Ankylosing spondylitis (AS) is a progressive, chronic inflammatory illness with an unclear etiology ¹ . Prolonged inflammation can cause vertebral fusion, which may result in immobility ² . Clinically, the sickness manifests as pain and increasing rigidity of the spine ³ . The hallmarks of AS are inflammation of the sacroiliac joints (SIJ) and spine ⁴ . AS is directly proportional to the prevalence of human leukocyte antigen (HLA)-B27 in a given population, and has been estimated to range between 0.1 and 1.4% in various parts of the world ⁵ . In Iraq, the estimated frequency of AS is 0.9 per 100,000, while HLA-B27 positivity was elicited in 55% of the population investigated ⁶ . In Arab people, between 2% and 5% of individuals are HLA-B27 positive, which is prevalent in 64% of AS patients ⁷ . TNF-α is a highly potent pro-inflammatory molecule and a critical component of the immune system's signaling cascade that is strongly activated in response to infection or tissue injury. Patients with AS, in particular, respond well to TNF antagonist therapy ⁸ . Etanercept (ETN) has been examined in various rheumatologic disorders ⁹ . ETN is the only soluble TNF receptor the Food and Drug Administration (FDA) has licensed for therapeutic use. It is typically administered in weekly doses of 50 mg subcutaneously (or 25 mg twice weekly), either by self-injection or by a caregiver ¹⁰ .

Since the promoter region of the TNF-α gene would seem to be remarkably polymorphic, genetic variation is one of the most significant factors in the response ¹¹ . In contrast to numerous aspects that could potentially influence or change the ETN response, genetic manifestations do not change during a patient's lifetime ¹² . Consequently, polymorphism testing assists in dividing individuals with just a high response from those with an inappropriate or poor response. Accordingly, early identification of individuals who would not adhere to biological treatments allows a quicker transfer to another type of medicine, increasing the patient's likelihood of achieving therapeutic purposes more quickly ¹³ . The goal of pharmacogenomic and pharmacogenetic research is to use available knowledge about gene variants that influence the medication response to develop personalized treatment methods that enhance therapeutic effectiveness and safety ¹⁴ . Numerous single nucleotide polymorphisms (SNPs), primarily in the TNF promoter, are susceptible to rheumatoid arthritis (RA) and spondyloarthritis, and related to tumor necrosis factor inhibitor (TNFi) response. Genetic variations in cytokine genes may affect cytokine gene transcription and secretion, modulating cardiovascular disease development risk ¹⁵ . In addition, these promoter SNPs are thought to contribute to treatment responses by modifying TNF gene expression ¹⁶ . Identifying pharmacogenetic markers, enabling treatment only for those patients who will respond without the risk of unwanted effects, would considerably improve treatment efficacy and reduce expenses ¹⁷ . Previous studies have not examined the impact of the rs4248158C/T polymorphism on AS patients' response to ETN. No prior research has been conducted in Iraq to explore the influence of SNPs within the TNF-α promoter region upon the propensity to respond to ETN in AS patients. This current research study aimed to determine whether the presence of SNPs in the promoter region of the TNF-α gene at positions -1031T/C (rs199964), -857C/T (rs1799724), and -806C/T (rs4248158) in a sample of Iraqi AS patients could influence the patients' outcomes with ETN.

Demographic data and clinical characteristics variables of the research participants are expressed in Table 3 and Table 4.

As shown in Table 5, there was a high prevalence of CC genotype for both rs1799724 C/T and rs4248158C/T in contrast to a low proportion of mutant C allele (2%) in rs1799964T/C. At the same time, the wild T allele in rs1799964T/C was more frequent and predominant in over half of patients (73%).

Furthermore, highlighting the disparity in genotypes frequency between responder and non-responder groups, the analysis of this study showed a significant difference in the TT genotype of rs1799964 (p-value = 0.02) in the responder group, in contrast to the TC genotype of rs1799964, which was significantly more frequent in the non-responder group (p-value = 0.01), as revealed in Table 6.

Table 7 shows the significant and non-significant association with each studied SNP in the TNF-α promoter regain gene by binary logistic regression. The TC-rs1799964 genotype significantly reduced the tendency to be a responder to ETN (OR=0.208, 95%CI:0.057-0.762, p=0.018); this indicates that the heterozygote TC of rs1799964 is a predictor for non-responsiveness to ETN in AS patients.

Similarly, the phi correlation coefficient (phi) analysis was shown to verify the strength of association between each genotype and the predisposition to be a responder to ETN. For example, Table 8 shows that the homozygote wild TT genotype of rs1799964 seemed to enhance the probability of being a responder to ETN in AS patients. Nevertheless, the heterozygote TC genotype of rs1799964 showed a negative and significant correlation for responsiveness to ETN.

