Introduction

AA is an infrequent, life-threatening problem of haemopoiesis characterized by pancytopenia and hypocellular bone marrow in the absence of an abnormal infiltrate, with no increase in reticulin [1,2,3]. According to the modified Camitta classification criteria for diagnosis, AA is clinically classified into three types based on the severity of the disease; severe aplastic anemia (SAA), non-severe aplastic anemia (NSAA) and very-severe aplastic anemia (VSAA) [4]. The incidences of AA vary geographically, and the precise incidence of AA in India is not well known due to lack of epidemiological studies. There is a biphasic age distribution with peaks between the ages of 15 and 25 years and a second smaller peak in incidence was noted after 60 years, with no significant difference in incidence between males and females [5].

The IL-1family comprises three related genes, namely IL-1α, IL-1β and IL-Ra, which encode the pro-inflammatory cytokine IL-1α and IL-1β and their receptor IL-1R [6, 7]. The IL-1 plays a major role in inflammation-mediated autoimmune diseases. The IL-1Ra is a natural anti-inflammatory molecule that neutralizes the pro-inflammatory effect of IL-1β, and thus plays a role in maintaining homeostasis [8]. It might be possible that polymorphism in IL-1β-511 and IL-1β-3954 may lead to changes in the production of IL-1β, hence disturbing the homeostasis and making individuals more susceptible towards developing AA. 1L-1β is a cytokine protein which is encoded by the IL-1β gene in humans and has a molecular weight of 17.5 kDa [9]. It is known to be a strong proinflammatory cytokine with multiple biological effects including cell proliferation, differentiation, and apoptosis [10]. It exists in three polymorphic forms IL-1β-31C/T, IL-1β-511T/C and IL-1β-3954 C/T [11, 12].

It is evident from various studies that cytokine genes are polymorphic in nature [13]. The promoter sequence is a potential source of polymorphism affecting gene expression. Several single nucleotide polymorphisms (SNPs) have been reported in the regulatory region of cytokine genes, and some of them were associated with altered gene expression [6]. Various studies from across the world have shown that genetic polymorphism plays an essential role in development of AA [14, 15]. However, genetic associations found in one population need not necessarily hold true in another population with a different ethnic background. Therefore, in the present study, we identified three isoforms of IL gene (IL-1β-31 C/T, IL-1β-511T/C and IL-1β-3954 C/T) and their occurrence, which might increase susceptibility towards developing AA [16].

Materials and Methods

Study Subjects

The present study involved two subject groups: cases and controls. The case group included 120 AA patients who were enrolled irrespective of their age and gender, at the Department of Clinical Hematology, King George's Medical University (KGMU) Lucknow, Uttar Pradesh, India. The control group included 120 healthy individuals not suffering from any disease during the same period. The control group included both the subjects from the general population and those from the hospital.

Data Collection

The data pertaining to all the AA patients includes various clinico-pathological parameters, demographic variables, and these were obtained and evaluated from the patient medical records, pathology reports, questionnaires and also from the personal interviews with the patients and their guardians (for those who were illiterate or unable to communicate). All the patients and their guardians were informed about the study and their willingness to participate in this study was documented using a predesigned questionnaire, and the same procedure was followed for the controls. All the procedures pertaining to the study subjects including sample procurement and the data collection were carried out in accordance with the ethical standards laid down by the Institutional Ethics Committee, KGMU, India [17].

Patients and Clinical Examination

The diagnosis, classification (severe AA, non-severe AA and very-severe AA) and the response assessment of AA were made according to the standard guidelines [4, 18, 19]. Samples were collected during March 2015 to March 2018 from 120 cases and equivalent number of controls after obtaining their written informed consent. For immuno suppressive therapy (IST) out of thetotal of 120 patients included for this study, (21) 17.50% received Anti-Thymocyte Globulin + cyclosporine (ATG + CsA) and 99 (82.5%) received cyclosporine only and follow up was conducted every month for 1 year.

Sample Preparation and Genotyping

Five ml of peripheral blood was collected by venipuncture, under aseptic condition, from each individual of the case and control group in ethylene-diamine-tetra-acetic-acid (EDTA) tubes (ADS Hitech Polymers) and stored at − 80 °C until further use [20]. The genomic DNA was extracted from the blood specimens using Qiagen Blood DNA Mini Kit, Hilden, Germany according to the manufacturers’ instructions. The extracted DNA was stored at − 20 °C for further use. The qualitative analysis of the extracted genomic DNA samples was carried out by a UV–visible spectrophotometer (O.D. at 260 nm and 280 nm) and the quantitative analysis by agarose gel electrophoresis.

PCR amplification was carried out in a final volume of 20 μl (3 μl DNA, 10 μl Top Taq PCR Master Mix, 1 μl of forward and reverse primer and 5 μl distilled water). The PCR reaction was carried out in the DNA thermal cycler (Eppendorf® Mastercycler® Nexus Thermal Cyclers, Hamburg, Germany) [21]. The computerized thermal cycler was programmed for the following conditions:

  • IL-1β-31C/T polymorphism denaturation at 94 °C for 10 min, then 30 cycles at 94 °C for 30 s, 50.2 °C for 45 s, 72 °C for 45 s, and finally 72 °C for 10 min.

