Introduction

Catechol O-methyltransferase (COMT, EC 2.1.1.6) plays an important role in the metabolism of catecholamine neurotransmitters.¹ COMT catalyzes the O-methylation of catechol compounds, with S-adenosyl-L-methionine (AdoMet) as the methyl donor. COMT substrates include not only neurotransmitters such as norepinephrine, epinephrine and dopamine, but also catecholestrogens and catechol drugs that are used to treat hypertension, asthma and Parkinson's disease.^1,2 COMT is present in mammalian cells as both membrane-bound (MB) and soluble (S) cytosolic forms.³ The enzyme is ubiquitously expressed, although S-COMT is expressed at higher levels in most tissues than MB-COMT.⁴

Family studies of red blood cell (RBC) COMT activity showed a bimodal frequency distribution, and the results of segregation analysis were consistent with the inheritance of level of activity as a result of two autosomal codominant alleles.^5,6,7,8 Levels of RBC COMT activity were correlated with levels of activity in other tissues such as kidney, lung and liver.^9,10 In addition, the trait of a low level of COMT activity was associated with decreased enzyme thermal stability, suggesting that the COMT polymorphism might result from an alteration in amino-acid sequence.^10,11,12 Subsequently, the human COMT cDNA and gene were cloned.^4,13,14 COMT is encoded by a single gene with six exons that maps to chromosome 22q11.21.¹⁵ The phenotype of low levels of COMT activity has been shown to be due mainly to a single nucleotide polymorphism (SNP) that changes the amino acid at codon 108 in S-COMT (codon 158 in MB-COMT) from Val to Met.¹⁶ The frequency of the low-activity allele that encodes Met 108/158 ranges from approximately 50% in Caucasian subjects to 20–30% in East Asians, with some populations having even lower allele frequencies, for example, 6% in Ghana.^17,18

The Val108/158Met genetic polymorphism has been the subject of intense molecular epidemiologic study because of the important role of COMT in catecholamine metabolism, and has been reported to be associated with risk for schizophrenia, obsessive–compulsive disorder, bipolar disorder, Parkinson's disease and breast cancer.^{19,20,21,22,23} Many of these associations have proven difficult to confirm,^24,25,26 but, based on this type of data, a study of COMT inhibitors in schizophrenic patients who have been stratified for the codon 108/158 polymorphism is currently underway.²⁷ However, not all COMT phenotypic variance can be attributed to the Val108/158Met polymorphism, indicating that additional genetic variants may contribute to this phenotype.¹⁶ Although several additional COMT gene sequence variants have been identified, none of them has been reported to be of functional significance. In the present study, we set out to systematically resequence the exons, splice junctions and promoter for the portion of this gene encoding S-COMT using DNA samples from both African-American (AA) and Caucasian-American (CA) subjects. Functional genomic studies were then performed for all nonsynonymous cSNPs observed—including that responsible for the Val108/158Met polymorphism. In the course of these experiments, we discovered and characterized a novel, common nonsynonymous cSNP that was observed only in DNA from AA subjects. We also determined that the mechanism responsible for the functional effects of the common—and intensely studied—Val108/158Met COMT polymorphism was a decrease in the quantity of COMT protein.

Materials and methods

DNA samples

DNA samples from 60 AA and 60 CA subjects (sample sets HD100AA and HD100CAU) were obtained from the Coriell Institute Cell Repository (Camden, NJ, USA). These samples had been anonymized prior to deposit, and all subjects had provided written consent for the use of their DNA for experimental purposes. DNA was also extracted from 33 anonymized liver biopsy samples studied previously by Lachman et al.¹⁶ using the QIAamp DNA mini kit (QIAGEN, Valencia, CA, USA). Our experiments had been reviewed and approved by the Mayo Clinic Institutional Review Board.

