Penetrance and expressivity of mitochondrial variants in a large clinically unselected population.

Whole genome sequencing (WGS) from large clinically unselected cohorts provides a unique opportunity to assess the penetrance and expressivity of rare and/or known pathogenic mitochondrial variants in population. Using WGS from 179 862 clinically unselected individuals from the UK Biobank, we performed extensive single and rare variant aggregation association analyses of 15 881 mtDNA variants and 73 known pathogenic variants with 15 mitochondrial disease-relevant phenotypes. We identified 12 homoplasmic and one heteroplasmic variant (m.3243A>G) with genome-wide significant associations in our clinically unselected cohort. Heteroplasmic m.3243A>G (MAF = 0.0002, a known pathogenic variant) was associated with diabetes, deafness and heart failure and 12 homoplasmic variants increased aspartate aminotransferase levels including three low-frequency variants (MAF ~0.002 and beta~0.3 SD). Most pathogenic mitochondrial disease variants (n = 66/74) were rare in the population (<1:9000). Aggregated or single variant analysis of pathogenic variants showed low penetrance in unselected settings for the relevant phenotypes, except m.3243A>G. Multi-system disease risk and penetrance of diabetes, deafness and heart failure greatly increased with m.3243A>G level ≥ 10%. The odds ratio of these traits increased from 5.61, 12.3 and 10.1 to 25.1, 55.0 and 39.5, respectively. Diabetes risk with m.3243A>G was further influenced by type 2 diabetes genetic risk. Our study of mitochondrial variation in a large-unselected population identified novel associations and demonstrated that pathogenic mitochondrial variants have lower penetrance in clinically unselected settings. m.3243A>G was an exception at higher heteroplasmy showing a significant impact on health making it a good candidate for incidental reporting.

Dynamics of the most common pathogenic mtDNA variant m.3243A > G demonstrate frequency-dependency in blood and positive selection in the germline.

The A-to-G point mutation at position 3243 in the human mitochondrial genome (m.3243A > G) is the most common pathogenic mtDNA variant responsible for disease in humans. It is widely accepted that m.3243A > G levels decrease in blood with age, and an age correction representing ~ 2% annual decline is often applied to account for this change in mutation level. Here we report that recent data indicate that the dynamics of m.3243A > G are more complex and depend on the mutation level in blood in a bi-phasic way. Consequently, the traditional 2% correction, which is adequate 'on average', creates opposite predictive biases at high and low mutation levels. Unbiased age correction is needed to circumvent these drawbacks of the standard model. We propose to eliminate both biases by using an approach where age correction depends on mutation level in a biphasic way to account for the dynamics of m.3243A > G in blood. The utility of this approach was further tested in estimating germline selection of m.3243A > G. The biphasic approach permitted us to uncover patterns consistent with the possibility of positive selection for m.3243A > G. Germline selection of m.3243A > G shows an 'arching' profile by which selection is positive at intermediate mutant fractions and declines at high and low mutant fractions. We conclude that use of this biphasic approach will greatly improve the accuracy of modelling changes in mtDNA mutation frequencies in the germline and in somatic cells during aging.

Mitochondrial DNA disorders: from pathogenic variants to preventing transmission.

Mitochondrial DNA (mtDNA) disorders are recognized as one of the most common causes of inherited metabolic disorders. The mitochondrial genome occurs in multiple copies resulting in both homoplasmic and heteroplasmic pathogenic mtDNA variants. A biochemical defect arises when the pathogenic variant level reaches a threshold, which differs between variants. Moreover, variants can segregate, clonally expand, or be lost from cellular populations resulting in a dynamic and tissue-specific mosaic pattern of oxidative deficiency. MtDNA is maternally inherited but transmission patterns of heteroplasmic pathogenic variants are complex. During oogenesis, a mitochondrial bottleneck results in offspring with widely differing variant levels to their mother, whilst highly deleterious variants, such as deletions, are not transmitted. Complemented by a complex interplay between mitochondrial and nuclear genomes, these peculiar genetics produce marked phenotypic variation, posing challenges to the diagnosis and clinical management of patients. Novel therapeutic compounds and several genetic therapies are currently under investigation, but proven disease-modifying therapies remain elusive. Women who carry pathogenic mtDNA variants require bespoke genetic counselling to determine their reproductive options. Recent advances in in vitro fertilization techniques, have greatly improved reproductive choices, but are not without their challenges. Since the first pathogenic mtDNA variants were identified over 30 years ago, there has been remarkable progress in our understanding of these diseases. However, many questions remain unanswered and future studies are required to investigate the mechanisms of disease progression and to identify new disease-specific therapeutic targets.