  • IL-1β-511T/C polymorphism annealing 49 °C for 45 s (rest all other conditions were similar)

  • IL-1β-3954C/T polymorphism annealing 54 °C for 45 s (rest all other conditions were similar).

Digestion of the Amplified Product by Specific Restriction Enzyme for Each Polymorphism

Amplified PCR product (10 μl) mixed with (1 μl) restriction enzyme (New England Biolabs, UK) was used in the reaction. The reaction mixture was incubated for 2 h at 37 °C. The digested products undergo gel electrophoresis in the range of 1.5–3%. Further, the separated fragments were stained with EtBr and visualized along with ladder using the molecular imager gel doc XR System (Bio-Rad, Hercules, CA). The details of restriction enzymes and their resulting base pair length are shown in Table 1.

Table 1 Genotyping information of IL-1β-31 and IL-1β-511 and IL-1β-3954 single nucleotide polymorphisms
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Measurement of Plasma IL-1β

Venous blood was collected in commercially available EDTA tubes. Plasma was separated using a refrigerated centrifuge for 15 min at 2000 × g. Plasma of IL-1β level was determined by ELISA kit (Bioassay Technology Laboratory).

Data Analysis

Allele counting method was used to determine the allele and genotype frequencies. Odds ratio (OR) was determined and an assessment of confidence intervals (95%) was made. All these statistical calculations were carried out using software (Graph Pad Software ver. 3.05, San Diego, USA). p value found to be (< 0.05) was considered significant. To better define the association between the genotype and aplastic anemia, three genetic models (dominant, over-dominant, recessive) were analyzed. In the dominant model, wild type homozygous was compared with the pair of mutant homozygous and heterozygous. In genetic models, the over dominant model includes wild type homozygous plus heterozygous that was compared with homozygous mutant type. In the recessive model, homozygous type mutant was compared with the pair of wild type homozygous and heterozygous.

Results

In this study 240 participants (patients-120 and healthy control-120) were enrolled. The male to female ratio in AA patients was 70:30 and control was 51:49. The percentage of AA patients with severe, non-severe and very-severe was 44.2, 46.7 and 9.1 respectively. Demographic details of aplastic anemia patients and those of healthy controls are given in (Table 2).

Table 2 Demographic detail of acquired aplastic anemia patients and healthy control subjects:
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Occurrence of IL-1β-31, IL-1β-511 and IL-1β-3954 Polymorphisms and their Susceptibility to Developing AA

IL-1β-31polymorphismshowedhigher susceptibility to develop AA in over-dominant model (CC + TC vs TT), but was not significantly associated in any of the model with AA. The genotypic variants IL-1β-511did not show any significant susceptibility, although high occurrence was noticed inmutant variant (CC) and recessive model (CC vs TT + CT) of IL-1β-511 was found in AA cases (Table 3). The occurrence of homozygous (TT) variant of IL-1β-3954 had significantly higher susceptibility to developing AA when compared with the healthy controls (OR = 2.35, 95% CI 1.10–5.00, p = 0.03) (Table 3). Furthermore, the occurrence of mutant allele of IL-1β-3954 showed a higher risk of developing AA. Table 3 depicts the genetic models of polymorphisms out of which a IL-1β-3954 showed significant protective association in the over dominant model (CC + CT vs TT), (OR = 0.39, 95% CI 0.19–0.81, p = 0.01) (Table 3). However, recessive model (TT vs CC + CT) was also found to be significantly associated with the disease (OR = 2.5, 95% CI 1.22–5.11, p = 0.01).

Table 3 Genotype and allele frequencies of the IL-1β-31, IL-1β-511 and IL-1β-3954 cytokine gene among acquired aplastic anemia cases and controls and their risk associations with acquired aplastic anemia
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Occurrence of IL-1β-31, IL-1β-511 and IL-1β-3954 Polymorphisms in Non-severe, Severe and Very-Severe AA

The mutant variant ofIL-1β-31showed higher risk with the severity index of SAA and NSAA patients when compared with those of controls (Table 4). The frequency of homozygous mutant (TT) and heterozygous (CT) variant of IL-1β-511 showed significant association with the NSAA (OR = 0.31, 95% CI 0.12–0.76, p = 0.018and OR = 0.88, 95% CI 0.43–1.82, p = 0.002) (Table 4). The recessive model (TT vs CC + CT) of IL-1β-511showed significant correlation with disease severity in SAA and VSAA patients compared with healthy controls (OR = 3.01, 95% CI 1.31–6.87, p = 0.007 and OR = 4.7, 95% CI 1.21–18.27, p = 0.03) (Table 4). The heterozygous variant (CT) of IL-1β-3954 showed a higher risk association with VSAA patients, but was not found significant (Table 4).

