The adult-aged patients or older of both genders were estimated for competence. Schizophrenic patients diagnosed based on the DSM-IV (Diagnostic and Statistical Manual of Mental Disorders) (Tandon et al. 2013). After completing a written consent form that contained a full explanation of the study’s objective and a request to complete a specially created questionnaire, all participants were enrolled in the study. Patients were receiving 10 mg /day olanzapine from 6 months up to years with no additional disorder were included in this study. Patients with pervious hyperglycemia/diabetes, hypotension, weight gain, hyperprolactinemia, tardive dyskinesia before taking olanzapine or receiving treatment or any other medicine that interacts with olanzapine such as bromocriptine, levodopa, methyldopa were excluded from the study.

The case-control study was performed from December 2022 to Abril 2023 in Al-Hassan Al-Mojtaba hospital. The 100 patients consisting of both male and female, aged between 20 and 65 years were recruited from Psychiatry Outpatient Department and 50 apparently healthy without any disease comprising both males and females aged 20 to 63 years, were also enrolled, and served as a control group. The study was approved by the scientific and ethical committee, Kerbala University, college of pharmacy and Ministry of Health of Iraq- Karbala health department with the project being assigned No: 244.

The Positive and Negative Syndrome Scale (PANSS), which has three parts: Positive (P), Negative (N), and cognitive or General Psychopathology (G) was utilized by the psychiatrist to gauge the illness’s severity and the patient’s reaction to olanzapine. The General Psychopathology subscale has 16 items with a strong focus on cognition (G1–G16), while the Positive and Negative subscales each have seven items (P1–P7, N1–N7). This information is shown in Table 1. Each component is given a score between 1 and 7 according to severity, with 1 denoting absence, 2; minimum, 3; mild, 4; moderate, 5; moderate-severity, 6; severity, and 7; extremeness. A thorough definition and exact standards for each of the seven rating points are provided for each item on the PANSS. The minimum scores required by this scoring approach are 7 points for each of the Positive and Negative subscales and the cognitive symptoms had 16 points, for a total of at least 30 points. Positive, Negative, and Cognition each have maximum scores of 49, 49, and 112 points, respectively, for a combined maximum score of 210 points (Shankar and Nate 2007).

Positive syndrome is characterized by symptoms like hallucinations, delusions, and disorganized thought. Cognitive, affective, and social deficiencies, such as deflection of emotion and passive disinterest, are characteristics of the negative syndrome. Many cognitive problems, including confusion, inadequate attention, inability to understand, and purposeful avoiding of people, make up general psychopathology.

The genomic DNA was purified from 2 ml whole blood taken from schizophrenic patients and healthy volunteers using the phenol/chloroform method. According to a pervious study, the genotyping for D₂ receptor A-241G (rs1799978) polymorphisms was detected using the a two-step nested polymerase chain reaction (PCR) (Zahari et al. 2011). The procedure used an overall volume of 25.0 L, contained 200 ng of DNA template, 1.0 mM MgCl2, 0.2 mM dNTPs (Promega, Madison, Wisconsin, USA), 0.5 U of Biotool DNA Taq Polymerase (B&M Labs, Madrid, Spain), and 1 Biotool PCR buffer (B&M Labs, Madrid, Spain), and produced equal amplification of all alleles. The concentrations of the best primer (Invitrogen, California, USA) were discovered to be 0.15 to 0.40 M. Standard 0.2 mL Eppendorf PCR tubes were used for all PCRs, which were then run through an Eppendorf Mastercycler Gradient Cycler (Eppendorf, Hamburg, Germany). The initial PCR amplified a section of DRD2 from exons 3 to 4. Prior to the cycling program, at first, The DNA was denatured for two minutes at 94 °C. Thereafter, a total of 35 cycles of DNA annealing at 65 °C for one minute, extension at 72 °C for two minutes, and a final extension session at 72 °C for five minutes were carried out. To examine the PCR data, a 2.0% agarose gel (LE, analytical grade; Promega, Madison, Wisconsin, USA) stained in ethidium bromide in a 1 Tris-borate-EDTA (TBE) buffer was utilized. This process took 90 minutes at 130 V. The first PCR, which employed the primer sets, produced 276 bp-sized fragments. After the first PCR was successful, 2.0 L of the diluted PCR output was utilized as a template for a second PCR to identify wild-type or mutant alleles. The reaction mixture used for the second PCR was the same as the one used for the first. The second PCR consisted of 15 cycles with DNA being denaturized for 1 minute at 94 °C, annealed for 1 minute at 63 °C, and extended for 2 minutes at 72 °C. Following that, a 2.0% agarose gel and 1 TBE were used to analyze 10 mL of the second PCR result for 90 minutes at 130 V. The predicted fragment size of the products is 252 bp. The PCR primers sequence used for detection of the D₂ receptor gene A-241G (rs 1799978) were illustrated in Table 2.

