Low Enzymatic Activity Haplotypes of the Human Catechol-O-Methyltransferase Gene: Enrichment for Marker SNPs Andrea G. Nackley 1 , Svetlana A. Shabalina 2 , Jason E. Lambert 3 , Mathew S. Conrad 1 , Dustin G. Gibson 1 , Alexey N. Spiridonov 4 , Sarah K. Satterfield 1 , Luda Diatchenko 1 * 1 Center for Neurosensory Disorders, School of Dentistry, University of North Carolina, Chapel Hill, North Carolina, United States of America, 2 National Center for Biotechnology Information, National Institutes of Health, Bethesda, Maryland, United States of America, 3 Department of Endodontics, School of Dentistry, University of North Carolina, Chapel Hill, North Carolina, United States of America, 4 Department of Mathematics, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America Abstract Catechol-O-methyltransferase (COMT) is an enzyme that plays a key role in the modulation of catechol-dependent functions such as cognition, cardiovascular function, and pain processing. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous (val 158 met) position, designated as low (LPS), average (APS), and high pain sensitive (HPS), are associated with experimental pain sensitivity and risk of developing chronic musculoskeletal pain conditions. APS and HPS haplotypes produce significant functional effects, coding for 3- and 20-fold reductions in COMT enzymatic activity, respectively. In the present study, we investigated whether additional minor single nucleotide polymorphisms (SNPs), accruing in 1 to 5% of the population, situated in the COMT transcript region contribute to haplotype-dependent enzymatic activity. Computer analysis of COMT ESTs showed that one synonymous minor SNP (rs769224) is linked to the APS haplotype and three minor SNPs (two synonymous: rs6267, rs740602 and one nonsynonymous: rs8192488) are linked to the HPS haplotype. Results from in silico and in vitro experiments revealed that inclusion of allelic variants of these minor SNPs in APS or HPS haplotypes did not modify COMT function at the level of mRNA folding, RNA transcription, protein translation, or enzymatic activity. These data suggest that neutral variants are carried with APS and HPS haplotypes, while the high activity LPS haplotype displays less linked variation. Thus, both minor synonymous and nonsynonymous SNPs in the coding region are markers of functional APS and HPS haplotypes rather than independent contributors to COMT activity. Citation: Nackley AG, Shabalina SA, Lambert JE, Conrad MS, Gibson DG, et al. (2009) Low Enzymatic Activity Haplotypes of the Human Catechol-O- Methyltransferase Gene: Enrichment for Marker SNPs. PLoS ONE 4(4): e5237. doi:10.1371/journal.pone.0005237 Editor: Bernhard Baune, James Cook University, Australia Received November 24, 2008; Accepted March 17, 2009; Published April 13, 2009 Copyright: ß 2009 Nackley et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the NIH/NICHHD Roadmap K12 KL2 RR025746 and the NIH/OBSSR R24 DK067674 to A.N. and the NIH/NIDCR R01- DE016558, PO1-NS065685, and U01-DE017018 to L.D. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lbdiatch@email.unc.edu Introduction Catechol-O-methyltransferase (COMT) is a ubiquitously ex- pressed enzyme that maintains basic biologic functions by inactivating a broad range of catechol substrates, including catecholamines (epinephrine, norepinephrine, and dopamine) and catecholestrogens. The human COMT gene is located on chromosome 22, band q11.2 where it encodes two distinct proteins, soluble COMT (S-COMT) and membrane-bound COMT (MB-COMT), through the use of alternative promoters and translation initiation sites [1,2]. S-COMT is predominately expressed in peripheral tissues, while MB-COMT is predominatly expressed in brain. The specific cell and tissue distribution of these isoforms largely overlaps with that of its catechol substrates. To date, the role of COMT in catechol metabolism has prompted over 600 investigations of its variants in the etiology of numerous disorders. Functional polymorphisms in the COMT gene are associated with dopamine and norepinephrine-dependent neuropsychiatric disorders such as schizophrenia [3,4], bipolar disorder [5], obsessive compulsive disorder [6,7], anxiety disorders [8–10], attention deficit hyperactivity disorder [11,12], addiction [13], and anorexia nervosa [14,15] as well as neurodegenerative disorders such as Parkinson’s disease [16,17]. COMT polymor- phisms have also been associated with the development of disorders such as cardiovascular disease [18,19] and estrogen- induced hormonal cancers [20,21], which are characterized by increased levels of catecholamines and their reactive products in peripheral tissues. More recently, COMT has been implicated in the modulation of persistent pain. Studies show that reduced COMT activity results in increased pain sensitivity and proinflammatory cytokine production in animal models [22,23]. These results are consistent with clinical studies demonstrating that facial pain patients exhibit lower COMT activity relative to controls [24]. Furthermore, functional polymorphisms in the COMT gene resulting in reduced enzyme activity are associated with fibromyalgia [25,26], tempo- romandibular disorder (TMD) onset [27], experimental pain sensitivity [27,28], and altered morphine efficacy in cancer pain PLoS ONE | www.plosone.org 1 April 2009 | Volume 4 | Issue 4 | e5237