Abstract
Chronic Otitis Media (COM) is a chronic inflammation of the middle ear and mastoid with persistent membrane perforation and hearing loss. Osteoprotegerin (OPG) and NOD like receptor protein 3 (NLRP3) play an important role in bone metabolism. The aim of the study was to investigate the role of OPG and NLRP3 gene polymorphism on ossicular chain resorption in COM. Fourty COM patients and 20 healhty control group were included in the study. While 20 patients underwent ossiculoplasty, 20 patients underwent type 1 tympanoplasty. DNA was isolated from peripheral blood using the DNA kit. OPG gene c.226A > C (p.Thr76Pro) and NLRP3 gene c.592G > A (p. Val198Met) polymorphisms were genotyped using melting curve analysis technique. NLRP3 gene polymorphism were determined in 6 of 20 patients (30%) in ossiculoplasty group, 4 of 20 patients (20%) in type 1 tympanoplasty group and 3 of 20 patients (15%) in control group. OPG gene polymorphism were determined in 5 of 20 patients (25%) in ossiculoplasty group, 3 of 20 patients (15%) in type 1 tympanoplasty group and 1 of 20 patients (5%) in control group, respectively. There was no statistically significant difference between groups regarding to results. Although the difference was not significant NLRP3 and OPG gene polymorphisms were higher in the ossiculoplasty group. Since NLRP3 and OPG gene polymorphisms were determined to be higher numerically in the ossiculoplasty group, OPG and NLRP3 gene regulation system may have an effect on ossicular chain destruction in COM.
Keywords: Tympanoplasty, Ossiculoplasty, Polymorphism, Osteoprotegerin, NOD like receptor protein 3
Introduction
Chronic otitis media (COM) is clinically manifests with tympanic membran perforation, otorrhea and hearing loss; it is a chronic infection of mucosa which is laying the middle ear and mastoid [1]. Osteoprotegerin is a member of tumor necrosis factor receptor family, also known as osteoclastogenesis inhibitory factor (OCIF), is a protein that inhibits bone destruction caused by osteoclasts. NLRP3(NOD like receptor protein 3) inflammasome is a group of inflammasome which increases bone destruction with activation in the presence of inflammation [2, 3].
COM is seen with otorrhea and hearing lossdue to tympanic membran perforation or/and irreversible resorption of the ossicular chain [1]. There are medical and/or surgical treatment options in COM patients. The first aim of all treatment options is the eradication of the infection, that is followed by hearing restoration. COM is oftenly caused to ossicular chain damage. When this situtation is occured, it effects the mechanism of the sound transmission and caused to hearing loss. 3 steps must be re-performed in order to provide for healty ear and also hearing function. These are intact tympanic membrane, a well-ventilated middle ear cavity and intact and working ossicular chain. Tympanoplasty and/or ossiculoplasty may be preferred according to situation of the middle ear in order to provide hearing reconstruction in COM patients [4–7].
During the intraopreative assessment of patients underwent surgery due to COM without cholesteatoma, some of them had defects in the ossicular chain, while others had intact/normal ossicules. Ossicular chain system can be destroyed due to the duration of the disease and also the number of recurrent acute inflammatory attacks. Destruction of ossicular chain is frequently seen in COM patients with cholesteatoma but also it can be seen without cholesteatoma [5, 6, 8]. Even though cholesteatoma is a major factor in ossicular chain destruction development, other factors partaking in ossicular chain destruction development need to be examined as well. The levels of the genes effective on the inflammatory process can be affected the middle ear microenvironment. While OPG acts like an anti-osteoclastogenic factor and prevents bone mass from reduction, NLRP3 increases bone resorption during inflammation [2, 3]. However; pathophysiology of genetic basis of ossicular chain destruction in COM patients without cholesteatoma remains uncharted to this day and there is insufficient knowledge about it. In patients with this condition, maybe a relationship between genetic basis and ossicular destruction. If this mechanism is understood, genetic treatments may be become the main subject of future research. Gene therapy may be affective to recover inflamatuar effects and prevent ossicular damage [6–21].
