Opioid use disorder is considered a long-term relapsing brain disease marked by obsessive drug seeking. Numerous epidemiological studies have demonstrated that opioid use disorder is a complicated and highly heritable illness, with multiple genes believed to have weak to moderate impacts on its pathogenesis [1-4].

The glutamatergic system is regarded as a key mediator of neuronal transmission, synaptic plasticity, learning, and memory functions [5,6]. Increasing evidence has indicated that the pathogenesis of drug addiction may be influenced by abnormal glutamate neurotransmission [6-8]. The N-methyl-D-aspartate (NMDA) receptor belongs to a significant class of ionotropic glutamate receptors; GRIN2A codes for subunit 2A of this receptor. Several lines of evidence suggest that the NMDA receptor containing the 2A subunit regulates reward-related learning, memory, and behavioral plasticity in individuals with drug addiction [6,9-11]. Long-term potentiation in the hippocampal region, spatial learning, and conditioned place preference for drugs have been found to be diminished in GRIN2A knockout mice [12,13]. Chronic drug abuse has been shown to alter brain GRIN2A expression [14-16]. Furthermore, heroin has the potential to modify glutamate transmission, either by inhibiting GABAergic interneurons or through dopaminergic pathways [6,7]. Blocking glutamatergic receptors in the ventral tegmental area can attenuate the reward effects of drugs [8-10]. Abnormal function of the NMDA receptor containing the 2A subunit leads to alterations in glutamate signaling, which regulates the reward effects of drugs [10,11]. These findings imply that GRIN2A plays a crucial role in the development of drug addiction [11,17].

Studies have demonstrated that the (GT)n repeat polymorphism (rs3219790) in the promoter of GRIN2A may modulate the gene expression level by inhibiting transcriptional activity in a length-dependent manner, with longer repeats leading to lower promoter activity [18,19]. This finding was supported by a receptor-binding assay in postmortem brains, which revealed a negative correlation between NMDA receptor activity and long (GT)n alleles [18]. Additionally, according to magnetic resonance imaging findings, longer (GT)n repeats are linked to smaller volumes in areas such as the hippocampus and amygdala, which are the primary regions affected by NMDA receptors [20]. In line with these findings, the (GT)n polymorphism in the GRIN2A promoter has been found to be associated with an increased risk for psychiatric disorders, such as schizophrenia and bipolar disorder, with a greater proportion of longer alleles in patients with these conditions [18,21-23]. Evidence suggests that there are extensive interactions between the dopamine and glutamate systems. Dysfunctions in these systems are closely linked to the pathophysiology of various psychiatric disorders, with hyperdopaminergic signaling in the striatum having been observed in GRIN2A-deficient mice [24]. Therefore, functional (GT)n repeats may be a significant candidate risk factor for psychiatric disorders, including drug addiction.

One case-control study showed evidence of an association between the (GT)n repeat polymorphism and alcohol dependence, with longer alleles overrepresented in patients with alcoholism [25]. Similarly, two studies revealed that longer alleles of (GT)n repeats were significantly more frequent among individuals with opioid use disorder in the Chinese Han population [26,27]. One report demonstrated that the frequency of (GT)₂₆ repeats in 210 heroin-dependent patients was significantly greater than that in 205 healthy individuals [26], while another revealed that the frequency of (GT)₂₆ repeats in the heroin-dependent group (405 patients) was significantly greater that in the control group (397 participants) [27]. Considering the extensive genetic heterogeneity of complex human diseases, coupled with population stratification and environmental factors, it is necessary to validate these findings to determine their significance. However, no studies have reported a relationship between the (GT)n polymorphism in GRIN2A and heroin craving. This study aimed to verify the association between the (GT)n polymorphism (rs3219790) in the GRIN2A promoter and opioid use disorder among the Chinese Han population, as well as to evaluate the relationship between the GRIN2A (GT)n polymorphism and heroin cravings.

