Novel MTND1 mutations cause isolated exercise intolerance, complex I deficiency and increased assembly factor expression

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  We sought to assess in vivo and vitro mitochondrial function and evaluate the molecular mechanisms underlying a purely muscular phenotype in two adults whose clinical pictures were dominated by progressive exercise intolerance, lactic acidaemia and severe isolated mitochondrial CI deficiency in muscle.

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  We describe the detailed clinical, physiological, biochemical and molecular characterization of these patients who we show to harbour novel heteroplasmic mutations in the mtDNA-encoded MTND1 gene.

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  We demonstrate for the first time that mitochondrial super-complex reorganization occurs as a response to a compensatory mechanism to partially extricate the clinical phenotype involving up-regulation of CI assembly factors, a phenomenon not previously reported with MTND1 mutations.

Isolated deficiencies of human complex I (CI) often cause severe early-onset progressive disease, but the spectrum of disorders associated with this biochemical abnormality is expanding. In particular, mutations in the ND1 subunit of CI [MIM #252010] are associated with Leigh syndrome [MIM #256000]; cardiomyopathy; epilepsy; encephalopathy; mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) [MIM #54000]; Leber hereditary optic neuropathy (LHON) [MIM #535000] and an overlap syndrome comprising clinical features of both LHON and MELAS [1–7]. Exercise intolerance is a common symptom of mitochondrial disorders that can occur in isolation or as part of a multi-system disorder and has been associated with mutations in many genes encoding subunits of various complexes [8–11]. However, the relationship between the pathogenic mtDNA mutation and the biochemical and phenotypic expression of the defect remains poorly understood.

In the present paper, we describe two unrelated adult patients with severe isolated CI deficiency in skeletal muscle, progressive exercise intolerance, myopathy (without cardiomyopathy) and persistent lactic acidaemia. Both patients harbour novel heteroplasmic MTND1 (NADH dehydrogenase subunit 1) gene [MIM #516000] mutations. We have characterized VO₂ (oxygen uptake) kinetics during graded aerobic exercise, assessed in vivo mitochondrial function using phosphorus MR spectroscopy and evaluated the molecular mechanisms underlying this purely muscular phenotype to understand the impact of both mutations on CI biogenesis.

Local study approval was granted (NRES Committee North East- Newcastle & North Tyneside 1) and written informed consent from both patients was obtained prior to study inclusion. All clinical investigations were evaluated according to the Declaration of Helsinki.

Patient 1 presented at age 16 years with mild exercise intolerance and prominent fatigue following a viral illness. She was diag-nosed with chronic fatigue syndrome. At age 25 years she presented with progressive exertion-related dyspnoea and palpitations and was provisionally diagnosed with asthma. By 28 years of age, muscle weakness and fatigue with exercise-induced headache, vomiting, cardiac palpitations and syncope were prominent and a metabolic acidosis with elevated serum lactate was detected (Table 1). At this stage a neuromuscular opinion was sought. The clinical picture has progressed rapidly over the last 2 years with exercise tolerance reduced to less than 50 m. She has developed alcohol intolerance and postural orthostatic tachycardia syndrome (POTS). Patient 2 presented at age 22 years of age to a neurologist with life long indolent exertion-related muscle weakness and pain, dyspnoea, cardiac palpitations and syncope. There was no family history of muscle disease or parental consanguinity in either case.

Cardiopulmonary exercise testing (CPET) was performed in both subjects as described elsewhere [12]. Anaerobic threshold (AT) was determined using the V-slope method, as previously described [13].

Muscle phosphorus magnetic resonance spectroscopy (³¹P-MRS) was performed on the left calf, as detailed previously, in Patient 1 to evaluate in vivo mitochondrial function relative to an age matched reference group [14].

Standard histological [H&E (haematoxylin and eosin), modified Gomori trichrome] and histochemical analyses of skeletal muscle biopsies were performed on fresh-frozen skeletal muscle sections (10 μm) [15]. Mitochondrial respiratory chain complex activities were determined in skeletal muscle homogenates and expressed relative to the activity of the matrix marker enzyme citrate synthase [16].

