Michael D. Köhnke

A genotype-controlled analysis of plasma dopamine β-hydroxylase in healthy and alcoholic subjects: evidence for alcohol-related differences in noradrenergic function

BACKGROUND Norepinephrine and dopamine mediate important aspects of alcoholism and alcohol withdrawal. Dopamine-beta-hydroxylase (DbetaH) converts dopamine to norepinephrine. A recent study demonstrated a strong association between variance in plasma DbetaH activity and a novel polymorphism (DBH-1021C-->T) at the structural locus (DBH) encoding DbetaH protein. METHODS Our study investigated whether the DBH-1021C-->T polymorphism and plasma DbetaH activity were associated with alcoholism or with delirium tremens (DT) during alcohol withdrawal by analyzing 207 German alcoholic and 102 healthy control subjects. We also examined the influence of the polymorphism on enzyme activity. RESULTS Mean (+SD) plasma DbetaH activity measured in alcoholic subjects abstinent was significantly lower than that observed in control (27.7 + 16.7 vs. 35.6 + 18.8; p =.01). It did not differ between subjects with DT during withdrawal and subjects with mild withdrawal symptoms. The T allele of the DBH-1021C-->T polymorphism was significantly associated with lower plasma DbetaH activity. None of the alleles or genotypes were associated with alcoholism or DT. CONCLUSIONS The data indicate that the alcoholism-related reduction in plasma DbetaH activity is independent of genotype at DBH-1021C-->T and replicate the finding that DBH-1021C-->T is strongly associated with plasma DbetaH activity in a native Western European population.

A Single Nucleotide Polymorphism at DBH, Possibly Associated with Attention-Deficit/Hyperactivity Disorder, Associates with Lower Plasma Dopamine β-Hydroxylase Activity and is in Linkage Disequilibrium with Two Putative Functional Single Nucleotide Polymorphisms

Background The DBH gene regulates plasma dopamine β-hydroxylase activity (pDβH). Two single nucleotide polymorphisms (SNPs), −1021C→T (rs1611115; SNP1) and +1603C→T (rs6271; SNP3), independently influence pDβH. Another SNP, commonly known as DBH Taq1A (rs2519152; SNP2) is associated with attention-deficit/hyperactivity disorder (ADHD) in some (but not all) studies. We tested whether 1) SNP2 associates with pDβH; and 2) whether linkage disequilibrium (LD) between SNP2 and the other SNPs explains that association. Methods Plasma dopamine β-hydroxylase activity and genotypes at the SNPs were determined in Caucasian subjects ( n = 418). Associations to pDβH were examined using analyses of variance (ANOVAs) and LD among the SNPs using estimation maximization. Results 1) Each polymorphism analyzed alone associated with pDβH; 2) SNP2 was in strong LD with SNP1 and SNP3, respectively, but there was no significant LD between SNP1 and SNP3; and 3) analyzed jointly, each SNP contributed significantly and uniquely to plasma DβH activity. Conclusions 1) SNP2 associates with pDβH; 2) SNP2 shows LD with SNP1 and SNP3; 3) most of the association between SNP2 and pDβH simply reflects that LD; however, 4) SNP2 also appears to exert a small independent effect on pDβH, suggesting that SNP2, or another variant in LD with it, uniquely influences pDβH.

Haplotype-controlled analysis of the association of a non-synonymous single nucleotide polymorphism at DBH (+ 1603C T) with plasma dopamine β-hydroxylase activity

The DBH locus controls plasma dopamine β‐hydroxylase activity (pDβH). A 5′‐upstream single nucleotide polymorphism (SNP) at DBH (−1021C → T) explains ∼45% of the variance in pDβH, and a non‐synonymous SNP in exon 11 (+ 1603C → T) an additional 2%. However, that regression result underestimates the effect of + 1603C → T because of its low minor allele frequency. We estimated the biological effect of + 1603C → T on pDβH by comparing subjects of identical −1021CγT genotype, in a diagnostically heterogeneous group of subjects of European origin (N = 367). + 1603C → T genotype associated with pDβH within groups of identical genotype at −1021 C → T, accounting for 5%–16% of the variance. There was no significant linkage disequilibrium between −1021C → T and + 1603C → T (D = 0.0058, D′ = 0.4774, d2 = 0.0011, P > 0.05), confirming the validity of assessing the two polymorphisms independently. These results suggest that altered homospecific activity of the enzyme can contribute to variation in pDβH. This conclusion informs how associations between DBH and psychiatric disorders should be approached.

Plasma homovanillic acid: a significant association with alcoholism is independent of a functional polymorphism of the human catechol-O-methyltransferase gene.

