Eur Arch Otorhinolaryngol DOI 10.1007/s00405-015-3839-7

OTOLOGY

Mitochondrial DNA deletions in patients with chronic suppurative otitis media Arzu Tatar1 • Sener Tasdemir2 • Ibrahim Sahin2 • Ceyda Bozoglu3 Haktan Bagis Erdem2 • Ozgur Yoruk1 • Abdulgani Tatar2

•

Received: 21 August 2015 / Accepted: 18 November 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract The aim of this study was to investigate the 4977 and 7400 bp deletions of mitochondrial DNA in patients with chronic suppurative otitis media and to indicate the possible association of mitochondrial DNA deletions with chronic suppurative otitis media. Thirty-six patients with chronic suppurative otitis media were randomly selected to assess the mitochondrial DNA deletions. Tympanomastoidectomy was applied for the treatment of chronic suppurative otitis media, and the curettage materials including middle ear tissues were collected. The 4977 and 7400 bp deletion regions and two control regions of mitochondrial DNA were assessed by using the four pair primers. DNA was extracted from middle ear tissues and peripheral blood samples of the patients, and then polymerase chain reactions (PCRs) were performed. PCR products were separated in 2 % agarose gel. Seventeen of 36 patients had the heterozygote 4977 bp deletion in the middle ear tissue but not in peripheral blood. There wasn’t any patient who had the 7400 bp deletion in mtDNA of their middle ear tissue or peripheral blood tissue. The patients with the 4977 bp deletion had a longer duration of chronic suppurative otitis media and a higher level of hearing loss than the others (p \ 0.01). Long time chronic suppurative otitis media and the reactive oxygen species

& Arzu Tatar [email protected]; [email protected] 1

Department of Otorhinolaryngology, Head and Neck Surgery, Faculty of Medicine, Ataturk University, Erzurum, Turkey

2

Department of Medical Genetics, Faculty of Medicine, Ataturk University, Erzurum, Turkey

3

Department of Molecular Biology, Faculty of Science, Ataturk University, Erzurum, Turkey

can cause the mitochondrial DNA deletions and this may be a predisposing factor to sensorineural hearing loss in chronic suppurative otitis media. An antioxidant drug as a scavenger agent may be used in long-term chronic suppurative otitis media. Keywords mtDNA  4977 bp deletion  7400 bp deletion  Chronic otitis media  Hearing loss

Introduction Chronic suppurative otitis media (CSOM) is a chronic inflammation of the middle ear and mastoid cavity, and a major cause of hearing loss (HL). CSOM is characterized by persistent drainage from the middle ear, through a tympanic perforation. Especially in developing countries, CSOM is a common cause of preventable HL. It has been reported that 96 % of ears with CSOM had a 26 dB or over of HL [1]. After a long period of inflammation, conductive type hearing loss progresses to the sensorineural type. Although the relationship between otitis media and sensorineural hearing loss (SNHL) is not very clear, the predominant opinion is that SNHL may occur in patients with CSOM [1]. Mitochondria are known as the manufacturers of adenosine-50 -triphosphate (ATP) in the cells, and also have a regulating role in cell proliferation and cell death. Mitochondria have their own inheritance material, mitochondrial DNA (mtDNA), and each mitochondrion contains between 2 or 10 copies of mtDNA which encodes 13 essential subunits of the oxidative phosphorylation (OXPHOS) system, ribosomal RNA genes, and transfer RNA genes. During OXPHOS, some reactive oxygen species (ROS) are also produced in mitochondrial inner

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pathologies [13, 14]. MT-RNR1 (encoding mitochondrial 12S ribosomal RNA) and MT-TS1 (encoding mitochondrial transfer RNA serine 1) are the two hot spot genes in which pathogenic variants are currently known to cause nonsyndromic mitochondrial HL [15]. mtDNA deletions, especially the common mtDNA4977 deletion, are shown to be associated with HL [16–18]. In the present study, the mtDNA deletions in the middle ear tissue of patients with HL resulting from CSOM were investigated. We tried to make a possible correlation between mtDNA deletions and HL and possibly contribute to the explanation of hearing loss mechanism in CSOM.