AS is a chronic inflammatory condition with an unknown cause. In contrast to other systemic autoimmune diseases, the innate immune system plays a dominant role in AS, which is characterized by the aberrant activity of innate and innate-like immune cells, such as T cells, group 3 innate lymphoid cells, neutrophils, mucosal-associated invariant T cells, and mast cells, at disease-prone sites ²³ .

The first biological DMARDs considered second-line therapy are TNFi. Adalimumab, certolizumab pegol, ETN, golimumab, and infliximab, as well as its biosimilars, are now indicated. Anti-TNF medication is quite successful in AS, with significant improvements in disease activity and functional scores following six months of treatment ²⁴ . These drugs should be used according to their indications, contraindications, and patient comorbidities. No TNFi are recommended for preferred efficacy ²⁵ . In large, randomized, controlled clinical trials, TNF-α inhibitors have shown efficacy and are well-tolerated. However, a considerable minority of individuals do not respond to these medications, and their use may be associated with severe adverse drug reactions.

It is worth mentioning that the most reported adverse drug reactions to biological drugs in a study performed in Iraq were related to general disorders and administration site conditions, followed by skin and subcutaneous disorders, respiratory disorders, and gastrointestinal disorders ²⁶ . In the current research, the mean age in the responder and non-responder groups was not significantly different (p = 0.559). The study results revealed that there was a significant difference (p < 0.005) in gender distribution, in which the percentage of males in the responder group was 97%, whereas it was 70.4% in the non-responder group, while the percentage of females in the responder and non-responder groups was 3% and 29.6%, respectively; this result is consistent with various previous studies ^{27– 29} .

The serum TNF-α and high-sensitivity C-reactive protein (hs-CRP) levels were also shown to vary significantly between the responder and non-responder groups. The mean BASDAI (4.8 ± 1.1) and BASFI) 5.0 ± 1.4) of the non-responder group treated with ETN was higher than the responder group, and this difference was statistically significant (p-value < 0.001). Additionally, the mean serum level of ESR after treatment with ETN was higher in the non-responder group (31.2 ± 17.5) than the responder group (16.8 ± 10.9), and this difference was statistically significant (p-value < 0.001). This finding is close to the result of a previously performed study by Albagoa et al. ³⁰ .

Prior studies conducted in Iraq examined the association between genetic variants in different genes and the prevalence and severity of AS ^{31– 33} . This observational study is the first study in Iraq that has analyzed the association of SNPs in the TNF-α promoter region gene -1031T/C (rs1799964), -857C/T (rs1799724) and -806C/T (rs 4248158) with outcomes of treatment in 60 Iraqi patients with AS taking ETN.

Numerous global researchers have investigated the influence of SNPs in the different genes on the TNFi response ^{34– 37} . Regarding the frequency of the prevalence of -1031T/C (rs1799964) in all AS patients, the present study showed that the wild TT homozygote in rs1799964T/C was more frequent and predominant in over half of patients (73%), followed by heterozygote TC (25%) and a low proportion of mutant CC homozygote (2%). Moreover, the T allele was found in over 85% of patients, but the C allele was present in only about 14% of patients. There has been no previous research conducted in Iraq that closely examined -1031T/C on AS or any other disease to match it with; hence, the outcomes seemed to be equivalent to those of Han Chinese and Korean people with AS, demonstrated in previous research.

This result is consistent with the Sheng et al. ³⁸ study, where the TT, TC, and CC genotypes were found in approximately 76%, 19%, and 4% of AS patients, respectively, with the proportion of T allele to C allele being 86 % and 14%, respectively. Similarly, Chung et al. ³⁹ . showed a higher proportion of wild TT homozygote genotype in about 59 patients, followed by 18 patients with the TC heterozygote genotype and no patients with the CC genotype. Regarding the difference in -1031T/C (rs1799964) genotypes frequency between responders and non-responders, the analysis of this study showed a significant difference in the TT genotype of -1031T/C polymorphism (rs1799964)( p-value = 0.02) in the responder group, in contrast to the TC genotype, which was significantly more frequent in the non-responder group (p-value = 0.01), while there was no significant difference in the availability of the CC genotype between responder and non-responder groups. Similar findings were seen in a study by Tong et al ^{. 40} , who observed that the TT genotype had a better treatment outcome compared with the CT and CC genotypes.

The current research findings could be attributed to the fact that the availability of the -1031TT genotype diminished TNF-α concentrations in the serum. Regarding the frequency of the prevalence of -857 C/T (rs1799724) in all AS patients, the current data show that there was a high prevalence of CC genotype, occurring in more than half the patients. Besides that, the C allele was found in over 80% of patients, but the T allele was present in only about 18% of patients.