COMT resequencing

Seven PCR amplifications were performed for each of the 120 human DNA samples using primers that flanked COMT exons 2–6, as well as the proximal promoter region (intron 2) for S-COMT (PCR primer sequences and detailed reaction conditions are available online in Supplementary Information Table 1). Locations of primers were chosen to avoid repetitive sequence and to ensure amplification specificity. M13 tags were added to the 5'-ends of each forward and reverse primer to make it possible to use dye-primer sequencing chemistry to enhance our ability to identify heterozygous bases.²⁸ The G472A SNP responsible for the Val108/158Met polymorphism was also determined in 33 DNA samples from human liver biopsies by amplifying and sequencing COMT exon 4 only. Amplicons were sequenced on both strands in the Mayo Clinic Molecular Biology Core Facility with an ABI 377 DNA sequencer using BigDye™ dye-primer chemistry. Samples with ambiguous chromatograms or those with SNPs that were observed in only a single DNA sample were subjected to an independent amplification, followed by sequencing. DNA sequencing chromatograms were analyzed using the PolyPhred 3.0²⁹ and Consed 8.0 and GCG programs.^30,31 The consensus sequence used was the COMT gene sequence in the RefFeq chromosome 22 draft sequence (NT_011519).

COMT expression

A cDNA encoding S-COMT was amplified from a human liver Marathon-Ready cDNA library (BD Biosciences Clontech, Palo Alto, CA, USA). S-COMT cDNA sequences encoding polymorphic amino acids were created using site-directed mutagenesis performed with overlap extension as described by Ho et al.³² The wild type (WT) and variant S-COMT cDNAs were then cloned into the eukaryotic expression vector pCR3.1 (Invitrogen, Carlsbad, CA, USA) and were verified by DNA sequencing. These expression constructs were used to transfect COS-1 and HEK293 cells with the TransFast™ transfection reagent as suggested by the manufacturer (Promega, Madison, WI, USA). pSV--galactosidase (Promega) was cotransfected with each COMT expression construct in a 1 : 1 ratio to make it possible to correct for transfection efficiency. Transfected cells were harvested after 48 h, and high-speed supernatant (HSS) cytosol preparations were prepared as described previously.³³ Hepatic cytosol was also prepared from the 33 human liver biopsy samples as described by Lachman et al.¹⁶ Aliquots of these preparations were stored at -80°C prior to assay.

Enzyme activity and protein assays

COMT enzyme activity was measured using the assay of Raymond and Weinshilboum³⁴ as modified by Boudíková et al.¹⁰ This assay is based on the transfer of a radioactive methyl group from [¹⁴C-methyl]-S-adenosyl-L-methionine (AdoMet) to the catechol substrate 3,4-dihydroxybenzoic acid (DBA). Blank samples did not contain DBA. Endogenous COMT activity was never more than 1% of the activity observed after transfection with the WT construct. Apparent K_m values for DBA were determined with substrate concentrations that varied from 5 to 300 M. A series of AdoMet concentrations (1.3–88 M) was also tested with each allozyme to determine apparent K_m values for the cosubstrate. Enzyme activity was expressed as nanomoles of methylated product formed per minute of incubation per microgram of protein (ie, U/g protein). Enzyme thermal stability was tested by preincubating each recombinant allozyme for 15 min at temperatures that ranged from 37 to 65°C. -Galactosidase activity was measured spectrophotometrically using the -Galactosidase Enzyme Assay System (Promega). Protein concentration was determined with the dye-binding method of Bradford³⁵ using BIORAD Protein Assay Dye reagent (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin (BSA) as the standard.

