Genes that modulate or mediate dopaminergic neurotransmission : expression of the human prodynorphin gene and association studies with candidate genes in an alcohol patient sample

Sammanfattning: The development of alcoholism may at least partly be attributed to the effect of ethanol on reward and reinforcement. As a genetic influence in the vulnerability to alcoholism and drug abuse has been documented, it is of interest to investigate genes active in brain structures which mediate reward and reinforcement. The mesolimbic and mesocortical dopamine pathways constitute the neural sites most frequently indicated to mediate reward. Both pathways originate in the ventral tegmental area (VTA) and mediate dopaminergic neurotransmission to the nucleus accumbens (NA) and the prefrontal cortex, respectively. They are influenced by prodynorphin neuropeptides. The purpose of this study was to identify alleles relevant to alcoholism. The studies focused on genes that mediate or modulate dopaminergic neurotransmission. A patient sample (74) from the Magnus Huss Clinic, Karolinska Hospital was assessed according to the criteria of DSM-III-R alcohol dependence and compared with non-alcoholic controls with regard to the distribution of alleles defined by polymorphisms occurring in, or adjacent to, the dopamine D2, D3 and D4 receptors (DRD2, DRD3, DRD4), the tyrosine hydroxylase (TH) and the prodynorphin genes. The controls were derived from 108 individuals mainly recruited among students and hospital staff living in Stockholm. The patients were also divided into severe or moderate dependence (investigated only with regard to the DRD2 and DRD3 polymorphism), parental (at least one alcoholic parent) or non-parental alcoholism, early onset (onset of drinking habits which interfered with their daily life before the age of 25) or late onset (onset after 25 years of age). An additional patient sample (19) where alcoholism during life was assessed from medical records, data from the criminal and social security registers and alcohol-related tissue damage was investigated only with regard to the DRD2 polymorphism. Investigation of genotype prevalence or allele frequencies in the alcoholic patient samples did not yield evidence of a major/moderate predisposition for alcoholism being mediated by alleles defined by the investigated polymorphisms. However, an increased prevalence (p=0.041, not significant after correction for multiple testing) of carriers of one of the TH alleles, TH-K3, among alcoholic patients characterized by late onset implies an association with a phenotype aggregating in this patient group and may need further study. The human prodynorphin gene was also investigated with regard to structure and expression using Northern blot, RNase protection assay and polymerase chain reaction based partial cDNA cloning followed by sequencing. Transcription is initiated 0.12-0.14 bp upstream of intron A. The sequence of the initiation site to approximately 0.8 kb upstream is essential for the expression of human prodynorphin, i.e. it includes the prodynorphin promotor. The data also suggest that further upstream sequences include inhibitory cis-acting factors. However, these data are valid only for prodynorphin gene expression in the human adult brain. The studies show that the tumor cell lines SK-N-MC and SH-SYSY are suitable models for studies of the human adult brain prodynorphin gene expression when combined with transfection/expression vector techniques. Other types of transcription mechanisms were indicated in the developing human brain, the testis and the adrenal gland. Potential polymorphisms in the prodynorphin promotor/exon 1 and exon 4 regions were investigated. Sequencing of polymerase chain reaction fragments derived from different individuals revealed a biallelic polymorphism characterized by a G/A bp exchange. This polymorphism occurs at 301 b upstream of the exon l/intron A boundary, coinciding with the location of the human adult brain prodynorphin gene promotor region.