Heteroplasmic Mitochondrial DNA Research Articles

Efficient elimination of MELAS-associated m.3243G mutant mitochondrial DNA by an engineered mitoARCUS nuclease

Nuclease-mediated editing of heteroplasmic mitochondrial DNA (mtDNA) seeks to preferentially cleave and eliminate mutant mtDNA, leaving wild-type genomes to repopulate the cell and shift mtDNA heteroplasmy. Various technologies are available, but many suffer from limitations based on size and/or specificity. The use of ARCUS nucleases, derived from naturally occurring I-CreI, avoids these pitfalls due to their small size, single-component protein structure and high specificity resulting from a robust protein-engineering process. Here we describe the development of a mitochondrial-targeted ARCUS (mitoARCUS) nuclease designed to target one of the most common pathogenic mtDNA mutations, m.3243A>G. mitoARCUS robustly eliminated mutant mtDNA without cutting wild-type mtDNA, allowing for shifts in heteroplasmy and concomitant improvements in mitochondrial protein steady-state levels and respiration. In vivo efficacy was demonstrated using a m.3243A>G xenograft mouse model with mitoARCUS delivered systemically by adeno-associated virus. Together, these data support the development of mitoARCUS as an in vivo gene-editing therapeutic for m.3243A>G-associated diseases.

High-throughput single-cell analysis reveals progressive mitochondrial DNA mosaicism throughout life.

Heteroplasmic mitochondrial DNA (mtDNA) mutations are a major cause of inherited disease and contribute to common late-onset human disorders. The late onset and clinical progression of mtDNA-associated disease is thought to be due to changing heteroplasmy levels, but it is not known how and when this occurs. Performing high-throughput single-cell genotyping in two mouse models of human mtDNA disease, we saw unanticipated cell-to-cell differences in mtDNA heteroplasmy levels that emerged prenatally and progressively increased throughout life. Proliferating spleen cells and nondividing brain cells had a similar single-cell heteroplasmy variance, implicating mtDNA or organelle turnover as the major force determining cell heteroplasmy levels. The two different mtDNA mutations segregated at different rates with no evidence of selection, consistent with different rates of random genetic drift in vivo, leading to the accumulation of cells with a very high mutation burden at different rates. This provides an explanation for differences in severity seen in human diseases caused by similar mtDNA mutations.

Mitochondrial genome study in blood of maternally inherited ALS cases

BackgroundALS is a heterogeneous disease in which different factors such as mitochondrial phenotypes act in combination with a genetic predisposition. This study addresses the question of whether homoplasmic (total mitochondrial genome of a sample is affected) and/or heteroplasmic mutations (wildtype and mutant mitochondrial DNA molecules coexist) might play a role in familial ALS. Blood was drawn from familial ALS patients with a possible maternal pattern of inheritance according to their pedigrees, which was compared to blood of ALS patients without maternal association as well as age-matched controls. In two cohorts, we analyzed the mitochondrial genome from whole blood or isolated white blood cells and platelets using a resequencing microarray (Affymetrix MitoChip v2.0) that is able to detect homoplasmic and heteroplasmic mitochondrial DNA mutations and allows the assessment of low-level heteroplasmy.ResultsWe identified an increase in homoplasmic ND5 mutations, a subunit of respiratory chain complex I, in whole blood of ALS patients that allowed maternal inheritance. This effect was more pronounced in patients with bulbar onset. Heteroplasmic mutations were significantly increased in different mitochondrial genes in platelets of patients with possible maternal inheritance. No increase of low-level heteroplasmy was found in maternal ALS patients.ConclusionOur results indicate a contribution of homoplasmic ND5 mutations to maternally associated ALS with bulbar onset. Therefore, it might be conceivable that specific maternally transmitted rather than randomly acquired mitochondrial DNA mutations might contribute to the disease process. This stands in contrast with observations from Alzheimer’s and Parkinson’s diseases showing an age-dependent accumulation of unspecific mutations in mitochondrial DNA.

