Mitochondria, the “Powerhouses of the cell”, are double-membrane organelles that are able to generate 90% of cell energy in the form of adenosine triphosphate (ATP), through the oxidative phosphorylation (OXPHOS) process in mammalian cells . They are maternally inherited, present in elevated numbers within cells and have their own DNA - called mitochondrial DNA (mtDNA) - that makes them interesting to study due to its importance in mitochondrial functionality and to its singular characteristics. In fact, mtDNA is different from nuclear DNA (nDNA) and similar to a bacterial chromosome: circular, close and double-stranded .
Furthermore, mitochondria have their own replication, transcription and transduction mechanisms, being reflected by their gene composition. The human mtDNA is small (16.5 kilobase pairs) and contains only 37 genes: 2 ribosomal RNA (rRNA) subunits, 22 transfer RNA (tRNA) genes and other 13 genes encoding protein subunit (included the enzyme complex OXPHOS) . In contrast to nDNA, in mtDNA there are no introns and everything is coding, except for the displacement loop sequence (D-loop) which acts as a promoter for both strands and it is also a hot spot for mutations . Also in tRNA, rRNA and protein-coding genes have been identified mtDNA mutations, and they invariably compromise mitochondrial gene expression causing various degrees of OXPHOS deficiency .
MtDNA mutations are essentially point mutations, although single, large-scale deletions are a known cause of sporadic diseases. They can be generated by spontaneous errors during replication and also by incorrect repair of damaged DNA bases. In fact, mtDNA’s repair mechanism is limited compared to nDNA’s one, and this makes mtDNA more susceptible to damage .
Furthermore, the high percentage of coding sequences in mtDNA reflects the evidence that mutations arising in mtDNA are much more likely to have pathological impact than mutations arising in the nDNA, where the majority of sequences are intronic or intergenic . Indeed, mtDNA mutation have been associated to different human diseases, starting from mitochondrial-related syndromes, such as the Leber hereditary optic neuropathy (LHON) or the mitochondrial encephalomyopathy with lactic acidosis and stroke like episodes (MELAS), and to “common” diseases such as heart disease, Alzheimer, Parkinson, hypertension, cancer and also diabetes .
To better understand the heterogeneous spectrum of consequences related to mitochondrial dysfunction, it is important to specify that each cell contains a huge number of mitochondria and each of them contains a multicopy of mtDNA . Due to this multicopy nature, mutation can be present in all mtDNA copies (homoplasmy) or in a subset of all copies (heteroplasmy). While homoplasmy for disease-causing mutations is rare because severe mtDNA mutations are unlikely to be tolerated in all the copies, heteroplasmy is more common. Heteroplasmic pathogenic mutations have a mechanism similar to “recessiveness”: they cause mitochondrial deficiency only when present above a certain threshold (cut-off effect) .
MtDNA mutations can be inherited from the mother (mitochondria are contained in the egg cell and absent from the head of sperm cells). Therefore, the mutation can randomly segregate between primary oocytes and between different tissues. On the other hand, a mtDNA mutation can be also sporadically acquired, arising in a single mtDNA molecule in a single cell. Here instead, the mutation is clonally expanded and accumulates in specific tissues .
Sequencing and variant interpretation
Studying mutations associated with mitochondrial disorders is extremely challenging due to phenotypic variability and genetic heterogeneity among individuals. Specific combinations of single nuclear polymorphisms (SNPs) in mtDNA inherited from a common ancestor define mitochondrial haplogroups, i.e.: the collections of similar haplotypes, formed as a result of the sequential accumulation of mutations through maternal lineages. The presence of these mutations can be a confounding effect, since some mutations can have a pathogenic effect for specific haplogroups and benign for others.
Nowadays, Next-generation sequencing (NGS) techniques are used to address the challenges of mitochondrial variant identification. Apart from detecting common and uncommon point mutations and deletions, NGS enables a sensitive measurement of heteroplasmy level .
There are two main different NGS methods for mtDNA analysis, depending on sequencing goal: whole genome sequencing (WGS) allows to discover de-novo mutations, while targeted gene sequencing enables to find a specific mutation (in order to diagnose a mitochondrial-related pathology, for example). A recent survey of 2021 tested the two methods for mtDNA sequencing and reported that WGS is expensive and time consuming for a small amount of DNA such as mitochondrial one. This disadvantage promotes the use of targeted sequencing, described as a more affordable approach .
Beyond mitochondrial variant identification issues, variant interpretation remains an open challenge. An explicative case: the point mutation m.3243A>G is associated with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) in the main genomic databases. The presence of this mutation is responsible for 80% of MELAS cases. Actually, only 10% of the patients with the mutation develop this phenotype, 30% instead have maternally inherited diabetes and deafness (MIDD), others a mixture of both the conditions, and eventually 9% have no clinical features .
This example raises awareness on the breadth of the pathogenic conditions caused by a single mtDNA mutation, going through the well known problem of variant interpretation. Additionally, tissue-specific differences in heteroplasmy levels for a given variant are a common source of potential misinterpretation for mtDNA variant pathogenicity classification. In this case ACMG/AMP germline variant interpretation criteria are not applicable to mtDNA variant analysis due to all the features that distinguish mtDNA from nDNA. For this reason, an international working group of mtDNA experts was assembled within the Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium to critically review mtDNA variant in the context of the current ACMG/AMP guidelines, in order to give specifications for mitochondrial variant interpretation .
To conclude, mtDNA analysis is a current subject of discussion, because of the unique characteristics of the mitochondrial genome: lack of introns, maternal inheritance, multiple copy number per cell, complex heteroplasmy and threshold effect. As it was mentioned, mtDNA sequencing is a fundamental instrument to identify mutations due to diagnose pathological conditions, but also to study the genome itself, discovering new variants. However, there are some challenges remaining: the extensive heterogeneity of clinical symptoms, the non-specific nature of many phenotypes and the poorly understood genotype–phenotype correlation. For these reasons, it is indispensable to develop accurate tools for variant interpretation, in order to overcome the challenges and to ameliorate the knowledge of the mitochondrial genome itself.
Written by Martina Cattane