The allelic distribution of the (GT)n polymorphism (rs3219790) in the GRIN2A promoter in the opioid use disorder and control groups was determined (Figure 1 and Table 1). The genotype distributions were consistent with Hardy-Weinberg equilibrium in both the case and control groups (p>0.05). The dominant allele was (GT)₂₆, with a repeat number ranging from 19 to 36, similar to the findings of previous studies [26,27]. Although there was no significant difference in the global distribution between the two groups, the frequency of the (GT)₂₆ allele in patients was significantly greater than that in the controls (p=0.029, odds ratio [OR]=1.264, 95% confidence interval [CI]=1.025-1.560), in agreement with previous findings [26,27]. The frequency of the (GT)₂₄ allele was considerably lower in the case group than that in the control group (p=0.006, OR=0.674, 95% CI=0.506-0.891).

Moreover, the average number of repeats was nearly identical: 25.70±2.28 in the case group vs. 25.65±2.24 in the control group (p>0.05). The alleles were dichotomized into two categories according to length: short alleles (S≤25 repeats) and long alleles (L≥26 repeats), referring to previous analysis [20]. The samples were genotyped as LL homozygotes, SS homozygotes, or LS heterozygotes. No deviation from the Hardy-Weinberg equilibrium was observed in any group (p>0.05). Overall, there was no significant difference in the distributions of these genotypes or alleles between the case and control groups (Table 2).

Next, the drug use urge questionnaire scores and the S/L genotypes were compared (Table 3). Compared with homozygous SS individuals, carriers of the L alleles demonstrated significantly stronger drug cravings (p<0.05). Supplementary Table 1 shows the individual characteristics of the patients with opioid use disorder.

Our findings indicated a greater presence of the long (GT)₂₆ repeat of the functional polymorphism (rs3219790) in the GRIN2A gene in patients with opioid use disorder than in controls. In addition, individuals carrying long alleles of the (GT)n repeats showed markedly stronger drug cravings compared to those with homozygous short alleles. These findings reveal that the (GT)n polymorphism (rs3219790) may play a role in the pathophysiology of opioid use disorder, suggesting that decreasing GRIN2A receptor function may contribute to the onset of opioid use disorder and cravings.

According to the guidelines of the Clinical Pharmacogenetics Implementation Consortium (CPIC), the clinical effects and safety of opioid drugs should take into account the impact of the CYP2D6, OPRM1, and COMT genotypes [31]. These CPIC’s guidelines will be used to enhance the therapeutic treatment for opioid use disorder. Therefore, it is conceivable that the rs3219790, as one of the genetic markers for the risk of opioid use disorder, may be considered in future clinical practice.

Drug cravings are considered a key factor in inducing drug relapse, and determining how to reduce cravings is crucial in preventing relapse. For the first time, we demonstrated that the (GT)n polymorphism (rs3219790) in GRIN2A is associated with heroin cravings, with longer alleles linked to stronger heroin cravings. Understanding the genetic mechanisms underlying drug cravings can help us more effectively reduce cravings in the treatment and prevention of opioid use disorder.

Glutamate is the primary excitatory neurotransmitter in the central nervous system. NMDA receptors as an ionotropic subtype of glutamate receptors have been proposed to mediate various neuropsychiatric processes, such as brain plasticity, learning, and memory [5,6]. NMDA receptor dysfunction leads to decreased dopamine D1 receptor activity in the cortex and excessive dopamine D2 receptor stimulation in the mesolimbic region. Moreover, overstimulating D2 receptor further prevents the effects of NMDA receptor in the cortex and striatum. Therefore, it is possible that abnormalities in glutamate and dopamine transmissions may contribute to the development of psychiatric disorders [32]. Evidence from pharmacological, animal, and clinical studies indicates that NMDA receptors may be involved in the pathophysiology of several psychiatric disorders [33,34].

