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1.  Low Enzymatic Activity Haplotypes of the Human Catechol-O-Methyltransferase Gene: Enrichment for Marker SNPs 
PLoS ONE  2009;4(4):e5237.
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 (val158met) 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.
doi:10.1371/journal.pone.0005237
PMCID: PMC2664927  PMID: 19365560
2.  A Universal Trend of Reduced mRNA Stability near the Translation-Initiation Site in Prokaryotes and Eukaryotes 
PLoS Computational Biology  2010;6(2):e1000664.
Recent studies have suggested that the thermodynamic stability of mRNA secondary structure near the start codon can regulate translation efficiency in Escherichia coli, and that translation is more efficient the less stable the secondary structure. We survey the complete genomes of 340 species for signals of reduced mRNA secondary structure near the start codon. Our analysis includes bacteria, archaea, fungi, plants, insects, fishes, birds, and mammals. We find that nearly all species show evidence for reduced mRNA stability near the start codon. The reduction in stability generally increases with increasing genomic GC content. In prokaryotes, the reduction also increases with decreasing optimal growth temperature. Within genomes, there is variation in the stability among genes, and this variation correlates with gene GC content, codon bias, and gene expression level. For birds and mammals, however, we do not find a genome-wide trend of reduced mRNA stability near the start codon. Yet the most GC rich genes in these organisms do show such a signal. We conclude that reduced stability of the mRNA secondary structure near the start codon is a universal feature of all cellular life. We suggest that the origin of this reduction is selection for efficient recognition of the start codon by initiator-tRNA.
Author Summary
Synonymous mutations are mutations that change the nucleotide sequence of a gene without changing the amino-acid sequence. Because these mutations don't alter the expressed protein, they are frequently also called silent mutations. Yet increasing evidence demonstrates that synonymous mutations are not that silent. In particular, experimental work in Escherichia coli has shown that the choice of synonymous codons near the start codon can greatly influence protein production. Codons that allow the mRNA to fold into a stable secondary structure seem to inhibit efficient translation initiation. This observation suggests that selection should prefer reduced mRNA stability near the start codon in many organisms. Here, we show that this prediction generally holds true in most organisms, including bacteria, archaea, fungi, plants, insects, and fishes. In birds and mammals it doesn't hold true genome-wide, but it does hold true in the most GC-rich genes. In all organisms, the extent to which mRNA stability is reduced increases with increasing GC content. In prokaryotes, it also increases with decreasing optimal growing temperature. Thus, it seems that all organisms have to optimize their synonymous sites near the start codon to guarantee efficient protein translation.
doi:10.1371/journal.pcbi.1000664
PMCID: PMC2816680  PMID: 20140241
3.  Comt1 Genotype and Expression Predicts Anxiety and Nociceptive Sensitivity in Inbred Strains of Mice 
Genes, brain, and behavior  2010;9(8):933-946.
Catechol-O-methyltransferase (COMT) is an ubiquitously expressed enzyme that maintains basic biologic functions by inactivating catechol substrates. In humans, polymorphic variance at the COMT locus has been associated with modulation of pain sensitivity (Andersen & Skorpen, 2009) and risk for developing psychiatric disorders (Harrison & Tunbridge, 2008). A functional haplotype associated with increased pain sensitivity was shown to result in decreased COMT activity by altering mRNA secondary structure-dependent protein translation (Nackley et al., 2006). However, the exact mechanisms whereby COMT modulates pain sensitivity and behavior remain unclear and can be further studied in animal models. We have assessed Comt1 gene expression levels in multiple brain regions in inbred strains of mice and have discovered that Comt1 is differentially expressed among the strains, and this differential expression is cis-regulated. A B2 Short Interspersed Element (SINE) was inserted in the 3′UTR of Comt1 in 14 strains generating a common haplotype that correlates with gene expression. Experiments using mammalian expression vectors of full-length cDNA clones with and without the SINE element demonstrate that strains with the SINE haplotype (+SINE) have greater Comt1 enzymatic activity. +SINE mice also exhibit behavioral differences in anxiety assays and decreased pain sensitivity. These results suggest that a haplotype, defined by a 3′ UTR B2 SINE element, regulates Comt1 expression and some mouse behaviors.
doi:10.1111/j.1601-183X.2010.00633.x
PMCID: PMC2975805  PMID: 20659173
4.  Catechol O-Methyltransferase Pharmacogenomics: Human Liver Genotype-Phenotype Correlation and Proximal Promoter Studies 
Pharmacogenetics and genomics  2009;19(8):577-587.
Objectives
Catechol O-methyltransferase (COMT) is expressed as both soluble (S) and membrane-bound (MB) isoforms, with S-COMT predominantly expressed in liver. A common nonsynonymous SNP, 472G>A (108/158Val>Met, S/MB), has been associated with variation in levels of COMT enzyme activity and thermal stability. We set out to test the hypothesis that additional COMT polymorphisms might also be associated with phenotypic variation.
Methods
We phenotyped 268 liver biopsy samples for S-COMT activity and thermal stability, resequenced a portion of the gene that had not previously been resequenced, and genotyped DNA from these same samples for 16 COMT polymorphisms.
