Muscle mitochondrial DNA deletion and 31P-NMR spectroscopy alterations in a migraine patient.

A 40-year-old female suffering from recurrent migrainous strokes is reported. She did not show any muscle weakness or wasting. Ragged red and cytochrome c oxidase negative fibers were present in the muscle biopsy. Muscle mitochondrial DNA analysis showed a 5 kb deletion, without a point mutation at nucleotide pair 3243 in the mitochondrial tRNALeu(UUR) gene. Phosphorus nuclear magnetic resonance spectroscopy of brain and gastrocnemius muscle showed a defective energy metabolism in both organs. An increased inorganic phosphate to phosphocreatine ratio due to a decreased phosphocreatine content was found in the occipital lobes, while an abnormal work-energy cost transfer function and a low rate of phosphocreatine post-exercise recovery were found in the muscle.     

Brain energy metabolism studied by 31P-MR spectroscopy in a case of migraine with prolonged aura

The brain and skeletal muscle oxidative metabolism of a patient with prolonged aura was studied by phosphorus magnetic resonance spectroscopy. We found that the phosphocreatine to ATP ratio in brain was reduced, while the inorganic phosphate to phosphocreatine ratio and the calculated ADP concentration were increased. The phosphorylation potential and percentage of maximal rate of ATP synthesis were also altered. Intracellular pH and inorganic phosphate concentration were normal. In muscle we found a low post-exercise recovery of phosphocreatine. These data indicate an impairment of energy oxidative metabolism both in brain and muscle.     

Early-onset cerebellar ataxia,myoclonus and hypogonadism in a case of mitochondrial complex III deficiency treated with vitamins K3 and C

A 16-year-old girl presented with early-onset cerebellar ataxia, myoclonus, elevated lactic acidosis and hypogonadotropic hypogonadism. Muscle biopsy specimens revealed fibres with a ragged appearance with increased mitochondria and lipid droplets. Biochemical investigation revealed a deficiency of complexbc ₁ (complex III) of the mitochondrial respiratory chain. Genetic analysis did not show either deletions or known mutations of mitochondrial DNA (mtDNA). Phosphorus magnetic resonance spectroscopy (³¹P-MRS) showed defective energy metabolism in brain and gastrocnemius muscle. A decreased phosphocreatine (PCr) content was found in the occipital lobes accompanied by normal inorganic phosphate (Pi) and cytosolic pH. These findings represented evidence of a high cytosolic adenosine diphosphate concentration and a relatively high rate of metabolism accompanied by a low phosphorylation potential. Muscle³¹P-MRS showed a high Pi content at rest, abnormal exercise transfer pattern and a low rate of PCr post-exercise recovery. These findings suggested a deficit of mitochondrial function. Therapy with vitamins K₃ and C normalized brain³¹P-MRS indices, whereas it did not affect muscle bioenergetic metabolism. In this patient, the endocrinological disorder is putatively due to a mitochondrial cytopathy. Although an unknown mtDNA mutation cannot be ruled out, the genetic defect may lie in the nuclear genome.     

Lipoic (thioctic) acid increases brain energy availability and skeletal muscle performance as shown by in vivo31P-MRS in a patient with mitochondrial cytopathy

A woman affected by chronic progressive external ophthalmoplegia and muscle mitochondrial DNA deletion was studied by phosphorus magnetic resonance spectroscopy (³¹P-MRS) prior to and after 1 and 7 months of treatment with oral lipoic acid. Before treatment a decreased phosphocreatine (PCr) content was found in the occipital lobes, accompanied by normal inorganic phosphate (Pi) level and cytosolic pH. Based on these findings, we found a high cytosolic adenosine diphosphate concentration [ADP] and high relative rate of energy metabolism together with a low phosphorylation potential. Muscle MRS showed an abnormal work-energy cost transfer function and a low rate of PCr recovery during the post-exercise period. All of these findings indicated a deficit of mitochondrial function in both brain and muscle. Treatment with 600 mg lipoic acid daily for 1 month resulted in a 55% increase of brain [PCr], 72% increase of phosphorylation potential, and a decrease of calculated [ADP] and rate of energy metabolism. After 7 months of treatment MRS data and mitochondrial function had improved further. Treatment with lipoate also led to a 64% increase in the initial slope of the work-energy cost transfer function in the working calf muscle and worsened the rate of PCr resynthesis during recovery. The patient reported subjective improvement of general conditions and muscle performance after therapy. Our results indicate that treatment with lipoate caused a relevant increase in levels of energy available in brain and skeletal muscle during exercise.     

