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 atro- phy, 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 oligosympto- matic 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 gener- alised myoclonic seizures. Brain MRI per- formed at the age of 18 showed a severe atrophy of the cerebral cortex and subcortical white matter, as well as of the semiovale cen- tres 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 atro- phy 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 per- formed 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 * <t8;2.'.,r dehydrogenase stainling on the proband's second muscle biopsy (x 175) shows a small artery with densely stained granules in its wall. t ;A_;S.+X9. / (D) Electronmicrography I X,ttt; 0S of the proband's second mtiuscle biopsy, shows aggregates 4 A; i 4\$S ! ! of ;i i.ntermyofibrillar .j.l, mitochondria containing paracrystalline inclusions ( x 4200). http://jnnp.bmj.com/ 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.