Background

Tuberculosis (TB) is a communicable curable infectious disease caused by Mycobacterium tuberculosis (MTB) which is a highly host adapted intracellular bacterial pathogen of macrophages. TB generally affects the lungs (pulmonary tuberculosis “PTB”), but can also affects any organ of the body (extrapulmonary TB “EPTB”) [1]. Although MTB has infected around a third of the world’s population, only 3–10% develop active disease during their lifetime [2, 3]. This could be due to complex interactions of MTB with environmental (e.g., microbial), nongenetic host factors (e.g., acquired immunodeficiency) and host genetic factors that play a critical role in TB development [4].

Tuberculosis remains a public health problem in Egypt despite the efforts to decrease its spread in the community. According to WHO report in 2019, about 10 million globally fell ill with TB where 1.2 million died [5]. The current estimated incidence of TB in Egypt is 12 (11–14) cases/100,000 population, while mortality rate is 0.43 (0.39–0.48) cases/100,000 population [5].

Various genetic polymorphisms have been found in many studies to be associated with the development of active TB; however, the results are variable between the studies [3, 6,7,8]. Interestingly, the human P2X7 (purinergic receptor P2X, ligand-gated ion channel 7) gene, which encodes the P2X7 receptor, is highly expressed on macrophages. P2X7 receptor gene contains 13 exons and is localized on chromosome 12q24 [9]. The activation of P2X7 receptor by ATP opens cation-selective channel triggers the influx of Ca2+ and Na+ and the efflux of K+ to the macrophage, thus inducing caspase cascade with subsequent apoptosis as well as activating phospholipase D promoting phagosome–lysosome fusion and elimination of MTB [10].

P2X7 is a highly polymorphic gene with over 680 known single-nucleotide polymorphisms (SNPs), 11 of which have been documented as loss-of-function mutations. One of the most common SNPs is the substitution of adenine to cytosine in exon 13 in the position 1513 (A1513C, rs3751143) which changes glutamic acid at position 496 to alanine, resulting in the complete loss of function of the protein product in homozygotes and partial loss in heterozygotes [11]. Various studies throughout the world reported that P2X7 polymorphisms contributed to the susceptibility to TB [9, 12, 13], while others found that P2X7 polymorphism did not contribute to TB susceptibility [14]. However, the results are variable between the studies which could be attributed to various factors as the studied ethnic group, the variable genomic technologies used in each study, and the lack of proper study design in some studies that need more investigation and further studies [3]. To our knowledge, there are no similar studies done in the Egyptian population aiming to identify the genetic susceptibility of the development of TB as well as minimal data available from the Arabic area (Middle East and North Africa) with their different ethnic groups [3].

Hence, we aimed to evaluate the allelic distribution of P2X7 receptor gene polymorphism 1513A/C among PTB and EPTB patients in a cohort of Egyptian population and to investigate the association between this genetic polymorphism and the susceptibility to develop active TB disease.

Methods

Study design

A case–control study that enrolled 50 patients newly diagnosed as tuberculosis (PTB and EPTB) presented to Main Alexandria University hospital, Maamora chest hospital or referred from primary care outpatients’ clinics of ministry of health of Alexandria city of Egypt as well as 25 healthy control subjects. The study was conducted in Alexandria city of Egypt, and the patients were collected between over 12 months. The study was approved by the local ethical committee of Alexandria University according to Declaration of Helsinki standards, and a consent was signed by all participants.

Study population

The study included 50 patients with new diagnosis of TB who were divided into 2 groups: 25 patients with PTB diagnosed by positive smear using direct Ziel Neelsen (ZN) staining or positive culture for TB and 25 patients with EPTB diagnosed by pathological, histochemical, bacteriological and immunological studies according to the site of the disease. The patients with family history of TB, those receiving immunosuppressive medications as corticosteroids, and those with human immunodeficiency virus (HIV) positive were excluded from the study. Regarding the control group, 25 subjects were invited to participate in the study. The control subjects were healthy, with no history of TB or evidence of respiratory disease, had normal chest x-ray and normal laboratory testing.

Characteristics of the participants

All participants were subjected to detailed history taking (including age, gender, smoking history, respiratory symptoms, comorbidities, history of old TB, drug history and family history of TB), complete clinical examination, and routine laboratory investigations (including complete blood count, liver enzymes (ALT and AST), bilirubin (total and direct), renal function (urea and creatinine) and fasting blood glucose. Adenosine deaminase (ADA) measurement in the pleural fluid was used to assist the diagnosis of tuberculous pleural effusion [15]. Plain chest x-ray was performed to all participants. Computed Tomography (CT) scan of the chest was performed if needed following the physician recommendation. Magnetic resonance imaging of the vertebral column for selected cases with suspected TB of the vertebrae was requested. Fine needle aspiration from lymph nodes or tissue biopsies from pleura, genitalia, kidney and gastrointestinal tract were performed for selected cases of EPTB according to their clinical presentation as TB was diagnosed if caseating granuloma was identified.

