Cholinergic neurotransmission in the central and autonomic nervous systems regulates immediate variations in and longerterm maintenance of cardiovascular function with acetylcholinesterase (AChE) activity that is critical to temporal responsiveness. Butyrylcholinesterase (BChE), largely confined to the liver and plasma, subserves metabolic functions. AChE and BChE are found in hematopoietic cells and plasma, enabling one to correlate enzyme levels in whole blood with hereditary traits in twins. Using both twin and unrelated subjects, we found certain single nucleotide polymorphisms (SNPs) in the ACHE gene correlated with catalytic properties and general cardiovascular functions. SNP discovery from ACHE resequencing identified 19 SNPs: 7 coding SNPs (cSNPs), of which 4 are nonsynonymous, and 12 SNPs in untranslated regions, of which 3 are in a conserved sequence of an upstream intron. Both AChE and BChE activity traits in blood were heritable: AChE at 48.8 Ϯ 6.1% and BChE at 81.4 Ϯ 2.8%. Allelic and haplotype variations in the ACHE and BCHE genes were associated with changes in blood AChE and BChE activities. AChE activity was associated with BP status and SBP, whereas BChE activity was associated with features of the metabolic syndrome (especially body weight and BMI). Gene products from cDNAs with nonsynonymous cSNPs were expressed and purified. Protein expression of ACHE nonsynonymous variant D134H (SNP6) is impaired: this variant shows compromised stability and altered rates of organophosphate inhibition and oxime-assisted reactivation. A substantial fraction of the D134H instability could be reversed in the D134H/R136Q mutant. Hence, common genetic variations at ACHE and BCHE loci were associated with changes in corresponding enzymatic activities in blood.
The identification of common genetic variants such as single nucleotide polymorphisms (SNPs) in the human genome has become central in human population genetics and evolution studies, as well as in the study of the genetic basis of complex traits and diseases. Crucial for the accurate identification of genetic variants is the availability of high quality genomic DNA (gDNA). Since popular sources of gDNA (buccal cells, lymphocytes, hair bulb) often do not yield sufficient quantities of DNA for molecular genetic applications, whole genome amplification methods have recently been introduced to generate a renewable source of double-stranded linear DNA. Here, we assess the fidelity of one method, multiple displacement amplification (MDA), which utilizes bacteriophage Phi29 DNA polymerase to generate amplified DNA from an original source of gDNA, in a representative SNP discovery and genetic association study at the melanocortin 1 receptor (MC1R) locus, a highly polymorphic gene in humans involved in skin and hair pigmentation. We observed that MDA has high fidelity for novel SNP discovery and can be a valuable tool in generating a potentially indefinite source of DNA. However, we observed an allele amplification bias that causes genotype miscalls at heterozygous sites. At loci with multiple polymorphic sites in linkage disequilibrium, such as at MC1R, this bias can create a significant number of heterozygote genotype errors that subsequently misrepresents haplotypes.
Nicotinic acetylcholine receptors (nAChRs) are combinations of subunits arranged as pentamers encircling a central cation channel. At least nine ␣ and four  subunits are expressed in the central and peripheral nervous systems; their presence in autonomic ganglia, the adrenal medulla, and central nervous system, with accompanying responses elicited by nicotinic agonists, point to their involvement in cardiovascular homeostasis. nAChRs formed by ␣3, ␣5, and 4 subunits may regulate blood pressure (BP) by mediating release of catestatin, the endogenous nicotinic antagonist fragment of chromogranin A (CHGA) and potent inhibitor of catecholamine secretion. Genes encoding these subunits (CHRNA3, CHRNA5, and CHRNB4) are clustered on human chromosome 15q24. Because variation in this cluster may alter autonomic regulation of BP, we sequenced ϳ15 kilobase pairs in 15q24 containing their coding and 5Ј-and 3Ј-untranslated regions in 80 individuals. We identified 63 variants: 25 in coding regions of CHRNA3, CHRNA5, and CHRNB4 and 48 noncoding single-nucleotide polymorphisms (SNPs). Haplotype frequencies varied across ethnic populations. We assessed the contribution of six SNPs in the putative catestatin binding region of CHRNA3 and CHRNB4 to autonomic traits. In twins, catestatin and BP were heritable. CHRNA3 SNPs and haplotypes containing K95K (G285A) associated with circulating plasma catestatin, epinephrine levels, as well as systolic BP, suggesting altered coupling of the nAChRs to BP. Studies of chromaffin cells in vitro reveal that nicotinic agonist stimulation releases catecholamines and CHGA, a process augmented by overexpression of CHRNA3 and blocked by catestatin. These cellular events suggest a homeostatic mechanism underlying the pleiotropic actions of CHRNA3 genetic variation on autonomic function observed in twins.Nicotinic acetylcholine receptors (nAChRs) are homomeric and heteromeric assemblies of five subunits (Grutter and Changeux, 2001;Karlin, 2002). These subunits are encoded by homologous genes expressed in nerve, muscle, and endocrine cells. The subunits surround an agonist-mediated ion channel that gates currents generated by the flow of cations (Na ϩ and Ca 2ϩ ) from extracellular to intracellular space. Agonist association at specific subunit interfaces gives rise to a concerted conformational change that propagates to the transmembrane spanning region. Rapid gating of cations causes transient depolarization of the cell membrane. In preganglionic sympathetic neurons, depolarization enhances
