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Mitochondrial DNA m.3243A>G heteroplasmy affects multiple aging phenotypes and risk of mortality
Mitochondria contain many copies of a circular DNA molecule (mtDNA), which has been observed as a mixture of normal and mutated states known as heteroplasmy. Elevated heteroplasmy at a single mtDNA site, m.3243A>G, leads to neurologic, sensory, movement, metabolic, and cardiopulmonary impairments. We measured circulating mtDNA m.3243A>G heteroplasmy in 789 elderly men and women from the bi-racial, population-based Health, Aging, and Body Composition Study to identify associations with age-related functioning and mortality. Mutation burden for the m.3243A>G ranged from 0-19% and elevated heteroplasmy was associated with reduced strength, cognitive, metabolic, and cardiovascular functioning. Risk of total, dementia and stroke mortality was significantly elevated for participants in the highest tertiles of m.3243A>G heteroplasmy. These results indicate that the accumulation of a rare genetic disease mutation, m.3243A>G, manifests as several aging outcomes and that some diseases of aging may be attributed to the accumulation of mtDNA damage.
Within each mitochondrion there are thousands of maternally inherited loops of mtDNA coding for mitochondrial genes critical to oxidative phosphorylation (or OXPHOS). Each of these loops can contain either normal or mutated DNA, a mixture known as heteroplasmy1. Studies of elderly populations demonstrate an age-related increase in heteroplasmic load due to mtDNA mutations and rearrangements in heart, central nervous system, brain and skeletal muscle post-mitotic tissues. An increased heteroplasmic load leads to reduced OXPHOS enzymatic activity and may be responsible for age-related functional decline 2 3 . The greatest impact of this bioenergetics defect has been shown in organs with high energy requirements such as skeletal muscle, retina, auditory neuroepithelia, brain and heart 4, 5, 6, 7, 8.
Because mitochondrial function is cell-type specific, a single mtDNA mutation may lead to a variety of mitochondrial diseases, depending on the tissue in which the mutation is expressed 9. Bioenergetic impairment of mitochondria through high heteroplasmic load (>80% burden of a pathogenic mtDNA mutation) may play a large role in disease initiation or progression 10. We previously reported associations between elevated mtDNA heteroplasmy levels and reduced neurosensory and mobility function in older persons 11. Average heteroplasmy levels among 20 candidate mutations detected in this earlier study of elderly participants ranged from 5-32% 11.
Mitochondrial diseases resulting from mtDNA mutations often involve dysfunctions across multiple functional domains, including: neurologic, sensory, movement, metabolic, and cardiopulmonary outcomes 10. Among the most thoroughly studied and best characterized of these pathogenic mutations is m.3243A>G, which causes several mitochondrial diseases and physiological dysfunctions, including: Mitochondrial Encephalopathy, Lactic Acidosis, Stroke-like episodes (MELAS), Mitochondrial Myopathy, Leigh syndrome, Chronic Progressive External Ophthalmoplegia (CPEO), Maternally Inherited Deafness and Diabetes (MIDD), hypertrophic cardiomyopathy (HCM), kidney dysfunction, migraine, bowel dysmotility, muscle stiffness, and diabetes 10. Heterogeneity in the phenotypic expression related to m.3243A>G mutation ranges from mild to severe symptoms (e.g. mild deafness to stroke-like episodes) 12, 13.
To date, m.3243A>G heteroplasmy has not been examined for clinical associations in an aging population. In the current study we quantified m.3243A>G heteroplasmy in a community-based cohort of men and women over age 70 years old and tested associations with age-related functioning and mortality.
A total of 789 Health ABC participants of African and European ancestry yielded complete mtDNA sequences for analysis, analysis including 371 men and 418 women aged 74.1 (mean) ±2.9 (SD) years. Baseline values for measures of strength, movement, and cognitive, metabolic, and cardiovascular function are detailed in Table 1. Numbers for mortality events are also detailed in Table 1. Mean sequencing coverage for m.3243A>G was 961X with a lower limit of 82X. Heteroplasmy at m.3243A>G ranged from 0-19% with a mean (SD) of 5.55% (3.6) and a median of 5.11%. Heteroplasmy detected in this study is comparable to that from previous chip- 14 and NGS-based 15 platforms.
We identified statistically significant cross-sectional associations with one measurement from each of the subsets of 9 phenotypes examined (strength and movement, cognitive-, metabolic-, and cardiovascular-function). The linear associations presented herein achieved nominal significance (p<0.05): grip strength (p=0.02), Digit Symbol Substitution Test (DSST) (p=0.04), fasting insulin (p=0.04), and pulse wave velocity (PWV) (p=0.008). No statistically significant associations with other phenotypes examined in this study: 400 meter (m) walk (p=0.94), Modified Mini-Mental State Examination (3MS) (p=0.49), fasting glucose (p=0.90), Systolic blood pressure (SBP) (p=0.30), and resting heart rate (RHR) (p=0.13).
