D2 dopamine receptor gene (DRD2) Taq1A (rs1800497) affects bone density

Schizophrenia patients are susceptible to lower bone mineral density (BMD). However, studies exploring the genetic effects are lacking. Genes that affect the activity of antipsychotics may be associated with BMD, particularly in patients receiving long-term antipsychotic treatment. We aimed to explore the relationship between the dopamine receptor D2 (DRD2) gene Taq1A (rs1800497) polymorphism and BMD in chronic schizophrenia patients. We recruited schizophrenia patients (n = 47) and healthy controls (n = 39) from a medical center in Taiwan and collected data that may affect BMD. Patients’ BMD was measured by dual-energy X-ray absorptiometer (DEXA). DRD2 rs1800497 was genotyped through polymerase chain reaction–Restriction Fragment Length Polymorphism (PCR–RFLP). Among all participants, subjects with DRD2 rs1800497(T;T) allele had lower DEXA T score and DEXA Z score compared to those with rs1800497(C;T) and rs1800497(C;C) alleles (p = 0.008, 0.003, respectively). In schizophrenia patients, subjects with rs1800497(T;T) allele also had lower DEXA Z score compared to the other two alleles (p = 0.045). Our findings suggest that individuals with the DRD2 rs1800497(T;T) had lower BMD than those with the rs1800497(C;T) and rs1800497(C;C) genotypes. Therefore, genes should be considered as one of the risk factors of lower BMD.

Scientific RepoRtS | (2020) 10:13236 | https://doi.org/10.1038/s41598-020-70262-0 www.nature.com/scientificreports/ Dopamine is the predominant neurotransmitter in the central nervous system involved in a wide variety of physiological processes 12 . Dopamine has five different seven-transmembrane G protein-coupled receptors named D1 to D5 13 . The D2 receptor is mainly located in the striatum, olfactory tubercle and the core of nucleus accumbens 14 . It is known that D2 receptor is related to motor activity, reinforcement mechanisms, as well as learning and memory 13 . Some studies found that dopamine receptor D2 (DRD2) gene was involved in genetic susceptibility to posttraumatic stress disorder, risk factors associated with smoking and schizophrenia [15][16][17] . The DRD2 rs1800497 polymorphisms were found to be related with body weight, pathological eating behavior and risk of alcoholism 18,19 . In the genetic aspects, several studies have suggested that individuals with DRD2 rs1800497 T allele have a higher plasma prolactin level under antipsychotic use [20][21][22] .
So far, few studies have explored possible genetic effects on BMD. The latent relationship between the DRD2 rs1800497 allele and BMD in schizophrenia patients remains unclear. Therefore, in this study, we aimed to clarify the relationship between DRD2 rs1800497 gene and bone mineral density.

Methods
Setting. This study was performed in the outpatient department and chronic inpatient units of Kaohsiung Chang Gung Memorial Hospital, a major psychiatric center in south Taiwan. The hospital offers inpatients a balanced diet that contains 600 mg calcium and approximately 2000 cal/day. The study was approved by the Institutional Review Board of Kaohsiung Chang Gung Memorial Hospital, and all participants provided their written informed consent in accordance with the Declaration of Helsinki after being provided a complete description of the study.
Patients. We enrolled patients diagnosed with chronic schizophrenia by research psychiatrists based on the DSM-IV criteria (American Psychiatric Association, 1994). All patients had been clinically ill but stable in psychotic symptoms. They had been treated with unchanged antipsychotics and doses for at least 6 months. According to the antipsychotic regimens at the time of enrollment, we separated patients into two groups: patients taking first-generation antipsychotics, risperidone, paliperidone, amisulpride, or ziprasidone were defined as the prolactin-releasing (PR) group; while those taking clozapine, olanzapine, quetiapine, or aripiprazole were defined as the prolactin-sparing (PS) group. The various antipsychotic medications were converted and expressed as a chlorpromazine equivalent dose (mg/day) 23 . Such information as education duration, age of disease onset, disease duration, hospitalization duration, and antipsychotic treatment duration was also recorded. All participants were Han Chinese in Taiwan.

Controls.