Materials and methods Participants The study included a total of 36 patients (19 male and 17 female), who were referred for chronic suppurative otitis media. All patients provided written informed consent before participating in the study, which was approved by the Ethical Committee of the Ataturk University (decision number; 2/29). The ages of the patients with randomly selection method were between 15 and 53 years (mean ± SE; 29.19 ± 2.53 years). Subjects with cholesteatoma, previous tympanomastoid surgery, mental retardation, any mitochondrial disease, history of familiar mitochondrial disease, history of use of ototoxic drugs, congenital hearing loss, cranial radiotherapy, and aged more than 60 years were excluded. Duration of the CSOM was between 1 year and 30 years with non-periodic intervals. All subjects were first examined using an otoscope and confirmed clinically for CSOM. Bone- and air-conduction tests of all patients were performed using a pure tone audiometry at 500, 1000, 2000 and 4000 Hz before surgical operations (Fig. 1). Del (-) group

Del (+) group

50

Hearing loss (dB)

membrane and accumulated in mitochondrial matrix. Increased exposure of reactive oxidants and/or decreased protection from these oxidants can cause oxidative stress [2]. As a result of oxidative stress, oxidative damage occurs in the cell and the level of damage is related to the quantity of reactive oxygen species. Even though most ROS are generated in cells by the mitochondrial respiratory chain, polymorphonuclear activation in inflammation is the other main ROS source in mammalian cells [3, 4]. Inflammatory tissue contains a lot of polymorphonuclear neutrophils (PMNs), and the balance between production of ROS and protection from ROS is changed in favor of the production of ROS. The extension of the inflammation time results in the increase of ROS levels not only in PMN, but also in the host cells. High levels of ROS in chronic inflammatory diseases can cause mtDNA mutations, which play an important role in disease progression [5]. Chronic inflammation-related ROS and free radicals are capable of damaging DNA, breaking down lipid and protein molecules, and triggering cell death, all of which can contribute to the hair cell lesion in CSOM and loss of function [6]. The increase in the ROS level can cause oxidative damage in mitochondrial structures especially mtDNA; because mtDNA does not have a better protection and repair system and it is more susceptible [7]. Also mtDNA of nearby cells may be damaged during long-term chronic inflammation reactions due to ROS from PMNs [2]. mtDNA damage is especially prone to mis-pairing of repetitive elements and is correlated with deletions. The large-scale deletions in the mtDNA are associated with human diseases. So far, more than 100 deletions have been saved to GenBank database as related with various diseases (http://www.mitomap.org). In these deletions, a 4977 bp deletion called as ‘‘common deletion’’ and ‘‘age-related deletion’’ is the most common deletion in various tissues. The common deletion is caused by the two ‘‘hot spot’’ break points in mtDNA [8]. This mutation removes all or part of the genes encoding four complex I subunits, one complex IV subunit, two complex V subunits and five tRNA genes, therefore causing energy production catastrophes and abnormal reactive oxygen species generation [9]. The 7400 bp deletion, which is between the D-loop and the ATPase 6 genes of mtDNA, is flanked by direct repeats and it is associated with aging and cardiac problems [10]. In addition, there are some deletion sequences which are seen rarely in the other regions of mtDNA [11]. Notwithstanding HL is a heterogeneous trait with many known genetic and environmental causes, mutations in mtDNA are known to be associated with hereditary or acquired HL [12]. Mitochondrial deafness is thought to account for less than 1 % of HL; on the other hand, SNHL is present in 42–70 % of individuals with mitochondrial

40 30 20 10 0

١500 Hz

٢1000 Hz

٢2000 Hz

٢4000 Hz

٠Mean

Fig. 1 Comparison of sensorineural hearing loss levels of patients with and without mtDNA4977 deletion at four different frequencies and their mean levels. The means of the four measurements in patients with and without mtDNA4977 deletion were 26.10 ± 1.93 and 14.28 ± 0.81 dB, respectively. All of these comparisons were statistically significant (p \ 0.01)

Eur Arch Otorhinolaryngol

Surgical technique The study includes the patients who need surgical treatment for CSOM. Tympanomastoidectomy surgery was applied to the patients for curettage of infected tissue of the middle ear and mastoid cavity. Tissue samples of 36 patients were obtained during curative surgical operations by Department of Otorhinolaryngology. In addition, we took 2 ml of peripheral blood samples from the same patients. Collected middle ear tissue specimens and blood samples were stored at -20 °C until isolation of mtDNA. Investigation of mtDNA deletions In this study 4977 and 7400 bp mtDNA deletions have been studied. Four couple primers were used, of which the first (L.9590-H.10368) was to amplify the negative control sequence within the deleted sequence, the second (L.6251H.7261) was to amplify the positive control sequence out of the deleted sequence, the third (L.7901-H.13631) was to amplify the 4977 bp deletion region and the fourth (L.8531-H.381) was to amplify the 7400 bp deletion region (Table 1). The 1019 and 753 bp PCR products occur in case of 7400 and 4977 bp deletions of mtDNA, respectively. The 778 and 1010 bp PCR products represent negative control within deletion region and positive control out of deletion region, respectively.

