Association study of the low-activity allele of catechol-O-methyltransferase and alcoholism using a family-based approach

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Abstract

Catechol-O-methyltransferase (COMT) is a major component of the metabolic pathways of neurotransmitters such as dopamine, adrenaline, and noradrenaline. The activity of COMT is known to vary within the population; it exists in common high- and low-activity forms that are determined by a Val → Met polymorphism at amino acid position 108/158 (in soluble or membrane-bound COMT). Recently, the low-activity allele was reported to contribute to the development of late-onset alcoholism in men.1 The present study extends this study by utilizing a family-based association approach, and by including individuals with early-onset alcoholism. Although no significant transmission disequilibrium was found in the overall sample of 70 parent/offspring trios (TDT = 1.43, P = 0.23), we observed a preferential transmission of the low-activity allele to patients with an early onset of disease (n = 32, TDT = 4.83, P = 0.028). Our results provide further evidence for an involvement of the COMT low-activity allele in the development of alcoholism and demonstrate the need for further studies in large samples of alcoholic patients.

Main

Catechol-O-methyltransferase (COMT) is a biotransformation enzyme that inactivates biologically active or toxic catecholamines. It is a major component of the metabolic pathways of neurotransmitters such as noradrenaline, adrenaline and dopamine.2 A single gene encodes membrane-bound (MB-COMT) and soluble (S-COMT) forms of the enzyme, which differ by a 50-amino acid hydrophobic N-terminal sequence that is present in the MB form.3 A common genetic polymorphism in humans is associated with a three-to-four-fold variation in COMT enzyme activity and is also associated with individual variation in COMT thermal instability.456 It was shown that this polymorphism is due to a G → A transition at codon 108 (158) of S-COMT (MB-COMT), that leads to a valine → methionine substitution. The valine → methionine substitution results in a low-activity allele.67 In a global survey of populations, it was shown that frequencies of COMT high- and low-activity alleles vary greatly among ethnic groups.8

Tiihonen and colleagues recently,1 in this journal, reported a positive association between the COMT low-activity allele and development of alcoholism. By applying a case-control design, they studied genotype and allele frequencies in a sample of 123 Finnish male alcoholics with late onset (over 25 years) and two independent sets of controls (3140 Finnish blood donors and 267 race- and gender-matched controls). The low-activity allele was found to increase the risk for alcoholism, with an odds ratio of 2.51 (P = 0.006) for individuals homozygous for the low-activity allele vs those homozygous for the high-activity allele. Further support for a role of the COMT low-activity allele in drinking behavior was obtained in a population sample of 896 middle-aged Finnish men: men homozygous for the low-activity allele reported 27% higher weekly alcohol consumption compared with men from the two other genotype groups (P = 0.031).9 Taken together, these results indicate that the COMT low-activity allele may contribute to development of male late-onset alcoholism as well as drinking behavior in the general male population. However, there have also been few negative studies. No evidence of association with alcoholism was found in a case-control study from Japan10 and no association with personality traits conferring vulnerability to alcoholism was found in a community sample from Australia.11

To further investigate the role of the COMT low-activity allele in alcoholism and extend the analysis to alcoholism with early onset, we tested the transmission of low- vs high-activity alleles from parents to alcoholic offspring. The use of a family-based association approach avoids the problem of case-control association studies in which false positive results can be produced as a result of hidden population stratifications. The most widely used test for family-based association studies is the transmission disequilibrium test (TDT).12 The TDT was applied to a sample of 70 parent/offspring trios. Thirty-two patients had early-onset (<25 years) and 38 had late-onset (≥25 years) alcoholism.

The results of the TDT analysis are summarized in Table 1. Seventy of 140 parents were heterozygous for the low-activity allele. Although, in the overall sample, the low-activity allele was transmitted more often than the high-activity allele (40 transmissions vs 30 non-transmissions), the difference was far from statistically significant (TDT = 1.43, P = 0.23). Separate analysis of early- and late-onset individuals revealed a significant excess of the low-activity allele transmitted to early-onset offspring (24 transmissions vs 11 non-transmissions, TDT = 4.83, P = 0.028), while no preferential transmission of the low-activity allele was observed in the late-onset group (16 transmissions vs 19 non-transmissions, TDT = 0.26, P = 0.61). Subdivision of the sample with respect to sex-of-offspring revealed no significant findings (see Table 1). However, when the male and female groups were further subdivided with respect to age-of-onset, a significant excess of transmission of the low-activity allele was seen in male early-onset alcoholics (20 transmissions vs 9 non-transmissions, TDT = 4.17, P = 0.041).

