The Role of Mitochondrial DNA Mutations in Hearing Loss

Mutations in mitochondrial DNA (mtDNA) are one of the most important causes of hearing loss. Of these, the homoplasmic A1555G and C1494T mutations at the highly conserved decoding site of the 12S rRNA gene are well documented as being associated with either aminoglycoside-induced or nonsyndromic hearing loss in many families worldwide. Moreover, five mutations associated with nonsyndromic hearing loss have been identified in the tRNA^Ser(UCN) gene: A7445G, 7472insC, T7505C, T7510C, and T7511C. Other mtDNA mutations associated with deafness are mainly located in tRNA and protein-coding genes. Failures in mitochondrial tRNA metabolism or protein synthesis were observed from cybrid cells harboring these primary mutations, thereby causing the mitochondrial dysfunctions responsible for deafness. This review article provides a detailed summary of mtDNA mutations that have been reported in deafness and further discusses the molecular mechanisms of these mtDNA mutations in deafness expression.     

与耳聋相关的线粒体tRNA突变

线粒体tRNA基因突变是导致感音神经性耳聋的原因之一．有些tRNA突变可直接造成耳聋的发生,称之为原发突变．如tRNA^Leu(UUR) A3243G等突变与综合征型耳聋相关,而tRNA^Ser(UCN) T7511C等突变则与非综合征型耳聋相关．此外,继发突变如tRNA^Thr G15927A等突变则对原发突变起协同作用,影响耳聋的表型表达．这些突变可引起tRNA二级结构改变,从而影响线粒体蛋白质合成,降低细胞内ATP的产生,由此引起的线粒体功能障碍可导致耳聋的发生．主要讨论与耳聋相关的线粒体tRNA突变及其致聋机理．     

Coexistence of mitochondrial 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations in two Han Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing loss

Mutations in mitochondrial DNA are one of the important causes of hearing loss. We report here the clinical, genetic, and molecular characterization of two Han Chinese pedigrees with maternally transmitted aminoglycoside-induced and nonsyndromic bilateral hearing loss. Clinical evaluation revealed the wide range of severity, age-at-onset, and audiometric configuration of hearing impairment in matrilineal relatives in these families. The penetrances of hearing loss in these pedigrees were 20% and 18%, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss in these seven pedigrees were 10% and 15%. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the presence of the deafness-associated 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations. Their distinct sets of mtDNA polymorphism belonged to Eastern Asian haplogroup C4a1, while other previously identified six Chinese mitochondrial genomes harboring the C1494T mutation belong to haplogroups D5a2, D, R, and F1, respectively. This suggested that the C1494T or G7444A mutation occurred sporadically and multiplied through evolution of the mitochondrial DNA (mtDNA). The absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in their mtDNA suggest that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations in those Chinese families. However, aminoglycosides and other nuclear modifier genes play a modifying role in the phenotypic manifestation of the C1494T mutation in these Chinese families.     

Co-segregation of the T1095C with the A1555G mutation of the mitochondrial 12S rRNA gene in a patient with non-syndromic hearing loss

We reported here the clinical and molecular characterization of a Chinese subject with childhood-onset hearing impairment. Clinical evaluations showed that the patient suffered from profound and non-syndromic sensorineural hearing loss with flat configurations. Sequence analysis of the mitochondrial 12S rRNA and tRNA^Ser(UCN) genes led to the identification of double deafness-associated mutations of A1555G and T1095C in the 12S rRNA gene which apparently in the homoplasmic forms. In additional, there was no other functionally significant nucleotide variants found in this subject. As previous studies have indicated that the A1555G mutation was a primary contributing factor underlying the development of deafness but not sufficient to produce clinical phenotype, the co-segregation of two mitochondrial DNA mutations raises the possibility that the T to C transition at position 1095 plays a role in the phenotypic expression of deafness-associated A1555G mutation. Actually, the T1095C mutation disrupted an evolutionarily conserved base-pair at stem-loop of helix 25 of 12S rRNA, resulting in impaired translation in mitochondrial protein synthesis and a significant reduction of cytochrome c oxidase activity. As a result, it may enhance the biochemical defect in patient carrying the A1555G mutation, thus changing the age of onset and the severity of hearing impairment.     

