Human Catechol-O-Methyltransferase Haplotypes Modulate Protein Expression by Altering mRNA Secondary Structure

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Science  22 Dec 2006:
Vol. 314, Issue 5807, pp. 1930-1933
DOI: 10.1126/science.1131262


Catechol-O-methyltransferase (COMT) is a key regulator of pain perception, cognitive function, and affective mood. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous position, code for differences in COMT enzymatic activity and are associated with pain sensitivity. Haplotypes divergent in synonymous changes exhibited the largest difference in COMT enzymatic activity, due to a reduced amount of translated protein. The major COMT haplotypes varied with respect to messenger RNA local stem-loop structures, such that the most stable structure was associated with the lowest protein levels and enzymatic activity. Site-directed mutagenesis that eliminated the stable structure restored the amount of translated protein. These data highlight the functional significance of synonymous variations and suggest the importance of haplotypes over single-nucleotide polymorphisms for analysis of genetic variations.

The ability to predict the downstream effects of genetic variation is critically important for understanding both the evolution of the genome and the molecular basis of human disease. The effects of nonsynonymous polymorphisms have been widely characterized; because these variations directly influence protein function, they are relatively easy to study statistically and experimentally (1). However, characterizing polymorphisms located in regulatory regions, which are much more common, has proved to be problematic (2). Here, we focus on the mechanism whereby polymorphisms of the cathechol-O-methyltransferase (COMT) gene regulate gene expression.

COMT is an enzyme responsible for degrading catecholamines and thus represents a critical component of homeostasis maintenance (3). The human COMT gene encodes two distinct proteins: soluble COMT (S-COMT) and membrane-bound COMT (MB-COMT) through the use of alternative translation initiation sites and promoters (3). Recently, COMT has been implicated in the modulation of persistent pain (47). Our group demonstrated that three common haplotypes of the human COMT gene are associated with pain sensitivity and the likelihood of developing temporomandibular joint disorder (TMJD), a common chronic musculoskeletal pain condition (4). Three major haplotypes are formed by four single-nucleotide polymorphisms (SNPs): one located in the S-COMT promoter region (A/G; rs6269) and three in the S- and MB-COMT coding region at codons his62his (C/T; rs4633), leu136leu (C/G; rs4818), and val158met (A/G; rs4680) (Fig. 1A). On the basis of subjects' pain responsiveness, haplotypes were designated as low (LPS; GCGG), average (APS; ATCA), or high (HPS; ACCG) pain sensitive. Individuals carrying HPS/APS or APS/APS diplotypes were nearly 2.5 times as likely to develop TMJD. Previous data further suggest that COMT haplotypes code for differences in COMT enzymatic activity (4); however, the molecular mechanisms involved have remained unknown.

Fig. 1.

Common haplotypes of the human COMT gene differ with respect to mRNA secondary structure and enzymatic activity. (A) A schematic diagram illustrates COMT genomic organization and SNP composition for the three haplotypes. Percent frequency of each haplotype in a cohort of healthy Caucasian females, and percent independent SNP contribution to pain sensitivity, are indicated. (B) The local stem-loop structures associated with each of the three haplotypes are shown. Relative to the LPS and APS haplotypes, the HPS local stem-loop structure had a higher folding potential. (C and D) The LPS haplotype exhibited the highest, while the HPS haplotype exhibited the lowest enzymatic activity and protein levels in cells expressing COMT. ***P <0.001, ≠ LPS. +++P< 0.001, ≠ APS.

A common SNP in codon 158 (val158met) has been associated with pain ratings and μ-opioid system responses (7) as well as addiction, cognition, and common affective disorders (3, 810). The substitution of valine (Val) by methionine (Met) results in reduced thermostability and activity of the enzyme (3). It is generally accepted that val158met is the main source of individual variation in human COMT activity; numerous studies have identified associations between the low-activity met allele and several complex phenotypes (3, 8, 10). However, observed associations between these conditions and the met allele are often modest and occasionally inconsistent (3). This suggests that additional SNPs in the COMT gene modulate COMT activity. Indeed, we found that haplotype rather than an individual SNP better accounts for variability in pain sensitivity (4). The HPS and LPS haplotypes that both code for the stable val158 variant were associated with the two extreme-pain phenotypes; thus, the effect of haplotype on pain sensitivity in our study cannot be explained by the sum of the effects of functional SNPs. Instead, the val158met SNP interacts with other SNPs to determine phenotype. Because variation in the S-COMT promoter region does not contribute to pain phenotype (Fig. 1A), we suggest that the rate of mRNA degradation or protein synthesis is affected by the structural properties of the haplotypes, such as haplotype-specific mRNA secondary structure.

Previous reports have shown that polymorphic alleles can markedly affect mRNA secondary structure (11, 12), which can then have functional consequences on the rate of mRNA degradation (11, 13). It is also plausible that polymorphic alleles directly modulate protein translation through alterations in mRNA secondary structure, because protein translation efficiency is affected by mRNA secondary structure (1416). To test these possibilities, we evaluated the affect of LPS, APS, and HPS haplotypes on the stability of the corresponding mRNA secondary structures (17).

