Hearing loss is one of the most common human health problems, affecting one in 700-1000 newborns [1]. Deafness can be caused by gene alternations and environmental factors including the ototoxic drugs such as aminoglycoside antibiotics. Of the hereditary factors, variants in mitochondrial DNA (mtDNA), especially in
With the aim of elucidating the molecular basis of hearing loss, an extensive mutational screening for mitochondrial
As a part of genetic screening program for hearing loss, a three-generation Han Chinese family (as shown in Figure 1) was found at the Department of Otolaryngology, Hangzhou First People’s Hospital, Zhejiang Province, PRC. Informed consent was obtained from the participants. Blood samples were obtained from all participants prior to their participation in the study, in accordance with the Ethics Committee of Hangzhou First People’s Hospital. In addition, a comprehensive history and physical examination were performed to identify any syndromic findings, the history of the use of aminoglycosides, as well as the genetic factors related to the hearing impairment in members of this pedigree. An age-appropriate audiological examination was performed, and this examination included pure tone audiometry (PTA) and auditory brainstem response (ABR), immittance testing and distortion product otoacoustic emissions. The PTA was calculated from the sum of the audiometric thresholds at 500, 1000, 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. Moreover, DNA was obtained from 200 control subjects from a panel of unaffected Han Chinese subjects from the same region who were clinically tested.
A three-generation Han Chinese family with AINHL. Hearing loss individuals are indicated by filled symbols. The arrow denotes the proband. Asterisks denote individuals who have a family history of exposure to amino glycosides.Figure 1
Genomic DNA was isolated from whole blood of participants using the Puregene DNA Isolation Kits (Gentra Systems, Minneapolis, MN, USA). First, three matrilineal relatives (I-2, II-5, III-2) and control subject’s DNA fragments spanning the mitochondrial
A total of 17 vertebrates’ mitochondrial genome sequences were used in the interspecific analysis. These include
The DNA fragments spanning the entire coding region of the
A previous study showed that the
All patients from the Han Chinese family lived in Hangzhou City of Zhejiang Province. The proband (III-2) was an infant born in Hangzhou First People’s Hospital. As shown in Table 1 and Figure 2, the proband exhibited bilateral hearing impairment (90 dB right ear and 95 dB left ear). A comprehensive history and physical examination were performed to identify any syndromic findings, and the history of use of aminoglycosides. Moreover, we noticed that the proband’s mother (II-5), a young woman at the age of 26 years; had been administered gentamicin (5 mg/kg/dose, 10 days) for fever when she was 18-years-old. She developed the profound hearing loss 2 months after the drug administration. It is interesting to note that two matrilineal relatives (I-2, II-5), who had a history of exposure to gentamicin and streptomycin, exhibited a severe hearing impairment in this maternal kindred, suggesting that the aminoglycosides may play an important role in this disorder.
Summary of clinical data for several members of this family PTA: pure tone audiometry; dB: decibel.Subjects I-2 II-1 II-5 III-2 II-4 Gender female male female male male Age when tested 50 26 30 1 39 Age at onset 46 18 25 1 – Use of aminoglycoside yes no yes no no PTA (dB) right ear 90 90 100 85 25 PTA (dB) left ear 92 85 100 75 25 Level of hearing loss profound profound severe severe normal
Air conduction audiogram of family members with the mitochondrial C1494T and G7444A pathogenic variants, subject II-4 was used as a control. Symbols: X: left ear, O: right ear.Figure 2
The maternal transmission of hearing loss in this family suggested a mitochondrial involvement and led us to analyze the mitochondrial genome of matrilineal relatives (I-2, II-5, III-2) and the healthy subjects. We first examined the known mtDNA pathogenic variants associated with deafness by PCR amplification (A1555G, C1494T, A7445G, T7510C and T7511C). As shown in Figure 3, the PCR-Sanger sequencing identified two known pathogenic variants: the C1494T in the
Identification G7444A pathogenic variant in the Figure 3
To elucidate the molecular basis for maternally transmitted deafness, 24-overlapping DNA fragments spanning the entire mitochondrial genome were PCR-amplified and sequenced. The comparison of the resultant sequence with the Cambridge consensus sequence identified a set of polymorphisms, as shown in Table 2. Among these, there were five variants in the D-loop, two known variants in the
mtDNA sequence variants in this family with hearing impairment. Conservation of amino acid for polypeptides or nucleotide for RNAs in human (H), bovine (B), mouse (M), and Xenopus laevis (X). See the online mitochondrial genome database (Gene Position Replacement Conservation Previously Reported 73 A>G – yes 152 T>C – yes 263 A>G – yes 16223 C>T – yes 16519 T>C – yes 827 A>G – yes 1438 A>G – yes 1494 C>T C/C/C/C yes 2706 A>G A/G/A/A yes 3010 G>A G/G/A/A yes 3497 C>T (Ala→Val) – yes 3970 C>T – yes 4883 C>T – yes 7444 G>A (Term→Lys) – yes 8860 A>G (Thr→Ala) – yes 10398 A>G (Thr→Ala) – yes 10400 C>T – yes 11719 G>A – yes 12705 C>T – yes 15301 G>A – yes 15426 A>G (Thr→Ala) T/M/I/I yes
To examine the role of the
In this study, we have performed clinical, genetic and molecular characterization of a three-generation Han Chinese family with AINHL. Hearing impairment as a sole clinical phenotype was mostly present in the maternal lineage of this pedigree, suggesting that the mtDNA variant was the molecular basis for this disorder. As shown in Figure 1, this family exhibited a high penetrance of hearing loss, in particular, the penetrance of hearing loss in this family was 80.0 and 40.0%, when aminoglycoside was included and excluded.
Sequence analysis of the mitochondrial genome showed the presence of C1494T pathogenic variant in the
In addition, the mitochondrial haplotype has been shown to influence the penetrance of hearing loss associated with mtDNA primary mutations. In particular, mtDNA variants at positions 4216 and 13708, acting as second Lebers’ hereditary optic neuropathy (LHON) variants, were implicated to increase the penetrance of the deafness-associated A7445G pathogenic variant [19]. Moreover, the T5628C variant in tRNAAla was thought to have a modifying role in the phenotypic manifestation of the C1494T pathogenic variant in a Han Chinese family [20]. In this study, the sequence analysis of the entire mitochondrial genome identified a set of polymorphisms, apart from C1494T and G7444A pathogenic variants, other variants in the mitochondrial genome showed no evolutionary conservation. As shown in Figures 3 and 4, the G7444A pathogenic variant resulted in a read-through of the stop condon AGA of the COI message, thereby adding three amino acids (Lys-Gln-Lys) to the C-terminal of the polypeptide. Thus, the mutated polypeptide may retain a partial function. Alternatively, the G7444A pathogenic variant was adjacent to the site of 3′ end endonucleolytic processing of the L-strand RNA precursor, spanning tRNASer(UCN) and
Location of deafness-associted mutations in tRNASer(UCN) and adjacent COI. The arrow indicates the A7445G and G7444A pathogenic variants in the precursor of this tRNA and adjacent sequence of COI from wild-type (WT) and mutant (MT).Figure 4