Discussion:
Substance use disorders are chronic brain diseases that have serious effects on people’s health and socioeconomic status worldwide. Genetic, epigenetic, and environmental variables all play a role in the neuropsychiatric state of drug dependence 7, 32, 33. The DA system is the most well-known neurobiological component of substance use disorders. DA primarily exerts its influence by interacting with and activating dopamine receptors, which belong to the G-protein-coupled receptor superfamily. To date, few studies have examined the synergistic impact of genetic-epigenetic (G×E) regulatory variables on the symptoms in patients with MAUD. Here, we discovered that DRD4 rs1800955-CpG2.3 and COMT rs4818-CpG51.52 interactions were associated with paranoid and motor impulsive symptoms of MAUD, respectively.
DRD4, a member of Gi protein-coupled subtype that is expressed in areas of the limbic system associated with cognitive and emotional function, inhibits adenosine cyclase, thereby reducing the concentration of the second intracellular messenger cAMP. According to our findings (Table 2, Figure 1A, B), individuals with the DRD4 rs1800955 C allele, but not DRD4 rs747302 or rs3758653, have a decreased prevalence of paranoid symptoms. The polymorphism rs1800955 (-521 T>C) is located at the 5’-promoter region of DRD4; in this gene, the T allele decreases the transcriptional efficiency by 40% compared with the C allele34, which may be related to differences in psychological and behavioral symptoms. The DRD4 rs1800955 TT polymorphism was associated with higher rates of avoidant and obsessive personality disorder symptomatology 35. When exposed to neutral or negative (but not positive) social stimuli, DRD4 rs1800955 CC carriers show higher mean positive affect ratings than T carriers36. Although the DRD4 gene’s SNP rs1800955 C allele showed a nominal association with heroin dependence37, it was strongly related to cigarette smoking in comparisons of heavy smokers to non-smokers and light smokers to non-smokers 38. The DRD4 rs1800955 TT and CT genotypes were associated with low reward dependence in juniors (p<0.001) and seniors (p=0.010), respectively39. In contrast, there was no significant connection between the DRD4 rs1800955 SNPs and the progression of attention-deficit /hyperactivity disorder 40. However, the prefrontal cortex-controlled executive function of humans was significantly impacted by the combination of two SNPs COMT rs4680 and the DRD4 rs1800955 41.
As a major epigenetic regulatory pathway that controls gene expression, DNA methylation is involved in adaptive changes in neuroplasticity after prolonged drug use 42, 43. Despite the lack of significant correlations between overall methylation levels in the DRD4 promoter regions and paranoid symptoms in MAUD patients (Figure 1C, D), the methylation degree at the DRD4 CpG2.3 unit was significantly lower in this group.
Similar to our study, several studies have discovered a connection between drug addiction and DRD4 methylation. According to Duan’s research, MA users have greater levels of DRD4 CpG1 and CpG4 methylation than controls 44. Elevated DRD4 promoter methylation raises the risk of Alzheimer’s disease in men 45. A study revealed that there were several correlations of DRD4 rs3758653 and rs11246226 with the methylation levels of some CpG loci in the same gene 46. Consistent with the SNP sequencing results, the DRD4 rs1800955 C allele had high efficiency in gene transcription, while also exhibiting a lower methylation degree at the DRD4 CpG2.3 unit, DNA methylation is generally thought to inhibit gene transcription. This result suggests that the combination of the methylation and SNPs contributes to the paranoid symptoms (Figure 1F).
The COMT enzyme methylates and thereby inactivates catecholamine neurotransmitters, such as norepinephrine, epinephrine, and DA. COMT rs4818 is associated with cognitive processing, psychological function, pain modulation, brain metabolic activity, etc. According to our data (shown in Table 3 and Figure 2), MAUD patients who were COMT rs4818 G carriers had higher motor impulsivity scores. It has been reported that COMT rs4818 GG carriers have higher levels of COMT in the prefrontal cortex than CC carriers 47. In addition, COMT gene expression in the prefrontal cortex differs significantly between GG and CC carriers 48. Moreover, rs4818 GG carriers had an increased level of CHRM1 expression in the human dorsolateral prefrontal cortex 49. These changes may influence the modulation of human cognitive ability. COMT SNP rs4818 is associated with variation in performance on cognitive tasks, including the Stockings of Cambridge and the Iowa Gambling Task 50. The Pivac team reported that the presence of the G allele or GG genotype of COMT rs4818 was associated with an increase in several dimensions of negative symptoms and anhedonia 51. Patients receiving treatment for persistent low back pain displayed a substantial association between COMT rs4818 and their baseline level of impairment (p = 0.02)52.
We also examined the methylation status of the COMT promoter region (Supplementary Figure 2). The results demonstrated that the COMT rs4818 CC genotype was correlated with a greater overall methylation level and greater methylation degree at COMT CpG51.52 in MAUD patients (Figure 2B, 2C). In contrast, the COMT rs4818 GG genotype was strongly associated with a higher level of methylation in children with ADHD53. Although the methylation status of the COMT promoter region did not correlate with the motor impulsivity symptom in MAUD patients (Figure 2C, D), when combined with the COMT rs4818 CC genotype, the decreased methylation level of the COMT promoter region and decreased methylation degree at COMT CpG51.52 unit elevated the motor impulsivity scores (Figure 3E, F). COMT rs4818 G allele and lower level of methylation at the promoter region are both associated with higher levels of COMT, which may result in decreased DA regulation activity in the brain synaptic networks, and thus lay the molecular foundation for the heightened motor impulsive symptoms.
In summary, based on data from 189 MAUD patients, our investigation discovered that DRD4 rs1800955-CpG2.3 and COMT rs4818-CpG51.52 are related to the paranoid and motor impulsivity symptom of MAUD, respectively. Our findings demonstrated that certain genetic-epigenetic regulatory factor combinations may function as biological markers for early illness detection and a target for therapy.