David J. Vandenbergh

A common silencer element in the SCG10 and type II Na+ channel genes binds a factor present in nonneuronal cells but not in neuronal cells

We have localized a cell type-specific silencer element in the SCG10 gene by deletion analysis. This neural-restrictive silencer element (NRSE) selectively represses SCG10 expression in nonneuronal cells and tissues. The NRSE contains a 21 bp region with striking homology to a sequence present in a silencer domain of the rat type II sodium channel (NaII), another neuron-specific gene. We have identified a sequence-specific protein(s) that binds the SCG10 NRSE, as well as the homologous element in the NaII gene. A point mutation in the NRSE that abolishes binding of this neural-restrictive silencer-binding factor (NRSBF) in vitro also eliminates silencing activity in vivo. NRSBF is present in nuclear extracts from nonneuronal cells but not in extracts from neuronal cells, suggesting that the neuron-specific expression of SCG10 reflects, at least in part, the absence or inactivity of this protein. These data identify the NRSE as a potentially general DNA element for the control of neuron-specific gene expression in vertebrates.

High-Activity Catechol-O-Methyltransferase Allele is More Prevalent in Polysubstance Abusers

Allelic variants at the catechol-O-methyltransferase (COMT) locus are candidates to contribute to genetic components of interindividual differences in vulnerability to substance abuse. COMT plays a prominent role in dopaminergic circuits important for drug reward, and COMT alleles encode enzymes whose activities vary from three- to four-fold. We compared COMT allele frequencies in control research volunteers reporting insignificant lifetime use of addictive substances with those in volunteers reporting substantial polysubstance use. Homozygosity for the high-activity COMT allele was found in 18% of controls, 31% of volunteers with high lifetime substance use, and 39% meeting DSMIII-R substance abuse criteria [odds ratio (relative risks) 2.0 (control vs. use; 95% confidence interval 1.2-3.5; P < 0.013) and 2.8 (control vs. DSM; 1.3-6.1; P < 0.008)]. Individuals with the high-activity COMT variant may have greater genetic vulnerability to drug abuse.

Human dopamine transporter gene: coding region conservation among normal, Tourette's disorder, alcohol dependence and attention-deficit hyperactivity disorder populations.

The dopamine transporter (DAT) provides major regulation of the synaptic levels of dopamine and is a principal target of psychostimulant drugs. Associations between DAT gene polymorphisms and human disorders with possible links to dopaminergic neurotransmission, including attention-deficit/hyperactivity disorder (ADHD) and consequences of cocaine and alcohol administration, have been reported. We now report approximately 60000 bp of genomic sequence containing the entire DAT gene. This sequence was used to amplify each of the 15 DAT gene exons and several introns and analyze these amplification products by single-stranded sequence conformation (SSCP) and/or direct sequencing. These results define silent allelic single nucleotide sequence variants in DAT gene exons 2, 6, 9 and 15. Rare conservative mutations are identified in amino acids encoded by DAT exons 2 and 8. Analyses of the common nucleotide variants and the previously reported VNTR in the non-coding region of exon 15 define the pattern of linkage disequilibrium across the DAT locus. These comprehensive analyses, however, fail to identify any common protein coding DAT sequence variant in more than 150 unrelated individuals free of neuropsychiatric disease, 109 individuals meeting City of Hope criteria for Tourettes syndrome, 64 individuals with DSM-IV diagnoses of ethanol dependence, or 15 individuals with ADHD. These data are consistent with substantial evolutionary conservation of the DAT protein sequence. They suggest that gene variants that alter levels of DAT expression provide the best current candidate mechanism for reported associations between DAT gene markers, ADHD and other more tentatively associated neuropsychiatric disorders.

No association between novelty seeking and dopamine D4 receptor (D4DR) exon III seven repeat alleles in Baltimore Longitudinal Study of Aging participants.

