Ulrich Finckh

Association of a CB1 Cannabinoid Receptor Gene (CNR1) polymorphism with severe alcohol dependence

Due to the involvement of the endogenous cannabinoid system in brain reward mechanisms a silent polymorphism (1359G/A; Thr453Thr) in the single coding exon of the CB1 human cannabinoid receptor gene (CNR1) was analysed in 121 severely affected Caucasian alcoholics and 136 most likely non-alcoholic controls. The observed frequency of the A allele was 31.2% for controls and 42.1% for alcoholics with severe withdrawal syndromes (P=0.010). Post-hoc exploration indicated that this allelic association resulted from an excess of the homozygous A/A genotype in patients with a history of alcohol delirium (P=0.031, DF 2), suggesting s an increased risk of delirium (OR=2.45, 95% CI 1.14--5.25). This finding suggests that the homozygous genotype CNR1 1359A/A confers vulnerability to alcohol withdrawal delirium.

Dopamine D1, D2 and D3 receptor genes in alcohol dependence.

Hereditary factors play a substantial role in the etiology of alcohol dependence. Alcohol mediates its reinforcing effects by an activation of the mesolimbic dopamine system. These findings suggest that the genes encoding the dopamine receptor (DR) subtypes represent high-ranking candidates for susceptibility genes to addictive disorders. Our present population-based association study investigated whether sequence variants of the dopamine D1, D2 and D3 receptor genes confer susceptibility to alcohol dependence in 278 alcoholics, and clinically more homogeneous subgroups ascertained through positive family history, early age at onset, delirium, withdrawal seizures and antisocial tendencies. No evidence for an allelic association was found for the PCR-based TaqA RFLP of the DRD2 gene and a Bsp1286I RFLP of the DRD1 gene. Without correction for multiple testing, we found a significantly increased allele frequency of a common DRD3 gene variant expressing a serine at position 9 in the extracellular N-terminal part of the receptor protein in 55 alcohol-dependent individuals with delirium (X2 = 4.1, df = 1, p = 0.042). Further studies have to examine whether this amino acid substitution or a nearby mutation confers genetic susceptibility to at least a subgroup of alcohol-dependent individuals with delirium.

Association of the dopamine D2 receptor gene with alcohol dependence: haplotypes and subgroups of alcoholics as key factors for understanding receptor function.

Objectives The dopamine D2 receptor (DRD2) plays an important role in the reinforcing and motivating effects of ethanol. Several polymorphisms have been reported to affect receptor expression. The amount of DRD2, expressed in a given individual, is the result of the expression of both alleles, each representing a distinct haplotype. We examined the hypothesis that haplotypes composed of polymorphisms, associated with reduced receptor expression, are more frequent in alcoholics compared with healthy individuals. Methods The polymorphisms −141ins/del, C957T, A1385G, and TaqIA were genotyped in a case–control sample comprising 360 alcoholics and 368 controls, and in a family-based sample of 65 trios. To investigate more homogenous groups, we constructed two subgroups with respect to age at onset and antisocial personality disorder. In addition, a subgroup with positive family history of alcoholism was investigated. Results The haplotypes I-C-G-A2 and I-C-A-A1 occurred with a higher frequency in alcoholics [P = 0.026, odds ratio (OR): 1.340; P = 0.010, OR: 1.521, respectively]. The rare haplotype I-C-A-A2 occurred less often in alcoholics (P = 0.010, OR: 0.507), and was also less often transmitted from parents to their affected offspring (1 vs. 7). Among the subgroups, I-C-G-A2 and I-C-A-A1 had a higher frequency in Cloninger 1 alcoholics (P = 0.083 and 0.001, OR: 1.917, respectively) and in alcoholics with a positive family history (P = 0.031, OR: 1.478; P = 0.073, respectively). Cloninger 2 alcoholics had a higher frequency of the rare haplotype D-T-A-A2 (P<0.001, OR: 4.614) always compared with controls. In patients with positive family history haplotype I-C-A-A2 (P = 0.004, OR: 0.209), and in Cloninger 1 alcoholics haplotype I-T-A-A1 (P = 0.045, OR: 0.460) were less often present. Conclusion We confirmed the hypothesis that haplotypes, which are supposed to induce a low DRD2 expression, are associated with alcohol dependence. Furthermore, supposedly high-expressing haplotype weakened or neutralized the action of low-expressing haplotypes.

