Mitochondrial Trnaleu(Uur) May Cause an MERRF Syndrome

Home , MELAS syndrome, MERRF syndrome, Mitochondrial myopathy

J7ournal ofNeurology, Neurosurgery, and Psychiatry 1996;61:47-51 47

The A to G transition at nt 3243 of the J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.61.1.47 on 1 July 1996. Downloaded from mitochondrial tRNALeu(uuR) may cause an MERRF syndrome

Gian Maria Fabrizi, Elena Cardaioli, Gaetano Salvatore Grieco, Tiziana Cavallaro, Alessandro Malandrini, Letizia Manneschi, Maria Teresa Dotti, Antonio Federico, Giancarlo Guazzi

Abstract Two distinct maternally inherited encephalo- Objective-To verify the phenotype to myopathies with ragged red fibres have been genotype correlations of mitochondrial recognised on clinical grounds: MERRF, DNA (mtDNA) related disorders in an which is characterised by myoclonic epilepsy, atypical maternally inherited encephalo- skeletal myopathy, neural deafness, and optic myopathy. atrophy,' and MELAS, which is defined by Methods-Neuroradiological, morpholog- stroke-like episodes in young age, episodic ical, biochemical, and molecular genetic headache and vomiting, seizures, dementia, analyses were performed on the affected lactic acidosis, skeletal myopathy, and short members of a pedigree harbouring the stature.2 Molecular genetic studies later con- heteroplasmic A to G transition at firmed the nosological distinction between the nucleotide 3243 of the mitochondrial two disorders, showing that MERRF is strictly tRNAI-u(UR), which is usually associated associated with two mutations of the mito- with the syndrome of mitochondrial chondrial tRNALYs at nucleotides 83443 and encephalomyopathy, lactic acidosis, and 8356,4 and MELAS with three point muta- stroke-like episodes (MELAS). tions of the mitochondrial tRNAIxu(UUR) at Results-The proband was affected by a nucleotides 3243, 3271, and 3291; the 3243 A fullblown syndrome of myoclonic to G transition (A3243G) is the most common epilepsy with ragged red fibres cause of MELAS.5 (MERRF), severe brain atrophy, and We discuss here the clinical, neuroradiolog- basal ganglia calcifications, without the ical, morphological, biochemical, and molecu- MRI T2 hyperintense focal lesions which lar genetic findings in the affected members of are pathognomonic of MELAS. Oligo- a clinically atypical pedigree harbouring the symptomatic relatives were variably heteroplasmic A3243G "MELAS" mutation. affected by lipomas, goitre, brain atrophy, and basal ganglia calcifications. Muscle biopsies in the proband and his Patients and methods mother showed a MELAS-like pattern PEDIGREE ANALYSIS http://jnnp.bmj.com/ with cytochrome c oxidase hyperreactive Family member III-1 proband ragged red fibres and strongly succinate The proband, an 18 year old man, was the dehydrogenase reactive vessels. firstborn of non-consanguineous parents (fig Quantification of the A3243G mutation 1). Since the age of 5 years, he had had disclosed 78% and 70% of mutated mtDNA in the muscle of the severely affected proband and of his oligosymptomatic mother respectively. Nucleotide on September 27, 2021 by guest. Protected copyright. sequencing of the Istituto di Scienze mitochondrial Neurologiche, tRNAIeu(UUR) and tRNALYs in the proband's Universita di Siena, muscle failed to show any additional Italy nucleotide change which could account G M Fabrizi E Cardaioli for the clinical oddity of this pedigree by G S Grieco modulating the expression of the primary A Malandrini pathogenic mutation. L Manneschi M T Dotti Conclusion-So far, MERRF has been A Federico associated with mutations of the G Guazzi mitochondrial tRNALYS, and MELAS with Istituto di Neurologia, mutations of the mitochondrial Universita di Verona, tRNALeu(UuR). Now MERRF may also be Italy considered among the clinical syndromes T Cavallaro I Correspondence to: associated with the A to G transition at nt Dr G M Fabrizi, Istituto di 3243 of the tRNALeu(uuR). Scienze Neurologiche, Universiti di Siena, Viale Bracci, 53100 Siena, Italy. (7 Neurol Neurosurg Psychiatry 1996;61:45-5 1) Figure 1 Pedigree: black symbol indicates the severely Received 31 October 1995 affected proband; grey symbols indicate the and in final revised form oligosymptomatic members. Percentage of the mutated 19 February 1996 mtDNA in muscle is given as m; percentage ofthe mutated Accepted 23 February 1996 Keywords: MERRF; MELAS; mitochondrial DNA mtDNA in blood is given as b. 48 Fabrizi, Cardaioli, Grieco, Cavallaro, Malandrini, Manneschi, et al

