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Study of modifiers factors associated to mitochondrial mutations in individuals with hearing impairment
Biochemical and Biophysical Research Communications 381 (2009) 210–213
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Study of modifiers factors associated to mitochondrial mutations in individuals with hearing impairment Vanessa Cristine Sousa de Moraes a, Fabiana Alexandrino a, Paula Baloni Andrade a, Marília Fontenele Câmara b, Edi Lúcia Sartorato a,* a b
Center of Molecular Biology and Genetic Engineering (CBMEG), Molecular Biology Laboratory, State University of Campinas—UNICAMP, Brazil Department of Phonoaudiology, University of Fortaleza-UNIFOR, Brazil
a r t i c l e
i n f o
Article history: Received 28 January 2009 Available online 12 February 2009
Keywords: Hearing loss Deafness Hearing impairment Nuclear modulator gene Nuclear modifier gene Aminoglycosides Mitochondrial mutation 12S rRNA MTRNR1 A827G A1555G MTO1 TRMU G28T
a b s t r a c t Hearing impairment is the most prevalent sensorial deficit in the general population. Congenital deafness occurs in about 1 in 1000 live births, of which approximately 50% has hereditary cause in development countries. Non-syndromic deafness can be caused by mutations in both nuclear and mitochondrial genes. Mutations in mtDNA have been associated with aminoglycoside-induced and non-syndromic deafness in many families worldwide. However, the nuclear background influences the phenotypic expression of these pathogenic mutations. Indeed, it has been proposed that nuclear modifier genes modulate the phenotypic manifestation of the mitochondrial A1555G mutation in the MTRNR1 gene. The both putative nuclear modifiers genes TRMU and MTO1 encoding a highly conserved mitochondrial related to tRNA modification. It has been hypothesizes that human TRMU and also MTO1 nuclear genes may modulate the phenotypic manifestation of deafness-associated mitochondrial mutations. The aim of this work was to elucidate the contribution of mitochondrial mutations, nuclear modifier genes mutations and aminoglycoside exposure in the deafness phenotype. Our findings suggest that the genetic background of individuals may play an important role in the pathogenesis of deafness-associated with mitochondrial mutation and aminoglycoside-induced. Ó 2009 Elsevier Inc. All rights reserved.
Introduction In the developed countries, the aminoglycoside antibiotics are mainly used in the treatment of hospitalized patients with aerobic Gram-negative bacterial infections, particularly in patients with chronic infections. [1]. However, in developing countries aminoglycosides are used even for relative minor infections. These drugs are known to exert their antibacterial effects by direct binding to 16S ribosomal RNA in the 30S subunit of the bacterial ribosome causing premature termination of protein synthesis [2]. The aminoglycosides may become concentrated in the perilymph and endolymph of the inner ear, potentially leading to ototoxicity [3].
* Corresponding author. Address: Laboratório de Genética Molecular Humana, UNICAMP/CBMEG, Cidade Universitária Zeferino Vaz s/n., Barão Geraldo, Campinas, SP 13083-970, Brazil. Fax: +55 19 3251 1089. E-mail address: [email protected] (E.L. Sartorato). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.02.014
In familial cases of ototoxic deafness, the aminoglycoside hypersensitivity is usually maternally transmitted, suggesting mitochondrial genome involvement [4]. A number of distinct mutations in the mitochondrial DNA (mtDNA) have been found to be associated with both syndromic and non-syndromic forms of hearing loss [4,5]. The most commonly reported SNHL associated with mtDNA mutations are A1555G, T1095C, C1494T and C insertion or deletion at position 961 in the rRNA gene [6–9], and the A7445G, 7472insC, T7510C and T1511C in the tRNASer(UCN) gene [10–13]. Of these, the homoplasmic A1555G mutation was associated with aminoglycoside-induced and non-syndromic deafness in many families from different ethnic backgrounds [14,15]. In absence of aminoglycosides, the A1555G mutation was responsible for a clinical phenotype that ranges from severe congenital deafness to moderate progressive hearing loss with later onset [16], to completely normal hearing [16,17]. Recently, A827G mutation in the 12S rRNA gene has been identified in a Chinese families with aminoglycoside-induced and SNHL, suggesting that mitochondrial 12S rRNA gene is a hot-spot
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for deafness-associated mutations [18,19]. There are proposed an interaction between nuclear and mitochondrial genes that modulate the phenotypic manifestation of the mitochondrial mutations. The nuclear modifier genes TRMU and MTO1 encode a highly conserved mitochondrial protein related to tRNA modification, developing an important role in the phenotypic expression of SNHL-induced by aminoglycosides associated mitochondrial mutations [20,21]. Therefore, we studied the mutations in mitochondrial genes, genes nuclear modulators and their interactions with the use of aminoglycoside antibiotics and/or ototoxic drugs. We performed a mutational screening of TRMU and MTO1 genes to examine the role of genes in the phenotypic expression mitochondrial mutations. In the present study, we reported the clinical and genetic findings in Brazilian patients with mitochondrial A827G mutation and aminoglycoside-induced SNHL. We have identified eight cases carrying the A827G mutation and were observed five cases with mutations in the TRMU and MTO1 nuclear genes.
