Molecular Epidemiology of Mitochondrial Cardiomyopathy: A Search Among Mitochondrial and Nuclear Genes

1. Introduction

Cardiomyopathies (CM) represent a wide group of human disorders, extending from isolated cardiac diseases to cardiac involvement in the context of complex diseases, such as neuromuscular disorders, mitochondrial pathology, and metabolic/infiltrative/storage disease [1,2]. An extensive clinical examination, comprising a systematic screening of several organs, should be, initially, conducted to define whether the cardiomyopathy is isolated or it is part of a multisystem disorder. Furthermore, the family history, specific symptoms, and the presence of typical clinical and instrumental findings may help the physicians in the differential diagnosis between mitochondrial cardiomyopathy (MCM) and sarcomere disease. Indeed, mitochondrial diseases (MDs) may manifest with structural and clinical features of hypertrophic (HCM), dilated (DCM), or restrictive (RCM) cardiomyopathy, in absence of concomitant coronary artery disease, valvular disease, or hypertension [3,4,5,6,7]. MDs are a group of rare disorders (prevalence 5–12/100,000), due to both mitochondrial DNA (mtDNA) and nuclear genome (nDNA) alterations, characterized by defect of oxidative phosphorylation (OXPHOS), impairing the ATP production [8,9,10,11,12] (Supplementary data: mitochondrial disease).

MDs show an extremely clinical, biochemical, and genetical heterogeneous phenotype, depending on the involved tissue, the specific nuclear or mitochondrial DNA mutation, and mtDNA heteroplasmic level. Several degenerative, neuromuscular, ophthalmological and gastroenterological disorders, deafness, and endocrine manifestations [13,14,15,16], such as diabetes mellitus, are frequently observed in the affected individuals [11,13,17,18,19,20]. In MDs the heart, being a high-energy demand tissue, is one of the main affected organs, [21]. In fact, cardiac involvement in patients with MDs has been estimated to be approximately 20–25%, and up to 40% in children [19,22] and 30% in adults [23].

In mitochondrial cardiomyopathies, the biogenesis of mitochondria represents an adaptive response to the mitochondrial defects. In these pathologies, the energy deficiency, mechanical interference due to sarcomere misalignment and uneven contraction, increased oxidative stress, and uncoupled respiration are possible harmful factors to myocyte function [24].

In particular, HCM due to inter-myofibrillary proliferation of mitochondria and resulting in mechanical damage and deficit of sarcomere function, is the most common cardiac manifestation of MDs, occurring in approximately 40–50% of MCM cases [7,16,19]. Dilated or restrictive cardiomyopathy and cardiac conduction defects are less commonly identified in MDs [25].

Although some MDs may affect a single organ, most of them interest multiple organs, displaying a clinical syndrome (Supplementary data: mitochondrial disease).

In many individuals, the diagnosis is complex and requires a multidisciplinary approach including clinical, biochemical, histochemical, immunocytochemistry, and neuropathological investigations. Finally, the molecular analysis, by mtDNA and nDNA sequencing, may reveal the genetic basis of the pathology. To date, besides the 13 OXPHOS system proteins, 22 tRNA, and 2 rRNA, overall encoded by the mitochondrial genome, about 1500 nuclear-genome encoded proteins, encompass the mammalian mitochondrial proteome. These proteins constitute the structural subunits of the OXPHOS system, are responsible of the mtDNA maintenance, replication, and function, as well as are involved in mitochondrial assembly and stability of the five OXPHOS complexes. [1,16,26,27]. Because of this great genetic heterogeneity, the MDs genetic testing, through the candidate gene sequencing approach, may not identify the molecular alteration, leaving many patients without diagnosis. In recent years, high-throughput sequencing strategies, such as next-generation sequencing (NGS), are proving to be fundamental for the diagnosis of MD patients and for mechanistic MDs studies [28,29,30].

