A case of mitochondrial myopathy and chronic progressive external ophthalmoplegia

Abstract

Mitochondrial myopathy is a group of multi-system diseases in which mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) defects lead to structural and functional dysfunction of mitochondria. The clinical manifestations of mitochondrial myopathy are complex and varied, and the testing for mtDNA and nDNA is not widely available, so misdiagnosis or missed diagnosis is common. Chronic progressive external ophthalmoplegia (CPEO) is a common type of mitochondrial myopathy, which is characterized by blepharoptosis. Here we report a 38-year-old female with mitochondrial myopathy presented with chronic numbness and weakness of the limbs, accompanied by blepharoptosis that was recently noticed. Laboratory and head magnetic resonance imaging (MRI) examinations showed no obvious abnormalities. Muscle and nerve biopsies showed characteristic ragged red fibers (RRFs) and large aggregates of denatured mitochondria. Testing for mtDNA and nDNA showed a known mutation c.2857C>T (p.R953C) and a novel variant c.2391G>C (p.M797I) in the polymerase gamma (POLG)gene, so the patient was diagnosed as mitochondrial myopathy. Clinicians should pay more attention to long-term unexplained skeletal muscle diseases with recent onset blepharoptosis. Histopathologic examination and genetic testing are of great value in the early diagnosis and therapeutic intervention.

Mitochondrial myopathy is a group of clinically heterogeneous muscle degenerative diseases caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA), mainly causing dysfunction of organ systems with high energy requirements, such as the skeletal muscle system^[1-2]. Mitochondrial myopathy often manifests as exercise intolerance, cramps, and fatigue, and can also present with a variety of neurological, muscular, liver, and gastrointestinal symptoms^[3]. Chronic progressive external ophthalmoplegia (CPEO) is a typical clinical manifestation of mitochondrial myopathy characterized by chronic, progressive, bilateral symmetrical extraocular muscle paresis and blepharoptosis^[4]. Here, we report a 38-year-old female with mitochondrial myopathy. She had long-term manifestations of limb weakness and numbness, which was not serious and did not attract attention.

1. Case presentation

A 38-year-old female presented with weakness and numbness of limbs for 3 years and blepharoptosis for 3 months. The patient developed weakness in the limbs and numbness mainly in the distal part of the limbs, but did not go to hospital for further examination. Three months prior to our initial assessment, bilateral ptosis appeared (Figure 1) and could not be alleviated, so she was admitted to our hospital. She had no family history of disease and her parents were healthy. The patient’s consciousness was clear, the answer was fluent, cognitive function was normal. Physical examination revealed that the patient had bilateral drooping eyelids and difficulty opening eyes. Bilateral pupil diameters were both 3 mm, in round with the same size and normal light reflection. Bilateral eye abduction was limited, the rest of the eye movement was normal, with mild intermittent diplopia, without hemianopsia. Examination of the other cranial nerves showed no abnormalities. The muscle strength examination showed weakness of 4 limbs with a Medical Research Council (MRC) scale of V- in the proximal, V- in the distal upper extremities, V- in the proximal, and IV+ in distal in the lower extremities. Muscle tone was normal. Sensory examination showed mild impairment on pain and temperature over both fingers and 5 cm above the ankle joint. Bilateral deep sensation was generally normal, and complex sensation was present and symmetrical in both sides. There was no spinal sensory levels and signs of ataxia. The deep tendon reflexes were hypoactive. No ankle clonus or knee clonus was found, and pathological reflex was negative.

Figure 1. Bilateral ocular ptosis.

