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Severe encephalomyopathy in a patient with homoplasmic A5814G point mutation in mitochondrial tRNACys gene
Neuromuscular Disorders 17 (2007) 258–261 www.elsevier.com/locate/nmd
Case report
Severe encephalomyopathy in a patient with homoplasmic A5814G point mutation in mitochondrial tRNACys gene Carmela Scuderi a,*, Eugenia Borgione b, Sebastiano Musumeci a, Maurizio Elia a, Filippa Castello b, Marco Fichera c, Guido Davidzon d, Salvatore DiMauro d a Dipartimento di Neurologia, IRCCS Oasi Maria SS, Troina, Italy Laboratorio di Neuropatologia Clinica, IRCCS Oasi Maria SS, Troina, Italy c Laboratorio di Diagnosi Genetica, IRCCS Oasi Maria SS, Troina, Italy Department of Neurology, Columbia University Medical Centers, New York, NY 10032, USA b
d
Received 4 May 2006; received in revised form 13 November 2006; accepted 23 November 2006
Abstract We report a patient with severe encephalomyopathy and homoplasmic A5814G point mutation in the mitochondrial DNA tRNA gene for cysteine. This mutation had been reported in heteroplasmic condition in patients with different clinical phenotypes. Our results confirm the pathogenicity of the mutation and support the concept that homoplasmic mutations in tRNA genes can be responsible for mitochondrial disorders with variable penetrance. This report also extends the clinical spectrum associated with the A5814G mutation. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Mitochondrial DNA; tRNACys; Homoplasmic mutation; Nuclear factors
1. Introduction Mitochondrial encephalomyopathies are a heterogeneous group of diseases often associated with multisystemic presentations [1]. The most severely affected organs are those with high oxidative metabolism, such as brain, skeletal and cardiac muscle, and kidney [1]. Diseases due to mutations in mitochondrial DNA (mtDNA) are usually transmitted by maternal inheritance and are heteroplasmic, i.e., mutant and wild-type mtDNAs coexist in tissues. Since 1988, over 150 point mutations in mitochondrial DNA have been associated with human diseases, many involving mitochondrial tRNA genes [1]. Pathogenic tRNA mutations typically impair mtDNA transla-
*
Corresponding author. E-mail address: [email protected] (C. Scuderi).
0960-8966/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2006.11.006
tion, resulting in disruption of protein synthesis and reduction in the activities of respiratory chain complexes containing mtDNA-encoded polypeptides. Here, we describe a woman with severe encephalomyopathy and her oligosymptomatic relatives, who harbor an apparently homoplasmic A5814G mutation in the tRNACys gene of mtDNA. 2. Case report The family tree is shown in Fig. 1A. The proband (III-1) was a 30-year-old woman, born at term by normal delivery and after an uneventful pregnancy. In the first months of life, the mother noted that she did not follow objects and interacted poorly with the environment. She controlled her head at 2 years of age, sat without support at 4, but could never stand without support or walk, and she never learned to speak. At age 8 years, she developed hand tremor and at age 17 she manifested
C. Scuderi et al. / Neuromuscular Disorders 17 (2007) 258–261
Fig. 1. (A) Pedigree tree. Individuals that present A5814G mutation are represented by solid gray symbols. (B) PCR/RFLP analysis. MM, molecular marker; U, uncut PCR product; C, control; B, blood; M, muscle; R, rectal mucose. The 298 bp PCR amplified fragment is normally cleaved by the endonuclease XcmI in two fragments sized 259 and 39 bp. The A5814G mutation creates a new restriction site at nt 5821. Thus, XcmI cleaves mutant mtDNA into three fragments sized 137, 122 and 39 bp.
