Deletion of Mitochondrial DNA in Patients with Combined Features of Kearns-Savre and MELAS Sindrornes Mary L. Zupanc, MD,* Carlos T. Moraes, MSc,t Sara Shanske, PhD,S Craig B. Langman, MD,§ Emma Ciafaloni, MD,$ and Salvatore DiMauro, MD$ A 9-year-old girl and an 11-year-old boy had ptosis, progressive external ophthalmoplegia, pigmentary retinopathy, and sensorineural hearing loss. T h e girl had diabetes mellitus and the boy had hypoparathyroidism. Both children also developed recurrent vomiting and cerebral infarcts with lactic acidosis. Muscle biopsy specimens showed ragged-red fibers and Southern analysis demonstrated a distinct heteroplasmic deletion of muscle mitochondrial DNA in each patient but no evidence of the point mutation in the transfer R.NAh(UUR) gene recently identified in mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). These 2 children had combined features of Kearns-Sayre syndrome and MELAS, suggesting that mitochondrial DNA deletions occasionally can have pleomorphic clinical expression. Zupanc ML, Moraes CT, Shanske S, Langman CB, Ciafaloni E, DiMauro S. Deletion of mitochondrial DNA in patients with combined features of Kearns-Sayre and MELAS syndromes. Ann Neurol 1991;29:680-683 ~ Among the “mitochondrial encephalomyopathies,” the following three syndromes have been identified on the basis of characteristic constellations of signs and symptoms: Kearns-Sayre syndrome (KSS); mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS); and myoclonic epilepsy and raggedred fibers (MERRF) {I}. This classification has generated a spirited controversy between “splitters,” who consider the subdivision not only useful but indicative of separate etiologies 121, and “lumpers,” who consider it artificial and believe that patients with mitochondrial encephalomyopathies share the same symptoms and signs, although in different proportions and combinations [3}. Overlap patients, characterized by the simulFrom the Departments of “Pediatrics and Neurology, University of Wisconsin, Madison, WI; tGenetics and Development and SNeurology, Columbia Presbyterian Medical Center, New York, NY; and SPediarrics, Northwestern University, Chicago, IL. Received Oct 8, 1990, and in revised form Dec 24. Accepted for publication Dec 26, 1990. Address correspondence to Dr Z u p n c , Department of Neurology, University of Wisconsin Hospitals and Clinics, 600 Highland Ave, Madison, WI 53792-0001. taneous occurrence of major features of two syndromes in the same patient, are considered rare by splitters, common by lumpers. In view of recent dramatic advances in o u r understanding of the genetic bases of all three syndromes {4-61, it is especially instructive to study overlap patients by molecular genetic analysis. We report such studies in 2 children who appeared to have a combination of KSS and MELAS syndromes. Patient Reports Patzent 1 A 9-year-old girl was healthy and developmentally normal until 3% years of age, when she was diagnosed with insulindependent diabetes mellitus. Bilateral sensorineural highfrequency hearing loss was noted at age 5 years and required the use of hearing aids. At age 7 years, she developed progressive ataxia, poor coordination, and tremor; 1 year later, pigmentary degeneration of the retina was recognized. Through the years, she also developed episodic nausea and vomiting, which were not related to her diabetes rnellitus. These episodes were accompanied by exercise intolerance, weakness, and mood changes. Since age 8% years, she has had several episodes of hemiplegia. The first episode consisted of transient right hemiplegia, associated with headache, and lasted less than 24 hours. It was not associated with hypoglycemia. At the age of 9 years, there was acute onset of left hemiplegia and obtundation, which lasted for approximately 3 days. This episode was associated with mild lactic acidosis. Since then, the girl has had several more episodes of transient herniplegia associated with nausea, vomiting, general malaise, and headache. Physical examination between episodes of hemiplegia showed a