COMT antibodies and Western blot analysis

Polyclonal antibodies to COMT were produced in rabbits. As a first step, two peptides were synthesized that corresponded to S-COMT amino acids 1–25 and 125–148, with an additional cysteine residue at the amino terminus of each peptide. These peptides differed from comparable regions of other known methyltransferase enzymes and did not correspond to any other known human proteins. The synthetic peptides were conjugated to keyhole limpet hemocyanin, and these conjugates were used to generate rabbit polyclonal antibodies (ResGen, Huntsville, AL, USA). The rabbit polyclonal anti-COMT antibodies were tested against pooled human liver cytosol and were found to react with a 25 kDa protein—the anticipated M_r value for COMT—with little cross-reactivity. Furthermore, this 25 kDa protein was detected in COS-1 and HEK293 cells only after transfection of the cells with a COMT expression construct. The antibodies were used to perform quantitative Western blot analyses with recombinant COMT allozymes. In those experiments, the quantity of COS-1 or HEK293 cytosol loaded on a 12% acrylamide gel was adjusted so that each lane had been corrected for transfection efficiency. After transfer to a PVDF membrane, the blots were probed with rabbit antiserum, followed by secondary antibody (goat anti-rabbit horseradish peroxidase) and bound antibody was detected using the ECL Western Blotting system (Amersham Pharmacia, Piscataway, NJ, USA). The AMBIS Radioanalytic Imaging System, Quant Probe Version 4.31 (Ambis Inc., San Diego, CA, USA) was used to quantitate the immunoreactive protein in each lane, and the data were expressed as a percentage of the intensity of WT COMT protein bands on the same gel. When human hepatic cytosol was used to perform Western analysis, the quantity of cytosol loaded in each lane was adjusted to achieve equivalent quantities of total cytosol protein.

Data analysis

Apparent K_m and V_max values were calculated using the method of Wilkinson³⁶ with a computer program written by Cleland.³⁷ These data were compared statistically using ANOVA (StatView program version 4.5; Abacus Concepts, Inc., Berkeley, CA, USA). Linear regression analysis was used to determine T₅₀ values (GraphPad Prism, version 3.03, GraphPad Software, San Diego, CA, USA). Linkage analysis was performed by calculating D' values for all possible pairwise combinations of polymorphisms. D' is a method for calculating linkage analysis that is independent of allele frequency.^38,39 Haplotypes were inferred using a program based on the EM algorithm.^40,41,42 Values for , the average heterozygosity per site and , the population mutation parameter, as well as Tajima's D-test for neutrality were determined using the Arlequin (2.0) software developed by Schneider et al.⁴³

Results

COMT gene resequencing

Human COMT was resequenced by amplifying exons 2–6, intron–exon splice junctions and the entire 'proximal promoter' (intron 2). Unfortunately, exon 1 proved difficult to amplify, probably because of its high GC content, so it was not resequenced. A total of approximately 816 kb of DNA sequence was analyzed for the 120 human DNA samples studied. All samples were sequenced on both strands. A total of 24 polymorphisms were observed in the 240 alleles analyzed, including eight ORF SNPs, 15 SNPs located within intronic and flanking regions and one insertion/deletion in the 3'-UTR (Figure 1 and Table 1). The numbering scheme for polymorphisms located within exons and in 5'- and 3'-untranslated regions is based on the MB-COMT cDNA sequence, with the 'A' in the translation initiation codon designated (+1). Nucleotides located 5' to that position were assigned negative numbers, while those located 3' were assigned positive numbers. Positions within introns were numbered relative to splice junctions, with the initial 5' nucleotide in an intron designated (+1). In all, 13 of the SNPs were present in samples from both AA and CA subjects, but allele frequencies often differed in the two populations. Most of the polymorphisms had frequencies greater than 1% and, as a result, would be considered 'common' in these populations. Nine of the 24 polymorphisms that we identified had been described previously in publicly available databases such as LocusLink, dbSNP and the EST database.

Figure 1.

Human COMT polymorphisms. Schematic representation of genetic variations within exons 2–6 of the COMT gene that were observed during our resequencing studies. The diagram is not to scale. Locations of polymorphisms are indicated by arrows. Red arrows represents frequencies greater than 10%, blue frequencies between 1 and 10%, and black frequencies below 1%. 'CA' indicates data for the DNA samples from Caucasian-American and 'AA' data from African-American subjects.

Full figure and legend (110K)

Table 1 - Human COMT polymorphisms.