In this article, we reviewed the current genetic knowledge on MDs, focusing on diagnosis and management of MCM and MDs showing cardiac involvement. We searched for publications on mitochondrial and nuclear genes involved in mitochondrial cardiomyopathies, and identified a number of targeted genes, which should be included in genetic-panel for the molecular diagnosis of the mitochondrial cardiomyopathies, by using Next Generation Sequencing.

2. Mutations in Nuclear and Mitochondrial Genes

Several variations in mitochondrial and OXPHOS-related nuclear protein coding genes cause a reduction in enzyme function in one or more of the RC complexes, while mt-tRNA mutations may impair mitochondrial translation, by decreasing the availability of functional mt-tRNAs [31]. These dysfunctions, overall, cause defects of the oxidative phosphorylation system and subsequently impair the ATP production, leading to different phenotypes, depending on the tissue involved, type of pathogenic mutations and heteroplasmy level [10,11]. Estimates suggest that about 15–20% of RC defects are due to mtDNA mutations, while the remaining are caused by defects in nuclear genes [19,32].

Mitochondrial mutations may be inherited (~75% cases) or may occur de novo (~25% cases) [33]. MtDNA point mutations represent the most frequent causes of mtDNA alterations [8,11]. MtDNA point mutations (including small indel mutations) are pretty frequent as they are found in 1:200 newborns; however many individuals carry low heteroplasmic level of the mutant gene, thus they remain asymptomatic [34]. On the contrary, large-scale mtDNA deletions are generally rare (1.5/100,000) and typically arise as de novo mutations during embryonic development. Conversely to nDNA rearrangements, large-scale mtDNA deletions have a low recurrence risk (<10% risk of transmission), although the identification of the same deletion in mother and son suggests a matrilineal transmission [35,36,37]. In a more recent classification, the primary mitochondrial diseases (PMD) are caused by germline mutations, in mtDNA and/or nDNA genes encoding RC proteins or in nDNA genes involved in the complex protein-organization, needed for mitochondrial RC [38]. Conversely, secondary mitochondrial dysfunction (SMD) refers to abnormal mitochondrial function caused by mutations in non-OXPHOS genes affecting the mitochondrial dynamics and its capacity to produce ATP. SMD can be inherited or acquired (due to non-genetic causes such as environmental factors) which is an important distinction from PMD, which can only be inherited.

Furthermore, in addition to ATP deficiency, mitochondrial dysfunction, contributing to pathogenesis of MDs, may also affect calcium handling, ROS production, apoptosis dysregulation and nitric oxide (NO) deficiency [39].

Although the production of ROS and accumulation of their active metabolites is reported as the major known cause of mtDNA mutations [40], recent studies have reported mitochondria fission and fusion (mitochondrial dynamics) imbalance in cardiomyopathies [41]. In particular, the processes of mitochondrial fusion, fission, biogenesis and mitophagy, involved in mitochondrial morphology, quality, and abundance, have recently been implicated in cardiovascular disease. Studies suggest that changes in mitochondrial morphology may be an adaptive mechanism during cardiac pathological remodeling [42].

Genetic deletions of mitochondrial fusion-promoting genes, Mitofusin 1 and 2 (MFN1, MFN2), have been shown to induce mitochondrial fragmentation, whereas deletions of mitochondrial fission-promoting gene (Dynamin-1-like DRP1) cause mitochondrial enlargement. Likewise, because the inner mitochondrial membrane fusion protein OPA1 (optic atrophy factor 1 OPA1 gene) is essential for maintaining the shape of the mitochondria and normal cristae structure, cardiomyopathies, caused by OPA 1 gene deletions may determine defective mitochondrial inner membrane architecture [43]. Chen et al. [44] described small and fragmented mitochondria in both human and rat models of heart failure, which were associated with decreased OPA1 levels.

Furthermore, nuclear gene mutations, regulating mtDNA replication and maintenance, such as mitochondrial transcription factor A (TFAM), mtDNA polymerase γ (POLG), and PEO1 (Twinkle) genes have been associated with cardiac diseases [45,46].

