Samples collection

Three milliliters of blood were collected, whereby 60 specimens of blood were obtained from a total of 30 patients and 30 individuals in good health. The blood was introduced directly into EDTA tubes. For the purpose of molecular investigations, the samples were transported from the hospital to the laboratory using a refrigerated container and subsequently conserved in a deep freeze set at -20°C.

DNA extraction and Molecular study

Following the manufacturer's instructions, DNA extraction and purification were conducted using the FAVORGEN kit. The primers for the MUC5B gene VNTR and IL-1 RN VNTR are provided in Table 1. After the reaction was done, the amplification products were checked by 2% electrophoresis agarose gel du point and visualized by staining with bromide ethidium. Amplified DNA fragments were compared according to the migration of the DNA marker 100 bp.    

Statistical analysis

Data were analyzed using version 22 of SPSS. Quantitative data were presented in the form of mean and standard deviation and qualitative data in frequency and percentage of the report. The independent-sample t-test was used to compare both groups, and the possible relationship between the ordinal or nominal variables about the current study was determined using the chi-square test. Significance was inferred if the p-value was ≤ 0.05.

Demographic characteristics of patients

A total of 74 patients were enrolled in the study. The demographic characteristics of patients are presented in Table 3, and those of control individuals are shown in Table 3. The study included 74 patients of various ages and genders, exhibiting otitis media symptoms. The maximum patient count (26) was observed in the 20-40 years age range, followed by less than 20 years (24) and more than 40 years (24).

Table 2 illustrates that the samples had a mean age (SD) of 33.47 (18.84) years, with a majority (35%) aged between 20-40 years. In terms of gender distribution, 44.5% were male, and 55.5% were female. Regarding residency, over three-quarters (75.7%) were from urban areas. The results showed a higher prevalence of ear infection among females than males, without a clear explanation for gender predilection in ear infections. The study found that 75.7% of patients resided in urban areas, while 24.3% lived in rural areas.

The study enrolled 74 patients with otitis media, with a relatively even distribution across age groups (less than 20 years, 20-40 years, and more than 40 years). Females exhibited a slightly higher prevalence of ear infection compared to males. The majority of the patients resided in urban areas. Despite observing a higher frequency of ear infections in females, the study did not identify a definitive reason for this gender difference.

Genotypic polymorphism of MUC5B gene

Genotypic polymorphism of the MUC5B gene was evaluated through PCR using primers designed to amplify the entire variable numbers of tandem repeats (VNTR) region. Agarose gel electrophoresis of PCR products for the MUC5B primer from both otitis media patients and control individuals is presented in Figure 1

Table 4 presents the frequency of MUC5B gene polymorphism genotypes and alleles. In this study, three genotypes of the MUC5B gene polymorphism were identified among patients with otitis media and control individuals. The genotypes 6/8, 8/8, and 8/9 had frequencies of 16.7%, 66.6%, and 16.7% in patients, and 16.7%, 76.7%, and 6.6% in controls, respectively. The most common genotype in both groups was 8/8, with frequencies of 66.6% in patients and 76.7% in controls.

The allele frequency of allele 8 was notably high, accounting for 83.4% in patients and 88.3% in controls. The other two alleles (6 and 9) had frequencies of 8.3% each in patients, and in controls, they were 8.3% and 3.3% respectively. The results suggest that carriers of allele 9 face an increased risk of developing otitis media. No significant difference was detected at a significance level of P ≤ 0.05 between the patients and controls.

This study investigated the association between genotypic polymorphism of the MUC5B gene and otitis media susceptibility. Three genotypes were identified (6/8, 8/8, and 8/9), with 8/8 being the most common in both patients and controls. While allele 8 was dominant in both groups, allele 9 potentially carries a slightly increased risk for otitis media. However, statistically significant differences in genotype or allele frequencies between otitis media patients and controls were not observed. Further studies with larger sample sizes might be needed to definitively assess the role of MUC5B polymorphisms in otitis media development.

Genotypic polymorphism of IL-1 RN gene 

Frequency of IL-1RN gene polymorphism genotypes and alleles is shown in Table 4. Four genotypes of IL-1RN gene polymorphism were identified among patients with otitis media, whereas two genotypes were observed in control individuals. The genotype frequencies in patients were: 1/1 (60.0%), 1/2 (16.7%), 1/3 (16.7%), and 2/2 (6.6%). In controls, the frequencies were: 1/1 (88.0%) and 1/3 (12.0%). The most prevalent genotype in both groups was 1/1 (60.0% in patients and 88.0% in controls). The allele frequency of allele 1 was notably high, at 76.7% in patients and 96.0% in controls. Allele 3 had a lower frequency of 8.3% in patients and 4.0% in controls. Allele 2 was exclusively found in patients, with a frequency of 15.0%. This reveals a nearly twofold increased risk for developing otitis media in allele two carriers.

