Pathogenic mitochondrial tRNA point mutations: nine novel mutations affirm their importance as a cause of mitochondrial disease.
ABSTRACT: Mutations in the mitochondrial genome, and in particular the mt-tRNAs, are an important cause of human disease. Accurate classification of the pathogenicity of novel variants is vital to allow accurate genetic counseling for patients and their families. The use of weighted criteria based on functional studies-outlined in a validated pathogenicity scoring system--is therefore invaluable in determining whether novel or rare mt-tRNA variants are pathogenic. Here, we describe the identification of nine novel mt--tRNA variants in nine families, in which the probands presented with a diverse range of clinical phenotypes including mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes, isolated progressive external ophthalmoplegia, epilepsy, deafness and diabetes. Each of the variants identified (m.4289T>C, MT-TI; m.5541C>T, MT-TW; m.5690A>G, MT-TN; m.7451A>T, MT-TS1; m.7554G>A, MT-TD; m.8304G>A, MT-TK; m.12206C>T, MT-TH; m.12317T>C, MT-TL2; m.16023G>A, MT-TP) was present in a different tRNA, with evidence in support of pathogenicity, and where possible, details of mutation transmission documented. Through the application of the pathogenicity scoring system, we have classified six of these variants as "definitely pathogenic" mutations (m.5541C>T, m.5690A>G, m.7451A>T, m.12206C>T, m.12317T>C, and m.16023G>A), whereas the remaining three currently lack sufficient evidence and are therefore classed as 'possibly pathogenic' (m.4289T>C, m.7554G>A, and m.8304G>A).
Project description:Confirming the pathogenicity of mitochondrial tRNA point mutations is one of the classical challenges in the field of mitochondrial medicine. In addition to genetic and functional studies, the evaluation of a genetic change using a pathogenicity scoring system is extremely useful to discriminate between disease-causing mutations from neutral polymorphisms. The pathogenicity scoring system is very robust for confirming pathogenicity, especially of mutations that show impaired activity in functional studies. However, mutations giving normal results using the same functional approaches are disregarded, and this compromises the power of the system to rule out pathogenicity. We propose to include a new criterion in the pathogenicity scoring systems regarding mutations which fail to show any mitochondrial defect in functional studies. To evaluate this proposal we characterized two mutations, m.8296A>G and m.8347A>G, in the mitochondrial tRNA(L) (ys) gene (MT-TK) using trans-mitochondrial cybrid analysis. m.8347A>G mutation severely impairs oxidative phosphorylation, suggesting that it is highly pathogenic. By contrast, the behavior of cybrids homoplasmic for the m.8296A>G mutation is similar to cybrids containing wild-type mitochondrial DNA (mtDNA). The results indicate that including not only positive but also negative outcomes of functional studies in the scoring system is critical for facilitating the diagnosis of this complex group of diseases.
Project description:Transfer RNAs (tRNAs) are essential for encoding the transcribed genetic information from DNA into proteins. Variations in the human tRNAs are involved in diverse clinical phenotypes. Interestingly, all pathogenic variations in tRNAs are located in mitochondrial tRNAs (mt-tRNAs). Therefore, it is crucial to identify pathogenic variations in mt-tRNAs for disease diagnosis and proper treatment. We collected mt-tRNA variations using a classification based on evidence from several sources and used the data to develop a multifactorial probability-based prediction method, PON-mt-tRNA, for classification of mt-tRNA single nucleotide substitutions. We integrated a machine learning-based predictor and an evidence-based likelihood ratio for pathogenicity using evidence of segregation, biochemistry and histochemistry to predict the posterior probability of pathogenicity of variants. The accuracy and Matthews correlation coefficient (MCC) of PON-mt-tRNA are 1.00 and 0.99, respectively. In the absence of evidence from segregation, biochemistry and histochemistry, PON-mt-tRNA classifies variations based on the machine learning method with an accuracy and MCC of 0.69 and 0.39, respectively. We classified all possible single nucleotide substitutions in all human mt-tRNAs using PON-mt-tRNA. The variations in the loops are more often tolerated compared to the variations in stems. The anticodon loop contains comparatively more predicted pathogenic variations than the other loops. PON-mt-tRNA is available at http://structure.bmc.lu.se/PON-mt-tRNA/.