small, cooperative child. She had mild intellectual delay by psychometric testing. Speech was ataxic. Ophthalmoscopic examination showed pigmentary retinopathy. Visual fields appeared to be full. There was bilateral ptosis and ophthalmoplegia in all directions of gaze, and mild facial diplegia. Motor examination showed mild proximal weakness and diffuse hypotonia. Deep tendon reflexes were hypoactive and plantars were flexor. Sensory examination was normal. Coordination testing showed truncal ataxia with titubation as well as pronounced bilateral dysmetria with mild intention tremor. Gait was wide based and ataxic, and tandem gait was impossible. The following laboratory tests were abnormal: serial magnetic resonance imaging (MRI) scans showed white matter disease in the cerebral hemispheres bilaterally, brainstem auditory evoked responses demonstrated abnormally long conduction time between wave I and wave V, somatosensory evoked potential showed long conduction times between the cervical and medullary pathways, and cerebrospinal fluid (CSF) protein was 177 mg/dl (normal, <55 mg/dl). Blood lactate was mildly elevated during the patient’s episode of severe left hemiplegia at the age of 9 years. The initial MRI scan showed only the findings previously described, but a second MRI scan obtained 2 days later showed mild cerebral edema in the right hemisphere, which was considered most compatible with an infarct. A repeat MRI scan several days later showed nearly complete resolution of the edema with return to baseline. 680 Copyright 0 1991 by the American Neurological Association Patient 2 An 11-year-old Chinese boy was the product of a term gestation, without prenatal or perinatal complications. At 3 days of age, he was lethargic, jaundiced, anemic, and acidotic and was admitted to the hospital. A presumptive diagnosis of tyrosinemia was made, and he was treated with a modified protein intake over the next 6 months. He was lost to follow-up until 5 years of age, when he presented with a hypocalcemic generalized seizure, and hypoparathyroidism was diagnosed. At age 8 years he had recurrent seizures, and a computed tomographic (CT) scan demonstrated temporal lobe and basal ganglia calcifications and a previously undiagnosed and clinically inapparent infarction around the left thalamus. An MRI scan demonstrated a lesion around the globus pallidus on the right. The boy had developed bilateral sensorineural hearing loss, bilateral ptosis, and external ophthalmoplegia. H e often had nausea and occasional vomiting unrelated to a defined biochemical abnormality, including hypoglycemia. At age 9 years, after 1 week of abdominal pain and nausea, the boy presented with obtundation, seizures, and severe electrolyte disturbances characterized by generalized renal Fanconi’s syndrome with hypokalemia, severe hypophosphatemia, hypercalcemia, and lactic acidosis. An electroencephalogram was abnormal, with generalized background slowing but no focal epileptiform discharges. Pigmentary degenerative changes of the retina were noted. During the next 2 years, the boy was admitted several times for abdominaI pain and dehydration with Fanconi’s syndrome but without lactic acidosis. At age 11 years, the boy presented with an acute left hemiplegia, obtundation, and lactic acidosis. Repeat CT scan and MRI studies demonstrated both peripheral white matter disease and new infarcts in the right brachium conjunctivum, and bilaterally in the thalami and t h e colliculi. CSF protein was 176 mgidl (normal, <55 mgidl). The boy developed nonoliguric acute renal insufficiency and systemic hypertension. He was treated with calcium channel antagonists, higher doses of his usual medication of thiamine, and administration of coenzyme Qlo.After 5 days the boy improved to a general level of consciousness, and his left hemiplegia resolved completely over the next 2 weeks. When last seen in follow-up, he had progressed back to his usual clinical and biochemical baseline status, but his statural growth and body weight have remained suboptimal and below the third percentile. Methods