A novel MT-CO1 m.6498C>A variation associated with the m.7444G>A mutation in the mitochondrial COI/tRNASer(UCN) genes in a patient with hearing impairment,diabetes and congenital visual loss

Mitochondrial diseases are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain. Sensorineural hearing loss (SNHL) has been described in association to different mitochondrial multisystem syndromes, often involving the central nervous system, neuromuscular, or endocrine organs. In this study, we described a Tunisian young girl with hearing impairment, congenital visual loss and maternally inherited diabetes. No mutation was found in the mitochondrial tRNA^Leu(UUR) and the 12S rRNA genes. However, we detected the m.7444G>A mutation in the mitochondrial COI/tRNA^Ser(UCN) genes. This mutation eliminates the termination codon of the MT-CO1 gene and extends the COI polypeptide by three amino acids (Lys–Gln–Lys) to the C-terminal. The whole mitochondrial genome screening revealed the presence of a novel mutation m.6498C>A (L199I) in the mitochondrial DNA-encoded subunit I of the cytochrome c oxidase (COX). This “probably damaging” transversion affects a highly conserved domain and it was absent in 200 Tunisian controls. The studied patient was classified under the haplogroup H2a.     

Mutation analysis of mitochondrial 12S rRNA gene in Polish patients with non-syndromic and aminoglycoside-induced hearing loss

Mutations in mitochondrial DNA have been reported as associated with non-syndromic and aminoglycoside-induced hearing loss. In the present study, we have performed mutational screening of entire 12S rRNA gene in 250 unrelated patients with non-syndromic and aminoglycoside-induced hearing loss. Twenty-one different homoplasmic sequence variants were identified, including eight common polymorphisms, one deafness-associated mutation m.1555 A>G and three putatively pathogenic variants: m.669 T>C, m.827 A>G, m.961 delT+C(n)ins. The incidence of m.1555 A>G was estimated for 3.6% (9/250); however, where aminoglycoside exposure was taken as a risk factor, the frequency was 5.5% (7/128). Substitution m.669 T>C was identified only in patients with hearing impairment and episode of aminoglycoside exposure, which may suggest that such additional risk factors must appear to induce clinical phenotype. Moreover, two 12S rRNA sequence variants: m.988 G>A and m.1453 A>G, localized at conserved sites and affected RNA secondary structure, may be new candidates for non-syndromic and aminoglycoside-induced hearing loss associated mutations.     

线粒体DNA G7444A突变可能影响A1555G突变的表型表达

线粒体12S rRNA和tRNASer(UCN) 基因是导致非综合征型听力损失的两个突变热点区域。作者收集了1个母系遗传感音神经性聋家系, 该家系同时携带线粒体DNA (mtDNA) A1555G和G7444A突变。临床资料分析表明, 该家系包括药物致聋的耳聋外显率(所有耳聋患者/所有母系成员)为58%, 而非药物致聋的耳聋外显率(非药物性聋患者/所有母系成员)为25%, 明显高于其他携带A1555G突变的耳聋家系。先证者的线粒体全序列分析表明, 该线粒体基因组共有28个多态位点, 属于东亚人群B4c1单体型。在这些多态位点中, 除A1555G和G7444A突变外, 未发现其他有功能意义的突变。这表明mtDNA G7444A突变可能加重由A1555G突变造成的线粒体功能缺失, 从而增加耳聋的外显率。     

Common molecular etiologies are rare in nonsyndromic Tibetan Chinese patients with hearing impairment

Background

Thirty thousand infants are born every year with congenital hearing impairment in mainland China. Racial and regional factors are important in clinical diagnosis of genetic deafness. However, molecular etiology of hearing impairment in the Tibetan Chinese population living in the Tibetan Plateau has not been investigated. To provide appropriate genetic testing and counseling to Tibetan families, we investigated molecular etiology of nonsyndromic deafness in this population.