Project description:Mutations of mitochondrial DNA are linked to many human diseases. Despite the identification of a large number of variants in the mitochondrially encoded rRNA (mt-rRNA) genes, the evidence supporting their pathogenicity is, at best, circumstantial. Establishing the pathogenicity of these variations is of major diagnostic importance. Here, we aim to estimate the disruptive effect of mt-rRNA variations on the function of the mitochondrial ribosome. In the absence of direct biochemical methods to study the effect of mt-rRNA variations, we relied on the universal conservation of the rRNA fold to infer their disruptive potential. Our method, named heterologous inferential analysis or HIA, combines conservational information with functional and structural data obtained from heterologous ribosomal sources. Thus, HIA's predictive power is superior to the traditional reliance on simple conservation indexes. By using HIA, we have been able to evaluate the disruptive potential for a subset of uncharacterized 12S mt-rRNA variations. Our analysis revealed the existence of variations in the rRNA component of the human mitoribosome with different degrees of disruptive power. In cases where sufficient information regarding the genetic and pathological manifestation of the mitochondrial phenotype is available, HIA data can be used to predict the pathogenicity of mt-rRNA mutations. In other cases, HIA analysis will allow the prioritization of variants for additional investigation. Eventually, HIA-inspired analysis of potentially pathogenic mt-rRNA variations, in the context of a scoring system specifically designed for these variants, could lead to a powerful diagnostic tool.
Project description:Mitochondrial DNA (mtDNA) mutations are well recognized as an important cause of inherited disease. Diseases caused by mtDNA mutations exhibit a high degree of clinical heterogeneity with a complex genotype-phenotype relationship, with many such mutations exhibiting incomplete penetrance. There is evidence that the spectrum of mutations causing mitochondrial disease might differ between different mitochondrial lineages (haplogroups) seen in different global populations. This would point to the importance of sequence context in the expression of mutations. To explore this possibility, we looked for mutations which are known to cause disease in humans, in animals of other species unaffected by mtDNA disease. The mt-tRNA genes are the location of many pathogenic mutations, with the m.3243A>G mutation on the mt-tRNA-Leu(UUR) being the most frequently seen mutation in humans. This study looked for the presence of m.3243A>G in 2784 sequences from 33 species, as well as any of the other mutations reported in association with disease located on mt-tRNA-Leu(UUR). We report a number of disease associated variations found on mt-tRNA-Leu(UUR) in other chordates, as the major population variant, with m.3243A>G being seen in 6 species. In these, we also found a number of mutations which appear compensatory and which could prevent the pathogenicity associated with this change in humans. This work has important implications for the discovery and diagnosis of mtDNA mutations in non-European populations. In addition, it might provide a partial explanation for the conflicting results in the literature that examines the role of mtDNA variants in complex traits.
Project description:Mitochondrial DNA variants associated with diseases are widely studied in contemporary populations, but their prevalence has not yet been investigated in ancient populations. The publicly available AmtDB database contains 1443 ancient mtDNA Eurasian genomes from different periods. The objective of this study was to use this data to establish the presence of pathogenic mtDNA variants putatively associated with mitochondrial diseases in ancient populations. The clinical significance, pathogenicity prediction and contemporary frequency of mtDNA variants were determined using online platforms. The analyzed ancient mtDNAs contain six variants designated as being "confirmed pathogenic" in modern patients. The oldest of these, m.7510T>C in the MT-TS1 gene, was found in a sample from the Neolithic period, dated 5800-5400 BCE. All six have well established clinical association, and their pathogenic effect is corroborated by very low population frequencies in contemporary populations. Analysis of the geographic location of the ancient samples, contemporary epidemiological trends and probable haplogroup association indicate diverse spatiotemporal dynamics of these variants. The dynamics in the prevalence and distribution is conceivably result of de novo mutations or human migrations and subsequent evolutionary processes. In addition, ten variants designated as possibly or likely pathogenic were found, but the clinical effect of these is not yet well established and further research is warranted. All detected mutations putatively associated with mitochondrial disease in ancient mtDNA samples are in tRNA coding genes. Most of these mutations are in a mt-tRNA type (Model 2) that is characterized by loss of D-loop/T-loop interaction. Exposing pathogenic variants in ancient human populations expands our understanding of their origin and prevalence dynamics.
