INTRODUCTION Genetic mutations can occur in nuclear or mitochondrial genes. The mutations in the mitochondrial DNA (mtDNA) are of exclusively maternal inheritance, being responsible for 0. 5% to 1% of the genetic causes of sensorineural auditory deficiency, but spontaneous mutations may occur. Morphologic, molecular and biochemical studies demonstrate that the oxidative phosphorylation normally declines with age. Moreover, deletions in the mitochondrial DNA have been found in some human tissues, with advanced age, and in tissues of patients with degenerative illnesses(1).
Studies demonstrate that gradual auditory deficiency, associated to age, is caused by a mitochondrial deletion of 4977 base pairs (bp), called del mtDNA
4977 being commonly related to the presbyacusis of spontaneous occurrence and without familial history(2,3). The presbyacusis is characterized by a gradual, insidious and bilateral deterioration of auditory sensitivity, generally associated to age, presenting a standard of descending curve, with loss higher than 2000Hz at the pure tone audiometric examinations, in the initial periods. It occurs, in part, as consequence of degenerative cochlear alterations. Approximately 23% of the population between 65 and 75 years of age and 40% of the population, above 75 years are affected, being this, the most common form of auditory deficiency in the United States(3).
It is classified into four distinct types by the correlation between the histopathological and audiometric aspects: sensorial, neural, strial or metabolic and conductive cochlear. Such classifications may have different molecular bases(4). In addition to these four classifications, there are two other more: mixed (association of one or more types) and undetermined (there is no possibility of correlation between the histopathological and audiometric criteria)(5). The objective of the present study is to verify, through PCR the technique, the frequency of the mitochondrial mutation del mtDNA
4977, in Brazilian patients with registered presbyacusis.
MATERIAL AND METHODS In the period between March and June 2006, a study of transversal cut was carried through, in which 100 patients were studied, at the Institution Otorhinolaryngology Clinic, of both genders (61 male and 39 female), with age between 48 years and 89 years, with registered presbyacusis. The patients were included in the study after being evaluated at least twice after the presbyacusis diagnosis. In all the included patients the clinical evaluations were always carried through by the same otorhinolaryngologist, audiometries in both ears by the same audiologist and the molecular evaluation of the mitochondrial DNA by the same researcher. The term of informed consent of all patients or tutors were obtained.
2.1 Clinical evaluation Each patient was submitted to complete anamnesis to investigate beginning age of the auditory deficiency, presence of other cases in the family and to exclude the possibility of environmental causes: perinatal complications, maternal-fetal infections, meningitis, use of ototoxic drugs, acoustic trauma, diabetes, etc. The physical, otorhinolaryngological and systemic examinations were carried through to abstain suggestive signals of syndrome forms of auditory deficiency (especially skull-face dysmorphism, tegument alterations, anomalies of branchial, cardiac, thyroidal origin, vision disorders, myopathies which are associated or not to diabetes, gait disorders, etc.). Moreover, the patients were submitted to ophthalmology evaluation (including funduscopy), vestibular tests and CT scan of temporal bone. These examinations were carried through to exclude patient with auditory deficiency caused by environmental factors, congenital malformations of internal ear or by genetic syndromes.
The patients were audiologically tested through immitanciometry, with the device Model ZODIAC 901
® - MIDDLE EAR - ANALYZER MEDSEN ELETRONIC (U.S.A.), and speech and pure-tone audiometries, with the audiometer of diagnosis - Model MAICO Me MA 41 (Maico Hearing Instruments INC., Minneapolis, U.S.A.), carried through in the Institution Phonology Clinic and those with the following audiometry parameters for the diagnosis and classification of presbyacusis(4,5) were enclosed: a) sensorial - drop in acute frequencies, generally with good discrimination, being able to save the frequencies of the conversation area. Audiogram in descending curve, of slow progression and the little affected speech recognition rate (SRR); b) neural: there is higher loss of vocal discrimination and lower loss of the tonal thresholds. Audiogram in descending curve and very low SRR; c) metabolic (strial): there is good vocal discrimination and the low SRR. Audiometric curve is plain, with loss in all frequencies; d) conductive cochlear (mechanics): audiogram in descending curve with generally preserved discrimination; e) mixing: association of different classifications; e) undetermined: compatible audiometric curve with the presbyacusis one, but without correlation with the classification ones.
