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vastus lateralis muscle were analysed; one had typical ragged red fibers and focal cytochrome c oxidase deficiency and the other was completely normal.
Copyright c Munksgaard 1999 ACTA NEUROLOGICA SCANDINAVICA IssN 0001-6314

A d a Neurol Scand 1999: 99: 125-129 Primed in U K - all rights reserved

Case report

Tissue mosaicism in the skeletal muscle and sural nerve biopsies in the MELAS syndrome Huang C-C, Chu C-C, Pang C-Y, Wei Y-H. Tissue mosaicism in the skeletal muscle and sural nerve biopsies in the MELAS syndrome. Acta Neurol Scand 1999: 99: 125-129. C Munksgaard 1999.

We describe a clinically full-blown MELAS patient, who had an A3243G point mutation of mitochondrial DNA (mtDNA) in muscle and blood cells, and his family members. From the proband two muscle biopsies from the vastus lateralis muscle were analysed; one had typical ragged red fibers and focal cytochrome c oxidase deficiency and the other was completely normal. He also had a peripheral neuropathy confirmed by nerve conduction velocity and sural nerve biopsy studies. Axonal degeneration, relative loss of large myelinated fibers and paracrystalline inclusion bodies in the Schwann cells were noted. Intriguingly, the A3243G mutation of mtDNA was not found in the sural nerve biopsy. Therefore, we conclude that tissue mosaicism is present in the muscle fibers and that the mtDNA mutation may not be detected in the nerve involved as proved by pathology. We also suggest that the involvement of specific tissues in patients with mitochondrial diseases should be further determined by single fiber mtDNA analysis.

Involvement of the peripheral nerves is not uncommon in mitochondrial encephalomyopathies including mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy and ragged red fibers (MERRF), Kearns-Sayre syndrome (KSS), and neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) (1-10). The peripheral neuropathy is usually axonal (3, S), sometimes demyelinating (2, 3 , 5, 11, 12) or occasionally hypertrophic, which suggests that mitochondrial dysfunction may exist in either axon, myelin or Schwann cells. The correlation among clinical features, nerve conduction study, sural nerve biopsy and mtDNA mutation has been rarely investigated. However, a positive correlation is usually speculated. In this communication, we report the clinical manifestation, the pathological changes in muscle and sural nerve biopsies and mtDNA study in a patient with full blown MELAS syndrome, and his family members.

C.-C. Huang’, C.-C. Chu’, C.-Y. Pang’, Y.-H.Wei’ ‘Department of Neurology, Chang Gung Memorial Hospital and Chang Gung University, ’Department of Biochemistry and Center for Cellular and Molecular Biology, National Yang-Ming University. Taipei. Taiwan

Key words MELAS, paracrystalline inclusions. molecular genetic study tissue mosaicism. sural nerve biopsy Chin Chang Huang, Department of Neurology, Chang Gung Memorial Hospital. 199 Tung Hwa North Road, Taipei. Taiwan Accepted for publication September 9.1998

Materials and methods Report of a case

The proband (Fig. 1, 111-1) is a 27-year-old man with body weight of 37 kg and height of 152 cm. In July 1993, he developed headache, nausea, vomiting and blurred vision. The symptoms disappeared after hospitalization for 10 days. In July 1995, another episode of nausea, vomiting, headache, blurred vision, disorientation to time and place, slurred speech and then conscious disturbance was noted. In September, generalized convulsion, renal function impairment, bilateral limb weakness and distal numbness developed. He was referred to our hospital in November and neurological examination revealed that he had decreased output of speech, and dull response to stimuli, but he could still understand verbal commands. In addition, he also had a right-sided hemiparesis, diminished tendon reflexes, bilateral neurosensory hearing impairment and urine and stool incontinence. 125

Huang et al. Table 1. Clinical manifestations and mitochondria1 DNA mutation in the subjects of

I

the MEIAS family ~~

Subjects

tl 0

@ M

3

11-3 b

b

b

4

111-1 r n

n

b

b

b

+

-

c 1159 bp c

591 bp 568 bD

Fig. I . Restriction analysis of mtDNAs from various tissues of the subjects in the MELAS family. A 1159 base pair (bp) polymerase chain reaction (PCR) product was obtained from each of the indicated tissues and a fraction of the PCR product could be cleaved into two comigrating fragments (591 bp and 568 bp) in the tissue of the family members of the maternal lineage except for subject 11-3 and the sural nerve biopsy of subject 111-1. b=blood m=muscle; n=sural nerve; +=positive control; -=negative control; M=marker.

