gastrointestinal encephalomyopathy (MNGIE): clinical, biochemical, and genetic features of an autosomal recessive mitochondrial disorder. Neurology 1994;44:721-727. 20. Uncini A, Servidei S, Silvestri G, et al. Ophthalmoplegia, demyelinating neuropathy, leukoencephalopathy, myopathy, and gastrointestinal dysfunction with multiple deletions of mitochondrial DNA a mitochondrial multisystem disorder in search of a name. Muscle Nerve 1994;17:667-674. 21. Johnston W, Karpati G, Carpenter S, Arnold D, Shoubridge EA. Late-onset mitochondrial myopathy. Ann Neurol 1995;37: 16-23. 22. Yuzaki M, Ohkoshi N, Kanazawa I, Kagawa Y, Ohta S. Multiple deletions in mitochondrial DNA at direct repeats of nonD-loop regions in cases of familial mitochondrial myopathy. Biochem Biophys Res Commun 1989;164:1352-1357. 23. Cormier V, Rotig A, Tardieu M, Colonna M, Saudubray JM, Munnich A. Autosomal dominant deletions of the mitochondrial genome in a case of progressive encephalomyopathy. Am J Hum Genet 1991;48:643-648. 24. Ohno K, Tanaka M, Sahashi K, et al. Mitochondrial DNA deletions in inherited recurrent myoglobinuria. Ann Neurol 1991;29:364-369. 25. Prelle A, Moggio M, Checcarelli N, et al. Multiple deletions of mitochondrial DNA in a patient with periodic attacks of paralysis. J Neurol Sci 1993;117:24-27. 26. Klopstock T, Naumann M, Schalke B, et al. Multiple symmetric lipomatosis: Abnormalities in complex IV and multiple deletions in mitochondrial DNA. Neurology 1994;44:862-866. 27. Casademont J, Barrientos A, Cardellach F, et al. Multiple deletions of mtDNA in two brothers with sideroblastic anemia and mitochondrial myopathy and in their asymptomatic mother. Hum Mol Genet 1994;3:1945-1949. 28. Mendell JR. Mitochondrial myopathy in the elderly: exaggerated aging in the pathogenesis of the disease. Ann Neurol 1995;37:3-4. 29. Mizusawa H, Watanabe M, Kanazawa I, et al. Familial mitochondrial myopathy associated with peripheral neuropathy: partial deficiencies of complex I and complex IV. J Neurol Sci 1988;86:171-184. 30. Suomalainen A, Paetau A, Leinonen H, et al. Inherited idiopathic cardiomyopathy with multiple deletions of mitochondrial DNA. Lancet 1992;340:1319-1320. 31. Suomalainen A, Kaukonen J, Amati P, et al. An autosomal locus predisposing to deletions of mtDNA. Nat Genet 1995;9: 146-151. A MERRFPEO overlap syndrome associated with the mitochondrial DNA 3243 mutation Ashok Verma, MD, DM; Carlos T. Moraes, PhD; Robert T. Shebert, MD; and Walter G. Bradley, DM, FRCP Article abstract-We describe a two-generation family with combined clinical features of myoclonic epilepsy, progressive external ophthalmoplegia (PEO), proximal myopathy, pigmentary retinopathy, progressive deafness, basal ganglia calcification, and ragged-red fibers in a muscle biopsy specimen. One family member died unexpectedly at age 22 years. The molecular tests revealed an A-to-G transition at nucleotide position 3243 of the mitochondrial tRNALeu(UUR) gene. No one in this family had stroke-like episodes. Although the propositus (a 28-year-old woman) had a significant number of white hairs, the percentage of mutant mtDNA in white-hair roots was not different from that in the colored-hair roots. Our findings suggest that the 3243 mutation can be associated with mixed clinical features of myoclonic epilepsy with ragged-red fibers (MERRF) and PEO and that a preferential increase in the levels of the mutant mtDNA is not related to graying of hair, and hence to the hypothesized production of premature aging of cells. NEUROLOGY 1996;46:1334-1336 Mitochondrial encephalomyopathies are a heterogeneous group of disorders that may affect several organ systems. Among the major distinct syndromes, Kearns-Sayre syndrome (KSS) and progressive external ophthalmoplegia (PEO) are generally associated with mitochondrial DNA (mtDNA) whereas mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS)4*5and myoclonic epilepsy with ragged-red fibers (MERRFY are characterized by point mutations involving the mitochondrial tRNALeu(UUR) and tRNALysgenes. The specific pathogenetic mechanisms in MELAS and MERRF are unknown. Theoretically, tRNA mutations could impair mitochondrial translation, or, by their peculiar (punctuated) position in mitochondrial genome, could alter the processing of primary polycistronic transcripts that include the flanking structural (rRNA and mRNA) gene^.