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Biomedical subjects

K Steven LaForge

Publications and source records attributed to K Steven LaForge.

16 recordsLinked to original sources
PubMed2006

Altered levels of basal cortisol in healthy subjects with a 118G allele in exon 1 of the Mu opioid receptor gene.

The mu opioid receptor is centrally involved in the development of the addictive diseases. It also modulates the stress responsive hypothalamic-pituitary-adrenal axis. Receptors encoded by the variant 118G polymorphism in exon 1 of the mu opioid receptor gene have a threefold increase in beta-endorphin binding and beta-endorphin is three times more potent in receptor-mediated activation of G protein-coupled inwardly rectifying potassium channels. Humans with this variant have increased stress response following opioid antagonism. Here, we study basal levels of adrenocorticotropic hormone and cortisol in subjects with this variant. In all, 59 healthy adults were genotyped and had morning levels of adrenocorticotropic hormone and cortisol measured following intravenous administration of saline placebo. Subjects with a 118G allele had significantly greater levels of cortisol than subjects with the prototype gene. Groups did not differ in levels of adrenocorticotropic hormone. A planned comparison revealed significantly greater cortisol in females with at least one copy of the 118G allele compared to females with the prototype gene. There was no significant effect of gender alone, nor was there a significant interaction between gender and genotype, on ACTH or cortisol. Subjects with at least one copy of the 118G allele have increased basal levels of cortisol, which may influence the susceptibility to and treatment of the stress responsive dyscrasia.

Adrenocorticotropic Hormone↗
PubMed2006

Association analysis of polymorphisms in serotonin 1B receptor (HTR1B) gene with heroin addiction: a comparison of molecular and statistically estimated haplotypes.

OBJECTIVES: 5-Hydroxytryptamine (serotonin)-1B receptors (HTR1B) may play an important role in psychiatric disorders and drug and alcohol dependence. In this study we report on genotype, molecular haplotype and statistically estimated haplotype analyses of previously identified polymorphisms in positions -261T>G, -161A>T, 129C>T, 861G>C and 1180A>G of the HTR1B gene in ethnically diverse populations (African-Americans, Caucasians, Hispanics and Asians) including 235 former heroin addicts and 161 control subjects from New York City. The objectives were to test for an association of molecular and statistically estimated haplotypes and genotypes in HTR1B gene with heroin addiction and to compare results provided by molecular and statistically estimated haplotyping methods. METHODS: Genotype analysis was performed using a standard TaqMan protocol. Molecular haplotype analysis of the subset of polymorphisms consisting of -261T>G, -161A>T and 129C>T was performed using a protocol specially designed by our group, using fluorescent PCR. This is based on use of allele-specific primers complementary to flanking polymorphisms and a fluorescently labeled sequence-specific TaqMan probe set complementary to an internal polymorphism of the haplotype region. Every individual's statistically inferred haplotype pair agreed with the individual's haplotype pair determined by molecular haplotyping. RESULTS AND CONCLUSION: A point-wise significant association of haplotype pairs containing allele G at position 1180 with protective effect from heroin addiction in Caucasians was found. A point-wise nominally significant association of allele 1180G with a protective effect from heroin addiction was found in Caucasians. Statistically significant differences across four ethnic groups in control subjects for allelic frequencies of -261T>G and -161A>T were found.

Black or African American↗
PubMed2005

Genetic influences on impulsivity, risk taking, stress responsivity and vulnerability to drug abuse and addiction.

Genetic variation may partially underlie complex personality and physiological traits--such as impulsivity, risk taking and stress responsivity--as well as a substantial proportion of vulnerability to addictive diseases. Furthermore, personality and physiological traits themselves may differentially affect the various stages of addiction, defined chronologically as initiation of drug use, regular drug use, addiction/dependence and potentially relapse. Here we focus on recent approaches to the study of genetic variation in these personality and physiological traits, and their influence on and interaction with addictive diseases.

Animals↗
PubMed2005

Increased attributable risk related to a functional mu-opioid receptor gene polymorphism in association with alcohol dependence in central Sweden.