Dopamine D2 receptor gene (DRD2) haplotypes in Caucasians

The human dopamine D2 receptor gene (DRD2) is considered a candidate gene for neuro-psychiatric diseases. We typed three new DNA sequence variants in DRD2 intron 4, intron 6 and exon 8, in combination with the known TaqI A restriction fragment length polymorphism (RFLP) and exon 7 311Ser/Cys in 106 unrelated psychiatrically healthy Caucasians. Based on the genotypic data we delineated 10 distinct DRD2 haplotypes and their genetic relationship. Our data provide evidence that the Taq A1 allele and the 311Cys variant are components of different groups of haplotypes though both variants have been speculated to be associated with alcoholism or schizophrenia in recent studies. Therefore we conclude that the prior knowledge of the frequencies and genetic relationships of DRD2 haplotypes will lead to the selection of more suitable intragenic markers for future association studies.

Simple PCR Amplification of the Entire Glucocerebrosidase Gene (GBA) Coding Region for Diagnostic Sequence Analysis

Mutations in the human glucocerebrosidase gene (GBA) may lead to Gaucher disease-an autosomal recessive, lysosomal storage disease. In about 15-25% of Caucasian patients with Gaucher disease yet the disease-causing mutations remain to be identified. There exists 16kb downstream from the functional GBA gene (chromosome 1q21) a highly homologous transcribed pseudogene (GBAP) with some sequence differences to GBA. These sequence differences might erroneously imitate a true mutation in the functional gene if an unintentional co-investigation of the pseudogene occurred. We describe a protocol which allows the selective analysis of a PCR-amplified 7.1 kb genomic GBA-fragment encompassing the entire GBA coding region. Direct, nonradioactive double stranded cycle-sequencing procedure of nested PCR fragments from this long range GBA-specific product allowed the sequencing of the coding exons including the flanking splice sites. Several, so far unknown coding mutation were identified in non-Jewish families with Gaucher disease. This protocol allows the rapid detection of new GBA mutations.

Chronic ethanol exposure changes dopamine D2 receptor splicing during retinoic acid-induced differentiation of human SH-SY5Y cells

There is evidence for ethanol-induced impairment of the dopaminergic system in the brain during development. The dopamine D2 receptor (DRD2) and the dopamine transporter (DAT) are decisively involved in dopaminergic signaling. Two splice variants of DRD2 are known, with the short one (DRD2s) representing the autoreceptor and the long one (DRD2l) the postsynaptic receptor. We searched for a model to investigate the impact of chronic ethanol exposure and withdrawal on the expression of these proteins during neuronal differentiation. RA-induced differentiation of human neuroblastoma SH-SY5Y cells seems to represent such a model. Our real-time RT-PCR, Western blot, and immunocytochemistry analyses of undifferentiated and RA-differentiated cells have demonstrated the enhanced expression of both splice variants of DRD2, with the short one being stronger enhanced than the long one under RA-treatment, and the DRD2 distribution on cell bodies and neurites under both conditions. In contrast, DAT was down-regulated by RA. The DAT is functional both in undifferentiated and RA-differentiated cells as demonstrated by [(3)H]dopamine uptake. Chronic ethanol exposure during differentiation for up to 4 weeks resulted in a delayed up-regulation of DRD2s. Ethanol withdrawal caused an increased expression of DRD2l and a normalization of DRD2s. Thus the DRD2s/DRD2l ratio was still disturbed. The dopamine level was increased by RA-differentiation compared to controls and was diminished under RA/ethanol treatment and ethanol withdrawal compared to RA-only treated cells. In conclusion, chronic ethanol exposure impairs differentiation-dependent adaptation of dopaminergic proteins, specifically of DRD2s. RA-differentiating SH-SY5Y cells are suited to study the impact of chronic ethanol exposure and withdrawal on expression of dopaminergic proteins during neuronal differentiation.

Methods in DNA amplification.

