Journal http://jcn.sagepub.com/ of Child Neurology
Three Patients With Lafora Disease: Different Clinical Presentations and a Novel Mutation Hatice Gamze Poyrazoglu, Emin Karaca, Hüseyin Per, Hakan Gümüs, Huseyin Onay, Mehmet Canpolat, Özlem Canöz, Ferda Ozkinay and Sefer Kumandas J Child Neurol published online 10 July 2014 DOI: 10.1177/0883073814535489 The online version of this article can be found at: http://jcn.sagepub.com/content/early/2014/07/10/0883073814535489
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Brief Communication
Three Patients With Lafora Disease: Different Clinical Presentations and a Novel Mutation
Journal of Child Neurology 1-5 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0883073814535489 jcn.sagepub.com
Hatice Gamze Poyrazog˘lu, MD1, Emin Karaca, MD2, Hu¨seyin Per, MD1, Hakan Gu¨mu¨s, MD1, Huseyin Onay, MD1, Mehmet Canpolat, MD1, ¨ zlem Cano¨z, MD3, Ferda Ozkınay, MD2, and Sefer Kumandas, MD1 O
Abstract Lafora disease is a rare, fatal, autosomal recessive hereditary disease characterized by epilepsy, myoclonus and progressive neurological deterioration. Diagnosis is made by polyglucosan inclusion bodies (Lafora bodies) shown in skin biopsy. Responsible mutations of Lafora disease involves either the EPM2A or NHLRC1 (EPM2B) gene. Mutations in the NHLRC1 gene are described as having a more benign clinical course and a later age of death compared with EPM2A mutations. We report 2 genetic mutations and clinical courses of Lafora disease in 3 adolescents with homozygote NHLRC1 mutation and novel homozygous EPM2A mutation. Keywords progressive myoclonic epilepsy, Lafora disease, genetic Received September 05, 2013. Received revised April 01, 2014. Accepted for publication April 14, 2014.
Lafora disease [LD] is a progressive, fatal disease that is inherited autosomal recessively and usually begins in the second decade. First symptoms of the disease are resistant myoclonic seizures, myoclonus, generalized or focal occipital seizures, and photosensitivity on electroencephalography (EEG) in patients with previously normal neurologic development.1 This is followed by not only worsening of seizures but also deterioration in cognitive and neurologic functions such as dysarthria, ataxia, and dementia. After the first symptoms, most patients die within 10 years of diagnosis, because of status epilepticus or complications associated with the degeneration of the nervous system.2 Polyglucosan inclusions (Lafora bodies) are observed in the typical periodic acid-Schiff–positive cytoplasm in the biopsy specimens. Although biopsy specimens could be taken from brain, spinal cord, skin, liver, and skeletal muscles, biopsy specimens from the axillary apocrine sweat glands is preferred mostly because it is less invasive.2-4 LD is caused by the mutation in EPM2A or EPM2B / NHLRC1 genes that encode laforin and malin, respectively.1,5 There is no significant difference clinically between patients with mutations in the EPM2A and NHLRC1 genes. However, it was reported that patients with NHLRC1 mutation had slowly progressive course and longer survival when compared with EPM2A mutation.5,6 In the present study, we report 3 patients one with identified homozygous mutation in the NHLRC1 gene who
experienced a fast and progressive clinical course and 2 siblings with identified homozygous mutation in the EPM2A genes who experienced slow progression with typical clinical signs.
Case Report Case 1 A 16-year-old female patient was evaluated for progressive myoclonus epilepsy. There were no problems at birth and during childhood, and her psychomotor development was normal and she was attended regular school. She had no health problems up to age 13 years. She presented with generalized 1
Department of Pediatrics, Division of Pediatric Neurology, Faculty of Medicine, Erciyes University, Kayseri, Turkey 2 Department of Medical Genetics, Faculty of Medicine, Ege University, _Izmir, Turkey 3 Department of Pathology, Faculty of Medicine, Erciyes University, Kayseri, Turkey Corresponding Author: Hatice Gamze Poyrazog˘lu, MD, Erciyes University, Faculty of Medicine, Department of Pediatrics, Division of Pediatric Neurology, 38039, Melikgazi, Kayseri, Turkey. Email: [email protected]
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Figure 1. Lafora bodies stain strongly positive with periodic acidSchiff, revealing numerous small ductlike granules in apocrine gland biopsy.
