Case report

A 22-year-old woman was referred to our respiratory outpatient clinic to undergo a cardiopulmonary exercise test (CPET), following presentation with a 2-year history of progressive unexplained exertional dyspnoea (ED). Specifically, she described dyspnoea precipitated by walking or running, which gradually improved with rest. She denied chest pain, cough, asthenia or muscular weakness. Until the age of 20, she practised regular non-competitive physical activity.

The patient had at term natural childbirth and normal psychophysical development. Menstrual period, started at the age of 14 years, was regular; no pregnancy, nor miscarriage. She was a lifelong non-smoker with no history of illicit substance use or occupational exposure (university student). Her past history included multinodular euthyroid goitre for which she was prescribed regular levothyroxine (75 μg/100 μg on alternate days). She also reported intermittent gastrointestinal discomfort, which had been ascribed to lactose intolerance for which, however, she was not prescribed a lactose-free diet. Her family history revealed an uncle deceased during childhood for ‘cardiac arrhythmia’.

Cardiorespiratory and neurological examination was unremarkable. She was normotensive (blood pressure (BP) 100/60 mm Hg, heart rate (HR) 98 bpm in sinus rhythm), not tachypnoeic (respiratory rate 16 breaths/min) and had preserved oxygen saturation (SatO₂ 98%). Her body mass index was 19.3.

Pulmonary function tests showed normal flow indices (FVC 3.52 L—88% of pred; FEV₁ 3.35 L—96% of pred; Tiffeneau index FEV1/FVC 0.95) and preserved lung volumes and gas transfer (total lung capacity 3.94 L—90% of pred; diffusing capacity of the lungs for carbon monoxide, 96% of pred). A high-resolution chest tomography showed no sign of lung disease.

Cardiological (including rest, stress and Holter ECG, as well as cardiac ultrasounds) and neurological (including electromyography) exams showed no abnormalities.

Routine blood tests (cell count, glucose and electrolyte levels, iron and ferritin profile, lipid screening, kidney and liver function), erythrocyte sedimentation rate, C reactive protein and muscular enzyme (creatine kinase with isoenzymes) values were within normal ranges. Thyroid hormones (measured during replacement therapy) were normal: thyroid-stimulating hormone (TSH) 0.31 µUI/mL, FT₃ 2.94 pmol/L, FT₄ 13.8 pmol/L. Autoimmune screening, including antinuclear antibodies, extractable nuclear antigens, anti-double stranded DNA antibodies, anti-β₂ glycoprotein I antibodies, anticardiolipin antibodies, lupus anticoagulant and antineutrophil cytoplasmic antibodies (pANCa, cANCA), was negative.

An incremental load (10 W/min) maximal (peak respiratory exchange ratio (RER) 1.6) CPET was conducted on a cycle ergometer till exhaustion for prevalent muscular fatigue (10-point Borg scale for rating of perceived exertion (RPE)1 at peak exercise: dyspnoea 9/10, leg fatigue 10/10), in the absence of cardiorespiratory symptoms (table 1).

The test showed a severe reduction in exercise capacity (VO_2peak 704 mL O₂/min or 12.8 mL O₂/min/kg, 38% pred) with impaired maximal workload (60 W, 35% pred) and work efficiency (VO₂/W slope 5.4 L/W/min, 52% pred).

The cardiac response revealed high resting hearth rate (110 bpm), with a rapid depletion of the HR reserve (176 bpm, 89% of max HR pred at 60 W) and a low O₂ pulse_peak value (4.0 mL O₂/bpm, 40% pred). Values of BP were within normal ranges both at rest and at peak of exercise. No arrhythmias, ventricular repolarisation abnormalities and signs of myocardial ischaemia were seen.

The analysis of the respiratory pattern during exercise revealed impaired ventilatory efficiency (VE/VCO_2LT 42, VE/VCO_2slope 39) with normal VE_peak value (46.8 L/min, 38% of maximum voluntary ventilation (MVV)) and preserved breathing reserve at exercise cessation with no significant desaturation. Of notice, ventilatory equivalents for O₂ at peak exercise reached very high values (VE/VO_2peak 65). At peak exercise, end-tidal oxygen tension (PetO₂) was high (127 mm Hg), while end-tidal carbon dioxide tension (PetCO₂) was low (29 mm Hg).

The study of the metabolic profile was determined using combined assessment of ‘V-slope’,‘ventilatory equivalents’ and ‘end-tidal pressure’ methods and highlighted an early lactic threshold (27% pred; normal values >35%).

Overall, the reduced CPET exercise tolerance, in the presence of a progressive rise of RER throughout exercise and a reduced anaerobic threshold (AT), suggested early onset of lactic acidosis. Observed high VE/VCO₂ and low PETCO₂ values might relate to increased activation of metabolic chemoreceptors within skeletal muscle; however, the exact underlying mechanism has not been yet fully elucidated. Furthermore, the abnormalities of O₂ pulse and the markedly impaired VO₂/W slope were indicative of a severe defect in O₂ delivery and/or muscle O₂ extraction.