Total DNA was extracted from available tissues including muscle, blood, buccal epithelia, urinary sediments, cultured myoblasts and fibroblasts. Muscle mtDNA rearrangements were investigated by long-range PCR strategies [17]. Direct sequencing of the entire mtDNA genome was undertaken [18]; alignment and variant calling were performed using SeqScape software (v2.1.1, Applied Biosystems) comparing changes to the GenBank reference sequence for human mtDNA (accession number NC_012920.1).

Heteroplasmic levels of the m.3365T>C and m.4175G>A mutations were determined in homogenate tissue and individual laser-microcaptured COX-positive and COX-positive ragged-red fibres (RRFs) by quantitative pyrosequencing. Quantification of the heteroplasmy level of each variant was achieved using Pyromark Q24 software [19].

Complex subunit immunohistochemistry was carried out on frozen tissue sections as described previously [20]. Primary antibodies and their dilutions used were: CI ND1, 1:200 (gift from Dr Anne Lombes), CI NDUFB8 1:50, CII SDHA (succinate dehydrogenase complex subunit A; 70 kDa flavoprotein subunit) 1:1000, CIII UQCRC2 (ubiquinone: cytochrome c reductase core protein 2) (Core 2) 1:1000, CIV MTCO1 (mitochondrial cytochrome c oxidase subunit 1) 1:1000, CIV MTCO4, 1:1000, Porin, 1:1000; all commercially available antibodies were purchased from Abcam. Primary antibodies were detected using a polymer detection system (Menarini Diagnostics) as per the manufacturer's instructions and visualized with 3,3′-diaminobenzidine (DAB; Sigma).

For BN (blue native)/PAGE, frozen skeletal muscle (10–50 mg) was homogenized and solubilized with 2% dodecylmaltoside (individual complexes and smaller super-complexes) or 4% digitonin (super-complexes) [21] and electrophoresed through precast 4–16% (dodecylmaltoside) or 3–13% gels (Invitrogen). Activities of CI, CII and CIV were estimated by in-gel assays as described in [22]. For 2D analysis, 1D lanes were incubated for 15 min in 1% SDS and 1% β-mercaptoethanol and separated in a 16% Tricine/SDS gel (BN-SDS/PAGE). For SDS/PAGE, proteins were extracted from enriched membranes (muscle) and separated by standard SDS/PAGE procedures. Proteins were transferred onto PVDF membranes (GE Healthcare) and processed for immunoblotting. Primary antibodies used were: NDUFB8 [NADH dehydrogenase (ubiquinone) 1β sub-complex 8], NDUFA9 [NADH dehydrogenase (ubiquinone) 1α sub-complex 9], Core2 (complex III Core protein 2), COXI, COXII, SDHA, CV subunits, TOM20 (translocase of outer mitochondrial membrane 20 kDa subunit) and porin (MitoSciences); AFG3L2 (AFG3-like AAA ATPase 2; Proteintech); LonP1 (Lon peptidase 1) and NDUFAF3 [NADH dehydrogenase (ubiquinone) complex I assembly factor; Sigma Life Science]; HSP60 (heat shock protein 60; BD Biosciences); NDUFAF1 and NDUFAF2 (gifts from Professor Leo Nijtmans) and ND1 (gift from Dr A. Lombes). Antibodies were detected with appropriate horseradish peroxidase (HRP)-conjugated immunoglobulins (Dako) and chemiluminescence detection reagents (GE Healthcare).

Total RNA from control (n=4) and patient muscle was extracted using TRIrizol Reagent (Life Technologies) and reverse transcribed (0.5–1 μg) with random hexamers (Superscript III First Strand Synthesis System, Life Technologies). Levels of mRNA expression of NDUFAF1 [MIM #606934], NDUFAF2 [MIM #609653], NDUFAF3 [MIM #612911] and ACTB (β-actin) [MIM #102630] were assessed in triplicate by real-time PCR using commercially-available TaqMan assays (assay IDs Hs00211245_m1, Hs02380072_u1, Hs00404252_g1 and Hs01060665_g1 respectively; Life Technologies). After normalization to ACTB, average expression levels of the CI assembly factor genes in patients were reported relative to control values, with differences assessed using a two-tailed Student's t test.