The proceedings of the 2nd International PCR (polymerase chain reaction) Symposium on Usage of PCR and Alternative Amplification Methods in Infectious and Genetic Diseases, held in Berlin, Germany, February 1993, provide lab-proven protocols from experienced scientists as a general introduction to a

Decreased Proopiomelanocortin mRNA in Lymphocytes of Chronic Alcoholics After Intravenous Human Corticotropin Releasing Factor Injection

BACKGROUND Alcohol abuse may involve an altered neuroendocrine response that mediates lymphocyte-derived proopiomelanocortin (POMC) production and inflammation. We investigated POMC messenger RNA (mRNA) expression in human lymphocytes ex vivo and their relation to plasma ACTH and immunoreactive beta-endorphin (IR-beta-EP) after intravenous injection of human corticotropin releasing factor (hCRF) in chronic alcoholics (n = 12) and nonalcoholics (n = 12) before surgery. Lipopolysaccharide-stimulated interleukin (IL)-1 receptor antagonist (IL-1 Ra) as a marker for chronic inflammation was determined. METHODS Chronic alcohol abuse was diagnosed according to DSM-IV criteria and alcohol consumption >60 g/day. A reverse transcription-polymerase chain reaction method with total RNA and subsequent solid phase minisequencing was used to quantify lymphocytic POMC mRNA after intravenous hCRF injection. Plasma ACTH, cortisol, and lipopolysaccharide-stimulated IL-1 Ra of monocytes were measured by enzyme-linked immunosorbent assay, and plasma IR-beta-EP was measured by using radioimmunoassay. RESULTS Baseline values of POMC mRNA content in lymphocytes and IL-1 Ra of chronic alcoholics were significantly increased compared with nonalcoholics (p < 0.01). Thirty minutes after intravenous hCRF injection, a significant increase of lymphocytic POMC mRNA was measured (p < 0.05) in nonalcoholics, whereas in chronic alcoholics a significant decrease was observed (p < 0.05). CONCLUSIONS Chronic alcoholic patients had an altered neuroendocrine immune axis before intravenous hCRF administration and were not able to adjust to intravenous CRF injection by increasing lymphocytic POMC mRNA expression.

Allele-specific PCR for simultaneous amplification of both alleles of a deletion polymorphism in intron 6 of the human dopamine 2 receptor gene (DRD2)

The human dopamine 2 receptor gene (DRD2) is an important candidate gene for drug addiction and alcoholism. So far, no mutations within the coding region of DRD2 have been found to be associated with addiction disorders. To identify sequence polymorphisms for further haplotype analyses and to analyze the importance of possible intron sequence variations of the human DRD2 gene (>260kb) in greater cohorts and in a routine manner we established an optimized methodological procedure for polymerase chain reaction (PCR) amplification and direct non-radioactive sequencing followed by a bidirectional allele-specific PCR protocol; the latter one allows the simultaneous amplification of several alleles in one reaction tube. Overall, the sequences of the DRD2 introns 3-7 are highly conserved. Nevertheless, in each of the analyzed intron sequences we found substitution variants as well as a one base-pair deletion polymorphism in intron 6. The allele-specific PCR allowed the reliable testing of 95 healthy control individuals and 270 alcoholics for analyzing a possible genetic association of this newly characterized polymorphic DRD2 marker with alcoholism in an ethnically and clinically homogenous group of patients. However, the observed allele frequencies for the 1bp deletion polymorphism were 15.9% for the alcoholics and 15.3% for the controls suggesting no association of the deletion to alcoholism.

Amplification of Nucleic Acids by Polymerase Chain Reaction: Overview on Principles and Applications

The polymerase chain reaction (PCR) is a powerfulin vitromethod in molecular biology for selective, highly specific and exceptionally efficient amplification of nucleic acid sequences. In the 10 years since the first publication on PCR (Saiki et al., 1985) this method has grown to rival in popularity traditional microbiological, genetic and technical procedures for cloning, sequencing, gene detection and related procedures. Furthermore, in the meantime PCR and all of its different applications are rapid and convenient alternatives to traditional procedures such as blotting technologies, conventional hybridization and molecular cloning. Initially, PCR was a rather complex and tricky generic procedure applied to basic research problems in molecular biology. It has developed into a simple, multipurpose procedure more or less optimized for diverse applications in nearly every biological discipline and commercial area. There are frequent instances of PCR techniques having passed into the service laboratory environment. These service laboratories are providing a broad range of diagnostic tests mainly covering medical and forensic applications, but also environmental, agricultural and veterinary topics.