Genome-wide Association Study Identifies a Regulatory Variant of RGMA Associated With Opioid Dependence in European Americans

Abstract

Background: Opioid dependence (OD) is at epidemic levels in the United States. Genetic studies can provide insight into its biology.

Methods: We completed an OD genome-wide association study in 3058 opioid-exposed European Americans, 1290 of whom met criteria for a DSM-IV diagnosis of OD. Analysis used DSM-IV criterion count.

Results: By meta-analysis of four cohorts, Yale-Penn 1 (n = 1388), Yale-Penn 2 (n = 996), Yale-Penn 3 (n = 98), and SAGE (Study of Addiction: Genetics and Environment) (n = 576), we identified a variant on chromosome 15, rs12442183, near RGMA, associated with OD (p = 1.3 × 10^-8). The association was also genome-wide significant in Yale-Penn 1 taken individually and nominally significant in two of the other three samples. The finding was further supported in a meta-analysis of all available opioid-exposed African Americans (n = 2014 [1106 meeting DSM-IV OD criteria]; p = 3.0 × 10^-3) from three cohorts; there was nominal significance in two of these samples. Thus, of seven subsamples examined in two populations, one was genome-wide significant, and four of six were nominally (or nearly) significant. RGMA encodes repulsive guidance molecule A, which is a central nervous system axon guidance protein. Risk allele rs12442183*T was correlated with higher expression of a specific RGMA transcript variant in frontal cortex (p = 2 × 10^-3). After chronic morphine injection, the homologous mouse gene (Rgma) was upregulated in C57BL/6J striatum. Coexpression analysis of 1301 brain samples revealed that RGMA messenger RNA expression was associated with that of four genes implicated in other psychiatric disorders, including GRIN1.

Conclusions: This is the first study to demonstrate an association of RGMA with OD. It provides a new lead into our understanding of OD pathophysiology.

Keywords: European Americans; Genome-wide association; Opioid dependence; Psychiatric genetics; RGMA; Regulatory variant.

Figure 1. Manhattan plots showing genome-wide association signals of opioid dependence in opioid-exposed European-Americans by meta-analysis

(A) Manhattan plot with one significant variant, rs12442183, on chromosome 15, in meta-analysis of four OD cohorts. The line in the plot represents the genome-wide significance cutoff (5×10^-8). (B) Regional Manhattan plot demonstrates rs12442183 is close to gene RGMA (and regulates RGMA expression; see Figure 3 and text). The light blue line and right Y-axis show the observed recombination rate in the 1000 Genomes Project European samples (EUR, hg19). The SNPs are colored in accordance to R² with rs12442183 (in purple), except for rs113200177, which is specifically highlighted in dark. rs113200177 (T/TG, meta-P=2×10^-3) is located in the promoter of RGMA and is predicted to be able to destroy the E-box motif (5′-CANNTG-3′) of transcription factor 3 or 4 (TCF3 or TCF4) by Haploreg4 (45). By adjusting the effect of the indel in conditional association analysis for rs12442183, the association signal of rs12442183 became stronger in Yale-Penn 1 taken individually (P=1.9×10^-8).

Figure 2. Forest plot for rs12442183 risk allele T in opioid dependence GWAS

Effect Beta and its 95% confidence interval are plotted with squares (proportional to sample size in each cohort) and horizontal lines with whiskers, respectively. The vertical line (beta=0) indicates no effect. Names of cohorts are shown on the left. Meta-analysis P-values and their corresponding effect betas (red diamonds) for European-American (EA) and African-American (AA) populations are also provided. The forest plot was generated by DistillerSR Forest Plot Generator from Evidence Partners (https://www.evidencepartners.com/resources/forest-plot-generator).

Figure 3. rs12442183 is a regulatory variant of RGMA and Rgma is an acute opioid response gene in mouse

(A) Six transcripts of RGMA and four exons used for alternative splicing analysis are mapped together. The relationship between the expression of these four exons and RGMA gene expression was determined by using a linear regression model implemented in R package ESLiMc. The difference between the observed exon expression and the predicted exon expression is named as residual regression score. The alternative splicing events of RGMA across 11 healthy human tissues are displayed in (B), with dot and error-bar represent mean and standard deviation of 3 replicates, respectively. Boxplots (C) are used to illustrate the association between rs12442183 genotype and the residual regression scores of four exons in brain tissue frontal cortex in European samples. Dot and line within each boxplot represent mean and median, respectively, and box region indicates the range from first quantile to third quantile. Up and down whiskers, as well as dots outside box region represent maximum value, minimum value, and outliers, respectively. (D) The expression of Rgma (homologous gene in mouse) among different mouse strains acutely after morphine injection (a single injection, 20 mg/kg) in comparison with chronic injections (repeated morphine administration, 10-40 mg/kg, 3 times daily for 5 days). X-axis shows the four inbred mouse strains (129P3/J, DBA/2J, C57BL/6J and SWR/J). Y-axis demonstrates the mRNA expression of mouse gene Rgma. The expression data were derived from NCBI Gene Expression Omnibus (GEO) dataset GSE7762. The standard deviation is at the top of each bar.