^^^ The clinical spectra of mitochondrial tRNA gene mutations can overlap between clinically distinct syndromes. We From the Department of Neurology, University of Miami, Miami, FL. Supported by a neurosciences fellowship of the Department of Veterans M a i r s (A.V.) and grants from the Muscular Dystrophy Association, the National Eye Institute, and the Pew Charitable Trust (C.T.M.). Received April 24, 1995. Accepted in final form August 29, 1995. Address correspondence and reprint requests to Walter G. Bradley, DM, FRCP, Department of Neurology, University of Miami School of Medicine, 1501 N.W. 9th Avenue, Miami, FL 33136. 1334 Copyright 0 1996 by the American Academy of Neurology describe a family with clinical features of MERRF and PEO. In addition, the index patient had a noticeable number of white hairs at a relatively young age. Because of recent reports implicating mtDNA mutations with the aging p r o c e s ~ , ~ weJ ~attempted to correlate the proportion of mutant mtDNA in hairs of different phenotypes. Patients. Index case (IZ-2). This 28-year-old woman had normal birth and childhood development. Progressive deafness was noted from age 21 years. At the same time, she developed occasional myoclonus, had a n abnormal EEG, and was treated with phenytoin. A brain CT showed basal ganglia calcification, and an MRI showed no additional abnormality. At age 24 years, she noted muscle cramps and fatigue. A right quadriceps muscle biopsy specimen a t age 26 showed myopathic changes with ragged-red fibers. The symptoms worsened slightly, over the next 2 years. Neurologic examination was significant for restriction of extraocular movements, with only 20" of upward gaze and 60" of lateral gaze in either direction for both eyes. Fundus examination showed bilateral pigmentary retinopathy, most pronounced a t the posterior pole. Bilateral predominantly high-tone neural deafness was noted. She had thin muscles, but no weakness except for slight difficulty with a deep knee bend. The tendon reflexes were 1+, plantar responses flexor, and sensory examination was normal. The following laboratory tests were normal: complete blood count, erythrocyte sedimentation rate, blood sugar, calcium, phosphorus, serum electrolytes, amylase, total proteins and albumin, lipid profile, total and direct bilirubin, alkaline phosphatase, thyroid function, urinalysis, ECG, and chest x-ray. Alanine aminotransferase was 60 U L (normal 7-56), aspartate aminotransferase 108 U L (5-40), LDH 1,345 U L (313-6181, CK 531 U/L (35-230), lactate 3.0 mEqL (0.5-2.21, total carnitine 25 nmol/mL (37-891, and free carnitine 19 nmol/mL (28-69). EEG showed frequent bursts of ill-formed spike-and-wave complexes at about 31/2 per second lasting 1 to 2 seconds. Electroretinogram and perimetry for separate rod- and cone-specific stimuli revealed a predominant rod dysfunction. I-1. The 52-year-old mother of case 11-2 had progressive deafness, muscle cramps, easy tiredness, PEO, and mild proximal muscle weakness for many years. Atypical pigmentary retinopathy was present. Muscle biopsy was not performed. ZZ-1. The 32-year-old sister of case 11-2 was asymptomatic, with normal neurologic examination. 11-3. The younger brother of case 11-2 was of small stature, with pectus excavatum since early childhood. He had deafness and retinal degeneration from about age 6 years and had seizures from age 20 years. He died unexpectedly in bed at age 22 years. Methods. Determination of the proportion of normal and mutant mtDNAs in MELAS patients. Approximately 1 pg of genomic DNA was submitted to polymerase chain reaction (PCR) amplification (1min 94 "C, 1 min 55 "C, 0.75 min 72 "C, 25 cycles) according to the manufacturer's instructions (Perkin-Elmer Cetus), using the following primers: light-strand positions 3116-3134 and heavy-strand I- 1 n ($ II-1 II-2 rI-3 -Wild Type 1 3 2 4 3 Mutant Figure. The A-G transition at mtDNA position 3243 in a family with MERRFIPEO syndrome. Mutant- and wildtype mtDNA were identified by PCRIRFLP as described in Methods. The individual lanes in the gel correspond to the family members shown in the upper panel. The tissues studied were blood (B), muscle (M), hair follicles (H), and muscle control (C). positions 3353-3333 (numbers according to reference 7). The DNA fragment produced by the PCR reaction was radiolabeled and digested with Hae I11 for 2 hours. The A-G transition a t position 3243 creates a new Hue I11 site that is diagnostic for the MELAS mutation. The digestion products were electrophoresed and the radioactive fragments quantitated as previously described.I1 Single-hair PCR. Isolated hair follicles were placed directly into 5 p1 of KOH/DTT solution. After incubation at 65 "C for 