psychomotor agitation and aggressiveness. At age 21, she had multiple brief seizures, characterized by loss of consciousness, lip cyanosis, extension of the right arm, followed by generalized jerks. At age 26, physical examination showed short stature (height 142 cm), low weight (46 kg), microcephaly (head circumference 49 cm), and coarse face. Neurological examination showed deafness, tetraplegia with flection of the limbs, plastic hypertonia of the arms and hypotonia of the legs, diffuse muscle wasting, postural and intentional gross tremor of the arms and fine tremor of the chin; tendon reflexes were decreased in the legs. At age 23, she had two episodes of bladder retention. When last seen at age 30, the patient had generalized spasticity and severe diffuse muscle wasting. Examination of the fundus, which had been normal when first examined at age 26, showed bilateral retinal dystrophy. Blood CK was elevated (350 IU/L, normal values 24– 173) only once. The following tests were normal: karyotype, plasma amino acids, lactate and pyruvate, liver function tests, complete blood count, lysosomal enzymes, ceruloplasmin, urinary polysaccharides, thyroid tests, cardiac, thyroid and hepatic ultrasonography. Urine organic acids showed increased lactic acid (201 lg/mg creatinine, normal values <50) and pyruvic acid (33 lg/mg creatinine, normal values <15). Electro-
259
Fig. 2. Brain magnetic resonance imaging of the patient III-1 (A and B) showing slight cerebral atrophy and her mother II-2 (C and D) revealing slight dilatation of the cerebral sulci and moderate atrophy of the cerebellar vermis.
cardiogram showed a left anterior atrioventricular block. Cognitive tests showed profound mental retardation. EEG showed almost continuous diffuse theta waves at 5 Hz and multifocal spikes prominent over the frontal regions and not correlated with rhythmic jerks of the arms. After a mitochondrial disease was diagnosed, valproic acid was replaced by lamotrigine and the patient had only rare partial complex and one tonic-clonic generalized seizures. Brain MRI showed slight cerebral atrophy (Figs. 2A and B). The 45-year-old proband’s mother (II-2) had only mild scoliosis. Her brain MRI revealed slight dilatation of the cerebral sulci and moderate atrophy of the cerebellar vermis (Figs. 2C and D). A 31-year-old maternal aunt (II-6) had mild diffuse hypotonia; brain MRI was normal. A 38-year-old maternal uncle (II-5) suffered from headache since his teens; his height was 166.6 cm (10th centile) and his head circumference was 53 cm (3rd centile). Brain T2-weighted MRI showed a small periventricular white matter hyperintensity. 3. Materials and methods 3.1. Morphologic and biochemical analysis Muscle specimens of the right quadriceps from the proband and her mother were immediately frozen in
260
C. Scuderi et al. / Neuromuscular Disorders 17 (2007) 258–261
liquid nitrogen-cooled isopentane. Cryostat sections were stained using hematoxylin and eosin and by a battery of standard histochemical stains. Mitochondrial enzyme activities in muscle extracts were determined by standardized techniques. 3.2. Molecular genetic analysis Samples of total genomic DNA isolated from frozen muscle specimens of the proband and her mother were screened for mtDNA large-scale rearrangements by Southern blot and long PCR and for the known common point mutations by RFLP analysis. We looked for the A5814G mutation by RFLP in blood DNA from the proband, her mother, and six members of the maternal lineage (I-2, II-3, II-5, II-6, III-2, III-3, Fig. 1). The digested products were separated on a non-denaturing polyacrylamide gel and subjected to autoradiography. Total mtDNA was amplified by polymerase chain reaction (PCR) using suitable oligonucleotide primers. The derived PCR fragments were subjected to direct sequencing with the Big Dye Terminator Cycle Sequencing Kit and an ABI PRISM 310 Genetic Analyzer (Perkin Elmer). 4. Results The proband’s muscle biopsy showed variation in fiber size, rare central nuclei, some esterase-positive hypotrophic fibers, type 2 fiber predominance and hypotrophy, some lipid accumulation, and a few fibers staining weakly with the cytochrome c oxidase (COX) reaction. The mother’s biopsy showed slight variation in fiber size, and modest fiber type 2 predominance and grouping. Biochemical analysis of muscle showed decreased activities of complexes I and IV of the respiratory chain in the patient but not in the mother (Table 1). Southern blot and long-PCR of muscle mitochondrial DNA excluded large-scale deletions or depletion. RFLP analysis revealed the presence of the A5814G mutation, which was homoplasmic in muscle and blood
Table 1 Activities of respiratory chain enzymes in muscle extract from the patient referred to activity of citrate synthase Enzymes
Patient III-1
Controls
Complex I Complex II Complex III Complex IV Complex V Citrate synthase
12.9 26.8 140.0 92.0 230.0 92.0
15–31 20–40 100–200 95–165 120–250 90–200
Values are expressed in nmol/min/mg.