Methods

A total of 114 unrelated deaf Tibetan children from the Tibet Autonomous Region were enrolled. Five prominent deafness-related genes, GJB2, SLC26A4, GJB6, POU3F4, and mtDNA 12S rRNA, were analyzed. Inner ear development was evaluated by temporal CT. A total of 106 Tibetan hearing normal individuals were included as genetic controls. For radiological comparison, 120 patients, mainly of Han ethnicity, with sensorineural hearing loss were analyzed by temporal CT.

Results

None of the Tibetan patients carried diallelic GJB2 or SLC26A4 mutations. Two patients with a history of aminoglycoside usage carried homogeneous mtDNA 12S rRNA A1555G mutation. Two controls were homozygous for 12S rRNA A1555G. There were no mutations in GJB6 or POU3F4. A diagnosis of inner ear malformation was made in 20.18% of the Tibetan patients and 21.67% of the Han deaf group. Enlarged vestibular aqueduct, the most common inner ear deformity, was not found in theTibetan patients, but was seen in 18.33% of the Han patients. Common molecular etiologies, GJB2 and SLC26A4 mutations, were rare in the Tibetan Chinese deaf population.

Conclusion

The mutation spectrum of hearing loss differs significantly between Chinese Tibetan patients and Han patients. The incidence of inner ear malformation in Tibetans is almost as high as that in Han deaf patients, but the types of malformation vary greatly. Hypoxia and special environment in plateau may be one cause of developmental inner ear deformity in this population.     

tRNASer(UCN)7472delC可能是耳聋与癫痫相关的线粒体基因新突变

线粒体DNA突变与许多人类疾病的发病机制相关。文章报道1例典型的患有耳聋与癫痫症状的具有母系遗传特征的中国家系。该家系共3代人, 其中14名母系成员中有3名耳聋患者, 3名癫痫患者, 而其他成员则无临床症状。线粒体全基因组序列分析表明, tRNA^Ser(UCN)基因7472delC新突变和33个多态位点属于东亚单体型B4b1a2。7472delC突变位于tRNA^Ser(UCN)高度保守的T-arm上。而在该区域的相同位点7472insC突变已在多个无遗传相关的家系中被发现与耳聋和癫痫相关。7472insC突变使tRNA代谢和线粒体功能产生缺陷。这样与7472insC突变相近的7472delC突变可能也会以相似机制引起线粒体功能障碍。同时, 在该家系中未发现GJB2基因及其他线粒体基因突变。因此, ^tRNASer(UCN) 7472delC可能是耳聋与癫痫相关的线粒体基因新突变。     

Mitochondrial 12S rRNA susceptibility mutations in aminoglycoside-associated and idiopathic bilateral vestibulopathy

The mitochondrial 12S rRNA is considered a hotspot for mutations associated with nonsyndromic (NSHL) and aminoglycoside-induced hearing loss (AIHL). Although aminoglycoside ototoxicity is the most common cause of bilateral vestibular dysfunction, the conceivable role of 12S rRNA mutations has never been systematically investigated. We sequenced the 12S rRNA of 66 patients with bilateral vestibulopathy (BV) with (n = 15) or without (n = 51) prior exposure to aminoglycosides, as well as 155 healthy controls with intact vestibular function (sport pilots), and compared these to 2704 published sequences (Human Mitochondrial Genome Database). No mutations with a confirmed pathogenicity were found (A1555G, C1494T), but four mutations with a hitherto tentative status were detected (T669C, C960del, C960ins, T961G). Due to their predominant occurrence in patients without aminoglycoside exposure, their detection in controls and a weak evolutionary conservation, their pathogenic role in vestibulocochlear dysfunction remains provisional.     