Background-Hypertension is a complex trait, with deranged autonomic control of circulation. Chromogranin B (CHGB) is the most abundant core protein in human catecholamine secretory vesicles, playing an important role in their biogenesis. Does common interindividual variation at the CHGB locus contribute to phenotypic variation in CHGB and catecholamine secretion, autonomic stability of circulation, or blood pressure (BP) in the population? Methods and Results-To probe interindividual variability in CHGB, we systematically studied polymorphism across the locus by resequencing CHGB (Ϸ6 kbp footprint spanning the promoter, 5 exons, exon/intron borders, untranslated regions) in 160 subjects (2nϭ320 chromosomes) of diverse biogeographic ancestries. We identified 53 single-nucleotide polymorphisms, of which 22 were common. We then studied 1182 subjects drawn from the most extreme BP values in the population (highest and lowest 5th percentiles), typing 4 common polymorphisms spanning the Ϸ14 kbp locus. Sliding-window haplotype analysis indicated BP associations peaking in the 5Ј/promoter region, most prominent in men, and a peak effect in the proximal promoter at variant A-261T (AϾT), accounting for Ϸ8/Ϸ6 mm Hg BP in males. The promoter allele (A-261) that was a predictor of higher diastolic BP and systolic BP was also associated with lower circulating/plasma CHGB concentration (CHGB 439 to 451 epitope) in twin pairs. In twins, the same CHGB variants that were predictors of lower basal CHGB secretion were also associated with exaggerated catecholamine secretion and BP response to environmental (cold) stress; likewise, women displayed increased plasma CHGB 439 to 451 but decreased catecholamine secretion as well as BP response to environmental stress. The effect of A-261T on CHGB expression was confirmed in chromaffin cells by site-directed mutagenesis on transfected CHGB promoter/luciferase reporter activity, and the allelic effects of A-261T on gene expression were directionally coordinate in cella and in vivo. To confirm these clinical associations experimentally, we undertook targeted homozygous (Ϫ/Ϫ) ablation of the mouse CHGB gene; knockout mice displayed substantially increased BP, by Ϸ20/Ϸ18 mm Hg, confirming the mechanistic basis of our findings in humans. Conclusion-Common genetic variation at the CHGB locus, especially in the proximal promoter, influences CHGB expression and later catecholamine secretion and the early heritable responses to environmental stress, eventuating in changes in resting/basal BP in the population. Both the early (gene expression) and late (population BP) consequences of CHGB variation are sex dependent. These results point to new molecular strategies for probing autonomic control of circulation and, ultimately, the susceptibility to and pathogenesis of cardiovascular disease states such as hypertension.
We are studying single nucleotide polymorphisms (SNPs) in the acetylcholinesterase (AChE) gene of a human population in relation to catalytic properties and cardiovascular (CV) function. Cholinergic proteins play an important regulatory role in homeostatic maintenance of CV function. AChE found in whole blood enabled a biochemical characterization in addition to correlating genotype with phenotypic physiologic responses. Enzymatic activity determined in whole blood from twin and general population subject registries revealed significant relationships between AChE activity and certain CV endpoints. Also, the analysis enabled us to ascertain the impact of genetic variation and environmental influences on AChE activity. To date, we identified 19 SNPs of which 4 are non‐synonymous coding SNPs (cSNPs). Mutagenesis was conducted to introduce the non‐synonymous cSNPs into a human AChE cDNA vector with subsequent transfection into HEK cells for protein expression. Ongoing characterization of the purified mutant enzymes revealed significant thermal stability differences when compared with the predominant AChE species. With the structure of cholinesterase enzyme proteins and genes delineated, this research is relevant particularly in CV diseases and individual risks associated with chemical agent exposure affecting cholinergic function. Supported by NIH R37‐GM18360
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