Abstract
Hearing loss (HL) is the most common sensory disorder in humans. Many patients with mitochondrial diseases have sensorineural HL (SNHL). The HL of these patients manifests as a consequence of either syndromic or nonsyndromic mitochondrial diseases. Furthermore, the phenotypes vary among patients even if they are carrying the same mutation. Therefore, these features make it necessary to analyze every presumed mutation in patients with hereditary HL, but the extensive analysis of various mutations is laborious. We analyzed 373 patients with suspected hereditary HL by using an extended suspension-array screening system for major mitochondrial DNA (mtDNA) mutations, which can detect 32 other mtDNA mutations in addition to the previously analyzed 29 mutations. In the present study, we detected 2 different mtDNA mutations among these 373 patients; m.7444G>A in the MT-CO1 gene and m.7472insC in the MT-TS1 gene in 1 patient (0.3%) for each. As these two patients had no clinical features other than HL, they had not been suspected of having mtDNA mutations. This extended screening system together with the previous one is useful for the genetic diagnosis and epidemiological study of both syndromic and nonsyndromic HL.
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Introduction
We can find many patients with hearing loss (HL) among those with mitochondrial DNA (mtDNA) mutations. These patients are classified into two categories; those having only HL (nonsyndromic HL) and those with HL plus other symptoms of mitochondrial disease (syndromic HL). Furthermore, as the severities and phenotypes of mitochondrial diseases vary from patient to patient, we often find mtDNA mutations in unexpected cases.1 The fact that there are many cases without any apparent family history makes it more difficult to diagnose mitochondrial diseases.2 Sensorineural HL (SNHL) is the most common sensory disorder in humans, having a prevalence of 2.7 per 1000 in children under 5 years of age.3 The frequency of patients with HL caused by mtDNA mutations increases with age, because mitochondrial diseases usually become aggravated with age. Therefore, it is necessary to analyze many different suspected mutations in mtDNA, but it is very exhaustive to examine these muations one by one.
Previously, we reported the results of extensive and rapid screening for major 29 major point mutations of mtDNA in patients with hereditary HL by using a suspension array technology.4 Our previous survey of 373 patients with suspected HL by use of this screening system revealed the m.1555A>G mutation in 11 patients, the m.3243A>G mutation in 9 patients, and the m.8348A>G, m.11778G>A and m.15498G>A mutations in 1 patient each. In the present extended study, we increased the number of mutations that could be detected from 29 to 61. We examined the applicability of this extended screening system for genetic diagnosis of hereditary HL by analyzing these same 373 patients with suspected hereditary HL.
Materials and methods
Patients
The study population included 373 unrelated Japanese patients with suspected hereditary HL, who visited the outpatient clinic of the Department of Otolaryngology, University Hospital of Medicine, Tokyo Medical and Dental University. The subjects included patients with a family history of HL and those with no apparent cause of HL, even though they did not have any apparent family history of HL. Their detailed demographic and audiometric features are shown in Table 1. The average age of them was 40 years, with a range between 1 and 77 years.
The study protocol complied with the Declaration of Helsinki, and it was also approved by the Committee on the Ethics of Human Research of the Tokyo Metropolitan Institute of Gerontology and the Institutional Review Board (IRB no. 68) of Tokyo Medical and Dental University. This study was carried out only after obtaining the written informed consent of each individual and/or the parents in the case of children.
Extended screening of mtDNA pathological mutation by use of suspension-array technology
DNA samples were purified from the blood by using a standard procedure. The mtDNA from each patient was analyzed with the previously described extended suspension array-based screening system.5 The targets of the present analysis were 32 mtDNA mutations in 15 genes: 1 in each of MT-TQ (tRNAGln), MT-TW (tRNATrp), MT-TC (tRNACys), MT-TH (tRNAHis), MT-TL2 (tRNALeu(CUN)), MT-ND6 and MT-CYB genes; 2 in each of MT-TN (tRNAAsn), MT-ATP6, MT-TG (tRNAGly) and MT-TE (tRNAGlu) genes; 3 in the MT-ND3 gene; 4 in the MT-CO1 gene; and 5 in each of the MT-TI(tRNAIle) and MT-TS1 (tRNASer(UCN)) genes as shown in Table 2. The mtDNA mutations reported in our previous study were within the DNA fragments amplified by multiplex PCR for mtDNA haplotyping, which was mainly designed for anthropological purposes. For the present study, we newly designed a second multiplex PCR system to analyze the 32 additional mtDNA mutations (including m.7445, which is known to cause of HL).