Blood routine tests showed elevated creatine kinase (443.3 U/L, reference range: 40.0-200.0 U/L) and other indicators, including lactate (1.80 mmol/L, reference range: 1.42-1.90 mmol/L) and antibodies to HIV and syphilis, were normal. Electromyography (EMG) showed widespread neurogenic changes. The nerve motor conduction velocity test showed that the bilateral tibial nerve conduction velocity was normal (left: 46.3 m/s; right: 46.3 m/s; reference value: >40 m/s) but the amplitude was low (left: 6.5 mV; right: 6.8 mV; reference value: >7 mV). No abnormality was observed in other parts. Sensory nerve conduction test of both sural, median, and ulnar nerves showed no responses (Table 1). F-wave study and repetitive nerve stimulation (RNS) were normal. Quantitative sensory test (QST) showed a decreased threshold of cold sensation in the left lower limb, suggesting that Aδ type nerve fibers may be damaged. The temperature threshold of the remaining limbs was normal. We performed a lumbar puncture and cerebrospinal fluid examination which showed elevated lactic dehydrogenase (LDH) (111.0 U/L; reference value: <40.0 U/L), but no other abnormalities were observed. Cerebrospinal fluid and serum paraneoplastic antibody, serum peripheral neuropathy antibody, blood and urine metabolism screening, serum-free light-chain (sFLC) , IgG4 and M protein were normal. Head MRI and diffusion weighted imaging (DWI) showed no obvious abnormalities. These results did not support the diagnosis of many diseases such as paraneoplastic syndrome, immune-mediated peripheral neuropathy, thyroid disease, and plasma cell disease. Neostigmine test was negative, indicating that the patient’s diagnosis of myasthenia gravis is not supported. Based on the above findings, we suspected that the patient was suffering from mitochondrial myopathy, and the recent blepharoptosis might be a manifestation of CPEO. Histopathologic examination and genetic tests were the next priority for this patient in order to further clarify the diagnosis and differential diagnosis.

Table 1.

Results of nerve conduction test

Nerves	Amplitude/mV	NCV/(m·s-1)
Motor studies
Median	12.7	56.5
Ulnar	17.0	55.8
Tibial (left)	6.5	46.3
Tibial (right)	6.8	46.3
Common peroneal (left)	7.0	45.9
Common peroneal (right)	6.9	49.8
Sensory studies
Median (left)	—	—
Median (right)	—	—
Ulnar (left)	—	—
Ulnar (right)	—	—
Sural (left)	—	—
Sural (right)	—	—

NCV: Nerve conduction velocity; —: Not elicited.

We performed gastrocnemius muscle and sural nerve biopsy after obtaining written consent from the patient. Under the light microscope, a hematoxylin and eosin (HE) staining of the gastrocnemius muscle showed significant differences in size of muscle fibers and visible nuclear aggregation. Gomori staining showed a small amount of RRFs. Reduced nicotinamide adenine dinucleotide (NADH) staining showed mesh-like structure disorder in some muscle fibers and a few target fibers. Succinate dehydrogenase (SDH) staining revealed ragged blue fibers (RBFs). Cytochrome C oxidase/succinate dehydrogenase (COX/SDH) double staining showed only occasional COX-negative and SDH-positive fibers in blue. Oil red O (ORO) staining showed slight increase of lipid droplets in a few muscle fibers (Figure 2A-2F). Under the electron microscope, muscle fibers and myofilaments were found to be necrotic, showing water waviness, and mitochondria in muscle fibers were pyknotic (Figure 3A). Electron microscopy of the nerve tissue showed a decrease in myelinated nerve fibers (Figure 3B). Some myelin sheaths were distorted and irregular and showed focal edema (Figure 3C). Mitochondrial edema was present in some myelinated axons and Schwann cells (Figure 3D).

Figure 2. Light microscopic biopsy sections of gastrocnemius.

A: Size of muscle fibers varies significantly, with small groups of atrophy and nuclear aggregation. B: Gomori staining showed a small amount of ragged red fibers (RRFs). C: Reduced nicotinamide adenine dinucleotide (NADH) staining showed mesh-like structure disorder in some muscle fibers and a few target fibers. D: Succinate dehydrogenase (SDH) staining revealed ragged blue fibers (RBFs). E: Cytochrome C oxidase/succinate dehydrogenase (COX/SDH) double staining showed only occasional COX-negative and SDH-positive fibers in blue. F: Oil red O (ORO) staining showed slight increase of lipid droplets in a few muscle fibers.

Figure 3. Electron microscopic biopsy sections of gastrocnemius (A) and sural nerves (B-D) .

A: Mitochondrial pyknosis in muscle fibers; B: Reduction of myelinated nerve fibers. C: Focal edema in some myelinated axons (asterisks) and mitochondrial edema in the axon (arrow). D: Mitochondrial edema of Schwann cells.

