Mitochondrial DNA mutations in rRNA and tRNA loci associated with maternally inherited deafness

Study design and participants

A cross-sectional genetic association study was designed throughout the period from January 2015 to December 2016. The research took place at a special education school for deaf students in Peshawar, KP Province, Pakistan, while working with the Department of Biotechnology and Genetic Engineering, Hazara University, Mansehra, Pakistan. The Human Genetics Laboratory at the Department of Biotechnology and Genetic Engineering, Hazara University, conducted all laboratory tests, including DNA extraction and polymerase chain reaction (PCR) amplification, and electrophoresis.

The research involved 71 students who were 12–16 years old (31 female and 40 Male) from a special education school for the deaf in Peshawar, Pakistan [Table 1]. The participants had bilateral symmetric sensorineural hearing loss, which was confirmed through school medical records and audiometric evaluations. The family histories obtained from parents/guardians were used to determine the maternal inheritance pattern, which was defined as multiple affected siblings or maternal relatives with hearing loss and no paternal transmission. Participants who had hearing loss due to environmental factors such as birth trauma, meningitis, or ototoxic drug exposure, or those with syndromic features, were excluded. This ensured a cohort enriched for maternally inherited deafness. The study included participants who met three conditions: (i) They had congenital or early-onset hearing loss confirmed by medical evaluation, (ii) they showed a clear pattern of deafness inheritance from their mothers, and (iii) they provided consent to participate, along with their guardians’ consent for minor participants. The study excluded participants who had deafness from environmental factors such as trauma, infection, or ototoxic drug use, those with unclear family history, and those who refused to consent. The saliva samples were obtained at the school location before freezing them at −20°C for transportation to the Human Genetics Laboratory, where phenol–chloroform extraction methods were used for DNA extraction from the saliva.

Sample collection and sequencing

In this study, 71 saliva samples from deaf student participants were obtained. Good-quality DNA from all samples was collected before successfully performing PCR amplification and Sanger sequencing. The sequencing process targeted a 795 base pair area that included the 3’ end of the 12S rRNA gene together with the complete mitochondrial transfer RNA for valine gene (MT-TV) and the 5’ end of the 16S rRNA gene. This specific DNA region was chosen because it has been linked to deafness that passes through maternal lines and mitochondrial disorders.

Reference alignment

The sequencing data were aligned to the revised Cambridge reference sequence (rCRS; GenBank Accession: NC_012920) using CLUSTALW. MitoTIP allowed the evaluation and annotation of tRNA variants after mutation detection.

DNA isolation and quality assessment

In the current study, we employed a modified phenol– chloroform extraction technique to extract DNA from their samples. The mixture containing 1 mL of saliva and 100 μL of lysis solution with 0.5% sodium dodecyl sulfate and 250 mM ethylenediaminetetraacetic acid (EDTA) and 500 mM Tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0) and 3 μL β-mercaptoethanol, and 10 μg proteinase K underwent 60°C incubation for 90 min with a second proteinase K treatment at the 30-min mark. The aqueous solution underwent phenol extraction followed by centrifugation before isopropanol precipitation at cold temperatures. The solution was then washed with 70% ethanol before air drying and dissolving in 40 μL of double-distilled water at 56°C.

DNA quality assessment

The spectrophotometer NanoDrop 2000 from Thermo Scientific measured DNA concentration and purity through absorbance readings at 260 nm and 280 nm. The A260/A280 ratio between 1.7 and 1.9 in DNA samples indicated suitable quality for PCR amplification. The DNA integrity assessment included 1% agarose gel electrophoresis with Tris-acetate-EDTA (TAE) buffer and ethidium bromide staining. The DNA bands became visible under Ultraviolet (UV) light after performing electrophoresis at 80–100 V.

Agarose gel electrophoresis of extracted DNA

The extracted DNA quality was evaluated using 1% agarose gel electrophoresis. The agarose solution needed TAE buffer to dissolve, while ethidium bromide served for detection purposes. The DNA samples underwent electrophoresis at 80–100 volts after being mixed with loading dye before being visualized under UV light.

