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Clinical evaluation and sequence analysis of the complete mitochondrial genome of three Chinese patients with hearing impairment associated with the 12S rRNA T1095C mutation
BBRC Biochemical and Biophysical Research Communications 325 (2004) 1503–1508 www.elsevier.com/locate/ybbrc
Clinical evaluation and sequence analysis of the complete mitochondrial genome of three Chinese patients with hearing impairment associated with the 12S rRNA T1095C mutation Lidong Zhaoa, Wie-Yen Younga,*, Roughua Lib, Qiuju Wanga, Yaping Qianb, Min-Xin Guanb,c,* a Institute of Otolaryngology, Chinese PLA General Hospital, Beijing, China Division and Program in Human Genetics and Center for Hearing and Deafness Research, Cincinnati ChildrenÕs Hospital Medical Center, Cincinnati, Ohio, USA Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA b
c
Received 21 October 2004 Available online 11 November 2004
Abstract Mutations in mitochondrial DNA (mtDNA), particularly those in the 12S rRNA gene, have been shown to be associated with sensorineural hearing loss. Here we report the clinical and sequence analysis of the entire mitochondrial genome in three Chinese subjects with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluation showed a variable phenotype of hearing impairment including the age of onset and audiometric configuration in these subjects. Sequence analysis of the complete mitochondrial genomes in three subjects showed the distinct sets of mtDNA polymorphism, in addition to the identical mitochondrial 12S rRNA T1095C mutation. This mutation was previously identified to be associated with hearing impairment in three families from different genetic backgrounds. The T1095C mutation was absent in 364 Chinese control. In fact, the occurrence of the T1095C mutation in these several genetically unrelated subjects affected by hearing impairment strongly indicates that this mutation is involved in the pathogenesis of hearing impairment. Among other nucleotide changes, the A2238G and T2885C mutations in the 16S rRNA, the I175V mutation in the CO2, the F16L mutation in the A6 and the V112M mutation in the ND6 exhibited a high evolutionary conservation. These data suggest that the T1095C mutation may be associated with aminoglycoside-induced and non-syndromic hearing impairments and A2238G and T2885C mutations in the 16S rRNA, the I175V mutation in the CO2, the F16L mutation in the A6 and the V112M mutation in the ND6 may contribute to the phenotypic expression of the T1095C mutation in these subjects. 2004 Elsevier Inc. All rights reserved. Keywords: Hearing loss; Mitochondrial DNA; 12S rRNA; Mutation; Aminoglycoside ototoxicity; T1095C; Chinese; Variant
Mitochondrial DNA (mtDNA) mutations have been found to be associated with sensorineural hearing loss [1,2]. In particular, the 12S rRNA gene has been shown to be a hot spot for aminoglycoside-induced and nonsyndromic hearing loss. Several deafness-associated mtDNA mutations have been identified in this gene. *
Corresponding authors. Fax: +1 513 636 2261 (M.-X. Guan). E-mail addresses: [email protected] (W.-Y. Young), min-xin. [email protected] (M.-X. Guan). 0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.10.199
Of those, the A1555G mutation in the highly conserved decoding site of the 12S rRNA has been associated with both aminoglycoside-induced and non-syndromic hearing loss in many families of different ethnic backgrounds [3–7]. Similarly, the C1494T mutation in the highly conserved decoding site of this rRNA has been associated with both aminoglycoside-induced and non-syndromic hearing loss in a large Chinese family [8]. In addition, a C-insertion or deletion at position 961 of the 12S rRNA gene has been shown to be associated only with
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L. Zhao et al. / Biochemical and Biophysical Research Communications 325 (2004) 1503–1508
aminoglycoside-induced deafness [9,10], while the novel T961G mutation has been implicated to be responsible for the non-syndromic hearing loss in five Caucasian pediatric subjects [11]. Furthermore, the T1095C mutation has also been shown to be associated with hearing impairment [12–14]. With the aim of identifying mtDNA mutations associated with hearing loss, a systematic and extended mutation screening of the mitochondrial 12S rRNA gene has been initiated in the large clinical population of Otology Clinic at the Chinese PLA General Hospital. As a consequence of this study, 34 pedigrees with a maternally inherited pattern of non-syndromic and aminoglycoside-induced hearing loss have been identified, including 15 pedigrees carrying the A1555G mutation and one pedigree carrying the C1494T mutation in the 12S rRNA gene [8]. In the present study, we have performed a clinical characterization and sequence analysis of the complete mitochondrial genome in three Chinese patients with hearing impairment associated with the T1095C mutation in the 12S rRNA gene.