The aim of this study is to investigate the role and basis of inflammatory gene polimorphisms on ossicular chain resorption in COM patients underwent type 1 tympanoplasty or ossiculoplasty.
Materials and Methods
A power analysis was made via G*Power (v.3.1.7) program in order to detect the sample figures prior to the study. As result of this power analysis, it was calculated that there be at least 60 person, 20 in each group to achieve 80% power at α = 0.05 level. Therefore 60 patient, 25 male and 35 female, between the ages 15–70(35.8 ± 14.2) were included in this study.
Surgery was performed in 40 patients with a diagnosis of COM who were included in the study. Types of surgeries were as follows: 20 ossiculoplasty (6 Partial ossicular replacement prosthesis, 6 Total ossicular replacement prosthesis, 8 applications of bone cement), 20 type 1 tympanoplasty. Besides that, twenty healthy controls were included in the study. An approval was received from the local ethical committee prior to the study (approval of the ethics committee: FSM EAH- KAEK 2016/60). The patients were informed about the study and gave informed consent to participate in the study.
During the study, the requirements of the Declaration of Helsinki developed by the World Medical Association were met.
Study was divided into 3 groups: first group is an ossiculoplasty group, consisted of twenty patients with COM who underwent ossiculoplasty. In this group, middle ear and mastoid are clean at Temporal Bone Computed Tomography (CT) and with middle ear ossicular defect findings in the surgery. Second group is a tympanoplasty group, consisted of twenty patients with COM underwent type 1 tympanoplasty. In this group, middle ear and mastoid are clean at Temporal Bone Computed Tomography (CT), without ossicular chain defect and observed to have a normal ossicular chain movement in the surgery. Third group is a control group, consisted of twenty healthy individuals with an intact tympanic membrane and hearing.
COM patients with cholesteatoma, undergoing mastoidectomy and undergoing surgery for revision were excluded from the study.
In order to perform the investigation of genetic polymorphism at the level of DNA, a peripheral blood sample was taken into a 10 mL EDTA container with a purple cap from patients participating in the study and the samples were stored at − 40 °C until the step of DNA isolation. Genomic DNA isolation of the samples was performed at the medical genetic laboratory.
Puregen Qiagen DNA isolation kit (Hilden, Germany) was used for DNA isolation. After taking blood sample into an Ependorf tube, peripheral blood DNA was isolated by using red blood cell lysis, cell lysis, protein lysis solutions and isopropyl alcohol respectively and by centrifugation of the sample each time. Then DNA was precipitated by using 70% ethanol alcohol. Thereafter, DNA was separated from alcohol and allowed to dry and then Tris–EDTA tampon solution was added and DNA was solved (Fig. 1 a–c).
Fig. 1.
DNA analysis from a peripheral blood sample a Precipitation including white blood cells at the bottom of the tube b Precipitation including white blood cells at the bottom of the tube following centrifugation after addition of lysis solutions c DNA solution collected at the top of Eppendorf tube
Gene sequences of Osteoprotegerin and NLRP3 genes planned to be investigated in the study were determined by using the Ensembl genome browser internet website (www.ensembl.org), designs of primers related to Osteoprotegerin and NLRP3 genes which would be used for investigation of polymorphisms were determined by using the OligoAnalyzer 3.1 program and investigated by using BLAST to prevent binding to different regions other than target regions. Later, 2 reverse primers and 2 forward primers were produced for OPG. Two forward primers and 3 reverse primers were produced for NLRP3 (Table 1). Primers [Qiagen Synthetic Oligonucleotide (200 nmol scales)/base (174 bases), Hilden, Germany] were supplied from service provider company (Genova Medikal, Istanbul, Turkey) by getting them synthesized in the manufacturing company.
Table 1.
