BRIEF COMMUNICATION
No evidence of a role for cystatin B gene in juvenile myoclonic epilepsy *1Laura Mumoli, †1Patrizia Tarantino, ‡Roberto Michelucci, §Amedeo Bianchi, *†Angelo Labate, ¶Silvana Franceschetti, #Carla Marini, **Pasquale Striano, *†Monica Gagliardi, *††Edoardo Ferlazzo, ‡‡Vito Sofia, **Loredana Pennese, †Grazia Annesi, *††Umberto Aguglia, #Renzo Guerrini, **Federico Zara, *†Antonio Gambardella, and on behalf of the Genetic Commission, Italian League Against Epilepsy Epilepsia, **(*):1–4, 2015 doi: 10.1111/epi.12944
SUMMARY
Laura Mumoli is a postdoctoral fellow with special interest in epilepsy.
Genetic factors play a major role in the etiology of juvenile myoclonic epilepsy (JME), a common form of idiopathic generalized epilepsy, but so far, genes related to JME remain largely unknown. JME shares electroclinical features with Unverricht-Lundborg disease (progressive myoclonic epilepsy type 1; EPM1), a form of progressive myoclonus epilepsy characterized by myoclonus, epilepsy, and gradual neurologic deterioration. EPM1 is caused by mutations in the gene that codes for cystatin B (CSTB), an inhibitor of cysteine protease. In the present study, we wished to investigate the role of the CSTB gene in patients with JME. Fifty-seven unrelated patients (35 women; mean age standard deviation [SD], 24.1 7.7; mean age SD at onset, 15.3 2.4) with JME were enrolled. Twenty-three of 57 patients were the probands of families with JME. The molecular diagnosis was carried out to identify the common dodecamer repeat expansion mutation or other disease-causing mutations in the CSTB gene. The molecular analysis did not depict mutations in any of the 57 patients with JME. Our study did not support a role for the CSTB gene in patients with familial or sporadic JME. KEY WORDS: Cystatin B gene, Juvenile myoclonic epilepsy, EPM1, Mutation, Genetics.
Mutations in the cystatin B (CSTB) gene on chromosome 21q22.3, coding for an inhibitor of cysteine proteases, are the major cause of Unverricht-Lundborg disease, progressive myoclonic epilepsy type 1 (EPM1; OMIM254800), an autosomal recessive neurodegenerative disorder with onset from 6 to 16 years.1 Most CSTB mutations consist of an unstable expansion of dodecamer repeat (50 -CCCCG CCCCGCG-30 ) in the promoter region that downregulates CSTB messenger RNA (mRNA).1,2 Other mutations involving the CSTB gene account for a few patients and affect splice sites that result in amino acid changes or predict truncated proteins.2 The EPM1 phenotype is typically characterized by action myoclonus and generalized myoclonic and tonic–clonic seizures, with progressive neurologic dysfunction, especially ataxia.3 The severity of disease does not seem to correlate with the number of repeats of dodecamer expansion mutation,2,4 whereas patients with compound
Accepted January 20, 2015. *Institute of Neurology, University Magna Græcia Catanzaro, Catanzaro, Italy; †Institute of Molecular Bioimaging and Physiology, Section of Germaneto, National Research Council, Catanzaro, Italy; ‡Division of Neurology, IRCCS—Institute of Neurological Sciences of Bologna, Bellaria Hospital, Bologna, Italy; §Division of Neurology, Hospital San Donato Arezzo, Arezzo, Italy; ¶Department of Neurophysiopathology and Epilepsy Centre, IRCCS Foundation C. Besta Neurological Institute, Milan, Italy; #Pediatric Neurology Unit, Neuroscience Department, Children’s Hospital A. Meyer-University of Florence, Firenze, Italy; **Pediatric Neurology and Muscular Diseases Unit, Department of Neurosciences Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, G. Gaslini Institute, University of Genoa, Genoa, Italy; ††Regional Epilepsy Center, Hospital of Reggio Calabria, Reggio Calabria, Italy; and ‡‡Section of Neurosciences, Department GF Ingrassia, University of Catania, Catania, Italy 1 These authors contributed equally to this study. Address correspondence to Antonio Gambardella, Institute of Neurology, University Magna Græcia of Catanzaro, Viale Europa, 88100 Catanzaro, Italy. E-mail: [email protected] Wiley Periodicals, Inc. © 2015 International League Against Epilepsy
1
2 L. Mumoli et al. heterozygous mutations seem to have a more severe phenotype.5 Growing evidence indicates that the EPM1 phenotype is more heterogeneous than previously assumed, even within the same family, and a substantial number of EPM1 patients display a mild phenotype, where the myoclonus is sufficiently moderate that the diagnosis is markedly delayed or the disorder misdiagnosed as juvenile myoclonic epilepsy (JME).3,6 It has been also claimed that a diagnosis of EPM1 should be considered during the course of JME, especially when the seizures are drug resistant or action myoclonus also occurs.3,6 Despite this overlap in clinical presentation between EPM1 and JME, to what extent the CSTB gene might contribute to JME phenotype remains to be elucidated. Because of the growing evidence of pleiotropic effects of homozygous and heterozygous mutations on epilepsy phenotypes, we hypothesized that the more common heterozygous mutations in the recessive gene CSTB might contribute to the development of JME in a subset of cases.