Subjects and methods Subjects and audiological examinations. As part of genetic screening program for the hearing impairment, three hearing-impaired subjects were ascertained at the Otology Clinic at PLA General Hospital. A comprehensive history and physical examination were performed to identify any syndromic findings or the history of the use of aminoglycosides, genetic factors related to the hearing impairment. An age-appropriate audiological examination was performed and this examination included pure-tone audiometry (PTA) and/or auditory brainstem response (ABR), immittance testing, and distortion product otoacoustic emissions (DPOAE). The PTA was calculated from the sum of the audiometric thresholds at 500, 1000 and 2000, 4000, and 8000 Hz. The severity of hearing impairment was classified into five grades: normal <26 dB; mild =26–40 dB; moderate =41–70 dB; severe =71–90 dB; and profound >90 dB. Informed consent was obtained from the participant prior to their participation in the study, in accordance with the Cincinnati ChildrenÕs Hospital Medical Center Institutional Review Board and Ethnic Committee of Chinese PLA General Hospital. Mutational analysis of mitochondrial genome. Genomic DNA was isolated from whole blood of participants using the Puregene DNA Isolation Kits (Gentra Systems). First, the subjectÕs DNA fragments spanning the entire mitochondrial 12S rRNA gene or tRNASer(UCN)
gene were amplified by PCR using oligodeoxynucleotides corresponding to the mitochondrial genome at positions 618–635 and 1988– 2007 [8,15] and 7148–7167 and 8076–8095 [16,17], respectively. Each fragment was purified and subsequently analyzed by direct sequencing in an ABI 3700 automated DNA sequencer using the Big Dye Terminator Cycle sequencing reaction kit. mtDNA sequence alignments were carried out using seqweb program GAP (GCG). The entire mitochondrial genomes of the subject carrying the T1095C mutation were PCR amplified in 24 overlapping fragments by use of sets of the light-strand and the heavy-strand oligonucleotide primers, as described elsewhere [18]. Each fragment was purified and subsequently submitted for sequence analysis as described above. The resultant sequence data were compared with the updated consensus Cambridge sequence (GenBank Accession No. NC_001807) [19].
Results Clinical presentation The subject #78 came to the otology clinic at the Chinese PLA General Hospital at the age of 9 years. He received a dose of gentamicin (40 mg) for fever at the age of 5. He began suffering bilateral hearing impairment one month after administration. As illustrated in Fig. 1, the audiological evaluation, including the pure-tone audiometry, immittance, and ABR, showed that he had moderate hearing impairment (62 dB at right ear, 55 dB at left ear) with flat-shaped pattern. The subject #081 complained of fluctuant hearing loss at the age of 10 years. As shown in Fig. 1, the audiological evaluation revealed that he suffered from moderate hearing impairment (58 dB at right ear, 58 dB at left ear) with U-shaped pattern. He did not have a history of aminoglycoside administration. The subject #101 is a male of 55 years. Although his hearing was normal according to the assessment criteria, pure-tone audiometry test showed a Hill-shaped pattern, as shown in Fig. 1. In particular, his hearing was reduced to 30 dB at 250 Hz and 55 dB at 8000 Hz. He has not received aminoglycoside administration. Comprehensive family medical histories of those three subjects showed no other clinical abnormalities, including diabetes, muscular diseases, visual problems, and neurological disorders. Furthermore, karyotype
Fig. 1. Air conduction audiogram of three affected subjects with the T0195C mutation. Symbols: (·) left and (s) right ear.