Accumulating evidence indicates that the GRIN2A gene, which encodes a subunit of NMDA receptor, plays a pivotal role in many neuropsychiatric disorders [35,36]. GRIN2A gene expression is altered in multiple brain regions in individuals chronically abusing morphine, methamphetamine, or cocaine [14-16]. Several studies have suggested that some polymorphisms in the GRIN2A gene may be associated with opioid use disorder [37,38]. The variable (GT)n repeats (rs3219790) in the GRIN2A promoter region can potentially modify the expression level of GRIN2A [18,19]. Mutations in regulatory elements of the promoter generated by repeat polymorphisms could form special structures, such as Z-DNA, which suppresses gene transcriptional activity [39]. Previous studies have also linked the long allele of the (GT)n polymorphism to several brain disorders [18-23,25-27]. This effect may be due to decreased transcription of GRIN2A, resulting in the reduction of NMDA receptor function.

In our study, the frequency of the (GT)₂₆ allele in patients with opioid use disorder (28.25%) was significantly greater than that in controls (23.75%), which aligned with the results of previous studies by Zhao et al. [26] and Zhong et al. [27] In Zhao et al.’s study [26], the frequencies were 23.81% and 14.40%, while in Zhong et al.’s study [27], the frequencies were 19.26% and 14.86%. Our study confirmed the association between the (GT)₂₆ allele and opioid use disorder. Notably, there were significant differences in the distribution of the (GT)₂₆ allele among healthy controls in the studies by Zhao et al. [26], Zhong et al. [27], and our own. In a study investigating the association between the (GT)n polymorphism and schizophrenia, Zhao et al. [40] found that the frequency of the (GT)₂₆ allele in controls from Shanghai, adjacent to Zhejiang, was 26.26%, nearly identical to our findings of 23.75%. The samples from Zhao et al. [26] and Zhong et al. [27] primarily originated from Northwest China, whereas those from Zhao et al. [40] and our study were mainly sourced from East China. Combining these data, the overall frequency of (GT)₂₆ allele at rs3219790 was found to be 19.82%±6.08% in the Han Chinese population. Thus, the discrepancy may be due to differences between ethnic groups residing in different regions of China. Furthermore, we found that the frequency of the (GT)₂₄ allele in the case group (9.85%) was significantly lower than that in the control group (14.00%); a result was not found in the studies of Zhao et al. [26] and Zhong et al. [27] Additionally, drug craving was associated with (GT)n repeats in our study. In general, these data suggest that the (GT)n polymorphism in the promoter of GRIN2A confers vulnerability to opioid use disorder.

Given that this repeat-number polymorphism modulates gene transcriptional activity in a length dependent manner, it is reasonable to expect the observation of a dose-response effect on the phenotype linked to this polymorphism. However, our data failed to demonstrate a linear relationship between GT repeat numbers and opioid use disorder. It is crucial to emphasize that the results indicating an association between the (GT)₂₆ variant and opioid use disorder may have arisen from statistical artifacts rather than true biological effects. Therefore, further experimental verification is needed to confirm the biological relevance of our findings.

The potential limitations of this study are as follows. First, the sample size in the current study was relatively small. The statistical power of this study was below 0.8, which indicated that the sample size of this study was insufficient. Consequently, our findings may have been affected by statistical fluctuations. Second, in genetic studies of addictive disorders, the most critical confounding variable is drug exposure opportunity. The optimal control group should consist of individuals who have been exposed to the drug but did not develop addiction. However, in the current study, we did not take into account differential drug exposure opportunities between groups, which may introduce environmental bias into our results. Additionally, this study did not consider several potentially relevant confounding factors (such as psychiatric comorbidities and the use of substances other than heroin), which could have led to some bias in our findings. Next, this study included only male Chinese Han subjects. Therefore, there are potential biases concerning the impact of sex and race. Additionally, considering that opioid use disorder is most likely influenced by numerous polymorphisms, the examination of only one gene polymorphism could account for limited genetic influence. Due to the above limitations, the present results should be interpreted with caution. Future research should include a larger cohort of females and different ethnic groups to validate these findings.

In conclusion, this study indicated that the (GT)n functional polymorphism (rs3219790) in the GRIN2A promoter could be an important predictor of susceptibility to opioid use disorder, suggesting that obtaining information about the GRIN2A genotype may provide useful insights for future therapy.