Comparison of results between suspension array and direct DNA sequencing
DNA sequencing was carried out by using an Applied Biosystems 3130 × l Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and Sequencher version 4.2.2 (Gene Codes, Ann Arbor, MI, USA) to compare the sequences with the revised Cambridge reference sequence,6, 7 while following the standard procedure.8, 9
Results and discussion
In the present extended study, 2 of the 32 mtDNA mutations, m.7444G>A and m.7472insC, were detected by the screening system, each in 1 patient out of the 373 patients with SNHL. The median fluorescent intensities for the m.7444G>A mutation and the 7472insC mutation are displayed in scatter diagrams (Figure 1). When the median fluorescent intensity values for the wild-type signals were below the cut-off values, we regarded the mutations as homoplasmic. The m.7444G>A mutation was homoplasmic and the 7472insC mutation was heteroplasmic. On the basis of the chromatogram, the mutation load was estimated as 59%. None of the other 30 mutations were detected in these patients with SNHL. We summarized the mtDNA mutations detected by the previous and the present analytical systems in Table 3.
SNHL is one of the most common disorder in patients with mitochondrial diseases,10 which is represented by the mutations of the homoplasmic m.1555A>G and the heteroplasmic m.3243A>G.11, 12, 13, 14, 15, 16 We previously reported that not only these mutations but also other mutations could be detected in the patients with either nonsyndromic or syndromic hereditary HL. The m.7444G>A mutation was earlier reported as a cause of aminoglycoside-induced and nonsyndromic HL.17 However, patient TMD278, carrying this mutation, had no history of aminoglycoside injection in her detailed clinical history. On the other hand, we should mention that this mutation characterizes haplogroup V7 and H40b, and there is no direct evidence that these haplogroups tend to have HL.18 Furthermore, it was also reported that the m.7444G>A mutation is a secondary mutation found in patients with Leber’s hereditary optic neuropathy (LHON) and that this mutation has an additional role in the pathogenesis of LHON.19, 20 Primary mutations, m.11778G>A, m.3460G>A and m.14484T>C can cause LHON. However, these mutations had already been examined in our previous study and the patient TMD278 was negative for them. With regard to her clinical data, she had high-tone SNHL as shown in Figure 2a, although she was 42 years of age. The onset of her HL occurred during her childhood, after which the HL became progressive. She had started wearing hearing aids 3 years before visiting our clinic. Her clinical feature seemed sporadic because she had neither other clinical disorders such as LHON nor a family history of HL.
The m.7472insC was reported as a pathogenic heteroplasmic mutation within the coding region of tRNASer(UCN). This mutation was previously reported to be associated with progressive myoclonus epilepsy or syndromic disorders including HL, ataxia and myoclonus in previous reports.21 The phenotypes of this mutation, however, vary even among individuals within the same family.22 The audiogram of TMD368 showed moderate SNHL (Figure 2b), although this patient was still just 35 years old. He had no other clinical disorders such as myoclonus or family history of HL or neurological disorders. It should also be noted that recently the m.7472insC mutation was identified in gastric cancer tissues.23 This report suggested that somatic mtDNA mutations may have an important role in the progression of gastric cancer.
In conclusion, the present extended screening system by use of a suspension array for major mtDNA mutations was demonstrated to be powerful, because we could detect both major causative and unexpected mtDNA mutations. The present system is helpful for both the diagnosis and epidemiological studies. Detecting mtDNA mutations in the early stage of HL could be meaningful both to select the optimal therapeutic strategies for the patients and to provide appropriate genetic counseling.
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Acknowledgements
We thank Y Abe for helpful discussions and excellent technical support. This work was supported in part by grants from the programs grants-in-aid for young scientists (B)-(no. 22791577 to TK), grants-in-aid for scientific research (B)-(no. 21390459 to KK), grants-in-aid for scientific research (C) (no. 18590317 to Y Nishigaki and no. 21590411 to HH) and grants-in-aid for scientific research (A-22240072, B-21390459 and C-21590411 to MT) from the Ministry of Education Culture, Sports, Science and Technology; by a grant-in-aid for scientific research from the Ministry of Health, Labor and Welfare of Japan (H23-kankaku-005 to KK); by grants-in-aid for the Research on Intractable Diseases (Mitochondrial Disease H23-016 and H23-119) from the Ministry of Health, Labor, and Welfare (to MT); and by grants for scientific research from the Takeda Science Foundation (to MT).
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Kato, T., Nishigaki, Y., Noguchi, Y. et al. Extended screening for major mitochondrial DNA point mutations in patients with hereditary hearing loss. J Hum Genet 57, 772–775 (2012). https://doi.org/10.1038/jhg.2012.109
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DOI: https://doi.org/10.1038/jhg.2012.109