L. Zhao et al. / Biochemical and Biophysical Research Communications 325 (2004) 1503–1508
analysis and a CT scan of temporal bones revealed no abnormal findings in those subjects. Mitochondrial genome analysis To elucidate the molecular basis of the hearing impairment, we have performed a mutational analysis of the mitochondrial genome in these subjects. First, DNA fragments spanning mitochondrial 12S rRNA and tRNASer(UCN) genes, which are the hot spots for deafness-associated mutations [1,2], were PCR amplified and each fragment was purified and subsequently analyzed by DNA sequencing. We failed to detect the presence of the A7445G, T7510C, 7472insC, and T7511C mutations in the tRNASer(UCN) gene and the A1555G, C1494T, and 961 mutations in this 12S rRNA gene [2]. However, the T1095C mutation in the 12S rRNA gene was found in these subjects apparently in the homoplasmy (Fig. 2). This mutation was absent in 364
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Chinese control [8]. This mutation has been found in an Italian family with auditory neuropathy, aminoglycoside-induced hearing loss, and Parkinsonism [13], and an other Italian family with maternally inherited hearing loss [12]. To understand the role of mitochondrial haplotype in the phenotypic expression of the T1095C mutation, we also performed a PCR-amplification of fragments spanning entire mitochondrial genome and subsequent DNA sequence analysis in these subjects. In addition to the identical T1095C mutation, as shown in Table 1, these subjects exhibited distinct sets of mtDNA polymorphism. Of other nucleotide changes in this mitochondrial genome, there are 19 variants in the D-loop and 20 silent mutations in the protein encoding genes. Furthermore, three subjects shared three variants in 12S rRNA gene and one variant in 16S rRNA gene, six known missense polymorphisms in the following protein coding genes: the A8108G (I175V) mutation in the CO2 gene [14], the A8701G (T59A) and A8860G (T112A) mutations in the A6 gene, the A10398G (T114A) in the ND3 gene, the G11969A (Ala-to-Thr) in the ND4, and the A15326G (A404T) in the cyto b gene. In addition, the subject #78 carries two known variants G1719A and T2885C, and a novel variant A2238G in 16S rRNA gene, the novel variant and the known variant A15236G (I164V) in the cyto b gene, while subjects #81 and #101 had the novel T14340C (V112M) variant in the ND6 gene. These variants were further evaluated by phylogenetic analysis of these mtDNA variants and mtDNAs from other organisms. The A2238G and T2885C mutations in the 16S rRNA, the I175V mutation in the CO2, the F16L mutation in the A6 and the V112M mutation in the ND6 are localized at sites which are highly conserved in human [19], mouse [21], bovine [22], and Xenopus [23], whereas the sites of other variants are not evolutionarily conserved.
Discussion
Fig. 2. Identification of T1095C mutation in the mitochondrial 12S rRNA gene. Partial sequence chromatograms of the 12S rRNA gene from control and affected subjects.
In the present study, we have performed the clinical and molecular characterization of three Chinese hearing-impaired subjects. The audiological studies, as shown in Table 2, revealed the variable degree of hearing impairment including the age of onset and audiometric configuration in these subjects, although these subjects share some common features: bilateral and sensorineural hearing impairment. Sequence analysis of the complete mitochondrial genomes in three subjects showed the distinct sets of mtDNA polymorphism, in addition to the identical mitochondrial 12S rRNA T1095C mutation. Indeed, this mutation was absent in 364 Chinese control [8]. Furthermore, this mutation has been identified in an Italian family with auditory neuropathy, aminoglycoside-induced hearing loss, and
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Table 1 mtDNA variants in three affected subjects with the T1095C mutation CRS