Full table (96K)

Two of the 24 COMT polymorphisms were nonsynonymous cSNPs, that is, they altered the encoded amino acid, resulting in two variant COMT allozyme sequences. One was the Val108/158Met polymorphism that had been described previously, with allele frequencies in these two population samples similar to those described previously.^18,44 The other, a novel nonsynonymous cSNP, resulted in an Ala52/102Thr change in the encoded amino acid and was observed only in AA subjects. A comparison of the human COMT amino acid sequence with those of chimpanzee, gorilla, orangutan, bonobo, dog, cat, rat, mouse and pig COMT showed that the amino acid encoded by codon 52/102 was Ala in all of these species except the pig. Pigs had the variant human amino acid, Thr, at this position. No other species among those studied had Met, the amino acid encoded by the variant human allele at codon 108/158, at that position. Neither of these nonsynonymous cSNPs altered amino acids which are thought to be involved in substrate binding on the basis of X-ray crystallography data,⁴⁵ and neither was located within the methyltransferase 'signature sequences' that have been designated regions I, II and III⁴⁶—observations relevant to our subsequent substrate kinetic studies. A third COMT nonsynonymous cSNP at amino acid 22/72 that had been reported previously^47,48 was not observed in either of our sample sets. That SNP had been observed in population samples of 48 Japanese subjects⁴⁸ and 51 individuals of mixed ethnicity.⁴⁷ Unfortunately, the frequency of the codon 22/72 SNP was not reported for either of these two population samples.

The region containing the proximal promoter for S-COMT—intron 2—was also of particular interest. Ten SNPs were observed within this region, one of which was located within the S-COMT core promoter.^4,49 The TRANSFAC database⁵⁰ was used to determine whether any of these intron 2 proximal promoter SNPs might be present within putative transcription factor recognition sequences. SNPs I2(521), I2(602) and I2(832) were located within putative transcription factor recognition sequences for TCF11 (also KCR-F1 and Nrf1), Sp1 and AP1 (Fos/Jun), respectively. In addition, the I2(521) polymorphism was located within an estrogen response element that had been characterized by Xie et al.⁴⁹ The SNP at I2(832) was present in both populations, while the other two polymorphisms were observed at low frequencies in only one ethnic group (Table 1). The SNP at I2(1140), within the S-COMT core promoter, was present at high frequency in both populations.

Of the 24 polymorphisms, 20 were observed in the DNA samples from AA subjects, and 17 were present in samples from CA subjects. The average number of polymorphisms in COMT was 6.7 per kb resequenced in the AA samples and 5.0 per kb in samples from CA subjects. These numbers are similar to a value of 4.6 SNPs per kb for 75 human genes reported by Halushka et al.⁵¹ 'Nucleotide diversity' is a measure of genetic variation—adjusted for the number of alleles studied. Two standard measures of nucleotide diversity are , average heterozygosity per site, and , a population mutation measure that is theoretically equal to the neutral mutation parameter.^52,53 In our populations, =3.51.1 10^-4 for AA and =3.0 1.0 10^-4 for CA subjects, while was 5.42.6 10^-4 for DNA from AA and 4.32.1 10^-4 for samples from CA subjects. These results are similar to data reported by Stephens et al.⁵² for 292 autosomal genes (average =9.6 10^-4 and average =5.8 10^-4). The difference between and is the basis for Tajima's D statistic. Under conditions of neutrality, it is assumed that =, with D=0. D was positive for both of our study populations, but neither value differed significantly from zero (data not shown).

Linkage and haplotype analysis

Linkage analysis performed for all possible pairwise combinations of COMT polymorphisms demonstrated that many of them were tightly linked (see Supplementary Information Table 2, which is available online). Patterns of linkage, like those for SNP frequencies, differed between the two populations studied. Tight linkage between SNPs located in the proximal promoter region and the common Val108/158Met polymorphism was more common in samples for the CA subjects (three SNPs) than in samples from AA (no SNPs).