We also enrolled 39 unrelated healthy volunteers from the community and staff of Kaohsiung Chang Gung Memorial Hospital. Ages ranged from 18 to 65 years old, and all participants were Han Chinese in Taiwan. Healthy volunteers were free from any axis I psychiatric disorder.
Exclusion criteria. Both patients and healthy individuals with the following physical or mental conditions that may have influenced BMD were excluded: eating disorder, substance abuse/dependence (including smoking and alcohol drinking, which are forbidden in hospitals and public places in Taiwan), renal function impairment, electrolyte imbalance, bone metabolism diseases, thyroid or parathyroid diseases, pituitary tumor, pregnancy or lactation, and co-medications known to influence BMD (e.g., glucocorticoids 24 , heparin 25 , and drugs for osteoporosis like parathyroid hormones 26 , alendronate 27 , selective estrogen receptor modulators 28 , and bisphosphonates, estrogens, and calcitonin 29 , with the exception of benzodiazepines and antidepressants, which have not been significantly associated with reduced BMD 8 ).
Assessments. We used measurements from the dual-energy X-ray absorptiometer (DEXA) 10,23,24 to assess bone marrow density at lumbar spine L2-L4 in a supine position. Professional radiologists, who were all blinded to the patients' clinical characteristics, evaluated these data.
An absolute BMD value T score between − 2.5 and − 1 was defined as osteopenia 25 , while a T score of − 2.5 or lower was defined as osteoporosis 26 . A T score of − 1 or less was considered LBMD (including osteopenia and osteoporosis) 25 . We also measured DEXA Z score to evaluate whether decreasing bone mineral density originated from aging 27 . A Z score of − 1 or less was considered bone mineral loss with causes except age 27 .
Blood samples were collected at 08:00 to assess bone remodeling-related factors, including complete blood and platelet count, serum estradiol, testosterone, LH, FSH, prolactin, cortisol, thyroid hormone, TSH, Free-T4, T3, blood urea nitrogen, creatinine, alkaline phosphatase, and calcium. Data from physical examinations, including body weight, height, and BMI, were also recorded.
We analyzed DRD2 rs1800497 genotype by polymerase chain reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). First, we designed a suitable sense primer and antisense primer to amplify the candidate gene (primers sequence, rs1800497_F: CCG TCG ACG GCT GGC CAA GTT GTC TA; rs1800497_R: CCG TCG ACC CTT CCT GAG TGT CAT CA). The total volume for PCR was 30 μl, including 1 μl DNA template, 3 μl of ten times PCR buffer, 1.5 μl DMSO, 0.75 μl dNTP (5 mM/μl), 0.45 μl sense primer (10 pmol/μl), 0.45 μl antisense primer (10 pmol/μl), 0.45 μl FastStar Taq DNA polymerase (5 U/μl), and 21.9 μl ddH2O. We used MJ PTC-200 for amplification. Reaction criteria for PCR were 95 °C for 5 min, 35 cycles of 95 °C for 30 s, 61 °C for 30 s, 72 °C for 30 s, and the last cycle of 72 °C for 7 min. We used 2% agarose gel to confirm the product, then cut it using a suitable limited-enzyme. The post-cut PCR product was then submitted to electrophoresis by 3% high resolution agarose and dyed with ethidium bromide to obtain the final genotype result. DRD2 Taq1A A1A1 referred to rs1800497(T;T), DRD2 Taq1A A2A2 referred to rs1800497(C;C), and DRD2 Taq1A A1A2 referred to rs1800497(C;T). Statistical analysis. We adopted the chi-squared test or Fisher's exact test as appropriate for between-group comparisons of categorical data. We used the t-test to analyze continuous data. Analysis of Variance (ANOVA) was used to determine the relationship between DRD2 rs1800497 genotype and BMD. We also performed post hoc analysis to assess the difference between rs1800497 genotypes. All tests were two-tailed, and statistical significance of tests was defined as p < 0.05. Data were analyzed with SPSS version 22.0 (SPSS Inc., USA).

