Diagnostic value of mitochondrial DNA mutation ...

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Oct 13, 2004 - in modified Gomori trichrome stain and cytochrome C- oxidase (COX)-negative fibers in COX stain. The ma- jority of these disorders is due to ...
Graefe’s Arch Clin Exp Ophthalmol (2005) 243:380–382

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DOI 10.1007/s00417-004-1000-1

Thorsten Okulla Wolfram S. Kunz Thomas Klockgether Rolf Schrder Cornelia Kornblum

Received: 8 April 2004 Revised: 14 July 2004 Accepted: 20 July 2004 Published online: 13 October 2004  Springer-Verlag 2004 T. Okulla · T. Klockgether · R. Schrder · C. Kornblum ()) Department of Neurology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany e-mail: [email protected] Tel.: +49-228-2875712 Fax: +49-228-2875024 W. S. Kunz Department of Epileptology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany

Diagnostic value of mitochondrial DNA mutation analysis in juvenile unilateral ptosis

Abstract Purpose: To highlight the diagnostic relevance of mitochondrial DNA (mtDNA) mutation analysis in acquired juvenile unilateral upper eyelid ptosis. Methods: A 13-year-old boy presented with acquired, slowly progressive unilateral ptosis. We performed ophthalmological and neurological examinations, laboratory testing, skeletal muscle biopsy including histological and histochemical investigations, biochemical analysis of respiratory chain enzymes in skeletal muscle homogenate and molecular genetic testing of skeletal muscle DNA. Results: Though clinical, laboratory, histological and biochemical analyses did not reveal any

Introduction Mitochondrial disorders are a heterogeneous group of diseases characterized by wide phenotypic variation ranging from severe childhood multiorgan failures to adultonset nonsyndromic disorders with tissue-specific involvement [4]. Mitochondrial dysfunction may result from abnormalities in nuclear genes or from primary mutations in the mitochondrial DNA (mtDNA) such as rearrangements or point mutations. Mitochondrial diseases have in common defective oxidative phosphorylation resulting in energy depletion of the cells. As a consequence, tissues with high-energy requirements such as the retina, central nervous system, and skeletal muscle are generally most affected [2]. Most common neuro-ophthalmic abnormalities seen in mitochondrial disorders are bilateral optic neuropathy, external ophthalmoplegia with ptosis, pigmentary retinopathy, and retrochiasmal visual loss [1].

hints suggesting a mitochondrial cytopathy, molecular genetic testing by Southern blot analysis of total DNA from skeletal muscle tissue showed a 5.8 kb mtDNA deletion thus proving the diagnosis of mitochondrial chronic progressive external ophthalmoplegia (CPEO). Conclusions: In patients with unexplained acquired juvenile unilateral ptosis, an underlying mitochondrial cytopathy should be considered even in cases of inconspicuous ancillary examinations comprising skeletal muscle histology and biochemistry. To establish the diagnosis, molecular genetic testing of DNA derived from skeletal muscle tissue is essential in those patients.

Classification of mitochondrial diseases may be made on the basis of the genetic defect alone or on the basis of the clinical presentation. Chronic progressive external ophthalmoplegia (CPEO) as a mitochondrial disease entity is characterized predominantly by a slowly progressive bilateral ocular immobility and ptosis and may be associated with other neurological or systemic symptoms (CPEO plus). In approximately 50% of CPEO patients, mtDNA analysis of skeletal muscle tissue reveals single large-scale mtDNA deletions that arise sporadically [1]. However, CPEO may be as well due to maternally inherited or sporadic mtDNA point mutations, duplications, or mutations in nuclear genes resulting in autosomal disorders of intergenomic communication [7]. Skeletal muscle histology and biochemistry normally show characteristic abnormalities in CPEO patients.

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Case report Material, methods, and results A 13-year-old boy presented to our department with a 12-month history of gradually progressive, nonfluctuating, unilateral upper eye lid ptosis. His past medical and family history were unremarkable. Neurological examination, cardiological workup, and extensive laboratory testing including creatine kinase serum levels and acetylcholine receptor autoantibodies were normal. Thyroid dysfunction and autoimmune thyroid disease were excluded. Magnetic resonance imaging of the brain and orbit were unremarkable. However, electromyography of the patient’s left vastus lateralis muscle showed positive sharp waves but no signs of myopathic or chronic neurogenic changes. Further neurophysiological workup including repetitive nerve stimulation as well as intravenous injection of edrophonium chloride (Tensilon test) gave no evidence of a neuromuscular junction disorder like myasthenia gravis. External ophthalmological examination revealed an upper lid ptosis of the left eye with the pupil covered more than 50% and the lid fold no longer visible. The function of left levator palpebrae was severely reduced. Both pupils were briskly reactive to light. Best-corrected Snellen visual acuity was 20/16 in the right eye (OD) and 20/20 in the left eye (OS) with holding upper lid. Anterior segment examination and dilated funduscopy were within normal limits. Muscle balance evaluated by the Cover test showed orthotropia. Eye motility examinations revealed a mild adduction deficit (score 1) more pronounced in the left eye (score from 0 to 4 with 0 indicating normal motility and 4 indicating no motility). The patient demonstrated full-range upgaze and downgaze motility (score 0). Horizontal saccades were normal except for the OS adduction saccade that was slowed slightly. Bell’s phenomenon was positive in both eyes. To address the issue of an underlying myopathy in otherwise unexplained ptosis, we performed open skeletal muscle biopsy from right vastus lateralis muscle followed by histological, histochemical, and biochemical workup. Histological and histochemical examinations were normal. Biochemical analysis of skeletal muscle homogenate showed regular respiratory chain enzyme activities. However, molecular genetic analysis of total DNA from skeletal muscle by Southern blot [8] revealed a heteroplasmic single largescale mtDNA deletion with a deletion size of 5.8 kb and a degree of heteroplasmy of 32% (Fig. 1).