Haplotype analysis was also performed. Unambiguous haplotypes, those with only a single heterozygous nucleotide, accounted for 28 and 86% of all alleles studied in the AA and CA population samples, respectively. There were also 45 inferred haplotypes for the AA and 15 for the CA subjects. Haplotypes, both inferred and unambiguous, that had frequencies of 2% or greater are listed in Table 2. The data in the table illustrate the differences in haplotype patterns between the two populations studied—most striking for ^*1A, the most common haplotype for CA subjects. However, this haplotype was neither observed nor inferred among the AA subjects. The data in Table 2 also serve to highlight the tight linkage observed between Met108/158 and SNPs located in the proximal promoter for DNA samples from CA subjects. Allele designations shown in the table were made on the basis of encoded amino acids, with the WT sequence at nucleotide 472 assigned ^*1 (ie Val108/158), while those with Met at codon 108/158 were assigned the designation ^*2. Letter designations were then assigned on the basis of descending allele frequencies. Although, as a result of its low frequency, we did not observe unequivocal haplotypes that contained Thr52/102, we assigned the designation ^*3 to alleles that included this genotype.

Table 2 - Human COMT haplotype analysis.

Full table (129K)

Recombinant COMT activity

The next series of experiments was designed to test the functional implications of the two nonsynonymous cSNPs observed during the gene resequencing studies. Expression constructs were created for the WT allele as well as alleles encoding the two COMT variant allozymes (Thr52/102 and Met108/158), and these constructs were used to transfect COS-1 and HEK293 cells transiently. Mammalian cells were used to ensure appropriate post-translational modification as well as the presence of mammalian protein degradation systems. Since S-COMT was expressed, the nomenclature for codon number used subsequently will be that which applies to S-COMT. Expression of the Met108 allozyme in both COS-1 and HEK293 cells resulted in a 35–40% decrease in activity (P<0.04) as compared to cells expressing the WT allozyme (Figure 2 and Supplementary Information Table 3, which is available online). These observations correlate with results anticipated on the basis of the association of this genotype with significantly decreased COMT activity in human tissue samples.¹⁶ The Thr52 allozyme displayed a slight, but not significant, increase in the level of activity when compared with the WT allozyme (Figure 2).

Figure 2.

Recombinant human COMT allozyme activity. Levels of activity for COMT allozymes expressed in COS-1 (N=12) or HEK293 (N=8) cells were determined with 300 M DBA as substrate. Activities (meanSEM) are shown relative to the activity of the WT allozyme after correction for transfection efficiency. ^*P<0.05 compared with cells transfected with the WT construct.

Full figure and legend (18K)

The changes in the level of COMT activity that we observed might have resulted from alterations in the kinetic properties of the enzyme, as has been observed for other genetically polymorphic enzymes.^54,55,56 However, apparent K_m values after expression in COS-1 cells—the only cell type in which the substrate kinetic studies were performed—showed only small differences for both cosubstrates, DBA and AdoMet (Table 3). Although the apparent K_m values did differ statistically for some of the comparisons, these small differences seemed unlikely to explain the differences in levels of activity observed with the saturating concentrations of substrate used to measure enzyme activity. Finally, thermal stability has often been used as an indirect indicator of alterations in the amino-acid sequence, and it was reported over 20 years ago that enzyme activity in subjects homozygous for the common genetic variant controlling low COMT activity was more thermolabile than activity in subjects homozygous for the WT (high activity) phenotype.¹¹ Therefore, we used recombinant protein to confirm that the Met108 allozyme had a significantly lower T₅₀ (temperature resulting in 50% inactivation) than either the WT or Thr52 allozymes (P<0.005; Table 3).

Table 3 - Recombinant human COMT allozyme substrate kinetics when expressed in COS-1 cells.