Results
There was a significant association between the two COMT phenotypes and genotype at the codon 108 SNP. A haplotype-based approach was then used to assess the possible association of other polymorphisms with phenotype. Specifically, codon 108 SNP explained 20.4% of variance in activity (P<10−6), and 59% of variance in thermal stability (P<10−6). Haplotypes that included SNPs at cDNA nucleotides 408 and 472 explained additional variance in enzyme activity (up to 24.4%), and the addition to the haplotype of a SNP at intron 2 (51) explained a total of 27.5% of the variance. However, no SNPs beyond that at nucleotide 472G>A polymorphism were associated with variation in thermal stability. We also observed a three-fold variation in the ability of reporter gene constructs for “proximal promoter” haplotypes to drive transcription.
Conclusions
The common COMT 108Val>Met polymorphism is associated with human liver S-COMT activity and thermal stability, but additional COMT SNPs also contribute to variation in activity.
doi:10.1097/FPC.0b013e32832c15c6
PMCID: PMC3052748  PMID: 19641441
catechol O-methyltransferase; COMT; genetic polymorphism; SNP; haplotype; thermal stability
5.  Association of COMT genotypes with S-COMT promoter methylation in growth-discordant monozygotic twins and healthy adults 
BMC Medical Genetics  2011;12:115.
Background
Catechol-O-Methyltransferase (COMT) plays a key role in dopamine and estrogen metabolism. Recently, COMT haplotypes rather than the single polymorphism Val158Met have been reported to underlie differences in protein expression by modulating mRNA secondary structure. So far, studies investigating the epigenetic variability of the S-COMT (soluble COMT) promoter region mainly focused on phenotypical aspects, and results have been controversial.
Methods
We assessed S-COMT promoter methylation in saliva and blood derived DNA with regard to early pre- and postnatal growth as well as to genotype for polymorphisms rs6269, rs4633, and rs4680 (Val158Met) in 20 monozygotic twin pairs (mean age 4 years), who were discordant for intrauterine development due to severe feto-fetal-transfusion syndrome. Methylation levels of two previously reported partially methylated cytosines were determined by the quantitative SIRPH (SNuPE- IP RP HPLC) assay.
Results
Overall, we observed a high variability of S-COMT promoter methylation, which did not correlate with individual differences in the pre- or postnatal growth pattern. Within the twin pairs however we noted a distinct similarity that could be linked to underlying COMT genotypes. This association was subsequently confirmed in a cohort of 93 unrelated adult controls. Interestingly, 158Val-alleles were found at both ends of the epigenotypical range, which is in accordance with a recently proposed model of COMT haplotypes corresponding to a continuum of phenotypical variability.
Conclusion
The strong heritable component of S-COMT promoter methylation found in our study needs to be considered in future approaches that focus on interactions between COMT epigenotype and phenotype.
doi:10.1186/1471-2350-12-115
PMCID: PMC3270002  PMID: 21884617
6.  Selection on synonymous codons in mammalian rhodopsins: a possible role in optimizing translational processes 
Background
Synonymous codon usage can affect many cellular processes, particularly those associated with translation such as polypeptide elongation and folding, mRNA degradation/stability, and splicing. Highly expressed genes are thought to experience stronger selection pressures on synonymous codons. This should result in codon usage bias even in species with relatively low effective population sizes, like mammals, where synonymous site selection is thought to be weak. Here we use phylogenetic codon-based likelihood models to explore patterns of codon usage bias in a dataset of 18 mammalian rhodopsin sequences, the protein mediating the first step in vision in the eye, and one of the most highly expressed genes in vertebrates. We use these patterns to infer selection pressures on key translational mechanisms including polypeptide elongation, protein folding, mRNA stability, and splicing.
Results
Overall, patterns of selection in mammalian rhodopsin appear to be correlated with post-transcriptional and translational processes. We found significant evidence for selection at synonymous sites using phylogenetic mutation-selection likelihood models, with C-ending codons found to have the highest relative fitness, and to be significantly more abundant at conserved sites. In general, these codons corresponded with the most abundant tRNAs in mammals. We found significant differences in codon usage bias between rhodopsin loops versus helices, though there was no significant difference in mean synonymous substitution rate between these motifs. We also found a significantly higher proportion of GC-ending codons at paired sites in rhodopsin mRNA secondary structure, and significantly lower synonymous mutation rates in putative exonic splicing enhancer (ESE) regions than in non-ESE regions.
Conclusions
By focusing on a single highly expressed gene we both distinguish synonymous codon selection from mutational effects and analytically explore underlying functional mechanisms. Our results suggest that codon bias in mammalian rhodopsin arises from selection to optimally balance high overall translational speed, accuracy, and proper protein folding, especially in structurally complicated regions. Selection at synonymous sites may also be contributing to mRNA stability and splicing efficiency at exonic-splicing-enhancer (ESE) regions. Our results highlight the importance of investigating highly expressed genes in a broader phylogenetic context in order to better understand the evolution of synonymous substitutions.
doi:10.1186/1471-2148-14-96
PMCID: PMC4021273  PMID: 24884412
Mutation-selection model; dN/dS; Codon-based likelihood models; Visual pigment evolution
7.  Pain modality- and sex-specific effects of COMT genetic functional variants 
Pain  2013;154(8):1368-1376.