Resolving complexity in mitochondrial disease: Towards precision medicine.

Mitochondrial diseases, caused by mutations in either the nuclear or mitochondrial genomes (mtDNA), are the most common form of inherited neurometabolic disorders. They are remarkably heterogeneous, both in their clinical presentation and genetic etiology, presenting challenges for diagnosis, clinical management and elucidation of molecular mechanism. The multifaceted nature of these diseases, compounded by the unique characteristics of mitochondrial genetics, cement their space in the field of complex disease. In this review we examine the m.3243A>G variant, one of the most prevalent mitochondrial DNA mutations, using it as an exemplar to demonstrate the challenges presented by these complex disorders. Disease caused by m.3243A>G is one of the most phenotypically diverse of all mitochondrial diseases; we outline known causes of this heterogeneity including mtDNA heteroplasmy, mtDNA copy number and nuclear genetic factors. We consider the impact that this has in the clinic, discussing the personalized management of common manifestations attributed to this pathogenic mtDNA variant, including hearing impairment, diabetes mellitus, myopathy, cardiac disease, stroke-like episodes and gastrointestinal disturbances. Future research into this complex disorder must account for this heterogeneity, benefitting from the use of large patient cohorts to build upon current clinical expertise. Through multi-disciplinary collaboration, the complexities of this mitochondrial disease can be addressed with the variety of diagnostic, prognostic, and treatment approaches that are moulded to best fit the needs of each individual patient.

Fit for purpose: Selecting the best mitochondrial DNA for the job.

The factors determining levels of pathogenic mitochondrial DNA in cells and tissues are critical to disease pathology but remain poorly understood and contentious. In Nature, Kotrys et al. published a single-cell-based analysis casting fresh light on this thorny problem and introduced a powerful new investigative tool.

The contributions of mitochondrial and nuclear mitochondrial genetic variation to neuroticism.

Neuroticism is a heritable trait composed of separate facets, each conferring different levels of protection or risk, to health. By examining mitochondrial DNA in 269,506 individuals, we show mitochondrial haplogroups explain 0.07-0.01% of variance in neuroticism and identify five haplogroup and 15 mitochondria-marker associations across a general factor of neuroticism, and two special factors of anxiety/tension, and worry/vulnerability with effect sizes of the same magnitude as autosomal variants. Within-haplogroup genome-wide association studies identified H-haplogroup-specific autosomal effects explaining 1.4% variance of worry/vulnerability. These H-haplogroup-specific autosomal effects show a pleiotropic relationship with cognitive, physical and mental health that differs from that found when assessing autosomal effects across haplogroups. We identify interactions between chromosome 9 regions and mitochondrial haplogroups at P < 5 × 10-8, revealing associations between general neuroticism and anxiety/tension with brain-specific gene co-expression networks. These results indicate that the mitochondrial genome contributes toward neuroticism and the autosomal links between neuroticism and health.

T cell differentiation drives the negative selection of pathogenic mitochondrial DNA variants.

Pathogenic mitochondrial DNA (mtDNA) single-nucleotide variants are a common cause of adult mitochondrial disease. Levels of some variants decrease with age in blood. Given differing division rates, longevity, and energetic requirements within haematopoietic lineages, we hypothesised that cell-type-specific metabolic requirements drive this decline. We coupled cell-sorting with mtDNA sequencing to investigate mtDNA variant levels within progenitor, myeloid, and lymphoid lineages from 26 individuals harbouring one of two pathogenic mtDNA variants (m.3243A>G and m.8344A>G). For both variants, cells of the T cell lineage show an enhanced decline. High-throughput single-cell analysis revealed that decline is driven by increasing proportions of cells that have cleared the variant, following a hierarchy that follows the current orthodoxy of T cell differentiation and maturation. Furthermore, patients with pathogenic mtDNA variants have a lower proportion of T cells than controls, indicating a key role for mitochondrial function in T cell homeostasis. This work identifies the ability of T cell subtypes to selectively purify their mitochondrial genomes, and identifies pathogenic mtDNA variants as a new means to track blood cell differentiation status.