Primer Sequences
| Name of Oligosaccharide | Base Sequence 5′-3′ | Number of Bases | Scale nmol) | W (g/mol) | Tm(C) | GC(%) | OD 260 nm | nmol | 100 µM Stock |
|---|---|---|---|---|---|---|---|---|---|
| NLRP3F | ATG ATG AGC ATT CTG AGC CTG | 21 | 200 | 6461 | 57.9 | 47.6 | 8.28 | 35.6 | 356 |
| NLRP3R | CTC TGT GTC ACA AGG CTC AC | 20 | 200 | 6053 | 59.4 | 55.0 | 7.79 | 37.7 | 377 |
| NLRP3-2F | ATC GGC AAG ACC AAG AC | 17 | 200 | 5197 | 52.8 | 52.9 | 8.81 | 43.9 | 439 |
| NLRP3-2R | TCC TGG CCA GGA TTG TTT T | 19 | 200 | 5801 | 54.5 | 47.4 | 7.67 | 39.6 | 396 |
| NLRP3-2R2 | TTC CTG GCC AGG ATT GTT TT | 20 | 200 | 6105 | 55.3 | 45.0 | 7.57 | 37.3 | 373 |
| OPGF | TCT CAT CAG CTG TTG TGT GA | 20 | 200 | 6114 | 55.3 | 45.0 | 7.79 | 37.3 | 373 |
| OPGR | TGC ACT CCT GCT TGA CGT A | 19 | 200 | 5755 | 56.7 | 52.6 | 7.62 | 39.4 | 394 |
| OPGF2 | ACC ACT ACT ACA CAG ACA G | 19 | 200 | 5735 | 54.5 | 47.4 | 8.57 | 39.6 | 396 |
| OPGR2 | AAG GTA GCG CCC TTC CTT G | 19 | 200 | 5780 | 58.8 | 57.9 | 7.79 | 39.8 | 398 |
NLRP NOD Like Receptor Protein, OPG Osteoprotegerin, MW Molecular Weight, Tm Melting Temperature,
GC Guanine-Cytosine Heterozygote, nmol Nanomole, μMMicromole, OD Optic Density
In this study; rs200071478 and rs121908147 polymorphic regions of OPG gene and NLRP3 gene were analyzed respectively [c.226A > C (p.Thr76Pro) polymorphic region for OPG gene and c.592G > A (p. Val198Met) polymorphic region for NLRP3 gene].
For PCR assay; patient samples were amplified by using Bio-Rad iTaq™ Universal SYBR® Green Supermix solution (California, USA), reverse and forward components of primer sequences, H2O and isolated DNA with Bio-Rad® GCFX96™ device (California, USA) (Fig. 2a).
Fig. 2.
Melting curves analysis of the samples a Amplification b Melting Curves analysis c Melting Knob at 82 °C
To be able to analyze OPG and NLRP3 gene polymorphisms, the samples were prepared as defined above with a ready PCR mixture including SYBER green fluorescent dye, reactions were realized at binding temperatures created and melting curve analysis were performed at the final step of the study (Fig. 2b). After melting curve analysis, it was determined that mutant samples showed melting knob ≤ 82 °C and wild type (without mutation) samples showed melting knob > 82 °C (Fig. 2c). In this case, mutations of the samples were determined with analysis.
Results
The results of the study were summarized in Tables 2 and 3.
Table 2.
Evaluation of Melting Temperature Parameters Between Ossiculoplasty, Type 1 Tympanoplasty and Control Groups
| Melting temperature | Group | p | |||
|---|---|---|---|---|---|
| O | T | C | |||
| n (%) | n (%) | n (%) | |||
| NLRP3 temperature | ≤ 82 °C (with mutation) | 6 (% 30) | 4 (% 20) | 3 (% 15) | 0.503 |
| > 82 °C (without mutation) | 14 (% 70) | 16 (% 80) | 17 (% 85) | ||
| OPG temperature | ≤ 82 °C (with mutation) | 5 (% 25) | 3 (% 15) | 1 (% 5) | 0.208 |
| > 82 °C (without mutation) | 15 (% 75) | 17 (% 85) | 19 (% 95) | ||
Chi-square test O Ossiculoplasty, T Type 1 Tympanoplasty, C Control, NLRP3 NOD Like Receptor Protein 3, OPG Osteoprotegerin
Table 3.