30 minutes, the solution was neutralized as described.I2The PCR amplification of the DNA fragment and RFLP analysis were performed as previously described." Statistical analysis (Student's t test) was performed using a Staview software package. Results. All three living family members (1-1, 11-1, 11-2) showed an A-to-G transition a t nucleotide position 3243 of the mitochondria1 tRNALeu(UUR) gene. The percentages of mutant mtDNA were as follows: 40% (lymphocytes), 62% (hair roots), and 60% (muscle) in the index case (11-2), and 27% and 18% in white blood cells of the mother (1-1) and sister (11-1)(figure). The percentages of mutant mitochondria in white-hair follicles (47.3 ? 4.1 SD; n = 14) and colored-hair follicles (52.8 ? 4.4 SD; n = 14) in the index case were not significantly different ( p = 0.36). Discussion. The clinical features of seizures, myoclonus, and hearing impairment, the maternal line of inheritance, and the presence of ragged-red fibers in the muscle biopsy sample suggested the diagnosis May lS96 NEUROLOGY 46 1336 of mutant mtDNA does not play a major role in ageof MERRF syndrome in this family. PEO, pigmenrelated hair discoloration, the possibility of other tary retinopathy, neural deafness, and short stature, concurrent mtDNA polymorphism contributing to and a possible sudden cardiac death in one member the hair phenotype changes cannot be ruled out. (11-3), are reminiscent of KSSPEO syndrome. However, neither large-scale mtDNA deletions found in References most KSSPEO cases nor tRNAL""'ULTR' gene point 1. Moraes CT, DiMauro S, Zeviani M, et al. Mitochondrial DNA mutations generally associated with MERRF syndeletions in progressive external ophthalmoplegia and drome were present in the family. The molecular syndrome. N Engl J Med 1989;320:1293-1299. tests did reveal a mutation in the tRNALeu(UUR) 2. Kearns-Sayre DiMauro S. Symposium on mitochondrial encephalopathies. gene, which is commonly associated with the MELAS Brain Pathol 1992;2:111-162. 3. Eymard B, Hauw J. Mitochondrial encephalopathy. Curr Opin phen~type,~.~.'' although no one in this family had Neurol Neurosurg 1992;5:909-916. stroke-like episodes. Some intrafamilial variability of 4. Got0 YI, Nonaka I, Horai S. A mutation in the tRNAL""'UUR' clinical symptoms in the previously described gene associated with the MELAS subgroup of mitochondrial MELAS families is attributable to tissue heteroenceDhaloDathies. Nature 1990:348:651-653. 5. Kobayashi Y, Momoi MY, Tominaga K, et al. A point mutation plasmy for the mitochondrial 3243 m ~ t a t i o n . ' ~ In -'~ in the mitochondrial tRNALeU"'UR'gene in MELAS (mitochonour family, certain clinical features present in all the drial myopathy, encephalopathy, lactic acidosis and strokefamily members (e.g., hearing impairment and retilike episodes). Biochem Biophys Res Commun 1990;173:816822. nopathy) suggest a single disorder, although individ6. Shoffner JM, Lott MT, Lezza A M S , et al. Myoclonic epilepsy ual family members also had extra and variable orand ragged-red fibers disease (MERRF) is associated with migan-system involvement. One sibling (11-1) was tochondrial DNA tRNALysmutation. Cell 1990;61:931-937. clinically asymptomatic, but bore a significant pro7. Anderson S, Bankier AT, Barrel1 BG, et al. Sequence and organisation of the human mitochondrial genome. Nature portion of the mutant mtDNA. 1981;290:457-465. What determines the occurrence, pattern, and 8. Attardi G. Human mitochondrial genetic system. In: Mitoclinical severity of symptoms in mitochondrial tRNA chondrial DNA in human pathology. DiMauro S, Wallace DC, mutations is poorly understood. A differential tissueeds. New York Raven Press, 1993:9-25. 9. Cortopassi G, Arnheim N. Accumulation of mitochondrial organ requirement of high-energy oxidative phosDNA mutation in normal aging brain and muscle. In: Mitophorylation metabolism, the heteroplasmic distribuchondrial DNA in human pathology. DiMauro S, Wallace DC, tion of mutant and wild-type mtDNA, and threshold eds. New York: Raven Press, 1993:125-136. 10. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants levels of mutant mtDNA are important factors that and the degenerative diseases of aging. Proc Natl Acad Sci could explain, at least in part, the variable expresUSA 1993;90:7915-7922. sion of a mitochondrial disease among the members 11. Ciafaloni E, Ricci E, Shanke S, et al. MELAS: clinical feaof an affected family.1-3J3-19 More than one mitochontures, biochemistry and molecular genetics. Ann Neurol 1992; 31~391598. drial tRNA mutation, like different mtDNA dele12. Li H, Cui H, Arnheim N. Analysis of DNA sequence variation tions,'+2may lead to somewhat similar physiologic in single cells. Methods: A companion to Methods in Enzymolconsequences and therefore overlapping clinical synogy 1991;2:49-59. 