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A case report of Leigh syndrome diagnosed by endomyocardial biopsy

Abstract Background Leigh syndrome is a neurodegenerative disorder caused by mitochondrial dysfunction with both phenotypic and genetic heterogeneity. Mitochondrial impairments are usually demonstrated by skeletal muscle biopsy. We report a case of Leigh syndrome diagnosed by endomyocardial biopsy (EMB), not by skeletal muscle biopsy. Case summary At aged 7 months, the patient had delayed motor development. He developed metabolic acidosis triggered by an infection with elevated lactate and pyruvate values in serum and cerebrospinal fluid when he was 1 year old. T2-weighted imaging on magnetic resonance imaging of the brain revealed bilateral hyperintensity in midbrain and dorsal pons. Biopsied skeletal muscle did not show evidence of mitochondrial disease. Left ventricular hypertrophy, bilateral putamen hyperintensity in T2-weighted imaging and a lactate peak in the right basal ganglia in single voxel spectroscopy, and a convulsive seizure appeared at the age of 12, 15, and 16, respectively. When he was 17 years old, biopsied myocardium showed cytoplasmic vacuolization and a marked proliferation of mitochondria within myofibrils pathologically. Respiratory chain enzyme activity of the biopsied myocardium showed decreased activity of complex I. Genetic testing revealed an m.14453 A>G mutation on the MT-ND6 gene. He was finally diagnosed with Leigh syndrome. Administration of oral 5-aminolevulinic acid reduced the frequency of seizures. Discussion EMB led to the diagnosis of Leigh syndrome. Efforts to find and conduct the biopsy of affected organs are important to diagnose mitochondrial disease. EMB is a useful diagnostic method when there is a difficulty in diagnosing mitochondrial disease by skeletal muscle biopsy.


Introduction
Leigh syndrome is a neurodegenerative disorder caused by mitochondrial dysfunction with both phenotypic and genetic heterogeneity. 1 Leigh syndrome is characterized by: (i) neurodegenerative disease with variable symptoms due to (ii) mitochondrial impairment caused by a hereditary genetic defect accompanied by (iii) bilateral central nervous system lesions that can be associated with further abnormalities in diagnostic imaging. 2 The term 'Leigh-like syndrome' should be used when these criteria are only partially met. 2 Mitochondrial impairments are demonstrated by mitochondrial respiratory chain dysfunction, pathological abnormalities on tissue biopsy, or gene mutation. 3 These abnormalities are usually found by skeletal muscle biopsy. We herein report a case of Leigh syndrome diagnosed by endomyocardial biopsy (EMB), not by skeletal muscle biopsy.

Case presentation
A 17-year-old boy was admitted to our hospital with left ventricular hypertrophy. Our patient was born from non-consanguineous parents, delivered vaginally at 38 weeks' gestational age weighing 2384 g with no neonatal asphyxia. He had no family history of cardiomyopathy or sudden cardiac death. Manifestations of neurological illness appeared in 7 months with delayed motor development. He developed metabolic acidosis triggered by a viral infection on three occasions when he was 1 year old. At this point, lactate and pyruvate values in serum and cerebrospinal fluid were elevated. T2-weighted imaging revealed bilateral hyperintensity in midbrain and dorsal pons in brain magnetic resonance imaging (MRI) (Figure 1). Leigh syndrome was suspected based on clinical course and brain images. Although skeletal muscle biopsy was performed, ragged red fibres were not identified pathologically, and respiratory chain enzyme activity of the biopsied skeletal muscle was normal. Point mutations associated with mitochondrial diseases at the mitochondrial DNA position 3243, 8344, 8993, and 9176 in the biopsied skeletal muscle were not identified. As the evidence of mitochondrial dysfunction was not shown, he was diagnosed as 'Leigh-like syndrome'. We started to supply thiamine and dichloroacetic acid as a treatment for mitochondrial impairments which was suspected at this time. The episode of metabolic acidosis did not occur after he was 4 years old. Although he had a delay in psychomotor development, he attended a school for disabled children and grew up with no regression. His activities of daily living such as functional mobility and self-feeding were independent, whereas bathing, dressing, and toilet hygiene needed partial assistance during childhood.
Left ventricular hypertrophy was identified on the electrocardiogram when he was 6 years old ( Figure 2). 4 Echocardiography showed left ventricular hypertrophy when he was 12 years old (Video 1). [5][6][7] Although there were no cardiovascular manifestations such as dyspnoea or palpitation, mitochondrial cardiomyopathy was suspected based on these cardiological findings and clinical course. When he was 15 years old, T2-weighted imaging showed hyperintensity in bilateral putamen and right cerebral peduncle, and cerebellar atrophy in brain MRI ( Figure 1). Single voxel spectroscopy demonstrated a lactate peak in the right basal ganglia (Figure 1). Although these MRI findings were suggestive of Leigh syndrome, a definitive diagnosis could not be made since we could not demonstrate evidence of mitochondrial dysfunction. The patient suffered a convulsive seizure when he

Age Events
At birth Delivered vaginally at 38 weeks' gestational age weighing 2348 g with no neonatal asphyxia.