#078
#081
#101
Previously reportedb,c
A T T C A A A T T A T T C A A T C C T
G
G C C T
G
T ACC CC C T A C
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes
A/A/G/T/T/T/T A/A/A/G
A T A
G C G
G/A/A/T A/A/A/A A/G/A/A T/T/T/T
G A A T
A G G C
A–G
A
CO1 6531 7028
C–T C–T
CO2 7642 8108
G–A A–G (Ile-Val)
A6 8602 8701 8860
T–C (Phe-Leu) A–G (Thr-Ala) A–G (Thr-Ala)
CO3 9540 9554 9950
T–C G–A T–C
ND3 10,235 10,398 10,400
T–C A–G (Thr-Ala) C–T
ND4 10,873 11,350 11,719 11,935
T–C A–G G–A T–C
Gene and position
Nucleotide change
D-loop 73 146 152 198 200 215 263 310 318 326 489 16,092 16,167 16,183 16,183 16,189 16,223 16,292 16,311
A–G T–C T–C C–T A–G A–G A–G T–CTC T–C A–G T–C T–C C–T A–ACC A–CC T–C C–T C–A T–C
12S rRNA 750 1095 1438
A–G T–C A–G
16S rRNA 1719 2238 2706 2885
G–A A–G A–G T–C
ND2 4769
Conservation (H/B/M/X)a
T G G CTC C C
G G CTC C G C
T
C T G G G CTC C G C C
T
C
G C G
G C G
Yes Yes Yes
A
Yes No Yes Yes
G
G
G
G
G
Yes
C C
T T
T T
T T
No Yes
I/I/I/I
G A
A G
A G
A G
Yes Yes
F/F/F/F T/S/L/Q T/A/A/T
T A A
C G G
G G
G G
No Yes Yes
T G T
C
C
C
C A C
T A C
C G T
G T
G T
T A G T
C
C
A
A C
C G A
T/T/T/A
Yes No Yes
No Yes Yes
Yes No Yes No (continued on next page)
L. Zhao et al. / Biochemical and Biophysical Research Communications 325 (2004) 1503–1508
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Table 1 (continued) Gene and position
Nucleotide change
Conservation (H/B/M/X)a
CRS
#078
#081
#101
Previously reportedb,c
11,969
G–A (Ala-Thr)
A/A/G/A
G
A
A
A
No
ND5 12,477 12,705 13,074
T–C C–T A–G
T C A
C T G
T G
T G
No Yes No
ND6 14,340 14,569
T–C (Val-Met) G–A
C A
T
No Yes
Cyto b 14,783 15,043 15,236 15,301 15,326
T–C G–A A–G (Ile-Val) G–A A–G (Thr-Ala)
C A
C A
A G
A G
Yes Yes Yes Yes Yes
T G
V/V/V/V
T G A G A
I/I/I/S T/M/I/I
C A G A G
Note. Conserved nucleotides and amino acids are boldfaced. a Conservation of amino acid for polypeptide in human (H), bovine (B), mouse (M), and Xenopus (X); CRS: Cambridge reference sequence. b See the online mitochondrial genome database MITOMAP [20]. c See Wang et al. [14].
Table 2 Summary of clinical and molecular data for three affected subjects with the T1095C mutation in the mitochondrial 12S rRNA gene Patient
Sex
Audiometric configuration
Usage of drug
Age of onset (years)
PTA (dB) right
PTA (dB) left
#078 #081 #101
M M M
Flat U-shaped Hill-shaped
Yes No No
4 10 35
61.6 58.3 11.6
55 58.3 11.6
Parkinsonism [13], an other Italian family with maternally inherited hearing loss [12] and a Chinese subject with auditory neuropathy [14]. In fact, the occurrence of the T1095C mutation in these several genetically unrelated subjects affected by hearing impairment strongly indicates that this mutation is involved in the pathogenesis of hearing impairment, including aminoglycoside ototoxicity. This T-to-C transition disrupted an evolutionarily conserved base-pair at stem loop of the helix 25 of 12S rRNA [24]. This nucleotide is also located at the P-site of ribosome, suggesting an important role in the initiation of mitochondrial protein synthesis [13]. The alteration of the tertiary or quaternary structure of this rRNA by the T1095C mutation may lead to impair mitochondrial protein synthesis, thereby causing the mitochondrial dysfunction associated with hearing impairment. Furthermore, there is an increasing evidence showing that the background sequences (haplotype) of the mtDNA modulate the severity and penetrance of the phenotypic expression of pathogenic mtDNA mutation(s) associated with some clinical abnormalities including hearing loss [16,17], blindness [25], and ageing [26]. Here, the A2238G and T2885C mutations in the 16S rRNA, the I175V mutation in the CO2, the F16L mutation in the A6, and the V112M mutation in the
ND6, showing high evolutionary conservation, may contribute to the phenotypic expression of the T1095C mutation in these subjects.
