Extremely low penetrance of hearing loss in four Chinese families with the mitochondrial 12S rRNA A1555G mutation

BBRC Biochemical and Biophysical Research Communications 328 (2005) 1244–1251 www.elsevier.com/locate/ybbrc

Extremely low penetrance of hearing loss in four Chinese families with the mitochondrial 12S rRNA A1555G mutation Wie-Yen Younga,*, Lidong Zhaoa, Yaping Qianb,c, Qiuju Wanga, Ning Lid, John H. Greinwald Jr.c, Min-Xin Guanb,c,* b c

a Institute of Otolaryngology, Chinese PLA General Hospital, Beijing, China Division and Program in Human Genetics, Cincinnati ChildrenÕs Hospital Medical Center, Cincinnati, OH, USA Center for Hearing and Deafness Research, Cincinnati ChildrenÕs Hospital Medical Center, Cincinnati, OH, USA d State Key Laboratory of Agricultural Biological Technique, China Agricultural University, Beijing, China

Received 17 January 2005 Available online 29 January 2005

Abstract Mutations in mitochondrial DNA (mtDNA) have been found to be associated with sensorineural hearing loss. We report here the clinical, genetic, and molecular characterization of four Chinese pedigrees with aminoglycoside-induced and nonsyndromic hearing impairment. Clinical evaluation revealed the variable phenotype of hearing impairment including audiometric configuration in these subjects, although these subjects share some common features: bilateral and sensorineural hearing impairment. Strikingly, these Chinese pedigrees exhibited extremely low penetrance of hearing loss (5.2%, 4.8%, 4.2%, and 13.3%, respectively, and with an average 8% penetrance). In particular, four of all five affected matrilineal relatives of these pedigrees had aminoglycoside-induced hearing loss. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the distinct sets of mtDNA polymorphism, in addition to the identical homoplasmic A1555G mutation, associated with hearing impairment in many families from different genetic backgrounds. The fact that mtDNA of those pedigrees belonged to different haplogroups R9a, N9a, D4a, and D4 suggested that the A1555G mutation occurred sporadically and multiplied through evolution of the mtDNA in China. However, there was the absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in these Chinese families. These data imply that the nuclear background or/and mitochondrial haplotype may not play a significant role in the phenotypic expression of the A1555G mutation in these Chinese pedigrees. However, aminoglycoside appears to be a major modifier factor for the phenotypic manifestation of the A1555G mutation in these Chinese families.
2005 Elsevier Inc. All rights reserved. Keywords: Hearing loss; Aminoglycoside ototoxicity; Mitochondrial DNA; Mutation; 12S rRNA; Penetrance; Modifier factor; A1555G; Nuclear background; Chinese; Nonsyndromic deafness

Mutations in mitochondrial DNA (mtDNA), particularly those in the 12S rRNA gene, have been shown to be one of the important causes of aminoglycoside-induced and nonsyndromic hearing loss [1,2]. Of those deafness-associated mtDNA mutations, the C1494T mutation in the highly conserved decoding site of 12S *

Corresponding authors. Fax: +1 513 636 3486. E-mail addresses: [email protected] (W.-Y. Young), min-xin.guan@ chmcc.org (M.-X. Guan). 0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.01.085

rRNA has been associated with both aminoglycoside-induced and nonsyndromic hearing loss in a large Chinese family [3]. Mutations at position 961 of the 12S rRNA have been implicated to be associated with aminoglycoside-induced deafness as well as nonsyndromic hearing loss [4–6]. Furthermore, the T1095C mutation in the same rRNA has also been shown to be associated with hearing impairment in several genetically unrelated families [7–9]. In contrast to those deafness-associated mtDNA mutations reported only in a small number of