were digested with a restriction enzyme HphI as described by Wang et al. (2006) [23]. The digested samples were then analyzed by electrophoresis through 1.5% agarose gel. Mutational screening of the mitochondrial A1555G mutation. The detection of the A1555G mitochondrial mutation was performed by PCR amplification followed by the digestion with restriction endonuclease BsmAI and according to the manufacturer’s recommended digestion condition [24,25]. Mutational analysis of the G28T mutation in the TRMU nuclear gene. The G28T variant was PCR amplified followed by digestion with the restriction enzyme Bsp1286I. The digested products were analyzed on 1.5% agarose gels [21]. Mutation analysis of the MTO1 nuclear gene. The 12 exons of MTO1 nuclear gene were screening by denaturing high-performance liquid chromatography (DHPLC) followed by direct sequencing in an ABI PRISMÒ 3700 DNA analyzer.
Subjects and methods
The Table 1 below summarizes the mutations found in different genes studied.
Subjects. A total of 85 Brazilian individuals were enrolled. Written informed consent was obtained from all the subjects included in the study or their parents. These subjects were divided in four groups in order to elucidate the etiology of their hearing impairment and the bases of aminoglycoside ototoxicity. Group A—25 newborns at risk with aminoglycoside-induced and sensorineural non-syndromic hearing impairment. Group B—25 newborns at risk with aminoglycoside-induced and normal hearing. Group C—25 individuals with sensorineural non-syndromic deafness and no history of ototoxy medication. Group D—10 adults individuals with sensorineural non-syndromic deafness and positive for A1555G mutation in the 12S rRNA mitochondrial gene and with no aminoglycoside history. The DNA samples of individuals of Groups A–C were obtained from Maternidade-Escola Assis Chateaubriand (Assis Chateaubriand Maternity-School) from Universidade Federal do Ceará (Federal University of Ceará). The samples of individuals of Group D were collected from Universidade Estadual de Campinas (State University of Campinas). After obtaining written informed consent, DNA samples were extracted from whole blood by standard phenol–chloroform method. The samples were tested for the presence of mutations in the coding region of GJB2, the two deletions affecting GJB6 [del(GJB6D13S1830), del(GJB6-D13S1854)], the A827G, C1494T and A1555G mitochondrial mutations in the 12S rRNA gene were analyzed. In addition, G28T mutation in the TRMU nuclear gene and mutations in the MTO1 nuclear gene were analyzed. Mutation analysis of the DFNB1 locus. The DNA fragments spanning the entire coding region of GJB2 gene were amplified by PCR and subsequent sequencing analysis were performed. The results were compared with the wild type GJB2 sequence (GenBank Acession No. GI62999485) to identify the mutations. To detect GJB6 deletions, a specific PCR assay was used, as described by [22]. Mutational screening of the mitochondrial A827G mutation. Genomic DNA was isolated from whole blood of participants using the standard phenol–chloroform method. The A827G variant was PCR amplified using the oligonucleotides corresponding to the mitochondrial genome at positions 611–1411. PCR conditions were follows as: initial denaturation at 94 °C for 5 min, followed by 32 three-step cycles (94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min), with a final extension at 72 °C for 10 min. The fragments were analyzed by direct sequencing in an ABI PRISMÒ 3700 DNA analyzer. Mutational screening of the mitochondrial C1494T mutation. For the detection of the C1494T mutation, the amplified segments
Results and discussion
Mutational analysis of DFNB1 locus To examine the role of GJB2 we also conducted the mutation screening of GJB2 gene in the 85 subjects. We detected five individuals homozygous for the 35delG mutation in the GJB2 gene. Mitochondrial DNA analysis To further elucidate the molecular basis of hearing loss, we have performed a mutational analysis of A827G, C1494T and A1555G in the 12S rRNA mitochondrial gene in 85 subjects. No one individual presented the C1494T mitochondrial mutation. The mutation A1555G was not found in group A–C. As mentioned, the group D was previously selected by the presence of A1555G mutation. The A827G mutation was found in all four groups. In group A two patient was detected positive for A287G, one of them has both A827G mitochondrial mutation