Arterial blood gas (ABG) sampling, breathing room air, showed a mixed acid–base disorder at rest (pO₂ 111 mm Hg, pCO₂ 35 mm Hg, pH 7.40, HCO_3- 21 mmol/L, lactate 2.2 mmol/L) and a significant metabolic acidosis at peak exercise (pO₂ 118 mm Hg, pCO₂ 26 mm Hg, pH 7.19, HCO_3- 9.9 mmol/L, lactate >20 mmol/L).

The above clinical and laboratory findings, as well as the CPET and ABG parameters, in the absence of pulmonary and cardiovascular diseases, were in favour of a diagnosis of a metabolic disorder characterised by altered skeletal muscle oxygen use. A muscular biopsy was therefore performed. According to the protocol adopted in our laboratory, the bioptic sample underwent a panel of histological, histochemical and immunohistochemical analyses, including H&E, Gomori trichrome, sequential cytochrome c oxidase/succinate dehydrogenase (COX/SDH) and ATPase stains. Immunohistochemical analyses with the following antibodies were also performed: fast, slow and neonatal myosin, dystrophin (N-terminus, rod domain, C-terminus), sarcoglycans (α, β, δ, γ), dysferlin, major histocompatibility complex class I, CD68. At histological analysis, the most peculiar feature was the presence of several fibres with increased subsarcolemmal intensity at COX/SDH stain, compatible with mitochondrial proliferation (figure 1A, B). There was only one COX-deficient fibre (characterised by a slight stain reduction), although COX-negative fibres were absent. Gomori trichrome stain did not show ragged red fibres. In addition, there was a slight variability in the fibre size, without muscle necrosis or degeneration (figure 1A, B). There were neither inflammatory infiltrates, nor endomysial fibrosis. Mitochondrial subsarcolemmal proliferation was confirmed by ultrastructural examination, which did not reveal cristae alterations and paracrystalline inclusions (data not shown). These findings suggested a diagnosis of a mitochondrial myopathy.

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Figure 1

Identification of the m.3250T>C mutation. (A) Histological analysis of the muscle biopsy from the proband showing only slight variation in fibre size (asterisk shows ipotrophic fibres; H&E, original magnification ×20). (B) Sequential cytochrome c oxidase (COX)/SDH histochemistry on skeletal muscle biopsy from the proband showing a COX-positive muscle fibre with increased subsarcolemmal staining (arrow), compatible with mitochondrial proliferation (original magnification ×20). (C) Sequence electropherogram of mt-tRNA^Leu(UUR) showing the m.3250T>C mutation in the proband's skeletal muscle (mutation is indicated by an asterisk). (D) Secondary structure of mt-tRNA^Leu(UUR) with the position of the m.3250T>C mutation highlighted in the D-loop. (E) Restriction fragment length polymorphism analysis of 3250T>C mutation. The restriction endonuclease NaeI cleaves wild-type sequences (size 231 bps) in two fragments (sized 212 and 19 bps; the latter is not visible in the figure). The mutation introduces a second NaeI recognition site that cleaves the 212 bps in two smaller fragments (sized 187 and 25 bps; the latter is not visible in the figure). Restriction fragments were separated using the Agilent 2100 bioanalyzer instrument with the 1000 LabChip kit. Left: LabChip gel image. Right: overlay of three different electropherograms (red=uncut; blue=wild type; green=3250T>C mutant). The bands are separated and identified by the software as separate peaks. WT, wild type; Pm, proband skeletal muscle; Pb, proband blood; Mb, =proband mother's blood.

To confirm the diagnosis, direct sequencing of whole mitochondrial DNA (mtDNA) from the muscle homogenate was performed, revealing the m.3250T>C mutation in the MTTL1 gene (figure 1C, D). Identical change was found in mtDNA from peripheral lymphocytes of the patient and of her mother, although clinically unaffected. Restriction fragment length polymorphism analysis showed that the m.3250T>C mutation was heteroplasmic, with a mutation load of ∼80% in the proband's muscle, ∼20% in the proband's blood and ∼10% in the mother's blood (figure 1E).

On the basis of the literature data, supplemental therapy with L-carnitine (5 g/day) and riboflavin (200 mg/day) was started, and the patient was asked to return for a 1-year reassessment.

At the follow-up visit, the patient reported a significant improvement in exercise capacity. A repeat maximal (RER peak 1.6) CPET (table 1), performed according to the same exercise protocol, continued to demonstrate impaired exercise capacity (VO_2peak 14.6 mL O₂/min/kg, 41% pred), low maximal work rate (60 W, 35% pred), early lactate threshold (30% pred), low O₂ pulse_peak value (4.6 mL O₂/bpm, 47% pred), mildly impaired ventilatory efficiency (VE/VCO_2slope 30) and high ventilatory equivalents for O₂ (VE/VO_2peak 56). However, resting HR was now within normal ranges (86 bpm) with a preserved heart rate reserve (HRR) (162 bpm, 82% of max HR pred at peak exercise). Borg scale scores for RPE were also significantly improved (at peak exercise: dyspnoea 4/10, leg fatigue 5/10). Peak exercise ABG sampling demonstrated significant improvement in peak exercise lactate: pH 7.27, HCO_3- 18 mmol/L, lactate 8.7 mmol/L.