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families from different ethnic origins, the homoplasmic A1555G mutation in the highly conserved decoding site of the 12S rRNA has been associated with both aminoglycoside-induced and nonsyndromic hearing loss in many families of different ethnic backgrounds [10–17]. In particular, matrilineal relatives of intra-families or inter-families carrying the A1555G mutation exhibited the variable severity and age-of-onset in hearing impairment [1,2]. Incomplete and variable penetrance of hearing loss as well as a mild biochemical defect associated with this mutation indicated that the A1555G mutation itself is not sufficient to produce the clinical phenotype [1,2,18,19]. Thus, aminoglycosides, nuclear modifier genes, and/or other mtDNA mutations/polymorphisms modulate the phenotypic variability and penetrance of deafness associated with the A1555G mutation [18–22]. However, less has been known about the pathogenesis of maternally inherited aminoglycoside-induced and nonsyndromic hearing loss in the Chinese population. Recently, 35 Chinese pedigrees with a maternally inherited pattern of aminoglycoside-induced and nonsyndromic hearing loss have been ascertained at Otology Clinic of the Chinese PLA General Hospital [3,9]. In the present study, we report the clinical, molecular, and genetic characterization of four Chinese pedigrees with aminoglycoside-induced and nonsyndromic hearing loss. Clinical and genetic evaluation revealed extremely low penetrance of hearing loss in these Chinese families. Molecular analysis has led to the identification of the A1555G mutation in those families. To elucidate the role of mitochondrial haplotype in the phenotypic expression of the A1555G mutation, we performed PCR-amplification of fragments spanning entire mitochondrial genome and subsequent DNA sequence analysis in the matrilineal relatives of those families. Subjects and methods Subjects and audiological examinations. As a part of genetic screening program for the hearing impairment, four Chinese families were ascertained through the Otology Clinic at Chinese PLA General Hospital. A comprehensive history and physical examination were performed to identify any syndromic findings, the history of the use of aminoglycosides, and genetic factors related to the hearing impairment in members of these pedigrees. An age-appropriate audiological

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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, and 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; profound >90 dB. Informed consent was obtained from participants 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 Puregene DNA Isolation Kits (Gentra Systems). SubjectÕs DNA fragments spanning the entire mitochondrial 12S rRNA gene were amplified by PCR using oligodeoxynucleotides corresponding to positions 618–635 and 1988– 2007 [23]. For the detection of the A1555G mutation, the amplified segments were digested with a restriction enzyme BsmAI [6,11]. Equal amounts of various digested samples were then analyzed by electrophoresis through 1.5% agarose gel. The proportions of digested and undigested PCR product were determined by laser densitometry after ethidium bromide staining to determine if the A1555G mutation is in homoplasmy in these subjects. The entire mitochondrial genomes of four probands carrying the A1555G mutation were PCR amplified in 24 overlapping fragments by the use of sets of light-strand and the heavy-strand oligonucleotide primers, as described elsewhere [23]. 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. The resultant sequence data were compared with the updated consensus Cambridge sequence (GenBank Accession No. NC_001807) [24].

Results Clinical presentation In family BJ101, the proband (III-7) was a 30-yearold man with congenital achondroplasia and dwarf of nonpituitary hyposecretion. His mother had fever in her early pregnancy. He received streptomycin (0.75 g/ dose for 7 days) for fever at the age of one. Since then, he had been administered gentamicin and kanamycin several times. He began suffering bilateral hearing impairment within 1 month after the first drug administration. As illustrated in Fig. 1, audiological evaluation showed that he had profound hearing impairment (>100 dB at right ear, >100 dB at left ear) with a flatshaped pattern. However, as shown in Fig. 2, familial history and clinical evaluation revealed that none of

Fig. 1. Air conduction audiogram of four affected subjects with the A1555G mutation and one control. Symbols: (·) left, (s) right ear.