We used the Qiagen QIAamp Mini Kit (Qiagen GmbH, Hilden Germany) for mtDNA isolation from middle ear tissues according to the suggestions of the supplier. For blood samples Qiagen robotic workstation (Qiagen GmbH, Hilden Germany) was used for automated purification of mtDNA using EZ1 kits. The PCR reactions were performed in 50 ll reaction mixture containing 18.5 ll PCR master mix (Qiagen

Primers

Agarose gel electrophoresis The 12 ll of each PCR products was separated together with a marker DNA (100 bp DNA ladder) on a 2 % agarose gel at 120 V for 120 min, and then analyzed by UV transilluminator after staining with 0.5 lg/ml ethidium bromide (Fig. 2). The gel patterns of the PCR products were photographed in a UV transillumination cabin. Statistical analysis All the statistical analyses including comparison of the demographic characteristics were performed using paired samples t test and SPSS version 20.0 for windows (SPSS Inc, Chicago, IL, USA). The p \ 0.01 value has showed that the correlation between mtDNA deletions and SNHL is statistically significant.

Results

DNA extraction and polymerase chain reaction (PCR)

Table 1 The primer sequences and the expected sizes of PCR products of the two fragments in the control regions and two fragments for deletion regions

GmbH, Hilden Germany), 2 ll of each primer, 0.5 ll Taq DNA polymerase, 17 ll H2O and 10 ll mtDNA in a thermal cycler (SensoQuest Labcycler, GmbH, Hilden, Germany) under the following conditions: 95 °C for 15 min (initial denaturation) followed by 32 cycles at 94 °C for 1 min (denaturation), 56 °C for 1 min (annealing), 72 °C for 80 s (extension), and a final extension at 72 °C for 15 min.

Firstly, we controlled the validity of mtDNA extraction and PCR procedure. In all samples the positive control region was determined. The mtDNA7400 deletion was not detected in any subject in both peripheral blood samples and middle ear tissues. The frequency of heterozygote mtDNA4977 deletion in middle ear tissues is 47.2 % (in 17 of the 36 patients). The 753 and 778 bp PCR products were detected on agarose gel in these patients (Fig. 2). In Fig. 1, the SNHL levels of patients

PCR product sizes (bp)

Control sequence (negative control)

778

Control sequence (positive control)

1010

4977

mtDNA region

7400

mtDNA region

deletion

Sequences of primer pairs L(9590) 50 -AGTCCCACTCCTAAACACATCCG-30 H(10368) 50 -AGGCCAGACTTAGGGCTAGGATGATG-30 L(6251) 50 -TAT AGT GGA GGC CGG AGC AG-30 H(7261) 50 -GAA TGA GCC TAC AGA TGA TA-30

753

L(7901) 50 -TGA ACC TAC GAG TAC ACC GA-30 H(13631) 50 -GGG GAA GCG AGG TTG ACC TG-30

deletion

1019

L(8531) 50 -ACG AAA ATC TGT TCG CTT CA-30 H(381) 50 -AAA TTT GAA ATC TGG TTA GG-30

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Fig. 2 PCR products were separated on 2 % agarose gel. ‘‘a’’ represented a 1019 bp PCR product occurring in case of 7400 bp deletion of mtDNA; ‘‘b’’ represented a 753 bp PCR product occurring in case of 4977 bp deletion of mtDNA; ‘‘c’’ represented a 778 bp PCR product, negative control within deletion region; ‘‘d’’ represented a 1010 bp PCR product, positive control out of deletion region;