The enzyme catechol-O-methyltransferase (COMT) metabolizes catecholamine neurotransmitters involved in a number of physiological functions including pain perception. Both human and mouse COMT genes possess functional polymorphisms contributing to inter-individual variability in pain phenotypes such as sensitivity to noxious stimuli, severity of clinical pain and response to pain treatment. In this study, we found that the effects of Comt functional variation in mice are modality-specific. Spontaneous inflammatory nociception and thermal nociception behaviors were correlated the most with the presence of the B2 SINE transposon insertion residing in the 3’UTR mRNA region. Similarly, in humans, COMT functional haplotypes were associated with thermal pain perception and with capsaicin-induced pain. Furthermore, COMT genetic variations contributed to pain behaviors in mice and pain ratings in humans in a sex-specific manner. The ancestral Comt variant, without a B2 SINE insertion, was more strongly associated with sensitivity to capsaicin in female versus male mice. In humans, the haplotype coding for low COMT activity increased capsaicin-induced pain perception in women, but not men. These findings reemphasize the fundamental contribution of COMT to pain processes, and provide a fine-grained resolution of this contribution at the genetic level that can be used to guide future studies in the area of pain genetics.
doi:10.1016/j.pain.2013.04.028
PMCID: PMC3700530  PMID: 23701723
8.  Genetic variation in the Catechol-O-Methyltransferase (COMT) gene and morphine requirements in cancer patients with pain 
Molecular Pain  2008;4:64.
Background
Genetic variation contributes to differences in pain sensitivity and response to different analgesics. Catecholamines are involved in the modulation of pain and are partly metabolized by the catechol-O-methyltransferase (COMT) enzyme. Genetic variability in the COMT gene may therefore contribute to differences in pain sensitivity and response to analgesics. It is shown that a polymorphism in the COMT gene, Rs4680 (Val158Met), influence pain sensitivity in human experimental pain and the efficacy for morphine in cancer pain treatment. In this study we wanted to investigate if variability in other regions in the COMT gene also contributes to interindividual variability in morphine efficacy.
Results
We genotyped 11 single nucleotide polymorphisms (SNPs) throughout the COMT gene, and constructed haplotypes from these 11 SNPs, which were in Hardy-Weinberg equilibrium. We compared both genotypes and haplotypes against pharmacological, demographical and patient symptoms measurements in a Caucasian cancer patient cohort (n = 197) receiving oral morphine treatment for cancer pain. There were two frequent haplotypes (34.5% and 17.8%) in our cohort. Multivariate analyses showed that patients carrying the most frequent haplotype (34.5%) needed lower morphine doses than patients not carrying the haplotype, with a reduction factor of 0.71 (p = 0.005). On the allele level, carriers of alleles for six of the SNPs show weak associations in respect to morphine dose and the alleles associated with the lowest morphine doses constitute part of the most frequent haplotype.
Conclusion
This study suggests that genetic variability in the COMT gene influence the efficacy of morphine in cancer patients with pain, and that increased understanding of this variability is reached by expanding from analyses of single SNPs to haplotype construction and analyses.
doi:10.1186/1744-8069-4-64
PMCID: PMC2644687  PMID: 19094200
9.  A B2 SINE insertion in the Comt1 gene (Comt1B2i) results in an overexpressing, behavior modifying allele present in classical inbred mouse strains 
Genes, Brain, and Behavior  2010;9(8):925-932.
Catechol-O-methyltransferase (COMT) is a key enzyme for dopamine catabolism and COMT is a candidate gene for human psychiatric disorders. In mouse it is located on chromosome 16 in a large genomic region of extremely low variation among the classical inbred strains, with no confirmed single nucleotide polymorphisms (SNPs) between strains C57BL/6J and DBA/2J within a 600-kB window. We found a B2 SINE in the 3′ untranslated region (UTR) of Comt1 which is present in C57BL/6J (Comt1B2i) and other strains including 129 (multiple sublines), but is not found in DBA/2J (Comt1+) and many other strains including wild-derived Mus domesticus, M. musculus, M. molossinus, M.castaneus and M. spretus. Comt1B2i is absent in strains closely related to C57BL/6, such as C57L and C57BR, indicating that it was polymorphic in the cross that gave rise to these strains. The strain distribution of Comt1B2i indicates a likely origin of the allele in the parental Lathrop stock. A stringent association test, using 670 highly outbred mice (Boulder Heterogeneous Stock), indicates that this insertion allele may be responsible for a difference in behavior related to exploration. Gene expression differences at the mRNA and enzyme activity level (1.7-fold relative to wild type) indicate a mechanism for this behavioral effect. Taken together, these findings show that Comt1B2i (a B2 SINE insertion) results in a relatively modest difference in Comt1 expression and enzyme activity (comparable to the human Val-Met polymorphism) which has a demonstrable behavioral phenotype across a variety of outbred genetic backgrounds.
doi:10.1111/j.1601-183X.2010.00614.x
PMCID: PMC3037563  PMID: 20618449
Behavior; comt; genetics; mouse; sine insertion
10.  A Nutrient-Driven tRNA Modification Alters Translational Fidelity and Genome-wide Protein Coding across an Animal Genus 
PLoS Biology  2014;12(12):e1002015.
Use of the nutrient queuine to modify tRNA anticodons can change the accuracy of certain codons during protein synthesis, resulting in evolutionary recoding of fruit fly genomes.