and G28T mutation in TRMU nuclear gene. In group B two individuals presented the A827G mutation and one of them presented the I392M mutation in the MTO1 nuclear gene too. In group C five individuals were detected positive for A827G, two of them presented two different alterations in the MTO1 nuclear gene. In group D one subject is positive for A1555G, A827G mutation in the 12S rRNA and G28T in the TRMU gene, as has been described. Mutation analysis of TRMU nuclear gene To determinate whether TRMU modulates the phenotypic expression of the A1555G or the A827G mutation, we performed a G28T mutation analysis of TRMU gene. We detected G28T mutation in three groups. In group A two cases of TRMU mutation were found, one of them has both G28T mutation in TRMU nuclear gene and A827G mitochondrial mutation in the 12S rRNA. In group C one patient was detected positive for G28T mutation. Finally, in group D one individual has three mutations, among them A1555G, A827G in the 12S rRNA gene and G28T. Mutation analysis of MTO1 nuclear gene To determinate whether MTO1 modulates the phenotypic expression of the A1555G or the A827G mutation, we performed mutation screening of MTO1 nuclear gene. We detected four different alterations in the MTO1 gene. We failed to detect mutations in
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Table 1 The different mutations found in all four groups. OD, ototoxicity drugs; (+), positive; ( ), negative; NP, not performed; N/N, normal homozygous individual; N, normal individual; M, mutant individual; N/M, heterozygous individual. Group
Subjects
OD
GJB2
GJB6
Mitochondrial mutations
35DelG
Others
Del 1
Del 2
A1555G
C1494T
A827G
TRMU G28T
MTO1
A
5 7 19 20
+ + + +
N/N N/N N/N N/N
N/N N/N N/N N/N
N/N N/N N/N N/N
N/N N/N N/N N/N
N N N N
N N N N
N M N M
N/M N/M N/M N/N
NP N/N NP N/N
B
7 20
+ +
N/N N/N
N/N N/N
N/N N/N
N/N N/N
N N
N N
M M
N/N N/N
I392M N/N
C
2 3 6 7 10 13 16 19 22 24
N/N N/N M/M M/M N/N M/M N/N N/N M/M M/M
N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N
N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N
N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N
N N N N N N N N N N
N N N N N N N N N N
M M N N M N M M N N
N/N N/N N/N N/N N/N N/N N/N N/M N/N N/N
N/N N/N NP NP IVS 11–7 T>G NP N/N rs4509087 exon 07 NP NP
D
1 2 3 4 5 6 9 10
N/N N/N N/N N/N N/N N/N N/N N/N
N/N N/N N/N N/N N/N N/N N/N N/N
N/N N/N N/N N/N N/N N/N N/N N/N
N/N N/N N/N N/N N/N N/N N/N N/N
M M M M M M M M
N N N N N N N N
N N N M N N N N
N/N N/N N/N N/M N/N N/N N/N N/N
rs4509087 rs7766570 rs7766570 rs4509087 rs4509087 rs7766570 rs4509087 rs4509087
the MTO1 nuclear gene in group A. In group B we found a I392M mutation, which changes the base C for the base G, in the MTO1 gene in one individual positive for A827G mitochondrial mutation. The I392M mutation, located in the exon 06 of the MTO1 gene, leads to the change of the isoleucine aminoacid for the methionine aminoacid. In group C we detected two alterations in the MTO1 gene, the first one is the SNP rs4509087 found in the exon 07 in one individual positive for both A827G mitochondrial mutation and G28T in the TRMU nuclear gene. This SNP rs4509087 is the change of the base T for the base C. The second alteration that was found in group C is the IVS 11–7 T>G, which consist of the change of the base T for the base G in the intron 11 of the MTO1 nuclear gene. The IVS 11–7 T>G was detected in two individuals, one of them positive for the A827G mitochondrial mutation. In group D we detected three alterations in the MTO1 nuclear gene, two of them are the SNP rs4509087 in the exon 07 found in four individuals and the IVS 11–7 T>G mutation in the intron 11 found in two individuals. The third mutation found is the SNP rs7766570, which changes the base G for the base A and it’s located in the intron 05 of the MTO1 gene. The SNP rs7766570 was found in five individuals of the group D. In group D one individual presented five mutations, among them A1555G, A827G in the 12S rRNA gene, G28T in the TRMU nuclear gene, SNP rs4509087 and the IVS 11–7 T>G in the MTO1 nuclear gene. In summary all the alterations observed in the MTO1 gene appear to be polymorphisms that are not related with the incomplete penetrance the mitochondrial mutations found. The TRMU gene has been show to modulate the severity of hearing loss associated with the mitochondrial mutation. This way, we performed a mutational screening of the TRMU gene to examine the role of this gene in the phenotypic expression of the A827G mutation in Brazilian patients. Sequence analysis revealed the presence of G28T mutation in the TRMU gene in three subjects carrying the A827G mutation in the 12S rRNA gene. Therefore, according the literature, G28T mutation in the TRMU gene showing to modulate the expression for the A827G mutation in the 12S rRNA