tonic-clonic seizures 3 years ago, and generalized myoclonic seizures were observed in later periods. Her seizures could not be controlled despite the antiepileptic drugs valproic acid, 2000 mg/d; clonazepam, 4 mg/d; levetiracetam, 1500 mg/d; and topiramate, 4 mg/kg/d (200 mg/d). Her 14-year-old brother had epilepsy history (case 2). Her parents were second-degree relatives. She was found to have disordered cognitive functions, perception, and speech as well as tremor in hands and gait disturbance in her neurologic examination. No pathology was found in her routine blood tests, ammonia, lactate and pyruvate ratios, quantification of amino acids of plasma and urine, and urine organic acid levels. Cerebrospinal fluid for white blood cell counts, glucose, protein, and measles immunoglobulin M and G levels were normal. Multifocal, from time-to-time generalized spike-wave activity, that suppressed with diazepam was observed with EEG. Mild cerebral and cerebellar atrophy were detected in her cranial magnetic resonance imaging (MRI). Eye examination and retinal examination were normal but electroretinogram latency delays were detected. Her antiepileptic therapy was rearranged. Topiramate and clonazepam were eliminated and zonisamide was added. Seizures were decreased significantly subsequent to zonisamide treatment. According to the clinical features, laboratory findings (blood test, EEG, and MRI), and ophthalmologic examination, we suggested the diagnosis of hereditary progressive myoclonus epilepsy. Periodic acid-Schiff–positive cytoplasmic polyglucosan inclusion bodies (termed as Lafora bodies) were identified in the apocrine sweat gland at axillary skin biopsy (Figure 1). Homozygous (c.336C>A) p.Y112X, a new mutation, in the EPM2A gene was detected in the genetic analysis.
Case 2 A 14-year-old male patient was evaluated for progressive myoclonus epilepsy. There were no problems at birth and during
childhood, and his psychomotor development was normal and he attended regular school. His general health was good. He presented at the age of 13 with myoclonic-astatic seizures. At the beginning of the disease, his seizures could be kept under control by valproic acid treatment but recently, frequency of seizures had increased and generalized tonic-clonic seizures were observed. His performance at school deteriorated. His parents were first-cousins as regards the degree of kinship. His sister, case 1, was also followed up with a diagnosis of intractable epilepsy. No pathologic disorder was found in neurologic examination of the patient, except mild disorder in cognitive functions at the time of admission. His routine blood tests, ammonia, lactate, pyruvate, and metabolic tests (tandem mass spectrometry analysis, urine and blood amino acids, and urine organic acid analysis) and cerebrospinal fluid analysis were normal. Multifocal, from time to time generalized, multiple spikes and spike-wave activity were observed in his EEG. No pathology was detected in electroretinogram, and his retinal examination was found normal. Mild cerebral and cerebellar atrophy was detected on cranial magnetic resonance imaging. The clinical features and pattern of inheritance in the family, EEG findings, and resistance to anticonvulsant treatment, as well as laboratory evaluations and ophthalmologic examination suggested the diagnosis of hereditary progressive myoclonus epilepsy. Periodic acid-Schiff-positive Lafora bodies in the cytoplasm were observed in his axillary skin biopsy. Homozygous (c.336C>A) p.Y112X, a novel mutation, in the EPM2A gene was detected in the genetic analysis.
Case 3 A 14-year-old female patient was evaluated for progressive myoclonus epilepsy. Her medical history showed no problems at birth and during childhood and psychomotor development was normal, and she attended regular school and her school performance was very good. Her general health was good. She presented at the age of 13 with generalized tonic-clonic seizures that began following episodic visual auras. She received valproic acid treatment. Much as there was a significant decrease in her seizures, it continued for a year after the treatment. One year later, segmental myoclonus started in addition to the occipital and generalized seizures. Clonazepam was added to her treatment; however, her seizures continued despite the dual treatment. Her parents had third degree of kinship and there was no similar case in the family. Motor and sensory impairment was not found in her neurologic examination and she had no gait disorder and her deep tendon reflexes were normal. No pathology was found in her routine blood tests, metabolic screening tests, and cerebrospinal fluid analysis. Her cranial magnetic resonance imaging was normal. Spike waves and multiple spike waves due to both 2 hemispheric centrotemporal and occipital regions were viewed in her EEG. Spontaneous myoclonus and myoclonic activities in the extremities, body ataxia, gait impairment, dysarthria, and significant learning difficulties began 6 months after admission.
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Figure 2. (A) Electropherogram of the Y112X heterozygous mutation in the EPM2A gene of the unaffected (carrier) father. (B) Electropherogram of the Y112X homozygous mutation in the EPM2A gene of the patient. (C) Electropherogram of the Y112X heterozygous mutation in the EPM2A gene of unaffected (carrier) mother. (The arrows show the position of the mutation.)