Natural selection favors efficient expression of encoded proteins, but the causes, mechanisms, and fitness consequences of evolved coding changes remain an area of aggressive inquiry. We report a large-scale reversal in the relative translational accuracy of codons across 12 fly species in the Drosophila/Sophophora genus. Because the reversal involves pairs of codons that are read by the same genomically encoded tRNAs, we hypothesize, and show by direct measurement, that a tRNA anticodon modification from guanosine to queuosine has coevolved with these genomic changes. Queuosine modification is present in most organisms but its function remains unclear. Modification levels vary across developmental stages in D. melanogaster, and, consistent with a causal effect, genes maximally expressed at each stage display selection for codons that are most accurate given stage-specific queuosine modification levels. In a kinetic model, the known increased affinity of queuosine-modified tRNA for ribosomes increases the accuracy of cognate codons while reducing the accuracy of near-cognate codons. Levels of queuosine modification in D. melanogaster reflect bioavailability of the precursor queuine, which eukaryotes scavenge from the tRNAs of bacteria and absorb in the gut. These results reveal a strikingly direct mechanism by which recoding of entire genomes results from changes in utilization of a nutrient.
Author Summary
Ribosomes translate mRNA into protein using tRNAs, and these tRNAs often translate multiple synonymous codons. Although synonymous codons specify the same amino acid, tRNAs read codons with differing speed and accuracy, and so some codons may be more accurately translated than their synonyms. Such variation in the efficiency of translation between synonymous codons can result in costs to cellular fitness. By favoring certain coding choices over evolutionary timescales, natural selection leaves signs of pressure for translational fidelity on evolved genomes. We have found that the way in which proteins are encoded has changed systematically across several closely related fruit fly species. Surprisingly, several of these changes involve two codons both read by the same tRNA. Here we confirm experimentally that the anticodons of these tRNAs are chemically modified—from guanine to queuosine—in vivo, and that the levels of this modification in different species track the differences in protein coding. Furthermore, queuosine modification levels are known to change during fruit fly development, and we find that genes expressed maximally during a given developmental stage have codings reflecting levels of modification at that stage. Remarkably, queuosine modification depends upon acquisition of its precursor, queuine, as a nutrient that eukaryotes must obtain from bacteria through the gut. We have thus elucidated a mechanism by which availability of a nutrient can shape the coding patterns of whole genomes.
doi:10.1371/journal.pbio.1002015
PMCID: PMC4260829  PMID: 25489848
11.  Characterization of NF-kB-mediated inhibition of catechol-O-methyltransferase 
Molecular Pain  2009;5:13.
Background
Catechol-O-methyltransferase (COMT), an enzyme that metabolizes catecholamines, has recently been implicated in the modulation of pain. Specifically, low COMT activity is associated with heightened pain perception and development of musculoskeletal pain in humans as well as increased experimental pain sensitivity in rodents.
Results
We report that the proinflammatory cytokine tumor necrosis factor α (TNFα) downregulates COMT mRNA and protein in astrocytes. Examination of the distal COMT promoter (P2-COMT) reveals a putative binding site for nuclear factor κB (NF-κB), the pivotal regulator of inflammation and the target of TNFα. Cell culture assays and functional deletion analyses of the cloned P2-COMT promoter demonstrate that TNFα inhibits P2-COMT activity in astrocytes by inducing NF-κB complex recruitment to the specific κB binding site.
Conclusion
Collectively, our findings provide the first evidence for NF-κB-mediated inhibition of COMT expression in the central nervous system, suggesting that COMT contributes to the pathogenesis of inflammatory pain states.
doi:10.1186/1744-8069-5-13
PMCID: PMC2662804  PMID: 19291302
12.  Widespread Positive Selection in Synonymous Sites of Mammalian Genes 
Molecular biology and evolution  2007;24(8):1821-1831.
Evolution of protein sequences is largely governed by purifying selection, with a small fraction of proteins evolving under positive selection. The evolution at synonymous positions in protein-coding genes is not nearly as well understood, with the extent and types of selection remaining, largely, unclear. A statistical test to identify purifying and positive selection at synonymous sites in protein-coding genes was developed. The method compares the rate of evolution at synonymous sites (Ks) to that in intron sequences of the same gene after sampling the aligned intron sequences to mimic the statistical properties of coding sequences. We detected purifying selection at synonymous sites in ∼28% of the 1,562 analyzed orthologous genes from mouse and rat, and positive selection in ∼12% of the genes. Thus, the fraction of genes with readily detectable positive selection at synonymous sites is much greater than the fraction of genes with comparable positive selection at nonsynonymous sites, i.e., at the level of the protein sequence. Unlike other genes, the genes with positive selection at synonymous sites showed no correlation between Ks and the rate of evolution in nonsynonymous sites (Ka), indicating that evolution of synonymous sites under positive selection is decoupled from protein evolution. The genes with purifying selection at synonymous sites showed significant anticorrelation between Ks and expression level and breadth, indicating that highly expressed genes evolve slowly. The genes with positive selection at synonymous sites showed the opposite trend, i.e., highly expressed genes had, on average, higher Ks. For the genes with positive selection at synonymous sites, a significantly lower mRNA stability is predicted compared to the genes with negative selection. Thus, mRNA destabilization could be an important factor driving positive selection in nonsynonymous sites, probably, through regulation of expression at the level of mRNA degradation and, possibly, also translation rate. So, unexpectedly, we found that positive selection at synonymous sites of mammalian genes is substantially more common than positive selection at the level of protein sequences. Positive selection at synonymous sites might act through mRNA destabilization affecting mRNA levels and translation.
doi:10.1093/molbev/msm100
PMCID: PMC2632937  PMID: 17522087
synonymous sites; nonsynonymous sites; positive selection; purifying selection; introns
13.  Comprehensive interrogation of CpG island methylation in the gene encoding COMT, a key estrogen and catecholamine regulator 
Background
The catechol-O-methyltransferase (COMT) enzyme has been widely studied due to its multiple roles in neurological functioning, estrogen biology, and methylation metabolic pathways. Numerous studies have investigated variation in the large COMT gene, with the majority focusing on single nucleotide polymorphisms (SNPs). This body of work has linked COMT genetic variation with a vast array of conditions, including several neurobehavioral disorders, pain sensitivity, and multiple human cancers. Based on COMT’s numerous biological roles and recent studies suggesting that methylation of the COMT gene impacts COMT gene expression, we comprehensively interrogated methylation in over 200 CpG dinucleotide sequences spanning the length of the COMT gene.