The mu-opioid receptor (MOR), through its effects on reward and stress-responsivity, modulates alcohol intake in both animal and human laboratory studies. We have previously demonstrated that the frequently occurring A118G single-nucleotide polymorphism (SNP) in exon 1 of the MORgene (OPRM1), which encodes an amino-acid substitution, is functional and receptors encoded by the variant 118G allele bind the endogenous opioid peptide beta-endorphin with three-fold greater affinity than prototype receptors. Other groups subsequently reported that this variant alters stress-responsivity in normal volunteers and also increases the therapeutic response to naltrexone (a mu-preferring opioid antagonist) in the treatment of alcohol dependence. We compared frequencies of genotypes containing an 118G allele in 389 alcohol-dependent individuals and 170 population-based controls without drug or alcohol abuse or dependence. The A118G SNP was present in the Hardy-Weinberg equilibrium with an overall frequency of the 118G allele of 10.9%. There was a significant overall association between genotypes with an 118G allele and alcohol dependence (p=0.0074). The attributable risk for alcohol dependence in subjects with an 118G allele was 11.1%. There was no difference in A118G genotype between type 1 and type 2 alcoholics. In central Sweden, the functional variant 118G allele in exon 1 of OPRM1 is associated with an increased attributable risk for alcohol dependence.

Adult↗
PubMed2005

Pharmacogenetics and human molecular genetics of opiate and cocaine addictions and their treatments.

Opiate and cocaine addictions are major social and medical problems that impose a significant burden on society. Despite the size and scope of these problems, there are few effective treatments for these addictions. Methadone maintenance is an effective and most widely used treatment for opiate addiction, allowing normalization of many physiological abnormalities caused by chronic use of short-acting opiates. There are no pharmacological treatments for cocaine addiction. Epidemiological, linkage, and association studies have demonstrated a significant contribution of genetic factors to the addictive diseases. This article reviews the molecular genetics and pharmacogenetics of opiate and cocaine addictions, focusing primarily on genes of the opioid and monoaminergic systems that have been associated with or have evidence for linkage to opiate or cocaine addiction. This evidence has been marshalled either through identification of variant alleles that lead to functional alterations of gene products, altered gene expression, or findings of linkage or association studies. Studies of polymorphisms in the mu opioid receptor gene, which encodes the receptor target of some endogenous opioids, heroin, morphine, and synthetic opioids, have contributed substantially to knowledge of genetic influences on opiate and cocaine addiction. Other genes of the endogenous opioid and monoaminergic systems, particularly genes encoding dopamine beta-hydroxylase, and the dopamine, serotonin, and norepinephrine transporters have also been implicated. Variants in genes encoding proteins involved in metabolism or biotransformation of drugs of abuse and also of treatment agents are reviewed.

Codeine↗
PubMed2004

Primary structure of guinea pig preprodynorphin and preproenkephalin mRNAs: multiple transcription initiation sites for preprodynorphin.

Preprodynorphin and preproenkephalin are protein precursors from which are derived two classes of opioid neurotransmitter peptides. Dynorphin A((1-17)) is produced by proteolytic processing of prodynorphin, and processing of proenkephalin yields the enkephalin peptides. We report here on the isolation and sequencing of multiple clones for these two mRNAs from a cDNA library. Two cDNA clones of preprodynorphin contained the full-length sequence (2.35 kb) with the primary structure predicted from the guinea pig gene sequence. In contrast, one clone encoded the full-length sequence but also an additional 192 nt at the 5' end. This sequence has high homology to the 5' flanking region of the human preprodynorphin gene, and RNase protection assays demonstrated that in addition to a primary initiation site, transcription of this mRNA is initiated at several sites 160-190 nt 5' with respect to the primary site. This difference may alter translational efficiency or mRNA stability. The sequence of preproenkephalin cDNA clones confirmed the structure predicted from the gene sequence. One clone, however, contained sequences encoded by exons 2 and 3, and initiated within the first intron (intron A) of the gene. We used RNase protection mapping to assess the abundance in the brain and pituitary of preproenkephalin transcripts that initiate within intron A. These studies confirmed that the primary transcription start site is 28 nucleotides downstream from the TATAA site, and that intron A sequences are not present in significant amounts in these tissues.

Animals↗
PubMed2004

Redefinition of the human kappa opioid receptor gene (OPRK1) structure and association of haplotypes with opiate addiction.