3. Mitochondrial Cardiomyopathy (MCM)

The most frequent cardiac disorders refereed to mitochondrial dysfunction are cardiomyopathies [3,16,25]. Although the true prevalence of mtDNA-related CM is unknown, based on the prevalence of mtDNA disease and frequency of cardiac manifestations, about 1/10,000–15,000 of the general population is affected [7]. Furthermore, several studies report different prevalence between pediatric and adult population, with cardiomyopathy estimated to occur in 20–40% of children with MDs [16,22,47,48].

Mitochondrial cardiomyopathy is a myocardial disorder, without concomitant coronary artery disease, valvular disease, congenital heart disease, and hypertension, characterized by abnormal myocardial structure and/or function, subsequent to genetic alterations and resulting in the impairment of the mitochondrial respiratory chain [4]. Hypertrophic cardiomyopathy, dilated cardiomyopathy, and left ventricular non compaction (LVNC) are the main manifestations of MCM. Histiocytoid cardiomyopathy (HICMP) (Purkinje fiber dysplasia) and restrictive cardiomyopathy, as well as Takotsubo syndrome (TTS) have been also observed [16,19].

Moreover, MCM, in addition to the myocardium, may affect other cardiac tissue, such as the cardiac conduction system, cardiac valves, aortic root, coronary arteries or the pericardium, and connective tissue, thus leading impulse generation/conduction insufficiency, valvular diseases, systolic dysfunction, or heart failure and pulmonary arterial hypertension (PAH). Conduction system disorders arise in 5–10% of patients with MD, especially in patients with associated neuromuscular disease [49].

The abnormal heart-muscle structure and/or function is due to genetic defects, impairing the functionality of the mitochondrial RC complexes, their assembly and stability factors, tRNAs, rRNAs, mtDNA maintenance, and coenzyme Q10 (CoQ10) synthesis [19,50].

The cardiac involvement in MDs may be already evident at onset, but in some cases, it may develop over time. More rarely, in patients without or with mild multisystem involvement, the heart may be the only or the main clinically affected organ.

The imbalance of mitochondrial function in MCM is supported by a large amount of scientific publications, investigating the cardiological aspect both as one of the manifestations of a syndromic mitochondrial disorder and, in a smaller number of cases, as an isolated cardiac disease. In particular, Whabi et al. showed that myocardial involvement in mtDNA diseases is progressive and, in association with other cardiovascular manifestations and risk factors (i.e., intraventricular conduction block, diabetes, premature ventricular contractions, and left ventricular hypertrophy-LVH), is an independent predictor of morbidity and early mortality [23].

4. Diagnosis

Diagnosis of MCM is particularly complex because of wide clinical and genetic heterogeneity and requires a multidisciplinary approach, including extensive medical, laboratory and neuroimaging investigations. Physicians should suspect a possible MCM in patients with signs and symptoms of cardiomyopathy and multisystem involvement without a clear cause and refer the patients to a geneticist or other specialists with MDs expertise.

Genetic counselling is a fundamental part of the diagnostic workup and should be performed by specialized personnel. A detailed pedigree of the patient’s family should be obtained, and all first-degree relatives should be referred to a specific cardiogenetic clinic. An integrated diagnostic approach, consisting of biochemical screening, histopathological studies, cardiological investigations, including magnetic resonance imaging, functional assays, and molecular genetic testing, should be conducted to reach a correct diagnosis. The identification of a causative mutation allows the presymptomatic identification and the follow up of family members. The advent of NGS techniques has revolutionized the diagnosis of MDs in terms of expanding the number of involved genes as well as the discovery of new genes potentially associated with the diseases.

5. Molecular Analysis of Mitochondrial Cardiomyopathies

Traditionally, for different pathologies as well as MDs, including MCM, molecular diagnosis is achieved through “candidate gene” studies. This approach is based on recognizable clinical manifestations and family history, followed by molecular testing performed by Sanger sequencing of known genes [133]. Indeed some MD phenotypes are linked to molecular defects in one or few mitochondrial and nuclear genes [12].

