Assessment of the Association of D2 Dopamine Receptor Gene and Reported Allele Frequencies With Alcohol Use Disorders

Key Points Question Is there a biological association between D2 dopamine receptor gene (DRD2) and alcohol use disorder? Findings This meta-analysis of 62 studies including 16 294 participants found that the association between DRD2 and alcohol and heterogeneity between studies are associated with spuriously low allele frequencies in positive studies rather than with any ability of the linked locus to drive transcription. Meaning These observations regarding the factors behind the association between alcohol use disorder and DRD2 and tactics to identify those factors may be relevant to other findings that are highly significant in meta-analyses but biologically meaningless and that may be associated with research and clinical care.

(Two HPA axis genes, CRHBP and FKBP5, interact with childhood trauma to increase the risk for suicidal behavior. Roy, 2012). All participants gave written informed consent to the study that was approved by the Institutional Review Boards (IRBs) of the Department of Veteran Affairs New Jersey Healthcare System (VANJHCS) and the University of Medicine and Dentistry, New Jersey Medical School (UMDNJ).
• Amount 583 genotyped subjects in the plots, 356 were males and 227 were females.

Array based genotyping
Array-based SNP genotyping was performed using Illumina GoldenGate protocols on 96-well format BioRealm Smokescreen arrays. Five hundred nanograms of genomic DNA was used per assay. Pre-PCR DNA processing was performed using a TECAN liquid handling robot running Illumina protocols. Arrays were imaged using an Illumina Beadstation GX500 and data analyzed using GenCall v6.2.0.4 and GTS Reports software v5.1.2.0 (Illumina). Genotype clusters were determined for a test dataset and this template was applied to all subsequent datasets. Data were polished by manual adjustment of the clustering for each SNP to correct for differences between datasets arising from sample integrity and concentration. The GenCall score is a value between 0 and 1 giving a confidence score for that genotype call (the higher the score the higher the confidence in the call) and is derived from the tightness of the clusters for a given locus and the position of the sample relative to its cluster. Loci with call rate <90% were determined to have failed and were excluded. At this point deviation from Hardy-Weinberg equilibrium was not used as an exclusion criterion because all datasets contained both case and control samples, potentially driving deviation from HWE.

Differential allelic expression (DAE) and SNP genotyping in human brain
Postmortem brain tissue was provided by the University of Miami Brain Bank. Specimens were obtained at autopsy from chronic cocaine and alcohol abusers and age-matched drug-and alcohol-free control subjects. All subjects died suddenly without a prolonged agonal state. Brain pH measures were done as quality control for each case with values > 6.0. Postmortem samples were genotyped for rs1800497 and rs62755, the latter being the reporter locus for DAE. These SNPs were PCR-amplified and genotyped in all subjects using custom 5' exonuclease primers and probes (Life Technologies). Genotypes were resolved by size using an ABI 3730 Capillary Sequencer and GeneMapper Software v4.0.

DRD2 differential allelic expression
To determine if there is a cis-acting eQTL affecting expression of DRD2, and if rs1800497 is a cis-acting eQTL, the expression of alleles for the reporter SNP rs62755 was compared in 28 heterozygous brain samples identified from a larger (N=82) number of brains. The reporter SNP was selected on the basis that it was exonic with an allele frequency approaching 0.5, thereby ensuring that multiple heterozygous samples would be available for analysis. The messenger RNA levels for both alleles were simultaneously quantified by real-time PCR on hippocampal SNPs are color coded according to linkage disequilibrium (LD) with rs1800497 (purple dot with red arrow) on a scale of r 2 0 to 1. Estimated recombination rates reflect local LD structure in the 600 kb buffer around rs1800497 in this African American population. rs1800497 allele frequencies (D) did not differ between cases and controls.