Patient 1 was of normal intelligence (Wechsler test of adult reading: thirty-seventh percentile), normal stature (1.7 m) and body habitus (68.7 kg). She had no ptosis and eye movements were normal. She did not have significant demonstrable clinical weakness at rest [Medical Research Council (MRC) 5/5]; however, subtle upper and lower limb proximal weakness (MRC 4+/5) was evident following minimal exertion (marching in place ten times). She also displayed marked evidence of increased ventilator rate (>30 breaths/min), sinus tachycardia (>120 beats/min) and bluish-hue discolouration to her digits. There was no evidence of a dysautonomia and no cardiac or respiratory muscle involvement. There was a persistent metabolic acidosis (serum bicarbonate 9–19 mmol/l; normal range 22–29 mmol/l), a high random serum lactate concentration [7.2–8.9 mmol/l; normal range < 1.7 mmol/l), markedly increasing post exertion up to 15.5 mmol/l and normal total creatine kinase activity (52–76 units/l; normal range <200 units/l). At the age of 27 years, there was electrophysiological evidence of a significant proximal myopathy with no findings of neuropathy. Conventional echocardiogram and cardiac MRI revealed no cardiac muscle abnormalities. She was treated with riboflavin (50 mg three times a day), high dose co-enzyme Q₁₀ (500 mg/day) and L-carnitine supplements with minimal symptomatic or objective improvement. Current treatment strategies include energy conservation with the use of a wheelchair and pharmacological management with ivabradine for POTS.

Patient 2 was of normal height (1.68 m) but thin body habitus (47 kg). He had subtle unilateral ptosis and eye movements were normal. His skeletal muscles were generally poorly developed but he did not have significant demonstrable clinical weakness at rest (MRC 5/5); however, subtle upper and lower limb proximal weakness (MRC 4+/5) was evident following moderate exertion (marching in place 20 times). There was no evidence of cardiac or respiratory muscle involvement. There was a persistently high resting serum lactate concentration (4.1–6.8 mmol/l; normal range <1.7 mmol/l), increasing post exertion [15.4 mmol/l; cycling for 5 min; peak power 53W; peak oxygen consumption (VO₂ peak) 14 ml/min per kg; AT 22% predicted VO₂ peak] and raised total creatine kinase activity (351–486 units/l; normal range < 320 units/l). Electrophysiological changes at the age of 11 years were compatible with a myopathy with no findings of neuropathy.

VO₂ peak, peak work capacity (power; W), AT and absolute AT (expressed as a percentage of predicted peak VO₂; ATVO₂), were markedly decreased in both subjects (Table 1). Plasma lactate concentrations were concomitantly raised during CPET (Patient 1: resting lactate: 4.4 mmol/l, peak lactate: 13.2 mmol/l; Patient 2: resting lactate: 6.8 mmol/l, peak lactate: 15.4 mmol/l), with slow lactate decay time to baseline (Patient 1: 60 min, Patient 2: 40 min).

³¹P-MRS examination showed that the ability of Patient 1’s muscle to reduce phosphocreatine (PCr) and accumulate ADP during exercise was normal. However, in recovery from muscle contraction, the main indicators of mitochondrial function demonstrated a severe impairment in oxidative metabolism (Table 1).

Oxidative enzyme reactions [succinate dehydrogenase (SDH) and cytochrome c oxidase (COX)] revealed numerous fibres with increased activity at the fibre periphery, confirmed by modified Gomori trichrome staining which showed sub-sarcolemmal accumulations typical of ‘ragged-red’ changes affecting >25% of all fibres in both patients (Figure 1A). Mitochondrial respiratory chain enzyme studies revealed isolated CI deficiency in skeletal muscle from both patients (Table 1), including a repeat muscle biopsy obtained from Patient 1 taken 2 years after her initial diagnostic biopsy. The biochemical defect in this patient was not expressed in cultured myoblasts (results not shown). Muscle analyses of Patient 1, shown in the present study, were performed on the first muscle biopsy. The activities of all other respiratory chain complexes were normal, confirming an isolated CI deficiency (Table 1). This was further confirmed by muscle immunohistochemistry in Patient 1 in which expression of both mtDNA-encoded (ND1) and nuclear-encoded (NDUFB8) CI structural components was markedly decreased in or absent from a vast number (>80%) of all muscle fibres (see Figures 1B,i and 1B,ii); protein components of CII, CIII and CIV showed normal expression (Figure 1B).