Acknowledgments This work was supported by the National Institutes of Health Grants DC04958 and DC05230 from the National Institute on Deafness and Other Communication Disorders to M.-X.G.
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Clinical evaluation and sequence analysis of the complete mitochondrial genome of three Chinese patients with hearing impairment associated with the 12S rRNA T1095C mutation Lidong Zhaoa, Wie-Yen Younga,*, Roughua Lib, Qiuju Wanga, Yaping Qianb, Min-Xin Guanb,c,* a Institute of Otolaryngology, Chinese PLA General Hospital, Beijing, China Division and Program in Human Genetics and Center for Hearing and Deafness Research, Cincinnati ChildrenÕs Hospital Medical Center, Cincinnati, Ohio, USA Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA b
c
Received 21 October 2004 Available online 11 November 2004
Abstract Mutations in mitochondrial DNA (mtDNA), particularly those in the 12S rRNA gene, have been shown to be associated with sensorineural hearing loss. Here we report the clinical and sequence analysis of the entire mitochondrial genome in three Chinese subjects with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluation showed a variable phenotype of hearing impairment including the age of onset and audiometric configuration in these subjects. Sequence analysis of the complete mitochondrial genomes in three subjects showed the distinct sets of mtDNA polymorphism, in addition to the identical mitochondrial 12S rRNA T1095C mutation. This mutation was previously identified to be associated with hearing impairment in three families from different genetic backgrounds. The T1095C mutation was absent in 364 Chinese control. In fact, the occurrence of the T1095C mutation in these several genetically unrelated subjects affected by hearing impairment strongly indicates that this mutation is involved in the pathogenesis of hearing impairment. Among other nucleotide changes, the A2238G and T2885C mutations in the 16S rRNA, the I175V mutation in the CO2, the F16L mutation in the A6 and the V112M mutation in the ND6 exhibited a high evolutionary conservation. These data suggest that the T1095C mutation may be associated with aminoglycoside-induced and non-syndromic hearing impairments and A2238G and T2885C mutations in the 16S rRNA, the I175V mutation in the CO2, the F16L mutation in the A6 and the V112M mutation in the ND6 may contribute to the phenotypic expression of the T1095C mutation in these subjects. 2004 Elsevier Inc. All rights reserved. Keywords: Hearing loss; Mitochondrial DNA; 12S rRNA; Mutation; Aminoglycoside ototoxicity; T1095C; Chinese; Variant
Mitochondrial DNA (mtDNA) mutations have been found to be associated with sensorineural hearing loss [1,2]. In particular, the 12S rRNA gene has been shown to be a hot spot for aminoglycoside-induced and nonsyndromic hearing loss. Several deafness-associated mtDNA mutations have been identified in this gene. *
Corresponding authors. Fax: +1 513 636 2261 (M.-X. Guan). E-mail addresses: [email protected] (W.-Y. Young), min-xin. [email protected] (M.-X. Guan). 0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.10.199
Of those, the A1555G mutation in the highly conserved decoding site of the 12S rRNA has been associated with both aminoglycoside-induced and non-syndromic hearing loss in many families of different ethnic backgrounds [3–7]. Similarly, the C1494T mutation in the highly conserved decoding site of this rRNA has been associated with both aminoglycoside-induced and non-syndromic hearing loss in a large Chinese family [8]. In addition, a C-insertion or deletion at position 961 of the 12S rRNA gene has been shown to be associated only with
1504
L. Zhao et al. / Biochemical and Biophysical Research Communications 325 (2004) 1503–1508
aminoglycoside-induced deafness [9,10], while the novel T961G mutation has been implicated to be responsible for the non-syndromic hearing loss in five Caucasian pediatric subjects [11]. Furthermore, the T1095C mutation has also been shown to be associated with hearing impairment [12–14]. With the aim of identifying mtDNA mutations associated with hearing loss, a systematic and extended mutation screening of the mitochondrial 12S rRNA gene has been initiated in the large clinical population of Otology Clinic at the Chinese PLA General Hospital. As a consequence of this study, 34 pedigrees with a maternally inherited pattern of non-syndromic and aminoglycoside-induced hearing loss have been identified, including 15 pedigrees carrying the A1555G mutation and one pedigree carrying the C1494T mutation in the 12S rRNA gene [8]. In the present study, we have performed a clinical characterization and sequence analysis of the complete mitochondrial genome in three Chinese patients with hearing impairment associated with the T1095C mutation in the 12S rRNA gene.