Feeding difficulties, loss of ability to walk without assistance, and dementia developed. The patient, whose seizures continued, was treated with zonisamide, and the generalized tonic-clonic seizures disappeared. Periodic acid-Schiff–positive Lafora bodies in cytoplasm were observed in her axillary skin biopsy. No mutations were found in her EPM2A gene in the genetic analysis. Homozygous (c.199 G>T) p.E67X mutation was detected in her NHLRC1 gene that resulted in a premature stop codon just at the beginning of exon 1. The general condition of the patient deteriorated 9 months after admission, and the patient who developed status epilepticus and pneumonia was taken in intensive care. In her follow-up, there was no improvement in clinical status, and she died in the 21st month approximately from the time of diagnosis.
Genetic Screening of the Cases The coding sequences from both EPM2A and NHLRC1 (EPM2B) were amplified by polymerase chain reaction as previously described.7,8 Polymerase chain reaction products were purified using the ZR-96 DNA Sequencing Clean-up Kit (Zymo Research Corp, Irvine, CA). Purified products were then sequenced and screened for mutations. This screening identified a novel homozygous (c.336C> A) p.Y112X mutation in genes for EPM2A 1 in cases 1 and 2 (Figure 2). This mutation
is thought to be overdetermined as it forms a stop codon early on and terminates the transcription. The father and mother were heterozygous carriers of the mutation (Figure 2). Genetic screening revealed a homozygous missense mutation (c.199 G> T) p.E67X for the NHLRC1 (EPM2B) gene and results in a premature stop codon at just the beginning of exon 1 in case 3.
Discussion Lafora body disease demonstrates progressive myoclonic epilepsy as an autosomal recessive hereditary disease. Clinical manifestations commence in early childhood and adolescent period (11-18 years) with generalized tonic-clonic, myoclonic occipital seizures, and visual hallucinations. Over time, the seizures get worse; dementia occurs that causes mental impairment leading to progression of ataxia and myoclonus and consequently death of the patient in approximately 10 years.5,9 In all of our 3 patients, clinical findings began after 13 years of age with myoclonic seizures whereas in 1 patient it started with visual auras accompanied by myoclonic seizures. Although patients were followed up with antiepileptic drug–resistant myoclonic epilepsy, over time they demonstrated a slowdown in the ground rhythm in EEG, spike waves and multi-spike waves increased, ataxia and significant deterioration occurred in mental functions, myoclonus of the extremities started, and they were unable to walk unassisted.
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It was considered as progressive myoclonic epilepsy. Their fundus examinations were normal. Patients were diagnosed with Lafora disease when they were evaluated by clinical and laboratory findings. Diagnosis of LD is made by detection of Lafora bodies in the axillary skin biopsy or genetic mutation studies along with clinical findings. Lafora bodies, which are the pathognomonic signs of the disease, are intracytoplasmic, basophilic, polyglucosan inclusion bodies that stain positively with periodic acidSchiff. These bodies can be found in skin, skeletal muscle, liver, sweat glands, and heart tissue. Skin biopsy is the least invasive method for pathologic diagnosis. However, the absence of Lafora bodies in skin biopsy does not eliminate the diagnosis of Lafora disease.4 Lafora bodies were demonstrated in the axillary skin biopsy of 3 of our patients. LD is caused by mutations in the EPM2A and EPM2B/ NHLRC1 genes. Mutation was detected in EPM2A genes in up to 97% (range 22%-97%) of families with Lafora disease.2,6,8 EPM2A gene encodes a protein of 331 amino acids (so-called laforin). The substrate and function of laforin have recently been explained. Laforin deficiency also leads to intracellular glycogen particles which are called Lafora bodies.10 On the other hand, EPM2B gene encodes E3 ubiquitin ligase, named malin, which interacts with laforin.11 The primary role of malin appears to be to protect tissues from accumulating polyglucosans by regulating proteins involved in glycogen metabolism. Malin deficiency causes