2.2 Molecular analysis The genomics DNA (nuclear and mitochondrial) was extracted of samples of leukocytes of peripheral blood collected from each selected patient, using the GFX
TM Genomic Blood DNA Purification Kit (Amersham Pharmacia Biotech Incorporation), according to the manufacturer protocol, being the procedure carried through in the Research Unit of Genetics and Molecular Biology (Unidade de Pesquisa em Genética e Biologia Molecular) (UPGEM) of the Institution.
To detect the deletion mtDNA
4977, fragments of mitochondrial DNA, which comprehends the deletion region, were amplified by the technique of Polymerase Chain Reaction (PCR), in which two pairs of starters or primers were used, which are synthetic oligonucleotide(6). Pair 1 was synthesized in way that comprehended the following positions of the mitochondrial DNA: primer foward (F1), position of 8247 to 8225 and primer reverse (R1), position of 13574 to 13551. The length of the nucleotide flanked by primers F1-R1 (pair 1) is of 5350 pb in the normal mitochondrial DNA. And the primer foward of pair 2 (F2), was synthesized in way that comprehended positions 13176 to 13198, and primer reverse (R2), the positions 13724 to 13705. The expected fragment size amplified by the PCR using primers F2-R2 (pair 2) is of 549 pb in the non-deleted mitochondrial DNA. One fragment of 373 pb is amplified, by primers F1-R1 (pair 1), in the presence of the 4977 pb deletion, commonly observed in the mutant mitochondrial DNA. The mtDNA
4977 deletion occurs between two repeated direct sequences (ACCTCCCTCACCA) in the mitochondrial DNA, in positions 8470-8482 and 13447-13459.
All samples were also analyzed for amplification of certain region of the cytochrome b gene. This specific region, an highly conserved area of the mitochondrial genome, acts as a control to verify the presence of the mitochondrial DNA, in the samples of the study. With specific primers for this region, a product of 161 pb is expected to be amplified after the PCR technique(7).
All primers used for the accomplishment of the PCR enclose regions, of the codified mitochondrial sequence, of the Human Mitochondrial DNA Revised Reference Cambridge Sequence(8) and all the PCR reactions had been processed in a thermocycler (Eppendorf
®, Model Mastercycler Personal, U.S.A.).
The products of the PCR reactions were analyzed by electrophoresis in agarose gel 2.0% in TBE 1X buffer, containing ethidium bromide, in the concentration of 0.5mg/mL, submitted to the ultraviolet illumination, to confirm the success of the reaction and of the gel, registered through pictures.
Approval Committee of Ethics The study was approved (Protocol number 083/2005) by the Committee of Ethics in Human Research of the present Institution and by the Comitê Nacional em Pesquisa Humana de Brasília (DF), Brasil (National Committee in Human Research of Brasilia (DF), Brazil).
Statistical study100 patients were evaluated, in pilot study, for proportion estimation (p) of carriers of the mitochondrial deletion mtDNA
4977 in this initial sample. At this proportion (p), after being determined, the statistical formula "sizing of sample with known population" was applied, getting the size of the final sample (n) statistically needed to represent the total population (Np) of patients assisted in the period determined for the accomplishment of the research (Np=123 patient). For the calculation of the final sample (n) the aforementioned statistical formula was used, with the following parameters: p=0,00 (estimated for the sample pilot); q=0,99; zc=3.00 (99.74% of confidence); e=0,03 (3% of error of estimative); Np=123 (size of the population in the period of the study).