Exercise-induced pain, stiffness, and tubular aggregation in skeletal muscle.

The case is described of a 36 year old man with exercise-induced pain, stiffness, and tubular aggregation in skeletal muscle. Persistent CPK elevation and a normal forearm ischaemic exercise test were demonstrated. Electromyography was normal. Muscle biopsy showed aggregates of tubular structures in most fibres by histochemical and ultrastructural analysis. A relationship of this syndrome to known defects in muscle energy metabolism is postulated.     

Mitochondrial dysfunction in bipolar disorder

Mitochondrial dysfunction is implicated in bipolar disorder based on the following lines of evidence: 1) Abnormal brain energy metabolism measured by 31 P‐magnetic resonance spectroscopy, that is, decreased intracellular pH, decreased phosphocreatine (PCr), and enhanced response of PCr to photic stimulation. 2) Possible role of maternal inheritance in the transmission of bipolar disorder. 3) Increased levels of the 4977‐bp deletion in mitochondrial DNA (mtDNA) in autopsied brains. 4) Comorbidity of affective disorders in certain types of mitochondrial disorders, such as autosomal inherited chronic progressive external ophthalmoplegia and mitochondrial diabetes mellitus with the 3243 mutation. Based on these findings, we searched for mtDNA mutations/polymorphisms associated with bipolar disorder and found that 5178C and 10398A polymorphisms in mtDNA were risk factors for bipolar disorder. The 5178C genotype was associated with lower brain intracellular pH. mtDNA variations may play a part in the pathophysiology of bipolar disorder through alteration of intracellular calcium signaling systems. The mitochondrial dysfunction hypothesis, which comprehensively accounts for the pathophysiology of bipolar disorder, is proposed.     

Rhabdomyolysis and acute encephalopathy in late onset medium chain acyl-CoA dehydrogenase deficiency.

A previously asymptomatic 30 year old man presented with rhabdomyolysis, muscle weakness, and acute encephalopathy after strenuous exertion in the cold without adequate food intake. Serum and muscle carnitine concentrations were decreased. Urinary excretion of carnitine and glycine esters and biochemical examination of skeletal muscle and fibroblasts led to the diagnosis of medium chain acyl-CoA dehydrogenase (MCAD) deficiency. A point mutation at nucleotide position 985 of the coding region of the MCAD gene was found. The MCAD protein was synthesised in the patient's fibroblasts at a normal rate, but was unstable. In general, patients in whom the 985 point mutation has been established show much more severe clinical symptoms and other symptoms than those seen in this patient. The relation of the 985 point mutation and the residual MACD activity to the symptoms is not as straightforward as previously thought.     

Lack of creatine in muscle and brain in an adult with GAMT deficiency

Guanidinoacetate methyltransferase deficiency, which so far has been exclusively detected in children, was diagnosed in a 26-year-old man. The full-blown spectrum of clinical symptoms already had been present since infancy without progression of symptoms during adolescence. Cranial magnetic resonance imaging showed normal findings. Ophthalmological examination showed no retinal changes. Besides creatine deficiency in the brain, a distinct lack of phosphocreatine in skeletal muscle was proved by (31)P magnetic resonance spectroscopy. Creatine substitution combined with a guanidinoacetate-lowering diet introduced first at the age of 26 years was shown to be effective by an impressive improvement of epileptic seizures, mental capabilities, and general behavior and by normalization of the (31)P spectrum in the skeletal muscle.     

Analysis of the tissue distribution and inheritance of heteroplasmic mitochondrial DNA point mutation by denaturing gradient gel electrophoresis in merrf syndrome

MERRF (Myoclonic Epilepsy and Ragged-Red Fibres) syndrome is one of the maternally inherited diseases for which a mitochondrial DNA (mtDNA) point mutation has recently been identified. The mutation is always heteroplasmic, that is normal and mutant mtDNA coexist within the same individual. We studied mtDNA heteroplasmy in two families with MERRF syndrome, using a denaturing gradient gel electrophoresis technique that avoids the errors in the evaluation of wild/mutant mtDNA ratios caused by restriction enzyme cutting in the situation of amplification of a heteroplasmic DNA. In two patients, the proportion of muscle mutant mtDNA was in agreement with the severity of muscle mitochondrial proliferation, energy defect and fibre type I predominance. In nine patients from three generations of one family, mutant mtDNA proportion in leukocytes was in relative agreement with the clinical severity of the disease. Transmission of mutant mtDNA through these three generations did not show any tendency toward homoplasmy. Homogeneity of the mutant mtDNA proportion among different tissues from one patient was demonstrated in brain, liver, muscle and heart but a possibility of divergence of the mutant mtDNA proportion during mitosis was documented in cultured skin fibroblasts.     