Subjects and methods Subjects and audiological examinations. As part of genetic screening program for the hearing impairment, three hearing-impaired subjects were ascertained at the Otology Clinic at PLA General Hospital. A comprehensive history and physical examination were performed to identify any syndromic findings or the history of the use of aminoglycosides, genetic factors related to the hearing impairment. An age-appropriate audiological examination was performed and this examination included pure-tone audiometry (PTA) and/or auditory brainstem response (ABR), immittance testing, and distortion product otoacoustic emissions (DPOAE). The PTA was calculated from the sum of the audiometric thresholds at 500, 1000 and 2000, 4000, and 8000 Hz. The severity of hearing impairment was classified into five grades: normal <26 dB; mild =26–40 dB; moderate =41–70 dB; severe =71–90 dB; and profound >90 dB. Informed consent was obtained from the participant prior to their participation in the study, in accordance with the Cincinnati ChildrenÕs Hospital Medical Center Institutional Review Board and Ethnic Committee of Chinese PLA General Hospital. Mutational analysis of mitochondrial genome. Genomic DNA was isolated from whole blood of participants using the Puregene DNA Isolation Kits (Gentra Systems). First, the subjectÕs DNA fragments spanning the entire mitochondrial 12S rRNA gene or tRNASer(UCN)
gene were amplified by PCR using oligodeoxynucleotides corresponding to the mitochondrial genome at positions 618–635 and 1988– 2007 [8,15] and 7148–7167 and 8076–8095 [16,17], respectively. Each fragment was purified and subsequently analyzed by direct sequencing in an ABI 3700 automated DNA sequencer using the Big Dye Terminator Cycle sequencing reaction kit. mtDNA sequence alignments were carried out using seqweb program GAP (GCG). The entire mitochondrial genomes of the subject carrying the T1095C mutation were PCR amplified in 24 overlapping fragments by use of sets of the light-strand and the heavy-strand oligonucleotide primers, as described elsewhere [18]. Each fragment was purified and subsequently submitted for sequence analysis as described above. The resultant sequence data were compared with the updated consensus Cambridge sequence (GenBank Accession No. NC_001807) [19].
Results Clinical presentation The subject #78 came to the otology clinic at the Chinese PLA General Hospital at the age of 9 years. He received a dose of gentamicin (40 mg) for fever at the age of 5. He began suffering bilateral hearing impairment one month after administration. As illustrated in Fig. 1, the audiological evaluation, including the pure-tone audiometry, immittance, and ABR, showed that he had moderate hearing impairment (62 dB at right ear, 55 dB at left ear) with flat-shaped pattern. The subject #081 complained of fluctuant hearing loss at the age of 10 years. As shown in Fig. 1, the audiological evaluation revealed that he suffered from moderate hearing impairment (58 dB at right ear, 58 dB at left ear) with U-shaped pattern. He did not have a history of aminoglycoside administration. The subject #101 is a male of 55 years. Although his hearing was normal according to the assessment criteria, pure-tone audiometry test showed a Hill-shaped pattern, as shown in Fig. 1. In particular, his hearing was reduced to 30 dB at 250 Hz and 55 dB at 8000 Hz. He has not received aminoglycoside administration. Comprehensive family medical histories of those three subjects showed no other clinical abnormalities, including diabetes, muscular diseases, visual problems, and neurological disorders. Furthermore, karyotype
Fig. 1. Air conduction audiogram of three affected subjects with the T0195C mutation. Symbols: (·) left and (s) right ear.
