16 year male with ptosis first noted in school photographs 5 years earlier. He was an otherwise healthy
young man with a history of learning difficulties requiring special education. The ptosis persisted
despite attempted surgical repair X 2. Ophthalmologic examination uncovered concomitant ophthalmoplegia
and retinal pigment changes. Mitochondrial DNA studies on his blood were normal. The ophthalmologist
then referred him to a geneticist who suggested a muscle biopsy to further evaluate his neuromuscular
Mitochondrial Muscle Pathology
- Light microscopy (frozen muscle):
- Gomori trichrome – scattered ragged red fibers accounting for 3-5% of all myofibers; no endomysial fibrosis
- Oxidative enzymes: Increased subsarcolemmal NADH and SDH staining scattered fibers; no increased vascular SDH staining; COX staining negative in 80% of fibers
- Additional findings not included in photographs:
- PAS - Increased glycogen in many fibers
- ORO - coarse lipid droplets in increased numbers in many fibers
- ATPase – 75% type 2 fiber predominance
- Electron microscopy: Normally formed sarcomeres. Extensively abnormal mitochondria including concentric whorls and parallel packed arrays of cristae and paracrystalline inclusions
The ragged-red fibers (RRFs), and partial COX deficiency in light microscopy, and the mitochondrial
paracrystalline inclusions noted on electron microscopic exam provide strong evidence for a mitochondrial
myopathy. RRFs occur however in a spectrum of mitochondrial and non-mitochondrial disorders and do not
point to a specific diagnosis (Table 1). The partial deficiency in COX staining, a finding indicative of
respiratory chain complex IV malfunction, does little to narrow the differential. These findings
combined with the clinical picture of chronic external ophthalmoplegia, ptosis, and retinitis pigmentosa
are however virtually diagnostic of a limited spectrum of disorders including Kearns-Sayre syndrome and
chronic external ophthalmoplegia (CPEO). Mitochondrial enzyme and DNA studies performed on snap frozen
muscle tissue revealed low levels of respiratory chain complex I, III and IV activity and a 5 kb deletion
of mitochondrial DNA to add confirmatory evidence for this diagnosis.
Ragged Red Fibers
|KSS ||Myositis |
|CPEO ||Dystrophies |
|MERRF ||AZT Rx |
|MELAS ||Chronic Steroids|
|Pearson Syndrome||Aging |
Diagnosis - Kearns-Sayre syndrome
The clinicopathologic triad of ptosis, ophthalmoplegia,
and ragged-red fiber myopathy identifies a group of disorders included under the rubric of CPEO
syndromes1 (Table 2). Mitochondrial DNA deletions are frequently identified in these
patients1,2; less commonly they may manifest mtDNA duplications or point
mutations1. Of note, mitochondrial DNA deletions, unlike point mutations, are rarely
detectable in blood samples.
|Onset <20 years|
One of following:
Complete heart block
CSF protein >100mg/dl
|Onset adolescent or young adult|
Extraocular symptoms variable
Lack KSS defining symptoms
Sensorineural Hearing Loss
The straightforward diagnosis rendered in this case is unfortunately not the norm for pediatric
muscles biopsied to evaluate for mitochondrial myopathies. RRFs occur rarely in young children and COX
enzyme negative fibers are minimally more common3,4. Less specific histologic features such
as increased subsarcolemmal oxidative enzyme (NADH, SDH) staining, increased lipid and/or glycogen and
type 1 or 2 fiber predominance may suggest a mitochondrial abnormality4, but are as likely to
occur in other forms of muscle/metabolic disease. The histopathology of the muscle biopsy material may
also be entirely normal4; in our own material 50% of cases with clinical and respiratory
enzyme chain analysis evidence of a mitochondrial disorder displayed entirely normal muscle
morphology. The histologic evaluation of muscle therefore may, if one is lucky, provide evidence to
support a diagnosis of mitochondrial myopathy but absence of such finding cannot be used to exclude the
Electron microscopy has classically played a major role in the pathologic evaluation for mitochondrial
disease with variety of ultrastructural changes described in mitochondrial disorders 5,6(Table
3). These ultrastructural changes are in fact identified in a higher number of suspected mitochondrial
myopathy cases than are the light microscopic findings of RRFs and deficient COX staining4,7.
Unfortunately these changes are not as specific as one would like and similar changes have been observed
in muscular dystrophy, neurogenic atrophy, myositis and in otherwise seemingly normal
muscle4. Ultrastructural findings therefore cannot be considered diagnostic of
mitochondrial disorders but can severe as another piece of evidence in the quest for a firm diagnosis.