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240 mg gentamicin. However, as shown in Fig. 2, 11 matrilineal relatives of this family had normal hearing. In family BJ103, the proband (III-9) was a young man at the age of 22 years. He had been administered kanamycin and gentamicin for pneumonia at the age of 3 years. He exhibited hearing impairment within 1 month after administration of the drugs. As shown in Fig. 1, he had severe hearing loss (87 dB at right ear, 82 dB at left ear) with a slope-shaped pattern. As shown in Fig. 2, the remaining 19 matrilineal relatives of this family exhibited normal hearing. In family BJ104, the proband (III-7)Õs hearing was evaluated by the Otology Clinic of Chinese PLA General Hospital at the age of 23 years. She began suffering hearing loss after receiving kanamycin and gentamicin at the age of 7 years. In fact, she had profound hearing loss (92 dB at right ear, 92 dB at left ear) with a slopeshaped pattern, as shown in Fig. 1. Familial history and clinical examination, as shown in Fig. 2, exhibited that none of 13 other matrilineal relatives including the probandÕs mother in this family had hearing failure. Comprehensive family medical histories of those subjects and other members of these families showed no other clinical abnormalities, including diabetes, muscular diseases, visual dysfunction, and neurological disorders. Furthermore, karyotype analysis and a CT scan of temporal bones revealed no abnormal findings in those subjects. Mitochondrial genome analysis

Fig. 2. Four Chinese pedigrees with aminoglycoside-induced and nonsyndromic hearing impairment. Hearing impaired individuals are indicated by filled symbols. Arrowhead denotes probands. Asterisks denote individuals who had a history of exposure to aminoglycosides.

other 14 matrilineal relatives including the probandÕs mother in this family had hearing defect. In family BJ102, the proband (III-4) came to the Otology Clinic of Chinese PLA General Hospital at the age of 27 years. She exhibited bilateral hearing impairment at the age of 7 years without any obvious cause. She did not have a history of aminoglycoside administration. As illustrated in Fig. 1, the audiological examination revealed that she suffered from severe hearing impairment (77 dB at right ear, 68 dB at left ear) with a slope-shaped pattern. Her elder brother (III-1) had received gentamicin (3–5 mg/kg/dose every 8 h) for dysentery at the age of 3 years. He had complained of hearing loss after being administered a total dose of

To elucidate the molecular basis of hearing impairment, we have performed a mutational analysis of the mitochondrial genome in these families. First, DNA fragments spanning mitochondrial 12S rRNA and tRNASer(UCN) genes, which are the hot spots for deafness-associated mutations [2,25], were PCR amplified from each proband. Each fragment was purified and subsequently analyzed by DNA sequencing. We failed to detect the presence of the T7510C, T7511C, 7472insC, and A7445G mutations in the tRNASer(UCN) gene and mutations at positions 961, 1095, and 1494 in the 12S rRNA gene [2]. However, the A1555G mutation in the 12S rRNA gene was found in these subjects. The restriction enzyme digestion and subsequent electrophoresis analysis indicated that the A1555G mutation was indeed present in homoplasmy in those subjects and other matrilineal relatives of these families (Fig. 3). To understand the role of mitochondrial haplotype in the phenotypic expression of the A1555G mutation, we performed a PCR-amplification of fragments spanning entire mitochondrial genome and subsequent DNA sequence analysis in these probands. In addition to the identical A1555G mutation, as shown in Table 1, these subjects exhibited distinct sets of mtDNA polymorphism. Of other nucleotide changes in these mitochon-

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whereas the sites of other missense variants in proteinencoding genes, the G10427A in the tRNAArg and six variants in rRNA, are not evolutionarily conserved.

Discussion

Fig. 3. Qualification of the A1555G mutation of four Chinese subjects with aminoglycoside-induced and nonsyndromic hearing loss. PCR products around A1555G region of mitochondrial genome were digested with BsmAI and analyzed by electrophoresis in a 1.5% agarose gel stained with ethidium bromide. A3 and A4 are Chinese control subjects [3].