M represented a marker DNA to detect the size of fragments. The PCR products of middle ear tissues (753 bp) of three patients with mtDNA4977 deletion had run on the left of marker DNA (M) (1b, 2b and 3b), and the PCR products of peripheral blood (no band) of the same patients had run on the right of marker DNA (4b, 5b and 6b)

with and without mtDNA4977 deletion at four different frequencies have been compared. Mean SNHL levels of patients with mtDNA4977 deletion were 20.00 ± 3.03, 20.88 ± 3.40, 33.82 ± 4.23 and 29.71 ± 3.87 dB at 500, 1000, 2000 and 4000 Hz, respectively, and the mean of the four measurements were 26.10 ± 1.93 dB (Fig. 1). Mean SNHL levels of patients without mtDNA4977 deletion were 11.84 ± 1.34, 12.36 ± 1.59, 17.63 ± 1.50 and 15.26 ± 1.77 dB at 500, 1000, 2000 and 4000 Hz, respectively, and the mean of the four measurements were 14.28 ± 0.81 dB (Fig. 1). All of these comparisons were statistically significant for patients with and without mtDNA4977 deletion (p \ 0.01). The mtDNA4977 deletion was not detected in peripheral blood samples of the subjects. In addition, the mean durations of the disease were 10.82 ± 1.60 (mean ± SE) years at the patients with mtDNA4977 deletions and 6.97 ± 1.86 (mean ± SE) years at the patients without mtDNA4977 deletions (p \ 0.01). Mean ages of the patients with deleted mtDNA and nondeleted mtDNA were 29.18 ± 2.73 years and 29.21 ± 2.34, respectively (Table 2).

It is obvious now that mtDNA mutations are associated with some SNHL, such as a point mutation in MT-RNR1 gene (m.1555A[G) [12, 15, 21]. Unlike point mutations, mtDNA deletions usually are not germline and are limited to an organ tissue. Germline mitochondrial deletions have also been reported with a mixed population of normal and deleted mitochondrial genomes and heterozygote mutations [22]. The gross deletions may include thousands of nucleotides and affect more mitochondrial genes which are necessary to mitochondrial OXPHOS and so they are not compatible with life [23]. However, the tissue limited deletions result in local cell destruction and loss of function, which is permanent in post-mitotic tissues. The mtDNA4977 deletion is called the age-related mutation due to accumulation of mutations during the lifetime and it has been detected in the post-mitotic tissues of elderly individuals such as neuronal and muscular tissues [24]. We also reported mtDNA4977 mutation in epithelial tissue in our previous study [25]. Taylor et al. interpreted the etiology of acquired mitochondrial deletions with two mechanisms; the somatic generation of the deletions and clonal expansion to phenotypic levels [26]. Chronic inflammation is also a cause of the somatic generation of the deletion in high turnover cells [25]. Therefore, the mtDNA4977 was detected in several ageindependent diseases including some chronic inflammatory diseases [23, 27]. mtDNA deletions, especially the common mtDNA4977 deletion, are shown to be associated with HL [16–18]. Ueda et al. detected mtDNA4977 mutation in 75 % of patients with bilateral SNHL and mentioned that the percentage of mutant mtDNA reached clinical

Discussion Hearing loss is the one of the most complicated topics of medicine in terms of inheritance. A wide range of genes, nuclear or mitochondrial genes, plays a role at the hearing mechanism and so the different inheritance mechanisms are described [14, 19]. Hearing impairment has been shown at many clinical pictures with mitochondrial heredity [20].

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Eur Arch Otorhinolaryngol Table 2 Descriptive analysis of CSOM patients with and without mtDNA4977 deletion Deletion

Number of patients (%)

Age (years) (mean ± SE)

Duration (years) (mean ± SE)

SNHL levels (db) (mean ± SE)

p value

mtDNA4977 deletion-positive patients

17 (47.2 %)

29.18 ± 2.73

10.82 ± 1.60

26.10 ± 1.93

\0.01

mtDNA4977 deletion-negative patients

19 (52.8 %)