Evidence for a relationship between body mass and energy metabolism in the human brain

Cerebral energy metabolism has been suggested to have an important function in body weight regulation. We therefore examined whether there is a relationship between body mass and adenosine triphosphate (ATP) metabolism in the human brain. On the basis of our earlier findings indicating a neuroprotective preferential energy supply of the brain, as compared with peripheral muscle on experimentally induced hypoglycemia, we examined whether this physiological response is preserved also in low-weight and obese participants. We included 45 healthy male subjects with a body mass index (BMI) ranging from 17 to 44 kg/m². Each participant underwent a hypoglycemic glucose-clamp intervention, and the ATP metabolism, that is, the content of high-energy phosphates phosphocreatine (PCr) and ATP, was measured repeatedly by ³¹phosphor magnetic resonance spectroscopy (³¹P-MRS) in the cerebral cortex and skeletal muscle. Results show an inverse correlation between BMI and high-energy phosphate content in the brain (P<0.01), whereas there was no such relationship found between skeletal muscle and BMI. The hypoglycemic clamp intervention did not affect the ATP metabolism in both tissues. Our data show an inverse correlation between BMI and cerebral high-energy phosphate content in healthy humans, suggesting a close relationship between energetic supply of the brain and body weight regulation.     

Lack of paternal inheritance of muscle mitochondrial DNA in sporadic mitochondrial myopathies

In 2002, paternal inheritance of muscle mitochondrial DNA (mtDNA) was reported in a patient with exercise intolerance and a mitochondrial DNA (mtDNA) mutation restricted to skeletal muscle. To evaluate whether paternal inheritance is a common phenomenon, we studied 10 sporadic patients with skeletal muscle-restricted mtDNA mutations: five harbored mtDNA point mutations in protein-coding genes and five had single mtDNA deletions. We performed haplotype analysis and direct sequencing of the hypervariable regions 1 and 2 of the D-loop in muscle and blood from the patients and, when available, in blood from their parents. We did not observe paternal inheritance in any of our patients.     

Mitochondrial dysfunction in myotonic dystrophy type 1

The pathophysiological mechanism linking the nucleotide expansion in the DMPK gene to the clinical manifestations of myotonic dystrophy type 1 (DM1) is still unclear. In vitro studies demonstrate DMPK involvement in the redox homeostasis of cells and the mitochondrial dysfunction in DM1, but in vivo investigations of oxidative metabolism in skeletal muscle have provided ambiguous results and have never been performed in the brain. Twenty-five DM1 patients (14M, 39 ± 11years) underwent brain proton MR spectroscopy (¹H-MRS), and sixteen cases (9M, 40 ± 13 years old) also calf muscle phosphorus MRS (³¹P-MRS). Findings were compared to those of sex- and age-matched controls. Eight DM1 patients showed pathological increase of brain lactate and, compared to those without, had larger lateral ventricles (p < 0.01), smaller gray matter volumes (p < 0.05) and higher white matter lesion load (p < 0.05). A reduction of phosphocreatine/inorganic phosphate (p < 0.001) at rest and, at first minute of exercise, a lower [phosphocreatine] (p = 0.003) and greater [ADP] (p = 0.004) were found in DM1 patients compared to controls. The post-exercise indices of muscle oxidative metabolism were all impaired in DM1, including the increase of time constant of phosphocreatine resynthesis (TC PCr, p = 0.038) and the reduction of the maximum rate of mitochondrial ATP synthesis (p = 0.033). TC PCr values correlated with the myotonic area score (ρ = 0.74, p = 0.01) indicating higher impairment of muscle oxidative metabolism in clinically more affected patients. Our findings provide clear in vivo evidence of multisystem impairment of oxidative metabolism in DM1 patients, providing a rationale for targeted treatment enhancing energy metabolism.     

Ca2+-ATPase deficiency in a patient with an exertional muscle pain syndrome.