13. Moraes CT, Ricci E, Bonilla E, et al. The mitochondrial dromes. The unique (punctuated) organization of tRNALeU'UUR' mutations in MELAs: genetic, biochemical and tRNA genes in the mitochondrial genome and the morphological correlations in skeletal muscle. Am J Hum unusual (polycistronic) mode of expression of mitoGenet 1992;50:934-949. chondrial genes suggest that the tRNA genes might 14. Crimmins D, Morris JGL, Waler GL, e t al. Mitochondrial encephalomyopathy: variable clinical expression in a single kinhave more than one function, including the followdred. J Neurol Neurosurg Psychiatry 1993;56:900-905. ingS,20,21: encoding the tRNAs, signal for processing of 15. de Vries D, de Wijs I, Ruitenbeek W, et al. Extreme variability polycistronic transcripts, and possibly the regulation of clinical symptoms among sibs in a MELAS family correof initiationhermination of transcription. The phenolated with heteroplasmy for the mitochondrial A3243G mutation. J Neurol Sci 1994;124:77-82. typic spectrum of tRNA mutation may stem from 16. Moraes CT, Ciacci F, Silvestri G, et al. Atypical presentations impairment of one or more of these several functions. associated with the MELAS mutations a t position 3243 of Detailed analysis of these overlap syndromes is imhuman mitochondrial DNA. Neuromuscul Disord 1993;3:4350. portant to understand the pathogenesis of common 17. Macmillan C, Lach B, Shoubrid e EA Variable distribution of symptoms that in typical cases help divide the mitomitochondrial DNAs (tRNALeU'&') in tissues of symptomatic chondrial syndromes into distinct clinical entities relatives with MELAS: the role of mitotic segregation. Neuroland in overlap patients cast doubt on the nosologic O ~ Y1993;43:1589-1590. 18. Zupanc ML, Moraes CT, Shanske S, et al. Deletion of mitodistinctions. chondrial DNA in patients with combined features of KearnsMutations of mtDNA accumulate in aging mamSayre and MELAS syndromes. Ann Neurol 1991;29:680-683. malian tissues to a greater extent than mutations in 19. Fang W, Huang C, Lee C, et al. Ophthalmologic manifestations in MELAS syndrome. Arch Neurol 1993;50:977-980. nuclear DNA.gJOThe question whether mtDNA mu20. King MP, Koga Y, Davidson M, Schon EA. Defects in mitotations in tRNA genes accelerate aging in patients chondrial protein synthesis and respiratory chain activity segwith MERRF and MELAS has not been previously regate with the tRNAL"U'UUR' mutation associated with mitoaddressed. The index case (11-2) in this family had chondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes. Mol Cell Biol 1992;12:480-490. many white hairs at age 28, but the percentage of 21. Hess JF, Parisi MA, Bennett JL, Clayton DA. Impairment of mutant mtDNA in white-hair follicles was not signifmitochondrial transcription termination by a point mutation icantly different from that in the dark-hair follicles. associated with the MELAS subgroup of mitochondrial enAlthough these results suggest that the percentage cephalopathies. Nature 1991;351:236-239. 1336 NEUROLOGY 46 May 1996 A MERRF/PEO overlap syndrome associated with the mitochondria1 DNA 3243 mutation Ashok Verma, Carlos T. Moraes, Robert T. Shebert, et al. Neurology 1996;46;1334 DOI 10.1212/WNL.46.5.1334 This information is current as of May 1, 1996 Updated Information & Services including high resolution figures, can be found at: http://www.neurology.org/content/46/5/1334.full.html References This article cites 19 articles, 1 of which you can access for free at: http://www.neurology.org/content/46/5/1334.full.html##ref-list-1 Citations This article has been cited by 2 HighWire-hosted articles: http://www.neurology.org/content/46/5/1334.full.html##otherarticl es Permissions & Licensing Information about reproducing this article in parts (figures,tables) or in its entirety can be found online at: http://www.neurology.org/misc/about.xhtml#permissions Reprints Information about ordering reprints can be found online: http://www.neurology.org/misc/addir.xhtml#reprintsus Neurology ® is the official journal of the American Academy of Neurology. Published continuously since 1951, it is now a weekly with 48 issues per year. Copyright Copyright 1996 by the American Academy of Neurology. All rights reserved. Print ISSN: 0028-3878. Online ISSN: 1526-632X.