We further examined tertiles of m.3243A>G heteroplasmy for associations with grip strength, DSST score, fasting insulin levels, and PWV (Table 2, Figure 1). Tertiles of m.3243A>G heteroplasmy (%) included: low (0.0-≤4%), middle (>4-≤6%), and high (>6-≤19%). Grip strength (p=0.027) and DSST score (p=0.009) were significantly higher for participants in the lowest tertile when compared with those in the middle and high tertiles. Fasting insulin levels were significantly lower (p=0.006) for participants in the lowest tertile when compared with those in the middle and highest tertiles. PWV was significantly lower (p=0.032) for participants in the lowest tertile when compared with those in the highest tertile.
Increased risks of all-cause (p=0.046), dementia (p=0.02), and stroke (p=0.05) mortality were observed for participants in the highest tertile of heteroplasmy when compared with those in the lowest tertile (Table 3). Kaplan-Meier curves demonstrate the cumulative incidence of all-cause (Figure 2), dementia (Figure 3), and stroke (Figure 4) mortality. No statistically significant associations with cardiovascular (p=0.32) and cancer (p=0.16) mortality were identified.
Here we present novel findings on the role of sub-clinical levels of m.3243A>G heteroplasmy in an elderly population. Elevated m.3243A>G levels were associated with significantly impaired strength, cognition, cardiovascular, metabolic function, and mortality consistent with the diverse mitochondrial diseases and phenotypes typically associated with the m.3243A>G heteroplasmic load 10, 12, 13, 16, 17. In the present study of non-disease presenting participants, circulating m.3243A>G heteroplasmy ranged from 0-19% with clear impairment at the highest tertiles. In the context of mitochondrial diseases where m.3243A>G mutation burden can reach levels over 90%, variation in the intracellular percentage of m.3243A>G mutation burden is associated with phenotypic heterogeneity and severity 18, 19. This previous work has shown that m.3243A>G heteroplasmy levels of 10–30% are associated with type I and type II diabetes and autism; 50–90% heteroplasmy levels are associated with encephalomyopathies including MELAS; and 90– 100% heteroplasmy levels lead to Leigh syndrome or perinatal lethality 18.
The biochemical and physiological consequences of the m.3243A>G mutation have been extensively studied using in vitro cybrid models, with the majority of studies using cybrids with high heteroplasmic loads. King (25) created cybrids from clinically symptomatic MELAS patients with m.3243A>G mutation loads at >95%. These cybrids exhibited a severe respiratory chain deficiency and oxygen consumption of the mutant cybrid lines was 74% of the wild-type cybrids. The cybrids in the King study also demonstrated a decrease in the synthesis of mtDNA encoded mitochondrial proteins (especially COX). In a similar cybrid study 20, the mutation produced a severe respiratory chain defect which was shown to be caused by impaired assembly of the mtDNA-encoded OXPHOS complexes ( I, III, IV and V). Ouweland et al. 21 found that cybrids with a 98% load showed higher lactic acid production rate and impaired cellular respiration. Yasukawa et al (2008) attributed reduced OXPHOS activity in cybrids to mistranslation of OXPHOS proteins by mutant tRNA (deficient in uridine modification at the wobble position). Yasukawa et al (2008) also demonstrated that respiratory deficiency caused by this mutation is not likely due solely to decreased protein synthesis (which was moderate) but due to much lower mtDNA OXPHOS enzymatic activity. This study also showed that respiratory impairment was attributed to lack of protein assembly into respiratory chain complexes as well as reduced enzymatic activity of these assembled complexes.
More recent in vitro cybrid studies examining lower m.3243A>G mutation loads have also shown biochemical impacts. Cybrid cells harboring 20% of the m.3243A>G mutation showed a 25% reduction in cell volume, rounded mitochondria, and elevated mtDNA density when compared with cybrids carrying only wild-type mtDNAs 18. Among cybrids carrying the m.3243A>G mutation, the proportion of cellular transcripts of mtDNA origin differed as did mRNA:tRNA ratio. The proportion of cellular transcripts of mtDNA origin was 23.0% for wild-type cybrids as compared with 9.7% for cybrids carrying 20% m.3243A>G mutant mtDNAs 18. In comparison to wild-type cybrids where tRNAs made up 1.5% of the mitochondrial transcriptome, tRNAs made up 3.0% of the mitochondrial transcriptome in cybrids carrying 20% m.3243A>G mutant mtDNAs 18. Variation in mtDNA m.3243A>G mutation burden also activates cellular metabolic pathways differentially. For example, at 20-30% m.3243A>G heteroplasmy levels mitochondrial SOD2 and mitochondrial chaperones are induced while mTOR and growth signaling pathways are suppressed.