increased laforin, increased laforin-binding glycogen, leading to the formation of Lafora bodies. Moreover, increased levels of laforin, which binds to glycogen, causes Lafora bodies.10,12-14 Genetic allelic heterogeneity is present in LD associated with mutations in EPM2B. Patients with mutations in EPM2A and EPM2B express similar clinical manifestation, although patients with EPM2B-associated LD seem to have a slightly milder clinical course.15,16 Baykan et al17 have reported that 4 brothers diagnosed with LD (average 19.5 years of age) had milder disease course, and homozygous p.D146N mutation was identified in their NHLRC1 gene. In another study, 7 of 17 Italian patients diagnosed with mutation in their NHLRC1 (EPM2B) gene could maintain their daily living activities and social interactions for a period of 4 years from the onset of the disease, and the disease had a slow progression.6 Brackmann et al18 described a rapidly progressive phenotype of Lafora disease in an adolescent patient with a novel NHLRC1 mutation (ending in a stop at position 39) who developed severe disability and dementia less than 2 years after the onset of signs. In the third patient in our study, a homozygous c.199G>T (p.E67X) mutation was detected in her NHLRC1 gene and it resulted in a premature stop codon at just the beginning of exon 1. Consequently, NHLRC1 protein was not synthesized. Thus, we suggest that her disease was a rapidly progressive phenotype of LD. The starting age of these cases, with observation of myoclonic seizures as the only sign for a long time in the first long-term period and its having similar features with juvenile myoclonic epilepsy detected in EEG, may cause it to be diagnosed as Juvenile myoclonic epilepsy in the initial period. However, failure
to show adequate response to pharmacotherapy, monitoring of additional pathologies, including impairment of the ground rhythm in EEG, cognitive impairment, and addition of other types of seizure brings progressive myoclonic epilepsy to the mind first. As in our cases, especially LD should be considered in the differential diagnosis in progressive myoclonic epilepsies seen in adolescence. There is no specific treatment for LD. However, valproic acid used in the treatment of myoclonus at the early stages of the disease and some antiepileptic drugs such as levetiracetam may provide temporary symptomatic cure. Some antiepileptics such as phenytoin, carbamazepine, and vigabatrin can cause an increase in myoclonus.4 Patients with progressive epilepsy whose clinical diagnosis does not improve despite appropriate antiepileptic therapy must be evaluated strictly in terms of the possibility of progressive myoclonic epilepsia, and skin biopsy must be conducted even in cases of mild cognitive impairment; slowing ground rhythm in EEG and genetic analysis should be done for LD. Acknowledgment All authors thank all the patients and family members for their participation in this study.
Author Contributions HGP conceived the study; HG, HP and Ek reviewed the literature; SK provided investigation support; and HGP performed literature review and wrote the manuscript. MC and FO reviewed the manuscript. OC was demonstrated Lafora body in axillary skin biopsy. EK and HO demonstrated genetic mutations of patients. All authors were responsible for patient management.
Declaration of Conflicting Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The authors received no financial support for the research, authorship, and/or publication of this article.
Ethical Approval Informed consent was obtained from the patient’s parents to publish this report.
References 1. Kecmanovic´ M, Jovic´ N, Cukic M, et al. Lafora disease: severe phenotype associated with the homozygous deletion of the NHLRC1 gene. J Neurol Sci. 2013;325:170-173. 2. Minassian BA. Lafora’s disease: towards a clinical, pathologic, and molecular synthesis. Pediatr Neurol. 2001:25:21-29. 3. Ki CS, Kong SY, Seo DW, et al. Novel Mutations in the gene in a Korean patient with EPM2A Lafora’s progressive myoclonus epilepsy. J Hum Genet. 2003;48:51-54. 4. Bocella P, Striano P, Zara F, et al. Bioptically demonstrated Lafora disease without EPM2A mutation: a clinical and neurophysiological study of two sisters. Clin Neurol Neurosurg. 2003;106:55-59.