Having excluded mtDNA rearrangements, we determined the mtDNA sequence in muscle from both patients identifying novel candidate pathogenic MTND1 mutations; Patient 1 harboured a novel m.3365T>C variant [predicting p.(Leu20Pro)] which was present at high levels of heteroplasmy (82% in biopsy 1; 86% in biopsy 2) in skeletal muscle, but undetectable in all other tissue samples tested (Figures 2A and 2B). Patient 2 was shown to harbour a novel m.4175G>A MTND1 variant predicting p.(Trp290*) and premature truncation of the ND1 protein. Quantitative pyrosequencing showed that the m.4175G>A mutation was present at high levels of heteroplasmy in skeletal muscle (90% mutant load); at low levels (5% mutant load) in a urinary sediment-derived DNA sample, but undetectable in all other DNA samples (Figure 2C). Concurrent studies in the mothers of both patients failed to detect the respective mtDNA mutation suggesting de novo mutation events.

The m.3365T>C and m.4175G>A mutations were not previously reported on online databases of pathogenic mtDNA mutations [23,24] or within our own database of >980 human mtDNA sequences. Using quantitative pyrosequencing, we detected a trend towards higher levels of the m.3365T>C mutation in RRF (72.2±24.4%, n=10) than non-RRF (56.5±28.8%, n=10, P=0.2049, two-tailed Student's t test). For the m.4175G>A mutation, significantly higher levels of mutation were detected in COX-positive RRF (89.0±24.2% n=21) than in COX-positive non-RRF (39.6±44.2%, n=11) (P=0.0025, two-tailed Student's t test), confirming segregation of the m.4175A genotype with a histopathological abnormality.

The m.3365T>C; p.(Leu20Pro) mutation is located within the first transmembrane domain of the ND1 protein, whereas the m.4175G>A; p.(Trp290*) mutation occurs within the hydrophilic loop that faces the mitochondrial matrix between transmembrane domains G and H (Figure 2D). Western blot analysis of muscle from both patients failed to detect ND1 protein and identified markedly decreased levels of the nuclear-encoded CI subunit NDUFB8, whereas steady-state levels of other respiratory chain complex structural subunits were unaffected (Figures 3A and 3B). BN/PAGE analysis with subsequent Western blot analyses showed a fully assembled CI could be detected in both patients, albeit at very low levels, whereas steady-state levels of fully-assembled CII, CIII and CIV appeared unchanged (Figure 3C). This observation was accurately reflected in the in-gel activity assays which indicated some residual CI activity (Figure 3D), in agreement with immunocytochemical studies (Figure 1B).

BN/PAGE, followed by Western blot and in-gel-activity analyses of control muscle samples, showed a small amount of fully assembled CI in its monomeric form, whereas the majority of assembled complex was associated with other complexes building the SC2 (supercomplex 2) [CI + CIII₂ (CIII dimer)] and SC3 (CI + CIII₂ + CIV_(1–4)) super-complexes (Figure 3E). Levels of fully assembled CI were strongly decreased in patient samples, with residual CI associated, in the most part, with CIII in the SC2 (CI+CIII₂) super-complex (Patient 1) and with CIV in the SC3 (CI + CIII₂ + CIV_(1–4)) super-complex (Patient 2; Figures 3E and 3F).