29.21 ± 2.34

6.97 ± 1.86

14.28 ± 0.81

efficiency levels, over time [17]. Markaryan et al. reported three additional deletions except the common mtDNA4977 deletion in the cochlear tissue of two patients with SNHL [11]. We detected mtDNA4977 deletion in 47.2 % of patients. In our study, the mean age of the patients with deleted mtDNA was 29.18 ± 2.73 years and the oldest patient was 53 years (Table 2). These are early ages for the occurrence of age-related mutations. In our patients hearing loss was thought to be acquired but not congenital because there was no apparent familial history. Furthermore, we detected this deletion in middle ear tissue but not peripheral blood, supporting the idea of the deletion was not germline and restricted to the middle ear and most probably the inner ear. Furthermore, we detected a statistically significant correlation between the level of sensorineural hearing loss and the presence of mtDNA4977 deletion. The heterozygote mtDNA deletion may contribute to SNHL because the mean level of SNHL was 26.10 ± 1.93 dB in deleted patients and statistically different from patients without mtDNA4977 deletion. We also assessed the mtDNA7400 deletion but it could not be detected in any patient. The middle ear has epithelial tissue with high cellular turnover, and in fact mtDNA deletion due to increased mitochondrial ROS is expected at lower quantities [18]. The proliferation of the cells in an inflammatory environment with high levels of ROS means the new cells are exposed to these toxic agents from the beginning of their life. Therefore, somatic mtDNA mutations are transmitted increasingly to the new cells, and this supports clonal expansion over time. Mechanisms regulating polymorphonuclear neutrophil (PMN) oxidative metabolism are not sufficiently understood, but it is known that neutrophils maintain a function in an environment of high oxidative stress such as in purulent secretions [28]. Besides, ROS levels also increase in host tissue cells. The conductive hearing loss is related especially to tympanic perforation and middle ear destruction. Hearing loss in otitis media patients progresses from conductive type to sensorineural type in the upcoming years. Longterm inflammation in the middle ear may affect the inner ear and may contribute to SNHL in CSOM. Some reason for this may be cellular damage in the inner ear due to the long-term deleterious inflammation. It is known that

cochlear hair cells and stria vascularis include many mitochondria and need OXPHOS for energy production. The ROS originating from inflammation may affect the limited hair cell of the organ of corti but not the auditory neuron. Auditory degeneration develops slowly as seconder to cochlear degeneration [29]. Cochlear dysfunction without any pathology in auditory nerve is sufficient to hearing loss, which may be detected in the point mutations of mtDNA [30]. Zhong et al. showed that the common deletion is the most frequent type of mtDNA deletions, exceeding 67.86 % in inner ear of aging rats with HL [31]. In the study of Menardo et al., they showed chronic inflammation and oxidative stress may lead to mitochondrial dysfunction and this may contribute to HL and cochlear degeneration in rats [32]. Bai et al. reported that even minor mtDNA abnormalities might disrupt the function of the inner ear [18]. In addition, the increase of ROS related with chronic inflammation, besides mitochondrial OXPHOS, may affect auditory neurons in the long term. Hence, the hearing loss level in CSOM may be related to the duration of the disease [33]. While our patients with deleted mtDNA suffered from CSOM for 11 years (mean ± SE; 10.82 ± 1.60 years), duration of disease in patients without deleted mtDNA was 7 years (mean ± SE; 6.97 ± 1.86 years). These data support the idea that mitochondrial dysfunction might be linked to excessive formation of ROS and ototoxicity. Recently, a novel treatment to protect against the adverse effects of OXPHOS, through the use of antioxidants, has been discussed [7, 34]. Previous studies have reported the scavenger effect of some antioxidant agents on hearing loss-related ROS [35, 36]. Likewise, Mukherjea et al. discussed the use of different antioxidants and ROS scavengers, such as sodium thiosulfate, alpha-lipoic acid, NAC, gingko biloba extract, anti-inflammatory drugs, etc., to protect the organs of corti and auditory neurons from noise and ototoxic agents, which are the causes of acquired SNHL [37]. Antioxidants or ROS scavengers may be tested for reducing the effects of some ototoxic agents [38]. Potentially, one of the targets of antioxidant treatments will be CSOM after studies of antioxidant treatments have been finished persuasively. The study should be validated with animal models and further studies need to be carried out in order to highlight the role of mtDNA in HL.

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Conclusions CSOM is an important medical problem because of its results in addition to being common in the world. Permanent hearing impairment is one of the most important outcomes. In the present research, we studied the mtDNA4977 bp and mtDNA7400 bp deletions in patients diagnosed with CSOM, and we detected a significant correlation between mtDNA4977 bp deletion in the middle ear tissue and the level of SNHL. Audiologic and molecular biologic findings of these patients suggest that the common deletion may be a predisposing factor in sensorineural hearing loss in CSOM. We concluded that the increase of chronic inflammation-related ROS may cause mtDNA damage and this may contribute to SNHL and an antioxidant agent may be added to the CSOM treatment to protect the mtDNA from ROS. Acknowledgments We thank Prof. Hasan Turkez, PhD, from Erzurum Technical University, for statistical analysis of this study. Compliance with ethical standards Conflict of interest

11.

12.

13. 14.

15.

16.

17.

18.

The authors declare no conflict of interest. 19.

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