Biochemical studies and Southern blot analysis were performed as described 14). An A to G transition at nucleotide 3,243 of the mitochondrial DNA (mtDNA) has been reported in numerous patients with MELAS [GI; to detect this point mutation, we have amplified by polymerase chain reaction (PCR) a DNA fragment (using primers corresponding to positions nt 2164-2187 (F) and 4544-4523 (B), spanning the putative mutation, which was digested with Apa I. The point mutation creates an additional Apa I site, thus providing a simple molecular test for this genetic defect. Results In Patient 1, abnormal mitochondria were seen by electron microscopy in the cells of the buffy coat and in cultured skin fibroblasts, with intramitochondrial myelin figures and electron-dense inclusion. In both patients, muscle biopsy specimens showed ragged-red fibers, and biochemical analysis of muscle extracts showed that mitochondria1 enzyme activities for cytochrome c oxidase, reduced nicotinamide adenine dinucleotide ( N A D H ) coenzyme Q oxidoreductase, succinate cytochrome c reductase, NADH dehydrogenase, succinate dehydrogenase, and citrate synthase were all within the normal range. Southern blot analysis of muscle m t D N A from Patient 1 showed two populations, o n e of which (representing 64% of total m t D N A ) harbored a 7,521-base pair deletion. Similarly, a population of muscle mtDNA from Patient 2 harbored a 6,903-base pair deletion (39% of total m t D N A ) (Fig 1A). The deletion break points have been previously determined for both patients by sequence analysis (Fig 1B), and o u r Patients 1 and 2 correspond to Patients K8 and M13 in the report by Mita and colleagues [bf. In both of o u r patients, the deletion encompassed structural genes encoding subunits of complexes I, 111, IV,and V of the respiratory chain and several transfer RNAs (tRNAs). A deletion identical to that found in muscle was also observed in leukocytes, platelets, and cultured fibroblasts of Patient 1, but the proportion of “deleted” m t D N A s was lower than in muscle (see Fig 1A). Apa 1 digestion of a PCR fragment encompassing tRNALeU(”” failed to show the point mutation gene recently documented by Goto and colleagues [ 7 ) in 26 of 31 patients with MELAS. Discussion O u r patients had the obligatory clinical triad of KSS [2f, that is, onset before age 20 years, progressive external ophthalmoplegia (PEO), and pigmentary retinopathy; Patient 1 also had progressive cerebellar ataxia, and both had CSF protein greater than 100 mg/ dl. Other less specific manifestations of KSS included short stature, sensorineural hearing loss, insulindependent diabetes mellitus, and hypoparathyroidism. Additionally, however, they also had several episodes of transient hemiplegia associated with lactic acidosis, a feature not usually found in patients with KSS, but that was reminiscent of patients with MELAS 181. MELAS is characterized by recurrent episodes of prolonged migrainous headaches accompanied clinically by repetitive vomiting and lethargy, and metabolically by lactic acidosis. Partial seizures with prolonged partial status epilepticus and repeated (sometimes reversible) cerebral infarctions accompany these episodes. Many patients are left with residual neurological deficits such as cortical blindness, spastic quadriparesis o r hemiparesis, or dementia {8, 91. In both patients with KSS and patients with MELAS, muscle biopsy specimens typically show ragged-red hbers (RRE). Electron microscopy confirms the presence of mitochondria that are increased in number and Brief Communication: Zupanc et al: mtDNA in Kearns-Sayre and MELAS 681 Fig 1. (A) Autoradiograms of Southern blot hybridization of mitochondrial D N A from various tissues of Patient I and from muscle of Patient 2. Total D N A was digested with Pvu 11, electrophoresed through O.Sfj& agarose gel, transferred to nitmcellulose. and probed with purijed human mitochondrial DNA labeled with j2P as previowly described (5). Lane 1 , control muscle: lane 2, Patient I muscle; lane 3, Patient I leukocytes; lane 4, Patient I