Formula: (n = zc2 × p × q × Np/e2 × (Np - 1) + zc2 × p × q)
Their average and percentages with standard deviations were calculated, and the results were respectively expressed in %, (SD
%) and, (SD).
RESULTS Out of the 100 Brazilian patients, 61 (61%, SD
%=4.87) are male and 39 (39%, SD
%=4.87) female. In relation to age, it varied from 48 to 76 years for men, being the average 70.35 years (SD = 1.02) and for women the age varied from 54 to 89 years, being the average 74.03 years (SD = 1.06).
The following results are presented as they were obtained after the molecular techniques performance: genomic extraction of DNA (nuclear and mitochondrial) and PCR technique, with posterior electrophoresis in agarose gel.
Extraction of the genomic DNA:The extraction of the DNA of all the samples of the study (100%) was possible based on leukocytes of peripheral blood.
PCR Technique for amplification of the cytochrome b gene:It was carried through to confirm the presence or absence of mitochondrial DNA in the samples. The amplification of the cytocrome b gene was possible, in fragment of 161 pb, confirming it presence of mitochondrial DNA in all study samples (100%).
PCR Technique for amplification of fragment that comprehends the deletion region of 4977 pb: It was carried through to verify the presence or absence of the deletion. The 373 pb fragment, which would be amplified in case there was deletion was not identified in all the samples (100%). Therefore, 5350 pb fragment was observed indicating that the samples of the study do not present the cited deletion.
PCR Technique for amplification control: It was carried through as control of amplification of the PCR, which encloses the region of the deletion, in order to confirm the presence or not of the deletion in the samples, once the product of this PCR, one 549 pb fragment amplifies in the normal mitochondrial genomes, what does not occur in the deleted ones. The amplification of 549 pb fragments was possible, thus confirming the absence of the deletion of 4977 pb in all samples of the study (100%).
The results related to the classification of the presbyacusis, according to the audiometric criteria, are presented in Table 1:
DISCUSSION This study provided, through the technique of the Polymerase Chain Reaction (PCR), the molecular analysis of the mitochondrial DNA in order to investigate the frequency of the deletion mtDNA
4977 in Brazilian patients with presbyacusis.
Since the concept of associated auditory deficiency to the age was introduced in the end of 19th century, extensive studies have been carried through for the etiological, pathological, histological and epidemiology diagnosis of the presbyacusis and, although it is common in industrialized countries, is rare in other countries, which makes this difference be attributed environmental, dietary differences and genetic factors(2,9,10).
Once it is impossible to analyze the common deletion of aging from samples extracted from temporal bone of living individuals, some studies have used the PCR technique to verify the presence of deletions in the mitochondrial DNA, not only filed in samples of temporal human bone(2,10-12), but also in human leukocytes(6,13), as it was carried through in the present study, even not being able to reflect the amount of existing mitochondria in the sensorial auditory tissue(6).
In performed studies, the deletion mtDNA
4977 and the mutation mtDNA A3243G in Japanese patients with sensorineural deafness were found, thus revealing that it is possible to identify deletions and/or mutations in the mitochondrial DNA through the analysis in leukocytes, finding, respectively, the frequency of 75% (45/60)(6) and 15% (3/20)(13) in the analyzed cases.
The number samples of the present study, 100 cases, was very superior to the studies carried through both in histological cuts of human being cochlea or of rats and in human leukocytes, varying from 3 to 60 cases(2,6,7,12,13). The methodology used in the present study was similar to the described ones in literature(6,7), thus allowing the amplification of the non-deleted mitochondrial DNA and the specific region of the cytocrome b gene, a highly conserved area of the mitochondrial genome acting as a control to verify the mitochondrial presence of DNA in the samples.