Methods
Using saliva-derived DNA from a non-clinical sample of human subjects, we tested for associations between COMT CpG methylation and factors reported to interact with COMT genetic effects, including demographic factors and alcohol use. Finally, we tested associations between COMT CpG methylation state and COMT gene expression in breast cancer cell lines. We interrogated >200 CpGs in 13 amplicons spanning the 5’ UTR to the last exon of the CpG dinucleotide-rich COMT gene in n = 48 subjects, n = 11 cell lines and 1 endogenous 18S rRNA control.
Results
With the exception of the CpG island in the 5’UTR and 1st exon, all other CpG islands were strongly methylated with typical dynamic ranges between 50-90%. In the saliva samples, methylation of multiple COMT loci was associated with socioeconomic status or ethnicity. We found associations between methylation at numerous loci and genotype at the functional Val 158 Met SNP (rs4680), and most of the correlations between methylation and demographic and alcohol use factors were Val 158 Met allele-specific. Methylation at several of these loci also associated with COMT gene expression in breast cancer cell lines.
Conclusions
We report the first comprehensive interrogation of COMT methylation. We corroborate previous findings of variation in COMT methylation with gene expression and the Val 158 Met genotype, and also report novel associations with socioeconomic status (SES) and ethnicity at several methylated loci. These results point to novel mechanisms for COMT regulation, which may have broad therapeutic implications.
doi:10.1186/1755-8794-7-5
PMCID: PMC3910242  PMID: 24460628
14.  Epistasis between COMT and MTHFR in Maternal-Fetal Dyads Increases Risk for Preeclampsia 
PLoS ONE  2011;6(1):e16681.
Preeclampsia is a leading cause of perinatal morbidity and mortality. This disorder is thought to be multifactorial in origin, with multiple genes, environmental and social factors, contributing to disease. One proposed mechanism is placental hypoxia-driven imbalances in angiogenic and anti-angiogenic factors, causing endothelial cell dysfunction. Catechol-O-methyltransferase (Comt)-deficient pregnant mice have a preeclampsia phenotype that is reversed by exogenous 2-methoxyestradiol (2-ME), an estrogen metabolite generated by COMT. 2-ME inhibits Hypoxia Inducible Factor 1α, a transcription factor mediating hypoxic responses. COMT has been shown to interact with methylenetetrahydrofolate reductase (MTHFR), which modulates the availability of S-adenosylmethionine (SAM), a COMT cofactor. Variations in MTHFR have been associated with preeclampsia. By accounting for allelic variation in both genes, the role of COMT has been clarified. COMT allelic variation is linked to enzyme activity and four single nucleotide polymorphisms (SNPs) (rs6269, rs4633, rs4680, and rs4818) form haplotypes that characterize COMT activity. We tested for association between COMT haplotypes and the MTHFR 677 C→T polymorphism and preeclampsia risk in 1103 Chilean maternal-fetal dyads. The maternal ACCG COMT haplotype was associated with reduced risk for preeclampsia (P = 0.004), and that risk increased linearly from low to high activity haplotypes (P = 0.003). In fetal samples, we found that the fetal ATCA COMT haplotype and the fetal MTHFR minor “T” allele interact to increase preeclampsia risk (p = 0.022). We found a higher than expected number of patients with preeclampsia with both the fetal risk alleles alone (P = 0.052) and the fetal risk alleles in combination with a maternal balancing allele (P<0.001). This non-random distribution was not observed in controls (P = 0.341 and P = 0.219, respectively). Our findings demonstrate a role for both maternal and fetal COMT in preeclampsia and highlight the importance of including allelic variation in MTHFR.
doi:10.1371/journal.pone.0016681
PMCID: PMC3031618  PMID: 21304959
15.  Effects of Catechol-O-Methyltransferase on Normal Variation in the Cognitive Function of Children 
The American journal of psychiatry  2009;166(8):909-916.
Objective
Genetic variants that contribute to the risk of psychiatric disorders may also affect normal variation in psychological function. Indeed, the behavioral effects of many genetic variants may be better understood as process-specific rather than disease-specific. A functional valine-to-methionine (Val158Met) polymorphism in the catechol-O-methyltransferase (COMT) gene has been associated with cognitive function and brain metabolic activity accompanying such tasks. Not all studies are consistent, and less is known about the effect of this polymorphism during development. The authors tested the hypothesis that a more informative COMT haplotype predicts normal cognitive development in a large population-based cohort of children enrolled in the Avon Longitudinal Study of Parents and children.