7-month-old
Delayed motor development appeared.
1-year-old Metabolic acidosis triggered by a viral infection occurred. Lactate and pyruvate values in serum and cerebrospinal fluid were elevated.
T2-weighted imaging on magnetic resonance imaging (MRI) showed bilateral hyperintensity in midbrain and dorsal pons.
Biopsied skeletal muscle did not show ragged red fibres or decreased respiratory chain enzyme activity. Thiamine and dichloroacetic acid replacement therapy was initiated.
6-year-old Left ventricular hypertrophy was identified on the electrocardiogram.
12-year-old Echocardiography showed left ventricular hypertrophy. 15-year-old T2-weighted imaging on MRI showed hyperintensity in bilateral putamen and right cerebral peduncle, and cerebellar atrophy.
Single voxel spectroscopy demonstrated a lactate peak in right basal ganglia.
17-year-old He was diagnosed with Leigh syndrome and mitochondrial cardiomyopathy by endomyocardial biopsy.
Genetic testing revealed an m.14453 A>G mutation on the MT-ND6 gene. 21-year-old 5-aminolevulinic acid was initiated, which reduced the frequency of seizures. was 16 years old. Levocarnitine supplementation as an additional treatment for suspected mitochondrial impairments and anticonvulsants were started. When he was 17 years old, he was admitted to our hospital to investigate the cause of left ventricular hypertrophy. His blood pressure was 108/60 mmHg. Pulse rate was 53/min and regular. Heart sounds were regular, and he had no murmur. Neurological examination showed mild muscle weakness with normal deep tendon reflexes. Blood analysis showed normal brain natriuretic peptide value of less than 5.8 pg/mL (reference range: 0-18.4 pg/mL). EMB was performed by cardiac catheterization and biopsy samples were obtained at the ventricular septum in the right ventricle. Light microscopy of the biopsied myocardium showed cytoplasmic vacuolization (Figure 3), and electron microscopy showed a marked proliferation of mitochondria within myofibrils ( Figure 3). Using electron microscopy with quantitative analysis, the volume density of mitochondria within  cardiac muscle cells was 39%, which indicated the possibility of mitochondrial cardiomyopathy. 8 Respiratory chain enzyme activity of the biopsied myocardium and cultured fibroblasts obtained from the biopsied skin showed decreased activity of complex I (Table 1). 9,10 Oxygen consumption rate of skin fibroblasts measured by Seahorse XF96 apparatus was 92% of control in glucose medium and 64% of normal in galactose medium, which indicated oxidative phosphorylation impairment. 11,12 Immunohistopathological analysis with respiratory chain enzyme antibodies of biopsied myocardium showed decreased signal intensity of complex I compared with complex II (22% of normal) and normal signal intensity of complex IV compared with complex II (116% of normal) ( Figure 4). 8 Genetic testing revealed an m.14453 A>G mutation on the MT-ND6 gene, with a heteroplasmy rate of the blood sample 60% and the cardiac tissue 83%. This mutation was not shown in his parents, suggesting that it was a de novo mutation. Association of mutations in MT-ND6 gene with Leigh syndrome has been reported previously. 13,14 This m.14453 A>G mutation has been reported in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, 15 whereas it has not been reported in Leigh syndrome previously. Taken together with clinical course and laboratory findings, this mutation was considered the causative agent. He was finally diagnosed with Leigh syndrome and mitochondrial cardiomyopathy. As the frequency of seizures gradually increased and his motor function gradually regressed, we started oral 5-aminolevulinic acid as a treatment when he was 21 years old. After the initiation of oral 5aminolevulinic acid, the frequency of seizures decreased, whereas his motor function gradually continued to regress. In the most recent follow-up at the age of 22, he had no life-threatening complications and was able to walk slowly, whereas his activities of daily living such as functional mobility and self-feeding required partial assistance.

