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. 2015 Oct;156(10):2072-83.
doi: 10.1097/j.pain.0000000000000273.

COMT Gene Locus: New Functional Variants

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Free PMC article

COMT Gene Locus: New Functional Variants

Carolina B Meloto et al. Pain. .
Free PMC article

Abstract

Catechol-O-methyltransferase (COMT) metabolizes catecholaminergic neurotransmitters. Numerous studies have linked COMT to pivotal brain functions such as mood, cognition, response to stress, and pain. Both nociception and risk of clinical pain have been associated with COMT genetic variants, and this association was shown to be mediated through adrenergic pathways. Here, we show that association studies between COMT polymorphic markers and pain phenotypes in 2 independent cohorts identified a functional marker, rs165774, situated in the 3' untranslated region of a newfound splice variant, (a)-COMT. Sequence comparisons showed that the (a)-COMT transcript is highly conserved in primates, and deep sequencing data demonstrated that (a)-COMT is expressed across several human tissues, including the brain. In silico analyses showed that the (a)-COMT enzyme features a distinct C-terminus structure, capable of stabilizing substrates in its active site. In vitro experiments demonstrated not only that (a)-COMT is catalytically active but also that it displays unique substrate specificity, exhibiting enzymatic activity with dopamine but not epinephrine. They also established that the pain-protective A allele of rs165774 coincides with lower COMT activity, suggesting contribution to decreased pain sensitivity through increased dopaminergic rather than decreased adrenergic tone, characteristic of reference isoforms. Our results provide evidence for an essential role of the (a)-COMT isoform in nociceptive signaling and suggest that genetic variations in (a)-COMT isoforms may contribute to individual variability in pain phenotypes.

Conflict of interest statement

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Figures

Figure 3
Figure 3
Predicted (a)S-COMT crystal structure and docking poses of dopamine and measured substrate concentration–COMT velocity curves. (A) Crystallographic structure of S-COMT (red), computationally determined structural model of (a)S-COMT as extracted from DMD simulations (blue), and superimposition of both. (B) Zoomed view of best dopamine docking solutions for S-COMT and (a)S-COMT (carbon atoms of ligands, SAM, and (a)S-COMT residues are represented in green, grey, and white, respectively. Catalytic Mg2+ ion and Mg-coordinating conserved water molecule are represented as a pink and red sphere, respectively; see Supplementary Table S7 (available online as Supplemental Digital Content at http://links.lww.com/PAIN/A120) for chemical structures of catecholamines and docking energy values). (C-F) Cell lysates from Be2C cells transfected with vectors expressing (C) MB-COMT or (D) S-COMT, or their alternative counterparts (a)-COMT isoforms, (E) (a)MB-COMT or (F) (a)S-COMT were tested using DHBA, dopamine (DA), norepinephrine (NE), and epinephrine (E). The fitted kinetic values with 95% confidence limits are given in Supplementary Table S7 (available online as Supplemental Digital Content at http://links.lww.com/PAIN/A120).
Figure 4
Figure 4
Relative mRNA expression, enzymatic activity, and protein expression levels of reference and alternative COMT isoforms and their allelic variants. Fold change in relative mRNA expression level of (A) S-COMT and its alternative counterparts (a)S-COMT-G and (a)S-COMT-A, and of (B) MB-COMT and its alternative counterparts (a)MB-COMT-G and (a)MB-COMT-A. Enzymatic activity with DHBA (normalized to mRNA amount) of (C) of S-COMT and its alternative counterparts (a)S-COMT-G and (a)S-COMT-A, and of (D) MB-COMT and its alternative counterparts (a)MB-COMT-G and (a)MB-COMT-A. Enzymatic activity with DHBA of (E) (a)S-COMT and (F) (a)MB-COMT isoforms only, as extracted from (C) and (D). Protein expression of (G) (a)S-COMT and (H) (a)MB-COMT isoforms. Data are expressed as mean 6 SEM. *P < 0.05, **P < 0.01, ****P < 0.001, and ****P < 0.0001.
Figure 1
Figure 1
Association analysis results and genomic localization of SNP rs165774 (G>A, minor allele frequency: 0.315). (A) Forest plot depicting odds ratios (OR; with 95% confidence intervals) for COMT SNP rs165774 and risk of TMD; (B) Forest plot depicting regression coefficient (β; with 95% confidence intervals) for COMT SNP rs165774 and pressure pain. The analysis was performed in TMD case–control and OPPERA cohorts; CI, 95% confidence interval; L95, lower bound of 95% confidence interval; Meta, meta-analysis; OR, odds ratio; U95, upper bound of 95% confidence interval; β, regression coefficient. (C) Alternative (a)MB-COMT transcript (CR616943 and BX449536.2) shares its 5′ sequence with MB-COMT—one of the 2 reference COMT transcripts. At base pair (bp) position 865, transcription leaks through intron 5 until polyadenylation site, creating an alternative splice variant. Red star: alternatively spliced COMT transcript; purple star: reference MB-COMT isoform; green star: reference S-COMT isoform.
Figure 2
Figure 2
Expression levels of COMT isoforms. The relative expression level (%) of the (a)MB-COMT vs the MB-COMT is compared between primates and tissue types. (A) The expression levels are estimated from mRNA-Seq data (GEO set GSE30352), the region highlighted in grey is assessed. The raw mRNA-Seq data pileup (clipped at height 25) are shown for cerebellum (CB) and testis (TS), in both male (M) and female (F) individuals from human (top), chimp (middle), and gorilla (bottom). The exon tracks for the reference S-COMT, reference MB-COMT, and (a)MB-COMT isoform are shown. The last exon—unique for (a)MB-COMT isoform—is colored in red; the last exon—unique for reference isoforms—is colored in yellow. (B) Z scores are computed from the distribution of expression levels across tissues in a given species. Tissue types include cerebellum (CB, magenta), testis (TS, blue), brain minus cerebellum, heart, kidney, and liver (Oth, grey).

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