However, even with a bigger casuistry and rigorously similar methodology to the studies of reference(6,7) the deletion mtDNA
4977 was not found in all the analyzed samples, differently from the revised studies that had found the deletion in a 47% to 66% frequency of the cases, including analyses in histological cuts of cochlea and leukocytes, as it has been previously mentioned(2,6,7,12,13). Exception to the only described study(2) in which the authors had analyzed filed histological cuts of temporal bone of three patients with presbyacusis, finding a patient without the deletion mtDNA
4977.
In the present study, the phenotypes of the patients with presbyacusis are in accordance with the classification of Schuknecht et al. (1993)(4,5), following the standards of the audiometric curve. Even with most of the samples having presented the most common sensorial, neural and metabolic types, molecular relation in the different phenotypes found in the sample was not established, although the criteria of election of the patients and phenotypic results of the present study to have been similar to the ones of literature, in which a heredity succession relation was determined, that is, patients with the sensorial or strial types presented the deletion, allowing the authors to establish a molecular relation with the existing histopathological alterations in the several types of found presbyacusis(14,15).
The differences in the molecular results obtained in the present study could be explained by some reasons that perhaps justified why these patients with presbyacusis did not present the deletion: the different classifications of the presbyacusis, its genetic heterogeneity and the fact it is a multifactorial process influenced by hereditary and changeable environmental factors(7), in addition to the fact that ethnic composition of the Brazilian population is highly heterogeneous, resulting in different prevalence rates between the Brazilian regions, which justifies, as described in the literature, that the presbyacusis is part of multifactorial processes, because of which etiological differences between different societies may occur(7,11,16).
Probably, the susceptibility to the accumulation of mitochondrial deletions varies among individuals and among different populations. Perhaps in some people the accumulation of deletions, during the aging process, can occur more slowly, thus contributing, together with other mutagenic alterations in the mitochondrial DNA such as the injuries induced by the free radicals, for the loss of the mitochondrial bioenergetic production. Therefore, the amount of deletions and the threshold for the expression of the mitochondrial illnesses can vary between individuals and populations(17).
Many deletions can contribute for the presbyacusis and the mitochondrial deletion mtDNA
4977 can be only one of them. The identification of underlying genetic causes of the presbyacusis is basic to assist in the counceling of families and to allow precocious rehabilitation having, as consequence, the inclusion and/or re-inclusion in the social and professional activities, besides propitiating a worthy familiar living to all these patients.
There is the necessity of a multicentre study to determine the current prevalence of the MtDNA
4977 deletion in Brazilian population and, also, to investigate other mutations that can be associated to the aging and the presbyacusis.
CONCLUSION The absence of the mutation del mtDNA
4977, in the mitochondrial genome, does not discard the possibility of the existence of mutations in other genes in the patients with presbyacusis, which suggests the necessity of posterior studies.
The Polymerase Reaction Chain technique (PCR), with the used protocol, is a method of easy execution for tracking of the mitochondrial deletion mtDNA
4977 collaborating, therefore, for the molecular ivestigation of the presbyacusis.
ACKNOWLEDGEMENTSWe thank the patients without whose consent and cooperation, this study would not be possible. This contribution is extremely important to allow the continuity of the scientific research in order to provide a better future to the Brazilian people. We thank our friend and translator Cecília Meneguette Blacksmith, for her considerable aid.
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1. Master (Aid Professor)
2. Professor (Chief of Otorhinolaryngology and Head and Neck Surgery Departament)
3. Doctor Professor (Aid Professor)
4. Master (Chief of Phonology Clinic)
5. MAster (Head Nurse at Otorhinolaryngology Clinic)
6. Doctor Professor (Biologist)
7. Senior Medicine Undergraduate
Faculdade de Medicina de São José do Rio Preto, SP - FAMERP
Address: Av. Brigadeiro Faria Lima, nº 5416, Vila São Pedro, São José do Rio Preto, SP, Brazil. Cep:15090-000. Phone: +55-17-32015747.
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This article was submitted to SGP (Sistema de Gestão de Publicações) of R@IO on February 21,2007 and approved on March 29, 2007 at 22:31:47.
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