exon 07 and rs7766570 intron 05 intron 05 intron 05 exon 07 and IVS 11–7 T>G exon 07 intron 05 exon 07, rs7766570 intron 05 and IVS 11–7 T>G exon 07 and rs7766570 intron 05
gene in three subjects in this study and one subject carrying the A1555G mutation too. Aminoglycoside antibiotics are clinically important drugs used worldwide for controlling bacteria-related infections, especially in developing countries [3]. The aminoglycosides exert their antibacterial effects by directly binding to 16S rRNA of the bacterial ribosome [2]. As the human mitochondrial share many similarities to bacterial ribosomes, it is proposed that one of the primary targets for the aminoglycoside antibiotics in mammalian cells is the small ribosomal RNA (12S rRNA) of mitochondrial ribosome [26]. The C1494T, A1555G and A827G mitochondrial mutations are located at the A-site of the mitochondrial 12S rRNA gene. It is possible that the alteration of the tertiary or quaternary structure of the rRNA by these mitochondrial mutations may lead to mitochondrial dysfunction, thereby playing a role in the pathogenesis of hearing loss and/or aminoglycoside hypersensitivity [19]. Accumulation of aminoglycosides in cochlear mitochondria would lead to an inhibition of protein synthesis by interacting with the 12S rRNA carrying these mitochondrial mutations. As a result of this mitochondrial translation defect, the ATP production declines and the generation of reactive oxygen species increases, consequently damaging hair cells and giving rise to hearing impairment [26]. We have identified a homoplasmic A827G transition in the mitochondrial 12S rRNA gene. This mutation has been implicated to be associated with both aminoglycoside ototoxicity and nonsyndromic hearing loss in a few genetically unrelated individuals [27], indicating that A827G mutation is involved in the pathogenesis of hearing impairment. Finally, the A827G mutation is located at the A-site of the mitochondrial 12S rRNA gene which is highly evolutionary conserved in human [27]. It is possible that the alteration of the tertiary or quaternary structure of this rRNA by the A827G mutation may lead to mitochondrial dysfunction, thereby playing a role in the pathogenesis of hearing loss and aminoglycoside hypersensitivity. The incomplete penetrance indicates that the A827G mutation alone is not sufficient to produce clinical phenotype.
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Conclusion In all four groups studied, the mutations found seem not to be sufficient to cause clinical symptoms. Probably the SNPs in MTO1 gene or in other nuclear genes may be involved to explain the phenotype. Therefore, previous studies is necessary to showed that the expression of clinical phenotype of non-syndromic deafness-associated mitochondrial mutations required for the contribution of modulating factors including aminoglycosides, mitochondrial haplotypes or nuclear modifier genes. References [1] M.A. Sande, G.L. Mandell, The Pharmacological Basis of Therapeutics, eight ed., Pergamon Press, New York, 1990. [2] Z. Li, R. Li, J. Chen, Z. Liao, Y. Zhu, Y. Qian, S. Xiong, S. Heman-Ackah, J. Wu, D.I. Choo, M.X. Guan, Mutational analysis of the mitochondrial 12S rRNA gene in Chinese pediatric subjects with aminoglycoside-induced and non-syndromic hearing loss, Hum. Genet. 117 (1) (2005) 9–15. [3] N. Fischel-Ghodsian, Genetic factors in aminoglycoside toxicity, Pharmacogenomics 6 (2005) 27–36. [4] N. Fischel-Ghodsian, Mitochondrial deafness mutations reviewed, Hum. Mutat. 13 (1999) 261–270. [5] G. Van Camp, R.J. Smith, Maternally inherited hearing impairment, Clin. Genet. 