unsteadiness of gait and limb tremors. At the proic acid and phenobarbitone. At the age of age of 6, he was admitted for generalised 10, when the patient was evaluated by us, J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.61.1.47 on 1 July 1996. Downloaded from seizures; examination disclosed mental retar- weight and height were below the 100 per- dation, generalised ataxia, intentional tremor centile. His IQ was 62. An ECG showed a and diffuse muscle weakness, and hypotrophy. block of the right bundle branch; an echocar- An EEG showed bilateral irritative elements diogram was normal. There was generalised and generalised slow activity. An EMG had a muscle wasting, gait ataxia and dysmetria, myopathic pattern in the proximal limb mus- intention myoclonus, nystagmus, hyperreflexia cles. Brain CT showed bilateral calcifications of the lower limbs, bilateral optic atrophy, and of the basal ganglia and atrophy of the fron- cervical lipoma. An EEG showed synchronous totemporal cortical and subcortical regions (fig bursts of sharp waves and spike wave com- 2A). At the age of 7, generalised myoclonic plexes on the temporal regions. Audiometric jerks appeared, sometimes followed by grand tests disclosed neural deafness. Fundoscopy mal attacks; seizures were frequent (five- disclosed bilateral optic atrophy with a normal 10/day) and were partially responsive to val- electroretinogram. Proteins in CSF were 103 mg% with a normal IgG/albumin ratio. Abnormal laboratory investigations included Figure 2 (A) Proband's blood lactate (405 mg/dl, normal 2 7-11 7 CT (6 years of age) mg/dl), pyruvate (115 mg/dl, normal 026-07 showing calcifications of the lactate to pyruvate ratio. basal ganglia and atrophy mg/dl), and increased of the frontotemporal Parathyroid hormone (amino acids 1-84) was cortical and subcortical 29 pg/ml (normal range 5-10 pg/ml) and cal- regions. (B) proband's Ti caemia was 9-1 mg/dl. Brain MRI disclosed weighted MRI coronal section (18 years of age), diffuse supratentorial and infratentorial atro- showing a severe phy of the cortical and subcortical regions. supratentorial and The syndrome evolved towards a severe mental infratentorial cortical and subcortical atrophy. deterioration with spastic tetraparesis, dysarthria, dysphagia, and recurrent generalised myoclonic seizures. Brain MRI performed at the age of 18 showed a severe atrophy of the cerebral cortex and subcortical white matter, as well as of the semiovale centres and corpus callosum. Cerebellar hemi- spheres and the vermis were also atrophic (fig 2B). Family member II-1: mother of the proband The mother of the proband, 43 years old, had a negative neurological history. Since the age of 30 she had had multinodular euthyroid goitre which was treated with thyroxine. Clinical

examination disclosed a cervical lipoma. An http://jnnp.bmj.com/ EMG was normal. Among relevant laboratory investigations, blood lactate, pyruvate, T3, T4, thyroid stimulating hormone, and parathyroid hormone (amino acids 1-84) were in the normal ranges. Brain CT disclosed bilateral basal ganglia calcifications and atrophy of the frontotemporal cortex, cerebellum, on September 27, 2021 by guest. Protected copyright. .:..i.. and pons. Ophthalmoscopy and audiometric 'A. tests normal results. "' gave Non-examined maternal members member rX The proband's grandmother, family I-1, aged 65, had had hyperthyroidism since the age of 25. At the age of 50 fluorangiogra- phy had shown bilateral drusen of the optic disc and degeneration of the macular epithe- lium of the left eye. The proband's sister, family member 111-2, 14 years old, had headache and nasal speech.