We evaluated the steady-state levels of different CI assembly factors including NDUFAF3 (early-stage factor), NDUFAF1 (involved in the middle stages) and NDUFAF2 (involved in the late stages of CI assembly) and mitochondrial proteases including AFG3L2, LonP1 and HSP60. We observed no changes in the expression levels of any proteases; however, steady-state levels of all three CI assembly factors were markedly increased in patient muscle samples suggestive of a compensatory response to the ND1 defect (Figure 4A). Confirmatory real-time PCR analy-ses showed increased NDUFAF1 [MIM #606934], NDUFAF2 [MIM #609653] and NDUFAF3 [MIM #612911] transcript levels in patient muscle (Figure 4B).

BN-SDS/PAGE analysis of digitonin-solubilized control and patient muscle samples showed that the majority of NDUFA9 was associated with the SC2 (CI + CIII₂) and SC3 (CI + CIII₂ + CIV_(1–4)) super-complexes in controls (Figure 4C). Both NDUFAF1 and NDUFAF2 were undetectable in control muscle. In the patient muscle samples, the majority of NDUFA9 was detected in association with the SC2 (CI + CIII₂) and SC3 (CI + CIII₂ + CIV_(1–4)) super-complexes. However, smaller sub-complexes were also observed in both patients. We found that NDUFAF1 was associated with two complex I intermediates of ∼500 and ∼800 kDa, whereas NDUFAF2 was detected within higher-molecular-mass associations corresponding to complexes of 800 and 1500 kDa in muscle of both patients (Figure 4C). In the patient samples, we observed higher amounts of CV in higher supramolecular organizations (as dimer V₂ and tetramer V₄; Figure 4C), whereas the majority of mt-ATP synthase was found as monomers in controls.

We describe novel sporadic MTND1 mutations in two unrelated patients with isolated CI deficiency characterized clinically by marked exercise intolerance and in vivo mitochondrial dysfunction. The clinical phenotypes, severity of the biochemical defect and the relatively indolent nature of the disease are conspicuously similar in both patients. Neurological examination was essentially normal in the resting state; however, response to exercise in both cases was indicative of a mitochondrial myopathy. Both cases highlight the importance of serum lactate testing in cases of persistent unexplained exertional weakness or dyspnoea. Indeed, mitochondrial disorders are recognized as a common cause of referral for unexplained dyspnoea, with a period prevalence of up to 8.5% [25].

Several strands of evidence indicate that the MTND1 mutations are pathogenic, causing the clinical phenotype. The m.3365T>C and m.4175G>A mutations are novel and absent from available databases of characterized mtDNA mutations, whereas both MTND1 variants were heteroplasmic and segregate with disease in muscle and, for Patient 2, with the histopathological abnormality (COX-positive RRF). The undetectable steady-state levels of ND1 protein suggest that both MTND1 mutations affect production and/or stability of the protein with dramatic effects on CI assembly and degradation of other CI subunits, including NDUFB8. Intriguingly, ND1 protein deficiency does not totally preclude assembly of the holocomplex, similar to that observed in a patient with progressive encephalopathy due to a B17.2L (NDUFAF2) [MIM #609653] null mutation [26].

Smaller CI subunits were also detectable with several intermediate complexes clearly seen in association with CI assembly factors. Accumulation of stalled intermediates of ∼500 kDa and ∼800 kDa has previously been reported in patients with various CI assembly pathway mutations who also exhibit reduced assembly of the holoenzyme complex [26–31]. Steady-state levels of the mitochondrial quality-control machinery including the AAA protease AFG3L2, LonP1 protease and HSP60 remained unchanged in association with up-regulation of assembly factor for intermediate-stage (NDUFAF1) and late-stage (NDUFAF2) CI assembly in patient muscle, suggesting a shift in this dynamic equilibrium state that favours assembly and stability of respiratory complex formation over degradation.

We speculate that mutation load and tissue-specific effects in CI formation and stability, mediated through assembly factor up-regulation, act as a compensatory response to the deleterious MTND1 genetic defect, with an absence of secondary defects in CII, CIII and CIV further facilitating complex stabilization. Taken together, our findings all support assembly factor up-regulation in isolated CI deficiency as an adaptive process to stabilize the respiratory complexes, a phenomenon not previously reported with MTND1 mutations [32] and that the pathogenic role of these mutations at a molecular level is dependent on the stage of CI assembly. Mitotic segregation has long been recognized to contribute to phenotypic variability that may explain the isolated muscle defects in these patients, although we concede that definitive exclusion of CNS involvement cannot be definitively made based on current evaluations.