Methods Patients The study group included 57 unrelated patients (35 women with mean age standard deviation [SD], 24.1 7.7; mean age SD at onset, 15.3 2.4) who had a diagnosis of JME based on comprehensive clinical and laboratory evaluations, according to the criteria of the International League Against Epilepsy (ILAE, 1989).7 A detailed history of the type and frequency of seizures was obtained from patients, parents, and other relatives at the time of investigation and from a review of the patients’ medical records. All patients had extensive awakening and sleeping electroencephalography (EEG) recordings. All patients were Caucasian and were born in Italy. Seventeen of these 57 patients were the probands of unrelated JME families of Italian origin that had already been part of a previous study, and their electroclinical features were reported in greater detail elsewhere.8 The transmission of the disease in most families suggests an autosomal dominant pattern of inheritance with incomplete penetrance. Additional 16 of 57 patients had a family history of seizures or febrile convulsions in one or more first- to second-degree relatives. All patients enrolled had classic JME phenotype, as they had a history of unprovoked myoclonic jerks, mostly affecting upper limbs on awakening, with or without generalized tonic seizures and\or typical absence seizures. None of the patients had additional clinical manifestations such as action or reflex sensitive myoclonus, or intellectual or neurologic deficit. In all patients, the interictal EEG showed generalized multiple spike or polyspike and wave complexes on a normal background. At the time of investigation, the most utilized antiepileptic drugs (AEDs) were valproate or levetiracetam, usually in monotherapy. AEDs were usually effective in controlling seizures including myoclonic jerks in all of them but nine (15%), who had persisting Epilepsia, **(*):1–4, 2015 doi: 10.1111/epi.12944
seizures, mainly myoclonic jerks, despite adequate therapy and lifestyle. Seventy-five healthy, unrelated subjects (40 women; mean SD age, 28.46 10.96), with the same ethnic background, were also screened for mutations of the CSTB gene only to test the occurrence of the sequence variants identified in the JME screening sample. All patients or their parents/guardians, in case of minors with JME, and controls signed an informed consent prior to participation in the genetic studies. The human research ethics committee of the University Magna Græcia, Catanzaro, Italy approved the study. Molecular study Screening for mutations in the CSTB gene was performed in all index patients and controls. Genomic DNA was extracted from venous blood samples using standards methods. The CSTB gene was investigated as previously described.2 Polymerase chain reaction (PCR) amplification of the large and normal alleles was performed in a 20 lL volume containing 15 ng DNA, 0.5 mM of each nucleotide, 0.5 lM of each primer, 0.12 U Taq long-template PCR system (Roche Diagnostic, Mannheim, Germany), 19 bovine serum albumin, 5% dimethyl sulfoxide (DMSO), 1.0 M GC melt mix (Clontech, Palo Alto, CA, U.S.A.). Primers 2f: (50 -CCCGGAAAGACGATACCAG-30 ) and 1R (50 -GAG GAGGCACTTTGGCTTC-30 ) were used. After the initial denaturation at 94°C for 2 min a 40-cycle reaction (94°C 10 min, 57°C 45 s, and 68°C 8 min) and a final extension at 68°C for 7 min. The PCR fragments were detected by electrophoresis analysis. For mutation scanning, 2,949 bp of the CSTB gene (GenBank AF208234), including the exons, the introns, and the 50 and 30 untranslated regions, was amplified in overlapping fragments (Fig. 1) using the following primers: 1F (50 AAA CGC AAA TTC CAC CAG AG-30 ) and 1R (50 -GAG GAG GCA CTT TGG CTT C-30 ), 2F (50 -CCC GGA AAG ACG ATA CCA G-30 ) and 2R (50 -CGG CTT CTT TCG CTC CAG-30 ), 3F (50 -GCC GAG ACC CAG CAC ATC-30 ) and 3R (50 -CCT GTG GAC CTT TTA TGC AG-30 ), 4F (50 -GCA AGA GGT CCC CAG TGA TA-30 ) and 4R (50 -TGA CAC GGC CTT AAA CAC AG-30 ), 5F (50 -ACC GTA CCC AGC TGG AAC TGT-30 ) and 5R (50 -GCT TAT CTC AGG GGG CAG CCA CAG-30 ), 6F (50 -GTA GAG TGT GGG CCT CAG GA-30 ) and 6R (50 -AAG CCT CTG ATC CCA AGT CA-30 ), and 7F (50 -ATT GTC TTC AGC TGG CTG CTA AT-30 ) and 7R (50 -AAG ATC ACC TAT TGG GAA GGA AAG A-30 ). The PCR fragments were sequenced by an ABI 3130 DNA analyzer (Applied Biosystems, Foster City, CA, U.S.A.).