In vivo measures show that MELAS patients can have lower, tissue-specific, mutation loads (31-40%) 21, 22. Functional, biochemical, and structural effects of them.3243A>G mutation have been demonstrated in skeletal muscle 23, 24, 25, 26, 27, neurons 28 , and cerebral vasculature 28. Although cybrid studies find respiratory impairment only at high levels of heteroplasmy, >95% 18, 29, respiratory chain dysfunction has been reported in skeletal muscle 30 and brain 31 samples carrying m.3243A>G mutation levels under 10%. It has been proposed, however, that that cybrids, with their aneuploid and genetically unstable nature, may not show an accurate picture of clinical pathology 20. Nevertheless, the results presented here support the idea that respiratory chain impairment can occur at low levels of heteroplasmy.
Numerous studies have demonstrated biochemical, transcriptional, and structural perturbations resulting from the m.3243A>G mutation involving muscular, neuronal and vascular brain effects which may explain associations with all-cause, dementia, and stroke mortality. The m.3243A>G mutation is located in the tRNAleu gene and also occurs in a specific region responsible for regulation and control of transcription termination at the end of the 16S rRNA gene 32, 33. The m.3243A>G mutation produces quantitative differences in processing of the tRNAleu which leads to amino acid misincorporation as well as impaired mitochondrial protein synthesis and assembly resulting in electron transport chain deficiency 20, 29. In vitro studies 34 show that this mutation decreases life span of mutant tRNAleu resulting in lower overall steady state amounts. Biochemical studies of muscle in MELAS have shown isolated defects of complex I 23 as well as combined defects of complexes I, III, and IV 24, 25. The m.3243A>G mutation is also associated with cytochrome c oxidase deficient muscle fibers and the focal accumulation of m.3243A>G causes significant impairment of mitochondrial function in individual cells 26. Computed tomography (CT) and electromyography of skeletal muscle show structural abnormalities and prominent muscle weakness in m.3243A>G carriers 27. Levels of the pathogenic m.3243A>G mutation increase with age in the human putamen 35 and the stroke-like episodes in MELAS involve defects in both neuronal metabolism and cerebral vasculature 28. This widespread cellular dysfunction observed in MELAS results from high m.3243A>G mutation burden across neuronal, endothelial, and smooth muscle cells of cerebral blood vessels 28.
A low-level of mtDNA heteroplasmy is commonly found in human populations. Despite protective maternal mechanisms intended to minimize the transmission of mutated mtDNA36, 37, heteroplasmy has been measured both in young children and during early adulthood. It remains uncertain whether the presence of heteroplasmy early in life is due to maternally transmitted mtDNA mutation1 or mutation acquisition during development 38 39 40. Hetroplasmic load may accrue without consequence in post-mitotic tissues until a tissue-dependent threshold in the proportion of normal to mutated mtDNA is breached and the cells become bioenergetically deficient 41. Within the same individual, specific tissues may vary considerably with respect to their heteroplasmy load and variant frequencies42, 43, 44. The mechanisms responsible for inconsistent loading of mitochondrial heteroplasmy among specific cells 38, 39, 40 and tissues is unknown; possibly genetic drift 45 or selection against a particular mutation 46, 47 plays a role.
A better understanding of the mechanisms driving the expansion of mtDNA mutations and
The current study has a number of strengths including the use of NGS sequencing and a platform designed specifically for complete mtDNA sequencing, and a large, community-based, well-characterized, biracial, longitudinal, cohort. Additionally, we were able to test our specific hypothesis that increased mtDNA heteroplasmy at the m.3234>G mutation, across multiple phenotypes, would be associated with impaired function consistent with known mitochondrial disease impairments previously associated with this single mutation.
Although the rate of mitochondrial heteroplasmy accumulation is unknown in this population, we associated the measurements taken in the clinic with the mtDNA collected on a same-day visit thus ensuring that mortality was prospective and that the associations reported here are cross-sectional. Although the observed effect sizes for individual clinical measures associated with heteroplasmy are only moderately clinically significant, the ability to identify predictors of functional decline is critical to refining the associations between future disease onset and these clinical measures (e.g. of strength with disability or DSST with dementia). A limitation to this study is its absence of independent replication; the lack of associations for a number of phenotypes may be attributed to a limitation on sample size and tissue types (e.g. phenotypic examination may not have included all relevant tissues). In order to confirm these findings, further population-level research including appropriate phenotypes, biospecimens, and design is necessary.