The kappa opioid receptor (KOR) plays a role in stress responsivity, opiate withdrawal and responses to cocaine. KOR activation by its endogenous ligand dynorphin A(1-17) decreases basal and drug-induced striatal levels of dopamine. The complete structure of the human KOR gene (hOPRK1) has not been previously determined. This study: (i) characterized the genomic structure of the hOPRK1 gene; (ii) identified single nucleotide polymorphisms (SNPs) in the hOPRK1 gene; and (iii) investigated possible associations of these variants with vulnerability to develop heroin addiction. Analysis of 5'-RACE cDNA clones revealed the presence of a novel exon 1 ranging in length from 167 to 251 nucleotides in the 5' 5'-untranslated region of the hOPRK1 mRNA. We found that the hOPRK1 gene has four major exons and three introns, similar to rodent OPRK1 genes. Direct sequencing of amplified DNA containing all four exons and intron 1 of the hOPRK1 gene were evaluated for polymorphisms in 291 subjects (145 former heroin addicts and 146 controls). Twelve SNPs were identified, nine novel variants and three previously reported SNPs. Using logistic regression with opioid dependence as the dependent variable, the 36G>T SNP exhibited a point-wise significant association (P = 0.016) with disease status. The number of haplotypes seen in the three ethnic groups were nine, six and five for African-Americans, Caucasians, and Hispanics, respectively, with corresponding significance levels for differences in haplotype frequencies between cases and controls of P = 0.0742, 0.1015 and 0.0041. Combining ethnicities by Fisher's method yields an empirical significance level of P = 0.0020.

5' Untranslated Regions↗
PubMed2004

Genes associated with addiction: alcoholism, opiate, and cocaine addiction.

Drug addiction is a complex disorder that has a large spectrum of causes. Vulnerability to addiction has been shown in twin studies to have a robust genetic component. This genetic basis for addiction has general and specific components for each drug abused. Although many genes have been implicated in drug addiction, only a handful have either been replicated to have an association or to have an identified functional mechanism related to specific effects of abused drugs. A few selected genetic variants that currently look promising for the study of alcohol, opiate, and cocaine addiction are discussed in this article.

Alcoholism↗
PubMed2003

Acute 'binge' cocaine administration elevates dynorphin mRNA in the caudate putamen of C57BL/6J but not 129/J mice.

Preprodynorphin mRNA was measured in the nucleus accumbens (NAc) and caudate putamen (CPu) after 3-day 'binge' pattern cocaine administration in C57BL/6J and 129/J mice, strains which differ in behavior and in dopamine increases in the CPu after 'binge' cocaine. In the CPu, there was increased preprodynorphin mRNA in C57BL/6J (P<0.05), but not in 129/J mice, with no differences in the NAc. Thus, 129/J mice are hyporesponsive to the preprodynorphin activating effects of acute 'binge' cocaine in the CPu.

Animals↗
PubMed2003

Quantification of multiple mRNA levels in rat brain regions using real time optical PCR.

TaqMan is significantly more sensitive than other methods of mRNA quantification and makes possible the simultaneous analysis of numerous genes in small brain regions. This technique was used to quantify levels of mRNAs of 21 genes in tissue extracts from caudate putamen and nucleus accumbens from individual rats after 1 day 'binge' cocaine or saline administration. Expression of glyceraldehyde-3-phosphate-dehydrogenase, cyclophilin and actin mRNAs as well as 18S ribosomal RNA were evaluated for normalization of levels of gene expression.

Animals↗
PubMed2003

The Kreek-McHugh-Schluger-Kellogg scale: a new, rapid method for quantifying substance abuse and its possible applications.

The new Kreek-McHugh-Schluger-Kellogg scale ('KMSK scale') is designed to quantify self-exposure to opiates, cocaine, alcohol, and/or tobacco. Each section of the KMSK scale assesses the frequency, amount, and duration of use of a particular substance during the individual's period of greatest consumption. The scale also assesses the mode of use, whether the substance use is current or past, and whether each substance is the substance of choice. The administration time is under 5 min. In an initial validation study of this scale, 100 human subjects were administered the KMSK scale concurrently with the Structured Clinical Interview for DSM-IV (SCID-I DSM-IV version). The sensitivity and specificity were very good for opiates, cocaine, and alcohol use. In addition, the correlations between KMSK scores and the number of SCID-I criteria items met were excellent for opiates and cocaine and good for alcohol use. Nicotine dependence was not assessed in this study as there is no SCID-I nicotine criteria. These preliminary results show that the KMSK scale may have both construct validity similar to that of other established self-report measures and the potential to be an effective screening instrument for the assessment of a lifetime diagnosis of alcohol, opiate, or cocaine dependence.

Adult↗
PubMed2003

Optimizing primer--probe design for fluorescent PCR.