The current study achieved a statistically significant association between the polymorphism of the interleukin-1 receptor antagonist gene and susceptibility to otitis media. Genotypes with allele 2 were significantly more in numbers concerning the otitis media group as compared to the control. Furthermore, allele 2 was exclusively present among the group of patients and may, therefore, be considered possibly responsible for the etiology of the disease. The homozygotes which may be at an almost 1.9-fold increased risk of otitis media compared with this allele. However, available data are limited, nuanced analysis should be directed towards a better understanding of the functional consequence of IL-1RN gene polymorphisms needed for one to know their role in the pathogenesis of otitis media.

Figures & Tables

Discussion

Otitis is characterized by inflammation and infection of the membrane lining the middle ear. The presence of inflammation, as well as drainage and perforation of the tympanic membrane, can increase the incidence of otitis media (OM) which can present as an acute and chronic condition. This condition has the potential to result in lasting complications, such as conductive and sensorineural hearing impairments [14].The current investigation is centered on MUC5B and IL-1RN polymorphisms, delving into the significance of variations and alterations in genes associated with innate immunity.

The higher prevalence of ear infections in women is consistent with the findings of Aleneji et al., and Yadava [15,1 However, these results differ from those reported in other studies. The study of Addas et al., [17] and Osazuwa et al., [18] reported that ear infections were more common in men. However, there are no well-documented genetic or physiological differences between the sexes in hearing-related issues [19].

In contrast, another study demonstrated that the prevalence of the illness was higher among communities characterized by inadequate personal hygiene, substandard housing conditions, and limited literacy levels. This discrepancy may be ascribed to the fact that a significant portion of the subjects in this examination were affiliated with a particular urban demographic subgroup, which experienced superior living conditions in comparison to others. This assertion was corroborated by investigations carried out among urban communities by Ahmed & Akaiduzzaman [20], as well as Uddin et al., [21]. Comparable results are also documented by Biswas et al., [22] and Anggraeni et al., [23].

MUC5B is classified in the group of secreted gel-forming mucins. Chromosome 11 is a gene located on band p15.5, which resides in a 400 kb segment of genomic DNA along with MUC6, MUC2, and MUC5AC, which also secretes gel-forming mucins Major properties of in mucus and mucins sequences are often and differ among different mucins. Detailed MUC5B genomic and cDNA sequences provide a valuable framework for investigating the developmental regulation of a wide range of mucins [26].  Ahn and colleagues identified seven variable numbers of tandem repeats (VNTRs; minisatellites) within the complete MUC5B region. These minisatellites have the potential to serve as markers for paternity mapping and DNA fingerprinting [25].

Our findings align with those of Ubell et al., who also did not observe significant differences between otitis media patients and controls regarding the size of MUC5B genes determined through polymerase chain reaction [7]. This could be attributed to the relatively small sample size of patients. Recent studies propose that targeted treatment strategies focused on MUC5B might be beneficial for addressing otitis media with effusion (OME) [8,27]. MUC5B stands out as the primary mucin in conditions like cystic fibrosis, chronic obstructive pulmonary disease, and middle ear effusion and mucosa during OME [28, 29].

Genes that control the production of cytokines are commonly identified as strong candidates in the development of chronic otitis media (COM), as well as various chronic inflammatory and autoimmune human conditions. Numerous research studies have utilized the IL-1 RN VNTR primer to serve as an indicator of the host's regulation of immunity. Although many investigations have suggested a link between the IL-1 gene cluster and a predisposition to specific inflammatory diseases, only a limited number have focused on otitis media [31]. Our findings are consistent with those of Zivkovic et al., who investigated the relationship between chronic otitis media and variations in genes within the IL-1 RN gene associated with innate immunity and inflammatory processes. The carriage of this allele has been found in their study to be positively associated with chronic otitis media [32]. The genotypes of IL-1RN-VNTR 1/2 and 2/L were significantly higher in another study in patients with cutaneous melanoma (43.6% and 45.1%, respectively) compared to healthy subjects [33].

In conclusion, our study established an association between polymorphisms in the MUC5B gene and IL-1 RN gene with the severity of otitis media and hearing loss. Testing for these polymorphisms could aid in the early prediction of disease progression and identify patients who might benefit from introducing antagonists into their treatment regimen. This indicates that targeted treatment strategies based on specific genes could hold potential benefits for managing OME. 

Conflict of Interest

The author declare that there is no conflict of interest regarding the publication of this paper.

Author Contributions

Kawther: Refinement of study design , supervision and technical support.

Safa Amer: Sample collection, laboratory work and writing.

References