's^ MORPHOLOGICAL STUDIES The proband's biopsy specimens were obtained under local anaesthesia from the right deltoid muscle at the age of 10 and from the left vastus lateralis muscle at the age of 18; a biopsy of the deltoid muscle was also performed in the 43 year old mother. Specimens for histopathological study were frozen in The A to G transition at nt 3243 of the mitochondrial tRNA'-LtU-RI may cause a MERRF syndrome 49

Figure 3 Modified

Gomori trichrome stain *\.\t J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.61.1.47 on 1 July 1996. Downloaded from (A x 140) and cytochrome c oxidase .. ,.. 0. ,.V, te,. 0.s_ ,*;v :. _ reaction (B x 140) in the *X.,.)iM--_ 4 * wit;;ie. ¢/e'; mother's deltoid mnuscle; asterisks indicate the faU' correspondingfibres in serial sections. Ragged red %;r A. fibres have a strong lp 0 ...:A,, cytochrome c oxidase activity which is more .E I '. Or_).;8 intense at the periphery. I (C) Succinate *

isopentane cooled with liquid nitrogen. Serial MOLECULAR GENETIC ANALYSIS 8 pum thick transverse sections were stained Total DNA was extracted from blood of family with haematoxylin and eosin, modified members III-1, II-1, and I-1 and from 50 mg Gomori trichrome, and a battery of histo- of frozen bioptic muscle of members III-1 and chemical methods including succinate dehy- II-1, according to standard procedures. The drogenase, routine ATPase, ATPase with presence of the following pathogenic point preincubation at pH 4,6 and 43, and mutations of mtDNA were investigated on the on September 27, 2021 by guest. Protected copyright. cytochrome c oxidase. Small pieces of the proband's muscle mtDNA, by standard specimens were fixed in 2-5% glutaraldehyde RFLP-PCR (polymerase chain reaction) and postfixed in 1% 0S04 in 01- M phosphate analysis: MERRF A8344G and T8356C at buffer. After fixation the specimens were dehy- the tRNALYS; MELAS A3243G, T3271C, drated and embedded in epoxy resin for elec- T3291 C at the tRNALcu(UUR); MELAS T9957C tron microscopy at the COXIII gene; and NARP (neurogenic ataxia and retinitis pigmentosa) T8993G at BIOCHEMICAL STUDIES the ATPase6 gene. The mitochondrial fraction was isolated from Quantification of the A3243G mutation in 200-300 mg of frozen muscle samples from blood from I-1, II-1, and III-1 and muscle family members III-1 and II-1 and the activi- from HI-1 and I-1 was obtained by a routine ties of the following respiratory chain enzymes ApaI-RFLP analysis on the PCR amplified were determined spectrophotometrically as mitochondrial tRNAIU(ULUR), modified accord- previously described6: NADH cytochrome c ing to Tanno et a17 to reduce the underestima- reductase, succinate cytochrome c reductase, tion of mutated mtDNA caused by the and cytochrome c oxidase. Values were formation of heteroduplexes. Direct sequenc- expressed as ratios with citrate synthase, an ing of both strands of the mitochondrial enzyme of the mitochondrial matrix that is a tRNAI-eu(UUR) and tRNALys was performed on reliable index of mitochondrial protein con- polymerase chain reaction (PCR) amplified tent. fragments corresponding to nucleotides 50 Fabrizi, Cardaioli, Grieco, Cavallaro, Malandrini, Manneschi, et al