Results
Patients and controls. This study consisted of a total of 47 patients and 39 controls. Participants' demographic and clinical characteristics are shown in Table 1. Age, free T4, TSH, renal function (BUN, creatinine), and BMD (DEXA T score, DEXA Z score) were similar between patients and controls. The schizophrenia group had more men than women compared to the control group. Patients with schizophrenia had significantly higher levels of BMI, T3, ALK-P, prolactin, and FSH than the controls. The mean estradiol level of schizophrenia patients was significantly lower than that of controls. www.nature.com/scientificreports/ Genotype and bone mineral density. The relationships between DRD2 rs1800497 genotype and BMD (including DEXA T, DEXA Z, and BMD scores) were analyzed by ANOVA and Bonferroni correction ( Table 2). For all participants (both schizophrenia patients and controls), the mean bone densities were different among different rs1800497 genotypes (p = 0.008, 0.003 and 0.014 for DEXA T, DEXA Z and BMD scores, respectively (Fig. 1). Post hoc analysis with Bonferroni correction revealed lower bone density in the rs1800497(T;T) allele group than in the (C;T) allele group in DEXA T (p = 0.006), DEXA Z (p = 0.002), and BMD (p = 0.011) scores. We observed no significant differences in bone density between (T;T) allele versus (C;C) allele and (C;T) allele versus (C;C) allele (Table 2). Notably, the aforementioned tests did not reach statistical significance when applying the multiple testing corrections. In schizophrenia patients, the rs1800497(T;T) group had lower DEXA Z scores (p = 0.045) than the rs1800497(C;C) and (C;T) groups ( Table 3). The rs1800497(T;T) group also had lower DEXA T scores (p = 0.070) and BMD scores (p = 0.075) than the other two genotypes. In the controls, rs1800497(T;T) group had lower  We also analyzed the relationship between different rs1800497 genotypes and prolactin level in all individuals. As shown in Table 4, after adjusting gender, we found that prolactin levels were different among PR group, PS group, and controls in the rs1800497 (T;T) genotype (p = 0.000023) and the (C;T) genotype (p = 0.000003), respectively. In the rs1800497(T;T) group, post hoc analysis with Bonferroni correction showed higher prolactin level in PR group comparing to PS group and controls (p = 0.000059, 0.000027, respectively). Similarly, in the rs1800497(C;T) group, post hoc analysis with Bonferroni correction showed higher prolactin level in PR group comparing to PS group and controls (p = 0.000029, 0.000011, respectively). In the rs1800497(C;C) group, there was no significant difference in prolactin level among subgroups. We further examined the effect of rs1800497 genotypes, gender and prolactin level on DEXAZ score using multiple linear regression model. We found that rs1800497 was associated with DEXAZ score while adjusting gender and prolactin level (p = 0.001) ( Table 5).
To test the potential confounders that influence the genetic effect on BMD, variables including gender, BMI, T3, alkaline phosphatase, estradiol, and prolactin, which significantly differed between schizophrenia patients and controls, were adjusted in the analysis of covariance. The DEXAZ scores were still different between the rs1800497(T;T) genotype and (C;T) genotype (p = 0.049) after adjusting the aforementioned variables. Regarding DEXAT scores and BMD scores, the differences in bone density between the rs1800497(T;T) genotype and (C;T) genotype did not reach statistical significance (0.055 and 0.055, respectively) (Supplementary Table S1).

Discussion
In this study, we found that participants with the DRD2 rs1800497(T;T) genotype had lower bone density than those with (C;C) or (C;T) genotypes. The trend remained similar after adjusting potential covariates. The findings suggest that individuals with DRD2 rs1800497(T;T) genotype might be prone to have lower bone density than the other two genotypes. To the best of our knowledge, this study is the first to demonstrate the possible relationship between DRD2 rs1800497 polymorphism and bone density. www.nature.com/scientificreports/ There was not yet definite mechanism for the relationship between DRD2 rs1800497 and BMD so far. However, several recent studies found that DRD2 had impact on bone homeostasis. Hanami K et al. found that dopamine decreased osteoclast differentiation via D2-like receptor expressed on human osteoclast precursor cells 30 . Besides, dopamine D2-like receptor signaling can inhibit osteoclastogenesis and bone resorption in vitro 30 . In mice study, administration of D2 receptor antagonist amisulpride to diabetic mice restored trabecular bone structure to near normal and partially reversed downregulation of lysyl oxidase 31 . Moreover, bromocriptine, a potent D2 agonist has been widely used in the treatment of bone metastatic prostate cancer 32 . Several studies have found that people with the DRD2 rs1800497 T allele had a higher prolactin level during antipsychotics use 20 . A recent meta-analysis of patients with schizophrenia showed that prolactin levels were significantly higher in rs1800497 T carriers than T non-carriers 21 . Previous studies explored the relationship between antipsychotics-induced hyperprolactinemia and BMD loss, which have resulted in inconsistent findings 33 . Many factors contribute to the regulation of prolactin level, including environment, physiological stimuli, sexual hormones, medication, etc. 34 . Whether DRD2 rs1800497 controls bone density through elevated prolactin level or other mechanisms unrelated to hyperprolactinemia requires further investigation. A larger-scale study is warranted to clarify the relationship between DRD2 rs1800497 and prolactin level.