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5. Lesca G, Boutry-Kryza N, de Toffol B, et al. Novel mutations in EPM2A and NHLRC1 widen the spectrum of Lafora disease. Epilepsia. 2010;51:1691-1698. 6. Franceschetti S, Gambardella A, Canafoglia L, et al. Clinical and genetic findings in 26 Italian patients with Lafora disease. Epilepsia. 2006;47:640-643. 7. Gomez-Garre P, Sanz Y, Rodriguez De Cordoba SR, Serratosa JM. Mutational spectrum of the gene in progressive myoclonus epilepsy EPM2A of Lafora: high degree of allelic heterogeneity and prevalence of deletions. Eur J Hum Genet. 2000; 8:946-954. 8. Singh S, Suzuki T, Uchiyama A, et al. NHLRC1 Mutations in the gene are the common cause for Lafora disease in the Japanese population. J Hum Genet. 2005;50:347-352. 9. Koc¸ AF, Bozdemir H, Zorludemir S, et al. Lafora body disease: clinical, electrophysiological and histopathological findings. Turk J Med Sci. 2004;34:379-384. 10. Ferna´ndez-Sa´nchez ME, Criado-Garcı´a O, Heath KE, et al. Laforin, the dual-phosphatase responsible for Lafora disease, interacts with R5 (PTG), a regulatory subunit of protein phosphatase-1 that enhances glycogen accumulation. Hum Mol Genet. 2003;12: 3161-3171. 11. Tiberia E, Turnbull J, Wang T, et al. Increased laforin and laforin binding to glycogen underlie Lafora body formation
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in malin-deficient Lafora disease. J Biol Chem. 2012;287: 25650-25659. Vilchez D, Ros S, Cifuentes D, et al. Mechanism suppressing glycogen synthesis in neurons and its demise in progressive myoclonus epilepsy. Nat Neurosci. 2007;10:1407-1413. Solaz-Fuster MC, Gimeno-Alcan˜iz JV, Ros S, et al. Regulation of glycogen synthesis by the laforin-malin complex is modulated by the AMP-activated protein kinase pathway. Hum Mol Genet. 2008;17:667-678. Worby CA, Gentry MS, Dixon JE. Malin decreases glycogen accumulation by promoting the degradation of protein targeting to glycogen (PTG). J Biol Chem. 2008;283:4069-4076. Go´mez-Abad C, Go´mez-Garre P, Gutie´rrez-Delicado E, et al. Lafora disease due to EPM2B mutations. A clinical and genetic study. Neurology. 2005;64:982-986. Chan EM, Andrade DM, Franceschetti S, Minassian B. Progressive myoclonus epilepsies: EPM1, EPM2A, EPM2B. Adv Neurol. 2005;95:47-57. Baykan B, Striano P, Gianotti S, et al. Late-onset and slowprogressing Lafora disease in four siblings with EPM2B mutation. Epilepsia. 2005;46:1695-1697. Brackmann FA., Kiefer A, Agaimy A, et al. Rapidly progressive phenotype of Lafora disease associated with a novel NHLRC1 mutation. Pediatr Neurol. 2011;44:475-477.
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Three Patients With Lafora Disease: Different Clinical Presentations and a Novel Mutation Hatice Gamze Poyrazoglu, Emin Karaca, Hüseyin Per, Hakan Gümüs, Huseyin Onay, Mehmet Canpolat, Özlem Canöz, Ferda Ozkinay and Sefer Kumandas J Child Neurol published online 10 July 2014 DOI: 10.1177/0883073814535489 The online version of this article can be found at: http://jcn.sagepub.com/content/early/2014/07/10/0883073814535489
Published by: http://www.sagepublications.com
Additional services and information for Journal of Child Neurology can be found at: Email Alerts: http://jcn.sagepub.com/cgi/alerts Subscriptions: http://jcn.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> OnlineFirst Version of Record - Jul 10, 2014 What is This?
Downloaded from jcn.sagepub.com at University of Waikato Library on July 15, 2014
Brief Communication
Three Patients With Lafora Disease: Different Clinical Presentations and a Novel Mutation
Journal of Child Neurology 1-5 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0883073814535489 jcn.sagepub.com
Hatice Gamze Poyrazog˘lu, MD1, Emin Karaca, MD2, Hu¨seyin Per, MD1, Hakan Gu¨mu¨s, MD1, Huseyin Onay, MD1, Mehmet Canpolat, MD1, ¨ zlem Cano¨z, MD3, Ferda Ozkınay, MD2, and Sefer Kumandas, MD1 O
Abstract Lafora disease is a rare, fatal, autosomal recessive hereditary disease characterized by epilepsy, myoclonus and progressive neurological deterioration. Diagnosis is made by polyglucosan inclusion bodies (Lafora bodies) shown in skin biopsy. Responsible mutations of Lafora disease involves either the EPM2A or NHLRC1 (EPM2B) gene. Mutations in the NHLRC1 gene are described as having a more benign clinical course and a later age of death compared with EPM2A mutations. We report 2 genetic mutations and clinical courses of Lafora disease in 3 adolescents with homozygote NHLRC1 mutation and novel homozygous EPM2A mutation. Keywords progressive myoclonic epilepsy, Lafora disease, genetic Received September 05, 2013. Received revised April 01, 2014. Accepted for publication April 14, 2014.