31P Magnetic resonance spectroscopy studies were carried out in vivo on skeletal muscle of a patient with verapamil-responsive, chronic, progressive post-exertional muscle pain. A sister suffered from a similar complaint. The results showed that the muscle: (1) decreased its high energy phosphate content more rapidly than normal during exercise, indicating either increased utilisation or decreased production of ATP; (2) acidified more rapidly than normal during exercise suggesting an increased glycolytic rate; (3) continued in some studies to acidify markedly during the first minute after exercise, indicating that glycolysis remained active into the recovery period; (4) had phosphocreatine and ADP recovery rates consistent with normal rates of oxidative phosphorylation. On the basis of these results, it was proposed that the patient suffers from a defect in Ca2+ handling in the muscle. Subsequently, direct measurement of Ca2+-ATPase activity in the sarcoplasmic reticulum fraction from a muscle biopsy sample showed that the activity of this enzyme was reduced by about 90%.     

Toxic myopathy with multiple deletions in mitochondrial DNA associated with long‐term use of oral anti‐viral drugs for hepatitis B: A case study

Oral nucleoside analogs (NAs) reduce hepatitis B virus (HBV) replication by inhibiting HBV DNA polymerase. However, NAs can also affect human mitochondrial DNA (mtDNA) polymerase, which can lead to mtDNA depletion (quantitative abnormality). Indeed, several mitochondrial myopathy cases have been reported in which a reduced mtDNA copy number was induced by oral NAs for hepatitis B. Herein, we report a case of toxic myopathy with multiple mtDNA deletions (qualitative abnormality) associated with long‐term use of NAs for hepatitis B. A 68‐year‐old woman, who underwent long‐term treatment with lamivudine and adefovir for chronic hepatitis B, developed proximal muscle weakness in the four extremities. Neurological examination showed mild proximal muscle weakness and atrophy in the four extremities. Upon admission to our hospital, her blood lactate/pyruvate ratio during an aerobic exercise test was elevated. Myogenic patterns were observed in lower limb muscles on electromyographic examination. Muscle magnetic resonance imaging revealed diffuse atrophy of proximal muscles in the four extremities with no signal changes. A biopsy from the biceps brachii muscle showed an abnormally large variation in fiber size, scattered muscle fibers with decreased cytochrome c oxidase activity, and ragged‐red fibers. Analysis of mtDNA from skeletal muscle revealed no decrease in copy number but increased incidence of multiple deletions, including a deletion of 4977 base pairs (known as the common deletion) reflecting oxidative stress‐induced mtDNA damage. This case study indicates that long‐term oral antiviral therapy for hepatitis B can induce chronic oxidative damage to mtDNA resulting in qualitative mtDNA abnormalities and toxic myopathy.     

Leber's hereditary optic neuropathy: genetic, biochemical, and phosphorus magnetic resonance spectroscopy study in an Italian family

Three siblings of a family affected with Leber's hereditary optic neuropathy (LHON) showed a mitochondrial DNA mutation at position 11778. The lactate response to a standardized effort was increased in only one case. Muscle biopsies and biochemistry of muscle and platelet mitochondrial enzymes were normal. All patients showed an altered energy metabolism during exercise and during recovery after exercise on phosphorus 31-magnetic resonance spectroscopy (31P-MRS) of muscle. Brain 31P-MRS showed a decreased energy reserve (decreased PCr/Pi ratio) in all patients. 31P-MRS noninvasively demonstrated an altered mitochondrial energy metabolism in muscle and, for the first time, in the brains of LHON patients.     

Ekbom's syndrome of photomyoclonus, cerebellar ataxia and cervical lipoma is associated with the tRNALys A8344G mutation in mitochondrial DNA

We have investigated nine maternal offsprings to patients with a hereditary syndrome of cerebellar ataxia, photomyoclonus, skeletal deformities and lipoma, originally described by Ekbom. The nine family members underwent a thorough neurological examination, neurophysiological investigations and molecular genetic analysis of mtDNA from lymphocytes and muscle. Clinical examination showed a partial syndrome in one relative and minor signs and symptoms in three additional offsprings. We found the heteroplasmic tRNA^LyS A8344G point mutation in mtDNA in all investigated maternal offsprings. The fraction of mutated mtDNA ranged from 33 to 87% in lymphocytes and from 59 to 92% in muscle tissue. Analysis of mtDNA from a lipoma showed a high level (96%) of the tRNA^LyS A8344G mutation. We conclude that Ekbom's syndrome is a mitochondrial encephalomyopathy associated with the same heteroplasmic tRNA mutation as seen in myoclonus epilepsy with ragged-red fiber (MERRF) syndrome.     