57 (2000) 589–646. [6] C. Bacino, T.R. Prezant, X. Bu, P. Fournier, N. Fischel-Ghodsian, Susceptibility mutations in the mitochondrial small ribosomal RNA gene in aminoglycoside induced deafness, Pharmacogenetics 5 (1995) 165–172. [7] M. Yoshida, T. Shintani, M. Hirao, T. Himi, A. Yamaguchi, K. Kikuchi, Aminoglycoside-induced hearing loss in a patient with the 961 mutation in mitochondrial DNA, ORL J. Otorhinolarybgol Relat. Spec. 64 (2002) 219–222. [8] R. Li, G. Xing, M. Yan, X. Cao, X.Z. Liu, X. Bu, M.X. Guan, Cosegragation of Cinsertion at position 961 with the A1555G mutation of mitochondrial 12S rRNA gene in a large Chinese family with maternally hearing loss, Am. J. Med. Genet. A 124 (2004) 113–117. [9] H. Zhao, R. Li, Q. Wang, Q. Yan, J.H. Deng, D. Han, Y. Bai, W.Y. Young, M.X. Guan, Maternally inherited aminoglycoside-induced and nonsyndromic deafness is associated with the novel C1494T mutation in the mitochondrial 12S rRNA gene in a large Chinese family, Am. J. Med. Genet. A 74 (2004) 139–159. [10] T.P. Hutchin, N.J. Lench, S. Arbuza, A.F. Markham, R.F. Mueller, Maternally inherited hearing impairment in a family with mitochondrial DNA A7445G mutation, Eur. J. Hum. Genet. 9 (2001) 56–58. [11] T.P. Hutchin, N.C. Navarro-Coy, G. Van Camp, V. Tiranti, M. Zeviani, M. Schuelke, M. Jaksch, V. Newton, R.F. Mueller, Multiple origins of the mtDNA 7472insC mutation associated with hearing loss neurological dysfunction, Eur. J. Hum. Genet. 9 (2001) 384–387. [12] T.P. Hutchin, M.J. Parker, I.D. Yong, A.C. Davis, L.J. Pulleyn, J. Deeble, N.J. Lench, A.F. Markham, R.F. Muelle, Anovel mutation in the mitochondrial t RNA Ser(UCN) gene in a family with nonsyndromic sensorineural hearing impairment, J. Med. Genet. 37 (2000) 692–694. [13] E. Chapiro, D. Feldmann, F. Denoyelle, D. Sternberg, C. Jardel, M.M. Eliot, D. Bouccara, D. Weil, E.N. Garabedian, R. Couderc, C. Petit, S. Marlin, Two large French pedigrees with non syndromic sensorineural deafness and the mitochondrial DNA T7511C mutation: evidence for a modulatory factor, Eur. J. Hum. Genet. 10 (2002) 851–856.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Study of modifiers factors associated to mitochondrial mutations in individuals with hearing impairment Vanessa Cristine Sousa de Moraes a, Fabiana Alexandrino a, Paula Baloni Andrade a, Marília Fontenele Câmara b, Edi Lúcia Sartorato a,* a b
Center of Molecular Biology and Genetic Engineering (CBMEG), Molecular Biology Laboratory, State University of Campinas—UNICAMP, Brazil Department of Phonoaudiology, University of Fortaleza-UNIFOR, Brazil
a r t i c l e
i n f o
Article history: Received 28 January 2009 Available online 12 February 2009
Keywords: Hearing loss Deafness Hearing impairment Nuclear modulator gene Nuclear modifier gene Aminoglycosides Mitochondrial mutation 12S rRNA MTRNR1 A827G A1555G MTO1 TRMU G28T
a b s t r a c t Hearing impairment is the most prevalent sensorial deficit in the general population. Congenital deafness occurs in about 1 in 1000 live births, of which approximately 50% has hereditary cause in development countries. Non-syndromic deafness can be caused by mutations in both nuclear and mitochondrial genes. Mutations in mtDNA have been associated with aminoglycoside-induced and non-syndromic deafness in many families worldwide. However, the nuclear background influences the phenotypic expression of these pathogenic mutations. Indeed, it has been proposed that nuclear modifier genes modulate the phenotypic manifestation of the mitochondrial A1555G mutation in the MTRNR1 gene. The both putative nuclear modifiers genes TRMU and MTO1 encoding a highly conserved mitochondrial related to tRNA modification. It has been hypothesizes that human TRMU and also MTO1 nuclear genes may modulate the phenotypic manifestation of deafness-associated mitochondrial mutations. The aim of this work was to elucidate the contribution of mitochondrial mutations, nuclear modifier genes mutations and aminoglycoside exposure in the deafness phenotype. Our findings suggest that the genetic background of individuals may play an important role in the pathogenesis of deafness-associated with mitochondrial mutation and aminoglycoside-induced. Ó 2009 Elsevier Inc. All rights reserved.
Introduction In the developed countries, the aminoglycoside antibiotics are mainly used in the treatment of hospitalized patients with aerobic Gram-negative bacterial infections, particularly in patients with chronic infections. [1]. However, in developing countries aminoglycosides are used even for relative minor infections. These drugs are known to exert their antibacterial effects by direct binding to 16S ribosomal RNA in the 30S subunit of the bacterial ribosome causing premature termination of protein synthesis [2]. The aminoglycosides may become concentrated in the perilymph and endolymph of the inner ear, potentially leading to ototoxicity [3].