To date, for patients suspected of having maternally inherited mtDNA disorders, the molecular diagnosis is still initially based on the mtDNA Sanger sequencing. In adult onset cases, mtDNA aetiology is recurrent and mtDNA sequencing for point mutations or the whole mitochondrial genome, when common point mutations are not detected, remains the first approach [134].

Moreover, Sanger sequencing does not detect either heteroplasmic mutations below 15–20% or allow measuring the degree of heteroplasmy. It also does not show large deletions, which are, conversely, detectable by other different technologies (i.e., array comparative genome hybridization) [135,136,137].

The genetic diagnosis of many MDs, including MCM, is further impaired by numerous nuclear genes whose defects may cause mitochondrial diseases [28].

Nowadays, for the above mentioned reasons, the diagnosis of MCM based on clinical/biochemical and instrumental examinations and sequencing of candidate genes one by one, is not a practical approach because it leaves many undiagnosed cases.

Advances in genomics and high-throughput sequencing technologies, such as NGS, have revolutionized the diagnostic setting of MDs, allowing identification of a large number of nuclear genes simultaneously, but also providing the identification and an accurate measure of heteroplasmy, in case of mtDNA mutations [29,30,138,139,140,141,142,143]. For making diagnosis of many complex diseases, characterized by a wide clinical spectrum and heterogeneous genetic involvement, next generation sequencing based approach is available and sometimes necessary [144,145].

NGS screening encompasses different configurations, ranging from a small number of genes (targeted gene panels: i.e., Complex I genes) to hundred or more genes (targeted exome sequencing: i.e., Mito Exome, that includes about 1500 genes related to mitochondrial structure and function), and even up to Whole Exome Sequencing (WES) [122,146,147]. The choice depends by clinical, laboratory, and instrumental data, based on the clinical indication, family history, biochemical, and histopathological findings and imaging studies. The costs, time, acceptable depth of coverage and the management of off-target findings have to be also evaluated.

6. Literature Review

We reviewed and collected the literature data from July 1990 to October 2020, (one papers published in January 2021) by searching publications including genetic data from patient’s cohort showing mitochondrial cardiomyopathy or mitochondrial disorder with cardiac involvement. Database searching and Inclusion/Exclusion Criteria are reported in Supplementary data: online database. The objective was to investigate the genotype/phenotype correlation in several studies, including patients with syndromic and non-syndromic features of mitochondrial cardiomyopathy. For this aim we investigated (accessed on 2 October 2020) the most known databases, available online.

Furthermore, in recent years, especially since the introduction of NGS technology, several studies, using extensive genetic analysis, highlighted the main disease-genes associated with MCM and MDs showing cardiomyopathy; this has contributed to increase our knowledge on the molecular background of these diseases.

The second aim of this review was to identify disease-genes, by searching publications of the last ten years, in order to prove the utility of genetic tests in MCM diagnosis in patients with syndromic or non-syndromic MDs and to identify a targeted gene panel for the molecular diagnosis.

7. Discussion

CMs are reported by the European Society of Cardiology (ESC) Working Group on Myocardial and Pericardial Diseases as structurally and functionally myocardial disorders, in the absence of coronary artery disease, hypertension, valvular heart disease, and congenital heart disease explaining the myocardial defect [177].

The contribution of clinical molecular biology in the evaluation and prevention of cardiological risk, in particular sudden cardiac death, and in the therapeutic approach [178], both in patients and in healthy subjects and athletes, is an important goal that involves many areas of the medical profession [179,180,181,182,183].

MCM, mainly HCM followed by DCM and LVNC or, more rarely, RCM and HICMP, are common cardiac symptoms of MDs and should be considered in patients with multi-organ involvement. Cardiac abnormalities, such as cardiomyopathy, arrhythmias, and conduction defects are, indeed, present in up to 60% of patients with MD and represent often a major determinant of morbidity and mortality [53]. The wide genetic heterogeneity associated with mitochondrial cardiomyopathies, supports the opinion that the “cardiological phenotype” should often be considered as a common feature of many mitochondrial disorders and more rarely may be the first, or even the only, clinical manifestation.