Respiratory chai'n enzyme activities in skeletal mluscle mitochontdria ACTIVITIES OF RESPIRATORY CHAIN ENZYMES J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.61.1.47 on 1 July 1996. Downloaded from NADH Succiniate Biochemical analysis on the mitochondrial cytochromle cytochronie Cytochrortie Citr-ate fraction (table) showed a significant decrease c reductase c reductase c oxidase sywthase of the activities of the NADH cytochrome c Pedigree nmemibers reductase (complexes I + III) and cytochrome c III-1 0 15 0 10 0Q49 719 muscle of II-1 019 013 062 946 oxidase (complex IV) in the skeletal controls (n = 50) 0-24 (0 08) 0 19 (0 09) 1.2 (0 45) 690 (229) the proband III-1. Values in the mother's mean (SD) muscle were in the normal range. Values are nmol/min/mg of mitochondrial proteins. MOLECULAR ANALYSIS OF MITOCHONDRIAL DNA 3150-3369 for the tRNAL' LUU3R) and nucleo- PCR-RFLP analysis with Apa I showed the tides 8184-8478 for the tRNA"-S, using the heteroplasmic A to G transition at nt 3243 of "Sequenase PCR product sequencing kit" the mitochondrial tRNA'uUUR) in the pro- (Amersham). band's muscle. Quantitative analysis (fig 4) showed that the percentage of the mutated Results DNA in muscle was 78% in the severely MORPHOLOGICAL FINDINGS IN SKELETAL affected proband and 70% in the oligosympto- MUSCLE BIOPSIES matic mother. Percentages of mutated DNA The proband's first muscle biopsy (deltoid) in blood were 30%, 20%, and 5% in the showed 8%-10% of ragged red fibres. In the proband, mother, and grandmother respec- second biopsy (vastus lateralis), ragged red tively. fibres were reduced to 1%-2%, but in many Direct nucleotide sequencing of the fibres the succinate dehydrogenase staining proband's muscle mtDNA confirmed the was stronger than that of normal fibres. The presence of the heteroplasmic A3243G muta- oligosymptomatic mother's muscle biopsy tion and failed to show any other nucleotide showed 8%-10% of ragged red fibres. Type I change of the tRNA''-'I' and tRNA'. fibres were preferentially involved in mitochondrial proliferation. Some ragged red fibres were completely deficient in cytochrome c oxidase, but most showed cytochrome c oxidase Discussion hyperreactivity, which was more evident in the The present pedigree harboured the hetero- periphery of the fibres (fig 3 A,B). Blood ves- plasmic A to G transition at nucleotide 3243 sels stained darkly with succinate dehydroge- of the mtDNA, which is typical of MELAS, nase (fig 3C), and were hyperreactive to although the proband was affected by a full- cytochrome c oxidase. Ultrastructural exami- blown MERRF. The syndrome was charac- nation showed intermyofibrillar and sub- terised by seizures, intention myoclonus, sarcolemmal aggregates of abnormal ataxia, psychomotor delay, optic atrophy, mitochondria containing paracrystalline inclu- neural deafness, and cervical lipoma, without sions (fig 3D). An increased number of mito- any sign or symptom of stroke episodes. A chondria were also present in the pericytes of detailed CT and MRI follow up disclosed

capillaries, endothelial cells, and smooth mus- basal ganglia calcifications and severe, pro- http://jnnp.bmj.com/ cle cells of the small arteries. gressive atrophy of the cortex, subcortical white matter, corpus callosum, and semiovale centres. Two MRIs, performed at different stages of disease, did not show the T2 hyperintense focal lesions which are pathognomonic of MELAS.5 Basal ganglia calcifications, although often encountered in MELAS, may be found in various mitochondrial encephalo- on September 27, 2021 by guest. Protected copyright. myopathies, and they are not specific for the disease.' The A3243G mutation was first described in MELAS, but it may also occur in association with other two syndromes: chronic progressive external ophthalmoplegia and maternally inherited diabetes with deafness.') I" The same mutation has been also identified in a few cases of MERRF/MELAS overlap." In these cases, myoclonus or ataxia were always associated with clinical or radiological evi- dence of stroke-like episodes. More recently, the A3243G mutation was found in a MERRF pedigree,'5 but the occurrence of focal cerebral lesions could not be ruled out in the report because of the absence of MRI studies. Clinical findings in the oligosymptomatic Figure 4 Autoradiogramti of PCR products after digestion with Apa I. M anid B indicate members of the pedigree also showed atypical mtiuscle and blood of the examined pedigree mnembers. Wild type mitDNA corresponds to the features. The proband's mother had cervical 428 bpfragment, which is resolved inzto two fragments of 315 and 113 bp wheni the lipoma, euthyroid goitre, basal ganglia calcifi- A3243G mutation is present. A small percentage (5%) of mutant DNA present in blood from I-1 is not visible on the photographic reproduction. cations, and atrophy of the frontotemporal The A to G transition at nt 3243 of the mitochondrial tRNA'- UUR may cause a MERRF syndromne 51