L. Zhao et al. / Biochemical and Biophysical Research Communications 325 (2004) 1503–1508
analysis and a CT scan of temporal bones revealed no abnormal findings in those subjects. Mitochondrial genome analysis To elucidate the molecular basis of the hearing impairment, we have performed a mutational analysis of the mitochondrial genome in these subjects. First, DNA fragments spanning mitochondrial 12S rRNA and tRNASer(UCN) genes, which are the hot spots for deafness-associated mutations [1,2], were PCR amplified and each fragment was purified and subsequently analyzed by DNA sequencing. We failed to detect the presence of the A7445G, T7510C, 7472insC, and T7511C mutations in the tRNASer(UCN) gene and the A1555G, C1494T, and 961 mutations in this 12S rRNA gene [2]. However, the T1095C mutation in the 12S rRNA gene was found in these subjects apparently in the homoplasmy (Fig. 2). This mutation was absent in 364
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Chinese control [8]. This mutation has been found in an Italian family with auditory neuropathy, aminoglycoside-induced hearing loss, and Parkinsonism [13], and an other Italian family with maternally inherited hearing loss [12]. To understand the role of mitochondrial haplotype in the phenotypic expression of the T1095C mutation, we also performed a PCR-amplification of fragments spanning entire mitochondrial genome and subsequent DNA sequence analysis in these subjects. In addition to the identical T1095C mutation, as shown in Table 1, these subjects exhibited distinct sets of mtDNA polymorphism. Of other nucleotide changes in this mitochondrial genome, there are 19 variants in the D-loop and 20 silent mutations in the protein encoding genes. Furthermore, three subjects shared three variants in 12S rRNA gene and one variant in 16S rRNA gene, six known missense polymorphisms in the following protein coding genes: the A8108G (I175V) mutation in the CO2 gene [14], the A8701G (T59A) and A8860G (T112A) mutations in the A6 gene, the A10398G (T114A) in the ND3 gene, the G11969A (Ala-to-Thr) in the ND4, and the A15326G (A404T) in the cyto b gene. In addition, the subject #78 carries two known variants G1719A and T2885C, and a novel variant A2238G in 16S rRNA gene, the novel variant and the known variant A15236G (I164V) in the cyto b gene, while subjects #81 and #101 had the novel T14340C (V112M) variant in the ND6 gene. These variants were further evaluated by phylogenetic analysis of these mtDNA variants and mtDNAs from other organisms. The A2238G and T2885C mutations in the 16S rRNA, the I175V mutation in the CO2, the F16L mutation in the A6 and the V112M mutation in the ND6 are localized at sites which are highly conserved in human [19], mouse [21], bovine [22], and Xenopus [23], whereas the sites of other variants are not evolutionarily conserved.
Discussion
Fig. 2. Identification of T1095C mutation in the mitochondrial 12S rRNA gene. Partial sequence chromatograms of the 12S rRNA gene from control and affected subjects.
In the present study, we have performed the clinical and molecular characterization of three Chinese hearing-impaired subjects. The audiological studies, as shown in Table 2, revealed the variable degree of hearing impairment including the age of onset and audiometric configuration in these subjects, although these subjects share some common features: bilateral and sensorineural hearing impairment. Sequence analysis of the complete mitochondrial genomes in three subjects showed the distinct sets of mtDNA polymorphism, in addition to the identical mitochondrial 12S rRNA T1095C mutation. Indeed, this mutation was absent in 364 Chinese control [8]. Furthermore, this mutation has been identified in an Italian family with auditory neuropathy, aminoglycoside-induced hearing loss, and
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L. Zhao et al. / Biochemical and Biophysical Research Communications 325 (2004) 1503–1508
Table 1 mtDNA variants in three affected subjects with the T1095C mutation CRS
#078
#081
#101