Mitochondrial DNA 13513G>A Mutation Causing Leber Hereditary Optic Neuropathy Associated With Adult-Onset Renal Failure.

Leber hereditary optic neuropathy (LHON) is one of the more common mitochondrial diseases and is rarely associated with mitochondrial renal disease. We report 3 unrelated patients with a background of adult-onset renal failure who presented to us with LHON and were shown to have a heteroplasmic mitochondrial DNA mutation (m.13513G>A). Retrospective chart review. All 3 patients had a background of chronic renal failure and presented to us with bilateral optic neuropathy (sequential in 2) and were found to have heteroplasmic m.13513G>A mutations in the MT-ND5 gene. Two of the patients were females (aged 30 and 45 years) with chronic kidney disease from their 20s, attributed to pre-eclampsia, one of whom also had diabetes and sudden bilateral hearing loss. One patient was a male (aged 54 years) with chronic kidney disease from his 20s attributed to IgA nephropathy. His mother had diabetes and apparently sudden bilateral blindness in her 70s. Renal biopsy findings were variable and included interstitial fibrosis, acute tubular necrosis, focal segmental glomerulosclerosis, and IgA/C3 tubular casts on immunofluorescence. Mild improvements in vision followed treatment with either idebenone or a combination supplement including coenzyme Q10, alpha-lipoic acid, and B vitamins. Our cases expand the clinical syndromes associated with m.13513G>A to include bilateral optic neuropathy and adult-onset renal disease. This highlights that in patients with bilateral, especially sequential, optic neuropathy a broad approach to mitochondrial testing is more useful than a limited LHON panel. Mitochondrial diseases present a diagnostic challenge because of their clinical and genetic variability.

Mitochondrial DNA heteroplasmy distinguishes disease manifestation in PINK1/PRKN-linked Parkinson's disease.

Biallelic mutations in PINK1/PRKN cause recessive Parkinson's disease. Given the established role of PINK1/Parkin in regulating mitochondrial dynamics, we explored mitochondrial DNA integrity and inflammation as disease modifiers in carriers of mutations in these genes. Mitochondrial DNA integrity was investigated in a large collection of biallelic (n = 84) and monoallelic (n = 170) carriers of PINK1/PRKN mutations, idiopathic Parkinson's disease patients (n = 67) and controls (n = 90). In addition, we studied global gene expression and serum cytokine levels in a subset. Affected and unaffected PINK1/PRKN monoallelic mutation carriers can be distinguished by heteroplasmic mitochondrial DNA variant load (area under the curve = 0.83, CI 0.74-0.93). Biallelic PINK1/PRKN mutation carriers harbour more heteroplasmic mitochondrial DNA variants in blood (P = 0.0006, Z = 3.63) compared to monoallelic mutation carriers. This enrichment was confirmed in induced pluripotent stem cell-derived (controls, n = 3; biallelic PRKN mutation carriers, n = 4) and post-mortem (control, n = 1; biallelic PRKN mutation carrier, n = 1) midbrain neurons. Last, the heteroplasmic mitochondrial DNA variant load correlated with IL6 levels in PINK1/PRKN mutation carriers (r = 0.57, P = 0.0074). PINK1/PRKN mutations predispose individuals to mitochondrial DNA variant accumulation in a dose- and disease-dependent manner.

INHERITANCE OF A HETEROPLASMIC mtDNA MUTATION ASSOCIATED WITH LEIGH SYNDROME

Reproductive options to prevent maternal transmission of heteroplasmic mitochondrial (mt)DNA mutations to children are limited due to complex and often unpredictable inheritance patterns. We sought to evaluate transmission of pathogenic 13513G>A mtDNA associated with Leigh Syndrome from a heteroplasmic mother to her children with the intent to develop prenatal diagnostics.

Heteroplasmic mitochondrial DNA variants in cardiovascular diseases.