cortex and cerebellum. The grandmother had The financial support of Telethon-Italy is gratefully aknowl- J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.61.1.47 on 1 July 1996. Downloaded from retinopathy and hyperthyroidism. So far, lipo- edged (grants to GCG, AF, and GMF). 1 Fukuhara N, Tokiguchi S, Shirakawa K, Tsubaki T. mas have been related to the A to G transition Myoclonus epilepsy associated with ragged-red fibers at nt 8344 of the tRNALs,l7 in association (mitochondrial abnormalities): disease entity or a syndrome? Light- and electron-microscopic studies of two with MERRF or with Ekbom's syndrome cases and review of literature. J Neurol Sci 1980;47: (MERRF plus lipomas, ataxia, and neuropa- 117-33. 2 Pavlakis SG, Phillips PC, DiMauro S, DeVivo DC, thy), as well as to the autosomal dominant Rowland LP. Mitochondrial myopathy, encephalopathy, multiple deletions of the mtDNA." Thyroid lactic acidosis, and stroke-like episodes. Ann Neurol 1984;16:481-8. dysfunction has been reported in a MERRF 3 Shoffner JM, Lott MT, Lezza AM, Seibel P, Ballinger SW, case4 and in a MERRF/MELAS pedigree,'" Wallace DC. Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial both associated with a T to C transition at nt DNA tRNALys mutation. Cell 1991,61:931-7. 8356 of the tRNA'-s. We conclude that lipo- 4 Silvestri G, Moraes CT, Shanske S, Oh SJ, DiMauro S. A new mtDNA mutation in the tRNALys gene associated mas and thyroid dysfunction occur in a wide with myoclonic epilepsy and ragged-red fibers range of molecular lesions of the mtDNA and (MERRF). Am J Husn Genet 1992;51:1213-7. 5 Goto Y. Clinical features of MELAS and mitochondrial they are not specific of any syndrome. DNA mutations. Muscle Nerve 1995;3(suppl): 107-12. Unlike the clinical phenotype, the morpho- 6 Federico A, Manneschi L, Paolimi E. Biochemical differ- logical abnormalities in muscle followed the ences between intermyofibrillar and subsarcolemmal mitochondria from human muscle. _7 Inherit Metab Dis pattern seen with the A3243G mutation, 1987;1O(suppl):242-6. 7 Tanno Y, Yoneda M, Nonaka I, Tanaka K, Miyatake T, which is characterised by ragged red fibres Tsuji S. Quantitation of mitochondrial DNA carrying the hyperreactive to cytochrome c oxidase and tRNALys mutation in MERRF patients. Biochenm Biophys reactive to succinate Res Commun 199 1;179:880-5. vessels strongly dehydro- 8 Matthews PM, Phil D, Tampieri D, et al. Magnetic reso- genase.2' nance imaging shows specific abnormalities in the MELAS syndrome. Neurology 1991 ;41:1043-6. Quantification of the A3243G mutation 9 Hammans SR, Sweeney MG, Hanna MG, Brockington M, showed 78% of mutated muscle mtDNA in Morgan-Hughes JA, Harding AE. The mitochondrial DNA transfer RNALeu(UUR) A to G (3243) mutation. the proband and 70% in the oligosymptomatic A clinical and genetic study. Brain 1995;118:721-34. mother. Percentages of mutated mtDNA in 10 Mariotti C, Savarese N, Soumalainen A, et al. Genotype to phenotype correlations in mitochondrial encephalomy- blood were 30%, 20%, and 5% in the opathies associated with the A3243G mutation of mito- proband, mother, and grandmother respec- chondrial DNA. Neurol 1995;242:304-12. 11 Ciafaloni E, Ricci E,_ Shanske S, et al. MELAS: clinical fea- tively. Recent surveys showed that the per- tures, biochemistry, and molecular genetics. Anni Neurol centages of the A3243G mutation did not 1992;31:391-8. 