Method
Effects on verbal and performance IQ as well as verbal inhibition were assessed at age 8, and effects onworking memory were assessed at age 10. From the five COMT single nucleotide polymorphisms (SNPs) genotyped, the effect of a functional three-SNP haplotype consisting of Val158Met and two synonymous SNPs (rs6269 and rs4818), which together exert a major influence on the level of COMT expression and enzyme activity, was evaluated.
Results
This three-SNP haplotype predicted both verbal inhibition and working memory, and there was a genotype-by-sex interaction on verbal IQ. The effect of COMT genotype (diplotype) on cognition was curvilinear, which is consistent with the “inverted U” model of dopamine effect on frontal cortical efficiency. In addition, the SNP rs2075507 (previously rs2097603) was independently associatedwith verbal inhibition, while rs165599 showed no main cognitive effects. However, rs165599 showed a genotype-by-sex interaction with working memory.
Conclusions
Genetic variation at several loci in the COMT gene affects normal cognitive function in children.
doi:10.1176/appi.ajp.2009.08081251
PMCID: PMC2768274  PMID: 19605537
16.  A Transposon in Comt Generates mRNA Variants and Causes Widespread Expression and Behavioral Differences among Mice 
PLoS ONE  2010;5(8):e12181.
Background
Catechol-O-methyltransferase (COMT) is a key enzyme responsible for the degradation of dopamine and norepinephrine. COMT activity influences cognitive and emotional states in humans and aggression and drug responses in mice. This study identifies the key sequence variant that leads to differences in Comt mRNA and protein levels among mice, and that modulates synaptic function and pharmacological and behavioral traits.
Methodology/Principal Findings
We examined Comt expression in multiple tissues in over 100 diverse strains and several genetic crosses. Differences in expression map back to Comt and are generated by a 230 nt insertion of a B2 short interspersed element (B2 SINE) in the proximal 3′ UTR of Comt in C57BL/6J. This transposon introduces a premature polyadenylation signal and creates a short 3′ UTR isoform. The B2 SINE is shared by a subset of strains, including C57BL/6J, A/J, BALB/cByJ, and AKR/J, but is absent in others, including DBA/2J, FVB/NJ, SJL/J, and wild subspecies. The short isoform is associated with increased protein expression in prefrontal cortex and hippocampus relative to the longer ancestral isoform. The Comt variant causes downstream differences in the expression of genes involved in synaptic function, and also modulates phenotypes such as dopamine D1 and D2 receptor binding and pharmacological responses to haloperidol.
Conclusions/Significance
We have precisely defined the B2 SINE as the source of variation in Comt and demonstrated that a transposon in a 3′ UTR can alter mRNA isoform use and modulate behavior. The recent fixation of the variant in a subset of strains may have contributed to the rapid divergence of inbred strains.
doi:10.1371/journal.pone.0012181
PMCID: PMC2923157  PMID: 20808911
17.  A Single-Nucleotide Polymorphism in the Fetal Catechol-O-methyltransferase Gene is Associated With Spontaneous Preterm Birth in African Americans 
Reproductive Sciences  2012;19(2):135-142.
Catechol-O-methyltransferase (COMT) activity has been reported to be higher in African Americans (AA) than Caucasians (Cau). COMT converts 2- and 4-hydroxy (OH) estrogens to 2- and 4-methoxyestrogens, respectively, and can increase estrogenic milieu locally in tissues. To assess whether the increased incidence of preterm birth (PTB) among AA women is associated with single-nucleotide polymorphism (SNP) in the COMT gene, we examined variations in maternal and fetal COMT genes and their association with pregnancy outcomes (term vs preterm pregnancies) using 4 functional SNPs: rs4633, rs4680, rs4818, and rs6269 in both AA and Cau. We analyzed samples from 267 AA women (191 term and 76 preterm pregnancies) and 339 Cau (194 term and 145 preterm pregnancies) in this study. The results showed a significant difference (P < .05) in allele and genotype frequencies between term and preterm AA and Cau women in 3 SNPs in both maternal and fetal DNA. The analysis revealed that in AA fetal COMT genes, SNP rs4818 is associated with PTB at the allele (C; P < .001), genotype (C/C; P < .01), and 2- (P < .03) and 3 (P < .04)-window haplotype levels. Multidimensionality reduction analysis also showed a significant (P < .01) association between rs4818 and PTB. In conclusion, our study demonstrated that a synonymous polymorphism, rs4818 in the fetal COMT gene, is associated with PTB in AA.
doi:10.1177/1933719111417885
PMCID: PMC3343133  PMID: 22158829
preterm birth; African Americans; Caucasians; single-nucleotide polymorphism; catechol-O-methyltransferase
18.  Catechol-O-Methyltransferase (COMT) Single Nucleotide Polymorphisms and Haplotypes Are Not Major Risk Factors for Polycystic Ovary Syndrome 
Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects 5–8% of reproductive age women. The primary features of PCOS are hyperandrogenemia, chronic anovulation and infertility. It has been suggested that defects in ovarian steroid metabolism contribute to the follicular growth arrest and abnormal production of ovarian steroid hormones that are characteristic of PCOS. 2-methoxyestradiol (2-ME) is formed by the action of catechol-O-methyltransferase (COMT) on 2-hydroxyestradiol. COMT expression is increased in the follicles and ovarian stroma of women with PCOS. Moreover, 2-ME decreases granulosa cell proliferation and steroidogenesis, raising the possibility that ovarian dysfunction associated with PCOS is due, in part, to increased synthesis of 2-ME resulting from increased COMT activity. Four single-nucleotide polymorphisms (SNPs) (rs6269, rs4633, rs4818, rs4680) in the COMT gene characterize haplotypes, which are associated with large variations in COMT enzymatic activity. The aim of this study was to determine whether individual COMT SNPs and the COMT haplotypes are associated with PCOS using a family-based test of association and linkage. Additionally, we examined the relationships between COMT SNPs and haplotypes with quantitative variables usually assessed in the evaluation of women with PCOS. There were no significant correlations between genotype and total testosterone, non-SHBG bound testosterone and BMI. However, we found that the prolactin level in women with PCOS varied significantly with COMT haplotype, and suggest that this association reflects a genetic factor influencing the stress response. Our findings suggest that common variants and haplotypes of the COMT gene are not major contributors to risk for PCOS, but that COMT genotype may influence prolactin levels.
