as a New Strategy for the Treatment of Acromegaly?

CONTEXT Targeted secretion inhibitors (TSIs), a new class of recombinant biotherapeutic proteins engineered from botulinum toxin, represent a novel approach for treating diseases with excess secretion. They inhibit hormone secretion from targeted cell types through cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor-activating protein receptor) proteins. qGHRH-LH(N)/D is a TSI targeting pituitary somatotroph through binding to the GHRH-receptor and cleavage of the vesicle-associated membrane protein (VAMP) family of SNARE proteins. OBJECTIVE Our objective was to study SNARE protein expression in pituitary adenomas and to inhibit GH secretion from somatotropinomas using qGHRH-LH(N)/D. DESIGN We analyzed human pituitary adenoma analysis for SNARE expression and response to qGHRH-LH(N)/D treatment. SETTING The study was conducted in University Hospitals. PATIENTS We used pituitary adenoma samples from 25 acromegaly and 47 nonfunctioning pituitary adenoma patients. OUTCOME Vesicle-SNARE (VAMP1-3), target-SNARE (syntaxin1, SNAP-23, and SNAP-25), and GHRH-receptor detection with RT-qPCR, immunocytochemistry, and immunoblotting. Assessment of TSI catalytic activity on VAMPs and release of GH from adenoma cells. RESULTS SNARE proteins were variably expressed in pituitary samples. In vitro evidence using recombinant GFP-VAMP2&3 or pituitary adenoma lysates suggested sufficient catalytic activity of qGHRH-LH(N)/D to degrade VAMPs, but was unable to inhibit GH secretion in somatotropinoma cell cultures. CONCLUSIONS SNARE proteins are present in human pituitary somatotroph adenomas that can be targeted by TSIs to inhibit GH secretion. qGHRH-LH(N)/D was unable to inhibit GH secretion from human somatotroph adenoma cells. Further studies are required to understand how the SNARE proteins drive GH secretion in human somatotrophs to allow the development of novel TSIs with a potential therapeutic benefit.