Aminoglycoside-induced and non-syndromic hearing loss is associated with the G7444A mutation in the mitochondrial COI/tRNASer(UCN) genes in two Chinese families

We report here the clinical, genetic, and molecular characterization of two Chinese families with aminoglycoside induced and non-syndromic hearing impairment. Clinical and genetic evaluations revealed the variable severity and age-of-onset in hearing impairment in these families. Strikingly, there were extremely low penetrances of hearing impairment in these Chinese families. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the distinct sets of mtDNA polymorphism, in addition to the identical G7444A mutation associated with hearing loss. Indeed, the G7444A mutation in the CO1 gene and the precursor of tRNASer(UCN) gene is present in homoplasmy only in the maternal lineage of those pedigrees but not other members of these families and 164 Chinese controls. Their mitochondrial genomes belong to the Eastern Asian haplogroups C5a and D4a, respectively. In fact, the occurrence of the G7444A mutation in these several genetically unrelated subjects affected by hearing impairment strongly indicates that this mutation is involved in the pathogenesis of hearing impairment. However, there was the absence of other functionally significant mtDNA mutations in two Chinese pedigrees carrying the G7444A mutation. Therefore, nuclear modifier gene(s) or aminoglycoside(s) may play a role in the phenotypic expression of the deafness-associated G7444A mutation in these Chinese pedigrees.     

Biochemical characterization of the mitochondrial tRNASer(UCN) T7511C mutation associated with nonsyndromic deafness

We report here the biochemical characterization of the deafness-associated mitochondrial tRNA^Ser(UCN) T7511C mutation, in conjunction with homoplasmic ND1 T3308C and tRNA^Ala T5655C mutations using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from an African family into human mtDNA-less (ρ°) cells. Three cybrids derived from an affected matrilineal relative carrying the homoplasmic T7511C mutation, exhibited ~75% decrease in the tRNA^Ser(UCN) level, compared with three control cybrids. This amount of reduction in the tRNA^Ser(UCN) level is below a proposed threshold to support a normal rate of mitochondrial protein synthesis in lymphoblastoid cell lines. This defect is likely a primary contributor to ~52% reduction in the rate of mitochondrial protein synthesis and marked defects in respiration and growth properties in galactose-containing medium. Interestingly, the T5655C mutation produces ~50% reduction in the tRNA^Ala level in mutant cells. Strikingly, the T3308C mutation causes a significant decrease both in the amount of ND1 mRNA and co-transcribed tRNA^Leu(UUR) in mutant cells. Thus, mitochondrial dysfunctions caused by the T5655C and T3308C mutations may modulate the phenotypic manifestation of the T7511C mutation. These observations imply that a combination of the T7511C mutation with two mtDNA mutations accounts for the high penetrance of deafness in this family.     

Novel nucleotide changes in mutational analysis of mitochondrial 12SrRNA gene in patients with nonsyndromic and aminoglycoside-induced hearing loss

Mitochondria have essential role in cellular energy metabolism and defects in their function lead to many metabolic diseases. Mitochondrial DNA (mtDNA) mutations have been associated with number diseases such as nonsyndromic and aminoglycoside-induced hearing loss. Mutational screening of entire 12SrRNA and tRNA ^{ser (UCN)} genes in 107 unrelated Iranian patients with amino glycoside-induced and nonsyndromic bilateral hearing loss by direct sequencing analysis method were performed. Twenty different homoplasmic sequence variants were identified; including fifteen common polymorphisms, two putatively pathogenic variants: m.921T>C and m.1005T>C, one 12SrRNA sequence variant m.739C>T and two nucleotides substitution; m.1245T>C and m.1545T>C. Deafness-associated mutation, m.1555A>G, was not found. In our patients we found the mutation 1005 was associated with R haplogroup. These finding show that m.1555A>G mutation is not important in our population. Nucleotide change, m.739C>T, previously reported with very low frequency. We suggested the variation of two nucleotides 1245 and 1545 that localized at conserved site of 12SrRNA may be new candidate for amino glycoside-induced and nonsyndromic hearing impairment associated mutations. However, aminoglycoside exposure is a risk factor for clinical phenotype appearance of these mutations.     