Previously reportedb,c
A T T C A A A T T A T T C A A T C C T
G
G C C T
G
T ACC CC C T A C
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes
A/A/G/T/T/T/T A/A/A/G
A T A
G C G
G/A/A/T A/A/A/A A/G/A/A T/T/T/T
G A A T
A G G C
A–G
A
CO1 6531 7028
C–T C–T
CO2 7642 8108
G–A A–G (Ile-Val)
A6 8602 8701 8860
T–C (Phe-Leu) A–G (Thr-Ala) A–G (Thr-Ala)
CO3 9540 9554 9950
T–C G–A T–C
ND3 10,235 10,398 10,400
T–C A–G (Thr-Ala) C–T
ND4 10,873 11,350 11,719 11,935
T–C A–G G–A T–C
Gene and position
Nucleotide change
D-loop 73 146 152 198 200 215 263 310 318 326 489 16,092 16,167 16,183 16,183 16,189 16,223 16,292 16,311
A–G T–C T–C C–T A–G A–G A–G T–CTC T–C A–G T–C T–C C–T A–ACC A–CC T–C C–T C–A T–C
12S rRNA 750 1095 1438
A–G T–C A–G
16S rRNA 1719 2238 2706 2885
G–A A–G A–G T–C
ND2 4769
Conservation (H/B/M/X)a
T G G CTC C C
G G CTC C G C
T
C T G G G CTC C G C C
T
C
G C G
G C G
Yes Yes Yes
A
Yes No Yes Yes
G
G
G
G
G
Yes
C C
T T
T T
T T
No Yes
I/I/I/I
G A
A G
A G
A G
Yes Yes
F/F/F/F T/S/L/Q T/A/A/T
T A A
C G G
G G
G G
No Yes Yes
T G T
C
C
C
C A C
T A C
C G T
G T
G T
T A G T
C
C
A
A C
C G A
T/T/T/A
Yes No Yes
No Yes Yes
Yes No Yes No (continued on next page)
L. Zhao et al. / Biochemical and Biophysical Research Communications 325 (2004) 1503–1508
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Table 1 (continued) Gene and position
Nucleotide change
Conservation (H/B/M/X)a
CRS
#078
#081
#101
Previously reportedb,c
11,969
G–A (Ala-Thr)
A/A/G/A
G
A
A
A
No
ND5 12,477 12,705 13,074
T–C C–T A–G
T C A
C T G
T G
T G
No Yes No
ND6 14,340 14,569
T–C (Val-Met) G–A
C A
T
No Yes
Cyto b 14,783 15,043 15,236 15,301 15,326
T–C G–A A–G (Ile-Val) G–A A–G (Thr-Ala)
C A
C A
A G
A G
Yes Yes Yes Yes Yes
T G
V/V/V/V
T G A G A
I/I/I/S T/M/I/I
C A G A G
Note. Conserved nucleotides and amino acids are boldfaced. a Conservation of amino acid for polypeptide in human (H), bovine (B), mouse (M), and Xenopus (X); CRS: Cambridge reference sequence. b See the online mitochondrial genome database MITOMAP [20]. c See Wang et al. [14].
Table 2 Summary of clinical and molecular data for three affected subjects with the T1095C mutation in the mitochondrial 12S rRNA gene Patient
Sex
Audiometric configuration
Usage of drug
Age of onset (years)
PTA (dB) right
PTA (dB) left
#078 #081 #101
M M M
Flat U-shaped Hill-shaped
Yes No No
4 10 35
61.6 58.3 11.6
55 58.3 11.6
Parkinsonism [13], an other Italian family with maternally inherited hearing loss [12] and a Chinese subject with auditory neuropathy [14]. In fact, the occurrence of the T1095C mutation in these several genetically unrelated subjects affected by hearing impairment strongly indicates that this mutation is involved in the pathogenesis of hearing impairment, including aminoglycoside ototoxicity. This T-to-C transition disrupted an evolutionarily conserved base-pair at stem loop of the helix 25 of 12S rRNA [24]. This nucleotide is also located at the P-site of ribosome, suggesting an important role in the initiation of mitochondrial protein synthesis [13]. The alteration of the tertiary or quaternary structure of this rRNA by the T1095C mutation may lead to impair mitochondrial protein synthesis, thereby causing the mitochondrial dysfunction associated with hearing impairment. Furthermore, there is an increasing evidence showing that the background sequences (haplotype) of the mtDNA modulate the severity and penetrance of the phenotypic expression of pathogenic mtDNA mutation(s) associated with some clinical abnormalities including hearing loss [16,17], blindness [25], and ageing [26]. Here, the A2238G and T2885C mutations in the 16S rRNA, the I175V mutation in the CO2, the F16L mutation in the A6, and the V112M mutation in the
ND6, showing high evolutionary conservation, may contribute to the phenotypic expression of the T1095C mutation in these subjects.
Acknowledgments This work was supported by the National Institutes of Health Grants DC04958 and DC05230 from the National Institute on Deafness and Other Communication Disorders to M.-X.G.
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