Mitochondria are implicated in the pathogenesis of cardiovascular diseases (CVDs) but the reasons for this are not well understood. Maternally-inherited population variants of mitochondrial DNA (mtDNA) which affect all mtDNA molecules (homoplasmic) are associated with cardiometabolic traits and the risk of developing cardiovascular disease. However, it is not known whether mtDNA mutations only affecting a proportion of mtDNA molecules (heteroplasmic) also play a role. To address this question, we performed a high-depth (~1000-fold) mtDNA sequencing of blood DNA in 1,399 individuals with hypertension (HTN), 1,946 with ischemic heart disease (IHD), 2,146 with ischemic stroke (IS), and 723 healthy controls. We show that the per individual burden of heteroplasmic single nucleotide variants (mtSNVs) increases with age. The age-effect was stronger for low-level heteroplasmies (heteroplasmic fraction, HF, 5–10%), likely reflecting acquired somatic events based on trinucleotide mutational signatures. After correcting for age and other confounders, intermediate heteroplasmies (HF 10–95%) were more common in hypertension, particularly involving non-synonymous variants altering the amino acid sequence of essential respiratory chain proteins. These findings raise the possibility that heteroplasmic mtSNVs play a role in the pathophysiology of hypertension.

Mitochondrial DNA heteroplasmy is modulated during oocyte development propagating mutation transmission.

Heteroplasmic mitochondrial DNA (mtDNA) mutations are a common cause of inherited disease, but a few recurrent mutations account for the vast majority of new families. The reasons for this are not known. We studied heteroplasmic mice transmitting m.5024C>T corresponding to a human pathogenic mutation. Analyzing 1167 mother-pup pairs, we show that m.5024C>T is preferentially transmitted from low to higher levels but does not reach homoplasmy. Single-cell analysis of the developing mouse oocytes showed the preferential increase in mutant over wild-type mtDNA in the absence of cell division. A similar inheritance pattern is seen in human pedigrees transmitting several pathogenic mtDNA mutations. In m.5024C>T mice, this can be explained by the preferential propagation of mtDNA during oocyte maturation, counterbalanced by purifying selection against high heteroplasmy levels. This could explain how a disadvantageous mutation in a carrier increases to levels that cause disease but fails to fixate, causing multigenerational heteroplasmic mtDNA disorders.

The relevance of mitochondrial DNA variants fluctuation during reprogramming and neuronal differentiation of human iPSCs.

SummaryThe generation of inducible pluripotent stem cells (iPSCs) is a revolutionary technique allowing production of pluripotent patient-specific cell lines used for disease modeling, drug screening, and cell therapy. Integrity of nuclear DNA (nDNA) is mandatory to allow iPSCs utilization, while quality control of mitochondrial DNA (mtDNA) is rarely included in the iPSCs validation process. In this study, we performed mtDNA deep sequencing during the transition from parental fibroblasts to reprogrammed iPSC and to differentiated neuronal precursor cells (NPCs) obtained from controls and patients affected by mitochondrial disorders. At each step, mtDNA variants, including those potentially pathogenic, fluctuate between emerging and disappearing, and some having functional implications. We strongly recommend including mtDNA analysis as an unavoidable assay to obtain fully certified usable iPSCs and NPCs.

Mitochondrial targeted meganuclease as a platform to eliminate mutant mtDNA in vivo

Diseases caused by heteroplasmic mitochondrial DNA mutations have no effective treatment or cure. In recent years, DNA editing enzymes were tested as tools to eliminate mutant mtDNA in heteroplasmic cells and tissues. Mitochondrial-targeted restriction endonucleases, ZFNs, and TALENs have been successful in shifting mtDNA heteroplasmy, but they all have drawbacks as gene therapy reagents, including: large size, heterodimeric nature, inability to distinguish single base changes, or low flexibility and effectiveness. Here we report the adaptation of a gene editing platform based on the I-CreI meganuclease known as ARCUS®. These mitochondrial-targeted meganucleases (mitoARCUS) have a relatively small size, are monomeric, and can recognize sequences differing by as little as one base pair. We show the development of a mitoARCUS specific for the mouse m.5024C>T mutation in the mt-tRNAAla gene and its delivery to mice intravenously using AAV9 as a vector. Liver and skeletal muscle show robust elimination of mutant mtDNA with concomitant restoration of mt-tRNAAla levels. We conclude that mitoARCUS is a potential powerful tool for the elimination of mutant mtDNA.