Results No pathologic (≥30) dodecamer repeat expansions or other pathogenic CSTB gene mutations were detected in any
3 No Role for CSTB in JME
Figure 1. Gel electrophoresis showing PCR-amplified dodecamer repeat expansions of normal CTSB alleles. Lanes 2 and 3, homozygous patients with two copies of the repeat; lanes 4 and 5, homozygous patients with three copies of the repeat; lanes 6 and 7, compound heterozygous (two different mutant alleles of the disease gene that are present in a patient affected by a recessive disease) with two and three copies of the repeat; lanes (1 and 8) ladder. Epilepsia ILAE
of 57 patients with JME. Figure 1 shows the typical electrophoresis result. In detail, almost all (56 of 57) JME patients had normal alleles, with 2–3 dodecamer repeats (genotype 2/2: 12 of 57 [21.0%] patients; genotype 2/3: 14 of 57 [24.6%]; genotype 3/3: 30 of 57 [52.6%]). One affected individual carried an allele with four copies (genotype 3/4), which represents a very rare normal variant.9 In the healthy control individuals, the analysis revealed the commonly observed two and three copy repeat alleles (genotype 2/2: 16 of 75 [21.3%] subjects; genotype 2/3: 20 of 75 [26.7%]; genotype 3/3: 39 of 75 [52.0%]).
Discussion Juvenile myoclonic epilepsy is a common form of idiopathic generalized epilepsy characterized by symmetric, myoclonic jerks, mostly affecting upper limbs, generalized tonic–clonic seizures and, more rarely, absence seizures. Clinical genetic studies support a high genetic predisposition, and positive family history of epilepsy can be obtained in 30–50% of patients. The mechanisms and pathophysiology of JME are not yet clear, but growing evidence indicates that JME is typically a polygenic disorder with heterogeneous genetic and pathologic signatures.8 Mutations in ion channel genes have been occasionally associated with autosomal dominant JME. Nonetheless, a more common pathogenic mechanism may involve abnormalities in neuronal connectivity that are caused by subtle structural changes in the brain.10,11 Indeed, some genes associated with both
sporadic and familial JME, such as Myoclonin1/EFHC1 and BRD2, play key roles in the development and maintenance of normal neuronal structures, through cell growth and proliferation, differentiation, survival, and apoptosis that would disrupt the normal balance between excitatory and inhibitory circuits.10,11 In addition, the CSTB gene plays a role in the development and maintenance of normal neuronal structures through cell-survival and cell-death pathways.12 Therefore, EPM1 and JME might share, at least in part, a common pathogenic mechanism that involves subtle structural changes in the brain with consequent abnormalities in neuronal connectivity. Subtle increases in specific populations of neurons may occur in JME,11 whereas a defect in apoptosis with increased neuronal cell death may be associated with EPM1.12 On this basis, because EPM1 and JME also share several electroclinical features, the CSTB gene might play a pathogenic role in a subset of patients with JME. In this study, we have explored this hypothesis, but no disease-causing mutation in CSTB was found in our patients with familial or sporadic JME, including those with a more severe phenotype. Therefore, our findings strongly indicate that JME and EPM1 are distinct clinical and genetic entities. The diagnosis of