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Low enzymatic activity haplotypes of the human catechol-O-methyltransferase gene: enrichment for marker SNPs.

Nackley AG, Shabalina SA, Lambert JE, Conrad MS, Gibson DG, Spiridonov AN, Satterfield SK, Diatchenko L - PLoS ONE (2009)

Bottom Line: 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.These data suggest that neutral variants are carried with APS and HPS haplotypes, while the high activity LPS haplotype displays less linked variation.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurosensory Disorders, School of Dentistry, University of North Carolina, Chapel Hill, North Carolina, 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.

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Effect of minor SNPs linked to the APS and HPS haplotypes on COMT RNA abundance, protein expression, and enzymatic activity.(A) The relative abundance of RNA was uniform among cells transfected with constructs corresponding to the LPS, APS, and HPS haplotypes. Inclusion of the 800A mutation in the APS construct or the 417T, 422A, or 641T mutation in the HPS construct did not alter RNA abundance relative to that exhibited by the respective parent haplotypes. Data from (B) independent and (C) pooled Western blot experiments reveal that in cells expressing COMT, the HPS haplotype exhibited a significant reduction in protein expression compared to the LPS haplotype. This effect was not altered by inclusion of the 417T, 422A, or 641T mutations. (D) Both the APS and HPS haplotypes showed reduced enzymatic activity compared to the LPS haplotype. The modest reduction in enzymatic activity displayed by the APS haplotype and the marked reduction displayed by the HPS haplotype was not altered by inclusion of individual allelic variants of minor SNPs. Data are Mean±SEM. *P<0.05 and **P<0.01 different from the LPS haplotype.
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pone-0005237-g002: Effect of minor SNPs linked to the APS and HPS haplotypes on COMT RNA abundance, protein expression, and enzymatic activity.(A) The relative abundance of RNA was uniform among cells transfected with constructs corresponding to the LPS, APS, and HPS haplotypes. Inclusion of the 800A mutation in the APS construct or the 417T, 422A, or 641T mutation in the HPS construct did not alter RNA abundance relative to that exhibited by the respective parent haplotypes. Data from (B) independent and (C) pooled Western blot experiments reveal that in cells expressing COMT, the HPS haplotype exhibited a significant reduction in protein expression compared to the LPS haplotype. This effect was not altered by inclusion of the 417T, 422A, or 641T mutations. (D) Both the APS and HPS haplotypes showed reduced enzymatic activity compared to the LPS haplotype. The modest reduction in enzymatic activity displayed by the APS haplotype and the marked reduction displayed by the HPS haplotype was not altered by inclusion of individual allelic variants of minor SNPs. Data are Mean±SEM. *P<0.05 and **P<0.01 different from the LPS haplotype.

Mentions: In agreement with results from previous reports [27], [37], LPS, APS, and HPS haplotypes exhibited uniform levels of COMT RNA abundance (Fig. 2A). Inclusion of the 800A minor allele in the APS haplotype or the 417T, 422A, or 641T minor alleles in the HPS haplotype did not alter RNA abundance relative to that exhibited by the respective parent haplotypes. As previous studies demonstrated that COMT haplotype-dependent RNA abundance does not parallel protein expression or enzyme activity [27], [37], minor SNPs may produce effects downstream of RNA transcription.


Low enzymatic activity haplotypes of the human catechol-O-methyltransferase gene: enrichment for marker SNPs.

Nackley AG, Shabalina SA, Lambert JE, Conrad MS, Gibson DG, Spiridonov AN, Satterfield SK, Diatchenko L - PLoS ONE (2009)

Effect of minor SNPs linked to the APS and HPS haplotypes on COMT RNA abundance, protein expression, and enzymatic activity.(A) The relative abundance of RNA was uniform among cells transfected with constructs corresponding to the LPS, APS, and HPS haplotypes. Inclusion of the 800A mutation in the APS construct or the 417T, 422A, or 641T mutation in the HPS construct did not alter RNA abundance relative to that exhibited by the respective parent haplotypes. Data from (B) independent and (C) pooled Western blot experiments reveal that in cells expressing COMT, the HPS haplotype exhibited a significant reduction in protein expression compared to the LPS haplotype. This effect was not altered by inclusion of the 417T, 422A, or 641T mutations. (D) Both the APS and HPS haplotypes showed reduced enzymatic activity compared to the LPS haplotype. The modest reduction in enzymatic activity displayed by the APS haplotype and the marked reduction displayed by the HPS haplotype was not altered by inclusion of individual allelic variants of minor SNPs. Data are Mean±SEM. *P<0.05 and **P<0.01 different from the LPS haplotype.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664927&req=5

pone-0005237-g002: Effect of minor SNPs linked to the APS and HPS haplotypes on COMT RNA abundance, protein expression, and enzymatic activity.(A) The relative abundance of RNA was uniform among cells transfected with constructs corresponding to the LPS, APS, and HPS haplotypes. Inclusion of the 800A mutation in the APS construct or the 417T, 422A, or 641T mutation in the HPS construct did not alter RNA abundance relative to that exhibited by the respective parent haplotypes. Data from (B) independent and (C) pooled Western blot experiments reveal that in cells expressing COMT, the HPS haplotype exhibited a significant reduction in protein expression compared to the LPS haplotype. This effect was not altered by inclusion of the 417T, 422A, or 641T mutations. (D) Both the APS and HPS haplotypes showed reduced enzymatic activity compared to the LPS haplotype. The modest reduction in enzymatic activity displayed by the APS haplotype and the marked reduction displayed by the HPS haplotype was not altered by inclusion of individual allelic variants of minor SNPs. Data are Mean±SEM. *P<0.05 and **P<0.01 different from the LPS haplotype.
Mentions: In agreement with results from previous reports [27], [37], LPS, APS, and HPS haplotypes exhibited uniform levels of COMT RNA abundance (Fig. 2A). Inclusion of the 800A minor allele in the APS haplotype or the 417T, 422A, or 641T minor alleles in the HPS haplotype did not alter RNA abundance relative to that exhibited by the respective parent haplotypes. As previous studies demonstrated that COMT haplotype-dependent RNA abundance does not parallel protein expression or enzyme activity [27], [37], minor SNPs may produce effects downstream of RNA transcription.

Bottom Line: 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.These data suggest that neutral variants are carried with APS and HPS haplotypes, while the high activity LPS haplotype displays less linked variation.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurosensory Disorders, School of Dentistry, University of North Carolina, Chapel Hill, North Carolina, 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.

Show MeSH
Related in: MedlinePlus