This study recruited 52 unrelated patients (27 males and 25 females) who attended public and private psychiatry clinics in Amman and Irbid City of Jordan. The patients were diagnosed as having OCD by a psychiatrist or a licensed mental health specialist. A healthy control group, matched by age and gender, consisting of 27 males and 27 females, was included. None of the control participants had clinical evidence of OCD, family history of OCD, or other autoimmune disorders. Participants who volunteered to participate were informed about the study's goal and significance. Subjects were assured that the collected information would be safe and available only for the research purposes and not to be accessed by anyone except the researchers. The individuals were also notified of their rights to drop out of the research.

Each competent participant was allowed to hear and sign an informed consent before participating in the study. Each participant was assigned a code number for transcription. A specially designed questionnaire was created, and a direct interview was conducted with each patient and control by a researcher trained for this purpose. The questionnaire included information on demographic characteristics (gender and family history) as well as lifestyle habits, severity of the disease, and response to medication ). All participants’ questions were answered honestly and completely. Given the topic's sensitive nature, participants were protected since the questionnaire did not ask for any identifiable data to ensure that the personal information remained concealed.

Strict criteria for inclusion and exclusion were used in this study to meet the following inclusion criteria: (1) All the OCD-affected patients were between 16 and 55 years old and (2) the patient is diagnosed pure contamination OCD, which is one of the most usual subtypes of OCD. It is identified by fear of becoming contaminated with something and compulsive behaviors that are played to remove the risk of contamination that intervenes with the patient’s daily routine.

Exclusion criteria included: (1) any OCD symptoms including harm or checking, unacceptable thoughts or mental compulsions, symmetry, or ordering, or other subtypes was excluded, (2) any serious disease or genetic syndromes and the absence of well-documented medical records, (3) patients diagnosed with dual diagnosis (OCD and any severe disease or genetic syndromes) were excluded, and (4) the inability of a patient to respond to questions of the survey.

Early studies on the genetics of OCD concentrated on candidate gene association studies [29]. Until recent times, Genome-wide association studies (GWAS) of OCD have not disclosed any genome-wide significant SNPs connected with this disorder in case–control analyses. The SNPs investigated in this research were selected based on potential cytokine genes for OCD from a few studies available in the published literature [15, 30, 31]. IL-1β, IL-6, and TNF-α are among the most widely studied cytokines in OCD [15]. Some functional polymorphisms in the promoter region of the TNF-α gene have been described to be associated with OCD vulnerability [28, 32].

Figure 1 presents a list of cytokine candidate genes and the polymorphisms that have been chosen for them, together with the SNPs, reference SNP ID numbers (rsIDs), chromosomal placements, and strength of relation with OCD [19].

Schematic representation shows the chromosome location, cytokine gene under study, the relative position of the SNP, and its reference ID number

A venous blood sample (3 ml) was obtained from each individual in a sterile vacutainer tube containing EDTA. In the field, the blood samples were immediately placed on ice directly after their collection.

The genomic DNA was extracted by using a Blood DNA Preparation-Solution Kit (Jena Bioscience, Germany) according to the manufacturer’s protocol. The concentration and purity of the extracted samples were evaluated by spectrophotometry on a Multiskan SkyHigh Microplate Spectrophotometer using the µDrop™ Plate (Thermo Fisher Scientific, USA). The pure DNA was eluted with 100 μl Nuclease Free Water (NFW, Sigma-Aldrich, USA) and stored at − 20 °C until use.

The genomic region containing the polymorphic site at a specific position of the corresponding cytokine gene was amplified using specific primers under the conditions specified in Table 1. Each cytokine's DNA fragment was amplified under the manufacturer's recommendations. Amplification was carried out by Swift™ MaxPro Thermal Cycler (Esco, Singapore). The PCR mix was prepared using 15ul GoTaq® Green Master Mix, 2X (Promega, USA), 10.5 ul (NFW), 1.5 ul (5 μmol) of each primer designed by IDT (Integrated DNA Technologies, USA), and 1.5 μl volume of 30 µL, 1.5 µL (50 to 200 ng) genomic DNA.