Project description:Pathogenic mitochondrial DNA (mtDNA) mutations leading to mitochondrial dysfunction can cause cardiomyopathy and heart failure. Owing to a high mutation rate, mtDNA defects may occur at any nucleotide in its 16?569?bp sequence. Complete mtDNA sequencing may detect pathogenic mutations, which can be difficult to interpret because of normal ethnic/geographic-associated haplogroup variation. Our goal is to show how to identify candidate mtDNA mutations by sorting out polymorphisms using readily available online tools. The purpose of this approach is to help investigators in prioritizing mtDNA variants for functional analysis to establish pathogenicity. We analyzed complete mtDNA sequences from 29 Italian patients with mitochondrial cardiomyopathy or suspected disease. Using MITOMASTER and PhyloTree, we characterized 593 substitution variants by haplogroup and allele frequencies to identify all novel, non-haplogroup-associated variants. MITOMASTER permitted determination of each variant's location, amino acid change and evolutionary conservation. We found that 98% of variants were common or rare, haplogroup-associated variants, and thus unlikely to be primary cause in 80% of cases. Six variants were novel, non-haplogroup variants and thus possible contributors to disease etiology. Two with the greatest pathogenic potential were heteroplasmic, nonsynonymous variants: m.15132T>C in MT-CYB for a patient with hypertrophic dilated cardiomyopathy and m.6570G>T in MT-CO1 for a patient with myopathy. In summary, we have used our automated information system, MITOMASTER, to make a preliminary distinction between normal mtDNA variation and pathogenic mutations in patient samples; this fast and easy approach allowed us to select the variants for traditional analysis to establish pathogenicity.
Project description:Pathogenic mitochondrial tRNA (mt-tRNA) gene mutations represent a prominent cause of primary mitochondrial DNA (mtDNA)-related disease despite accounting for only 5%-10% of the mitochondrial genome.(1,2) Although some common mt-tRNA mutations, such as the m.3243A>G mutation, exist, the majority are rare and have been reported in only a small number of cases.(3) The MT-TP gene, encoding mt-tRNA(Pro), is one of the less polymorphic mt-tRNA genes, and only 5 MT-TP mutations have been reported as a cause of mitochondrial muscle disease to date (table e-1 at Neurology.org/ng, P6-10). We report 5 patients with myopathic phenotypes, each harboring different pathogenic mutations in the MT-TP gene, highlighting the importance of MT-TP mutations as a cause of mitochondrial muscle disease and the requirement to study clinically relevant tissue.
Project description:Mitochondrial transfer RNA (mt-tRNA) mutations are the commonest sub-type of mitochondrial (mtDNA) mutations associated with human disease. We report a patient with multisytemic disease characterised by myopathy, spinal ataxia, sensorineural hearing loss, cataract and cognitive impairment in whom a novel m.7539C>T mt-tRNA(Asp) transition was identified. Muscle biopsy revealed extensive histopathological findings including cytochrome c oxidase (COX)-deficient fibres. Pyrosequencing confirmed mtDNA heteroplasmy for the mutation whilst single muscle fibre segregation studies revealed statistically significant higher mutation loads in COX-deficient fibres than in COX-positive fibres. Absence from control databases, hierarchical mt-tRNA mutation segregation within tissues, and occurrence at conserved sequence positions, further confirm this novel mt-tRNA mutation to be pathogenic. To date only three mt-tRNA(Asp) gene mutations have been described with clear evidence of pathogenicity. The novel m.7539C>T mt-tRNA(Asp) gene mutation extends the spectrum of pathogenic mutations in this gene, further supporting the notion that mt-tRNA(Asp) gene mutations are associated with multisystemic disease presentations.