We also performed mtDNA and nDNA tests on gastrocnemius muscle from patients, mtDNA was detected by Long-Range PCR and Whole-exome sequencing (WES) techniques, and nDNA was detected by WES. A known mutation c.2857C>T (p.R953C) and a novel variant c.2391G>C (p.M797I) in the polymerase gamma (POLG) gene were found. In addition, we found no duplication or deletion of mitochondrial genes. The patient’s parents agreed to carry out genetic testing of blood and the result showed c.2857C>T mutation in father and c.2391G>C mutation in mother (Figure 4). c.2857C>T (p.R953C) was low frequency (<0.5%) in the public genome databases (Exome Aggregation Consortium or Genome Aggregation Database) and in healthy controls (PM2). This mutation has been reported^[5-7] in several cases related to mitochondrial diseases, in which the homozygous variant was detected, or the variant was found to be compound heterozygous with other pathogenic variants (PM3). The pathogenic mutation p.Y955C near p.R953C has been reported^[8-9] in several clinical cases of mitochondrial disease (PM1). The deleterious effects of this variant on genes or gene products were predicted by a variety of statistical methods, including conservative predictions (PP3). Thus mutation c.2857C>T (p.R953C) was classified as likely pathogenic (PM1+PM2+PM3+PP3) according to the American College of Medical Genetics and Genomics (ACMG) criteria^[10]. c.2391G>C (p.M797I) was neither reported in public genomic databases nor detected in healthy controls (PM2). The deleterious effects of this variant on genes or gene products were predicted by a variety of statistical methods, including conservative predictions (PP3). Thus, this mutation c.2391G>C was graded as variant of uncertain significance (VUS) (PM2+PP3) based on the standard of ACMG. Based on the above clinical symptoms, histopathologic findings and genetic test results, the patient was diagnosed as mitochondrial myopathy.

Figure 4. Pedigree of the family and sequencing results.

A: Pedigree of the family affected with mitochondrial myopathy; B: Sequencing results of the polymerase gamma (POLG) gene mutation.

We then administered relevant symptomatic supportive treatment to the patient, such as diet therapy, drug therapy, physical therapy. After 1 year of follow-up, the patient’s eyelid ptosis was slightly relieved, the limb weakness and numbness were less severe than previously reported, and the mood was more stable than before. No progression of mitochondrial myopathy-related complications was found. We will continue to follow-up the patient and adjust the treatment plan according to changes in her condition.

2. Discussion

The patient suffered from weakness of limbs and numbness for a long time, but the mild degree did not attract attention. When the blepharoptosis appeared recently, she came to our hospital and was diagnosed as mitochondrial myopathy through muscle and nerve biopsy and genetic test.

Mitochondrial myopathy affects not only skeletal muscle, but also multiple organ systems, such as cardiomyopathy, epilepsy, or stroke-like episodes, whose clinical presentation varies with age, course, and severity of disease^[11]. Symptoms are varied and often non-specific. Therefore, before the appearance of characteristic symptoms, clinicians may miss the diagnosis. POLG mutations are common causes of mitochondrial diseases, especially mitochondrial epilepsy, polyneuropathy, ataxia, and progressive external ophthalmoplegia (PEO)^[12]. Due to the significant heterogeneity and diversity of POLG-related phenotypes or diseases, we summarized some common phenotypes associated with POLG gene mutations according to the age of onset associated with POLG phenotypes, which will help to identify and diagnose POLG-related diseases (Supplementary Table 1, https://doi.org/10.11817/j.issn.1672-7347.2023.220605T1). CPEO is a syndrome of mitochondrial myopathy with specific symptom. Ptosis is often the first symptom of CPEO, and as the disease progresses, defects in eye movement may occur^[13]. The patient reported herein showed limitation of abduction in addition to ptosis on admission physical examination, which was consistent with the above characteristics. CPEO may also present some of the manifestations of peripheral nerve involvement commonly seen in mitochondrial diseases, such as axon-type nerve fiber injury, mainly demyelination, or axon-demyelination mixed type involvement^[14-15]. A sural nerve biopsy may show axonal degeneration or demyelination^[16]. We also biopsied the sural nerve of the patient and found partial myelin irregular and deformed, as well as local myelin edema and axonal mitochondrial edema. Study^[17] investigated 43 patients with CPEO and found that 8% of them had peripheral neuropathy. A prospective investigation^[14] of peripheral nerve involvement in 33 patients with CPEO found that most of them had abnormalities in electrophysiology of sensory nerves, complex action potential (CAP) amplitude and nerve conduction velocity. The patient we reported also had decreased neuromotor conduction amplitude and a decreased threshold of cold sensation in the left lower limb. Alternative diagnoses including myasthenia and paraneoplastic, dysimmune, or nutritional neuropathies were excluded with appropriate testing as described above.