from the proband and her mother and in blood from all six maternal relatives (Fig. 1B). Sequence analysis of the proband’s muscle mt-DNA confirmed the presence of the A5814G mutation and revealed 14 homoplasmic polymorphisms already reported as non-pathogenic changes in the MITOMAP database (www.mitomap.org). We also found four previously unreported homoplasmic changes, two in the 16S rRNA gene (A2355G, T2442C), one in the ND2 gene (A5181A), and one in the cyt b gene (T15674C). These four positions are not conserved in mammalian species and the same changes were found in all other family members. 5. Discussion The clinical presentation of our patient did not correspond to any defined syndrome, but was highly suggestive of a mitochondrial encephalomyopathy because of the complex neurological picture, which included mental retardation, epilepsy, tetraplegia, cerebellar and extrapyramidal signs, and muscle atrophy. In addition, this patient showed deafness, retinal dystrophy, short stature, microcephaly, dysmorphic features, recurrent vomiting, and bladder dysfunction. Brain MRI showed nonspecific diffuse cerebral atrophy, which is not incompatible with mitochondrial encephalopathy. Onset in infancy, mental retardation, and dysmorphic features are not common in disorders due to mtDNA mutations, but have been reported in some patients [2]. The diagnosis of mitochondrial encephalopathy is also supported by the increased urinary excretion of lactate and pyruvate, and by muscle morphology and biochemistry in the muscle of the proband. Intriguingly, the A5814G mutation was homoplasmic in the patient and in all maternal relatives examined, although these presented only mild clinical signs, such as short stature, microcephaly, headache, hypotonia, and asymptomatic cerebellar atrophy. In contrast, the A5814G mutation was heteroplasmic in three previously described unrelated patients with different clinical presentations [3–5]. The first patient was a 5-year-old girl with a MELAS-like picture, who developed episodic nausea and vomiting, tonic-clonic seizures, and hemiparesis at age three years [3]. The second patient was a 33year-old woman, who at age 25 developed progressive external ophthalmoparesis (PEO), gait ataxia, myoclonic jerks, generalized tonic-clonic seizures, lactic acidosis, and abnormal cardiac conduction [4]. The third patient was a 5-year-old boy with developmental delay, myopathy and cardiomyopathy [5]. These previous reports support the pathogenicity of A5814G mutation, according to several canonical criteria, including the following: (i) the A-to-G transition at nt 5814 alters a base-pair in the D-stem of tRNACys, which is highly conserved among species; (ii) the muta-
C. Scuderi et al. / Neuromuscular Disorders 17 (2007) 258–261
tion was absent in healthy controls; and (iii) the mutation segregated in the maternal lineage and healthy or oligosymptomatic relatives had lower mutation loads than patients [3,4]. Heteroplasmy has been traditionally considered important evidence for the pathogenicity of mtDNA mutations, but clinical heterogeneity is only partially explained by heteroplasmy. Conversely, there are pathogenic mtDNA mutations that are homoplasmic and generally involve protein-coding genes [6], rRNA genes [7], or – less commonly – tRNA genes [8,9]. These mutations are considered relatively mild and may require additional factors to produce a clinical phenotype. One contributing factor might be the age-related decline in respiratory chain function, which, however, cannot explain the extremely early clinical presentation in our patient. Exposure to environmental toxic agents can also unmask the effects of an otherwise silent homoplasmic mutation [7], but the clinical history of our patient did not