Project description:Although over 200 pathogenic mitochondrial DNA (mtDNA) mutations have been reported to date, determining the genetic aetiology of many cases of mitochondrial disease is still not straightforward. Here, we describe the investigations undertaken to uncover the underlying molecular defect(s) in two unrelated Caucasian patients with suspected mtDNA disease, who presented with similar symptoms of myopathy, deafness, neurodevelopmental delay, epilepsy, marked fatigue and, in one case, retinal degeneration. Histochemical and biochemical evidence of mitochondrial respiratory chain deficiency was observed in the patient muscle biopsies and both patients were discovered to harbour a novel heteroplasmic mitochondrial tRNA (mt-tRNA)(Ser(AGY)) (MTTS2) mutation (m.12264C>T and m.12261T>C, respectively). Clear segregation of the m.12261T>C mutation with the biochemical defect, as demonstrated by single-fibre radioactive RFLP, confirmed the pathogenicity of this novel variant in patient 2. However, unusually high levels of m.12264C>T mutation within both COX-positive (98.4 ± 1.5%) and COX-deficient (98.2 ± 2.1%) fibres in patient 1 necessitated further functional investigations to prove its pathogenicity. Northern blot analysis demonstrated the detrimental effect of the m.12264C>T mutation on mt-tRNA(Ser(AGY)) stability, ultimately resulting in decreased steady-state levels of fully assembled complexes I and IV, as shown by blue-native polyacrylamide gel electrophoresis. Our findings expand the spectrum of pathogenic mutations associated with the MTTS2 gene and highlight MTTS2 mutations as an important cause of retinal and syndromic auditory impairment.
Project description:Mitochondrial disorders are clinically and genetically diverse, with mutations in mitochondrial or nuclear genes able to cause defects in mitochondrial gene expression. Recently, mutations in several genes encoding factors involved in mt-tRNA processing have been identified to cause mitochondrial disease. Using whole-exome sequencing, we identified mutations in TRMT10C (encoding the mitochondrial RNase P protein 1 [MRPP1]) in two unrelated individuals who presented at birth with lactic acidosis, hypotonia, feeding difficulties, and deafness. Both individuals died at 5 months after respiratory failure. MRPP1, along with MRPP2 and MRPP3, form the mitochondrial ribonuclease P (mt-RNase P) complex that cleaves the 5' ends of mt-tRNAs from polycistronic precursor transcripts. Additionally, a stable complex of MRPP1 and MRPP2 has m(1)R9 methyltransferase activity, which methylates mt-tRNAs at position 9 and is vital for folding mt-tRNAs into their correct tertiary structures. Analyses of fibroblasts from affected individuals harboring TRMT10C missense variants revealed decreased protein levels of MRPP1 and an increase in mt-RNA precursors indicative of impaired mt-RNA processing and defective mitochondrial protein synthesis. The pathogenicity of the detected variants-compound heterozygous c.542G>T (p.Arg181Leu) and c.814A>G (p.Thr272Ala) changes in subject 1 and a homozygous c.542G>T (p.Arg181Leu) variant in subject 2-was validated by the functional rescue of mt-RNA processing and mitochondrial protein synthesis defects after lentiviral transduction of wild-type TRMT10C. Our study suggests that these variants affect MRPP1 protein stability and mt-tRNA processing without affecting m(1)R9 methyltransferase activity, identifying mutations in TRMT10C as a cause of mitochondrial disease and highlighting the importance of RNA processing for correct mitochondrial function.