Bacteriological evaluation

Early morning sputum sample obtained over 3 consecutive days was collected for bacteriological evaluation. In case of no spontaneous sputum, induced sputum or bronchoalveolar lavage was performed. In case of EPTB, urine specimen was collected in case of renal TB; pus specimen in tuberculous abscess; pleural fluid in pleural TB; and cerebrospinal fluid analysis in case of meningitis. The samples were examined microscopically after being stained by ZN stain according to the criteria defined by the American Thoracic Society (ATS) in order to detect acid fast bacilli (AFB) [4]. ATS quantitation scale for AFB was considered [4]. TB culture was followed for all samples for isolation of MTB and drug sensitivity testing using BACTEC MGIT 960 system (BD life-science, New Jersey, USA). Further, samples were examined by Gene-Xpert MTB/RIF system (Cepheid smart-cycler® system, California, USA) in Maamora chest hospital for rapid diagnosis of cases and early identification of relevant Rifampicin resistance.

Genetic evaluation

Genomic DNA extraction

A blood sample was taken from all patients on admission to hospital before starting of therapy and from all healthy control subjects for P2X7 receptor 1513A/C polymorphism evaluation. All the samples were stored at − 18 °C for further analysis. DNA concentration was evaluated as µg/µl using spectrophotometer. DNA purity was measured as optical density at 260/280 nm. The readings should be greater than 1.8 for more accuracy. DNA quality and quantity of samples were assured through electrophoresis power supply EPS 500/400 (Pharmacia fine chemical, Uppsala, Sweden) on agarose gel 1.5℅ using 1 Kb DNA Ladder (Fermentas, EU) as standard.

Genotyping of the 1513 A/C polymorphism

The 1513A → C SNP was genotyped by PCR-restriction fragment length polymorphism (RFLP) with the following primers: 5′-AGA CCT GCG ATG GAC TTC ACA G-3′ (forward) and 5′-AGC GCC AGC AAG GG CTC-3′ (reverse) [16]. The PCR device used was Peqlab-Primus-25 (LabMakelaar Benelux B.V., Zevenhuizen, The Netherlands). The PCR conditions were: initial denaturation at 95 °C for 5 min; 36 cycles of 95 °C for 30 s, 36 cycles of 66.3 °C for 30 s, 36 cycles of 72 °C for 40 s, and final elongation at 72 °C for 7 min. The PCR products were digested at 37 °C for 4 h with 5.0 U of HaeII (Promega). The digested products were run on a 3% agarose gel and visualized with 10 ng/ml ethidium bromide. The PCR products were separated by electrophoresis using 15 µl of PCR product and 10 µl of Molecular marker (O´ Gene Ruler ™ 1 Kb; Fermantas, EU) as standard. The run was performed at 55 V in (Wealtac-Corp) submarine (20 × 20 cm) for 2 h. Bands were detected on ultraviolet Transilluminator, photographed by digital camera (Panasonic).

Statistical analysis

The data were expressed as mean ± standard deviation (SD) or numbers and percentage (%) for quantitative and qualitative data, respectively. Student-t test, Fisher’s exact test, and Monte Carlo test were used as appropriate. Hardy–Weinberg equation [17] was used to calculate the expected common homozygotes, expected heterozygotes, expected rare homozygotes and the frequency range of the alleles from the observed genotypes. The equation is an expression of the principle known as Hardy–Weinberg equilibrium, which states that the amount of genetic variation in a population will remain constant from one generation to the next in the absence of disturbing factors. Two-tailed p value ≤ 0.05 was considered as significant. All the statistical analysis was done using SPSS statistics (version 17.0. Chicago: SPSS Inc.).

Results

Study population

Table 1 describes the frequencies of the sites affected in EPTB patients included in the current study and their tools of diagnosis. The majority of EPTB patients had tuberculous pleural effusion (32%) followed by tuberculous lymphadenitis (20%). Table 2 shows the demographic and clinical characteristics of the participants of the studied population. The male: female ratio was 1.03:1, whereas 52% of the participants were in the 3rd decade of age. There was no statistically significant difference between the 3 groups (PTB, EPTB, and controls) regarding age, gender, and smoking history (p > 0.05, Table 2). Sixteen percent of EPTB had old TB history; 16% of EPTB and 28% of PTB patients had associated comorbidities as diabetes mellitus, hypertension, and/or chronic obstructive pulmonary disease (COPD) (p = 0.017, Table 2).