reveal any obvious exposure to noxious factors, although we cannot exclude pre- or perinatal distress. Concurrent mtDNA mutations can increase the risk of expression of a homoplasmic mtDNA mutation, as illustrated by ‘‘secondary mutations’’ associated with Leber’s hereditary optic atrophy (LHON) [6]. While we did identify numerous polymorphisms in the mtDNA of our patient, the presence of only subtle clinical signs in our patient’s relatives harbouring the same polymorphisms militates against this explanation. More likely, the different phenotypes manifested by patients homoplasmic for the A5814G mutation are due to modifying nuclear genes. Nuclear genes encode most polypeptides of the respiratory chain and all other mitochondrial proteins. Nuclear gene variants have been hypothesized to explain the variable expression of LHON, of sensorineural deafness in families with homoplasmic 12S rRNA gene mutation [7] and of the A3243G MELAS mutation [10]. Also, cybrid studies, in heteroplasmic condition, showed that nuclear background influences the proportion of mutant mtDNA [11] and, in the case of homoplasmic mutation, the steady state levels of mt-tRNA [9,12]. In conclusion, this report extends the clinical spectrum associated with the A5814G mutation. This mutation, observed in both heteroplasmic and homoplasmic patients, appears to be poorly pathogenic by itself, but has the potential of becoming severely pathogenic in
261
some conditions. What these conditions are remains to be determined.
Acknowledgements This work was supported by NIH Grants HD32062 and NS11766, by a grant from the Muscular Dystrophy Association, and by the Marriott Mitochondrial Disorder Clinical Research Fund (MMDCRF).
References [1] DiMauro S, Hirano M. Mitochondrial encephalomyopathies: an update. Neuromusc Disord 2005;15:276–86. [2] Tulinius MH, Holme E, Kristiansson B, Larsson NG, Oldfors A. Mitochondrial encephalomyopathies in childhood. II. Clinical manifestations and syndromes. J Pediatr 1991;119:251–9. [3] Manfredi G, Schon EA, Bonilla E, Moraes CT, Shanske S, DiMauro S. Identification of a mutation in the mitochondrial tRNACys gene associated with mitochondrial encephalopathy. Hum Mut 1996;7:158–63. [4] Santorelli F, Siciliano G, Casali C, et al. Mitochondrial tRNA(Cys) gene mutation (A5814G): a second family with mitochondrial encephalomyopathy. Neuromusc Disord 1997;7:156–9. [5] Karadimas CL, Tanji K, Geremek M, et al. A5814G mutation in mitochondrial DNA can cause mitochondrial myopathy and cardiomyopathy. J Child Neurol 2001;16:531–3. [6] Carelli V. Leber’s hereditary optic neuropathy. In: Schapira AHV, DiMauro S, editors. Mitochondrial Disorders in Neurology 2. New York: Butterworth-Heinemann; 2002. p. 115–42. [7] Prezant TR, Agapian JV, Bohlman MC, et al. Mitochondrial ribosomal RNA mutation associated with both antibiotic-induced and non-syndromic deafness. Nat Genet 1993;4:289–93. [8] Santorelli FM, Schlessel JS, Slonim AE, DiMauro S. Novel mutation in the mitochondrial DNA tRNA glycine gene associated with sudden unexpected death. Pediatr Neurol 1996;15:145–9. [9] McFarland R, Schaefer AM, Gardner A, et al. Familial myopathy: New insights into the T14709C mitochondrial tRNA mutation. Ann Neurol 2004;55:478–84. [10] Chinnery PF, Zwijnenburg PJ, Walker M, et al. Nonrandom tissue distribution of mutant mtDNA. Am J Med Genet 1999;85:498–501. [11] Dunbar DR, Moonie PA, Jacobs HT, Holt IJ. Different cellular backgrounds confer a marked advantage to either mutant or wildtype mitochondrial genomes. Proc Natl Acad Sci USA 1995;92:6562–6. [12] Taylor RW, Giordano C, Davidson MM, et al. A homoplasmic mitochondrial transfer ribonucleic acid mutation as a cause of maternally inherited cardiomyopathy. J Am Coll Cardiol 2003;41:1786–96.