EPM1 in patients with the JME phenotype should be considered only when additional clinical manifestations such as action or reflex sensitive myoclonus occur, as they are distinctive features of progressive myoclonus epilepsy. To the best of our knowledge, these findings provide substantial negative evidence for a role of CSTB gene in the pathogenesis of JME. In a large JME cohort, indeed, we did not identify any homozygous, compound heterozygous, or heterozygous mutations of CSTB. In accordance with our findings, a previous work provided exclusion for linkage to the EPM1 locus on chromosome 21q in a set of families segregating for JME or other idiopathic generalized epilepsies, which did not show linkage to markers on chromosome 6p.13 There is also a recent case report of a 30-year-old woman with a JME phenotype that was a compound heterozygote for two CSTB mutations: an expansion of the dodecamer repeat and a splice-site c.67-1G>C mutation in intron 1, predicting a deletion of the downstream exon 2 with inframe deletion of 34 amino acids (p.delV23_K56).14 Nevertheless, four siblings of her maternal grandmother had already received the diagnosis of EPM1.14 Of interest, an experimental study provided evidence that CSTB-deficient heterozygous mice showed a milder EPM1 phenotype with partial penetrance of EPM1-like symptoms (seizures, ataxia),15 suggesting pleiotropic effects of homozygous and heterozygous CSTB mutations. A recent example of different phenotypes with homozygous versus heterozygous mutations involves the progranulin gene (GRN, granulin precursor).16 Indeed, homozygous GRN mutations gave rise to the rare form of adult-onset neuronalceroid-lipofuscinosis, whereas heterozygous mutations in Epilepsia, **(*):1–4, 2015 doi: 10.1111/epi.12944
4 L. Mumoli et al. GRN are a major cause of frontotemporal lobar degeneration, which is the second most common early-onset dementia.16 Again, in the present series we did not identify any patients with sporadic or familial JME who carried heterozygous CSTB mutations, making the hypothesis of CSTB pleiotropism unlikely, with heterozygous CSTB mutations conferring susceptibility to a milder epilepsy phenotype, such as JME. Additional studies are needed to extend the molecular analysis in atypical patients who do not fit into the classic JME phenotype because of additional clinical manifestations, especially action or reflex sensitive myoclonus.
5.
6. 7. 8. 9.
Acknowledgment 10. We wish to thank Italian League Against Epilepsy (LICE) for supporting this work. 11.
Disclosure
12.
None of the authors has any conflicts of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
13.
References 1. Pennacchio LA, Lehesjoki AE, Stone NE, et al. Mutations in the gene encoding cystatin B in progressive myoclonus epilepsy (EPM1). Science 1996;271:1731–1734. 2. Joensuu T, Kuronen M, Alakurtii K, et al. Cystatin B: mutation detection, alternative splicing and expression in progressive myoclonus epilepsy of Unverricht-Lunborg type (EPM1) patients. Eur J Hum Genet 2007;15:185–193. 3. K€alvi€ainen R, Khyuppenen J, Koskenkorva P, et al. Clinical picture of EPM1-Unverricht-Lundborg disease. Epilepsia 2008;49:549–556. 4. Lalioti MD, Scott HS, Genton P, et al. A PCR amplification method reveals instability of the dodecamer repeat in progressive myoclonus
Epilepsia, **(*):1–4, 2015 doi: 10.1111/epi.12944
14.
15. 16.
