Joseph F. Cubells

Serotonin transporter protein (SLC6A4) allele and haplotype frequencies and linkage disequilibria in African- and European-American and Japanese populations and in alcohol-dependent subjects.

Abstract The SLC6A4 locus encodes the serotonin transporter, which in turn mediates the synaptic inactivation of the neurotransmitter serotonin. Two PCR-formatted polymorphisms at this locus have been described, the first of which is a variable number tandem repeat located in exon 2, and the second a repeat sequence polymorphism located in the promoter region. The latter polymorphism alters transcriptional activity of SLC6A4, and has been reported to be associated with anxiety and depression-related traits. We studied allele frequencies, and computed haplotype frequencies and linkage disequilibrium measures, for these two polymorphisms in European-American, African-American, and Japanese populations, and in a set of alcohol-dependent European-American subjects. Allele frequencies for both systems showed variation, with significant differences overall for each system, and significant differences between each pair of populations for both systems. Linkage disequilibrium also varied among the populations. There were no significant differences in allele or haplotype frequencies between the European-American population samples and alcohol-dependent subjects. The population differences demonstrate a potential for population stratification in association studies of either of these SLC6A4 polymorphisms. If genetic variation at this locus really is associated with behavioral variation, these results could reflect either different behavioral adaptations in different populations, or random genetic drift of a behaviorally important but selectively neutral polymorphism.

Population studies of polymorphisms of the serotonin transporter protein gene

The range of allele frequency variation in humans for any locus that may have functionally important genetic variation needs to be documented. Therefore, we tested two polymorphisms at the serotonin transporter protein locus (SLC6A4) in samples from seven specific populations from five continental regions. We studied the promoter polymorphism which is reported to have functional significance and to be associated with anxiety- and depression-related phenotypes [Lesch et al., 1996: Science 274:1527-1531], and the intron 2 VNTR polymorphism [Lesch et al., 1994: J Neural Transm 95:157-162]. Allele frequencies for both systems show significant global variation, and consequently so do haplotype frequencies. Linkage disequilibrium varied among the populations, being absent in some and highly significant in others. These differences further document a large potential for population stratification in association studies of either of these SLC6A4 polymorphisms.

A Quantitative-Trait Analysis of Human Plasma–Dopamine β-Hydroxylase Activity: Evidence for a Major Functional Polymorphism at the DBH Locus

Dopamine-beta-hydroxylase (D beta H) catalyzes the conversion of dopamine to norepinephrine and is released from sympathetic neurons into the circulation. Plasma-D beta H activity varies widely between individuals, and a subgroup of the population has very low activity levels. Mounting evidence suggests that the DBH structural gene is itself the major quantitative-trait locus (QTL) for plasma-D beta H activity, and a single unidentified polymorphism may account for a majority of the variation in activity levels. Through use of both sequencing-based mutational analysis of extreme phenotypes and genotype/phenotype correlations in samples from African American, European American (EA), and Japanese populations, we have identified a novel polymorphism (--1021C-->T), in the 5 flanking region of the DBH gene, that accounts for 35%--52% of the variation in plasma-D beta H activity in these populations. In EAs, homozygosity at the T allele predicted the very low D beta H-activity trait, and activity values in heterozygotes formed an intermediate distribution, indicating codominant inheritance. Our findings demonstrate that --1021C-->T is a major genetic marker for plasma-D beta H activity and provide new tools for investigation of the role of both D beta H and the DBH gene in human disease.

Genetics of two μ opioid receptor gene (OPRM1) exon I polymorphisms: population studies, and allele frequencies in alcohol- and drug-dependent subjects

The gene encoding the μ opioid receptor, OPRM1, contains at least two polymorphisms affecting protein sequence in exon 1, Ala6Val and Asp40Asn. In previous studies, each variant has been reported to be associated with some form of drug dependence. Although past reports have not been consistent, they have also not considered comparable populations. The goals of the present study were to delineate allele and haplotype frequencies of these variants in a range of populations, and in drug- or alcohol-dependent subjects deriving from some of those populations. We developed new PCR-RFLP methods to detect both of these polymorphisms and studied them in control and substance-dependent populations of African American (AA), European American (EA) and Hispanic origin, and in a series of populations differing in geographic origin (Japanese, Ethiopians, Bedouins, and Ashkenazi Jews), 891 subjects overall. We designed primers flanking the DNA segment containing both polymorphisms, each primer creating a different artificial restriction site, such that a single PCR reaction can be completed, then divided, and the PCR product digested with either of two enzymes to reveal both polymorphisms. We found that allele frequencies for both polymorphic systems were significantly different between AA and EA subjects, and there was significant heterogeneity among the more extensive set of populations. Furthermore, there were no significant differences in allele frequency by diagnosis; that is, neither polymorphism appears to be a direct risk factor for substance dependence. Finally, we demonstrated linkage disequilibrium between the two exon 1 markers, and a previously described short tandem repeat (STR) marker.