* Corresponding author. Address: Laboratório de Genética Molecular Humana, UNICAMP/CBMEG, Cidade Universitária Zeferino Vaz s/n., Barão Geraldo, Campinas, SP 13083-970, Brazil. Fax: +55 19 3251 1089. E-mail address: [email protected] (E.L. Sartorato). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.02.014
In familial cases of ototoxic deafness, the aminoglycoside hypersensitivity is usually maternally transmitted, suggesting mitochondrial genome involvement [4]. A number of distinct mutations in the mitochondrial DNA (mtDNA) have been found to be associated with both syndromic and non-syndromic forms of hearing loss [4,5]. The most commonly reported SNHL associated with mtDNA mutations are A1555G, T1095C, C1494T and C insertion or deletion at position 961 in the rRNA gene [6–9], and the A7445G, 7472insC, T7510C and T1511C in the tRNASer(UCN) gene [10–13]. Of these, the homoplasmic A1555G mutation was associated with aminoglycoside-induced and non-syndromic deafness in many families from different ethnic backgrounds [14,15]. In absence of aminoglycosides, the A1555G mutation was responsible for a clinical phenotype that ranges from severe congenital deafness to moderate progressive hearing loss with later onset [16], to completely normal hearing [16,17]. Recently, A827G mutation in the 12S rRNA gene has been identified in a Chinese families with aminoglycoside-induced and SNHL, suggesting that mitochondrial 12S rRNA gene is a hot-spot
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for deafness-associated mutations [18,19]. There are proposed an interaction between nuclear and mitochondrial genes that modulate the phenotypic manifestation of the mitochondrial mutations. The nuclear modifier genes TRMU and MTO1 encode a highly conserved mitochondrial protein related to tRNA modification, developing an important role in the phenotypic expression of SNHL-induced by aminoglycosides associated mitochondrial mutations [20,21]. Therefore, we studied the mutations in mitochondrial genes, genes nuclear modulators and their interactions with the use of aminoglycoside antibiotics and/or ototoxic drugs. We performed a mutational screening of TRMU and MTO1 genes to examine the role of genes in the phenotypic expression mitochondrial mutations. In the present study, we reported the clinical and genetic findings in Brazilian patients with mitochondrial A827G mutation and aminoglycoside-induced SNHL. We have identified eight cases carrying the A827G mutation and were observed five cases with mutations in the TRMU and MTO1 nuclear genes.
were digested with a restriction enzyme HphI as described by Wang et al. (2006) [23]. The digested samples were then analyzed by electrophoresis through 1.5% agarose gel. Mutational screening of the mitochondrial A1555G mutation. The detection of the A1555G mitochondrial mutation was performed by PCR amplification followed by the digestion with restriction endonuclease BsmAI and according to the manufacturer’s recommended digestion condition [24,25]. Mutational analysis of the G28T mutation in the TRMU nuclear gene. The G28T variant was PCR amplified followed by digestion with the restriction enzyme Bsp1286I. The digested products were analyzed on 1.5% agarose gels [21]. Mutation analysis of the MTO1 nuclear gene. The 12 exons of MTO1 nuclear gene were screening by denaturing high-performance liquid chromatography (DHPLC) followed by direct sequencing in an ABI PRISMÒ 3700 DNA analyzer.
Subjects and methods
The Table 1 below summarizes the mutations found in different genes studied.