The Isolation and Deep Sequencing of Mitochondrial DNA.

In recent years, next-generation sequencing (NGS) has become a powerful tool for studying both inherited and somatic heteroplasmic mitochondrial DNA (mtDNA) variation. NGS has proved particularly powerful when combined with single-cell isolation techniques, allowing the investigation of low-level heteroplasmic variants both between cells and within tissues. Nevertheless, there remain significant challenges, especially around the selective enrichment of mtDNA from total cellular DNA and the avoidance of nuclear pseudogenes. This chapter summarizes the techniques needed to enrich, amplify, sequence, and analyse mtDNA using NGS .

Mitochondrial DNA mutation exacerbates female reproductive aging via impairment of the NADH/NAD+ redox.

Mammals' aging is correlated with the accumulation of somatic heteroplasmic mitochondrial DNA (mtDNA) mutations. Whether and how aging accumulated mtDNA mutations modulate fertility remains unknown. Here, we analyzed oocyte quality of young (≤30 years old) and elder (≥38 years old) female patients and show the elder group had lower blastocyst formation rate and more mtDNA point mutations in oocytes. To test the causal role of mtDNA point mutations on infertility, we used polymerase gamma (POLG) mutator mice. We show that mtDNA mutation levels inversely correlate with fertility, interestingly mainly affecting not male but female fertility. mtDNA mutations decrease female mice's fertility by reducing ovarian primordial and mature follicles. Mechanistically, accumulation of mtDNA mutations decreases fertility by impairing oocyte's NADH/NAD+ redox state, which could be rescued by nicotinamide mononucleotide treatment. For the first time, we answer the fundamental question of the causal effect of age‐accumulated mtDNA mutations on fertility and its sex dependence, and show its distinct metabolic controlling mechanism.

Successful treatment of a patient with mitochondrial myopathy with alirocumab

A 48-year-old man presented to our lipid clinic with statin intolerance and elevated serum creatine kinase levels, being affected by mitochondrial myopathy because of heteroplasmic mitochondrial DNA missense mutation in MTCO1 gene (m.7671T>A). He had just been treated with a coronary artery bypass 4 years before because of acute coronary syndrome, and he had consistently high levels of both low-density lipoprotein cholesterol and triglycerides. Dyslipidemia was successfully treated using 75 mg of alirocumab subcutaneously every 2 weeks, 10 mg of ezetimibe daily, 2 g of marine omega-3 fatty acids daily, and 145 mg of micronized fenofibrate every 2 days. Although muscle weakness persisted, myalgia did not reoccur and serum creatine kinase levels remained almost stable over the time.

A Novel Pathogenic Variant in MT-CO2 Causes an Isolated Mitochondrial Complex IV Deficiency and Late-Onset Cerebellar Ataxia.

Both nuclear and mitochondrial DNA defects can cause isolated cytochrome c oxidase (COX; complex IV) deficiency, leading to the development of the mitochondrial disease. We report a 52-year-old female patient who presented with a late-onset, progressive cerebellar ataxia, tremor and axonal neuropathy. No family history of neurological disorder was reported. Although her muscle biopsy demonstrated a significant COX deficiency, there was no clinical and electromyographical evidence of myopathy. Electrophysiological studies identified low frequency sinusoidal postural tremor at 3 Hz, corroborating the clinical finding of cerebellar dysfunction. Complete sequencing of the mitochondrial DNA genome in muscle identified a novel MT-CO2 variant, m.8163A>G predicting p.(Tyr193Cys). We present several lines of evidence, in proving the pathogenicity of this heteroplasmic mitochondrial DNA variant, as the cause of her clinical presentation. Our findings serve as an important reminder that full mitochondrial DNA analysis should be included in the diagnostic pipeline for investigating individuals with spinocerebellar ataxia.

