title: A common genetic variant in the 3'-UTR of vacuolar H+-ATPase ATP6V0A1 creates a micro-RNA motif to alter chromogranin A processing and hypertension risk.
abstract: BACKGROUND
  The catecholamine release-inhibitor catestatin and its precursor chromogranin A (CHGA) may constitute "intermediate phenotypes" in the analysis of genetic risk for cardiovascular disease such as hypertension. Previously, the vacuolar H(+)-ATPase subunit gene ATP6V0A1 was found within the confidence interval for linkage with catestatin secretion in a genome-wide study, and its 3'-UTR polymorphism T+3246C (rs938671) was associated with both catestatin processing from CHGA and population blood pressure. We explored the molecular mechanism of this effect by experiments with transfected chimeric photoproteins in chromaffin cells.
  
  
  RESULTS
  Placing the ATP6V0A1 3'-UTR downstream of a luciferase reporter, we found that the C (variant) allele decreased overall gene expression. The 3'-UTR effect was verified by coupled in vitro transcription/translation of the entire/intact human ATP6V0A1 mRNA. Chromaffin granule pH, monitored by fluorescence of CHGA/EGFP chimera during vesicular H(+)-ATPase inhibition by bafilomycin A1, was more easily perturbed during coexpression of the ATP6V0A1 3'-UTR C-allele than the T-allele. After bafilomycin A1 treatment, the ratio of CHGA precursor to its catestatin fragments in PC12 cells was substantially diminished, though the qualitative composition of such fragments was not affected (on immunoblot or matrix-assisted laser desorption ionization (MALDI) mass spectrometry). Bafilomycin A1 treatment also decreased exocytotic secretion from the regulated pathway, monitored by a CHGA chimera tagged with embryonic alkaline phosphatase. 3'-UTR T+3246C created a binding motif for micro-RNA hsa-miR-637; cotransfection of hsa-miR-637 precursor or antagomir/inhibitor oligonucleotides yielded the predicted changes in expression of luciferase reporter/ATP6V0A1-3'-UTR plasmids varying at T+3246C.
  
  
  CONCLUSIONS
  The results suggest a series of events whereby ATP6V0A1 3'-UTR variant T+3246C functioned: ATP6V0A1 expression probably was affected through differential micro-RNA effects, altering vacuolar pH and consequently CHGA processing and exocytotic secretion.
authors.affiliation: Departments of Medicine and Pharmacology, Institute for Genomic Medicine, University of California at San Diego, the VA San Diego Healthcare System, La Jolla, USA.
authors.forename: Zhiyun
authors.initials: Z
authors.lastname: Wei
authors.forename: Nilima
authors.initials: N
authors.lastname: Biswas
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Maite
authors.initials: M
authors.lastname: Courel
authors.forename: Kuixing
authors.initials: K
authors.lastname: Zhang
authors.forename: Alex
authors.initials: A
authors.lastname: Soler-Jover
authors.forename: Laurent
authors.initials: L
authors.lastname: Taupenot
authors.forename: Daniel T
authors.initials: DT
authors.lastname: O'Connor
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D001665:Binding Sites; D053379:Chromogranin A / Q000378:metabolism*; D005089:Exocytosis; D014644:Genetic Variation; D006801:Humans; D006973:Hypertension / Q000235:genetics*; D035683:MicroRNAs; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; D053379:Chromogranin A; D035683:MicroRNAs; C529316:ATP6V0A1 protein, human; D025262:Vacuolar Proton-Translocating ATPases