Subjects. A total of 85 Brazilian individuals were enrolled. Written informed consent was obtained from all the subjects included in the study or their parents. These subjects were divided in four groups in order to elucidate the etiology of their hearing impairment and the bases of aminoglycoside ototoxicity. Group A—25 newborns at risk with aminoglycoside-induced and sensorineural non-syndromic hearing impairment. Group B—25 newborns at risk with aminoglycoside-induced and normal hearing. Group C—25 individuals with sensorineural non-syndromic deafness and no history of ototoxy medication. Group D—10 adults individuals with sensorineural non-syndromic deafness and positive for A1555G mutation in the 12S rRNA mitochondrial gene and with no aminoglycoside history. The DNA samples of individuals of Groups A–C were obtained from Maternidade-Escola Assis Chateaubriand (Assis Chateaubriand Maternity-School) from Universidade Federal do Ceará (Federal University of Ceará). The samples of individuals of Group D were collected from Universidade Estadual de Campinas (State University of Campinas). After obtaining written informed consent, DNA samples were extracted from whole blood by standard phenol–chloroform method. The samples were tested for the presence of mutations in the coding region of GJB2, the two deletions affecting GJB6 [del(GJB6D13S1830), del(GJB6-D13S1854)], the A827G, C1494T and A1555G mitochondrial mutations in the 12S rRNA gene were analyzed. In addition, G28T mutation in the TRMU nuclear gene and mutations in the MTO1 nuclear gene were analyzed. Mutation analysis of the DFNB1 locus. The DNA fragments spanning the entire coding region of GJB2 gene were amplified by PCR and subsequent sequencing analysis were performed. The results were compared with the wild type GJB2 sequence (GenBank Acession No. GI62999485) to identify the mutations. To detect GJB6 deletions, a specific PCR assay was used, as described by [22]. Mutational screening of the mitochondrial A827G mutation. Genomic DNA was isolated from whole blood of participants using the standard phenol–chloroform method. The A827G variant was PCR amplified using the oligonucleotides corresponding to the mitochondrial genome at positions 611–1411. PCR conditions were follows as: initial denaturation at 94 °C for 5 min, followed by 32 three-step cycles (94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min), with a final extension at 72 °C for 10 min. The fragments were analyzed by direct sequencing in an ABI PRISMÒ 3700 DNA analyzer. Mutational screening of the mitochondrial C1494T mutation. For the detection of the C1494T mutation, the amplified segments
Results and discussion
Mutational analysis of DFNB1 locus To examine the role of GJB2 we also conducted the mutation screening of GJB2 gene in the 85 subjects. We detected five individuals homozygous for the 35delG mutation in the GJB2 gene. Mitochondrial DNA analysis To further elucidate the molecular basis of hearing loss, we have performed a mutational analysis of A827G, C1494T and A1555G in the 12S rRNA mitochondrial gene in 85 subjects. No one individual presented the C1494T mitochondrial mutation. The mutation A1555G was not found in group A–C. As mentioned, the group D was previously selected by the presence of A1555G mutation. The A827G mutation was found in all four groups. In group A two patient was detected positive for A287G, one of them has both A827G mitochondrial mutation and G28T mutation in TRMU nuclear gene. In group B two individuals presented the A827G mutation and one of them presented the I392M mutation in the MTO1 nuclear gene too. In group C five individuals were detected positive for A827G, two of them presented two different alterations in the MTO1 nuclear gene. In group D one subject is positive for A1555G, A827G mutation in the 12S rRNA and G28T in the TRMU gene, as has been described. Mutation analysis of TRMU nuclear gene To determinate whether TRMU modulates the phenotypic expression of the A1555G or the A827G mutation, we performed a G28T mutation analysis of TRMU gene. We detected G28T mutation in three groups. In group A two cases of TRMU mutation were found, one of them has both G28T mutation in TRMU nuclear gene and A827G mitochondrial mutation in the 12S rRNA. In group C one patient was detected positive for G28T mutation. Finally, in group D one individual has three mutations, among them A1555G, A827G in the 12S rRNA gene and G28T. Mutation analysis of MTO1 nuclear gene To determinate whether MTO1 modulates the phenotypic expression of the A1555G or the A827G mutation, we performed mutation screening of MTO1 nuclear gene. We detected four different alterations in the MTO1 gene. We failed to detect mutations in
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Table 1 The different mutations found in all four groups. OD, ototoxicity drugs; (+), positive; ( ), negative; NP, not performed; N/N, normal homozygous individual; N, normal individual; M, mutant individual; N/M, heterozygous individual. Group
Subjects
OD
GJB2
GJB6
Mitochondrial mutations
35DelG
Others
Del 1
Del 2
A1555G
C1494T
A827G
TRMU G28T
MTO1
A
5 7 19 20
+ + + +
N/N N/N N/N N/N
N/N N/N N/N N/N
N/N N/N N/N N/N
N/N N/N N/N N/N
N N N N
N N N N
N M N M
N/M N/M N/M N/N
NP N/N NP N/N
B
7 20
+ +
N/N N/N
N/N N/N
N/N N/N
N/N N/N
N N
N N
M M
N/N N/N
I392M N/N
C
