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: 6.39). 06/2012; 21(19):4214-24. DOI: 10.1093/hmg/dds245
Source: PubMed


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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    • "Our results revealed identical homoplasmic variants in mtDNA sequence from blood-derived genomic DNA, which lend support to the idea that monozygotic twins may be highly similar in their mitochondrial genomic sequence, as they seem to be in nuclear genomic sequence (Baranzini et al. 2010; Jakobsen et al. 2011). The presence of shared variants in twins suggests that the variants were likely present since conception (Avital et al. 2012). As mtDNA variation impacts many biological processes and can affect disease, it presents important challenges to diagnostic capabilities and could inform us about mutation and selection. "
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    ABSTRACT: When applying genome-wide sequencing technologies to disease investigation, it is increasingly important to resolve sequence variation in regions of the genome that may have homologous sequences. The human mitochondrial genome challenges interpretation given the potential for heteroplasmy, somatic variation, and homologous nuclear mitochondrial sequences (numts). Identical twins share the same mitochondrial DNA (mtDNA) from early life, but whether the mitochondrial sequence remains similar is unclear. We compared an adult monozygotic twin pair using high throughput-sequencing and evaluated variants with primer extension and mitochondrial pre-enrichment. Thirty-seven variants were shared between the twin individuals, and the variants were verified on the original genomic DNA. These studies support highly identical genetic sequence in this case. Certain low-level variant calls were of high quality and homology to the mitochondrial DNA, and they were further evaluated. When we assessed calls in pre-enriched mitochondrial DNA templates, we found that these may represent numts, which can be differentiated from mtDNA variation. We conclude that twin identity extends to mitochondrial DNA, and it is critical to differentiate between numts and mtDNA in genome sequencing, particularly since significant heteroplasmy could influence genome interpretation. Further studies on mtDNA and numts will aid in understanding how variation occurs and persists.
    Full-text · Article · Sep 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.
    Full-text · Article · Aug 2013 · Genome Research
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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.
    Full-text · Article · Jul 2013 · Current Genetics
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