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Methodology for Accurate Detection of Mitochondrial DNA Methylation
Published on: May 20, 2018
Mitochondrial m.1584A 12S m62A rRNA methylation in families with m.1555A>G associated hearing loss
Mary O'Sullivan1, Paul Rutland1, Deirdre Lucas2
1Genetics and Genomic Medicine, UCL Institute of Child Health, London WC1N 1EH, UK.
Abstract:
The mitochondrial DNA mutation m.1555A>G predisposes to hearing loss following aminoglycoside antibiotic exposure in an idiosyncratic dose-independent manner. However, it may also cause maternally inherited hearing loss in the absence of aminoglycoside exposure or any other clinical features (non-syndromic hearing loss). Although m.1555A>G was identified as a cause of deafness more than twenty years ago, the pathogenic mechanism of this mutation of ribosomal RNA remains controversial. Different mechanistic concepts have been proposed. Most recently, evidence from cell lines and animal models suggested that patients with m.1555A>G may have more 12S rRNA N6, N6-dimethyladenosine (m(6) 2A) methylation than controls, so-called 'hypermethylation'. This has been implicated as a pathogenic mechanism of mitochondrial dysfunction but has yet to be validated in patients. 12S m(6) 2A rRNA methylation, by the mitochondrial transcription factor 1 (TFB1M) enzyme, occurs at two successive nucleotides (m.1584A and m.1583A) in close proximity to m.1555A>G. We examined m(6) 2A methylation in 14 patients with m.1555A>G, and controls, and found all detectable 12S rRNA transcripts to be methylated in both groups. Moreover, different RNA samples derived from the same patient (lymphocyte, fibroblast and lymphoblast) revealed that only transformed cells contained some unmethylated 12S rRNA transcripts, with all detectable 12S rRNA transcripts derived from primary samples m(6) 2A-methylated. Our data indicate that TFB1M 12S m(6) 2A rRNA hypermethylation is unlikely to be a pathogenic mechanism and may be an artefact of previous experimental models studied. We propose that RNA methylation studies in experimental models should be validated in primary clinical samples to ensure that they are applicable to the human situation.
Insights
The mitochondrial DNA mutation m.1555A>G causes hearing loss. Recent studies suggested hypermethylation of 12S rRNA as a cause, but this study found methylation in both patients and controls, refuting this mechanism.
Area of Science:
- Genetics
- Molecular Biology
- Otolaryngology
Background:
- The mitochondrial DNA mutation m.1555A>G is a known cause of maternally inherited hearing loss, often exacerbated by aminoglycoside antibiotics.
- The precise pathogenic mechanism of m.1555A>G, particularly concerning ribosomal RNA methylation, remains debated.
- Previous research suggested 'hypermethylation' of 12S rRNA as a potential mechanism, but this required validation in human patients.
Purpose of the Study:
- To investigate the role of 12S rRNA N6, N6-dimethyladenosine (m(6) 2A) methylation in patients with the m.1555A>G mitochondrial DNA mutation.
- To determine if increased m(6) 2A methylation, mediated by the TFB1M enzyme, is a pathogenic mechanism underlying hearing loss in these patients.
- To validate findings from experimental models in primary human clinical samples.
Main Methods:
- Analysis of m(6) 2A methylation levels in 12S rRNA from 14 patients carrying the m.1555A>G mutation and healthy controls.
- Comparison of methylation patterns across different primary (lymphocyte, fibroblast) and transformed (lymphoblast) cell types from the same individuals.
- Utilizing techniques to detect and quantify m(6) 2A methylation on 12S rRNA.
Main Results:
- All detectable 12S rRNA transcripts were found to be m(6) 2A-methylated in both m.1555A>G patients and controls.
- Transformed cell lines (lymphoblasts) showed some unmethylated 12S rRNA, while primary samples (lymphocytes, fibroblasts) from the same patients exhibited complete methylation.
- These findings indicate that TFB1M-mediated 12S m(6) 2A rRNA hypermethylation is unlikely to be the pathogenic mechanism.
Conclusions:
- TFB1M-mediated 12S m(6) 2A rRNA hypermethylation is unlikely to be the pathogenic mechanism for hearing loss associated with the m.1555A>G mutation.
- Observed hypermethylation in previous studies may be an artifact of experimental models, particularly transformed cell lines.
- Clinical validation of RNA methylation studies in primary human samples is crucial for accurate understanding of human disease mechanisms.
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