Histochemical studies of the left vastus lateralis muscle revealed normal fiber morphology. In addition, there were no ragged red fibers in Gomori-trichrome stains and hematoxylin-eosin stains. The cytochrome c oxidase and succinate dehydrogenase stains also showed normal activities in the muscle. However, when we reviewed the previous muscle biopsy performed from the right leg in another hospital 6 months ago, we found an increased variation of muscle fiber diameter and increased amount of connective tissue in endomysial space, suggesting myopathic changes. About 5 % of all muscle fibers containing ragged red fibers were identified with the Gomori-trichrome stains. Cytochrome c oxidase (COX) stains showed decreased activity in most muscle fibers indicating a focal COX deficiency. Electroencephalography (EEG) showed continuous diffuse theta waves. The brainstem auditory evoked potentials revealed absence of waves I and 111 following each ear stimulation indicating bilateral neurosensory hearing impairment. The motor nerve conduction study disclosed decreased amplitudes of compound muscle action potentials and bilateral slowing of nerve conduction velocities in the peroneal nerves. The sensory nerve conduction study showed decreased amplitudes of sensory nerve action potentials in the right median and ulnar nerves. These data indicated axonal degeneration in peripheral nerves. The brain computed tomography (CT) scan exhibited mild enlargement of lateral ventricles and prominent cerebral cortical and cerebellar atrophy. Furthermore, there were low density lesions in the right parietal lobe. The brain magnetic resonance images (MRI) revealed 126

Agelsex Height (cm) Weight (kgl Headache Vomiting Seizures Stroke-like episodes Hearing impairment Cortical blindness Hemiparesis Point mutation at mtDNA position 3243 Blood (Yo) Muscle (%) Nerve (%)

11-1 47/F 142

47

+

11-3

111-1

4O/F 158 59

27/M 141 45

+

+ + + + +

~~

111-2 25JF 152 45

+

111-3

111-4

23lM 163 63

8/F 113 20

+(24)* NA NA

+(66)* NA NA

+ +(23)* NA NA

i0)" NA NA

+

(35)" NA NA

"Numbers in parentheses represent percentages of the A3243G mutant mtDNA: NA=not available.

diffuse bilateral lesions in the frontal, parietal, temporal and occipital lobes with low signal intensities in TI-weighted images (T,W) and high signal intensities in T,-weighted images (T2W) and proton-weighted images (PW). There was a mild cortical enhancement after injection of the contrast medium. After anticonvulsant therapy and conservative treatment, the patient recovered slowly. On February 27, 1996, another episode of blurred vision developed again. Generalized tonic and clonic seizures were noted with focal onset appearing from his right hand. He was confused as far as time, place and person were concerned. Follow-up brain MRI scan on March 2, 1996 showed hypointensity lesions on T,W, and hyperintensity lesions on T,W of the cortex bilaterally and subcortical areas of the parietal and right occipital lobes. EEG showed moderate to marked abnormality with periodic lateralized epileptiform discharges over the right hemisphere. The brain MRI changes rapidly returned to normal 3 weeks later. A sural nerve biopsy was performed in the proband in November 1995. Family survey

Table 1 summarizes the clinical features of the MELAS family. The proband's mother (Fig. 1,II-1) is a 47-year-old short and thin woman. She had no other clinical features of MELAS except bilateral hearing impairment, headache and short stature with a height of 142 cm and body weight of 47 kg. The sister of the proband's mother (Fig. 1,II-3) is a

Tissue mosaicism in MELAS syndrome

40-year-old healthy woman without any MELAS features. The proband’s brother (111-3) and sisters (111-2 and 111-4) also did not have any feature of the MELAS syndrome. Methods

S u r d nerve biopsy - Approximately 2 cm of the nerve was obtained, fixed in a 3% glutaraldehyde solution and divided into 2 segments, one for epoxy sections and the other for teased fiber preparation. The first segment was washed in a buffer solution, fixed in 1YOosmium tetroxide, dehydrated and then embedded in epoxy resin. Semithin sections of 0.6 pm were stained with toluidine blue and examined under a light microscope. The size of each myelinated nerve fiber in these fascicles were measured in a morphometric analyzer (LEICA Q500MC image processing and analysis system). After a suitable field was determined, ultrathin sections were made by an ultramicrotome and then stained with uranyl acetate and lead citrate. The ultrathin sections were studied with a JOEL JEM200EXII electron microscope. The second segment was also fixed in 1% osmium tetroxide and dehydrated. About 100 nerve fibers were teased under a dissecting microscope and observed in a light microscope. Molecular analysis of miDNA - Total DNA was extracted from various tissues including blood, muscle and/or nerve of the family subjects in the MELAS family according to the standard method described previously (13, 14). For restriction analysis for the 3243-mutation, a 1159 base pair segment ranging from nt 2678 to nt 3836 was amplified by the polymerase chain reaction (PCR) technique. After amplification for 30 cycles, the PCR product was digested with the restriction enzyme ApaI which recognizes the nt 3243 mutation and gave rise to 2 additional fragments (591 and 568 bp) if the mutation is present. The ApaI-digested polymerase chain reaction products were subjected to electrophoresis on a 1.5% agarose gel at 100 V for 2 h. After electrophoresis, the gel was soaked in double distilled water containing 0.5 pg/ml ethidium bromide for 20 min and was subsequently destained in double distilled water with slow agitation. The gel was then photographed under transillumination with short wavelength UV radiation. Ethidium bromide is intercalated into DNA molecules and the complex thus formed emits fluorescence on UV radiation. The intensity of fluorescence is proportional to the total amount of DNA in each band in the agarose gel. Thus after photography the

negative film was scanned with a densitometer (Molecular Dynamics, Sunnyvale, CA), and the relative quantities of the mutant DNA (comigrating ApaI-digested fragments) and wild-type DNA (uncut fragment) were estimated by the ratios of their intensities on the negative film. Results