doi:10.1016/j.mce.2011.11.022
PMCID: PMC3264817  PMID: 22178088
Catechol-O-Methyltransferase; COMT; polycystic ovary syndrome; prolactin5
19.  Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression. 
Molecular and Cellular Biology  1987;7(8):2914-2924.
The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.
Images
PMCID: PMC367910  PMID: 2823108
20.  Codon-by-Codon Modulation of Translational Speed and Accuracy Via mRNA Folding 
PLoS Biology  2014;12(7):e1001910.
Secondary structure in mRNAs modulates the speed of protein synthesis codon-by-codon to improve accuracy at important sites while ensuring high speed elsewhere.
Rapid cell growth demands fast protein translational elongation to alleviate ribosome shortage. However, speedy elongation undermines translational accuracy because of a mechanistic tradeoff. Here we provide genomic evidence in budding yeast and mouse embryonic stem cells that the efficiency–accuracy conflict is alleviated by slowing down the elongation at structurally or functionally important residues to ensure their translational accuracies while sacrificing the accuracy for speed at other residues. Our computational analysis in yeast with codon resolution suggests that mRNA secondary structures serve as elongation brakes to control the speed and hence the fidelity of protein translation. The position-specific effect of mRNA folding on translational accuracy is further demonstrated experimentally by swapping synonymous codons in a yeast transgene. Our findings explain why highly expressed genes tend to have strong mRNA folding, slow translational elongation, and conserved protein sequences. The exquisite codon-by-codon translational modulation uncovered here is a testament to the power of natural selection in mitigating efficiency–accuracy conflicts, which are prevalent in biology.
Author Summary
Protein synthesis by ribosomal translation is a vital cellular process, but our understanding of its regulation has been poor. Because the number of ribosomes in the cell is limited, rapid growth relies on fast translational elongation. The accuracy of translation must also be maintained, and in an ideal scenario, both speed and accuracy should be maximized to sustain rapid and productive growth. However, existing data suggest a tradeoff between speed and accuracy, making it impossible to simultaneously maximize both. A potential solution is slowing the elongation at functionally or structurally important sites to ensure their translational accuracies, while sacrificing accuracy for speed at other sites. Here, we show that budding yeast and mouse embryonic stem cells indeed use this strategy. We discover that a codon-by-codon adaptive modulation of translational elongation is accomplished by mRNA secondary structures, which serve as brakes to control the elongation speed and hence translational fidelity. Our findings explain why highly expressed genes tend to have strong mRNA folding, slow translational elongation, and conserved protein sequences. The exquisite translational modulation reflects the power of natural selection in mitigating efficiency–accuracy conflicts, and our study offers a general framework for analyzing similar conflicts, which are widespread in biology.
doi:10.1371/journal.pbio.1001910
PMCID: PMC4106722  PMID: 25051069
21.  MANIPULATING THE GENETIC CODE FOR MEMBRANE PROTEIN PRODUCTION: WHAT HAVE WE LEARNT SO FAR? 
Biochimica et Biophysica Acta  2011;1818(4):1091-1096.
With synthetic gene services, molecular cloning is as easy as ordering a pizza. However choosing the right RNA code for efficient protein production is less straightforward, more akin to deciding on the pizza toppings. The possibility to choose synonymous codons in the gene sequence has ignited a discussion that dates back 50 years: Does synonymous codon use matter? Recent studies indicate that replacement of particular codons for synonymous codons can improve expression in homologous or heterologous hosts, however it is not always successful. Furthermore it is increasingly apparent that membrane protein biogenesis can be codon-sensitive. Single synonymous codon substitutions can influence mRNA stability, mRNA structure, translational initiation, translational elongation and even protein folding. Synonymous codon substitutions therefore need to be carefully evaluated when membrane proteins are engineered for higher production levels and further studies are needed to fully understand how to select the codons that are optimal for higher production.
doi:10.1016/j.bbamem.2011.08.018
PMCID: PMC3288168  PMID: 21884679
22.  A periodic pattern of mRNA secondary structure created by the genetic code 
Nucleic Acids Research  2006;34(8):2428-2437.