Table 1 The frequencies of various EPTB diseases among the studied population
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Table 2 Demographic and clinical characteristics of studied population
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Genetic evaluation and related correlations

Three genotypes of P2X7 1513A/C were detected: heterozygote AC genotype, homozygote wild type AA genotype and homozygote mutant type CC genotype (Fig. 1). The frequency of P2X7 1513AA genotype was significantly more prevalent in the control subjects versus all TB cases (17/ 25 patients (68%) versus 20 / 50 patients (40%), p = 0.036; Table 3). On the other hand, the frequency of P2X7 1513AC and CC genotypes was significantly higher in the all TB patients (48% and 12%, respectively) when compared to healthy control subjects (32% and 0%, respectively, p = 0.036; Table 3). Further, P2X7 1513AC genotype was significantly more frequent in EPTB (56%), while the 1513CC genotype was significantly more frequent among PTB group (16%, p = 0.048, Table 3).

Fig. 1
figure 1

1513A/C polymorphism in the P2X7 gene was detected by RFLP analysis. Heterozygote AC genotype produced products of 319, 200 and 119 base pair (bp), homozygote wild AA genotype produced digested products of 319 bp and homozygote mutant CC genotype produced digested products of 200 and 119 bp. Lane M, 100 bp marker

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Table 3 Association between P2X7 receptor 1513A/C polymorphism genotypes and the studied groups
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Considering the allelic frequency distribution analysis (using Hardy–Weinberg equilibrium), the 1513C loss-of-function allele among the TB groups (PTB and EPTB) was significantly more frequent than the controls (36% (36 alleles) versus 16% (8 alleles); p = 0.029); while the frequency of the 1513A allele was significantly higher in the control group when compared to the TB groups (84% (42 alleles) versus 64% (64 alleles); p = 0.029).

There was no statistically significant association between the 1513A/C genotypes and gender or history of old TB (p > 0.05, Fig. 2A). However, there was a statistically significant association between the P2X7 1513A/C genotypes and smoking status in x2 trend analysis (p = 0.036, Fig. 2B) as P2X7 receptor 1513AC and CC genotypes were more frequently observed in the smokers (active or former smoker) rather than the non-smokers (50% vs. 38.3% and 14.3% vs. 4.3%, respectively).

Fig. 2
figure 2

A P2X7 receptor 1513A/C genotypes frequency distribution among studied population regarding previous history of old TB; B P2X7 receptor 1513A/C genotypes distribution among smoker (either active or former smokers) and non-smoker subjects included in the study

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Discussion

In the current study, P2X7 1513AC and CC genotypes associated with partial and complete loss-of-function, respectively, were significantly frequent in active TB cases either PTB or EPTB. This polymorphism was significantly associated with active smoking status but not with history of old TB or gender. To our knowledge, this is the first study investigating the association between P2X7 genetic polymorphisms and susceptibility to different clinical forms of active tuberculosis in a cohort of Egyptian population.

Singla et al. [18], who studied two genetic polymorphisms of P2X7 gene (− 762T/C and 1513A/C) in an Indian population, found a significantly higher frequency of C allele in the disease groups than in the control group with a significant difference in the PTB versus EPTB. Similarly, Nino-Moreno et al. [16] found a significant association between P2X7 gene 1513A/C polymorphism rather than − 762T/C and NRAMP1/SLC11 A1 gene polymorphisms and the susceptibility of PTB in a Mexican population. Wu et al. [19] and Mokrousov et al. [20] found that the P2X7 gene 1513A/C polymorphism was significantly higher in PTB than the control group in Chinese and Slavic Russian populations, respectively. Further, Ben-Selma et al. [21] found that the frequency of 1513CC and AC genotypes were significantly more frequent in the EPTB group than in the control and PTB groups in Tunisian patients. Similarly, Fernando et al. [22] and Sharma et al. [23] found a significant association between the 1513C allele and active EPTB rather than PTB in Asian population. These results are in accordance with our current results and support the association between P2X7 gene 1513A/C polymorphism and various forms of active TB infection in different ethnic groups.