drial genomes, there are 21 variants in the D-loop, 36 silent mutations in the protein-encoding genes, three known variants in the 12S rRNA gene, and three known variants in the 16S rRNA gene [26]. Four probands shared six known variants including the A15326G (A164T) in the cytob gene. Interestingly, BJ101 pedigree carried more variants than other pedigrees. In particular, the novel G10427A variant in tRNAArg gene and the previous identified CO2/tRNALys intergenic 9 base pair deletion corresponding to mtDNA at position 8271–8279 [27] were identified in this pedigree. Furthermore, the BJ101 pedigree harbored eight missense variants in the protein-encoding genes: five novel variants: C5076T (L203F) in ND2, G5913A (D3N) in CO1, G10320A (V84I) in ND3, C10980G (P74R) and A10988C (I77L) in ND4, and three known variants: A3434G (Y33C) in ND1, T5442C (F325L) in ND2, and G13928C (S541T) in ND5. In BJ102 pedigree, there were five missense variants in the protein-encoding genes including three novel variants: G5046A (V193I) in ND2, C8532T (T56M) in A8, and T13856A (T508I) in ND5, and two known variants: A8860G (T111A) in A6 and A12358G (T6A) in ND5. BJ103 and BJ104 pedigrees shared three known missense variants in the protein-encoding genes: C5178A (L237M) in ND2, A8860G (T111A) in A6, and A10398G (T114A) in ND3. In addition, BJ103 pedigree carried the novel variant T10742G (H91Q) in ND4L and two known variants: T14979C (I78T) and A15758G (I338V) in cytob. These variants were further evaluated by phylogenetic analysis of these mtDNA variants and mtDNAs from other organisms. The L203F mutation in the ND2, the T56M mutation in the A8, the P74R mutation in the ND4, the T508I mutation in the ND5, and the I338V in cytob are localized at sites which are highly conserved from human [24], mouse [28], bovine [29], to Xenopus laevis [30],

In the present study, we have performed the clinical, genetic, and molecular characterization of four Chinese pedigrees with aminoglycoside-induced and nonsyndromic hearing impairment. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the distinct sets of mtDNA polymorphism, in addition to the identical homoplasmic A1555G mutation. Audiological studies, as showed in Table 2, revealed the variable phenotype of hearing impairment including audiometric configuration in these subjects, although these subjects shares some common features: bilateral and sensorineural hearing impairment. Most strikingly, these Chinese pedigrees exhibited extremely low penetrance of hearing loss. In particular, only one matrilineal relative each of BJ101, BJ103, and BJ104 pedigrees, and two matrilineal relatives of BJ102 pedigree developed hearing impairment (5.2%, 4.8%, 4.2%, and 13.3% penetrance, respectively, and with an average 8% penetrance). By contrast, many other pedigrees carrying the A1555G mutation revealed much higher penetrance of hearing loss than those Chinese pedigrees. For example, the average penetrance (including aminoglycoside-induced deafness) of three Mongolian pedigrees carrying the A1555G mutation is 62.5% [14], while 65% of matrilineal relatives from a large Arab-Israeli family without a history of exposure to aminoglycosides exhibited hearing impairment [10,31]. Furthermore, a large Chinese family carrying the C1494T mutation exhibited 31% penetrance excluding the effect of aminoglycosides, but 51% penetrance including aminoglycoside-induced deafness [3]. These various penetrances likely reflect the use of aminoglycosides, different nuclear backgrounds, and mitochondrial haplotypes between those four Chinese families and other pedigrees carrying the A1555G mutation. Indeed, the new G-C pair in the 12S rRNA created by the A1555G mutation facilitates the binding of aminoglycosides to the decoding region of 12S rRNA, accounting for the fact that exposure to aminoglycosides can induce or worsen hearing loss in individuals carrying the mutation [3,11,18,20]. Our previous biochemical data indicated that the A1555G mutation led to the sensitivity to aminoglycosides [18,20]. Here, four of five affected matrilineal relatives of four Chinese families suffered from aminoglycoside-induced hearing loss. Furthermore, almost half of affected matrilineal relatives in other three Chinese pedigrees carrying the A1555G mutation had aminoglycoside-induced hearing loss [17]. These observations suggest that the aminoglycoside

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Table 1 mtDNA variants in four Chinese families with nonsyndromic hearing loss Conservation (H/B/M/X)a

CRS

BJ101

BJ102

A C T G A A T G T C T C G A C C C A T T T

G T C

G T

Gene and position

Mutation

D-loop 73 150 152 207 249 263 310 316 489 495 16093 16111 16129 16220 16223 16257 16261 16265 16298 16362 16519