Lafora disease [LD] is a progressive, fatal disease that is inherited autosomal recessively and usually begins in the second decade. First symptoms of the disease are resistant myoclonic seizures, myoclonus, generalized or focal occipital seizures, and photosensitivity on electroencephalography (EEG) in patients with previously normal neurologic development.1 This is followed by not only worsening of seizures but also deterioration in cognitive and neurologic functions such as dysarthria, ataxia, and dementia. After the first symptoms, most patients die within 10 years of diagnosis, because of status epilepticus or complications associated with the degeneration of the nervous system.2 Polyglucosan inclusions (Lafora bodies) are observed in the typical periodic acid-Schiff–positive cytoplasm in the biopsy specimens. Although biopsy specimens could be taken from brain, spinal cord, skin, liver, and skeletal muscles, biopsy specimens from the axillary apocrine sweat glands is preferred mostly because it is less invasive.2-4 LD is caused by the mutation in EPM2A or EPM2B / NHLRC1 genes that encode laforin and malin, respectively.1,5 There is no significant difference clinically between patients with mutations in the EPM2A and NHLRC1 genes. However, it was reported that patients with NHLRC1 mutation had slowly progressive course and longer survival when compared with EPM2A mutation.5,6 In the present study, we report 3 patients one with identified homozygous mutation in the NHLRC1 gene who
experienced a fast and progressive clinical course and 2 siblings with identified homozygous mutation in the EPM2A genes who experienced slow progression with typical clinical signs.
Case Report Case 1 A 16-year-old female patient was evaluated for progressive myoclonus epilepsy. There were no problems at birth and during childhood, and her psychomotor development was normal and she was attended regular school. She had no health problems up to age 13 years. She presented with generalized 1
Department of Pediatrics, Division of Pediatric Neurology, Faculty of Medicine, Erciyes University, Kayseri, Turkey 2 Department of Medical Genetics, Faculty of Medicine, Ege University, _Izmir, Turkey 3 Department of Pathology, Faculty of Medicine, Erciyes University, Kayseri, Turkey Corresponding Author: Hatice Gamze Poyrazog˘lu, MD, Erciyes University, Faculty of Medicine, Department of Pediatrics, Division of Pediatric Neurology, 38039, Melikgazi, Kayseri, Turkey. Email: [email protected]
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Figure 1. Lafora bodies stain strongly positive with periodic acidSchiff, revealing numerous small ductlike granules in apocrine gland biopsy.
tonic-clonic seizures 3 years ago, and generalized myoclonic seizures were observed in later periods. Her seizures could not be controlled despite the antiepileptic drugs valproic acid, 2000 mg/d; clonazepam, 4 mg/d; levetiracetam, 1500 mg/d; and topiramate, 4 mg/kg/d (200 mg/d). Her 14-year-old brother had epilepsy history (case 2). Her parents were second-degree relatives. She was found to have disordered cognitive functions, perception, and speech as well as tremor in hands and gait disturbance in her neurologic examination. No pathology was found in her routine blood tests, ammonia, lactate and pyruvate ratios, quantification of amino acids of plasma and urine, and urine organic acid levels. Cerebrospinal fluid for white blood cell counts, glucose, protein, and measles immunoglobulin M and G levels were normal. Multifocal, from time-to-time generalized spike-wave activity, that suppressed with diazepam was observed with EEG. Mild cerebral and cerebellar atrophy were detected in her cranial magnetic resonance imaging (MRI). Eye examination and retinal examination were normal but electroretinogram latency delays were detected. Her antiepileptic therapy was rearranged. Topiramate and clonazepam were eliminated and zonisamide was added. Seizures were decreased significantly subsequent to zonisamide treatment. According to the clinical features, laboratory findings (blood test, EEG, and MRI), and ophthalmologic examination, we suggested the diagnosis of hereditary progressive myoclonus epilepsy. Periodic acid-Schiff–positive cytoplasmic polyglucosan inclusion bodies (termed as Lafora bodies) were identified in the apocrine sweat gland at axillary skin biopsy (Figure 1). Homozygous (c.336C>A) p.Y112X, a new mutation, in the EPM2A gene was detected in the genetic analysis.
Case 2 A 14-year-old male patient was evaluated for progressive myoclonus epilepsy. There were no problems at birth and during
childhood, and his psychomotor development was normal and he attended regular school. His general health was good. He presented at the age of 13 with myoclonic-astatic seizures. At the beginning of the disease, his seizures could be kept under control by valproic acid treatment but recently, frequency of seizures had increased and generalized tonic-clonic seizures were observed. His performance at school deteriorated. His parents were first-cousins as regards the degree of kinship. His sister, case 1, was also followed up with a diagnosis of intractable epilepsy. No pathologic disorder was found in neurologic examination of the patient, except mild disorder in cognitive functions at the time of admission. His routine blood tests, ammonia, lactate, pyruvate, and metabolic tests (tandem mass spectrometry analysis, urine and blood amino acids, and urine organic acid analysis) and cerebrospinal fluid analysis were normal. Multifocal, from time to time generalized, multiple spikes and spike-wave activity were observed in his EEG. No pathology was detected in electroretinogram, and his retinal examination was found normal. Mild cerebral and cerebellar atrophy was detected on cranial magnetic resonance imaging. The clinical features and pattern of inheritance in the family, EEG findings, and resistance to anticonvulsant treatment, as well as laboratory evaluations and ophthalmologic examination suggested the diagnosis of hereditary progressive myoclonus epilepsy. Periodic acid-Schiff-positive Lafora bodies in the cytoplasm were observed in his axillary skin biopsy. Homozygous (c.336C>A) p.Y112X, a novel mutation, in the EPM2A gene was detected in the genetic analysis.