Mitochondrial tRNA mutations associated with deafness

Mitochondrial tRNA mutations are one of the important causes of both syndromic and non-syndromic deafness. Of those, syndromic deafness-associated tRNA mutations such as tRNA(Leu(UUR)) 3243A>G are often present in heteroplasmy, while non-syndromic deafness-associated tRNA mutations including tRNA(Ser(UCN)) 7445A>G often occur in homplasmy or in high levels of heteroplasmy. These tRNA mutations are the primary mutations leading to hearing loss. However, other tRNA mutations such as tRNA(Thr) 15927G>A and tRNA(Ser(UCN)) 7444G>A may act in synergy with the primary mitochondrial DNA mutations, modulating the phenotypic manifestation of the primary mitochondrial DNA mutations. Theses tRNA mutations cause structural and functional alteration. A failure in tRNA metabolism caused by these tRNA mutations impaired mitochondrial translation and respiration, thereby causing mitochondrial dysfunctions responsible for deafness. These data offer valuable information for the early diagnosis, management and treatment of maternally inherited deafness.     

Unexpected heterogeneity due to recessive and de novo dominant mutations of GJB2 in an Iranian family with nonsyndromic hearing loss: implication for genetic counseling

Mutations in the GJB2 gene are the most common cause of nonsyndromic autosomal recessive sensorineural hearing loss (HL). A few mutations in GJB2 have also been reported to cause dominant nonsyndromic HL. Here we report a large inbred family including two individuals with nonsyndromic sensorineural hearing loss. A dominant GJB2 mutation, c.551G>A (p.R184Q), was detected in the proband, yet his parents were negative for the mutation. The second affected person had heterozygous c.35delG mutation, which was inherited from his father. Large deletions of the GJB6 gene were not detected in this family. This study highlights the importance of mutation analysis in all affected cases within a pedigree.     

Mitochondrial 12S rRNA variants in 1642 Han Chinese pediatric subjects with aminoglycoside-induced and nonsyndromic hearing loss

In this report, we investigated the frequency and spectrum of mitochondrial 12S rRNA variants in a large cohort of 1642 Han Chinese pediatric subjects with aminoglycoside-induced and nonsyndromic hearing loss. Mutational analysis of 12S rRNA gene in these subjects identified 68 (54 known and 14 novel) variants. The frequencies of known 1555A>G and 1494C>T mutations were 3.96% and 0.18%, respectively, in this cohort with nonsyndromic and aminoglycoside-induced hearing loss. Prevalence of other putative deafness-associated mutation at positions 1095 and 961 were 0.61% and 1.7% in this cohort, respectively. Furthermore, the 745A>G, 792C>T, 801A>G, 839A>G, 856A>G, 1027A>G, 1192C>T, 1192C>A, 1310C>T, 1331A>G, 1374A>G and 1452T>C variants conferred increased sensitivity to ototoxic drugs or nonsyndromic deafness as they were absent in 449 Chinese controls and localized at highly conserved nucleotides of this rRNA. However, other variants appeared to be polymorphisms. Moreover, 65 Chinese subjects carrying the 1555A>G mutation exhibited bilateral and sensorineural hearing loss. A wide range of severity, age-of-onset and audiometric configuration was observed among these subjects. In particular, the sloping and flat-shaped patterns were the common audiograms in individuals carrying the 1555A>G mutation. The phenotypic variability in subjects carrying these 12S rRNA mutations indicated the involvement of nuclear modifier genes, mitochondrial haplotypes, epigenetic and environmental factors in the phenotypic manifestation of these mutations. Therefore, our data demonstrated that mitochondrial 12S rRNA is the hot spot for mutations associated with aminoglycoside ototoxicity.     