This result was in agreement with a study by Mohammed et al. ⁴¹ , which covered 80 Iraqi RA patients and revealed the prevalence of the CC genotype, with the C allele found in 81.25%. In contrast, the T allele was found in only about 18% of participants. Similarly, an Italian study by Aita et al. ⁴² reported a high frequency of CC genotype (60%) compared with TC and TT genotypes in AS patients. In addition, the current investigation indicated that both CC and CT genotypes were distributed equally across study patients.

There was no significant difference statistically between the respondent and non-respondent groups, and without correlation with any response to ETN. The outcome seems to agree with the result of a previous study by Mohammed et al. ⁴¹ . However, the current findings disagree with the Chinese Han study by Tong et al., which revealed that -857CC genotypes can predict positive prognosis responsiveness to TNF-α inhibitors ⁴⁰ . Indeed, a meta-analysis of a variety of ethnic groupings highlighted a relationship between the -857 C allele and the sensitivity of TNF-inhibitors in Caucasians, which was not observed in Asians ³⁵ . Put another way, many investigations indicate that TNF-α -857CC improves the responsiveness to treatment (ETN), despite contradictory and insignificant evidence for this relationship ⁴³ .

This disparity in results comparing the current study to earlier research can be associated with the rarity of the T allele in patients with AS in Iraq who participated in the current research and may be because of genetic heterogeneity and different environmental factors in different populations. In the instance of -806C/T genotypes, the results of this investigation demonstrated that the CC genotype was widespread, in more than 90% of patients, followed by the CT genotype in about 8% of participants. More than 95% of patients carried the C allele, while just 4% carried the T allele.

This is the first study to examine the genotyping of -806C/T in AS patients from Iraq. Nevertheless, the results were equivalent to those from investigational research on the hazards of intracranial aneurysms among Chinese people ⁴⁴ . Furthermore, addressing the distinction in genotype frequency between the study groups, an analysis of this study showed a non-significant difference in CC and CT genotypes between the two groups. Similarly, while the CC genotype showed a positive relationship and the CT genotype showed a negative relationship, neither of them reached the statistical significance level. It is noteworthy that ours is the first study to investigate the association between the TNF-α -806C/T polymorphism and response to ETN in patients with AS. To investigate whether SNPs in the promoter region of the TNF-α gene alter the severity of harmful effects from ETN use, we looked at the relationship between genotype and the occurrence of these adverse events: infection, injection site reaction, headache, and rash.

These three SNPs were compared for each genotype between non-adverse and adverse events cases. The most frequently reported side effects were injection site reaction (21 patients, 35%), followed by infection (28.3%), then headache and rash, with an incidence of about 38%. The incidence of short-term and long-term adverse effects linked with TNF-blocker medication in Chinese Han patients with AS was investigated by Tong et al. ⁴⁵ , and they discovered that disease duration, ESR, and CRP levels in the serum were related to an increase in side effects when using TNF-blockers. Chronic therapy with infliximab correlated with more side effects than rhTNFR-Fc.

Moreover, Chou et al. ⁴⁶ found no severe adverse events in a prospective, open-label trial of ETN, involving 46 patients with AS from 60 Taiwan medical centers. However, SNPs investigated in this study cannot effectively predict the occurrence of drug-related adverse events. There are significant limits to the generalizability of our study's findings. First, the limited sample size is a drawback of this study. Specifically, even though ETN is a highly effective treatment for AS, its expense and stringent inclusion criteria are frequently the primary reasons why published observations in AS patients, as well as our study, typically have a small sample size. Next, the present research was conducted at one institute, and, while our institute serves Iraqi people from many cities, we recommend that further studies may be necessary prior to expressing the results among all Iraqi AS individuals.. Lastly, a tiny proportion of cases solely received ETN because of inadequate budget and resources. Regardless, it would be intriguing to increase the sample size to validate the result. Indeed, the small sample size and differences in genetic background amongst ethnic populations may explain the few contradictory findings.

The presence of the TT genotype of rs1799964 is associated with a higher likelihood of responding to ETN, suggesting that it is a useful diagnostic for predicting response in Iraqi AS patients. In contrast, the TC genotype of rs1799964 greatly increases the risk of becoming an ETN non-responder. These results suggest that the TC genotype of rs1799964 should be investigated in AS patients prior to delivery of ETN.

Ethical approval was obtained from the Scientific and Ethical Committee at the College of Pharmacy, Baghdad University, Iraq (approval number: RECACPUB-3102020D). This research was conducted in line with the Helsinki Declaration ¹⁸ .

Before data collection, written consent was obtained from each of the participants.