Interactions between nuclear and mitochondrial SNPs and Parkinson's disease risk.

Interactions between the products of the nuclear and mitochondrial genomes are critical for the function of most eukaryotic cells. Recently the introduction of mitochondrial replacement therapy has raised the question of incompatibilities between mitochondrial and nuclear variants, and their potential influence on the genetic makeup of human populations. Such interactions could also contribute to the variability of the penetrance of pathogenic DNA variants. This led us to investigate the frequencies of combinations of nuclear and mitochondrial SNP alleles (mitonuclear combinations) in healthy individuals (n = 5375) and in a cohort of patients with Parkinson's disease (PD, n = 2210). In the unaffected population, we were not able to find associations between nuclear and mitochondrial variants with a false discovery rate below 0.05 after accounting for multiple testing (i.e., the number of combinations examined). However, in the PD cohort, five combinations surpassed this threshold. Next, after combining both cohorts, we investigated whether these associations were modulated by disease status. All five combinations were significant (p < 10-3 for all tests). These combinations also showed significant evidence for an effect of the interaction between the mitochondrial and nuclear variants on disease risk. Their nuclear components mapped to TBCA, NIBAN3, and GLT25D1 and an uncharacterised intergenic region. In summary, starting from a single cohort design we identified combinations of nuclear and mitochondrial variants affecting PD disease risk.

The molecular pathology of pathogenic mitochondrial tRNA variants.

Mitochondrial diseases are clinically and genetically heterogeneous disorders, caused by pathogenic variants in either the nuclear or mitochondrial genome. This heterogeneity is particularly striking for disease caused by variants in mitochondrial DNA-encoded tRNA (mt-tRNA) genes, posing challenges for both the treatment of patients and understanding the molecular pathology. In this review, we consider disease caused by the two most common pathogenic mt-tRNA variants: m.3243A>G (within MT-TL1, encoding mt-tRNALeu(UUR) ) and m.8344A>G (within MT-TK, encoding mt-tRNALys ), which together account for the vast majority of all mt-tRNA-related disease. We compare and contrast the clinical disease they are associated with, as well as their molecular pathologies, and consider what is known about the likely molecular mechanisms of disease. Finally, we discuss the role of mitochondrial-nuclear crosstalk in the manifestation of mt-tRNA-associated disease and how research in this area not only has the potential to uncover molecular mechanisms responsible for the vast clinical heterogeneity associated with these variants but also pave the way to develop treatment options for these devastating diseases.

Imaging mass cytometry reveals generalised deficiency in OXPHOS complexes in Parkinson's disease.

Here we report the application of a mass spectrometry-based technology, imaging mass cytometry, to perform in-depth proteomic profiling of mitochondrial complexes in single neurons, using metal-conjugated antibodies to label post-mortem human midbrain sections. Mitochondrial dysfunction, particularly deficiency in complex I has previously been associated with the degeneration of dopaminergic neurons in Parkinson's disease. To further our understanding of the nature of this dysfunction, and to identify Parkinson's disease specific changes, we validated a panel of antibodies targeting subunits of all five mitochondrial oxidative phosphorylation complexes in dopaminergic neurons from Parkinson's disease, mitochondrial disease, and control cases. Detailed analysis of the expression profile of these proteins, highlighted heterogeneity between individuals. There is a widespread decrease in expression of all complexes in Parkinson's neurons, although more severe in mitochondrial disease neurons, however, the combination of affected complexes varies between the two groups. We also provide evidence of a potential neuronal response to mitochondrial dysfunction through a compensatory increase in mitochondrial mass. This study highlights the use of imaging mass cytometry in the assessment and analysis of expression of oxidative phosphorylation proteins, revealing the complexity of deficiencies of these proteins within individual neurons which may contribute to and drive neurodegeneration in Parkinson's disease.

Height as a Clinical Biomarker of Disease Burden in Adult Mitochondrial Disease.