Moreover, because MCM may also occur in non-syndromic MDs or in patients with only mild multisystem involvement and the differential diagnosis with non-mitochondrial cardiomyopathy through cardiological and imaging investigations is often difficult a considerable number of MCM remain undiagnosed or delayed [125]. An early recognition of MCM is crucial to initiate therapeutic procedures avoiding heart failure and arrhythmias as well as mitochondrial crisis.

Currently, the molecular screening for MCM, is a fundamental part of the management of MDs that, through an integrated multidisciplinary diagnostic approach allows getting to the diagnosis.

Recently, the Mitochondrial Medicine Society has provided consensus statements on the diagnosis and management of MDs, including measurements of mitochondrial biomarkers in plasma, urine, and spinal fluid [126].

Moreover, in patients with suspected of MD, biochemical screening, as well as histopathological studies and detailed clinical/cardiological evaluation should be followed by genetic investigation to make a definitive diagnosis.

Molecular analysis of mtDNA may be, initially performed when a clear maternally inheritance is observed, while both the mtDNA and nDNA should be considered if the inheritance pattern is uncertain.

The first objective of this review was to identify the major genes frequently associated with mitochondrial cardiomyopathies, in order to propose, by using Next Generation Sequencing technology, a novel targeted gene panel for the molecular diagnosis of the diseases. For this purpose, we explored literature and public data concerning the cardiac manifestations in mitochondrial diseases/disorders, in order to investigate the genotype/phenotype correlation in patients with MCM. This data collection allowed us to extract twenty-nine papers showing patients with mitochondrial cardiomyopathy and mtDNA and/or nDNA mutations.

The most of these papers report mtDNA mutations in association with the cardiological phenotype, in agreement with the higher frequency, in adults, of mutations in the mitochondrial genome. Moreover, many of these studies are not carried out with high-throughput genetic technologies that are able to highlight several disease-genes associated with MCM and MDs showing cardiomyopathy.

In this context, the next-generation sequencing technology has allowed, for several diseases, a huge expansion of the diagnostic screening, increasing the spectrum of involved genes and suggesting new genotype/phenotype correlations [184]. This correlation is particularly important in the diagnosis of MDs, including MCM and for the identification of pre-symptomatic family members to take in follow up.

The actual NGS strategies allow screening patients with suspected MD through a wide range of approaches, including targeted gene panel sequencing, mitochondrial gene panel, clinical exome panel and Whole Exome Sequencing (WES). The choice of the technique depends on several factors, such as funding, bioinformatics, and laboratory expertise.

WES or clinical exome panel may be considered for group of genetic disorders, which may be examined following the same pipeline. Then, the use of virtually assembled gene panel (i.e., Mitochondrial Cardiomyopathy gene panel, metabolic gene panel, etc.) allows to select only data in genes of interest and filtering for further genes or exome analysis, in a subsequent bioinformatics re-evaluation (i.e., if, in the selected panels, no mutations are found and the clinical suspicion is further deepened and confirmed). This is very important because the MD phenotype overlapped with other metabolic and neuromuscular disorders.

Although coverage for WES is constantly increasing and may be generally comparable to that obtained by targeted gene panel analysis, challenges in obtaining sufficient coverage are due to the need, for economic reasons, to collect a greater number of samples in the same analytical session.

Furthermore, the most challenging aspect of this approach is given by the large amount of unknown identified variants or novel disease genes, which need further investigation both by in silico analysis and through functional studies [185]. In this regards, the role of molecular biology research is fundamental to expand the list of pathogenic variants and increase our knowledge about MCM.

Conversely, smaller or targeted gene panels, such as the one proposed in this study, achieve a high diagnostic yield, at lower operating costs, in particular when they are designed to target the high yield genetic content, or most relevant and clinically associated genes.