epilepsy (EPM1) and no correlation between the size of the repeat and age at onset. Am J Hum Genet 1998;62:842–847. Canafoglia L, Gennaro E, Capovilla G, et al. Electroclinical presentation and genotype–phenotype relationships in patients with Unverricht-Lundborg disease carrying compound heterozygous CSTB point and indel mutations. Epilepsia 2012;53:2120–2127. de Haan GJ, Halley DJ, Doelman JC, et al. Univerricht-Lundborg disease: underdiagnosed in the Netherlands. Epilepsia 2004;45:1061– 1063. Commission on Classification and Terminology of the International League Against Epilepsy. Proposal for revised classification of epilepsies and epileptic syndromes. Epilepsia 1989;30:389–399. Annesi F, Gambardella A, Michelucci R, et al. Mutational analysis of EFHC1 gene in Italian families with juvenile myoclonic epilepsy. Epilepsia 2007;48:1686–1690. Horiuchi H, Osawa M, Furutani R, et al. Polymerase chain reactionbased analysis using deaminated DNA of dodecamer expansions in CSTB, associated with Unverricht-Lundborg myoclonus epilepsy. Genet Test 2005;9:328–333. Pal DK, Evgrafov OV, Tabares P, et al. BRD2 (RING3) is a probable major susceptibility gene for common juvenile myoclonic epilepsy. Am J Hum Genet 2003;73:261–270. Suzuki T, Delgado-Escueta AV, Aguan K, et al. Mutations in EFHC1 cause juvenile myoclonic epilepsy. Nat Genet 2004;8:842–849. Shannon P, Pennacchio LA, Houseweart MK, et al. Neuropathological changes in a mouse model of progressive myoclonus epilepsy: cystatin B deficiency and Unverricht-Lundborg disease. J Neuropathol Exp Neurol 2002;61:1085–1091. Rees M, Curtis D, Parker K, et al. Linkage analysis of idiopathic generalised epilepsy in families of probands with Juvenile Myoclonic Epilepsy and marker loci in the region of EPM 1 on chromosome 21q: Unverricht-Lundborg disease and JME are not allelic variants. Neuropediatrics 1994;25:20–25. Andermann E, Amrom D, Andermann F, et al. Mild case of Unverricht-Lundborg disease presenting as juvenile myoclonic epilepsy. Abstract/Program 3060T, presented at the 63rd Annual Meeting of the American Society of Human Genetics, October 24, 2013 in Boston, MA, USA. Kaasik A, Kuum M, Aonurm A, et al. Seizures, ataxia, and neuronal loss in cystatin B heterozygous mice. Epilepsia 2007;48:752–757. Smith KR, Damiano J, Franceschetti S, et al. Strikingly different clinicopathological phenotypes determined by progranulin-mutation dosage. Am J Hum Genet 2012;90:1102–1107.
No evidence of a role for cystatin B gene in juvenile myoclonic epilepsy *1Laura Mumoli, †1Patrizia Tarantino, ‡Roberto Michelucci, §Amedeo Bianchi, *†Angelo Labate, ¶Silvana Franceschetti, #Carla Marini, **Pasquale Striano, *†Monica Gagliardi, *††Edoardo Ferlazzo, ‡‡Vito Sofia, **Loredana Pennese, †Grazia Annesi, *††Umberto Aguglia, #Renzo Guerrini, **Federico Zara, *†Antonio Gambardella, and on behalf of the Genetic Commission, Italian League Against Epilepsy Epilepsia, **(*):1–4, 2015 doi: 10.1111/epi.12944
SUMMARY
Laura Mumoli is a postdoctoral fellow with special interest in epilepsy.
Genetic factors play a major role in the etiology of juvenile myoclonic epilepsy (JME), a common form of idiopathic generalized epilepsy, but so far, genes related to JME remain largely unknown. JME shares electroclinical features with Unverricht-Lundborg disease (progressive myoclonic epilepsy type 1; EPM1), a form of progressive myoclonus epilepsy characterized by myoclonus, epilepsy, and gradual neurologic deterioration. EPM1 is caused by mutations in the gene that codes for cystatin B (CSTB), an inhibitor of cysteine protease. In the present study, we wished to investigate the role of the CSTB gene in patients with JME. Fifty-seven unrelated patients (35 women; mean age standard deviation [SD], 24.1 7.7; mean age SD at onset, 15.3 2.4) with JME were enrolled. Twenty-three of 57 patients were the probands of families with JME. The molecular diagnosis was carried out to identify the common dodecamer repeat expansion mutation or other disease-causing mutations in the CSTB gene. The molecular analysis did not depict mutations in any of the 57 patients with JME. Our study did not support a role for the CSTB gene in patients with familial or sporadic JME. KEY WORDS: Cystatin B gene, Juvenile myoclonic epilepsy, EPM1, Mutation, Genetics.