Deep sequencing shows that oocytes are not prone to accumulate mtDNA heteroplasmic mutations during ovarian ageing.

Does ovarian ageing increase the number of heteroplasmic mitochondrial DNA (mtDNA) point mutations in oocytes? Our results suggest that oocytes are not subject to the accumulation of mtDNA point mutations during ovarian ageing. Ageing is associated with the alteration of mtDNA integrity in various tissues. Primary oocytes, present in the ovary since embryonic life, may accumulate mtDNA mutations during the process of ovarian ageing. This was an observational study of 53 immature oocyte-cumulus complexes retrieved from 35 women undergoing IVF at the University Hospital of Angers, France, from March 2013 to March 2014. The women were classified in two groups, one including 19 women showing signs of ovarian ageing objectified by a diminished ovarian reserve (DOR), and the other, including 16 women with a normal ovarian reserve (NOR), which served as a control group. mtDNA was extracted from isolated oocytes, and from their corresponding cumulus cells (CCs) considered as a somatic cell compartment. The average mtDNA content of each sample was assessed by using a quantitative real-time PCR technique. Deep sequencing was performed using the Ion Torrent Proton for Next-Generation Sequencing. Signal processing and base calling were done by the embedded pre-processing pipeline and the variants were analyzed using an in-house workflow. The distribution of the different variants between DOR and NOR patients, on one hand, and oocyte and CCs, on the other, was analyzed with the generalized mixed linear model to take into account the cluster of cells belonging to a given mother. There were no significant differences between the numbers of mtDNA variants between the DOR and the NOR patients, either in the oocytes (P = 0.867) or in the surrounding CCs (P = 0.154). There were also no differences in terms of variants with potential functional consequences. De-novo mtDNA variants were found in 28% of the oocytes and in 66% of the CCs with the mean number of variants being significantly different (respectively 0.321, SD = 0.547 and 1.075, SD = 1.158) (P < 0.0001). Variants with a potential functional consequence were also overrepresented in CCs compared with oocytes (P = 0.0019). N/A. Limitations may be due to the use of immature oocytes discarded during the assisted reproductive technology procedure, the small size of the sample, and the high-throughput sequencing technology that might not have detected heteroplasmy levels lower than 2%. The alteration of mtDNA integrity in oocytes during ovarian ageing is a recurring question to which our pilot study suggests a reassuring answer. This work was supported by the University Hospital of Angers, the University of Angers, France, and the French national research centers, INSERM and the CNRS. There are nocompeting interests.

Establishment of a heteroplasmic mouse strain with interspecific mitochondrial DNA haplotypes and improvement of a PCR-RFLP-based measurement system for estimation of mitochondrial DNA heteroplasmy.

Mitochondrial DNA segregation is one of the characteristic modes of mitochondrial inheritance in which the heteroplasmic state of mitochondrial DNA is transmitted to the next generation in variable proportions. To analyze mitochondrial DNA segregation, we produced a heteroplasmic mouse strain with interspecific mitochondrial DNA haplotypes, which contains two types of mitochondrial DNA derived from C57BL/6J and Mus spretus strains. The strain was produced on a C57BL/6J nuclear genomic background by microinjection of donor cytoplasm into fertilized eggs. The PCR-RFLP semi-quantitative analysis method, which was improved to reduce the effect of heteroduplex formation, was used to measure the proportion of heteroplasmic mitochondrial DNA in tissues. Founder mice contained up to approximately 14% of exogenous Mus spretus mitochondrial DNA molecules in their tails, and the detected proportions differed among tissues of the same individual. Heteroplasmic mitochondrial DNA is transmitted to the next generation in varying proportions under the maternal inheritance mode. This mitochondrial heteroplasmic mouse strain and the improved PCR-RFLP measurement system enable analysis of the transmission of heteroplasmic mitochondrial DNA variants between tissues and generations.

