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Updated: Nov 23, 2025
Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
Improvement of a Rapid and Highly Sensitive Method for the Diagnosis of the Mitochondrial m.1555A>G Mutation Based on
Yuichi Isaka1, Shin-Ya Nishio1,2, Eiji Hishinuma3,4,5
1Department of Hearing Implant Sciences and Shinshu University School of Medicine, Matsumoto, Japan.
Abstract:
Pathogenic variants in mitochondrial DNA are known to be associated with sensorineural hearing loss (SNHL) and aminoglycoside-induced HL. Among them, the m.1555A>G mutation is the most common. Thus, a rapid and easy companion diagnostic method for this mutation would be desirable to prevent HL caused by aminoglycoside therapy. In this study, we report an improved protocol for the single-stranded tag hybridization chromatographic printed-array strip (STH-PAS) method for identifying the m.1555A>G mutation. To evaluate the accuracy of a novel diagnostic for the m.1555A>G mutation we analyzed 378 DNA samples with or without the m.1555A>G mutation, as determined by Invader assay, and calculated the sensitivity, specificity, and false negative and false positive ratios of this new method. The newly developed protocol was robust; we, obtained the same results using multiple DNA concentrations, differing annealing temperatures, and different polymerase chain reaction thermal cyclers. The diagnostic sensitivity based on the STH-PAS method was 0.99, and the specificity was 1.00. The false negative and false positive ratios were 0 and 0.01, respectively. We improved the genotyping method for m.1555A>G mutations. This assays will be useful as a rapid companion diagnostic before aminoglycoside use.
Insights
A new diagnostic method accurately identifies the common m.1555A>G mitochondrial DNA mutation, which causes hearing loss. This rapid test can help prevent hearing loss in patients undergoing aminoglycoside therapy.
Area of Science:
- Genetics
- Pharmacogenomics
- Otolaryngology
Background:
- Pathogenic mitochondrial DNA variants are linked to sensorineural hearing loss (SNHL) and aminoglycoside-induced hearing loss (HL).
- The m.1555A>G mutation is the most prevalent cause of SNHL and aminoglycoside-induced HL.
- A rapid companion diagnostic is needed to prevent hearing loss associated with aminoglycoside therapy.
Purpose of the Study:
- To report an improved single-stranded tag hybridization chromatographic printed-array strip (STH-PAS) method for identifying the m.1555A>G mutation.
- To evaluate the accuracy and robustness of this novel diagnostic method.
Main Methods:
- An improved STH-PAS protocol was developed for m.1555A>G mutation detection.
- The accuracy of the STH-PAS method was evaluated by analyzing 378 DNA samples.
- Sensitivity, specificity, and false positive/negative ratios were calculated and compared to the Invader assay.
Main Results:
- The improved STH-PAS protocol demonstrated robustness across various DNA concentrations, annealing temperatures, and PCR thermal cyclers.
- The diagnostic sensitivity was 0.99, and specificity was 1.00.
- False negative and false positive ratios were 0 and 0.01, respectively.
Conclusions:
- The enhanced STH-PAS method provides a reliable and accurate genotyping assay for the m.1555A>G mutation.
- This improved diagnostic assay can serve as a valuable tool for rapid companion diagnostics prior to aminoglycoside administration.
- The method aids in preventing hearing loss in individuals susceptible to aminoglycoside-induced ototoxicity.