Mutations in the cystatin B (CSTB) gene on chromosome 21q22.3, coding for an inhibitor of cysteine proteases, are the major cause of Unverricht-Lundborg disease, progressive myoclonic epilepsy type 1 (EPM1; OMIM254800), an autosomal recessive neurodegenerative disorder with onset from 6 to 16 years.1 Most CSTB mutations consist of an unstable expansion of dodecamer repeat (50 -CCCCG CCCCGCG-30 ) in the promoter region that downregulates CSTB messenger RNA (mRNA).1,2 Other mutations involving the CSTB gene account for a few patients and affect splice sites that result in amino acid changes or predict truncated proteins.2 The EPM1 phenotype is typically characterized by action myoclonus and generalized myoclonic and tonic–clonic seizures, with progressive neurologic dysfunction, especially ataxia.3 The severity of disease does not seem to correlate with the number of repeats of dodecamer expansion mutation,2,4 whereas patients with compound
Accepted January 20, 2015. *Institute of Neurology, University Magna Græcia Catanzaro, Catanzaro, Italy; †Institute of Molecular Bioimaging and Physiology, Section of Germaneto, National Research Council, Catanzaro, Italy; ‡Division of Neurology, IRCCS—Institute of Neurological Sciences of Bologna, Bellaria Hospital, Bologna, Italy; §Division of Neurology, Hospital San Donato Arezzo, Arezzo, Italy; ¶Department of Neurophysiopathology and Epilepsy Centre, IRCCS Foundation C. Besta Neurological Institute, Milan, Italy; #Pediatric Neurology Unit, Neuroscience Department, Children’s Hospital A. Meyer-University of Florence, Firenze, Italy; **Pediatric Neurology and Muscular Diseases Unit, Department of Neurosciences Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, G. Gaslini Institute, University of Genoa, Genoa, Italy; ††Regional Epilepsy Center, Hospital of Reggio Calabria, Reggio Calabria, Italy; and ‡‡Section of Neurosciences, Department GF Ingrassia, University of Catania, Catania, Italy 1 These authors contributed equally to this study. Address correspondence to Antonio Gambardella, Institute of Neurology, University Magna Græcia of Catanzaro, Viale Europa, 88100 Catanzaro, Italy. E-mail: [email protected] Wiley Periodicals, Inc. © 2015 International League Against Epilepsy
1
2 L. Mumoli et al. heterozygous mutations seem to have a more severe phenotype.5 Growing evidence indicates that the EPM1 phenotype is more heterogeneous than previously assumed, even within the same family, and a substantial number of EPM1 patients display a mild phenotype, where the myoclonus is sufficiently moderate that the diagnosis is markedly delayed or the disorder misdiagnosed as juvenile myoclonic epilepsy (JME).3,6 It has been also claimed that a diagnosis of EPM1 should be considered during the course of JME, especially when the seizures are drug resistant or action myoclonus also occurs.3,6 Despite this overlap in clinical presentation between EPM1 and JME, to what extent the CSTB gene might contribute to JME phenotype remains to be elucidated. Because of the growing evidence of pleiotropic effects of homozygous and heterozygous mutations on epilepsy phenotypes, we hypothesized that the more common heterozygous mutations in the recessive gene CSTB might contribute to the development of JME in a subset of cases.
Methods Patients The study group included 57 unrelated patients (35 women with mean age standard deviation [SD], 24.1 7.7; mean age SD at onset, 15.3 2.4) who had a diagnosis of JME based on comprehensive clinical and laboratory evaluations, according to the criteria of the International League Against Epilepsy (ILAE, 1989).7 A detailed history of the type and frequency of seizures was obtained from patients, parents, and other relatives at the time of investigation and from a review of the patients’ medical records. All patients had extensive awakening and sleeping electroencephalography (EEG) recordings. All patients were Caucasian and were born in Italy. Seventeen of these 57 patients were the probands of unrelated JME families of Italian origin that had already been part of a previous study, and their electroclinical features were reported in greater detail elsewhere.8 The transmission of the disease in most families suggests an autosomal dominant pattern of inheritance with incomplete penetrance. Additional 16 of 57 patients had a family history of seizures or febrile convulsions in one or more first- to second-degree relatives. All