Case report
Severe encephalomyopathy in a patient with homoplasmic A5814G point mutation in mitochondrial tRNACys gene Carmela Scuderi a,*, Eugenia Borgione b, Sebastiano Musumeci a, Maurizio Elia a, Filippa Castello b, Marco Fichera c, Guido Davidzon d, Salvatore DiMauro d a Dipartimento di Neurologia, IRCCS Oasi Maria SS, Troina, Italy Laboratorio di Neuropatologia Clinica, IRCCS Oasi Maria SS, Troina, Italy c Laboratorio di Diagnosi Genetica, IRCCS Oasi Maria SS, Troina, Italy Department of Neurology, Columbia University Medical Centers, New York, NY 10032, USA b
d
Received 4 May 2006; received in revised form 13 November 2006; accepted 23 November 2006
Abstract We report a patient with severe encephalomyopathy and homoplasmic A5814G point mutation in the mitochondrial DNA tRNA gene for cysteine. This mutation had been reported in heteroplasmic condition in patients with different clinical phenotypes. Our results confirm the pathogenicity of the mutation and support the concept that homoplasmic mutations in tRNA genes can be responsible for mitochondrial disorders with variable penetrance. This report also extends the clinical spectrum associated with the A5814G mutation. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Mitochondrial DNA; tRNACys; Homoplasmic mutation; Nuclear factors
1. Introduction Mitochondrial encephalomyopathies are a heterogeneous group of diseases often associated with multisystemic presentations [1]. The most severely affected organs are those with high oxidative metabolism, such as brain, skeletal and cardiac muscle, and kidney [1]. Diseases due to mutations in mitochondrial DNA (mtDNA) are usually transmitted by maternal inheritance and are heteroplasmic, i.e., mutant and wild-type mtDNAs coexist in tissues. Since 1988, over 150 point mutations in mitochondrial DNA have been associated with human diseases, many involving mitochondrial tRNA genes [1]. Pathogenic tRNA mutations typically impair mtDNA transla-
*
Corresponding author. E-mail address: [email protected] (C. Scuderi).
0960-8966/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2006.11.006
tion, resulting in disruption of protein synthesis and reduction in the activities of respiratory chain complexes containing mtDNA-encoded polypeptides. Here, we describe a woman with severe encephalomyopathy and her oligosymptomatic relatives, who harbor an apparently homoplasmic A5814G mutation in the tRNACys gene of mtDNA. 2. Case report The family tree is shown in Fig. 1A. The proband (III-1) was a 30-year-old woman, born at term by normal delivery and after an uneventful pregnancy. In the first months of life, the mother noted that she did not follow objects and interacted poorly with the environment. She controlled her head at 2 years of age, sat without support at 4, but could never stand without support or walk, and she never learned to speak. At age 8 years, she developed hand tremor and at age 17 she manifested
C. Scuderi et al. / Neuromuscular Disorders 17 (2007) 258–261
Fig. 1. (A) Pedigree tree. Individuals that present A5814G mutation are represented by solid gray symbols. (B) PCR/RFLP analysis. MM, molecular marker; U, uncut PCR product; C, control; B, blood; M, muscle; R, rectal mucose. The 298 bp PCR amplified fragment is normally cleaved by the endonuclease XcmI in two fragments sized 259 and 39 bp. The A5814G mutation creates a new restriction site at nt 5821. Thus, XcmI cleaves mutant mtDNA into three fragments sized 137, 122 and 39 bp.