Discussion
Mitochondrial diseases are a genetically and clinically heterogeneous group of disorders due to dysfunctions of the mitochondrial oxidative phosphorylation. 16 They can be caused by mutations in either mitochondrial DNA or nuclear genes. 16 Mitochondrial DNA is usually all identical (homoplasmy), whereas individuals with mitochondrial diseases resulting from mutation of mitochondrial DNA may have a mixture of mutated and wild type mitochondrial DNA (heteroplasmy). 17 In the presence of heteroplasmy, there is a threshold effect and clinical phenotypes may vary depending on the percentage level of mutated mitochondrial DNA in each organ. 16 Mitochondrial diseases may present at any age. 18,19 Mitochondrial dysfunction is associated with a wide spectrum of diseases and clinical syndromes such as Kearns-Sayre syndrome, chronic progressive external ophthalmoplegia, MELAS, myoclonic epilepsy with ragged red fibres, neurogenic weakness with ataxia and retinitis pigmentosa, Leigh syndrome, or mitochondrial cardiomyopathy. 12,20 The diagnostic criteria for mitochondrial respiratory chain complex disorder is widely used for the diagnosis of oxidative phosphorylation disorder. 3 In this diagnostic criteria, ragged red fibres in pathological tissue of skeletal muscle are defined as diagnostic standard, whereas the diagnostic standard of pathological features in myocardial tissue is not defined. Therefore, in the case of mitochondrial cardiomyopathy alone, it is necessary to identify the decreased enzyme activity using myocardial tissue for diagnosis.
A clinical diagnosis of mitochondrial cardiomyopathy is usually made when cardiomyopathy appears as a complication of mitochondrial disease. A definitive diagnosis of mitochondrial cardiomyopathy is made by the trinity of genetic, pathological, and biochemical examinations ( Figure 5). 8,20 Typical phenotypes of mitochondrial cardiomyopathy are hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, and left ventricular noncompaction. 20 In practice, clinicians should be aware of mitochondrial cardiomyopathy as a differential diagnosis of unexplained cardiomyopathy, which can present for the first time in adulthood. The 'red flag' features which can point towards mitochondrial disease are unexplained cardiomyopathy, cardiac conduction disorder, maternal inheritance, diabetes mellitus, hearing impairment, neuromuscular features, and metabolic acidosis.
It has been reported that fusiform enlargement of affected myocytes around the peculiar region with cytoplasmic clearing and replacement of cross striae by fine granules in microscopy, and increased and larger mitochondria, abnormal cristae in the ultrastructural examination are found pathologically in cardiac specimens in mitochondrial cardiomyopathy. 21 In our previous study, we revealed that the volume density of mitochondria within cardiac muscle cells above 30% strongly suggests the possibility of mitochondrial cardiomyopathy. 8 Furthermore, immunohistopathological analysis with respiratory chain enzyme antibodies of biopsied myocardium of mitochondrial cardiomyopathy showed decreased signal intensity of complex I and/or complex IV compared with complex II. 8 All these pathological findings were found in our case. These pathological features in the myocardium may also be added as a diagnostic standard of mitochondrial respiratory chain complex disorder.
Although we could not diagnose as Leigh syndrome for 17 years as the evidence of mitochondrial dysfunction was not identified by skeletal muscle biopsy, EMB led to the diagnosis of mitochondrial cardiomyopathy and Leigh syndrome in our case. In the case of Leigh syndrome, Lee et al. 22 reported that muscle biopsies in 39 patients did not reveal any ragged red fibres and Sofou et al. 23 reported that ragged red fibres occurred in muscle biopsies in 57 out of 107 patients. A biopsy of the affected organs is essential for the diagnosis of mitochondrial disease. Thus, if a skeletal muscle is not affected, skeletal muscle biopsy has no diagnostic capability as in our case. Efforts to find affected organs and conducting a biopsy of those organs are important when there is a difficulty in diagnosing mitochondrial disease. For cases in which mitochondrial disease is suspected but cannot be diagnosed by skeletal biopsy, the cardiac examination should be performed, and if there is evidence of cardiomyopathy, EMB should be considered.
The main treatments for mitochondrial cardiomyopathy are cardioprotective and symptomatic therapy. 20,21 Cardiac manifestations vary from asymptomatic to severe heart failure or sudden death. 24 As it may cause the fatal outcomes, accurate diagnosis is important and careful follow-up is necessary.
Recently, it has been reported that 5-aminolevulinic acid and sodium ferrous citrate improved mitochondrial functions in skin fibroblasts from patients with mitochondrial diseases, which may become a new treatment option for mitochondrial diseases. 25 In our case, as the symptoms gradually worsened and there were no other specific treatment, we initiated 5-aminolevulinic acid as a treatment, which decreased frequency of seizures. Long-term follow-up is necessary to evaluate the efficacy of 5-aminolevulinic acid.

Conclusion
In summary, we reported a case of Leigh syndrome diagnosed by EMB. Efforts to find and conduct the biopsy of affected organs are important to diagnose mitochondrial disease. EMB is a useful diagnostic method in cases with cardiomyopathy when there is a difficulty in diagnosing mitochondrial disease by skeletal muscle biopsy. Accurate diagnosis is important to prevent fatal outcomes and to perform suitable treatment in mitochondrial disease.

Lead author biography
Dr Yuji Maruo received the MD degree from Hokkaido University Graduate School of Medicine (Sapporo, Japan) in 2015. He enrolled in the senior resident program of Department of Pediatrics, Hokkaido University Graduate School of Medicine in 2017. Since 2020, he has been working at the Department of Pediatrics, Japanese Red Cross Kitami Hospital as a paediatrician. His medical interest is in paediatric cardiology.