platelets; lane 5 , Patient 2 muscle. (B) Location of mitochondrial D N A deletion. The maps at the top represent the heavy (upper map) and light (lower map) strands of linearized mitochondrial DNA, showing the genes for the subunits of reduced nicotinamide adenine dinucleotide coenzyme Q oxidoreductase ( N D 1-6), cytochrome oxidase (CO I -3j, cytochrome b (CYT B), and adenosine triphosphate synthase (ATP, 8 and 61, andfor the 12s and 16S ribosomal RNAs. The transfer RNAs are denoted by small open boxes. The origins of heavy strand (OH) and light strand (OL) replication and the promotersforthe initiation of heavy strand (PHI and PH2) and light strand (PLj transcriptions are indicated by arrows. Intact region of mitochondrzd D N A shown a~solid area. Deleted regions s h u n as open (dotted area, with the size of the deletion denoted. abnormal in size or structure. The abnormal mitochondria may also be found in other tissues such as liver, brain, and leukocytes. In agreement with the suspected maternal inheritance of MELAS, Goto and colleagues [7} recently described a point mutation in the tRNAL""(UUR) gene of mtDNA. Thus, the molecular defect in patients with MELAS is similar to that pre682 Annals of Neurology Vol 29 No 6 June 1091 viously demonstrated in patients with MERRF, a point mutation affecting a different tRNA gene (tRNALys) 151. In contrast, large deletions of mtDNA have been found in almost all patients with KSS. Of 123 patients with mitochondrial myopathies or encephalomyopathies studied by Moraes and associates {41, 32 had mtDNA deletions; 18 of these had complete or incomplete KSS, whereas the others had ocular myopathy with RRF. The association of mtDNA deletions with PEO appears to be invariable; no deletions were found in patients with mitochondrial encephalomyopathies without PEO, including patients with MELAS [4]. Our patients showed large-scale deletions of mtDNA that were different from each other but did not differ significantly from those observed in patients with KSS or isolated ocular myopathy 141. In Patient 1, the mtDNA deletion was also found in several accessible nonmuscle tissues such as leukocytes, platelets, and cultured fibroblasts, although in smaller amounts than in muscle (approximately 10% of total mtDNA). The widespread tissue distribution of mtDNA deletions is also consistent with previous findings in patients with KSS {4, 10, l l ] . Additionally, almost all patients with KSS or ocular myopathy and mtDNA deletions are sporadic [4},suggesting spontaneous mu- tation of mtDNA in the oocyte or in the zygote. In contrast, MELAS is often familial; maternal inheritance has been suspected and is in accord with the recent demonstration of a mtDNA point mutation [7}. Our patients had no affected relatives, and a muscle biopsy specimen from the mother of Patient 1 showed no RRF and no detectable mtDNA deletion. Direct analysis of mtDNA excluded the presence of the point mutation in the tRNALeU(UUR) gene recently described {7]. The prominent ocular signs (PEO and pigmentary retinopathy), lack of family history, evidence of widespread mtDNA deletions, and lack of the point mutagene support the diagnosis of tion in the tRNALeu(UUR) KSS rather than MELAS. How, then, can we explain the stroke-like episodes? The stroke-like episodes in MELAS may not be the result of classical vascular problems because the cerebral “infarcts” often are not in the distribution of major vessels and there is usually no evidence of thromboembolisrn. It has been suggested that the smooth muscle cells of small and medium arterioles of the brain contain excessive numbers of abnormal mitochondria, resulting in intermittent abnormalities of autonomic vascular control [12, 131. If this hypothesis is correct, it is imaginable that an especially high number of deleted mtDNAs in the smooth muscle of the cerebral microvasculaturc could cause stroke-like episodes in patients with otherwise typical KSS. We think that molecular genetic analysis will confirm rather than confuse the distinction of the three major syndromes initially