PCR amplification

The 3’ end of the 12S rRNA and the complete tRNA-Val (MTTV) gene and the 5’ end of the 16S rRNA region in mitochondrial DNA were amplified using a specific primer pair. The forward primer sequence was 5’-CGATCAACCTCACCACCTCTT-3’ and the reverse primer sequence was 5’-CTTGGACAACCAGCTATCACCA-3’. National Center for Biotechnology Information (NCBI) reference mitochondrial genome (rCRS, NC_012920) was used to design new primers, which were tested for specificity through NCBI Primer-Basic Local Alignment Search Tool (BLAST) in silico analysis to prevent non-specific amplification and pseudogene (nuclear mitochondrial pseudogene) binding. The preliminary agarose gel electrophoresis test confirmed the presence of a single distinct amplification band that matched the expected 795 base pair (bp) fragment, which validated primer efficiency and specificity. The PCR mixture consisted of 25 μL with 2 μL deoxynucleotide triphosphates (dNTPs) and 2.5 μL MgCl₂ and 2.5 μL of ×10 Taq buffer and 2 μL of each primer and 0.5 μL of Taq DNA polymerase and 1.5 μL of template DNA, and 12 μL of ddH₂O. The thermal cycling process started with 5 minutes of denaturation at 94°C, followed by 35 cycles of denaturation at 94°C for 30 s and annealing at 53°C for 1 min, and extension at 72°C for 1 min before a final extension at 72°C for 5 min.

Agarose gel electrophoresis of the PCR product

The analysis of PCR products used 1% agarose gel following the procedures previously described. The samples received loading dye before undergoing 100-volt electrophoresis for 30 min. The UV light exposure revealed bands, which were then photographed.

Extraction of the desired PCR product from the gel

The PCR bands obtained from agarose gels were placed into microtubes with labels for storage at −20°C. The GeneAll Gel Extraction Kit from GeneAll Biotechnology Korea was used to purify DNA samples according to the manufacturer’s instructions. The NanoDrop spectrophotometer from Thermo Scientific verified DNA purity and concentration, while a 1% agarose gel electrophoresis confirmed DNA integrity by showing a single band without signs of degradation before sequencing.

Sequence analysis of the PCR product

The PCR products underwent Sanger sequencing at Microgen (Seoul, South Korea) through forward and reverse (bidirectional) sequencing to achieve accurate base-calling results. The sequence chromatograms received visual inspection to verify peak quality and the absence of background noise. The analysis included only reads that achieved a Phred quality score of 30 or higher (99.9% base-calling accuracy). The sequencing data reached an average coverage of ×2 per locus through forward and reverse reads. The obtained sequences underwent alignment against the rCRS with GenBank accession number NC_012920 using CLUSTALW software. The researchers used Mitochondrial DNA Mutation Database (MITOMAP) to annotate mutations and MitoTIP to evaluate their pathogenic potential.

Sequence alignment and variant annotation

Sequencing reads were aligned to the rCRS (rCRS; NC_012920) using CLUSTALW v2.1. Using the MitoTIP v3.0, which was available via the MITOMAP platform (MITOMAP; accessed March 2025), mitochondrial tRNA variants were annotated and scored. During the same access period, data on pathogenicity and variant frequency were compared to MITOMAP and related databases of mitochondrial variants.

Software and computational tools

Chromas Lite v2.6.6 and BioEdit version 7.2.5 were used to manually examine the sequence chromatograms. Microsoft Excel 2021 and GraphPad Prism v9.0 (GraphPad Software, USA) were used for statistical and data visualization analyses. The current study presented mutation data through counts and percentages, and proportions that spanned across three genomic areas, including 12S rRNA and 16S rRNA, and tRNA loci. The study did not perform formal statistical tests for subgroup differences between male and female participants or between transition and transversion mutations because it had only 50 successfully sequenced samples and an exploratory research design. The study did not execute any statistically significant subgroup analysis. The researchers determined mutation frequency through sequencing results of targeted mitochondrial loci without adjusting for complete mtDNA genome coverage because they analyzed a 795 bp segment instead of the full mtDNA genome. The method used for this study generated proportional mutation distribution data within the analyzed segment, which served as an appropriate method for detecting recurring mtDNA variants linked to maternal deafness.