Low dose resveratrol ameliorates mitochondrial respiratory dysfunction and enhances cellular reprogramming

Mitochondrial disease is associated with a wide variety of clinical presentations, even among patients carrying heteroplasmic mitochondrial DNA (mtDNA) mutations, probably because of variations in mutant mtDNA proportions at the tissue and organ levels. Although several case reports and clinical trials have assessed the effectiveness of various types of drugs and supplements for the treatment of mitochondrial diseases, there are currently no cures for these conditions. In this study, we demonstrated for the first time that low dose resveratrol (RSV) ameliorated mitochondrial respiratory dysfunction in patient-derived fibroblasts carrying homoplasmic mtDNA mutations. Furthermore, low dose RSV also facilitated efficient cellular reprogramming of the patient-derived fibroblasts into induced pluripotent stem cells, partly due to improved cellular viability. Our results highlight the potential of RSV as a new therapeutic drug candidate for the treatment of mitochondrial diseases.

Mitochondrial respiratory dysfunction disturbs neuronal and cardiac lineage commitment of human iPSCs

Mitochondrial diseases are genetically heterogeneous and present a broad clinical spectrum among patients; in most cases, genetic determinants of mitochondrial diseases are heteroplasmic mitochondrial DNA (mtDNA) mutations. However, it is uncertain whether and how heteroplasmic mtDNA mutations affect particular cellular fate-determination processes, which are closely associated with the cell-type-specific pathophysiology of mitochondrial diseases. In this study, we established two isogenic induced pluripotent stem cell (iPSC) lines each carrying different proportions of a heteroplasmic m.3243A>G mutation from the same patient; one exhibited apparently normal and the other showed most likely impaired mitochondrial respiratory function. Low proportions of m.3243A>G exhibited no apparent molecular pathogenic influence on directed differentiation into neurons and cardiomyocytes, whereas high proportions of m.3243A>G showed both induced neuronal cell death and inhibited cardiac lineage commitment. Such neuronal and cardiac maturation defects were also confirmed using another patient-derived iPSC line carrying quite high proportion of m.3243A>G. In conclusion, mitochondrial respiratory dysfunction strongly inhibits maturation and survival of iPSC-derived neurons and cardiomyocytes; our presenting data also suggest that appropriate mitochondrial maturation actually contributes to cellular fate-determination processes during development.

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

Gene mutation has been considered a research hotspot, and the rapid development of biomedicine has enabled significant advances in the evaluation of gene mutations. The advent of digital polymerase chain reaction (dPCR) elevates the detection of gene mutations to unprecedented levels of precision, especially in cancer-associated genes. dPCR has been utilized in the detection of tumor markers in cell-free DNA (cfDNA) samples from patients with different types of cancer in samples such as plasma, cerebrospinal fluid, urine and sputum, which confers significant value for dPCR in both clinical applications and basic research. Moreover, dPCR is extensively used in detecting pathogen mutations related to typical features of infectious diseases (e.g., drug resistance) and mutation status of heteroplasmic mitochondrial DNA, which determines the manifestation and progression of mtDNA-related diseases, as well as allows for the prenatal diagnosis of monogenic diseases and the assessment of the genome editing effects. Compared with real-time PCR (qPCR) and sequencing, the higher sensitivity and accuracy of dPCR indicates a great advantage in the detection of rare mutation. As a new technique, dPCR has some limitations, such as the necessity of highly allele-specific probes and a large sample volume. In this review, we summarize the application of dPCR in the detection of human disease-associated gene mutations.

Mitochondrial DNA mutations in iPS cells: mtDNA integrity as standard iPSC selection criteria?

Somatic cells harbor random heteroplasmic mitochondrial DNA mutations, which are considered to contribute to aging. In this issue of The EMBO Journal, Perales‐Clemente et al () show that mtDNA mutations, present at low levels in the starting fibroblasts, become enriched in iPS cells and lead to functional defects in iPS‐derived cells. In another recent study, Kang et al () demonstrated that accumulation of mtDNA mutations of somatic origin in iPSCs is age related.