Single-stranded mRNA molecules form secondary structures through complementary self-interactions. Several hypotheses have been proposed on the relationship between the nucleotide sequence, encoded amino acid sequence and mRNA secondary structure. We performed the first transcriptome-wide in silico analysis of the human and mouse mRNA foldings and found a pronounced periodic pattern of nucleotide involvement in mRNA secondary structure. We show that this pattern is created by the structure of the genetic code, and the dinucleotide relative abundances are important for the maintenance of mRNA secondary structure. Although synonymous codon usage contributes to this pattern, it is intrinsic to the structure of the genetic code and manifests itself even in the absence of synonymous codon usage bias at the 4-fold degenerate sites. While all codon sites are important for the maintenance of mRNA secondary structure, degeneracy of the code allows regulation of stability and periodicity of mRNA secondary structure. We demonstrate that the third degenerate codon sites contribute most strongly to mRNA stability. These results convincingly support the hypothesis that redundancies in the genetic code allow transcripts to satisfy requirements for both protein structure and RNA structure. Our data show that selection may be operating on synonymous codons to maintain a more stable and ordered mRNA secondary structure, which is likely to be important for transcript stability and translation. We also demonstrate that functional domains of the mRNA [5′-untranslated region (5′-UTR), CDS and 3′-UTR] preferentially fold onto themselves, while the start codon and stop codon regions are characterized by relaxed secondary structures, which may facilitate initiation and termination of translation.
doi:10.1093/nar/gkl287
PMCID: PMC1458515  PMID: 16682450
23.  Efficient translation initiation dictates codon usage at gene start 
Rare codons are enriched at gene start in many genomes. Genome analysis and experimental testing show that this enrichment evolved to keep the ribosome binding site free from stable mRNA structures, in order to facilitate efficient translation initiation.
The use of rare codons coincides with suppression of mRNA structures at the ribosome binding site across genomes.There is preferential selection for synonymous codons that reduce GC-content at the beginning of genes and a stronger pressure for rare codon usage in GC-rich organisms.Amino acids encoded by AU-rich codons are preferred at gene start.Experimental results show that mRNA structure at translation start strongly influences protein yield.
The genetic code is degenerate; thus, protein evolution does not uniquely determine the coding sequence. One of the puzzles in evolutionary genetics is therefore to uncover evolutionary driving forces that result in specific codon choice. In many bacteria, the first 5–10 codons of protein-coding genes are often codons that are less frequently used in the rest of the genome, an effect that has been argued to arise from selection for slowed early elongation to reduce ribosome traffic jams. However, genome analysis across many species has demonstrated that the region shows reduced mRNA folding consistent with pressure for efficient translation initiation. This raises the possibility that unusual codon usage is a side effect of selection for reduced mRNA structure. Here we discriminate between these two competing hypotheses, and show that in bacteria selection favours codons that reduce mRNA folding around the translation start, regardless of whether these codons are frequent or rare. Experiments confirm that primarily mRNA structure, and not codon usage, at the beginning of genes determines the translation rate.
doi:10.1038/msb.2013.32
PMCID: PMC3964316  PMID: 23774758
codon usage; mRNA structure; translation
24.  Departure from multiplicative interaction for catechol-O-methyltransferase genotype and active/passive exposure to tobacco smoke among women with breast cancer 
Background
Women with homozygous polymorphic alleles of catechol-O-methyltransferase (COMT-LL) metabolize 2-hydroxylated estradiol, a suspected anticarcinogenic metabolite of estrogen, at a four-fold lower rate than women with no polymorphic alleles (COMT-HH) or heterozygous women (COMT-HL). We hypothesized that COMT-LL women exposed actively or passively to tobacco smoke would have higher exposure to 2-hydroxylated estradiol than never-active/never passive exposed women, and should therefore have a lower risk of breast cancer than women exposed to tobacco smoke or with higher COMT activity.
Methods
We used a case-only design to evaluate departure from multiplicative interaction between COMT genotype and smoking status. We identified 502 cases of invasive incident breast cancer and characterized COMT genotype. Information on tobacco use and other potential breast cancer risk factors were obtained by structured interviews.
Results
We observed moderate departure from multiplicative interaction for COMT-HL genotype and history of ever-active smoking (adjusted odds ratio [aOR] = 1.6, 95% confidence interval [CI]: 0.7, 3.8) and more pronounced departure for women who smoked 40 or more years (aOR = 2.3, 95% CI: 0.8, 7.0). We observed considerable departure from multiplicative interaction for COMT-HL genotype and history of ever-passive smoking (aOR = 2.0, 95% CI: 0.8, 5.2) or for having lived with a smoker after age 20 (aOR = 2.8, 95% CI: 0.8, 10).
Conclusion
With greater control over potential misclassification errors and a large case-only population, we found evidence to support an interaction between COMT genotype and tobacco smoke exposure in breast cancer etiology.
doi:10.1186/1477-3163-5-3
PMCID: PMC1373621  PMID: 16417624
25.  Coding-sequence determinants of gene expression in Escherichia coli 
Science (New York, N.Y.)  2009;324(5924):255-258.
Synonymous mutations do not alter the encoded protein, but they can influence gene expression. To investigate the mechanisms, we engineered a synthetic library of 154 genes that vary randomly at synonymous sites, but all encode the same green fluorescent protein. When expressed in E. coli, GFP protein levels varied 250-fold across the library. GFP mRNA levels, mRNA degradation patterns, and bacterial growth rates also varied, but codon bias did not correlate with gene expression. Rather, the stability of mRNA folding near the ribosomal binding site explained over half the variation in protein levels. In our analysis, mRNA folding and associated rates of translation initiation play a predominant role in shaping expression levels of individual genes, whereas codon bias influences global translation efficiency and cellular fitness.
doi:10.1126/science.1170160
PMCID: PMC3902468  PMID: 19359587

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