Optimization of PCR conditions

In current research, we evaluated different primer sets to optimize the desired PCR product amplification. We optimized PCR amplification through modifications in annealing temperature and extension time to increase reaction efficiency. The results of PCR amplification for the mtDNA m.1555A>G primers, including specific bands observed during optimization and the final optimized PCR reaction, are presented in Figures 2 and 3.

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Figure 2:

Agarose gel electrophoresis for PCR optimization of a 795 bp mtDNA fragment spanning the 12S rRNA, MT-TV (tRNAVal), and 16S rRNA loci. Lane M: 100 bp DNA ladder; Lanes 1–8: PCR amplification under varying conditions; arrow indicates the expected 795 bp product.

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Figure 3:

Final PCR amplification of the 795 bp mtDNA fragment targeting the 12S rRNA, MT-TV, and 16S rRNA regions. Lane M: DNA ladder; Lanes 1–5: successful amplification; Lane N: negative control; arrow indicates the 795 bp product.

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Sequence analysis of nucleotide

In this study, we used the amplified PCR fragment from 71 participants to proceed with nucleotide sequencing. Fifty nucleotide sequences were retrieved after sequencing. The sequence peaks were analyzed and trimmed for further analysis.

Sequencing outcomes

All 71 saliva samples yielded high-quality DNA for analysis. The sequencing process yielded successful results for 50 out of 71 samples (70.4%). The remaining 21 samples failed sequencing because their DNA quality was insufficient, or their DNA had degraded during storage or PCR amplification produced weak or non-specific agarose gel bands. The extraction of DNA from field saliva samples faces typical difficulties because storage and transportation conditions differ between samples.

Mutation spectrum and novelty

The 50 successfully sequenced samples contained 78 mtDNA variants, which spread across the 12S rRNA, 16S rRNA, and tRNA-Val (MT-TV) regions. The MITOMAP database (accessed 2025) shows that m.1438A>G and m.1544A>T mutations in the 12S rRNA gene match known pathogenic variants that cause maternally inherited deafness. The MT-TV (tRNA-Val) gene variants m.1603A>C, m.1607T>G, and m.1623G>C appear to be discoveries because they do not exist in MITOMAP or the rCRS variant catalogs. The study-specific variants need additional population-based and functional analysis because they have not been documented in previous research [Table 2].

Across the 12S rRNA, 16S rRNA, and MT-TV (tRNA-Val) regions, 78 different forms of mtDNA were found. Because they were not included in MITOMAP (accessed March 2025) or other publicly accessible databases of mitochondrial variants, five of these were determined to be novel. These new variations included m.1492A>C* and m.1546A>T* in the 12S rRNA gene, as well as m.1603A>C*, m.1607T>G*, and m.1623G>C* in the MT-TV region. In addition, previously identified variants linked to deafness were found, such as m.1438A>G and m.1544A>T, which are known mutational hotspots in mitochondrial rRNA genes. These variations have been regularly documented in cases of ototoxicity caused by aminoglycosides and maternally inherited deafness. A number of novel mutations exhibited moderate-to-high predicted pathogenicity, according to MitoTIP scoring of the tRNA variants (e.g., m.1603A>C* = 61.4%; m.1623G>C* = 77.6%), suggesting possible functional relevance and requiring additional validation. Variants that are deemed novel in this study are indicated by asterisks (*) in Table 2.

Mitochondrial DNA variants identified

Multiple samples underwent mtDNA sequence analysis through comparison with the rCRS [Supplementary file Sample sequences]. We discovered different types of mutations throughout the 12S rRNA, 16S rRNA, and tRNA regions of the mitochondrial genome [Figure 4]. In the current study, we identified 78 mtDNA variants, which included m.1438A>G and m.1544A>T mutations in the 12S rRNA gene that have been linked to maternally inherited deafness. The MT-TV (tRNA-Val) region contains three newly discovered variants m.1603A>C, m.1607T>G, and m.1623G>C, which do not appear in MITOMAP or the rCRS variant databases. The newly discovered mutations received MitoTIP pathogenicity scores of 50% or higher, which indicates their potential functional impact and requires additional research. In this study, we grouped the genetic changes into two categories according to their nucleotide change type: Transitions and transversions. The rRNA and tRNA loci show mutations where 12S rRNA region: A large number of mutations were detected that include the frequent mutations at positions m.1438A>G, m.1544A>T, and m.1443T>G. The mutations occur in conserved domains of the 12S rRNA locus, which is necessary for both ribosome assembly and protein translation. tRNA region: The tRNA regions contained mutations at m.1603A>C, m.1607T>G, and m.1629A>C positions, where MitoTIP pathogenicity scores were between 9.9% and 77.6%.