A novel mitochondrial tRNA phenylalanine mutation presenting with acute rhabdomyolysis

We describe a patient who presented with acute rhabdomyolysis and had 68% cytochrome c oxidase (COX)-deficient fibers in skeletal muscle. Further investigations confirmed a respiratory chain defect that was associated with a novel heteroplasmic point mutation in the phenylalanine tRNA gene of the mitochondrial genome (mtDNA). Analysis of single muscle fibers revealed a significantly greater level of mutant mtDNA in COX-negative fibers. This is the first case of a mitochondrial tRNA gene point mutation presenting with acute rhabdomyolysis and recurrent myoglobinuria.     

Mutation in the mitochondrial tRNAIle gene causes progressive myoclonus epilepsy

PurposeThe group of the rare progressive myoclonic epilepsies (PME) include a wide spectrum of mitochondrial and metabolic diseases. In juvenile and adult ages, MERRF (myoclonic epilepsy with ragged red fibres) is the most common form. The underlying genetic defect in most patients with the syndrome of MERRF is a mutation in the tRNALys gene, but mutations were also detected in the tRNAPhe gene.MethodHere, we describe a 40 year old patient with prominent myoclonic seizures since 39 years of age without a mutation in the known genes who underwent intensive clinical, genetic and functional workup.ResultsThe patient had a slight mental retardation and a severe progressive hearing loss based on a defect of the inner ear on both sides. Ictal electroencephalography (EEG) showed bilateral occipital and generalized spikes and polyspikes induced and aggravated by photostimulation. A cranial magnetic resonance imaging (cMRI) detected a global cortical atrophy of the brain and mild periventricular white matter lesions. The electromyography (EMG) was normal but the muscle biopsy showed abundant ragged red fibres. Sequencing of the mitochondrial DNA from the skeletal muscle biopsy revealed a novel heteroplasmic mutation (m.4279A>G) in the tRNAIle gene which was functionally relevant as tested in single skeletal muscle fibre investigations.ConclusionMutations in tRNAIle were described in patients with chronic progressive external ophthalmoplegia (CPEO), prominent deafness or cardiomyopathy but, up to now, not in patients with myoclonic epilepsy. The degree of heteroplasmy of this novel mitochondrial DNA mutation was 70% in skeletal muscle but only 15% in blood, pointing to the diagnostic importance of a skeletal muscle biopsy also in patients with myoclonic epilepsy.     

Investigation of human mitochondrial myopathies by phosphorus magnetic resonance spectroscopy

Abnormal mitochondria are an increasingly recognized cause of neuromuscular disease. We have used phosphorus magnetic resonance spectroscopy to monitor noninvasively the metabolism of high-energy phosphates and the intracellular pH of human skeletal muscle in vivo in 12 patients with mitochondrial myopathy. At rest, an abnormality could be demonstrated in 11 of 12 patients. Ten patients had evidence of a reduced muscle energy state with at least one of the following abnormalities: low phosphorylation potential, low phosphocreatine concentration, high adenosine diphosphate concentration, or high inorganic phosphate concentration. Two patients had abnormal resting muscle intracellular pH. In some patients phosphocreatine concentration decreased to low values during exercise despite limited work output. This was not accompanied by particularly severe intracellular acidosis. Evidence of impaired rephosphorylation of adenosine diphosphate to adenosine triphosphate during recovery from exercise was found in approximately half the patients. Phosphorus magnetic resonance spectroscopy is useful in the noninvasive diagnosis of mitochondrial myopathies and in defining the pathophysiological basis of these disorders.     

The A8344G mutation in mitochondrial DNA associated with stroke-like episodes and gastrointestinal dysfunction

We report an unusual case of encephalo-entero-myopathy associated with the A8344G mutation in the tRNA(Lys) gene of mitochondrial DNA (mtDNA). This patient had mitochondrial myopathy, multiple lipomatosis, mild hearing loss, stroke-like episodes, and paralytic ileus, but she lacked the canonical clinical features of MERRF, myoclonus, epilepsy, or ataxia. We conducted genetic, biochemical, histochemical, and immunohistochemical studies in skeletal muscle, brain, intestine, and lipoma tissue. The mutation was abundant in all tissues, and cytochrome c oxidase (COX) activity was selectively decreased in brain and small intestine. COX deficiency was also documented histochemically and immunohistochemically in the small intestine, suggesting that mitochondrial dysfunction played a role in the pathogenesis of paralytic ileus. This case illustrates an unusual and dramatic clinical phenotype of the A8344G mutation, characterized by stroke-like episodes and acute ileus.