Table 4 Genotype distribution of the IL-1β-31, IL-1β-511 and IL-1β-3954 cytokine gene polymorphism among severe, non-severe and very-severe type of aplastic anemia versus control
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Occurrence Of IL-1β-31 , IL-1β-511 And IL-1β-3954 Polymorphisms With Response To Immunosuppressive Therapy

No significant association was found between IL-1β-31, IL-1β-511 and IL-1β-3954, with response to IST in any of the groups (Table 5).

Table 5 IL-1β-31, IL-1β-511 and IL-1β-3954 genotype distribution in responder (complete + partial responder) and non-responder
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Plasma Concentration of IL-1β in AA and in Controls

IL-1β level was elevated in blood plasma of 80.95% (34/42) of AA patients (3.37 ± 0.56 pg/ml) as compared to (p < 0.001) control subjects. Maximum increase in IL-1β concentration was observed in very-severe AA patients with a mean of 84.17 ± 2.1 pg/ml. Significant increase in IL-1β concentration was also observed in severe AA patients which was 35.73 ± 2.4 pg/ml. In non-severe AA patients, mean IL-1β concentration was 10.4 ± 0.62 pg/ml (Fig. 1).

Fig. 1
figure 1

a IL-1β-31T/C polymorphism; CC: wild homozygous (239 bp), TC: heterozygous (239 bp, 137 bp and 102 bp), TT: mutant (137 bp and 102 bp) MWL, molecular weight ladder (100 bp ladder). b IL-1β-511 C/T polymorphism; TT: wild homozygous (305 bp), CT: heterozygous (305 bp, 190 bp and 115 bp), CC: mutant (190 bp and 115 bp) MWL, molecular weight ladder (100 bp ladder). c IL-1β+3954 C/T polymorphism; CC: wild homozygous (152 bp), CT: heterozygous (152 bp, 88 bp and 64 bp), TT: mutant (88 bp and 64 bp) MWL, molecular weight ladder (50 bp ladder)

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Discussion

The pathogenesis of AA is very complex [22]. The pathogenesis of AA includes abnormal cellular immunity, gradual destruction of the hematopoietic stem cells, and hematopoietic failure, which can lead to a reduction in all blood cells [23, 24]. Recent studies have shown that cytokine gene polymorphisms enhance the susceptibility of the patients to develop AA [15, 25]. Scanty data is available on the role of cytokine gene polymorphisms in causation of AA in the Indian population. In this study, we identified the association of IL-1βpolymorphism with susceptibility and severity of AA, and also the effect of IST on AA. We also determined the expression of blood plasma level in AA patients (Fig. 2).

Fig. 2
figure 2

Box plot showing blood plasma levels of IL-1β in healthy controls and AA patients. The box includes observations from the 25th to 75th percentile, and the horizontal line within the box represents the median value. The upper and lower lines outside the box represent the highest and lowest values of NSAA, SAA and VSAA respectively. Significant increase in IL-1β concentration were observed in SAA and VSAA patients with respect to contol

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The pathophysiology of IL-1βinvolves several pathways which lead to a wide range of immunological and inflammatory effects [26]. The main role of IL-1β cytokine is to manage proinflammatory reactions in response to tissue injury. It stimulates and initiates other cytokines via accessory cells. Thus, it is the chief intermediate of innate immune reactions [27].

Different cellular signaling pathways may operate in response to varying levels of IL-1β leading to genotoxic damage, cell apoptosis or cell growth [28]. IL-1β represents the primary activator of early cytokines that facilitate the migration of leukocytes from blood vessels into the tissues [29]. A significant number of AA patients had increased levels of IL-1β in their blood plasma and these levels were significantly higher in patients in severe and non-severe group as compared to controls. This is the first study from India carried out by us showing elevated IL-1β levels in the blood of AA patients.

The results of IL-1β-31showed higher susceptibility in over-dominant model (Table 3). It also indicates that the mutant variant and over-dominant genetic model was higher in SAA and NSAA, but not significantly when compared with those of controls (Table 4). The homozygous mutant variant of IL-1β-511 and its recessive model showed higher trend towards susceptibility, although they were not found to be significantly associated (Table 3). The results of IL-1β-511 showed a relationship between polymorphism in recessive model of IL-1β-511 with severity of NSAA and VSAA patients (Table 4). The findings of this study also show that there is an association between polymorphism in homozygous mutant (TT) variant of IL-1β-3954 and susceptibility to developing AA (Table 3). Our results are in disagreement with the earlier studies where cytokines were found to be associated with other diseases and not with aplastic anemia, and this might probably be due to ethnic and geographical differences between the studied populations [30, 31]. To the best of our knowledge, this is the first study of IL-1β-3954polymorphism in cases of AA in India. Further, our findings are also in consonance with the earlier study, where polymorphism in the IL-1β-3954 was found to be associated in chronic periodontitis [32].

Our studies have thus shown that IL-1β-511 and IL-1β-3954 polymorphisms might play a role in the pathogenesis of AA. The limitation of our study is that the sample size was small; for better results, a larger sample size and different ethnic population are required. We have also demonstrated that elevated levels of IL-1β in plasma of AA patients show a direct correlation with severity of the disease, and thus play an important role in development of AA.