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Figure 4:

mtDNA gene map showing the amplified region (12S rRNA–tRNA-Val–16S rRNA) relative to the rCRS. Red marker indicates sequenced region.

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The tRNA-disrupting potential of m.1623G>C (77.6%) variants is higher than other variants. The results of mutation analysis were as follows: Transitions (e.g., A↔G, T↔C): The mutations occurred in large numbers as expected for mtDNA, which tends to produce such changes. Transversions (e.g., T↔G, A↔C): These mutations are less common, and they can have a larger impact on structure, especially when they occur in conserved regions.

The mutations detected at m.1438A>G and m.1544A>T positions in multiple samples indicate these regions function as mutation hotspots or hold critical functional positions under evolutionary selection. We used MitoTIP pathogenicity scores to evaluate the likelihood of tRNA mutations affecting mitochondrial function. The positions 1603 (61.4%), 1607 (56.3%), and 1629 (51.3%) had MitoTIP pathogenicity scores ranging from moderate to high, which suggests functional and clinical relevance [Table 2 and Figure 5].

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Figure 5:

Distribution of mtDNA mutations across genomic regions. Bars represent mutation counts, and orange bars denote the average MitoTIP pathogenicity score for tRNA variants.

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The research findings indicate that mtDNA mutations disrupt mitochondrial function when they target the rRNA regions. The mutations at positions m.1443T>G and m.1544A>T located in the 12S rRNA locus may destabilize ribosomes, which impairs mitochondrial protein synthesis. The disruptions impair the pathway of oxidative phosphorylation because the pathway supports tissues that require high energy levels, particularly the auditory system. Medical significance exists in tRNA locus mutations because their high MitoTIP scores can lead to mitochondrial diseases. The m.1603A>C mutation (61.4%) has previously been associated with deafness, together with other mitochondrial syndromes. The study results match previous research that shows tRNA mutations act as primary factors in mitochondrial dysfunction, which leads to neurological and auditory disorders. The m.1438A>G mutations found in several samples point toward these sites being evolutionary hotspots or under selective pressure. The widespread occurrence of these mutations in various samples suggests their potential involvement in mitochondrial adaptation or increased vulnerability to dysfunction. The hearing loss occurs because of mutations in the 12S rRNA locus, which contains vital ribosomal elements that also affect patients with aminoglycoside-induced ototoxicity or maternally inherited deafness. These detected mutations in the study could potentially contribute to the observed clinical manifestations. Collectively, the findings demonstrate a clear concentration of recurrent and novel mtDNA variants in critical rRNA and tRNA loci, underscoring their potential contribution to mitochondrial dysfunction and maternally inherited hearing loss.

Strengths and limitations

The research offers a detailed examination of 12S rRNA and 16S rRNA and MT-TV (tRNA-Val) mutations in Pakistani patients who inherited deafness from their mothers. The study used Sanger sequencing to achieve precise mutation detection and MitoTIP as a specialized tool to evaluate mttRNA variant pathogenicity through integration of structural and clinical data. The research method provides strong variant interpretation results while identifying specific areas where hearing loss mutations tend to occur.

The research contains multiple restrictions that affect its results. The research included only 71 participants but only 50 of them received successful sequencing results which made it difficult to detect rare genetic variations and reduced statistical power. The study lacks healthy control samples and family segregation analysis which prevents researchers from confirming the pathogenic nature of identified variants. The results should be interpreted with caution because this study was exploratory in nature and relied on descriptive analyses without formal statistical power calculations. Even though recurrent variations point to biologically significant patterns, these findings need to be confirmed by larger cohort studies and inferential statistical techniques. The study focused on a 795 bp mtDNA region but failed to detect mutations that could exist in other important genetic locations. The study did not perform biological tests to prove how high-scoring variants from MitoTIP affect cellular functions. Hence, further research requires additional studies with bigger participant groups across different populations and complete mitochondrial genome sequencing and biological tests to validate these results.