In summary, we report two novel MTND1 mutations that clinically manifest as isolated exercise intolerance and at a molecular level cause assembly factor up-regulation and sub-complex assembly acting to stabilize respiratory chain complexes and salvage assembly of the holoenzyme complex.

³¹P-MRS

phosphorus magnetic resonance spectroscopy

ACTB

β-actin

AFG3L2

AFG3-like AAA ATPase 2

AT

anaerobic threshold

BN

blue native

CI/CII/CIII/CIV/CV

complex I/II/III/IV/V

CIII₂

CIII dimer

COX

cytochrome c oxidase

CPET

cardiopulmonary exercise testing

H&E

haematoxylin and eosin

HSP60

heat shock protein 60

LHON

Leber hereditary optic neuropathy

LonP1

Lon peptidase 1

MELAS

mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes

MRC

Medical Research Council

NDUFA9

NADH dehydrogenase (ubiquinone) 1α sub-complex 9

NDUFAF

NADH dehydrogenase (ubiquinone) complex I assembly factor

NDUFB8

NADH dehydrogenase (ubiquinone) 1β sub-complex 8

PCr

phosphocreatine

POTS

postural orthostatic tachycardia syndrome

RRF

ragged-red fibre

SDH

succinate dehydrogenase

SDHA

succinate dehydrogenase complex subunit A

VO₂

peak, peak oxygen consumption

VO₂

oxygen uptake

The study was conceptualized and designed by Grainne Gorman, Emma Blakely, Hue-Tran Hornig-Do, Doug Turnbull, Robert McFarland and Robert Taylor. Acquisition of data was performed by Grainne Gorman, Emma Blakely, Hue-Tran Hornig-Do, Helen Tuppen, Laura Greaves, Langping He, Angela Baker, Gavin Falkous, Jane Newman, Michael Trenell, Bryan Lecky, Richard K. Petty, Doug Turnbull, Robert McFarland and Robert Taylor. Analysis and interpretation of data was done by Grainne Gorman, Emma Blakely, Hue-Tran Hornig-Do, Helen Tuppen, Laura Greaves, Langping He, Angela Baker, Gavin Falkous, Jane Newman, Michael Trenell, Bryan Lecky, Richard K. Petty, Doug Turnbull, Robert McFarland and Robert Taylor. The manuscript was drafted by Grainne Gorman, Emma Blakely, Hue-Tran Hornig-Do, Helen Tuppen, Laura Greaves and Robert Taylor. Critical revision of the manuscript for important intellectual content was done by Grainne Gorman, Emma Blakely, Hue-Tran Hornig-Do, Helen Tuppen, Laura Greaves, Michael Trenell, Bryan Lecky, Richard K. Petty, Doug Turnbull, Robert McFarland and Robert Taylor. The study was supervised by Grainne Gorman, Robert McFarland and Robert Taylor.

We thank the patients who have participated in this study. We also thank Ms Sarah J. Dowson and Ms Charlotte Blackburn for excellent technical support.

This work was supported by the Wellcome Trust [grant number 096919Z/11/Z (to D.M.T. and R.W.T.)]; the Medical Research Council [grant numbers 0800674 and M501700 (to D.M.T., R.M.F. and R.W.T.)]; the U.K. National Institute for Health Research (NIHR) Biomedical Research Centre for Ageing and Age-Related Diseases award to the Newcastle upon Tyne Hospitals NHS Foundation Trust (to D.M.T. and G.S.G.); the U.K. NHS Highly Specialized Rare Mitochondrial Disease of Adults and Children Service; the Deutsche Forschungsgemeinschaft for a postdoctoral fellowship (to H.-T. H.-D.); the U.K. Senior Fellowship (to M.I.T.); the Newcastle NIHR Biomedical Research Centre; and the North Tyneside Comprehensive Local Research Network.