psychomotor agitation and aggressiveness. At age 21, she had multiple brief seizures, characterized by loss of consciousness, lip cyanosis, extension of the right arm, followed by generalized jerks. At age 26, physical examination showed short stature (height 142 cm), low weight (46 kg), microcephaly (head circumference 49 cm), and coarse face. Neurological examination showed deafness, tetraplegia with flection of the limbs, plastic hypertonia of the arms and hypotonia of the legs, diffuse muscle wasting, postural and intentional gross tremor of the arms and fine tremor of the chin; tendon reflexes were decreased in the legs. At age 23, she had two episodes of bladder retention. When last seen at age 30, the patient had generalized spasticity and severe diffuse muscle wasting. Examination of the fundus, which had been normal when first examined at age 26, showed bilateral retinal dystrophy. Blood CK was elevated (350 IU/L, normal values 24– 173) only once. The following tests were normal: karyotype, plasma amino acids, lactate and pyruvate, liver function tests, complete blood count, lysosomal enzymes, ceruloplasmin, urinary polysaccharides, thyroid tests, cardiac, thyroid and hepatic ultrasonography. Urine organic acids showed increased lactic acid (201 lg/mg creatinine, normal values <50) and pyruvic acid (33 lg/mg creatinine, normal values <15). Electro-
259
Fig. 2. Brain magnetic resonance imaging of the patient III-1 (A and B) showing slight cerebral atrophy and her mother II-2 (C and D) revealing slight dilatation of the cerebral sulci and moderate atrophy of the cerebellar vermis.
cardiogram showed a left anterior atrioventricular block. Cognitive tests showed profound mental retardation. EEG showed almost continuous diffuse theta waves at 5 Hz and multifocal spikes prominent over the frontal regions and not correlated with rhythmic jerks of the arms. After a mitochondrial disease was diagnosed, valproic acid was replaced by lamotrigine and the patient had only rare partial complex and one tonic-clonic generalized seizures. Brain MRI showed slight cerebral atrophy (Figs. 2A and B). The 45-year-old proband’s mother (II-2) had only mild scoliosis. Her brain MRI revealed slight dilatation of the cerebral sulci and moderate atrophy of the cerebellar vermis (Figs. 2C and D). A 31-year-old maternal aunt (II-6) had mild diffuse hypotonia; brain MRI was normal. A 38-year-old maternal uncle (II-5) suffered from headache since his teens; his height was 166.6 cm (10th centile) and his head circumference was 53 cm (3rd centile). Brain T2-weighted MRI showed a small periventricular white matter hyperintensity. 3. Materials and methods 3.1. Morphologic and biochemical analysis Muscle specimens of the right quadriceps from the proband and her mother were immediately frozen in
260
C. Scuderi et al. / Neuromuscular Disorders 17 (2007) 258–261
liquid nitrogen-cooled isopentane. Cryostat sections were stained using hematoxylin and eosin and by a battery of standard histochemical stains. Mitochondrial enzyme activities in muscle extracts were determined by standardized techniques. 3.2. Molecular genetic analysis Samples of total genomic DNA isolated from frozen muscle specimens of the proband and her mother were screened for mtDNA large-scale rearrangements by Southern blot and long PCR and for the known common point mutations by RFLP analysis. We looked for the A5814G mutation by RFLP in blood DNA from the proband, her mother, and six members of the maternal lineage (I-2, II-3, II-5, II-6, III-2, III-3, Fig. 1). The digested products were separated on a non-denaturing polyacrylamide gel and subjected to autoradiography. Total mtDNA was amplified by polymerase chain reaction (PCR) using suitable oligonucleotide primers. The derived PCR fragments were subjected to direct sequencing with the Big Dye Terminator Cycle Sequencing Kit and an ABI PRISM 310 Genetic Analyzer (Perkin Elmer). 4. Results The proband’s muscle biopsy showed variation in fiber size, rare central nuclei, some esterase-positive hypotrophic fibers, type 2 fiber predominance and hypotrophy, some lipid accumulation, and a few fibers staining weakly with the cytochrome c oxidase (COX) reaction. The mother’s biopsy showed slight variation in fiber size, and modest fiber type 2 predominance and grouping. Biochemical analysis of muscle showed decreased activities of complexes I and IV of the respiratory chain in the patient but not in the mother (Table 1). Southern blot and long-PCR of muscle mitochondrial DNA excluded large-scale deletions or depletion. RFLP analysis revealed the presence of the A5814G mutation, which was homoplasmic in muscle and blood
Table 1 Activities of respiratory chain enzymes in muscle extract from the patient referred to activity of citrate synthase Enzymes
Patient III-1
Controls
Complex I Complex II Complex III Complex IV Complex V Citrate synthase
12.9 26.8 140.0 92.0 230.0 92.0
15–31 20–40 100–200 95–165 120–250 90–200
Values are expressed in nmol/min/mg.