A haplotype at the DBH locus, associated with low plasma dopamine β-hydroxylase activity, also associates with cocaine-induced paranoia

Low levels of dopamine β-hydroxylase (DβH) protein in the plasma or cerebrospinal fluid (CSF) are associated with greater vulnerability to positive psychotic symptoms in several psychiatric disorders. DβH level is a stable, genetically controlled trait. DBH, the locus encoding DβH protein, is the major quantitative trait locus controlling plasma and CSF DβH levels. We therefore hypothesized that DBH variants or haplotypes, associated with low levels of DβH in the plasma, would also associate with greater vulnerability to cocaine-induced paranoia. To test this hypothesis, we first showed that a di-allelic variant, DBH*5′-ins/del, located approximately 3u2009kb 5′ to the DBH transcriptional start site, significantly associates with plasma DβH activity in European-Americans (nu2009=u200966). Linkage disequilibrium analysis of that polymorphism and DBH*444g/a, another di-allelic variant associated with DβH levels, demonstrated that alleles of similar association to DβH levels are in positive disequilibrium. We then estimated DBH haplotype frequencies in cocaine-dependent European Americans rated for cocaine-induced paranoia (nu2009=u200945). As predicted, the low-DβH-associated haplotype, Del-a, was significantly more frequent (Pu2009=u20090.0003) in subjects endorsing cocaine-induced paranoia (nu2009=u200929) than in those denying it (nu2009=u200916). Comparison to control haplotype frequencies (nu2009=u2009145 healthy European-Americans) showed that the association predominantly reflected under-representation of Del-a haplotypes in those denying cocaine-induced paranoia. We conclude that: (a) the two DBH polymorphisms we studied are associated with plasma DBH levels; (b) those two polymorphisms are in significant linkage disequilibrium in European Americans, with alleles of similar association to DβH levels in positive disequilibrium; and (c) the haplotype associated with low DBH activity is also associated with cocaine-induced paranoia.

Dopamine β-hydroxylase: two polymorphisms in linkage disequilibrium at the structural gene DBH associate with biochemical phenotypic variation

Levels of the enzyme dopamine β-hydroxylase (DβH) in the plasma and cerebrospinal fluid (CSF) are closely related biochemical phenotypes. Both are under strong genetic control. Linkage and association studies suggest the structural gene encoding DβH (locus name, DΒH) is a major locus influencing plasma activity of DβH. This study examined relationships of DBH genotype determined at two polymorphic sites (a previously described GT repeat, referred to as the DBH STR and a single-base substitution at the 3’ end of DBH exon 2, named DBH*444 g/a), to CSF levels of DβH protein in European-American schizophrenic patients, and to plasma DβH activity in European-American patients with mood or anxiety disorders. We also investigated linkage disequilibrium (LD) between the polymorphisms in the pooled samples from those European-American subjects (n=104). Alleles of DBH*444 g/a were associated with differences in mean values of CSF DβH levels. Alleles at both polymorphisms were associated with plasma DβH activity. Significant LD was observed between respective alleles with similar apparent influence on biochemical phenotype. Thus, allele A3 of the DBH STR was in positive LD with DBH*444a, and both alleles were associated with lower plasma DβH activity. DBH STR allele A4 was in positive LD with DBH*444 g, and both alleles were associated with higher plasma DβH activity. The results confirm that DBH is a major quantitative trait locus for plasma DβH activity, and provide the first direct evidence that DBH also influences CSF DβH levels. Both polymorphisms examined in this study appear to be in LD with one or more functional polymorphisms that mediate the influence of allelic variation at DBH on DβH biochemical phenotypic variation

Population genetics of a functional variant of the dopamine β-hydroxylase gene (DBH)

Dopamine beta-hydroxylase (E.C. 1.14.17.1; protein abbreviation: DbetaH) catalyzes conversion of dopamine to norepinephrine. Previous work identified two expressed alleles of the gene encoding DbetaH (locus symbol DBH), containing either G or T at nucleotide position 910, resulting in specification by codon 304 of alanine (DBH*304A) or serine (DBH*304S), respectively. The current study employed denaturing gradient gel electrophoresis to identify these alleles, and after developing a PCR RFLP for rapid genotyping, estimated the frequencies of the alleles in African-Americans, European-Americans, and in several geographically dispersed populations (Mbuti, Danes, Adygei, Chinese, Japanese, Surui, Maya, and Nasioi). DBH*304A was the most common allele in all populations tested, with allele frequencies greater than 0.80 in each case. There was significant heterogeneity in allele frequency across population groups. The DBH*304S allele was most common in subjects of African descent, and least common in East Asians and individuals from indigenous populations of North and South America. The frequency of DBH*304S was significantly higher in African-Americans (0.16) than in European-Americans (0.06; P < 0.004). Of the four DBH*304S homozygotes observed, all were Europeans and three of the four were Danes. Based on empirical P-values generated by computer simulation, the observed proportions of DBH*304S homozygotes did not differ significantly from Hardy-Weinberg expectations in any of the populations after Bonferroni correction for multiple comparisons. The observation of significant heterogeneity in DBH*304S allele frequency across different population samples demonstrates the importance of controlling for population stratification in future studies testing for associations between DBH*304S and clinical phenotypes.