Case 3 A 14-year-old female patient was evaluated for progressive myoclonus epilepsy. Her medical history showed no problems at birth and during childhood and psychomotor development was normal, and she attended regular school and her school performance was very good. Her general health was good. She presented at the age of 13 with generalized tonic-clonic seizures that began following episodic visual auras. She received valproic acid treatment. Much as there was a significant decrease in her seizures, it continued for a year after the treatment. One year later, segmental myoclonus started in addition to the occipital and generalized seizures. Clonazepam was added to her treatment; however, her seizures continued despite the dual treatment. Her parents had third degree of kinship and there was no similar case in the family. Motor and sensory impairment was not found in her neurologic examination and she had no gait disorder and her deep tendon reflexes were normal. No pathology was found in her routine blood tests, metabolic screening tests, and cerebrospinal fluid analysis. Her cranial magnetic resonance imaging was normal. Spike waves and multiple spike waves due to both 2 hemispheric centrotemporal and occipital regions were viewed in her EEG. Spontaneous myoclonus and myoclonic activities in the extremities, body ataxia, gait impairment, dysarthria, and significant learning difficulties began 6 months after admission.
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Figure 2. (A) Electropherogram of the Y112X heterozygous mutation in the EPM2A gene of the unaffected (carrier) father. (B) Electropherogram of the Y112X homozygous mutation in the EPM2A gene of the patient. (C) Electropherogram of the Y112X heterozygous mutation in the EPM2A gene of unaffected (carrier) mother. (The arrows show the position of the mutation.)
Feeding difficulties, loss of ability to walk without assistance, and dementia developed. The patient, whose seizures continued, was treated with zonisamide, and the generalized tonic-clonic seizures disappeared. Periodic acid-Schiff–positive Lafora bodies in cytoplasm were observed in her axillary skin biopsy. No mutations were found in her EPM2A gene in the genetic analysis. Homozygous (c.199 G>T) p.E67X mutation was detected in her NHLRC1 gene that resulted in a premature stop codon just at the beginning of exon 1. The general condition of the patient deteriorated 9 months after admission, and the patient who developed status epilepticus and pneumonia was taken in intensive care. In her follow-up, there was no improvement in clinical status, and she died in the 21st month approximately from the time of diagnosis.
Genetic Screening of the Cases The coding sequences from both EPM2A and NHLRC1 (EPM2B) were amplified by polymerase chain reaction as previously described.7,8 Polymerase chain reaction products were purified using the ZR-96 DNA Sequencing Clean-up Kit (Zymo Research Corp, Irvine, CA). Purified products were then sequenced and screened for mutations. This screening identified a novel homozygous (c.336C> A) p.Y112X mutation in genes for EPM2A 1 in cases 1 and 2 (Figure 2). This mutation
is thought to be overdetermined as it forms a stop codon early on and terminates the transcription. The father and mother were heterozygous carriers of the mutation (Figure 2). Genetic screening revealed a homozygous missense mutation (c.199 G> T) p.E67X for the NHLRC1 (EPM2B) gene and results in a premature stop codon at just the beginning of exon 1 in case 3.
Discussion Lafora body disease demonstrates progressive myoclonic epilepsy as an autosomal recessive hereditary disease. Clinical manifestations commence in early childhood and adolescent period (11-18 years) with generalized tonic-clonic, myoclonic occipital seizures, and visual hallucinations. Over time, the seizures get worse; dementia occurs that causes mental impairment leading to progression of ataxia and myoclonus and consequently death of the patient in approximately 10 years.5,9 In all of our 3 patients, clinical findings began after 13 years of age with myoclonic seizures whereas in 1 patient it started with visual auras accompanied by myoclonic seizures. Although patients were followed up with antiepileptic drug–resistant myoclonic epilepsy, over time they demonstrated a slowdown in the ground rhythm in EEG, spike waves and multi-spike waves increased, ataxia and significant deterioration occurred in mental functions, myoclonus of the extremities started, and they were unable to walk unassisted.