Full table

Recombinant COMT Western blot analyses

Alterations in the amino-acid sequence as a result of genetic polymorphisms have often been found to be associated with changes in the levels of immunoreactive protein.^{54,55,56,57,58} Therefore, levels of COMT immunoreactive protein were also measured for the recombinant human COMT allozymes by Western blot analysis. A representative Western blot for COS-1 cell preparations is shown in Figure 3. Differences in the levels of immunoreactive protein for the three allozymes paralleled variations in the level of enzyme activity (Figure 4a). Specifically, average levels of COMT protein in both cell lines were decreased significantly in cells expressing Met108 (Figure 4a). Conversely, cells expressing Thr52 had increased level of COMT protein, significantly increased for COS-1 cells (P<0.05), when compared with those expressing the WT allozyme. It should be pointed out that the peptide used to generate the rabbit polyclonal antibody used to perform the Western blot analyses did not include the polymorphic amino acids. These observations were compatible with a growing body of evidence that alteration of only a single amino acid as a result of genetic polymorphisms can result in significant changes in levels of immunoreactive protein.^{54,55,56,57,58}

Figure 3.

COMT Western blot analysis. Western blots were used to analyze levels of COMT allozymes expressed in mammalian cells in culture as well as in 33 human liver biopsy samples. Representative Western blot data are shown for each allozyme expressed in COS-1 cells (left) and for livers from three individuals with the genotypes indicated at S-COMT codon 108 (right).

Full figure and legend (89K)

Figure 4.

Recombinant human COMT immunoreactive protein. (a) COMT immunoreactive protein expressed in COS-1 and HEK293 cells (N=6 independent transfections in each case) corrected for variation in transfection efficiency. Values shown are meanSEM relative to WT allozyme. ^*P<0.05 and ^**P<0.005 compared with cells transfected with the WT allozyme. (b) COMT immunoreactive protein in human liver biopsy samples. The gels were loaded with equal quantities of hepatic cytosol protein. A standard curve of WT COMT expressed in COS-1 cells was used to quantitate the data. ^*P<0.05 and ^**P<0.005 for the comparison indicated.

Full figure and legend (42K)

Human liver COMT genotype–phenotype correlation analysis

Our observation of a significant decrease in the level of immunoreactive protein for the recombinant Met108 allozyme after the transfection of both COS-1 and HEK293 cells suggested that a similar mechanism might occur in vivo. Therefore, we also performed genotype–phenotype correlation analysis for the level of COMT immunoreactive protein using 33 liver biopsy samples that had previously been genotyped for the codon 108/158 polymorphism and had been phenotyped for the level of enzyme activity.¹⁶ This group included 14 samples from heterozygous individuals, seven from subjects homozygous for the WT allele encoding Val108 and 12 samples from subjects homozygous for the Met108 allele. Equivalent concentrations of hepatic cytosol were loaded on the gels on the basis of protein concentration, and these samples were subjected to quantitative Western blot analysis. There was a significant decrease in the level of COMT protein in samples from subjects homozygous for the allele encoding Met108 when compared with heterozygous samples or samples from subjects homozygous for Val108 (P<0.002 by ANOVA; Figure 4b). These observations demonstrated that the major mechanism responsible for decreased COMT activity in COS-1 cells and HEK293—a decreased level of enzyme protein — was also associated with decreased enzyme activity in vivo.

Discussion

COMT plays an important role in the metabolism of endogenous catecholamines, catecholestrogens and catechol drugs. We previously identified a common genetic polymorphism that regulated the level of COMT enzyme activity,⁵ and later demonstrated that a common nonsynonymous cSNP that results in a Val108/158Met alteration in the encoded amino acid was associated with this polymorphism.¹⁶ However, that SNP did not explain all variation in the level of enzyme activity in 33 liver biopsy samples.¹⁶ We have now applied a genotype-to-phenotype research strategy in an attempt to identify additional DNA sequence variation that might contribute to individual differences in COMT activity or properties. As a first step, we resequenced COMT using DNA from 60 AA and 60 CA subjects. Regions of the gene sequenced included exons 2–6, exon–intron splice junctions and the promoter for S-COMT (intron 2). There were 23 SNPs in the 120 DNA samples (240 alleles) studied, including eight in the ORF and 15 in intronic and flanking regions. In addition, there was a single insertion/deletion located in the 3'-UTR. Two of the cSNPs were nonsynonymous: the common Val108/158Met polymorphism that we had described previously¹⁶ and a novel cSNP found only in the AA population that resulted in an Ala52/102Thr change in the encoded amino acid. An additional nonsynonymous cSNP within codon 22/72 that had been reported by Cargill et al⁴⁷ and Saito et al⁴⁸ was not observed in either of our population samples. In all, 20 of the 24 polymorphisms were present in samples from AA subjects and 17 were observed in DNA from CA subjects. Many of the polymorphisms were linked, with different linkage patterns in the two populations. Analysis of our data also resulted in the identification of a series of COMT haplotypes which also differed between the two populations (Table 2).