patients enrolled had classic JME phenotype, as they had a history of unprovoked myoclonic jerks, mostly affecting upper limbs on awakening, with or without generalized tonic seizures and\or typical absence seizures. None of the patients had additional clinical manifestations such as action or reflex sensitive myoclonus, or intellectual or neurologic deficit. In all patients, the interictal EEG showed generalized multiple spike or polyspike and wave complexes on a normal background. At the time of investigation, the most utilized antiepileptic drugs (AEDs) were valproate or levetiracetam, usually in monotherapy. AEDs were usually effective in controlling seizures including myoclonic jerks in all of them but nine (15%), who had persisting Epilepsia, **(*):1–4, 2015 doi: 10.1111/epi.12944
seizures, mainly myoclonic jerks, despite adequate therapy and lifestyle. Seventy-five healthy, unrelated subjects (40 women; mean SD age, 28.46 10.96), with the same ethnic background, were also screened for mutations of the CSTB gene only to test the occurrence of the sequence variants identified in the JME screening sample. All patients or their parents/guardians, in case of minors with JME, and controls signed an informed consent prior to participation in the genetic studies. The human research ethics committee of the University Magna Græcia, Catanzaro, Italy approved the study. Molecular study Screening for mutations in the CSTB gene was performed in all index patients and controls. Genomic DNA was extracted from venous blood samples using standards methods. The CSTB gene was investigated as previously described.2 Polymerase chain reaction (PCR) amplification of the large and normal alleles was performed in a 20 lL volume containing 15 ng DNA, 0.5 mM of each nucleotide, 0.5 lM of each primer, 0.12 U Taq long-template PCR system (Roche Diagnostic, Mannheim, Germany), 19 bovine serum albumin, 5% dimethyl sulfoxide (DMSO), 1.0 M GC melt mix (Clontech, Palo Alto, CA, U.S.A.). Primers 2f: (50 -CCCGGAAAGACGATACCAG-30 ) and 1R (50 -GAG GAGGCACTTTGGCTTC-30 ) were used. After the initial denaturation at 94°C for 2 min a 40-cycle reaction (94°C 10 min, 57°C 45 s, and 68°C 8 min) and a final extension at 68°C for 7 min. The PCR fragments were detected by electrophoresis analysis. For mutation scanning, 2,949 bp of the CSTB gene (GenBank AF208234), including the exons, the introns, and the 50 and 30 untranslated regions, was amplified in overlapping fragments (Fig. 1) using the following primers: 1F (50 AAA CGC AAA TTC CAC CAG AG-30 ) and 1R (50 -GAG GAG GCA CTT TGG CTT C-30 ), 2F (50 -CCC GGA AAG ACG ATA CCA G-30 ) and 2R (50 -CGG CTT CTT TCG CTC CAG-30 ), 3F (50 -GCC GAG ACC CAG CAC ATC-30 ) and 3R (50 -CCT GTG GAC CTT TTA TGC AG-30 ), 4F (50 -GCA AGA GGT CCC CAG TGA TA-30 ) and 4R (50 -TGA CAC GGC CTT AAA CAC AG-30 ), 5F (50 -ACC GTA CCC AGC TGG AAC TGT-30 ) and 5R (50 -GCT TAT CTC AGG GGG CAG CCA CAG-30 ), 6F (50 -GTA GAG TGT GGG CCT CAG GA-30 ) and 6R (50 -AAG CCT CTG ATC CCA AGT CA-30 ), and 7F (50 -ATT GTC TTC AGC TGG CTG CTA AT-30 ) and 7R (50 -AAG ATC ACC TAT TGG GAA GGA AAG A-30 ). The PCR fragments were sequenced by an ABI 3130 DNA analyzer (Applied Biosystems, Foster City, CA, U.S.A.).
Results No pathologic (≥30) dodecamer repeat expansions or other pathogenic CSTB gene mutations were detected in any
3 No Role for CSTB in JME
Figure 1. Gel electrophoresis showing PCR-amplified dodecamer repeat expansions of normal CTSB alleles. Lanes 2 and 3, homozygous patients with two copies of the repeat; lanes 4 and 5, homozygous patients with three copies of the repeat; lanes 6 and 7, compound heterozygous (two different mutant alleles of the disease gene that are present in a patient affected by a recessive disease) with two and three copies of the repeat; lanes (1 and 8) ladder. Epilepsia ILAE
of 57 patients with JME. Figure 1 shows the typical electrophoresis result. In detail, almost all (56 of 57) JME patients had normal alleles, with 2–3 dodecamer repeats (genotype 2/2: 12 of 57 [21.0%] patients; genotype 2/3: 14 of 57 [24.6%]; genotype 3/3: 30 of 57 [52.6%]). One affected individual carried an allele with four copies (genotype 3/4), which represents a very rare normal variant.9 In the healthy control individuals, the analysis revealed the commonly observed two and three copy repeat alleles (genotype 2/2: 16 of 75 [21.3%] subjects; genotype 2/3: 20 of 75 [26.7%]; genotype 3/3: 39 of 75 [52.0%]).