2 3 6 7 10 13 16 19 22 24
N/N N/N M/M M/M N/N M/M N/N N/N M/M M/M
N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N
N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N
N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N
N N N N N N N N N N
N N N N N N N N N N
M M N N M N M M N N
N/N N/N N/N N/N N/N N/N N/N N/M N/N N/N
N/N N/N NP NP IVS 11–7 T>G NP N/N rs4509087 exon 07 NP NP
D
1 2 3 4 5 6 9 10
N/N N/N N/N N/N N/N N/N N/N N/N
N/N N/N N/N N/N N/N N/N N/N N/N
N/N N/N N/N N/N N/N N/N N/N N/N
N/N N/N N/N N/N N/N N/N N/N N/N
M M M M M M M M
N N N N N N N N
N N N M N N N N
N/N N/N N/N N/M N/N N/N N/N N/N
rs4509087 rs7766570 rs7766570 rs4509087 rs4509087 rs7766570 rs4509087 rs4509087
the MTO1 nuclear gene in group A. In group B we found a I392M mutation, which changes the base C for the base G, in the MTO1 gene in one individual positive for A827G mitochondrial mutation. The I392M mutation, located in the exon 06 of the MTO1 gene, leads to the change of the isoleucine aminoacid for the methionine aminoacid. In group C we detected two alterations in the MTO1 gene, the first one is the SNP rs4509087 found in the exon 07 in one individual positive for both A827G mitochondrial mutation and G28T in the TRMU nuclear gene. This SNP rs4509087 is the change of the base T for the base C. The second alteration that was found in group C is the IVS 11–7 T>G, which consist of the change of the base T for the base G in the intron 11 of the MTO1 nuclear gene. The IVS 11–7 T>G was detected in two individuals, one of them positive for the A827G mitochondrial mutation. In group D we detected three alterations in the MTO1 nuclear gene, two of them are the SNP rs4509087 in the exon 07 found in four individuals and the IVS 11–7 T>G mutation in the intron 11 found in two individuals. The third mutation found is the SNP rs7766570, which changes the base G for the base A and it’s located in the intron 05 of the MTO1 gene. The SNP rs7766570 was found in five individuals of the group D. In group D one individual presented five mutations, among them A1555G, A827G in the 12S rRNA gene, G28T in the TRMU nuclear gene, SNP rs4509087 and the IVS 11–7 T>G in the MTO1 nuclear gene. In summary all the alterations observed in the MTO1 gene appear to be polymorphisms that are not related with the incomplete penetrance the mitochondrial mutations found. The TRMU gene has been show to modulate the severity of hearing loss associated with the mitochondrial mutation. This way, we performed a mutational screening of the TRMU gene to examine the role of this gene in the phenotypic expression of the A827G mutation in Brazilian patients. Sequence analysis revealed the presence of G28T mutation in the TRMU gene in three subjects carrying the A827G mutation in the 12S rRNA gene. Therefore, according the literature, G28T mutation in the TRMU gene showing to modulate the expression for the A827G mutation in the 12S rRNA
exon 07 and rs7766570 intron 05 intron 05 intron 05 exon 07 and IVS 11–7 T>G exon 07 intron 05 exon 07, rs7766570 intron 05 and IVS 11–7 T>G exon 07 and rs7766570 intron 05
gene in three subjects in this study and one subject carrying the A1555G mutation too. Aminoglycoside antibiotics are clinically important drugs used worldwide for controlling bacteria-related infections, especially in developing countries [3]. The aminoglycosides exert their antibacterial effects by directly binding to 16S rRNA of the bacterial ribosome [2]. As the human mitochondrial share many similarities to bacterial ribosomes, it is proposed that one of the primary targets for the aminoglycoside antibiotics in mammalian cells is the small ribosomal RNA (12S rRNA) of mitochondrial ribosome [26]. The C1494T, A1555G and A827G mitochondrial mutations are located at the A-site of the mitochondrial 12S rRNA gene. It is possible that the alteration of the tertiary or quaternary structure of the rRNA by these mitochondrial mutations may lead to mitochondrial dysfunction, thereby playing a role in the pathogenesis of hearing loss and/or aminoglycoside hypersensitivity [19]. Accumulation of aminoglycosides in cochlear mitochondria would lead to an inhibition of protein synthesis by interacting with the 12S rRNA carrying these mitochondrial mutations. As a result of this mitochondrial translation defect, the ATP production declines and the generation of reactive oxygen species increases, consequently damaging hair cells and giving rise to hearing impairment [26]. We have identified a homoplasmic A827G transition in the mitochondrial 12S rRNA gene. This mutation has been implicated to be associated with both aminoglycoside ototoxicity and nonsyndromic hearing loss in a few genetically unrelated individuals [27], indicating that A827G mutation is involved in the pathogenesis of hearing impairment. Finally, the A827G mutation is located at the A-site of the mitochondrial 12S rRNA gene which is highly evolutionary conserved in human [27]. It is possible that the alteration of the tertiary or quaternary structure of this rRNA by the A827G mutation may lead to mitochondrial dysfunction, thereby playing a role in the pathogenesis of hearing loss and aminoglycoside hypersensitivity. The incomplete penetrance indicates that the A827G mutation alone is not sufficient to produce clinical phenotype.
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