Identification and functional characterization of novel compound heterozygotic mutations in the TECTA gene

Mutations of the TECTA gene, which encodes alpha-tectorin, are associated with both dominant (DFNA8/A12) and recessive (DFNB 21) modes of inherited nonsyndromic sensorineural hearing loss, respectively. Although clinical data and genetic analysis for TECTA gene have been reported from different groups, there is no report that compound heterozygous mutations in the TECTA gene result in nonsyndromic sensorineural hearing loss. Here, we identified a missense mutation (p.C1691F) and a splicing mutation (c.6162 + 3insT), one in each TECTA allele, in the patient with hearing loss. Also, we demonstrated that the splicing mutation results in the abnormal skipping of an exon, which leads to a truncated protein as determined by exon-trapping analysis. To the best of our knowledge, this is the first report of an in vitro functional study of splice site mutations in the TECTA gene.     

Application of a New Genetic Deafness Microarray for Detecting Mutations in the Deaf in China

Objective

The aim of this study was to evaluate the GoldenGate microarray as a diagnostic tool and to elucidate the contribution of the genes on this array to the development of both nonsyndromic and syndromic sensorineural hearing loss in China.

Methods

We developed a microarray to detect 240 mutations underlying syndromic and nonsyndromic sensorineural hearing loss. The microarray was then used for analysis of 382 patients with nonsyndromic sensorineural hearing loss (including 15 patients with enlarged vestibular aqueduct syndrome), 21 patients with Waardenburg syndrome, and 60 unrelated controls. Subsequently, we analyzed the sensitivity, specificity, and reproducibility of this new approach after Sanger sequencing-based verification, and also determined the contribution of the genes on this array to the development of distinct hearing disorders.

Results

The sensitivity and specificity of the microarray chip were 98.73% and 98.34%, respectively. Genetic defects were identified in 61.26% of the patients with nonsyndromic sensorineural hearing loss, and 9 causative genes were identified. The molecular etiology was confirmed in 19.05% and 46.67% of the patients with Waardenburg syndrome and enlarged vestibular aqueduct syndrome, respectively.

Conclusion

Our new mutation-based microarray comprises an accurate and comprehensive genetic tool for the detection of sensorineural hearing loss. This microarray-based detection method could serve as a first-pass screening (before next-generation-sequencing screening) for deafness-causing mutations in China.     

The Deafness-Associated Mitochondrial DNA Mutation at Position 7445, Which Affects tRNASer(UCN) Precursor Processing, Has Long-Range Effects on NADH Dehydrogenase Subunit ND6 Gene Expression

The pathogenetic mechanism of the deafness-associated mitochondrial DNA (mtDNA) T7445C mutation has been investigated in several lymphoblastoid cell lines from members of a New Zealand pedigree exhibiting the mutation in homoplasmic form and from control individuals. We show here that the mutation flanks the 3′ end of the tRNA^Ser(UCN) gene sequence and affects the rate but not the sites of processing of the tRNA precursor. This causes an average reduction of ~70% in the tRNA^Ser(UCN) level and a decrease of ~45% in protein synthesis rate in the cell lines analyzed. The data show a sharp threshold in the capacity of tRNA^Ser(UCN) to support the wild-type protein synthesis rate, which corresponds to ~40% of the control level of this tRNA. Strikingly, a 7445 mutation-associated marked reduction has been observed in the level of the mRNA for the NADH dehydrogenase (complex I) ND6 subunit gene, which is located ~7 kbp upstream and is cotranscribed with the tRNA^Ser(UCN) gene, with strong evidence pointing to a mechanistic link with the tRNA precursor processing defect. Such reduction significantly affects the rate of synthesis of the ND6 subunit and plays a determinant role in the deafness-associated respiratory phenotype of the mutant cell lines. In particular, it accounts for their specific, very significant decrease in glutamate- or malate-dependent O₂ consumption. Furthermore, several homoplasmic mtDNA mutations affecting subunits of NADH dehydrogenase may play a synergistic role in the establishment of the respiratory phenotype of the mutant cells.