suggested by clinical criteria. It is, however, difficult to explain how genetic lesions of the mtDNA, which ought to have similar physiological consequences, usually cause dissimilar clinical pictures. Detailed study of overlap patients may help us understand the pathogenesis of those clinical differences that, in typical patients, separate KSS, M E W , and MELAS. References 1. DiMauro S, Bonilla E, Zeviani M, et al. Mitochondrial myopathies. Ann Neurol 1985;17:521-538 2. Pavlakis SG, Rowland LP, DeVivo DC, et al. Mitochondrial myopathies and encephalomyopathies. In: Plum F, ed. Advances in contemporary neurology. Philadelphia: Davis, 1988:95-133 3. Petty RKH, Harding AE, Morgan-Hughes JA. The clinical features of mitochondrial myopathy. Brain 1986;109:915-938 4. Moraes CT,DiMauo S, Zeviani M, et al. Mitochondrial DNA deletions in progressive external ophthalmoplegia and KearnsSayre syndrome. N Engl J Med 1989;320:1293-1299 5. ShoffnerJM, htt MT, Lezza AM, et al. Myoclonic epilepsy and r e e d red fiber disease (MERRF)is associated with a mitochondrial DNA tRNALysmutation. Cell 1990;61:931-937 6. Mita S, Rizzuto R, Moraes CT,et al. Recombination via flanking direct repeats is a major cause of large-scale deletions of human mitochondrial DNA. Nucleic Acid Res 1990;18:561-567 7. Goto Y,Nonaka I, Horai S. A mutation in the tRNALu‘UUR’ gene associated with the MELAS subgroup of mitochondrial encephalomyopathies. N a m e 1990;348:651-653 8. Pavlakis SG, Phillips PC, DiMauro S, et al. Mitochondrial myop- athy, encephalopathy, lactic acidosis, and stroke-like episodes: a distinctive clinical syndrome. Ann Neurol 1984;16:481-488 9. Montagna P, Gallassi R, Medori R, et al. MELAS syndrome: characteristic migrainous and epileptic features and maternal transmission. Neurology 1988;38:7 5 1-7 54 10. Shanske S, Moraes CT, h m b e s A, et al. Wide-spread tissue distribution of rnitochondrial DNA deletions in Kearns-Sayre syndrome. Neurology 1990;40:24-28 11. Zeviani M, Gellera C, Pannacci M, et al. Tissue distribution and transmission of rnitochondrial DNA deletions in mitochondrial myopathies. Ann Neurol 1990;28:94-97 12. Sakuta R, Nonaka I. Vascular involvement in mitochondrial myopathy. Ann Neurol 1989;25:594-601 13. Ohama E, Ohara SA, Tanaka K, et al. Mitochondrial angiopathy in cerebral blood vessels of mitochondrial encephalomyopathy. Acta Neuropathol 1987 ;?4:226-2 33 Fatal Disseminated Hemorrhagc Toxoplasmic EnceDhalitis As the-Initial Man;festation of AIDS Eelco F. M. Wijdicks, MD,*Jan C. C. Borleffs, MD,? Andy I. M. Hoepelman, MD,? and Gerard H. Jansen, MDS A 28-year-old woman presented with progressive coma after being asymptomatic for 1 year after the demonstration of seropositivity for human immunodeficiency virus and high serum immunoglobulin G toxoplasma titers. Computed tomographic scanning showed multiple rounded cerebral hemorrhages and massive cerebral edema. Postmortem examination disclosed Toxoplasma gondii cysts scattered throughout the brain. An overwhelming fatal toxoplasmosis associated with multiple cerebral hemorrhages may be the first presentation of the acquired immunodeficiency syndrome. We raise the possibility that frequent neuroradiological monitoring may be indicated in asymptomatic human immunodeficiency virus-infected patients with high immunoglobulin G toxoplasma titers. Wijdicks EFM, Borleffs JCC, Hoepelman AIM, Jansen GH. Fatal disseminated hemorrhagic toxoplasmic encephalitis as the initial manifestation of AIDS. Ann Neurol 1991;29:683-686 Central nervous system (CNS) toxoplasmosis as the initial manifestation of the acquired immunodeficiency From the Departments of *Neurology, +Internal Medicine, Section Immunology and Infection, and SNeuropathology, University Hospital Utrecht, Utrecht, The Netherlands. Received Oct 15, 1990, and in revised form Dec 18. Accepted for publication Dec 22, 1990. Address correspondence to D r Wijdicks, Department of Neurology, University Hospital Uuecht, Heidelberglaan 100,3584 CX Utrecht, The Netherlands. Copyright 0 1991 by the American Neurological Association 683