Discussion Isolated acquired unilateral ptosis in adolescence as seen in our 13-year-old patient is a relatively rare symptom when compared to congenital ptosis, bilateral ptosis in later adulthood, or ptosis associated with further neurological and systemic signs [5]. However, the common differential diagnoses of acquired unilateral ptosis (thyroid dysfunction, lesions of the third cranial nerve, Marcus-Gunn jaw-winking ptosis, Horner syndrome, myasthenia gravis, mechanical ptosis secondary to lid tumors or blepharophimosis syndrome, botulism) should be excluded in each patient. Since ptosis may be a symptom of an underlying neuromuscular disorder, particularly of a mitochondrial cytopathy, further neurological evaluation is required when symptoms remain unexplained. Routine laboratory

Fig. 1 Southern blot of PvuII-cleaved total DNA from skeletal muscle with a probe recognizing mitochondrial DNA (mtDNA). Blots were performed with about 1 mg total DNA isolated from skeletal muscle hybridized with a digoxigenin-labeled probe isolated from human skeletal muscle mitochondria. Lane 1 (M), molecular-weight markers (HindIII-cleaved l-DNA); lane 2 (arrow), patient DNA indicating a single mtDNA deletion with a deletion size of 5.8 kb and a grade of heteroplasmy of 32%; lane 3–6, normal controls

testing for mitochondrial disorders is limited [1]. Thus, diagnostic procedure in suspected mitochondrial myopathy usually comprises skeletal muscle biopsy followed by histological, histochemical, and biochemical workup of fresh muscle biopsy tissue. Skeletal muscle biopsies in patients with common mitochondrial disorders such as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy and ragged red fibers (MERRF), CPEO, or Kearns-Sayre syndrome (KSS) characteristically show ragged red fibers in modified Gomori trichrome stain and cytochrome Coxidase (COX)-negative fibers in COX stain. The majority of these disorders is due to tRNA point mutations or deletions of the mtDNA. Defective oxidative phosphorylation is mirrored in deficits of respiratory chain enzyme complex activities in biochemical analysis of skeletal muscle homogenate [8]. In our patient, histological and biochemical examinations of skeletal muscle biopsy were inconspicuous of a mitochondrial disease. In routine workup, diagnostic procedure would not have been continued beyond this point. Nevertheless, we completed diagnostic workup by molecular genetic analysis of skeletal muscle tissue that finally revealed a heteroplasmic, single, large-scale mtDNA deletion. Results of molecular genetic testing proved the diagnosis of a mitochondrial cytopathy in our patient, and we clinically suggested an early disease stage of CPEO. CPEO was first described in 1867 by Albrecht von Graefe as a clinical syndrome and disease entity [6]. Most cases of CPEO arise sporadically, and in approximately 50% of the affected patients, mtDNA analysis that

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is best performed in skeletal muscle tissue reveals a single deletion or duplication [1, 10]. CPEO patients are typically heteroplasmic for these rearrangements, and the mutant accounts for 20–90% of total skeletal muscle mtDNA [1]. mtDNA deletions can be found in patients with different clinical phenotypes, and different mtDNA rearrangements and grades of heteroplasmy have been shown in patients with similar clinical features [10]. Since mitochondrial disorders present with a broad clinical spectrum, often with a mild beginning, heterogeneous time course, and high genotype/phenotype variability, multiorgan affection is neither mandatory nor necessarily present at initial diagnosis [9]. As a consequence, the final correct diagnosis is often delayed. However, due to a remarkable progress in the understanding of etiology and molecular genetic background, mitochon-

drial cytopathies can now be diagnosed in the early stages of the disease [3]. In clinical practice, some of the most frequent and early presentations of mitochondrial disorders such as ptosis, pigmentary retinopathy, or eye motility deficits are first seen by ophthalmologists. In any case of unexplained acquired ptosis, mitochondrial cytopathy should be suspected. Appropriate diagnostic workup is important to allow accurate diagnosis early thus permitting adequate counseling on potentially life-threatening health issues such as cardiac complications. Our case indicates that molecular genetic testing of DNA derived from skeletal muscle tissue may be essential even in patients with mild or unusual clinical symptoms and unremarkable histological and biochemical skeletal muscle biopsy results in order to establish the correct diagnosis of CPEO.

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