We then performed experiments designed to study the possible functional implications of the two nonsynonymous COMT cSNPs that we had observed. Neither of these polymorphisms altered an amino acid located within substrate-binding sites on the basis of the crystal structure of the protein.⁴⁵ Perhaps for that reason, neither had major effects on the apparent K_m values of recombinant allozymes for either DBA or AdoMet (Table 3). However, thermal stability was decreased for the Met108 allozyme, as reported previously for both tissue preparations and bacterially expressed protein.^11,59 The thermal stability of the Thr52 allozyme did not differ significantly from that of the WT enzyme.

The decreased level of COMT activity that occurs in association with the codon 108/158 polymorphism has been well described over the past two decades.^{5,11,16,44,60} However, the underlying mechanism has remained elusive.⁶¹ After transient expression in COS-1 and HEK293 cells, we found that the level of activity for the Met108 allozyme was decreased by 35–40% compared to the WT allozyme. This effect resulted primarily from a decreased level of COMT immunoreactive protein. To determine whether that same phenomenon might occur in vivo, 33 liver biopsy samples that had previously been phenotyped for the level of activity and genotyped for the Val108Met polymorphism¹⁶ were used to perform quantitative Western blot analysis. The level of immunoreactive protein in these samples was also found to be significantly decreased in subjects homozygous for Met108. These observations were compatible with a growing body of evidence which shows that a change in only a single amino acid as a result of a genetic polymorphism can often result in a decreased level of immunoreactive protein.^{54,55,56,57,58} The most common underlying mechanism for this phenomenon is an increased rate of protein degradation—as a result of a ubiquitin-proteasome-mediated process—with the involvement of chaperone proteins.^62,63,64 Obviously, genetic polymorphisms in gene promoter and regulatory regions can also result in alterations in transcription and, thus, the level of expression.⁶⁵

In summary, we have identified a series of common genetic polymorphisms and haplotypes for the human COMT gene. Functional studies of variant allozymes confirmed in vivo data and demonstrated that the common Met108 allozyme results in decreased enzyme activity, primarily as a result of a decrease in the quantity of immunoreactive protein—both in human tissue and after transient expression in cultured mammalian cells. We found no evidence that the novel Thr52 allozyme which we observed in AA subjects greatly alters function—although both the level of activity and immunoreactive protein were slightly elevated (Figures 2 and 4). Future studies will be required to elucidate mechanism(s) responsible for the decreased level of this important neurotransmitter-metabolizing protein in response to the change in the amino acid at codon 108 from Val to Met. Finally, the present results represent a step towards understanding mechanisms that might contribute to interindividual differences in the biotransformation of catecholamine neurotransmitters and catechol drugs and, perhaps, in risk for the development of neuropsychiatric disease.

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Acknowledgements

We thank Dr Daniel Schaid for performing the linkage and haplotype analyses and Ms Luanne Wussow for her assistance with the preparation of this manuscript. This work was supported in part by NIH Grants RO1 GM28157 (RMW), RO1 GM35720 (RMW), PO1 CA82267 (RWM), UO1 GM61388 (RMW and EDW) and R25T CA92049 (AJS).