A-G C-T T-C G-A Del A A-G T-CTC Ins C T-C C-T T-C C-T G-A A-C C-T C-A C-T A-G T-C T-C T-C

12S rRNA 750 1382 1438 1555c

A-G A-C A-G A-G

A/A/G/A/A/A/G A/A/A/G A/A/A/A

16S rRNA 2706 3010 3206

A-G G-A C-T

ND1 3423 3434 3970

G-T A-G (Tyr-Cys) C-T

ND2 4769 4883 5046 5076 5178 5231 5442

A-G C-T G-A(Val-Ile) C-T (Leu-Phe) C-A(Leu-Met) G-A T-C(Phe-Leu)

MTNC 5885

G-A

CO1 5913 5978 6392 6710 7028

G-A (Asp-Asn) A-G T-C A-G C-T

COII 8020

G-A

G

MTNC7 8270

C-G

C

BJ103

BJ104

Previously reportedb

G

C

Ins C C

T A T

T

T

G C C

C

C

C C

Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes yes Yes Yes Yes Yes Yes Yes Yes

A A A A

G

G

G

G G

G G

G G

G C G G

Yes Yes Yes Yes

A/G/A/A G/G/A/A C/A/T/A

A G C

G

G

G A

Yes Yes Yes

Y/Y/Y/T

G A C

V/V/I/I L/L/L/L L/T/T/T

A C G C C G T

C

Yes Yes No No Yes Yes Yes

G

A

No

G A T A C

A G C T

No No Yes No Yes

A

Yes

F/F/M/L

D/N/N/T

C A

Del A G CTC

G CTC

C G C

G CTC

G

T A C

A T

T

Yes Yes Yes

G T

G

G

G T

T

A

A

A T A

G T

G

T

Yes

W.-Y. Young et al. / Biochemical and Biophysical Research Communications 328 (2005) 1244–1251

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Table 1 (continued) Conservation (H/B/M/X)a

CRS

BJ101

Gene and position

Mutation

8271–8279

9-bp del

A8 8414 8473 8532

C-T(Leu-Phe) T-C C-T(Thr-Met)

A6 8634 8701 8860 8964

T-C A-G (Thr-Ala) A-G (Thr-Ala) C-T

COIII 9296 9443 9540 9824 9947

C-T T-C T-C T-A G-A

ND3 10310 10320 10398 10400

G-A G-A(Val-Ile) A-G(Thr-Ala) C-T

V/L/M/I T/T/T/A

G G A C

A A

tRNAArg 10427

G-A

G/A/T/T

G

A

ND4L 10589 10640 10742

G-A T-C T-G (His-Gln)

H/Q/Q/F

G G T

ND4 10873 10894 10980 10988 11719 12007

T-C C-T C-G (Pro-Arg) A-C (Ile-Leu) G-A G-A

ND5 12358 12372 12468 12705 13856 13928

A-G (Thr-Ala) G-A T-C C-T T-A (Thr-Ile) G-C (Ser-Thr)

T/S/T/S

ND6 14384 14668

G-A (Ala-Val) C-T

A/L/I/I

Cytochrome b 14783 14979 15043 15301 15326 15758 15784

T-C T-C (Ile-Thr) G-A G-A A-G (Thr-Ala) A-G (Ile-Val) T-C

a b c

BJ102

BJ103

BJ104

9-bp del

L/F/M/W T/T/T/T

T/S/L/Q T/A/A/T

C T C

C T T T G

P/P/P/P I/L/L/L

T/T/T/T S/T/S/T

I/I/L/L

T/M/I/I I/I/I/I

T C C A G G

T G T C T G

Yes

T C

G

C G G

G G T

T C C

C A

A

G

C

C T G C A

G T

A C G

C

A A

T A

C

A

A

Yes No Yes Yes

G

Yes No No No Yes Yes

Yes Yes No Yes No Yes

C T

T

T

A T

No Yes

C

Yes Yes Yes Yes Yes Yes Yes

C

C

Yes No Yes Yes Yes

Yes No No

G A

G

No Yes Yes Yes

No

G C

T T G G A A T

Yes Yes No

T

T A A C

Previously reportedb

C C A A G G

A A G

Conservation of amino acid for polypeptide in human (H), bovine (B), mouse (M), and Xenopus laevis (X); CRS, Cambridge reference sequence. See the online mitochondrial genome database MITOMAP. Conserved nucleotides in RNA and residues are boldfaced.