Evaluation of Melting Temperature Parameters Between Undergoing Surgery (Ossiculoplasty and Type 1 Tympanoplasty) and Control Groups
| Melting temperature | Group | p | ||
|---|---|---|---|---|
| US | C | |||
| n (%) | n (%) | |||
| NLRP3 temperature | ≤ 82 °C (with mutation) | 10 (% 25) | 3 (% 15) | 0.375 |
| > 82 °C (without mutation) | 30 (% 75) | 17 (% 85) | ||
| OPG temperature | ≤ 82 °C (with mutation) | 8 (% 20) | 1 (% 5) | 0.125 |
| > 82 °C (without mutation) | 32 (% 80) | 19 (% 95) | ||
Chi-square test US Undergoing Surgery, C Control, NLRP3 NOD Like Receptor Protein 3, OPG Osteoprotegerin
Evaluation of melting temperature parameters between Ossiculoplasty, Type 1 Tympanoplasty and Control Groups
There was no statistically significant difference between groups regarding distributions of NLRP3 and OPG levels of patients (p > 0.05).
Evaluation of melting temperature parameters between groups undergoing surgery (Ossiculoplasty and Type 1 Tympanoplasty groups) and Control Group
There was no statistically significant difference between groups regarding distributions of NLRP3 and OPG levels of patients (p > 0.05).
Statistical Evaluations
In this study, IBM SPSS Statistics 22 (IBM SPSS, Turkey) program was used for the statistical analysis. Conformity of the parameters to the normal distribution was assessed by the Shapiro Wilks test. During the evaluation of the study data, regarding the comparisons of quantitative data as well as descriptive statistical methods (Mean, Standard deviation, and frequency), One-way Anova test was used for the intergroup comparisons of parameters with normal distribution and Tukey HDS test was used for the determination of the group causing the difference. The Kruskal–Wallis was used for the intergroup comparisons of parameters without normal distribution. Chi-Square test was used for comparison of qualitative data. Significance was evaluated at a level of p < 0.05.
Discussion
Ossicular destruction is frequently seen in COM patients with cholesteatoma than without cholesteatoma [4]. There are many studies about this in the literature. In a study performed by Albera et al., the authors thought that while ossicular chain erosion was seen more frequently especially in COM patients with cholesteatoma, it was observed also in COM patients without cholesteatoma [9]. Moreover other studies, Akarcay et al. [4] and Martins et al. [10], found similar results. But in the literature, there is no objective explicit knowledge about the pathophysiology of bone destruction in the COM without cholesteatoma. During the intraopreative assessment of patients undergoing surgery due to COM without cholesteatoma, while ossicular chain defect is present in some patients, ossicles are observed to be intact in others. Thus, this situation make us think of this question “While there is no cholesteatoma in both groups who underwent surgery, ossicular chain destruction is seen some of patients, but also we don’t see in some part of patients. What is the difference between, is there a genetic basis that initiating ossicular chain destruction?”. If the answer for this question is found, it may be important in terms of the treatment and algoritm of the disease.