The central dopamine system seems to influence addictive disorders. Plasma homovanillic acid (HVA) is an indicator of central dopaminergic activity. In this study the hypothesis that plasma HVA is associated with alcoholism or with delirium tremens (DT) during alcohol withdrawal was tested. A functional genetic polymorphism of the enzyme catechol-O-methyltransferase (COMT) that participates in converting dopamine into its final metabolite HVA was investigated for an association with alcoholism or DT during alcohol withdrawal. In addition, a relation between the functional polymorphism of COMT and plasma HVA concentrations was studied. Plasma HVA concentrations and COMT genotypes were determined in 142 German alcoholics and 101 German healthy controls. Alcoholic patients were examined after a minimum of 3 weeks after cessation of drinking. Mean plasma HVA concentrations were significantly lower in alcoholic patients compared to healthy controls. A group of alcoholics with a history of DT during alcohol withdrawal (n=62) did not differ significantly in plasma HVA concentrations from alcoholics with a history of only mild withdrawal symptoms (n=67). The functional polymorphism of the human COMT gene was neither significantly associated with the diagnosis of alcoholism or DT during alcohol withdrawal nor with plasma HVA concentrations.

DBH*444G/A polymorphism of the dopamine-β-hydroxylase gene is associated with alcoholism but not with severe alcohol withdrawal symptoms

Summary.As the enzyme dopamine-β-hydroxylase (DβH) converts dopamine to norepinephrine and both transmitters seem to be involved in the pathology of alcoholism and severe alcohol withdrawal symptoms, the gene encoding DβH (DBH) was applied to explore the genetic background of alcoholism and severe withdrawal symptoms. 102 healthy control subjects and 208 alcoholics, including 97 patients with a history of mild withdrawal symptoms, 57 with a history of alcohol withdrawal seizure (AWS) and 82 with a history of delirium tremens (DT) were genotyped for the DBH*444G/A polymorphism revealing a significantly elevated frequency of genotypes carrying the A-allele (p = 0.02; after Bonferroni adjustment for multiple tests) in alcoholics compared to healthy controls. Frequencies of alleles and genotypes of individuals with mild withdrawal symptoms did not differ significantly from those of patients with DT or AWS.

GENETIC STUDY: The polymorphism GABABR1 T1974C [rs29230] of the GABAB receptor gene is not associated with the diagnosis of alcoholism or alcohol withdrawal seizures

As the inhibitory neurotransmitter gamma aminobutyric acid (GABA) modulates ethanol consumption, alcohol withdrawal symptoms and seizure generation by interacting with the GABAB receptor, the genes encoding for the GABAB receptor can be considered as candidate genes for alcoholism and alcohol withdrawal seizures (AWS). As the polymorphism GABABR1 T1974C[rs29230] of the GABAB receptor gene had been associated with alcoholism and EEG abnormalities in prior studies, the present examination investigated if the polymorphism is associated with the diagnosis of alcoholism or AWS. After genotyping the allele and genotype frequencies of a group of alcoholics with a history of AWS (n = 69) were compared with the results of a group of alcoholics with only mild withdrawal symptoms (n = 97). Additionally a group of healthy controls (n = 101) was compared with individuals with the diagnosis of alcoholism (n = 220). As no significant differences were found between the compared groups, this study gave no further evidence for GABABR1 T1974C[rs29230] as a candidate for alcoholism or AWS.

Significant impact of MTHFR C677T polymorphism on plasma homovanillic acid (HVA) levels among alcohol-dependent patients.

The enzyme 5,10‐methylenetetrahydrofolate reductase (MTHFR) synthesizes 5‐methyltetrahydrofolate. It plays a critical role in homocysteine metabolism. A high impact of MTHFR C677T polymorphism on plasma homocysteine levels has been observed among alcoholics. Recent studies indicate that homocysteine has toxic effects on dopaminergic neurons. Thus it lowers levels of homovanillic acid (HVA) in the striatal region in rats. Alcoholics had significantly lower plasma HVA concentrations compared with healthy controls. Aim of this study is to elucidate whether HVA plasma levels in alcoholics are influenced by MTHFR C677T polymorphism. A total of 142 alcohol‐dependent patients and 101 healthy controls were examined regarding plasma HVA concentration and MTHFR C677T genotype. Blood samples of alcoholics were obtained after a minimum of 22 days of abstinence. Among alcohol‐dependent patients MTHFR C677T polymorphism was significantly associated with plasma HVA levels: carriers of MTHFR C677T T‐allele had significantly lower HVA plasma levels compared with homozygote carriers of C‐allele: 11.9 ng/ml versus 14.4 ng/ml (χ2: 5.39; P = 0.02). In healthy control subjects plasma HVA levels did not differ significantly between MTHFR C677T T‐allele carriers and homozygote carriers of C‐allele: 15.1 ng/ml versus 15.3 ng/ml (χ2: 0.04; P = 0.82). The data suggest an influence of MTHFR C677T polymorphism on plasma HVA among alcohol‐dependent patients. This might be due to neurotoxic effects of homocysteine on the dopaminergic system or direct impairment of monoamine metabolism. Future studies should try to elucidate whether this effect is reversible during alcohol abstinence.