The Health ABC Study cohort is well-characterized and specifically designed for studies of aging-related impairments. Participants were generally healthy at the start of the study and it is likely that results from a single population may not be applied to all possible populations. These results indicate that the accumulation of a rare genetic disease mutation manifests as several aging outcomes and that some diseases of aging may be attributed to the accumulation of mtDNA damage. With further validation, measures of circulating mtDNA heteroplasmy may prove to be a valuable biomarker for identifying at-risk individuals who may benefit from early mitochondrial health interventions as well as for monitoring patients receiving mitochondrial therapies.
The Health, Aging, and Body Composition (Health ABC) Study is a prospective cohort of 3,075 community-dwelling men and women aged 70–79 years at recruitment in 1996-1997 and living in Memphis, TN, or Pittsburgh, PA. Participants were recruited within designated zip code areas from a random sample of white and black Medicare-eligible individuals. Participants had to be free of life-threatening cancer diagnoses and report no difficulty with the following activities of daily living: climbing 10 steps without resting or walking a quarter of a mile. Of the 3,075 participants, 51% were female and 41% were black. All participants signed written informed consents approved by the institutional review boards at the clinical sites (University of Tennessee Health Science Center, Memphis and University of Pittsburgh) and the coordinating center (University of California, San Francisco). All research was performed in accordance with relevant guidelines and regulations of the institutional review boards at the clinical sites and the coordinating center.
Mitochondrial DNA sequencing
A total of 794 Health ABC participants were identified for mtDNA sequencing. Genomic DNA was extracted from buffy coat collected using PUREGENE® DNA Purification Kit from samples collected at the baseline visit (1997-1998). The entire mtDNA was sequenced using the Ovation® Human Mitochondrion Target Enrichment System (NuGEN, San Carlos, CA) on the Illumina MiSeq NGS platform. Briefly, DNA samples are first fragmented and end-repaired.
Barcoded sequencing adaptors are next ligated to the 5’ ends of the fragmented DNA and samples are combined for probe annealing and extension. Target enrichment prior to sequencing is accomplished with probes designed to independently target each strand of the mtDNA resulting in an enriched mtDNA library. After NGS sequencing, heteroplasmy was quantified for m.3243A>G by counting the number of reads for each of the ‘G’ minor allele (MA) and ‘A’ reference allele (RA) and calculated as MA/(MA+RA).
Strength and Mobility
Grip strength measured by handheld Jamar dynamometer 55 and a timed walk of 400 meter (m) were assessed at the first clinical visit.
Cognitive Function Testing
Two cognitive function tests were assessed among participants at the baseline clinical visit: Digit Symbol Substitution Test (DSST) and Modified Mini-Mental State Examination (3MS). DSST measures executive cognitive function 56, 57 and is calculated as the total number of items correctly coded in 90 seconds. 3MS is a general cognitive battery 58 with possible scores ranging from 0 to 100. Higher DSST and 3MS scores indicate better cognitive functioning.
Fasting insulin and glucose were measured at the first clinical visit. Fasting insulin (uIU/mL) was measured via Microparticle Enzyme Immunoassay; Abbott Laboratories Diagnostics Division, South Pasadena, CA. Fasting glucose (mg/dL) was measured using Vitros Glucose; Johnson & Johnson; Rochester, NY USA.
Systolic blood pressure (SBP), resting heart rate (RHR), and pulse wave velocity (PWV) were measured at the first clinical visit. Sitting SBP was computed as the average of 2 measurements in millimeters of mercury. RHR (beats/minute) was automatically measured using a 12‐lead electrocardiogram. PWV (cm/s), a measure of arterial stiffness, was assessed transcutaneous Doppler flow probes; Parks Medical Electronics, Aloha, OR.
Associations between m.3243A>G heteroplasmy and cognitive, movement and strength, cardiovascular, and metabolic function were examined using linear regression. Nominally significant linear associations (p<0.05) among continuous predictor and outcome variables were further compared among tertiles of m.3243A>G heteroplasmy using ANOVA and general linear models were used to test differences among tertiles of heteroplasmy.
Vital status and cause of death were confirmed according to death certificates and hospital discharge summaries (when available) over an average of 12.7 years of follow-up. Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals [CIs] among tertiles of heteroplasmy for all-cause and cause-specific mortality. Kaplan-Meier curves were used to assess the cumulative incidence of total and cause-specific mortality. All association and survival analyses were adjusted for age, sex, race, and clinic site using SAS version 9.4 (SAS Institute Inc, Cary, NC).