NLRP3 (NOD like receptor protein 3) inflammasome is a group of inflammasome including NLRP3, ASC, CARD, PYD and pro-caspase-1. NLRP3 plays an important role in innate immunity and inflammation as a receptor protein of the inflammasome complex. It is activated during chronic inflammation and causes bone destruction. Kariya et al. demonstrated that NLRP3 mRNA and consequently protein levels were increased cause of high expression of NLRP3 inflammasome gene in COM patients with cholesteatoma and simple chronic otitis media compared to individuals with normal middle ear mucosa [11]. These findings confirm that infiltrating inflammatory cells in cholesteatoma express NLRP3. Identified polymorphic alleles of NLRP3 (rs121908147) were defined to be the mutations reflecting protein structure (ncbi.nlm.nih.gov). It is considered that the mutation bearing polymorphic proteins contribute to the inflammatory process. While NLRP3 protein is in an inactive state in the absence of stimuli, it is activated in response to pathogen and/or outer inflammation signals. It is considered that mutation we are investigating activates the NLRP3-inflammasome process. NLRP3 inflammasome increases caspase-1 production. IL-1β and IL-18 production increase with stimulation of caspase-1 and so inflammatory and immune responses of NLRP3 inflammasome occurs in the presence of inflammation [12]. So when polymorphism is found in the NLRP3 gene, NLRP3 gene activation increases and caused to bone resorption. Because of these reasons NLRP3 inflammasome could be determined as a therapeutic target and used in the management of the disease in COM patients with cholesteatoma and simple COM. Although in our study, no statistically significant difference was observed between groups included in the study regarding distribution rates of patients (with and without mutation) according to NLRP3 melting temperature values (p > 0.05), NLRP3 gene polymorphism which is caused to activate NLRP3 and increased bone dectruction, was determined to be higher numerically in ossiculoplasty group than the other groups [6 of 20 patients (30%) in ossiculoplasty group, 4 of 20 patients (20%) in type 1 tympanoplasty group and 3 of 20 patients (15%) in control group], as we expected.
Osteoprotegerin, also known as tumor necrosis factor receptor superfamily member 11b (TNFRSF11B), is one of the preventive factor of bone tissue from destruction caused by osteoclasts. It is known that single-nucleotide variant of OPG gene (rs200071478) to be a clinically significant pathogenic mutation and determined in some bone diseases going along with bone resorption and bone formation like Paget’s disease [13]. In recent investigations, OPG has been demonstrated as a receptor for both RANKL and TNF-related apoptosis-inducing ligand (TRAIL). And when OPG binds to RANKL receptor, it inhibits differentiation and activation of the osteoclasts [14]. Polymorphisms developing in the transcription factor binding sites of the gene encoding OPG which have an important role in the prevention of osteoclast differentiation may cause a reduction in bone density or development of osteoporosis by changing the bone modeling [12, 15]. So when the OPG gene has polymorphism, the OPG gene cannot perform its normal function of protecting against bone destruction. In a study performed by Roshandel et al., the authors investigated the influence of genetic variation in the RANKL/RANK/OPG signaling pathway on bone turnover and bone mineral density in men, it was determined that OPG polymorphism caused an increase in bone turnover markers [16]. Soufi M et al. reported that OPG gene polymorphism lead to a predisposition to destructive bony lesions and/or pathological fractures [17]. In a study performed by Farrugia et al., the authors found that high levels of RANKL and low levels of OPG in multiple myeloma patients with lytic lesions [18]. Kao et al. demonstrated in their study performed in the murine ear that RANK and RANKL interaction in cochlear neurons inhibits neurite outgrowth besides OPG prevents RANKL-induced inhibition of neurite outgrowth [19]. In the study performed by Langdahl et al., the authors demonstrated that OPG disfunction due to OPG gene polimorphism was seen frequently in Danish patients with vertebral fractures [20]. Choi et al. reported that OPG genetic polymorphisms are related to low bone mineral density in postmenopausal Korean women and and this result indicates that OPG has a protective function on bone tissue [21]. As a result of our study, no statistically significant difference was observed between groups regarding distributions of patients according to melting temperature of OPG (patients with DNA showing melting knob ≤ 82 °C were considered to have mutation and the ones showing melting knob > 82 °C to have no mutation). But besides with that, OPG gene polymorphism which fails to perform anti-osteoclactic function, was determined to be higher numerically in ossiculoplasty group than the other groups [5 of 20 patients (25%) in ossiculoplasty group, 3 of 20 patients (15%) in type 1 tympanoplasty group and 1 of 20 patients (5%) in control group], as we expected.