A revised allele frequency estimate and haplotype analysis of the DBH deficiency mutation IVS1+2T --> C in African- and European-Americans.

Dopamine beta-hydroxylase (DBH; OMIM 223360) catalyzes the conversion of dopamine (DA) to norepinephrine (NE) in the central and peripheral nervous systems. DBH deficiency is a very rare genetic disorder characterized by severe sympathetic failure in which circulating levels ofNEandepinephrine (E)areundetectable, but DA levels are greatly elevated [Robertson et al., 1986]. The onset of symptoms occurs at birth or in early childhood and as adults all affected individuals suffer from profound postural hypotension and exercise intolerance. Only six patients from five families with the disorder, all of Western European descent, are well described in the literature [Robertson et al., 1986; Man in ’t Veld et al., 1987, 1988; Biaggioni et al., 1990; Mathias et al., 1990]. We recently reported the first putative disease-causing mutations in an analysis of the DBH locus in two unrelated patients and their families [Kim et al., 2002]. Both patients were compound heterozygotes who each carried a single copy of a T!C transition at the donor splice site of intron 1 (IVS1þ 2T!C) that resulted in an aberrantly spliced product containing a premature stop codon in an in vitro splicing assay. On the homologous chromosome, a total of three different missense mutations in the DBH gene were detected: one in patient 1 (exon 2) and two in patient 2 (exons 1and6). Themechanismbywhich these missensemutations produce a loss of DBH function is as yet unknown. In our initial analysis, we genotyped a small sample of 88 healthy European-American (EA) subjects, and found no carriers of any of the threemissensemutations [Kim et al., 2002]. However, we found two subjects heterozygous for IVS1þ2T!C, which yielded an unexpectedly high C allele frequency of 0.011 0.008. This suggested a large discrepancy between the prevalence of homozygotes for the C allele and individuals affected with DBH deficiency. One possible explanation is that these homozygotes are frequently embryonic lethal, which is consistent with the low survival rate observed inmouse embryos lackingDBH [Thomas et al., 1995]. Given our initial allele frequency estimate, these data indicated that DBH deficiency might thus be an epidemiologically significant cause of fetal demise. However, more extensive genotyping studies were clearly warranted in order to accurately estimate the frequency of DBH deficiency-related mutations in the general population.Herewe report frequencies for these mutations in a substantially larger sample of subjects of both European and African-American (AA) heritage. DNA samples from a total of 801 unrelated adults were collected in the course of several genetic studies as previously described [Zabetian et al., 2000; Kohnke et al., 2002] from the following groups: 122 AAs, 456 EAs, and223 individuals ofGermandescent.Thegroups included healthy individuals and those with psychiatric and substance-use disorders. However, none of these subjects reported severe disturbances in autonomic function and thus all were considered as controls for the purpose of this study. The EA and German population samples were combined to simplify data analysis and are abbreviated as ‘‘EA/G.’’ In order to supplement theAAsample,we includedDNAcollected froma total of 260 AA mothers and infants during an ongoing study of genetic risk factors for preterm labor. DNA from two EA patients with DBHdeficiency and their familymembers was obtained as previously reported [Kim et al., 2002]. None of the four putative disease-causing mutations (IVS1þ2T!C,V87M,D100E,D331N)weredetected in any of the 122 AA or 679 EA/G adult controls genotyped in this study. IVS1þ2T!C was also genotyped in the AA mother–infant group, and a single preterm infant was found to be heterozygous for the mutation. This suggests that the C allele frequency of IVS1þ2T!C that we initially reported in EAs [Kim et al., 2002] was Grant sponsor: NARSAD; Grant sponsor: NASA; Grant sponsor: the Nathan Blaser Shy-Drager Research Program; Grant sponsor: NIH; Grant sponsor: Tuebingen University; Grant sponsor: the US.

Central pontine myelinolysis in a normonatraemic alcoholic patient.

Central pontine myelinolysis (CPM) is a rare disease which has been associated with hyponatraemia and its rapid correction. We describe a malnourished 32‐year‐old alcohol‐dependent woman suffering from an infection who had developed CPM without any known prior derangement in serum electrolytes or its iatrogenic correction. Computerized tomography (CT) and magnetic resonance imaging (MRI) played an important role in the diagnosis. Even when an electrolyte derangement is absent, alcoholism, malnourishment and infection should be considered as important possible aetiological factors of CPM.