Enlarged abnormally shaped
Sparse, concentric or bizarre cristae
Crystalloid (paracrystalline) inclusions
A major purpose of the muscle biopsy in these cases is to obtain analysis of the mitochondrial
respiratory chain function and mtDNA. Although a finding of low respiratory chain enzyme activity seems
as though it should lead to a straightforward diagnosis of a mitochondrial myopathy, the respiratory
chain enzyme studies in reality aren't that straightforward. The most accurate and complete analysis
requires study of the respiratory chain function of whole mitochondrial harvested from fresh muscle
tissue8. This method is however possible only in a limited number of institutions and most
muscle is studied after snap freezing in liquid nitrogen. Mitochondrial enzyme activity displays a wide
range of normal values, but is rarely completely absent even in the face of significant
defects3. Defining the border between normal and abnormal thus becomes an often arbitrary
determination. Add to this a normally decreasing activity with age (e.g. 50% decrease in COX activity
between 4 and 19 years9) combined with an inherent difficulty in obtaining age-matched normal
control tissues. Finally factor in the variable handling muscle biopsy tissue undoubtedly receives
during removal from the patient, snap freezing, and transporting to the specialty laboratory performing
the testing and it is not hard to imagine how fraught with error the interpretation of mitochondrial
enzyme analysis can become.
The study of mitochondrial DNA from blood or sampled uscle could add specificity to the diagnosis.
Unfortunately only 10-20% of mitochondrial disorder in children are due to mtDNA
mutations10,11. Increasing numbers of nuclear DNA mutations resulting in mitochondrial
dysfunction are now being uncovered suggesting a potential for more readily availabe diagnostic studies
in the near future.
Given all these uncertainties, how can one arrive confidently at a diagnosis of mitochondrial
myopathy? A variety of diagnostic criteria, combining clinical, pathologic, mitochondrial enzyme
analysis, and mtDNA data have been proposed to address this difficulty7,12-14. The United
Mitochondrial Disease Foundation and allied groups ( www.mitosoc.org & www.mitoresearch.org) are
currently working on creating consensus criteria for diagnosing mitochondrial disorders – watch those
- Shoffner JM. Mitochondrial myopathy diagnosis. Neurol Clin 2000;18(1):105-123.
- Moraes CT, DiMauro S, Zeviani M, et al. Mitodchondrial DNA deletions in progressive external
ophthalmoplegia and Kearns-Sayre syndrome. NEJM 1989;320:1293-1299.
- Vogel H. Mitochondrial myopathies and the role of the pathologist in the molecular era. J
Neuropathol Exp Neurol 2001;60(1):217-227.
- Rollins S, Prayson RA, McMahon JT, Cohen BH. Diagnostic yield of muscle biopsy in pateints with
clinical evidence of mitochondrial cytopathy. Am J Clin Pathol 2001;116:326-330.
- Lindal S, Lund I, Torbergsen T, et al. Mitochondrial diseases and myopathies: a series of muscle
biopsy specimens with ultrastructural changes in the mitochondria. Ultrastruc Pathol 1992;16:263-275.
- Kyriacou K, Mikellidou C, Hadjianastasiou A, et al. Ultrastructural diagnosis of mitochondrial
encephatlomyopathies revisited. Ultrastruc Pathol 1999;23:163-170.
- Tulinius MH, Holme E, Kristiansson B, Larsson N-G, Oldfors A. Mitochondrial encephalomyopathies in
childhood. I. Biochemical and morphologic investigations. J Pediatr 1991;119:242-50.
- Thorburn DR, Smeitink J. Diagnosis of mitochondrial disorders: clinical and biochemical approach.
(Workshop report). J Inhert Meta Dis 2001;24:312-316.
- Lefai E, Terrier-Cayre A, Vincent A, et al. Enzymatic activities of mitochondrial respiratory
complexes from children muscular biopsies. Age-related evolutions. Biochim Biop;hys Acta
- Borchert A, Wolf NI, Wilichowski E. Current conceptgs of mitochondrial disorders in childhood. Sem
Pediatr Neurol 2002;9:151-159.
- Smeitink J, van den Heuvel L, DiMauro S. The genetics and pathology of oxidative phosphorylation.
Nat Rev Genet 2001;2:342-352.
- Walker UA, Collins S, Byrne E. Respiratory chain encephaloyopathies: a diagnostic classification.
Eur Neurol 1996;36:260-267.
- Bernier FP, Boneh A, Dennett X, et al. Diagnostic criteria for respiratory chain disorder in adultgs
and children. Neurol 2002;59:1406-1411
- Wolf NI, Smeitink JAM. Mitochondrial disorders. A proposal for consensus diagnositc criteria in
infants and children. Neurol 2002;59:1402-1405.