from the proband and her mother and in blood from all six maternal relatives (Fig. 1B). Sequence analysis of the proband’s muscle mt-DNA confirmed the presence of the A5814G mutation and revealed 14 homoplasmic polymorphisms already reported as non-pathogenic changes in the MITOMAP database (www.mitomap.org). We also found four previously unreported homoplasmic changes, two in the 16S rRNA gene (A2355G, T2442C), one in the ND2 gene (A5181A), and one in the cyt b gene (T15674C). These four positions are not conserved in mammalian species and the same changes were found in all other family members. 5. Discussion The clinical presentation of our patient did not correspond to any defined syndrome, but was highly suggestive of a mitochondrial encephalomyopathy because of the complex neurological picture, which included mental retardation, epilepsy, tetraplegia, cerebellar and extrapyramidal signs, and muscle atrophy. In addition, this patient showed deafness, retinal dystrophy, short stature, microcephaly, dysmorphic features, recurrent vomiting, and bladder dysfunction. Brain MRI showed nonspecific diffuse cerebral atrophy, which is not incompatible with mitochondrial encephalopathy. Onset in infancy, mental retardation, and dysmorphic features are not common in disorders due to mtDNA mutations, but have been reported in some patients [2]. The diagnosis of mitochondrial encephalopathy is also supported by the increased urinary excretion of lactate and pyruvate, and by muscle morphology and biochemistry in the muscle of the proband. Intriguingly, the A5814G mutation was homoplasmic in the patient and in all maternal relatives examined, although these presented only mild clinical signs, such as short stature, microcephaly, headache, hypotonia, and asymptomatic cerebellar atrophy. In contrast, the A5814G mutation was heteroplasmic in three previously described unrelated patients with different clinical presentations [3–5]. The first patient was a 5-year-old girl with a MELAS-like picture, who developed episodic nausea and vomiting, tonic-clonic seizures, and hemiparesis at age three years [3]. The second patient was a 33year-old woman, who at age 25 developed progressive external ophthalmoparesis (PEO), gait ataxia, myoclonic jerks, generalized tonic-clonic seizures, lactic acidosis, and abnormal cardiac conduction [4]. The third patient was a 5-year-old boy with developmental delay, myopathy and cardiomyopathy [5]. These previous reports support the pathogenicity of A5814G mutation, according to several canonical criteria, including the following: (i) the A-to-G transition at nt 5814 alters a base-pair in the D-stem of tRNACys, which is highly conserved among species; (ii) the muta-
C. Scuderi et al. / Neuromuscular Disorders 17 (2007) 258–261
tion was absent in healthy controls; and (iii) the mutation segregated in the maternal lineage and healthy or oligosymptomatic relatives had lower mutation loads than patients [3,4]. Heteroplasmy has been traditionally considered important evidence for the pathogenicity of mtDNA mutations, but clinical heterogeneity is only partially explained by heteroplasmy. Conversely, there are pathogenic mtDNA mutations that are homoplasmic and generally involve protein-coding genes [6], rRNA genes [7], or – less commonly – tRNA genes [8,9]. These mutations are considered relatively mild and may require additional factors to produce a clinical phenotype. One contributing factor might be the age-related decline in respiratory chain function, which, however, cannot explain the extremely early clinical presentation in our patient. Exposure to environmental toxic agents can also unmask the effects of an otherwise silent homoplasmic mutation [7], but the clinical history of our patient did not