TB patients with P2X7 gene 1513AC heterozygous genotype had significant impairment in macrophages’ ability to kill Mycobacterium Bovis BCG after ATP stimulation [22], while this ATP-mediated mycobacterial killing ability was totally ablated in macrophages obtained from P2X7 gene 1513CC homozygous patients with TB emphasize the complete loss of P2X7 function in leukocytes [24, 25]. Further, a deficiency of P2X7-mediated control of MTB infection within pulmonary macrophages may permit spread to extrapulmonary sites where the infection either progresses to post-primary TB disease, or is controlled by the emerging specific T-cell response. With later waning of T-cell immunity, reactivation of latent TB may result in increased frequency of EPTB in subjects with nonfunctioning SNPs in P2X7 [9, 22]. Additionally, signaling through P2X7 plays a more important role in the control of reactivated TB infection than in the initial containment of MTB within the lung. After reactivation of dormant bacilli, the absence of P2X7-mediated killing of mycobacteria in pulmonary macrophages could then result in spread to extrapulmonary sites [26].

On the other hand, Ozdemir et al. [27], Xiao et al. [28] and Li et al. [29] did not find significant associations between the P2X7 receptor 1513A/C polymorphism and different clinical forms of TB in Eastern Turkish population, Chinese Han population and Gambian population, respectively. This could be explained on the basis of various factors. Firstly, the ethnic origin of patients; it has been reported that ethnic-specific genetic variations may influence host immunity to TB, causing different tuberculosis susceptibilities [2] as we found low allele frequency of the 1513C allele in our Egyptian cohort as an example of African population. Secondly, sample size which may affect statistical calculations. Lastly, as genetic susceptibility to tuberculosis is polygenic, the other functional SNPs occurring in the P2X7 gene (as − 1269T/G, − 1140 C/T, − 838 C/T, − 762T/C and − 298 G/A) may be associated with susceptibility to active tuberculosis [29, 30]. In addition, interactions with other polymorphisms may be of great importance, at least in some populations, as variations in NRAMP1, MBL, VDR, P2X7 receptor, IL-10 and IL-1 receptor antagonist (IL-1Ra) are all significantly associated with TB susceptibility [2, 31, 32].

Further, Shemon et al. [33] described an association between the loss P2X7 receptor function in Thr357Ser homozygous polymorphism (located in gene 1096C – > G) in Caucasian population which led to impairment of ATP-induced apoptosis and MTB killing in macrophages. This Finding provides robust support for the concept of low P2X7 function in mediating TB susceptibility other than 1513C P2X7 polymorphism. Fairbairn et al. [34] also highlighted the role of P2X7 in lysosome–phagosome fusion in macrophages and TB bacteria killing through ATP stimulation; however, blocking of macrophage-phosphodiesterase D activity resulted in macrophage death but not TB bacteria.

Interestingly, P2X7 receptor 1513AC and CC genotypes were significantly frequent observed in the smokers rather than the non-smokers, suggesting that P2X7 receptor gene polymorphism could be associated with smoking. Tobacco smoke was shown to reduce alveolar macrophage activity, induce immune-depression of pulmonary lymphocytes, reduce the cytotoxic activity of natural killer cells, induce alteration of the activity of the pulmonary dendritic cells and mucociliary clearance [35], and may cause changes in chromosomes that affect gene activity and expression [36]. Further, active and passive smoking have been found to increase the risk of latent TB infection in previous study [37], increase PTB severity and relapse of both PTB and EPTB after treatment [38].

On the other hand, we found no statistically significant association between P2X7 receptor 1513A/C polymorphism and previous history of old TB. However, Fernando et al. [22] found that 35% of latent TB patients developed active EPTB disease and had a strong association with the P2X7 receptor 1513C allele.

We believed that the current results could be translated in the clinical care of TB especially among those with latent TB whom represent the majority of the infected TB patients [39]. Further, we thought that effective smoking cessation programs could be helpful in decreasing the genetic polymorphism and guard against development of active tuberculosis disease.

The current study has some limitations. Firstly, we did not analysis other genetic polymorphisms that could be linked also to disease susceptibility and affected with associated P2X7 receptor gene polymorphism. Secondly, the sample size was relatively small and coffined to one geographical area of Egypt. However, this is an early pilot study with promising results that could be a step for further investigation. Further, a meta-analysis is also still recommended to compare the current results with previous studies in the literature.

Conclusion

P2X7 gene receptor 1513A/C polymorphism positively associated with tuberculosis susceptibility in a cohort of Egyptian population. This polymorphism could be linked to ethnicity and exposure to smoking rather than previous history of TB; however, further studies with large scale are still required to confirm this association.