Statistical analysis will be performed using Statistical Package for Social Sciences (SPSS 26). Descriptive statistics for the numerical data were present as the mean and standard error of the mean (Mean ± SEM) and the non-numerical data were number and %. The normal distribution of data was tested with the aid of Shapiro – Wilk test. Numerical data will be analyzed by using an independent sample T-test and a one-way ANOVA-post-hoc-LSD test. Non-numerical data will be analyzed by using the Chi-square test. The multinomial logistic regression was used to assess the association of genetic variation with efficacy of olanzapine. The P values less than 0.05 will be considered statistically significant.

The demographic data including age, gender, and BMI were assessed in both healthy and schizophrenia volunteers. In terms of age and gender, there were no real distinctions (P > 0.05) between the healthy volunteers and those with schizophrenia as show in Table 3. Schizophrenic patients demonstrated significant weight gain, as evidenced by increased BMI 29.11 ± 0.64 in schizophrenic males and 30.01 ± 0.81 in schizophrenic females compared to the healthy individuals 24.52 ± 0.57 in males and 24.89 ± 0.69 in females (P < 0.05) as shown in Table 3.

The results of genotype D₂ receptor A-241G (rs1799978) genetic polymorphism showed a clear band with a molecular size 252 bps as presented in Fig. 1. The wild allele (AA) was predominated (100%) in healthy volunteers and about (78%) in schizophrenic patient, while the heterozygous type (AG) and mutant type (GG) were only presented in schizophrenic individuals with ratio of 14% and 8% respectively as shown in Fig. 2. The frequencies of the D₂ receptor alleles rs1799978 (A-241G) were significantly different between the healthy and schizophrenic volunteers (P < 0.05) as show in Table 4. There were significantly difference among male gender of healthy and schizophrenic individuals and There were no significantly difference among female gender of healthy and schizophrenic individuals regarding three different alleles of rs1799978 (A-241G) (P > 0.05) as explained in Tables 5, 6 and Fig. 3.

Figure 1. 

Genotyping of D₂ receptor genes A-241G (rs1799978).

Figure 2. 

The Prevalence of D₂ receptor alleles A-241G (rs1799978) among volunteers.

Figure 3. 

The Prevalence of D₂ receptor alleles A-241G (rs1799978) among both male and female volunteers.

The schizophrenic symptoms were represented by PANSS score which was significantly high in schizophrenic patients who taken olanzapine and had either heterozygous (AG) allele 146.93 ± 4.91 or mutant (GG) allele 197.13 ± 4.07 of A-241G (rs1799978) as compared to those with wild (AA) allele 70.54 ± 2.46 (P < 0.05) as shown in Table 7. There were significantly difference among schizophrenic volunteers who carried heterozygous (AG) allele and mutant (GG) allele of A-241G (rs1799978) regarding the PANSS score (P < 0.05) as shown in Table 7 and Fig. 4. There were significantly associated between response of schizophrenic patient according to PANSS and G allele of A-241G (rs1799978) (95% CI = 7.098, 9.067) for patients carry heterozygous (AG) allele and (95% CI = 7.006, 9.237) for patients carry mutant (GG) allele (P < 0.05) as explained in Table 8.

Figure 4. 

The PANSS score of schizophrenic and healthy volunteers.