reveal any obvious exposure to noxious factors, although we cannot exclude pre- or perinatal distress. Concurrent mtDNA mutations can increase the risk of expression of a homoplasmic mtDNA mutation, as illustrated by ‘‘secondary mutations’’ associated with Leber’s hereditary optic atrophy (LHON) [6]. While we did identify numerous polymorphisms in the mtDNA of our patient, the presence of only subtle clinical signs in our patient’s relatives harbouring the same polymorphisms militates against this explanation. More likely, the different phenotypes manifested by patients homoplasmic for the A5814G mutation are due to modifying nuclear genes. Nuclear genes encode most polypeptides of the respiratory chain and all other mitochondrial proteins. Nuclear gene variants have been hypothesized to explain the variable expression of LHON, of sensorineural deafness in families with homoplasmic 12S rRNA gene mutation [7] and of the A3243G MELAS mutation [10]. Also, cybrid studies, in heteroplasmic condition, showed that nuclear background influences the proportion of mutant mtDNA [11] and, in the case of homoplasmic mutation, the steady state levels of mt-tRNA [9,12]. In conclusion, this report extends the clinical spectrum associated with the A5814G mutation. This mutation, observed in both heteroplasmic and homoplasmic patients, appears to be poorly pathogenic by itself, but has the potential of becoming severely pathogenic in
261
some conditions. What these conditions are remains to be determined.
Acknowledgements This work was supported by NIH Grants HD32062 and NS11766, by a grant from the Muscular Dystrophy Association, and by the Marriott Mitochondrial Disorder Clinical Research Fund (MMDCRF).
References [1] DiMauro S, Hirano M. Mitochondrial encephalomyopathies: an update. Neuromusc Disord 2005;15:276–86. [2] Tulinius MH, Holme E, Kristiansson B, Larsson NG, Oldfors A. Mitochondrial encephalomyopathies in childhood. II. Clinical manifestations and syndromes. J Pediatr 1991;119:251–9. [3] Manfredi G, Schon EA, Bonilla E, Moraes CT, Shanske S, DiMauro S. Identification of a mutation in the mitochondrial tRNACys gene associated with mitochondrial encephalopathy. Hum Mut 1996;7:158–63. [4] Santorelli F, Siciliano G, Casali C, et al. Mitochondrial tRNA(Cys) gene mutation (A5814G): a second family with mitochondrial encephalomyopathy. Neuromusc Disord 1997;7:156–9. [5] Karadimas CL, Tanji K, Geremek M, et al. A5814G mutation in mitochondrial DNA can cause mitochondrial myopathy and cardiomyopathy. J Child Neurol 2001;16:531–3. [6] Carelli V. Leber’s hereditary optic neuropathy. In: Schapira AHV, DiMauro S, editors. Mitochondrial Disorders in Neurology 2. New York: Butterworth-Heinemann; 2002. p. 115–42. [7] Prezant TR, Agapian JV, Bohlman MC, et al. Mitochondrial ribosomal RNA mutation associated with both antibiotic-induced and non-syndromic deafness. Nat Genet 1993;4:289–93. [8] Santorelli FM, Schlessel JS, Slonim AE, DiMauro S. Novel mutation in the mitochondrial DNA tRNA glycine gene associated with sudden unexpected death. Pediatr Neurol 1996;15:145–9. [9] McFarland R, Schaefer AM, Gardner A, et al. Familial myopathy: New insights into the T14709C mitochondrial tRNA mutation. Ann Neurol 2004;55:478–84. [10] Chinnery PF, Zwijnenburg PJ, Walker M, et al. Nonrandom tissue distribution of mutant mtDNA. Am J Med Genet 1999;85:498–501. [11] Dunbar DR, Moonie PA, Jacobs HT, Holt IJ. Different cellular backgrounds confer a marked advantage to either mutant or wildtype mitochondrial genomes. Proc Natl Acad Sci USA 1995;92:6562–6. [12] Taylor RW, Giordano C, Davidson MM, et al. A homoplasmic mitochondrial transfer ribonucleic acid mutation as a cause of maternally inherited cardiomyopathy. J Am Coll Cardiol 2003;41:1786–96.