Mitochondrial diseases result from abnormalities in the mitochondrion, the essential organelle that provides energy in the form of ATP for normal heart function.1
Mitochondria are dynamic organelles that number from several to hundreds per cell with each mitochondrion containing multiple copies of mitochondrial DNA (mtDNA). Normal mitochondrial function relies on two types of inheritance: maternal inheritance of circular mtDNA sequences and biparental inheritance of nuclear DNA (nDNA) that has over 1000 genes for mitochondrial homeostasis. Consequently, mitochondrial dysfunction because of mtDNA defects or nDNA mutations can result in disease with cardiomyopathy and heart failure as the major features.2
mtDNA is the circular, 16
569 base sequence with 37 genes that encode for 13 proteins, 22 transfer RNAs (tRNAs) and 2 ribosomal RNAs.3
Heteroplasmy describes the state of a mixture of normal and mutant copies of mtDNA. Depending on the timing of origin, location and segregation of mutant mtDNA, an individual may have different ratios of mutant and normal mtDNA distributed amongst different tissues or organs that result in disease.4
Owing to these genetic factors, mitochondrial diseases leading to myocardial disease have much clinical heterogeneity in age of presentation and severity of symptoms.5
Cardiac features include concentric ventricular hypertrophy, dilated cardiomyopathy (DCM) and congestive heart failure.6, 7, 8
Cardiac conduction features include atrioventricular and bundle branch blocks.9
When mitochondrial disease primarily affects only the heart, hypertrophic cardiomyopathy (HCM) or dilated mitochondrial cardiomyopathies may be clinically indistinguishable from other cardiomyopathies.6, 7, 10
Diagnosis may be difficult requiring cardiac tissue for diagnosis; histopathological features include changes in mitochondrial shape or number, histochemical defects and abnormities in oxidative phosphorylation (OXPHOS) enzyme activities.5
Diagnosis of mitochondrial cardiomyopathies may be achieved by mtDNA sequence analysis for known pathogenic mtDNA mutations. For example, testing may be carried out for the m.3243A>G mutation in the tRNA leucine gene (MT-TL1
) associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke).11
The m.3243A>G mutation may also lead to severe, concentric HCM or DCM presenting in infancy or childhood.10, 12
Adults with m.3243A>G also may present with cardiomyopathy and progressive heart failure with extracardiac features of mitochondrial disease including deafness and diabetes.13, 14
A newly arising pathogenic mtDNA mutation will be heteroplasmic. As the percentage of mutant mtDNAs increases, the severity of the clinical phenotype at each percentage mutation will depend on the severity of the biochemical defect caused by the mutation. Severe mtDNA mutations will result in reproductive failure while still heteroplasmic. Hence, surviving patients with the mutation will be heteroplasmic. By contrast, milder mtDNA mutations may not cause clinical symptoms severe enough to affect reproduction until they reach homoplasmy. These later mutations have proven difficult to distinguish from neutral or adaptive polymorphisms.
It is now standard-of-care to sequence the mtDNAs of patients with complex diseases, such as cardiomyopathy associated with mitochondrial dysfunction.6, 7, 8, 15, 16, 17
The mtDNAs of over 500 patients with HCM or DCM patients have already been sequenced and the mtDNA variants that differ from a reference sequence have been compared with small samples of ‘control' cases. Those variants observed in the patient mtDNAs but not in the study controls have been considered to be potential disease causing mutations. Over 1000 variants have been reported in various cardiomyopathy cases, which encompass 200 different sequence variants.6, 7, 8, 15, 16, 17
However, population genetic studies have showed that the ‘normal' mtDNA sequence variation is very high. This is the result of ancient polymorphisms associated with distinct ethnic and/or geographic-associated maternal lineages, known as mtDNA haplogroups.18
Distinctive haplogroup lineages are descendent from a single founding maternal ancestor mtDNAs resulting in a discrete branch of the mtDNA phylogenetic tree which shares the variants of the founding mtDNA.19, 20, 21, 22
Therefore, if a patient is from a rare mtDNA haplogroup, then it is unlikely the control samples used in that study will also include a mtDNA from that haplogroup. Hence, many of the haplogroup-associated normal variants will be interpreted as potentially pathogenic.
This complexity can in part be resolved by comparing the mtDNA sequence variants of a patient with those of a large database of mtDNA sequences that have been delineated by haplogroup. If the database was exhaustive, then most of haplogroup-associated variants would be represented within the database, at frequencies consistent with the haplogroup distribution within the population. Therefore, variants associated with specific populations will be both routinely linked to other variants associated with that same haplogroup and will also be present at a significant frequency within the overall population of mtDNA sequences.
At the other extreme, a recently arising pathogenic variant will not be repeatedly linked to the same array of mtDNA variants and will be very rarely in the overall population, as it is continually being removed by selection. Therefore, novel mtDNA variants or one found very rarely in the population have a greater probability of being pathogenic. If the variant also affects a functionally important mitochondrial function and is heteroplasmic, this further increases the likelihood that the mutation is contributing to the disease.21
On the basis of this logic, we have developed a database of several thousand mtDNA sequences that encompasses much of the global mtDNA variation. In addition, we have developed a computer program, MITOMASTER,23
which will analyze a patient mtDNA sequence based on its deviations from a master reference sequence and then use this list of variants to deduce the patient's haplogroup. The variants are then compared with all other mtDNAs in that haplogroup and the common haplogroup variants identified. The frequency of each variant is also calculated. Rare, non-haplogroup-associated variants are then analyzed for the gene affected, sequence conservation, and functional consequences permitting assessment of the potential pathogenicity of the variant. To test the capacity of this system to help assess the pathogenic potential of mtDNA mutations, we now apply our MITOMASTER analysis system to a set of 29 mtDNA sequences from patients with mitochondrial cardiomyopathy and other mitochondrial clinical phenotypes. The systematic approach allowed us to prioritize mtDNA sequence variants for functional analysis to establish pathogenicity.