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Journal of Child Neurology
It was considered as progressive myoclonic epilepsy. Their fundus examinations were normal. Patients were diagnosed with Lafora disease when they were evaluated by clinical and laboratory findings. Diagnosis of LD is made by detection of Lafora bodies in the axillary skin biopsy or genetic mutation studies along with clinical findings. Lafora bodies, which are the pathognomonic signs of the disease, are intracytoplasmic, basophilic, polyglucosan inclusion bodies that stain positively with periodic acidSchiff. These bodies can be found in skin, skeletal muscle, liver, sweat glands, and heart tissue. Skin biopsy is the least invasive method for pathologic diagnosis. However, the absence of Lafora bodies in skin biopsy does not eliminate the diagnosis of Lafora disease.4 Lafora bodies were demonstrated in the axillary skin biopsy of 3 of our patients. LD is caused by mutations in the EPM2A and EPM2B/ NHLRC1 genes. Mutation was detected in EPM2A genes in up to 97% (range 22%-97%) of families with Lafora disease.2,6,8 EPM2A gene encodes a protein of 331 amino acids (so-called laforin). The substrate and function of laforin have recently been explained. Laforin deficiency also leads to intracellular glycogen particles which are called Lafora bodies.10 On the other hand, EPM2B gene encodes E3 ubiquitin ligase, named malin, which interacts with laforin.11 The primary role of malin appears to be to protect tissues from accumulating polyglucosans by regulating proteins involved in glycogen metabolism. Malin deficiency causes increased laforin, increased laforin-binding glycogen, leading to the formation of Lafora bodies. Moreover, increased levels of laforin, which binds to glycogen, causes Lafora bodies.10,12-14 Genetic allelic heterogeneity is present in LD associated with mutations in EPM2B. Patients with mutations in EPM2A and EPM2B express similar clinical manifestation, although patients with EPM2B-associated LD seem to have a slightly milder clinical course.15,16 Baykan et al17 have reported that 4 brothers diagnosed with LD (average 19.5 years of age) had milder disease course, and homozygous p.D146N mutation was identified in their NHLRC1 gene. In another study, 7 of 17 Italian patients diagnosed with mutation in their NHLRC1 (EPM2B) gene could maintain their daily living activities and social interactions for a period of 4 years from the onset of the disease, and the disease had a slow progression.6 Brackmann et al18 described a rapidly progressive phenotype of Lafora disease in an adolescent patient with a novel NHLRC1 mutation (ending in a stop at position 39) who developed severe disability and dementia less than 2 years after the onset of signs. In the third patient in our study, a homozygous c.199G>T (p.E67X) mutation was detected in her NHLRC1 gene and it resulted in a premature stop codon at just the beginning of exon 1. Consequently, NHLRC1 protein was not synthesized. Thus, we suggest that her disease was a rapidly progressive phenotype of LD. The starting age of these cases, with observation of myoclonic seizures as the only sign for a long time in the first long-term period and its having similar features with juvenile myoclonic epilepsy detected in EEG, may cause it to be diagnosed as Juvenile myoclonic epilepsy in the initial period. However, failure
to show adequate response to pharmacotherapy, monitoring of additional pathologies, including impairment of the ground rhythm in EEG, cognitive impairment, and addition of other types of seizure brings progressive myoclonic epilepsy to the mind first. As in our cases, especially LD should be considered in the differential diagnosis in progressive myoclonic epilepsies seen in adolescence. There is no specific treatment for LD. However, valproic acid used in the treatment of myoclonus at the early stages of the disease and some antiepileptic drugs such as levetiracetam may provide temporary symptomatic cure. Some antiepileptics such as phenytoin, carbamazepine, and vigabatrin can cause an increase in myoclonus.4 Patients with progressive epilepsy whose clinical diagnosis does not improve despite appropriate antiepileptic therapy must be evaluated strictly in terms of the possibility of progressive myoclonic epilepsia, and skin biopsy must be conducted even in cases of mild cognitive impairment; slowing ground rhythm in EEG and genetic analysis should be done for LD. Acknowledgment All authors thank all the patients and family members for their participation in this study.
Author Contributions HGP conceived the study; HG, HP and Ek reviewed the literature; SK provided investigation support; and HGP performed literature review and wrote the manuscript. MC and FO reviewed the manuscript. OC was demonstrated Lafora body in axillary skin biopsy. EK and HO demonstrated genetic mutations of patients. All authors were responsible for patient management.
Declaration of Conflicting Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The authors received no financial support for the research, authorship, and/or publication of this article.
Ethical Approval Informed consent was obtained from the patient’s parents to publish this report.
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