TaqMan, a variation of fluorescent PCR, is a powerful tool for gene expression and polymorphism studies. Here we describe the design and evaluation of 27 new TaqMan primer-probe sets for rat genes that play a key role in neural signaling. These newly designed and synthesized probes were tested and then used for quantification of RNA isolated from rat brain. The usual length of common TaqMan probes is 25 bases or less. In these studies we constructed probes with lengths of 25-39 bases to span exon-exon junctions of nucleic acids to avoid the influence of DNA contamination upon the RNA quantification. The specific sequences at these positions required probes of these lengths to optimize hybridization. We found that the relocation of the quencher from the traditional 3' position to an internal one increases the sensitivity of probe up to 30 fold. Substitution of 6-carboxyfluorescein with Alexa Fluor 488 as fluorophore and TAMRA with non-fluorescent quencher dabcyl was also investigated. We also describe the evaluation of part of a newly designed set of 27 TaqMan primer-probes for the measurement of differences in gene expression levels in samples from the caudate putamen region of rat brain after 'binge' paradigm cocaine administration. Cocaine-induced alterations in expression of c-fos and preprodynorphin mRNAs measured by TaqMan were confirmed by ribonuclease protection assay.

Animals↗
PubMed2003

"Binge" cocaine differentially alters preproenkephalin mRNA levels in guinea pig brain.

Male Hartley guinea pigs were administered i.p. injections of cocaine or saline for 2 or 7 days in a "binge" paradigm. RNA was isolated from dissected brain regions and levels of preproenkephalin mRNA and total RNA were quantified by RNase protection assays. Following 2 days of "binge" cocaine administration, no significant alterations in preproenkephalin mRNA levels were detected in six brain regions. Following 7 days of cocaine administration, however, lower levels of preproenkephalin mRNA were observed in the nucleus accumbens and hypothalamus of cocaine-treated animals and higher levels in the frontal cortex and amygdala. These findings differed from previous studies in the rat, so an additional experiment was performed with animals treated at the 7 day time point. For increased statistical power, data from the two experiments were combined and examined by two-way ANOVAs; in this combined analysis, increases in preproenkephalin mRNA were observed in frontal cortex, amygdala, and hippocampus, decreases were found in the nucleus accumbens and hypothalamus, with no change in thalamus, caudate putamen, or cerebellum. These observed differences between guinea pigs and rats make this species an interesting model for neurobiological studies of cocaine-induced alterations in neuropeptide gene expression in the mammalian brain.

Animals↗
PubMed2002

Potentially functional polymorphism in the promoter region of prodynorphin gene may be associated with protection against cocaine dependence or abuse.

It has been demonstrated that the opioid peptide dynorphin plays a role in modulating responses to several psychoactive substances including cocaine. Our laboratory and others have found that mRNA levels of dynorphin in the caudate and putamen are elevated after acute or chronic cocaine exposure in rats. Recently, a 68-base pair (bp) repeat polymorphism within the core promoter region of the human prodynorphin gene has been reported to occur in alleles containing one, two, three, or four copies. This repeat contains a putative AP-1 transcription factor binding site; reporter gene constructs with three or four, but not one or two, copies of the tandem repeats were shown to be associated with increases in transcriptional activation in in vitro cellular assays. We hypothesize that this polymorphism may be associated with individual differences in vulnerability to cocaine dependence or abuse. From an ongoing study of the genetics of addiction, 174 subjects were studied, including individuals with a primary diagnosis (DSM-IV criteria) of cocaine dependence (N = 61) or abuse (N = 22), and controls with no history of any substance dependence or abuse (N = 91). We designed primers for polymerase chain reaction (PCR) to amplify sequences of the promoter region of the prodynorphin gene containing the repeat element. The association of alleles containing three or four repeats with cocaine dependence/abuse was examined. With data stratified by ethnic group, pooled relative risk (RR) with Mantel-Haenszel Chi square was calculated: RR = 0.59 (95% confidence interval 0.37-0.95), chi2 (1) = 4.14, P = 0.042. Our results suggest that this allelic variation at the promoter region of the prodynorphin gene (alleles with three or four repeats), which may result in enhanced transcription of the gene, may contribute to relative protection and decrease individual vulnerability to develop cocaine dependence or abuse.

Alleles↗
PubMed2002

Pharmacotherapy of addictions.

Addiction to drugs, such as heroin, cocaine and alcohol, exacts great human and financial costs on society, but the development of pharmacotherapies for addiction has been largely neglected by the pharmaceutical industry. With advances in our understanding of the underlying biology of addictions now opening the door for the development of novel pharmacotherapies, it could be time for a reassessment of involvement in this increasingly important therapeutic area. Here, we summarize the current approved and implemented pharmacotherapeutic approaches to the treatment of addiction, and then highlight the most promising areas for future drug development from the perspective of our laboratory and our National Institutes of Health (NIH) National Institute on Drug Abuse (NIDA) Research Center.

Alcoholism↗