Discussion Juvenile myoclonic epilepsy is a common form of idiopathic generalized epilepsy characterized by symmetric, myoclonic jerks, mostly affecting upper limbs, generalized tonic–clonic seizures and, more rarely, absence seizures. Clinical genetic studies support a high genetic predisposition, and positive family history of epilepsy can be obtained in 30–50% of patients. The mechanisms and pathophysiology of JME are not yet clear, but growing evidence indicates that JME is typically a polygenic disorder with heterogeneous genetic and pathologic signatures.8 Mutations in ion channel genes have been occasionally associated with autosomal dominant JME. Nonetheless, a more common pathogenic mechanism may involve abnormalities in neuronal connectivity that are caused by subtle structural changes in the brain.10,11 Indeed, some genes associated with both
sporadic and familial JME, such as Myoclonin1/EFHC1 and BRD2, play key roles in the development and maintenance of normal neuronal structures, through cell growth and proliferation, differentiation, survival, and apoptosis that would disrupt the normal balance between excitatory and inhibitory circuits.10,11 In addition, the CSTB gene plays a role in the development and maintenance of normal neuronal structures through cell-survival and cell-death pathways.12 Therefore, EPM1 and JME might share, at least in part, a common pathogenic mechanism that involves subtle structural changes in the brain with consequent abnormalities in neuronal connectivity. Subtle increases in specific populations of neurons may occur in JME,11 whereas a defect in apoptosis with increased neuronal cell death may be associated with EPM1.12 On this basis, because EPM1 and JME also share several electroclinical features, the CSTB gene might play a pathogenic role in a subset of patients with JME. In this study, we have explored this hypothesis, but no disease-causing mutation in CSTB was found in our patients with familial or sporadic JME, including those with a more severe phenotype. Therefore, our findings strongly indicate that JME and EPM1 are distinct clinical and genetic entities. The diagnosis of EPM1 in patients with the JME phenotype should be considered only when additional clinical manifestations such as action or reflex sensitive myoclonus occur, as they are distinctive features of progressive myoclonus epilepsy. To the best of our knowledge, these findings provide substantial negative evidence for a role of CSTB gene in the pathogenesis of JME. In a large JME cohort, indeed, we did not identify any homozygous, compound heterozygous, or heterozygous mutations of CSTB. In accordance with our findings, a previous work provided exclusion for linkage to the EPM1 locus on chromosome 21q in a set of families segregating for JME or other idiopathic generalized epilepsies, which did not show linkage to markers on chromosome 6p.13 There is also a recent case report of a 30-year-old woman with a JME phenotype that was a compound heterozygote for two CSTB mutations: an expansion of the dodecamer repeat and a splice-site c.67-1G>C mutation in intron 1, predicting a deletion of the downstream exon 2 with inframe deletion of 34 amino acids (p.delV23_K56).14 Nevertheless, four siblings of her maternal grandmother had already received the diagnosis of EPM1.14 Of interest, an experimental study provided evidence that CSTB-deficient heterozygous mice showed a milder EPM1 phenotype with partial penetrance of EPM1-like symptoms (seizures, ataxia),15 suggesting pleiotropic effects of homozygous and heterozygous CSTB mutations. A recent example of different phenotypes with homozygous versus heterozygous mutations involves the progranulin gene (GRN, granulin precursor).16 Indeed, homozygous GRN mutations gave rise to the rare form of adult-onset neuronalceroid-lipofuscinosis, whereas heterozygous mutations in Epilepsia, **(*):1–4, 2015 doi: 10.1111/epi.12944
4 L. Mumoli et al. GRN are a major cause of frontotemporal lobar degeneration, which is the second most common early-onset dementia.16 Again, in the present series we did not identify any patients with sporadic or familial JME who carried heterozygous CSTB mutations, making the hypothesis of CSTB pleiotropism unlikely, with heterozygous CSTB mutations conferring susceptibility to a milder epilepsy phenotype, such as JME. Additional studies are needed to extend the molecular analysis in atypical patients who do not fit into the classic JME phenotype because of additional clinical manifestations, especially action or reflex sensitive myoclonus.
5.
6. 7. 8. 9.
Acknowledgment 10. We wish to thank Italian League Against Epilepsy (LICE) for supporting this work. 11.
Disclosure
12.
None of the authors has any conflicts of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
13.
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