Muscle biopsy is one of the important auxiliary examination methods for mitochondrial myopathy^[18-19]. Muscle biopsies have been found to be necessary in mitochondrial myopathy patients with muscle weakness, and up to 90% of patients have mitochondrial accumulation in muscle fibers composed of typical RRFs^[20-21]. The patient we report also had these typical findings in the gastrocnemius muscle. Due to the patient’s symptoms of numbness, physical examination revealed decreased sensation to large and small fiber modalities, and a nerve biopsy was performed. Study^[22] conducted morphological analysis of sural nerves of 15 patients with mitochondrial myopathy by electron microscopy and found that all patients had neuropathy, but did not have specific manifestations. Whether neuropathy is caused directly by mitochondrial dysfunction or by other pathologic genetic mechanisms remains uncertain. We observed the sural nerve of the patient under the electron microscope, and found that nerve fibers had different degrees of degeneration and mitochondria showed shrinkage and edema.

Genetic test is an important method for diagnosing of mitochondrial myopathy^[23-24]. We performed mtDNA and nDNA tests on the patient’s muscles and found a known mutation c.2857C>T (p.R953C) and a novel variant c.2391G>C (p.M797I) in the POLG gene. POLG is a nuclear DNA-coding enzyme responsible for mtDNA replication^[7]. Pathogenic mutations of POLG were first found in families with autosomal dominant or recessive CPEO and multiple deletions of muscle mitochondrial DNA, and were closely associated with ocular palsy^[25]. In 2004, Luoma et al^[5] identified a patient with c.C2857T mutation in the POLG gene, and the patient also had varying degrees of muscle weakness and blepharoptosis. Unlike our reported case, the patient developed ataxia^[5]. Although c.2391G>C is a novel variant, another similar mutation c.2391G>T was found in a family of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)^[26]. In this family, both the proband and his parents were found to carry the mutation, but their clinical presentations were quite different^[26]. They had varying degrees of muscle weakness, and the proband had bilateral optic nerve atrophy, but no ptosis and ophthalmoparesis^[26]. This suggests that the link between genotype and phenotype of mitochondrial myopathy is not absolutely consistent, and that clinical manifestations may vary widely even if the same mutation is carried in the same family. So, clinicians should adopt individualized diagnosis and treatment for mitochondrial myopathy patients, as different individuals receiving the same treatment may have different outcomes.

There are currently no Food and Drug Administration-approved drugs available to treat mitochondrial diseases^[27]. Current treatment for mitochondrial myopathy mainly focuses on early detection, symptomatic treatment, and delaying progression. For patients with mitochondrial myopathy, there are currently several approaches or clinical trials underway, such as ubiquinone and ubiquinone analogues, regulating lipid dynamics, nutritional supplements, exercise, which may be beneficial. Other alternative therapies, including at the molecular level, as well as reproductive options, are also being actively developed for patients with mitochondrial myopathy, which may offer hope for these patients in the future^[11].