However, there is no study performed in the literature related to the association between genetic basis and development of ossicular chain defect in COM and to the best of our knowledge our study is the first one investigates this subject. Besides that, while the levels of OPG and NLRP3 proteins were investigated in previous studies, in our study genetic structure differences which could provide a contribution to the inflammatory process were investigated.
Our results do not show a significant difference between the individuals with and without NLRP3 and OPG polymorphism regarding the occurrence of ossicular chain defect due to chronic otitis media (p > 0.05). Although the difference was not significant NLRP3 and OPG gene polymorphisms were higher numerically [6 of 20 patients (30%) and 5 of 20 patients (25%), respectively] in the ossiculoplasty group than the other groups. Also while our study includes patients with COM without cholesteatoma, other studies in the literature includes COM patients with cholesteatoma. Our hypothesis was also based on whether the presence of a mutation in OPG and NLRP3 genes those playing a role in osteoblastic and osteoclastic activity; respectively was a triggering factor or not in the occurrence of ossicular chain defect in patients with COM. There is also no significant difference between patients with and without ossicular chain defect in the group undergoing surgery in terms of the duration of disease. All of our patients are the ones treated with the surgical method without active infection and followed up with intact fascia. Therefore, there may be a silent difference that can not be determined statistically regarding the pathophysiology of the disease. As a matter of fact, while NLRP3 gene mutation indicating bone destruction activation was determined in 6 of 20 patients (30%) in the ossiculoplasty group, it was determined in 4 of 20 patients (20%) in type 1 tympanoplasty group. Also, in line to expectations; mutant type of OPG inhibiting osteoclast activation that was unable to perform this function was determined to be higher in ossiculoplasty group [5 of 20 patients (25%) in the ossiculoplasty group, 3 of 20 patients (15%) in type 1 tympanoplasty group]. Besides to these, NLRP3 and OPG genes mutation determined to be lowest in control group [3 of 20 patients (15%) and 1 of 20 patients (5%), respectively]. And this results support our hypothesis that there may be a genetic basis and relationship between for the development of ossicular chain erosion in COM patients without cholesteatoma.
Conclusion
This study was conducted in 3 groups, one of them is control group and the other 2 groups are surgery group. In undergoing surgery groups both groups did not have cholesteatoma and the difference between them is the need for ossicular chain reconstruction due to ossicular chain erosion. In this study, NLRP3 and OPG gene mutations were investigated to evaluate the presence of genetic basis playing a role ossicular chain defect in COM patients.
Although no statistically significant result was obtained regarding NLRP3 and OPG gene polymorphism, NLRP3 gene polymorphism resulting in bone resorption was found to be higher in the ossiculoplasty group with 6/20 patients (30%). Besides, OPG gene polymorphism associated with bone destruction was also found to be higher in the ossiculoplasty group with 5/20 patients (25%). This results shows that NLRP3 and OPG genes may have a role in the pathogenesis of development of ossicular chain destruction.
Additionally, even though the obtained difference is not statistically significant, the presence of higher osteoclast activity affected by the genes in the ossiculoplasty group with ossicular chain defect can be evaluated as a finding also NLRP3 and OPG may affect ossicular chain in COM. According to our opinion, new studies including a large number of patients and investigating the role of NLRP3 and OPG in the occurrence of ossicular chain defect in COM are needed.
Author Contributions
S. K.: Study design, experimental applications, statistics, literature research, writing. A. T.: Idea generation, study design, literature research, writing. P. A.: Study design, idea generation, experimental applications, writing.
Funding
None.
Declarations
Conflict of interest
There is no conflict of interest in the study.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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