This is an ideal NGS approach for the diagnosis of heterogeneous disorders, like MDs, involving dual genome.

In the post-genomic era, the benefits of NGS in molecular diagnosis of rare diseases are well established. However, the use of NGS-based approach in diagnostics is increasing the yield of VUS. The significance and possible consequences of these variants are difficult to establish. The possibility to study a specific VUS, in the context of a large pedigree by analyzing the variant’s segregation, as compared with the clinical phenotype may be useful; however, it is often unfeasible. Additionally, when clinical features suggest an underlying disease, the presence of a VUS may deserve reinterpretation regarding its pathogenicity during follow-up according to accumulating literature knowledge. We believe that the interpretation of variants should be verified approximately every 6 months. Indeed, it is not uncommon that the interpretation of variants changes after further studies are published over time. Moreover, we are fully aware that in silico predictions are not enough to establish the variants’ pathogenicity and in vitro functional studies would be needed to have more conclusive information on each identified variant.

Mitochondrial cardiomyopathies are very rare disorders; in this setting, despite laboratory capacity to generate sequence data has greatly increased, with the advent of high-throughput next generation sequencing, the capacity to interpret genomic data and to identify disease genes can be difficult. The literature review highlighted about thirty high-confidence genes known to cause mitochondrial diseases in large cohorts of MCM patients. At the same time, we selected many other genes rarely reported associated with MCM (<1% of cases, each), which are reported as potential disease-genes based on known gene function, segregation with disease in families, computational analysis, etc., although more information is needed so that they can be classified as clinically actionable genes. Consequently, the probability of finding pathogenic variants is higher in high confidence genes than in low confidence ones, using the updated guidelines for the clinical interpretation of sequence variants developed by the American College of Medical Genetics and Genomics (ACMG).

Employment, in a cohort of patients showing clinical features of MCM, of the genes panel designed by carefully selecting genes previously associated with MCM, will yield rapidly actionable results (variants in high-confidence genes) and also identify potentially causal variants (in low-confidence genes) that will require further information to be clinically actionable.

Although a lot of work is still needed to easily and quickly diagnose a Mitochondrial Cardiomyopathy, a significant number of undiagnosed patients may be identified by NGS technology.

In this review, we identified 130 disease-genes associated with MCM, by searching molecular data from papers published in the last ten years, reporting new sequencing technologies. These genes could be included as target genes to build a novel gene panel for the molecular diagnosis of patients with clinical suspect of MCM.

8. Conclusions

Mitochondrial cardiomyopathy is a challenging diagnosis, requiring wide medical, neuroimaging, and laboratory examinations. The cardiologists have to keep the degree of suspicion high, looking also for signs from other tissues involved and the family history. Echocardiography and neuroimaging findings are variable, but the hypertrophic cardiomyopathy is the most common phenotype. Moreover, the increasing use of Next Generation Sequencing technologies allows collecting more genetic data, continually improving the diagnostic success. This review collects the major genes associated with mitochondrial cardiomyopathy in recent decades, underlining how the link between research and diagnosis is crucial to expand the list of pathogenic variants and concomitantly enhance our knowledge of mitochondrial cardiomyopathy.

9. Limitation

This review has some limitations concerning both the possibility that the filters utilized for the literature and database research, the keywords used, and their association may not have been enough to avoid losing papers. Furthermore, we did not differentiate pediatric and adult studies. Finally, the panel of genes selected from such a large literature will have to be validated to know the diagnostic yield in mitochondrial cardiomyopathies.

Author Contributions

Conceived and designed the study, G.F. and C.M.; performed the data, N.T., B.L., and O.S.; collection reviewed the cardiological data, G.L. and M.C.; analyzed the data, B.M., F.B., G.F., and C.M.; wrote the paper, B.M., G.F., and C.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Research Projects of National Interest (PRIN) from the Italian Ministry of University and Research grant (20173ZWACS).

Data Availability Statement

Data is contained within the article or Supplementary material.

Conflicts of Interest

The authors declare no conflict of interest.