Recent studies by Ebstein et al1 and Benjamin et al2 found associations between long repeat polymorphisms in the D4 dopamine receptor gene (D4DR) and individual variation in a human personality trait, identified as ‘Novelty Seeking’(NS). Ebstein et al used the Tridimensional Personality Questionnaire (TPQ)3 to measure NS scores; Benjamin et al used the Revised NEO Personality Inventory (NEO-PI-R)4 to estimate NS scores. However, Malhotra et al5 failed to replicate the association between the direct measure of NS using the TPQ and the long alleles of the D4DR genotypes in two Finnish samples. In an attempt to confirm the association found by Benjamin et al using NEO-PI-R estimated NS, the present study used an alternative design extreme groups strategy to select high and low novelty seeking research volunteers from the Baltimore Longitudinal Study of Aging (BLSA).6 There were no significant associations between long alleles (7-repeat allele) and high novelty seeking groups. The findings of Ebstein and colleagues and those of Benjamin and colleagues do not generalize to this American middle-aged, mixed-gender sample, a conclusion also consistent with the findings of a recent Swedish study.7 Demographic factors such as the age and gender composition of the samples are important sources of variation in allelic association studies and future research must carefully address whether the D4DR genetic polymorphisms vary substantially across demographic groups.

Sex differences in voluntary oral nicotine consumption by adolescent mice: a dose-response experiment.

Recent studies with adolescent rodents offer valuable information regarding the neurochemical and behavioral effects of adolescent nicotine exposure. One hundred twenty-one male and 125 female adolescent (35 days of age) C57BL/6J mice were tested for voluntary nicotine consumption by providing 24-h access to both saccharin-only (SAC) and one of six nicotine-containing solutions [10, 25, 50, 75, 100, 200 ug (-)-freebase nicotine/ml in 2% SAC] in the home cage for 7 days. Although males and females drank similar volumes (ml) of nicotine, the female mice consumed more nicotine adjusted for body weight (mg/kg) and as a percentage of total fluid intake than did the male mice. In contrast, there was no sex difference in overall serum cotinine levels (adjusted for liver weight). For all mice, nicotine consumption and serum cotinine levels increased in a dose-dependent manner, and the volume of nicotine intake (ml), percent nicotine intake, and nicotine dosage (mg/kg) on the last day of the experiment were positively correlated with cotinine levels. Cotinine levels were inversely related to body weight only for females. Sex differences in nicotine consumption, but not in cotinine levels, suggest sex differences in pharmacokinetic processes that may contribute to oral nicotine consumption behavior during periadolescence.

Dopaminergic polymorphisms in Tourette syndrome: Association with the DAT gene (SLC6A3)

Tourette syndrome (TS) is a chronic neuropsychiatric disorder characterized by involuntary motor and phonic tics. The pattern of inheritance and associated genetic abnormality has yet to be fully characterized. A dopaminergic abnormality in this disorder is supported by response to specific therapies, nuclear imaging, and postmortem studies. In this protocol, dopaminergic polymorphisms were examined for associations with TS and attention‐deficit hyperactivity disorder (ADHD). Polymorphisms investigated included the dopamine transporter (DAT1 DdeI and DAT1 VNTR), dopamine receptor (D4 Upstream Repeat and D4 VNTR), dopamine converting enzyme (dopamine β‐hydroxylase), and the acid phosphatase locus 1 (ACP1) gene. DNA was obtained from 266 TS individuals ± ADHD and 236 controls that were ethnicity‐matched. A significant association, using a genotype‐based association analysis, was identified for the TS‐total and TS‐only versus control groups for the DAT1 DdeI polymorphism (AG vs. AA, P = 0.004 and P = 0.01, respectively). Population structure, estimated by the genotyping of 27 informative SNP markers, identified 3 subgroups. A statistical re‐evaluation of the DAT1 DdeI polymorphism following population stratification confirmed the association for the TS‐total and TS‐only groups, but the degree of significance was reduced (P = 0.017 and P = 0.016, respectively). This study has identified a significant association between the presence of TS and a DAT polymorphism. Since abnormalities of the dopamine transporter have been hypothesized in the pathophysiology of TS, it is possible that this could be a functional allele associated with clinical expression.