CONTEXT: Abnormal growth and short stature are observed in patients with mitochondrial disease, but it is unclear whether there is a relationship between final adult height and disease severity. OBJECTIVE: To determine whether patients with genetically confirmed mitochondrial disease are shorter than their peers and whether stature is related to disease severity. DESIGN: Analysis of final adult height in relation to disease severity as determined by the Newcastle Mitochondrial Disease Adult Scale (NMDAS). SETTING: UK Mitochondrial Disease Patient Cohort (Mito Cohort). PATIENTS: 575 patients were identified with recorded height, weight, and molecular genetic diagnosis of mitochondrial disease within the Mito Cohort. MAIN OUTCOME MEASURES: Adult height, body mass index (BMI), and their association with genetic subgroup and disease severity. RESULTS: Adults with mitochondrial disease were short, with a mean height of -0.49 SD (95% CI, -0.58 to -0.39; n = 575) compared with UK reference data. Patients were overweight, with a BMI SD of 0.52 (95% CI, 0.37 to 0.67; n = 472). The most common genetic subgroup (m.3243A>G mutation) had a height SD of -0.70 (95% CI, -0.85 to -0.54; n = 234) and a BMI SD of 0.12 (95% CI, -0.10 to 0.34; n = 212). NMDAS scores were negatively correlated with height SD (r = -0.25; 95% CI, -0.33 to -0.17; P < 0.001, n = 533). Rate of disease progression also correlated negatively with adult height (P < 0.001). CONCLUSION: Final height in mitochondrial disease reflects disease severity and rate of disease progression. Mitochondrial dysfunction and associated subclinical comorbidities affect growth plate physiology.

Phenotypic heterogeneity in m.3243A>G mitochondrial disease: The role of nuclear factors.

Objective: The pathogenic mitochondrial DNA m.3243A>G mutation is associated with a wide range of clinical features, making disease prognosis extremely difficult to predict. We aimed to understand the cause of this heterogeneity. Methods: We examined the phenotypic profile of 238 adult m.3243A>G carriers (patients and asymptomatic carriers) from the UK MRC Mitochondrial Disease Patient Cohort using the Newcastle Mitochondrial Disease Adult Scale. We modeled the role of risk factors for the development of specific phenotypes using proportional odds logistic regression. As mitochondria are under the dual control of their own and the nuclear genome, we examined the role of additive nuclear genetic factors in the development of these phenotypes within 46 pedigrees from the cohort. Results: Seizures and stroke-like episodes affect 25% and 17% of patients, respectively; more common features include hearing impairment, gastrointestinal disturbance, psychiatric involvement, and ataxia. Age, age-adjusted blood heteroplasmy levels, and sex are poor predictors of phenotypic severity. Hearing impairment, diabetes, and encephalopathy show the strongest associations, but pseudo-R2 values are low (0.14-0.17). We found a high heritability estimate for psychiatric involvement (h2=0.76, P = 0.0003) and moderate estimates for cognition (h2=0.46, P = 0.0021), ataxia (h2 = 0.45, P = 0.0011), migraine (h2 = 0.41, P = 0.0138), and hearing impairment (h2 = 0.40, P = 0.0050). Interpretation: Our results provide good evidence for the presence of nuclear genetic factors influencing clinical outcomes in m.3234A>G-related disease, paving the way for future work identifying these through large-scale genetic linkage and association studies, increasing our understanding of the pathogenicity of m.3243A>G and providing improved estimates of prognosis.

mtDNA heteroplasmy level and copy number indicate disease burden in m.3243A>G mitochondrial disease.

Mitochondrial disease associated with the pathogenic m.3243A>G variant is a common, clinically heterogeneous, neurogenetic disorder. Using multiple linear regression and linear mixed modelling, we evaluated which commonly assayed tissue (blood N = 231, urine N = 235, skeletal muscle N = 77) represents the m.3243A>G mutation load and mitochondrial DNA (mtDNA) copy number most strongly associated with disease burden and progression. m.3243A>G levels are correlated in blood, muscle and urine (R2 = 0.61-0.73). Blood heteroplasmy declines by ~2.3%/year; we have extended previously published methodology to adjust for age. In urine, males have higher mtDNA copy number and ~20% higher m.3243A>G mutation load; we present formulas to adjust for this. Blood is the most highly correlated mutation measure for disease burden and progression in m.3243A>G-harbouring individuals; increasing age and heteroplasmy contribute (R2 = 0.27, P < 0.001). In muscle, heteroplasmy, age and mtDNA copy number explain a higher proportion of variability in disease burden (R2 = 0.40, P < 0.001), although activity level and disease severity are likely to affect copy number. Whilst our data indicate that age-corrected blood m.3243A>G heteroplasmy is the most convenient and reliable measure for routine clinical assessment, additional factors such as mtDNA copy number may also influence disease severity.