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Table 1. Baseline characteristics among 789 sequenced Health ABC. Number of mortality events for a mean of 12.7 years of follow-up.
|Baseline age, years||74.1 (2.9)|
|Modified Mini-Mental State Examination (3MS)||91.1 (5.4)|
|Digit Symbol Substitution Test (DSST)||33.8 (14.2)|
|400m walk speed (m/s)||1.24 (0.21)|
|Grip Strength (kg)||29.3 (10.5)|
|Systolic blood pressure (mm Mercury)||136.5 (21.4)|
|Pulse wave velociy (cm/sec)||930.5 (421.9)|
|Resting heart rate (beats/min)||67.7 (10.7)|
|Fasting glucose (uIU/mL)||104 (30.8)|
|Fasting insulin (uIU/mL)||8.6 (8.36)|
|Mortality events (mean follow-up, 12.7 years)||n (%)|
Table 2. Cox proportional hazards models estimating hazard ratios and 95% confidence intervals among tertiles of m.3243 A>G heteroplasmy for total and cause-specific mortality. All analyses were adjusted for age, sex, race, and clinic site.
|m.3243 A>G heteroplasmy|
|Grip Strength (kg)||1.00||1.18 (0.94-1.47, p=0.16)||1.25 (1.01-1.56, p=0.046)|
|Digit Symbol Substitution Test Score||1.00||2.11 (0.85-5.26, p=0.11)||2.43 (1.00-5.97, p=0.05)|
|Fasting insulin (uIU/mL)||1.00||1.40 (0.88-2.23, p=0.17)||1.49 (0.96-2.32, p=0.08)|
|Pulse wave velociy (cm/sec)||1.00||1.30 (0.78-2.15, p=0.32)||1.28 (0.77-2.13, p=0.35)|
Grip strength (kg) was significantly higher (p=0.027) for participants in the lowest tertile (31.8±0.43), when compared with those in the middle (30.9±0.43), and high (30.4±0.43) tertiles. DSST score was significantly higher (p=0.009) for participants in the lowest tertile (35.4± 0.72) when compared with those in the middle (33.1±0.73) and highest (33.0±0.73) tertiles. Fasting insulin levels (uIU/mL) were significantly lower (p=0.006) for participants in the lowest tertile (7.65± (0.55) when compared with those in the middle (8.29±0.55) and highest (9.81±0.55) tertiles. PWV (cm/s) was significantly lower (p=0.032) for participants in the lowest tertile (890±27.8) when compared with those in the highest tertile (976±28.6).
Table 3. Cox proportional hazards models estimating hazard ratios and 95% confidence intervals among tertiles of m.3243 A>G heteroplasmy for total and cause-specific mortality. All analyses were adjusted for age, sex, race, and clinic site.
|m.3243 A>G heteroplasmy|
|Total mortality, HR (95% CI)||1.00||1.18 (0.94-1.47, p=0.16)||1.25 (1.01-1.56, p=0.046)|
|Dementia, HR (95% CI)||1.00||1.58 (0.89-2.82, p=0.12)||1.96 (1.11-3.44, p=0.02)|
|Stroke, HR (95% CI)||1.00||2.11 (0.85-5.26, p=0.11)||2.43 (1.00-5.97, p=0.05)|
|Cancer, HR (95% CI)||1.00||1.40 (0.88-2.23, p=0.17)||1.49 (0.96-2.32, p=0.08)|
|CVD, HR (95% CI)||1.00||1.30 (0.78-2.15, p=0.32)||1.28 (0.77-2.13, p=0.35)|
Figure 1. Mitochondrial m.3243A>G association with grip strength (linear regression p=0.018), digit symbol substitution test score (linear regression p=0.04), fasting insulin (linear regression p=0.04), and pulse wave velocity (PWV, linear regression p=0.008) compared across tertiles of m.3243A>G heteroplasmy. Values adjusted for age, sex, race, and clinic site.
Figure 2. Mitochondrial m.3243A>G association with total mortality. Survival was compared across tertiles of m.3243A>G heteroplasmy. Values adjusted for age, sex, race, and clinic site.
Figure 3. Mitochondrial m.3243A>G association with dementia-related mortality. Survival was compared across tertiles of m.3243A>G heteroplasmy. Values adjusted for age, sex, race, and clinic site.
Figure 4. Mitochondrial m.3243A>G association with stroke-related mortality. Survival was compared across tertiles of m.3243A>G heteroplasmy. Values adjusted for age, sex, race, and clinic site.