The specific amplicon (PCR product) digestion process was performed with a corresponding thermostable restriction enzyme (New England Biolabs, USA). Table 2 summarizes the resulting PCR DNA fragments after digestion by the specific restriction enzyme according to the manufacturer’s instructions for RFLP. A total volume of 20 ul reaction mixture consists of 12 ul PCR product, 5 ul NFW, 2 ul of 10X reaction buffer, and 1 ul restriction enzyme.

The restriction pattern was identified based on band size observed on agarose gel (at 90 V for 60 min) under UV light after staining with ethidium bromide. A corresponding DNA marker (50 bp) (GelPilot® DNA Molecular Weight Markers 50 bp (QIAGEN, Netherlands) was simultaneously included as a reference. Undigested control PCR product reaction mixtures with ddH2O substituted for the restriction enzyme. The gels were visualized with 0.5 μg/ml ethidium bromide (in 1X TAE (Tris–acetate-EDTA) buffer at 90 V for 1 h), and photographs of the gels were scanned and analyzed.

According to the manufacturer's recommendations, the digestion endonuclease cuts one of the two alleles. Genotypes were designated as follows: Homozygous mutant lost the restriction site for the endonuclease and thus remained a single undigested band, homozygous wild-type had one restriction site for the restriction enzyme and produced two-sized fragments, and heterozygous mutant had three fragments. To assess the reliability of genotyping, we performed double sampling RFLP-PCR in more than 10% of the samples. Two different readers interpreted the results, and no differences were found.

For technical reasons, we could not genotype the TNF-α gene. Instead, the DNA of the TNF-α gene from 35 patients (17 males and 18 females) was sequenced by the Sanger technique. A specialized technical company (Enzyme Company, Irbid/Jordan) carried out the direct Sanger sequencing protocol. Briefly, DNAs were amplified as described above. The PCR product was cleaned up for rapid turnaround times and to improve the efficiency of resource use, while delivering the same superior cleanup to eliminate excess NFW and unpaired primer. The sequencing cleanup step was accomplished following the protocol recommended by the manufacturer of the Biosystems™ BigDye XTerminator™ Purification Kit (Thermo Fisher Scientific, USA) and Terminator™ Purification Kit (Thermo Fisher Scientific, USA).

The sequencing step, the biosystems™ BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA), was applied according to the manufacturer’s protocol. After that, the reaction was sealed, spun down, and run in the thermal cycler at 4 °C overnight. Finally, capillary electrophoresis was carried out by the Applied Biosystems SeqStudio Genetic Analyzer (Thermo Fisher Scientific, USA). The obtained data were analyzed, and peaks were shown using the chromasPro 1.5 software. The nucleotide changes and the genotype frequencies were obtained from the discrete electropherograms formed for each of the SNPs. The homozygote showed a single peak on the electropherogram, while the heterozygous genotype revealed a double peak.

Each procedure in this study was repeated twice. Data analysis of data was performed by SPSS 25.0 statistical software (SPSS Inc., Chicago, IL, USA). The results were presented as mean ± standard deviation (mean ± SD) and percentages. Normality and homogeneity assumptions were tested and checked to be significant, and hence, the parametric tests were used. Before further genetic analysis, deviations of the observed frequencies of alleles and genotype distribution of cytokine genes in different groups from the HWE test using a goodness-of-fit Chi-squared (χ2) method with 1 degree of freedom. The strength of the association between cytokine gene polymorphism and the risk of OCD occurrence was assessed by the odds ratio (OR) with the corresponding 95% confidence interval (CI). The ORs and 95% CIs were calculated using logistic regression. The clinical variables related to gender were correlated and calculated for patients with different alleles and genotypes. Pearson’s two-tailed test and correlation were used to evaluate statistical significance. Probability (P) values of ≤ 0.05 were considered statistically significant.