Secondary structures of the full-length LPS, APS, and HPS mRNA transcripts were predicted by means of the RNA Mfold (18, 19) and A fold (20) programs. The mRNA folding analyses demonstrated that the major COMT haplotypes differ with respect to mRNA secondary structure. The LPS haplotype codes for the shortest, least stable local stem-loop structure, and the HPS haplotype codes for the longest, most stable local stem-loop structure in the val158 region for both S-COMT and MB-COMT. Gibbs free energy (ΔG) for the stem-loop structure associated with the HPS haplotype is ∼17 kcal/mol less than that associated with the LPS haplotype for both S-COMTand MB-COMT (Fig. 1B and fig. S1A). Additional evidence supporting predicted RNA folding structures was obtained by generating consensus RNA secondary structures based on comparative analysis of COMT sequences from eight mammalian species (fig. S2). The consensus RNA folding structures were LPS-like and did not contain highly stable local stem-loop structures analogous to the human HPS-like form. Thus, substantial deviation from consensus structure, as observed for the HPS haplotype, should have notable functional consequences. Additional studies were conducted to test this molecular modeling.

We constructed full-length S- and MB-COMT cDNA clones in mammalian expression vectors that differed only in three nucleotides corresponding to the LPS, APS, and HPS haplotypes (17, 21). Rat adrenal (PC-12) cells were transiently transfected with each of these six constructs. COMT enzymatic activity, protein expression, and mRNA abundance were measured. Relative to the LPS haplotype, the HPS haplotype showed a 25- and 18-fold reduction in enzymatic activity for S- and MB-COMT constructs, respectively (Fig. 1C and fig. S1B). The HPS haplotype also exhibited marked reductions in S- and MB-COMT protein expression (Fig. 1D and fig. S1C). The APS haplotype displayed a moderate 2.5- and 3-fold reduction in enzymatic activity for S- and MB-COMT constructs, respectively, while protein expression levels did not differ. The moderate reduction in enzymatic activity produced by the APS haplotype is most likely due to the previously reported decrease in protein thermostability coded by the met158 allele (3). These data illustrate that the reduced enzymatic activity corresponding to the HPS haplotype is paralleled by reduced protein levels, an effect that could be mediated by local mRNA secondary structure at the level of protein synthesis and/or mRNA degradation. Because total RNA abundance and RNA degradation rates did not parallel COMT protein levels (fig. S2), differences in protein translation efficiency likely results from differences in the local secondary structure of corresponding mRNAs.

To directly assess this hypothesis, we performed site-directed mutagenesis (17). The stable stem-loop structure of S- and MB-COMT mRNA corresponding to the HPS haplotype is supported by base pairs between several critical nucleotides, including 403C and 479G in S-COMT and 625C and 701G in MB-COMT (Fig. 2A and fig. S3A). Mutation of 403C to G in S-COMT or 625C to G in MB-COMT destroys the stable stem-loop structure and converts it into a LPS haplotype–like structure (HPS Lsm). Double mutation of mRNA in position 403C to G and 479G to C in S-COMT or 625C to G and 701G to C in MB-COMT reconstructs the original long stem-loop structure (HPS dm). The single- and double-nucleotide HPS mutants (HPS Lsm and HPS dm, respectively) were transiently transfected to PC-12 cells. As predicted by the mRNA secondary-structure folding analyses, the HPS Lsm exhibited increased COMT enzymatic activity and protein levels equivalent to those of the LPS haplotype, whereas the HPS dm exhibited reduced enzymatic activity and protein levels equivalent to those of the original HPShaplotype (Fig. 2, B and C; fig.S3, Band C). These data rule out the involvement of RNA sequence recognition motifs or codon usage in the regulation of translation. In contrast to the HPS haplotype, protein levels did not parallel COMT enzymatic activity for the APS haplotype and site-directed mutagenesis confirmed that the met158 allele, not a more stable mRNA secondary structure, drives the reduced enzymatic activity observed for the APS haplotype (fig. S4). This difference is moderate relative to the mRNA structure–dependent difference coded by LPS and HPS haplotypes. These results were verified by an alternate approach of modifying mRNA secondary structure (fig. S5).

Fig. 2.

Site-directed mutagenesis that destroys the stable stem-loop structure corresponding to the HPS haplotype restores COMT enzymatic activity and protein expression. (A) The mRNA structure corresponding to the HPS haplotype was converted to an LPS haplotype-like structure (HPS Lsm) by single mutation of 403C to G. The original HPS haplotype structure (HPS dm) was restored by double mutation of interacting nucleotides 403C to G and 479G to C. (B and C) The HPS Lsm exhibited COMT enzymatic activity and protein levels equivalent to those of the LPS haplotype, whereas the HPS dm exhibited reduced enzymatic activity. ***P <0.001, ≠ LPS.

Our data have very broad evolutionary and medical implications for the analysis of variants common in the human population. The fact that alterations in mRNA secondary structure resulting from synonymous changes have such a pronounced effect on the level of protein expression emphasizes the critical role of synonymous nucleotide positions in maintaining mRNA secondary structure and suggests that the mRNA secondary structure, rather than independent nucleotides in the synonymous positions, should undergo substantial selective pressure (22). Furthermore, our data stress the importance of synonymous SNPs as potential functional variants in the area of human medical genetics. Although nonsynonymous SNPs are believed to have the strongest impact on variation in gene function, our data clearly demonstrate that haplotypic variants of common synonymous SNPs can have stronger effects on gene function than nonsynonymous variations and play an important role in disease onset and progression.

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