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Table 2 Summary of clinical and molecular data for four probands carrying the A1555G mutation Subject

Gender

Audiometric configuration

Use of drug

Age of onset (year)

PTA (dB) right ear

PTA (dB) left ear

mtDNA haplogroup

BJ101-III-7 BJ102-III-4 BJ103-III-9 BJ104-III-7

M F M F

Flat Slope Slope Slope

Yes No Yes Yes

<2 7 3 7

>100 77 87 92

>100 68 82 92

R9a N9a D4a D4

is a major modifier factor for the phenotypic manifestation of the A1555G mutation in these Chinese families. Our previous genetic and biochemical data demonstrated that nuclear backgrounds play a determining role in the phenotypic expression of the A1555G mutation in a large Arab-Israeli family [18,19]. Recently, a genotyping analysis of the first nuclear modifier gene TRMU in 71 members of an Arab-Israeli family and 145 members of Spanish and Italian families carrying the A1555G mutation identified a missense mutation (G28T) altering an invariant amino-acid residue (A10S) in the evolutionally conserved N-terminal region of the Trmu protein (Yan et al. unpublished data). The A10S mutation occurs 10% in the Jewish and Caucasian populations but was absent in 142 Chinese controls (Yan et al. unpublished data). Further functional analysis revealed that resultant biochemical defects caused by the A10S mutation in TRMU aggravate the mitochondrial dysfunction associated with A1555G mutation, exceeding the threshold for expressing deafness phenotype. Thus, the mutated TRMU, acting as a modifier factor, modulates the phenotypic manifestation of deafness-associated A1555G mutation. Here, the low penetrance of hearing loss in those four Chinese families implies that nuclear modifier genes such as TRMU may not play a significant role in the phenotypic manifestation of the A1555G mutation. The background sequences (haplotype) of the mtDNA have been shown to influence the penetrance of hearing loss associated with primary mtDNA mutations. mtDNA mutations at positions 4216 and 13708 labeled as second LHON mutations were implicated to increase the penetrance of the deafness-associated A7445G mutation in the precursor of tRNASer(UCN) gene [32], while the ND1 T3308C and tRNAAla T5655C mutations likely contribute to the higher penetrance of deafness in an African pedigree than Japanese and French families carrying the tRNASer(UCN) T7511C mutation [33]. Furthermore, the A7444G mutation in the precursor of tRNASer(UCN) or C-insertion at position 961 of 12S rRNA may influence the phenotypic expression of the A1555G mutation [11,34]. However, the phylogenetic analysis of mtDNA haplotypes in 50 Spanish families carrying the A1555G mutation implied that mtDNA backgrounds may not play a significant role in the expression of the A1555G mutation [35]. Despite sharing five common variants including A263G,

T16362C, A750G, A1438G, and A15326G, these mitochondrial genomes of BJ101, BJ101, BJ103, and BJ104 pedigrees belong to different Eastern Asian haplogroups R9a, N9a, D4a, and D4, respectively [36]. This suggested that the A1555G mutation in those Chinese families, similar to the A1555G mutation in 50 Spanish and 13 Japanese families [35,37], occurred sporadically and multiplied through evolution of the mtDNA in China. However, there was the absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in these Chinese families. Despite the presence of several high evolutionary conservative variants in protein-encoding genes, the extremely low penetrance of hearing loss associated with the A1555G mutation implies that mtDNA haplotypes may not play an important role in the phenotypic expression of the A1555G mutation in those Chinese families.

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. and the Chinese National Nature Science Foundation Research Grant 30170530 to Y.W.Y. and 30470956 to Q.W.

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