Table 1 Transmission of COMT alleles to alcohol-dependent offspring

Our results suggest that the low-activity COMT allele plays a role in the development of early-onset alcoholism. To our knowledge, this is the first study to examine the association between the functional COMT polymorphism and early-onset alcoholism. Two previously published studies on the COMT polymorphism in alcoholism included no1 or only a small number10 of early-onset individuals. Early-onset alcoholism is thought to have a higher heritability than late-onset alcoholism13 which may favor this subtype for a gene identification approach. On the psychopathological level, early-onset alcoholism has been associated with increased aggressive behavior,13 a feature that has also been observed in heterozygous COMT-deficient mice.14

Our failure to detect preferential transmission of the low-activity allele to offspring with late-onset alcoholism may be secondary to insufficient power. Our sample had a power of only 23% to detect genotype-associated relative risks observed in the previous study (OR = 2.51 (CI 1.22–5.19) for homozygotes for the low-activity allele vs homozygotes for the high-activity allele).1 As a consequence of insufficient power, a true effect may have escaped detection in our study. There is clearly a need for larger studies to adequately address this issue. The sample size of our study reflects, however, the difficulty in obtaining large proband/parents samples for family-based association studies in alcoholism. This is not surprising given the problematic relationships that are often seen in families of alcoholic patients. We have not corrected our findings for multiple testing since this would have prevented detection of small to moderate genetic effects, given our sample size. However, it is clear that the chance of a type I error is increased and the results should be viewed with caution until independent replication.

A sexually dimorphic effect of COMT has been suggested on the basis of studies in COMT-deficient mice14 and family-based association studies in obsessive-compulsive disorder.15 It may therefore be worthwhile to separately analyze the effect of COMT in female and male alcoholics. The small number of females in our sample, however, did not allow us to obtain meaningful results regarding this subgroup. Future studies should address this issue and it will be interesting to see whether the COMT polymorphism is a source of vulnerability to alcoholism which is common to both sexes or is restricted to male alcoholism.

Materials and methods

Subjects

Seventy alcohol-dependent patients (54 males and 16 females) with both biological parents available were recruited from consecutive admissions to the Department of Psychiatry, University of Bonn. Written informed consent was obtained from all individuals participating in this study. All patients were interviewed using a semi-structured interview for axis I and II disorders and all fulfilled DSM-III-R criteria for alcohol dependence. Thirty-two patients had an age-of-onset <25 years, 38 patients ≥25 years. The distinction of early- and late-onset alcoholism is based on the typology proposed by Cloninger et al,16 in which alcoholics are classified into type 1 and type 2. Type 1 occurs in both men and women, has a relatively late age-of-onset, and little or no antisocial behavior. Type 2 is found mainly in men, has early age-of-onset, and is associated with antisocial behavior. All individuals were of German origin.

The study was approved by the local ethical committee of the University of Bonn, Germany.

Genotype analysis

Venous blood samples, anticoagulated with EDTA, were drawn from all individuals. Genomic DNA was isolated from peripheral blood lymphocytes by a standard salting-out method.

The COMT polymorphism is generated by the presence of a G or A encoding a valine or a methionine at codon 108 of the S-COMT, a codon equivalent to 158 of the MB-COMT.67 The COMT genotype was determined by restriction fragment length polymorphism (RFLP) analysis as previously described.17 Briefly, a 217-base pair (bp) PCR product was generated using primers 5′-TCGTGGACGCCGTGATTCAGG and 5′-AGGTCTGACAACGGGTCAGGC (nucleotides 1771–1791 and 1967–1987, GenBank accession number Z26491).3 The PCR product (5 μl) was treated with 5 U of NlaIII (New England BioLabs, Beverly, MA, USA) which cuts the Met-108 variant in addition to a constant cleavage site in the PCR product. The Val-108 variant is characterized by a 136-bp fragment, the Met-108 variant by two fragments of 40 and 96 bp. Cleavage of the constant restriction site results in a 81-bp fragment. The digested PCR products were separated on a 10% non-denaturing polyacrylamide gel (acrylamide:bisacrylamide = 29:1) followed by silver staining.

Statistical analysis

The transmission disequilibrium test (TDT)12 was applied for family-based association analysis. The TDT is based on the detection of disproportionate transmission of high- vs low-risk alleles by heterozygous parents to affected children. The Mendelian expectation under the null hypothesis of no association is that either allele carried by a heterozygote has a 50:50 chance of transmission to an affected child. If the allele plays a causal role in the development of the disorder, however, then its transmission should exceed 50%. The statistical significance level was set at P = 0.05.

A power analysis was calculated for the sample of late-onset patients. The a posteriori power (parental mating type) in the sample of 38 late-onset patients was 0.23, to detect genotype-associated relative risks observed in the previous study.1

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Acknowledgements

This work was supported by grant No. 01EB9418 (BMBF). TW received a stipend from the China Scholarship Council. We thank S Albrecht, K Meyer zur Kapellen, E Sohne and R Granath for data evaluation and excellent technical assistance. We express our gratitude to all participants for providing blood samples and to all psychiatrists involved in the study for their support in collecting data.

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Correspondence to M M Nöthen.

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Keywords

  • COMT
  • substance abuse
  • addiction
  • behavior genetics
  • candidate gene

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