DRD2 rs1800497 polymorphism is associated with various psychiatric diseases. Substance use disorders, such as cocaine, nicotine, opioid, and particularly alcohol dependence, were related to DRD2 35 . Large-scale meta-analysis has confirmed that DRD2 rs1800497 polymorphism was associated with alcohol dependence and even HIV positive alcohol abusers 36,37 . Furthermore, individuals with the rs1800497(C;C) allele have a higher likelihood of smoking cessation than those who carry the (T;T) or (C;T) allele 38 . In more recent studies, rs1800497 polymorphism was demonstrated in post-traumatic brain injury cognitive performance and Parkinson disease 39,40 .
As increasing research attempts to explore BMD via genetic-level and widespread genome-wide association studies (GWAS), several genes have been found to be associated with bone density. The key genes related to bone fragility include LDL receptor-related protein 5 (LRP5), Wnt Family Member 1 (WNT1), and Plastin 3 (PLS3) 41 . A recent systematic review and meta-analysis study demonstrated significant correlations between vitamin D receptor (VDR) polymorphisms, such as VDR ApaI and VDR FokI, and susceptibility to postmenopausal osteoporosis 42 . Such studies have offered us the opportunity to understand the bone density issue at the genetic level and increased our knowledge about osteoporosis 43 . A GWAS study integrating co-expression network data found that SPTBN1 (spectrin, beta, non-erythrocytic 1) and MARK3 (MAP/microtubule affinity-regulating kinase 3) were causal genes at BMD GWAS loci 44 . In addition to GWAS, genome-scale capture C based method Table 4. Analysis of variance (ANOVA) of prolactin level (ng/ml) and DEZAZ score among DRD2 rs1800497 genotypes in all individuals (schizophrenia patients and controls). The analyses were adjusted for gender. PR, prolactin-raising antipsychotics; PS, prolactin-sparing antipsychotics; ProL, prolactin level.   46 . To our knowledge, there is not yet publically available GWAS data investigating DRD2 rs1800497 polymorphism and bone density. So far, this research is the first to explore the possible relationship between DRD2 rs1800497 polymorphism and bone density. Our study has several limitations. First, the etiology of BMD loss is multifactorial, including age, gender, daily activities, sun exposure and others. Genetic effect is only one of many contributors. Furthermore, we only investigated one candidate gene in this study rather than using a large scale population-based GWAS study which may provide stronger evidence. Second, the sample size was relatively small. Under multiple testing corrections, the p values would not reach statistical significance since there were multiple tests. Third, such risk factors as diet, exercise, and sun exposure that may affect BMD were difficult to control strictly, particularly for outpatients, although we provided adequate education for every participant. Fourth, although we arranged physical and laboratory evaluations to exclude conditions that may affect BMD as much as possible, we were unable to completely exclude all physical conditions. Fifth, this study was a cross-sectional study. Further longitudinal studies are warranted to confirm our finding. Lastly, only Han Chinese participants were enrolled in this study. Further research with other ethnicities is required.
In summary, this study revealed for the first time that individuals with the DRD2 rs1800497(T;T) allele had lower bone density when compared to (C;C) and (C;T) alleles. Physicians should consider genetic effect when assessing the risk of osteoporosis or osteopenia. Future studies are required to elucidate the underlying mechanisms of DRD2 rs1800497′s influence on bone density.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.