Sural nerve biopsy

The sural nerve biopsy specimen consisted of 6 fasciculi. Light microscopic examination showed a reduction of fiber density especially in large myelinated fibers. There were some degenerating axons with dense axoplasm and some fibers containing thin myelin sheaths. A regenerating cluster was found. Morphometric analysis confirmed a decrease of myelinated fiber density (4650/ mm’, reference ranges 6000-10,000/mm2). At ultrastructural examination, an accumulation of variable sizes of mitochondria and paracrystalline inclusion bodies was seen in the Schwann cells (Fig. 2). rntDNA analysis

The mtDNA analysis revealed an A to G transition at nucleotide position 3243 (A3243G) in the blood cells and muscle of the proband. The proportions of the A3243G mutant mtDNA were 58% in blood cells and 41% in the muscle. However the mtDNA mutation was not found in the sural nerve specimen. The A3243G mutant mtDNA was found in the blood cells of the proband’s mother with a proportion of 23%. There was no A3243G mutant mtDNA in the blood cells of the subject 11-3. However, the blood cells contained the A3243G mutant mtDNA with a proportion of 35% in subject 111-2, 24% in subject 111-3 and 66% in subject 111-4 respectively. Discussion

The present study established a MELAS family with various clinical presentations from typical features of headache, seizures, strokes, hemiparesis, cortical blindness and hearing impairments to few or even no symptoms among the relevant family members. The proportion of the A3243G mutant mtDNA varied from 23% to 66% in the blood cells of the analysed individuals. The proband had peripheral neuropathy as judged from the clinical manifestations, nerve conduction study and sural nerve biopsy. The sural nerve biopsy also revealed a characteristic finding of paracrystalline inclusions in the cytoplasm of Schwann’s cells. Molecular genetic study revealed 127

Huang et al.

Fig. 2. Electronmicroscopic study of the sural nerve biopsy from the MELAS patient. (A) An accumulation of mitochondria with variable sizes and myelin degeneration, (B) paracrystalline inclusion bodies with vacuoles in the cytoplasm of a Schwann cell.

the MELAS-specific A3243G mutation of mtDNA in blood cells and in the muscle of the proband. However, this mtDNA mutation was not found in the biopsied sural nerve specimen. mtDNA analysis of peripheral nerve biopsies from patients with MELAS syndrome has rarely been reported (3, 10, 15, 16). Love et al. (15) demonstrated a MELAS patient with polyneuropathy and the A3243G mtDNA mutation with a proportion of 40% to 60% in the peripheral nerves. However, the electrophysiological and pathological studies were not described. Shiraiwa et al. (16) reported another MELAS patient with 90% mutant mtDNA in the sural nerve. Again, the clinical pictures and the pathological studies were not shown. In a recent study, we found that 80% of the mtDNA in the sural nerve of a MERRF patient, in whom a prominent axonal polyneuropathy was found, harboured the A8344G mutation (10). To the best of our knowledge, there has been no report of a patient as the one described here with polyneuropathy and paracrystalline inclusion bodies in the cytoplasm of Schwann’s cell but no mtDNA mutation in the sural nerve biopsy. The mechanisms of the involvement of peripheral nerves in mitochondrial encephalomyopathies are not fully understood. An increased number of mitochondria in capillary endothelial cells and 128

abnormal mitochondria with paracrystalline inclusion bodies in Schwann’s cells have been reported to play an important role in mitochondrial diseases (6). However, the discrepancy between the morphological changes and molecular genetic study in sural nerve biopsy of our patient is completely unknown. This kind of discrepancy had also been reported in a MELAS patient who had ragged red fibers (RRF) but had no mtDNA mutation in the muscle biopsies (17). Another interesting finding is that 2 muscle biopsies from different sites showed absolutely opposite findings in terms of the presence of R R F in 1 and absence of R R F in the other. In addition, the mtDNA mutation was still observed in the muscles which had normal morphology and biochemistry. These findings indicate that tissue mosaicism was present in the muscles. Furthermore, the A3243G MELAS mutation may not always be detected in all tissues involved. Further investigation involving single fiber analysis of mtDNA is warranted.

Acknowledgements The authors are grateful to Dr C.-C. Lee for providing the muscle biopsies and Ms Y.-C. Hsieh for typing the manuscript. The study was supported in part by a grant from the National Science Council (NSC 84-2331-B-182A-008) of Taiwan.

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