The Structure of Linkage Disequilibrium at the DBH Locus Strongly Influences the Magnitude of Association between Diallelic Markers and Plasma Dopamine β-Hydroxylase Activity

There is currently a great deal of interest in using linkage disequilibrium (LD) mapping to locate both disease and quantitative-trait loci on a genomewide scale. Recent findings suggest that much of the human genome is organized in discrete blocks of low haplotype diversity, but the utility of such blocks in identifying genes influencing complex traits is not yet known and must ultimately be tested empirically through use of real data. We recently identified a putative functional polymorphism (-1021C-->T) in the 5 upstream region of the DBH gene that accounted for 35%-52% of the total phenotypic variance in plasma dopamine beta-hydroxylase (DBH) activity in samples from three distinct populations. In the present study, we genotyped 11 diallelic markers at the DBH locus surrounding -1021C-->T in 386 unrelated individuals of European origin. We identified a single 10-kb block containing -1021C-->T, in which four haplotypes comprised 93% of the observed chromosomes. Only markers within the block were highly associated with phenotype (P< or =2.2 x 10(-10)), with one exception. In general, association with phenotype was strongly correlated with the degree of LD between each marker and -1021C-->T. Of four LD measures assessed, d(2) was the best predictor of this relationship. Had one attempted to map quantitative-trait loci for plasma DBH activity on a genomewide basis without prior knowledge of candidate regions and not included (by chance) markers within this haplotype block, the DBH locus might have been missed entirely. These results provide a direct example of the potential value of constructing a haplotype map of the human genome prior to embarking on large-scale association studies.

Genotype-controlled analysis of plasma dopamine β-hydroxylase activity in psychotic unipolar major depression

BACKGROUNDnPlasma activity of dopamine beta-hydroxylase (DbetaH), the enzyme that converts dopamine to norepinephrine, is reportedly lower in patients with unipolar major depression with psychotic features (UDPF) than in those with nonpsychotic unipolar major depression (UD). Plasma DbetaH is under genetic control by the structural locus encoding DbetaH protein, DBH. This study tested the hypothesis that diagnosis-specific allelic variation at DBH accounts for lower plasma DbetaH in UDPF.nnnMETHODSnPlasma DbetaH activity was measured in samples from patients with UDPF (n = 33) and UD (n = 45). Genotypes were determined at several functional DBH polymorphisms, including C-1021T, a single nucleotide polymorphism (SNP) in the proximal 5 region that associates with variation in plasma DbetaH activity.nnnRESULTSnMean plasma DbetaH activity was significantly lower in UDPF than in UD. Genotyping at DBH did not reveal genetic associations distinguishing UDPF from UD. A two-way analysis of variance showed significant effects of genotype and diagnostic group but no significant interaction.nnnCONCLUSIONSnAlthough the effects of the diagnosis of UDPF, and of DBH allele status, on plasma DbetaH activity were replicated, the lower plasma DbetaH in patients with UDPF was not accounted for by DBH genotype. Several explanations for this result are possible. First, other variants at DBH, or at other loci, could account for the findings. Second, nongenetic factors could account for the differences in plasma DbetaH. In this regard, we hypothesize that abnormal regulation of hypothalamic-pituitary-adrenal function in UDPF lowers expression of DbetaH protein, which could in turn alter the ratio of dopamine and norepinephrine in noradrenergic neurons, thereby promoting development of psychotic symptoms.

Comorbid psychiatric diagnoses and their association with cocaine-induced psychosis in cocaine-dependent subjects.

Comorbidity between drug abuse and mental illness is very common, but the association of such comorbidity with specific responses to drugs of abuse remains obscure. The current study examined the relationship between the presence of non-psychotic Axis I psychiatric diagnoses and the frequency and severity of cocaine-induced psychosis. We interviewed 243 unrelated cocaine-dependent adults [37% European American (EA), 52.3% African American (AA); 58.8% male] using the Semi-Structured Assessment for Drug Dependence and Alcoholism (SSADDA) to establish DSM-IV diagnoses, and two instruments for the identification of cocaine-induced paranoia, the Cocaine Experience Questionnaire (CEQ) and the Scale for Assessment of Positive Symptoms for Cocaine-Induced Psychosis (SAPS-CIP). Comorbid substance use and psychiatric disorders were common in this cocaine-dependent sample. Ninety percent of subjects met criteria for substance use disorders other than cocaine dependence; common non-substance-use disorders included antisocial personality disorder (ASPD), adult ASPD, major depression, and attention deficit-hyperactivity disorder (ADHD). Comorbid opioid dependence was more common in EA subjects than in AA participants. After correction for multiple comparisons, a lifetime diagnosis of ADHD was associated with the categorical presence of CIP (p = 0.007), as well as significantly more severe CIP symptoms. Comorbid substance use and psychiatric disorders are very common among individuals with cocaine dependence. Comorbid ADHD increases the odds of an individual endorsing CIP, suggesting some common basis for these phenomena.