Mitochondrial DNA heteroplasmy in diabetes and normal adults: role of acquired and inherited mutational patterns in twins

Department of Life Sciences, Faculty of Natural Sciences.
Human Molecular Genetics (Impact Factor: 6.68). 06/2012; 21(19):4214-24. DOI: 10.1093/hmg/dds245
Source: PubMed

ABSTRACT Heteroplasmy, the mixture of mitochondrial genomes (mtDNA), varies among individuals and cells. Heteroplasmy levels alter the penetrance of pathological mtDNA mutations, and the susceptibility to age-related diseases such as Parkinson's disease. Although mitochondrial dysfunction occurs in age-related type 2 diabetes mellitus (T2DM), the involvement of heteroplasmy in diabetes is unclear. We hypothesized that the heteroplasmic mutational (HM) pattern may change in T2DM. To test this, we used next-generation sequencing, i.e. massive parallel sequencing (MPS), along with PCR-cloning-Sanger sequencing to analyze HM in blood and skeletal muscle DNA samples from monozygotic (MZ) twins either concordant or discordant for T2DM. Great variability was identified in the repertoires and amounts of HMs among individuals, with a tendency towards more mutations in skeletal muscle than in blood. Whereas many HMs were unique, many were either shared among twin pairs or among tissues of the same individual, regardless of their prevalence. This suggested a heritable influence on even low abundance HMs. We found no clear differences between T2DM and controls. However, we found ∼5-fold increase of HMs in non-coding sequences implying the influence of negative selection (P < 0.001). This negative selection was evident both in moderate to highly abundant heteroplasmy (>5% of the molecules per sample) and in low abundance heteroplasmy (<5% of the molecules). Although our study found no evidence supporting the involvement of HMs in the etiology of T2DM, the twin study found clear evidence of a heritable influence on the accumulation of HMs as well as the signatures of selection in heteroplasmic mutations.

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Available from: Eitan Rubin, Dec 28, 2013
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    • "At least 1.6% of the reads (i.e., 0.8% from the reads of each of the strands, filter C) (He et al. 2010) and a minimum of five reads per strand at a given site (filter D) must have the RDD base. These stringent filters were imposed to minimize our false-discovery rate; however, most likely they led us to exclude some true RDD sites (Avital et al. 2012). By these criteria, we uncovered three mitochondrial RDD sites; two of these sites were present in all five individuals (positions 2617, A-to-U and A-to-G; 13710, A-to-U and A-to-G), and the third was found in two of the five individuals (position 295, C-to- U). "
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    ABSTRACT: RNA transcripts are generally identical to the underlying DNA sequences. Nevertheless, RNA-DNA differences (RDDs) were found in the nuclear human genome and in plants and animals but not in human mitochondria. Here by deep sequencing of human mitochondrial DNA (mtDNA) and RNA, we identified three RDD sites at mtDNA positions 295 (C-to-U), 13710 (A-to-U, A-to-G) and 2617 (A-to-U, A-to-G). Position 2617, within the 16S rRNA, harbored the most prevalent RDDs (more than 30% A-to-U and ~15% A-to-G of the reads in all tested samples). The 2617 RDDs appeared already at the precursor polycistrone mitochondrial transcript. Using traditional Sanger sequencing we identified the A-to-U RDD in 6 different cell lines and representative primates (Gorilla gorilla, Pongo pigmaeus and Macca mulatta), suggesting conservation of the mechanism generating such RDD. Phylogenetic analysis of more than 1700 vertebrate mtDNA sequences supported a thymine as the primate ancestral allele at position 2617, suggesting that the 2617 RDD recapitulates the ancestral 16S rRNA. Modeling U or G (the RDDs) at position 2617 stabilized the large ribosomal subunit structure in contrast to destabilization by an A (the pre-RDDs). Hence, these mitochondrial RDDs are likely functional.
    Genome Research 08/2013; 23(11). DOI:10.1101/gr.161265.113 · 13.85 Impact Factor
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    • "In contrast, 1.8 heteroplasmies per individual at [10 % MAF were identified using 40 immortalized lymphoblastoid cell line samples (Sosa et al. 2012). In recent reports (Andrew et al. 2011; Goto et al. 2011; Avital et al. 2012), variable heteroplasmy in the human body has been demonstrated to exist within a limited number of tissues such as blood, skeletal muscle and buccal epithelium, leaving the rest of the organism out of focus. As multiple tissue analysis in healthy individuals is highly complicated, data on whole body mtDNA heteroplasmy is still far from clear. "
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    ABSTRACT: Human mitochondrial DNA (mtDNA) research has entered a massively parallel sequencing (MPS) era, providing deep insight into mtDNA genomics and molecular diagnostics. Analysis can simultaneously include coding and control regions, many samples can be studied in parallel, and even minor heteroplasmic changes can be detected. We investigated heteroplasmy using 16 different tissues from three unrelated males aged 40–54 years at the time of death. mtDNA was enriched using two independent overlapping long-range PCR amplicons and analysed by employing illumina paired-end sequencing. Point mutation heteroplasmy at position 16,093 (m.16093T > C) in the non-coding regulatory region showed great variability among one of the studied individuals; heteroplasmy extended from 5.1 % in red bone marrow to 62.0 % in the bladder. Red (5.1 %) and yellow bone marrow (8.9 %) clustered into one group and two arteries and two aortas from different locations into another (31.2–50.9 %), giving an ontogenetic explanation for the formation of somatic mitochondrial heteroplasmy. Our results demonstrate that multi-tissue screening using MPS provides surprising data even when there is a limited number (3) of study subjects and they give reason to speculate that mtDNA heteroplasmic frequency, distribution, and even its possible role in complex diseases or phenotypes seem to be underestimated. Electronic supplementary material The online version of this article (doi:10.1007/s00294-013-0398-6) contains supplementary material, which is available to authorized users.
    Current Genetics 07/2013; 60(1). DOI:10.1007/s00294-013-0398-6 · 2.68 Impact Factor
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    ABSTRACT: Background Mitochondrial dysfunction is associated with various aging diseases. The copy number of mtDNA in human cells may therefore be a potential biomarker for diagnostics of aging. Here we propose a new computational method for the accurate assessment of mtDNA copies from whole genome sequencing data. Results Two families of the human whole genome sequencing datasets from the HapMap and the 1000 Genomes projects were used for the accurate counting of mitochondrial DNA copy numbers. The results revealed the parental mitochondrial DNA copy numbers are significantly lower than that of their children in these samples. There are 8%~21% more copies of mtDNA in samples from the children than from their parents. The experiment demonstrated the possible correlations between the quantity of mitochondrial DNA and aging-related diseases. Conclusions Since the next-generation sequencing technology strives to deliver affordable and non-biased sequencing results, accurate assessment of mtDNA copy numbers can be achieved effectively from the output of whole genome sequencing. We implemented the method as a software package MitoCounter with the source code and user's guide available to the public at
    BMC Genomics 12/2012; 13(Suppl 7). DOI:10.1186/1471-2164-13-S7-S5 · 4.04 Impact Factor
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