For patients with CPEO, surgery remains the primary treatment. In general, the surgical technique for repair of ptosis depends on the function of levator palpebrae superioris (LPS). In the presence of mild LPS injury, resection along the upper tarsal bone and/or advance of the levator tendon is preferred. For the more severe LPS injury common in CPEO patients, frontalis suspension, in which the upper eyelid is attached to the frontalis muscle using silicone or fascia lata suspensions, is preferred. However, non-surgical treatment such as eyelid crutches can be used for patients with CPEO, especially for visually significant ptosis^[28]. When binocular diplopia and strabismus occur, smaller angle ocular misalignments may be amenable to correction with prismatic glasses^[11]. In addition, we reviewed some recent clinical trials. KH176 acts as a potent intracellular redox modulating agent targeting the reactive oxygen species as demonstrated in a number of in vitro and in vivo assays^[29-30]. A phase II clinical trial evaluating the safety and efficacy of KH176 (100 mg, twice daily) in patients with CPEO has been initiated (clinical trials.gov NCT: 04604548). The magnetic levator prosthesis (MLP) is an external device that makes use of a newer class of permanent magnets [made of alloys of neodymium (Nd), iron (Fe) and boron (B)] to restore eyelid movement. The Kinesiotape Frontalis Sling (KTFS) involves the use of tape to help facilitate levator muscle contraction to open the eyelid. A randomized crossover trial comparing the 2 non-operative treatments for severe ptosis in CPEO patients is also ongoing (clinical trials.gov NCT:04678115). Therefore, for the patient we reported, in addition to symptomatic treatment to delay disease progression and long-term follow-up, we can also take a comprehensive approach based on surgical treatment to improve the visual function.

In conclusion, we report on a 38-year-old Chinese woman with weakness of limbs and numbness who recently developed a ptosis. Pathological biopsy and mtDNA and nDNA tests confirmed mitochondrial myopathy. Besides, we found a known mutation c.2857C>T (p.R953C) and a novel variant c.2391G>C (p.M797I) in the POLG gene in this patient. Clinicians should pay more attention to patients with long-term manifestations of unexplained systemic skeletal muscle involvement, whose blepharoptosis appears recently, and perform genetic testing as soon as possible.

Contributions: LIU Haokun

Drafted and revised the paper; Gao Ming Analyzed the clinical data, proofread and revised the paper; SUN Qiying, CHEN Si, YANG Huan, and LI Jing Collected and analyzed the clinical data; LUO Yuebei and LI Qiuxiang Collected and analyzed the pathological data; YANG Guang Designed the study, proofread and revised the paper. All authors have approved the final version of this manuscript.

Appendix.

Supplementary Table 1 Common POLG-related phenotypes or diseases according to age of onset

Common age of onset	Syndrome	References
Neonatal or infancy	MCHS	Wong et al[1] Hikmat et al[2]
Infancy or childhood	AHS	Harding et al[3] Wolf et al[4]
	Leigh syndrome	Scalais et al[5]
Adolescent or young adult	MELAS	Ohama et al[6] McPherson et al[7]
	MNGIE	Prasun et al[8] Huang et al[9]
	MIRAS	Palin et al[10] Mignarri et al[11]
	SCAE	Gramstad et al[12]
	MEMSA	Rajakulendran et al[13]
	Distal myopathy	Pitceathly et al[14] Giordano et al[15]
Adult	PEO (Autosomal dominant)	Bastian et al[16]
	PEO (Autosomal recessive)	Habek et al[17]
	PEO (sporadic)	Martikainen et al[18] Agostino et al[19]
	ANS	Lovan et al[20]
	SANDO	Li et al[21] McHugh et al[22]
Adult or elderly	Parkinsonism	Miguel et al[23] Davidzon et al[24] Rempe et al[25]
	Premature menopause	Pagnamenta et al[26] Duncan et al[27]
	Cataract	Castiglioni et al[28]

POLG: Polymerase gamma; MCHS: Myocerebrohepatopathy spectrum; AHS: Alpers-Huttenlocher syndrome; MELAS: Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes; MNGIE: Mitochondrial neurogastrointestinal encephalopathy; MIRAS: Mitochondrial recessive ataxia syndrome; SCAE: Spinocerebellar ataxia with epilepsy; MEMSA: Myoclonic epilepsy myopathy sensory ataxia; PEO: Progressive external ophthalmoplegia; ANS: Ataxia neuropathy spectrum; SANDO: Sensory ataxia neuropathy dysarthria and ophthalmoplegia.

Funding Statement

This work was supported by the Natural Science Foundation of Hunan Province (2021JJ41045, 2021JJ31093) and the Natural Science Foundation of Changsha (kq2014279), China.

Conflict of Interest

The authors declare that they have no conflicts of interest to disclose.

Note

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