title: Deep sequencing identifies novel regulatory variants in the distal promoter region of the dopamine-β-hydroxylase gene.
abstract: Dopamine-β-hydroxylase (DBH), an enzyme that converts dopamine into norepinephrine, is a drug target in cardiovascular and neuropsychiatric disorders. We aimed to identify functional variants in this gene by deep sequencing and enzyme phenotyping in an Indian cohort.
  
  
  
  Targeted resequencing of 12 exons and 10 kb upstream sequences of DBH in healthy volunteers (n=50) was performed using the Ion Personal Genome Machine System. Enzyme quantity and activity in their sera samples were determined by ELISA and ultra performance liquid chromatography, respectively. The association of markers with phenotypes was determined using Matrix eQTL. Global P-values for haplotypes generated using UNPHASED 3.1.5 were graphed using GrASP v.082 beta.
  
  
  
  Of the 49 variants identified, nine were novel (minor allele frequency≥0.01). Though individual markers associated with enzyme quantity did not withstand multiple corrections, a novel distal promoter block driven by rs113249250 (global P=1.5×10) was associated. Of the nine single nucleotide polymorphisms (SNPs) associated with enzyme activity, rs3025369, rs1076151 and rs1611115, all from the upstream region, withstood false discovery rate correction (false discovery rate=0.03, 0.03 and 2.9×10, respectively). Conditioning for rs1611115 identified rs1989787 also to affect activity. Importantly, we report an association of a novel haplotype block distal to rs1076151 driven by rs3025369 (global P=8.9×10) with enzyme activity. This regulatory SNP explained 4.9% of the total 46.1% of variance in DBH activity caused by associated SNPs.
  
  
  
  This first study combining deep sequencing and enzyme phenotyping identified yet another regulatory SNP suggesting that regulatory variants may be central in the physiological or metabolic role of this gene of therapeutic and pharmacological relevance.
authors.affiliation: aDepartment of Genetics, University of Delhi South Campus bDepartment of Psychiatry, Dr. Ram Manohar Lohia Hospital, New Delhi, India.
authors.forename: Toyanji J
authors.initials: TJ
authors.lastname: Punchaichira
authors.forename: Suman
authors.initials: S
authors.lastname: Prasad
authors.forename: Smita N
authors.initials: SN
authors.lastname: Deshpande
authors.forename: B K
authors.initials: BK
authors.lastname: Thelma
mesh_terms: D000328:Adult; D015331:Cohort Studies; D004299:Dopamine beta-Hydroxylase / Q000235:genetics*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D020022:Genetic Predisposition to Disease; D005838:Genotype; D006239:Haplotypes / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D015810:Linkage Disequilibrium; D008297:Male; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*
chemical_list: D004299:Dopamine beta-Hydroxylase