Tyrosine-533 of rat dopamine transporter : involvement in interactions with 1-methyl-4-phenylpyridinium and cocaine

To improve our understanding of structure-function relationships for neurotransmitter transporters, we performed site-directed mutagenesis of the rat dopamine transporter (DAT) and assessed the functions of the mutants in transiently-expressing COS cells. Tyrosine-533 of rat DAT lies in the 11th transmembrane region, where the corresponding amino acid of human DAT is phenylalanine. Alanine substitution of tyrosine-533 (Y533A) conferred an increased affinity for 1-methyl-4-phenylpyridinium (MPP+). Phenylalanine substitution of tyrosine-533 (Y533F) increased the velocity of MPP+ uptake but decreased DATs affinity for MPP+. Cocaines potency in inhibiting dopamine uptake was unchanged with Y533A, but increased with Y533F. Differences in the uptake kinetics and inhibitory potency of cocaine between rat and human DATs were similar to the differences observed between the wild-type and Y533F mutants DATs. Tyrosine-533 may be important for the DAT function and for species differences in transporter functions, including differential sensitivities to cocaine and 1-methyl-1,2,3,6-tetrahydropyridine (MPTP) in humans and rats.

Quantitative Trait Loci Analysis of Structural and Material Skeletal Phenotypes in C57BL/6J and DBA/2 Second-Generation and Recombinant Inbred Mice

QTL analyses identified several chromosomal regions influencing skeletal phenotypes of the femur and tibia in BXD F2 and BXD RI populations of mice. QTLs for skeletal traits co‐located with each other and with correlated traits such as body weight and length, adipose mass, and serum alkaline phosphatase.

Dose-dependent incidence of hepatic tumors in adult mice following perinatal exposure to bisphenol A.

Background: Bisphenol A (BPA) is a high production volume chemical with hormone-like properties that has been implicated as a potential carcinogen. Early-life exposure has been linked to increased risk for precancerous lesions in mammary and prostate glands and the uterus, but no prior study has shown a significant association between BPA exposure and cancer development. Objective: We explored the effects of BPA exposure during gestation and lactation on adult incidence of hepatic tumors in mice. Methods: Isogenic mice were perinatally exposed to BPA through maternal diets containing one of four environmentally relevant doses of BPA (0, 50 ng, 50 μg, or 50 mg per kilogram of diet), and we followed approximately one male and one female per litter until they were 10 months of age. Animals were tested for known risk factors for hepatocellular carcinoma, including bacterial and viral infections. Results: We found dose-dependent incidence of hepatic tumors in 10-month-old BPA-exposed mice. Of the offspring examined, 23% presented with hepatic tumors or preneoplastic lesions. We observed a statistically significant dose–response relationship, with an odds ratio for neoplastic and preneoplastic lesions of 7.23 (95% CI: 3.23, 16.17) for mice exposed to 50 mg BPA/kg diet compared with unexposed controls. Observed early disease onset, absence of bacterial or viral infection, and lack of characteristic sexual dimorphism in tumor incidence support a nonclassical etiology. Conclusions: To our knowledge, this is the first report of a statistically significant association between BPA exposure and frank tumors in any organ. Our results link early-life exposure to BPA with the development of hepatic tumors in rodents, and have potential implications for human health and disease. Citation: Weinhouse C, Anderson OS, Bergin IL, Vandenbergh DJ, Gyekis JP, Dingman MA, Yang J, Dolinoy DC. 2014. Dose-dependent incidence of hepatic tumors in adult mice following perinatal exposure to bisphenol A. Environ Health Perspect 122:485–491; http://dx.doi.org/10.1289/ehp.1307449

Long forms of the dopamine receptor (DRD4) gene VNTR are more prevalent in substance abusers: no interaction with functional alleles of the catechol-o-methyltransferase (COMT) gene.

Substance abuse is a complex behavior that is caused by both environmental and genetic factors. Work to understand the genetic factors has focused on genes related to dopamine activity because of its critical role in rewarding and reinforcing behaviors. The DRD3 and other dopamine receptor subtypes are expressed in many areas of the limbic system, and have been the objects of study for their possible roles in several neuropsychiatric disorders. Interest in variants of the D4 gene was heightened by reports that some alleles were more frequent in individuals who score high on Novelty Seeking, an aspect of personality that may be related to drug seeking behavior. We now show that the long form of the DRD4 gene is more frequent in individuals with high quantity/frequency of drug use compared to controls (chi(2) = 5.7, df = 1, P = 0.017, odds ratio = 1.89, CI = 1.1-3.2). There is no difference in DRD3 allele frequencies in these samples, and there is no interaction of DRD4 alleles with those of the catecholamine-o-methyl- transferase gene (COMT) that we previously identified to be more frequent in substance abusers than controls [Vandenbergh, et al.: 1997: Am. J. Med. Gen. 74:439-442].