12 Hirano M, Ricci E, Koenigsberg MR, et al. MELAS: correlate with the expression of specific an original case and clinical criteria for diagnosis. clinical syndromes, although, in the MELAS Neuromuscul Disord 1992;2: 125-35. 13 Chen R-S, Huang C-C, Lee C-C, et al. Overlapping syn- subgroup, the percentage of mutated mtDNA drome of MERRF and MELAS: molecular and neurora- correlated positively with the age of onset of diological studies. Acta Neurol Scand 1993;8:494-8. 14 Crimmins D, Morris JCL, Walker GL, et al. Mitochondrial disease.9 1" To explain these imperfect cor- encephalomyopathy: variable clinical expression within a relations, additional genetic factors involving single kindred. _7 Neurol Neurosurg Psyzchiatry 199356: 900-5. the mtDNA or the nuclear genome have been 15 Folgero T, Torbergsen T, 0ian P. The 3243 MELAS hypothesised to modulate the expression of mutation in a pedigree with MERRF. Eur Neurol 1995; 35:168-71. the primary pathogenic mutations of mtDNA.9 16 Larsson N-G, Tulinius MH, Holme E, Oldfors A. Nucleotide sequence analysis of the proband's Pathogenetic aspects of the A8344G mutation of mito- muscle mtDNA did not show any additional chondrial DNA associated with MERRF syndrome and multiple lipomas. Muscle Nerve 1995;3(suppl): 102-6. http://jnnp.bmj.com/ nucleotide change of the tRNAL-\s and 17 Calabresi P, Silvestri G, DiMauro S, Criggs C. Ekbom's syndrome: lipomas, ataxia, and neuropathy with tRNAI'u(UUR), which represent two genetic hot MERRF. Musclk Nerve 1994;17:943-5. spots in maternally inherited syndromes.2' We 18 Ciafaloni E, Shanske S, Apostolski S, et al. Multiple deletions of mitochondrial DNA. Neurology 1991 ;41 also excluded the T9957C mutation at the (suppl) :207. gene for subunit III of cytochrome c oxidase 19 Zeviani M, Muntoni F, Savarese N, et al. A MERRF/ which has been recently reported in MELAS.22 MELAS overlap syndrome associated with a new point mutation in the mitochondrial DNA tRNA'' gene. Eur]_ In conclusion, the presented pedigree Humn Genet 1992;1:80-7.

20 Moraes CT, Ricci E, Bonilla E, DiMauro S, Schon EA. on September 27, 2021 by guest. Protected copyright. challenges the known correlations between the The mitochondrial tRNALeu(UUR) mutation in mito- clinical phenotype and the genotype of the chondrial encephalomyopathy, lactic acidosis, and it indicates that stroke-like episodes (MELAS): genetic, biochemical, and mtDNA related disorders, and morphological correlations in skeletal muscle. Am 7 Hum a pure MERRF syndrome must be actually Genet 1992;50:934-49. 21 Moraes CT, Ciacci F, Bonilla E, et al. Two novel patho- considered as a possible phenotype of the genic mitochondrial DNA mutations affecting organelle A3243G mutation at the tRNAl-u(UUR). Further number and protein synthesis. Is the tRNALeu(UUR) gene an etiologic hot spot? _7 Clin Invest 1993;92: molecular genetic studies are needed to dis- 2906-15. close other alterations of the mtDNA which 22 Manfredi G, Schon EA, Moraes CT, et al. New mutation of the associated with MELAS is located in a mitochondrial could account for the clinical oddity DNA polypeptide-coding gene. N"euromuscul Disord 1995; described pedigree. 5:391-8.