Epilepsy Research (2014) 108, 1501—1510

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Late onset Lafora disease and novel EPM2A mutations: Breaking paradigms Aurelio Jara-Prado a, Adriana Ochoa a, María Elisa Alonso a, Gabriel A. Lima Villeda a, Francisca Fernández-Valverde b, nas b, Luis Ruano-Calderón b, Steven Vargas-Ca˜ Reyna M. Durón d, Antonio V. Delgado-Escueta c,d, Iris E. Martínez-Juárez e,∗ a

Neurogenetics and Molecular Biology Department, National Institute of Neurology and Neurosurgery of Mexico, Mexico City, Mexico b Experimental Pathology laboratory, National Institute of Neurology and Neurosurgery of Mexico, Mexico City, Mexico c Epilepsy Genetics/Genomics Laboratories and Epilepsy Center of Excellence, Neurology and Research Services, VA GLAHS, Los Angeles, CA, USA d David Geffen School of Medicine at UCLA, Los Angeles, CA, USA e Epilepsy Clinic and Professor of Mexico City, Mexico Received 10 April 2014; received in revised form 28 July 2014; accepted 21 August 2014 Available online 30 August 2014

KEYWORDS Lafora disease; Atypical phenotype; EPM2A; Slow progression; Late onset

Summary Lafora disease (LD) is an autosomal recessive progressive myoclonus epilepsy with classic adolescent onset of stimuli sensitive seizures. Patients typically deteriorate rapidly with dementia, ataxia, vegetative failure and death by 25 years of age. LD is caused by homozygous mutations in EPM2A or EPM2B genes. We found four novel mutations in EPM2A — three in exon 4 (Q247X, H265R G279C) and one in exon 1 (Y86D) — and a previously described mutation in exon 4 (R241X). These five EPM2A mutations were found in four index cases and affected relatives. Patient 1 with classic LD was doubly heterozygous for H265R and R241X in exon 4; while Patient 2, who also had classic LD, was homozygous for Q247X in exon 4. Patient 3 with classic LD was homozygous for Y86D in exon 1, but the same mutation in his affected brother manifested an atypical earlier childhood onset. For the first time, we describe a later onset and slower progression of EPM2A-deficient LD seen in Patient 4 and her three sisters who were doubly heterozygous for R241X and G279C in exon 4. In these sisters, seizures started later at 21 to 28 years of age and progressed slowly with patients living beyond 30 years of age.

∗ Corresponding author at: Insurgentes Sur #3877 Col. La Fama Del. Tlalpan, Mexico City 14269, Mexico. Tel.: +52 55 56063822x2052; fax: +52 55 56063822. E-mail address: [email protected] (I.E. Martínez-Juárez).

http://dx.doi.org/10.1016/j.eplepsyres.2014.08.017 0920-1211/© 2014 Elsevier B.V. All rights reserved.

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A. Jara-Prado et al. Our observations suggest that variations in phenotypes of EPM2A-deficient LD, like an earlier childhood or adolescent or later adult onset with a rapid or slower course, depend on a second modifying factor separate from pathogenicity or exon location of EPM2A mutations. A modifying gene amongst the patient’s genetic background or environmental factors may condition age of onset and rapid or slow progression of LD. © 2014 Elsevier B.V. All rights reserved.

Introduction Lafora type progressive myoclonic epilepsy (LD, OMIN 254780) is an autosomal recessive neurodegenerative disorder characterized by intractable stimuli-sensitive tonic—clonic, myoclonic and occipital seizures. Myoclonus start at 10 to 12 years of age followed by cerebellar ataxia, dysartria and cognitive decline, psychosis, amaurosis, mutism, muscle wasting, rapidly progressive dementia and respiratory failure. The onset in late childhood or adolescence with rapid deterioration and death between 17 and 24 years of age has been considered a characteristic feature of LD due to EPM2A mutations (Delgado-Escueta, 2007; Baykan et al., 2005; Ganesh et al., 2002). Histopathologically, LD is characterized by the presence of intracellular inclusion bodies, which are insoluble poorly branched hyperphosphorylated glycogen that precipitate and create Lafora bodies (LB) (Ganesh et al., 2006; Roach, 2011; Tagliabracci et al., 2011). LD is caused by mutations in at least two genes, EPM2A (Epilepsy Progressive Myoclonus type 2A) and EPM2B (Epilepsy Progressive Myoclunus type 2B) (Minassian et al., 1998; Serratosa et al., 1999; Chan et al., 2003). EPM2A is located on chromosome 6q24 and encodes laforin, a dualspecificity protein phosphatase having 331 amino acids and a functional carbohydrate-binding domain at the N-terminus. EPM2B is located on chromosome 6p22.3 and encodes malin, an E3 ubiquitin ligase of 395 amino acids with a RING finger domain at the N-terminus and six NHL domains in the C-terminal region. The onset and progression of the disease may vary due to genetic heterogeneity and diversity of mutations, making genotype—phenotype correlations difficult (Baykan et al., 2005; Ganesh et al., 2002). Ganesh et al. (2002) studied 22 patients with LD and described two clinical courses of the disease: (1) classic LD started in adolescence with stimuli sensitive myoclonic, tonic clonic, absence and occipital seizures followed by dementia and rapid progressive neurological deterioration. Classic LD was mainly associated with mutations in exon 4 of EPM2A; and (2) atypical LD starts in childhood with dyslexia and learning disorders followed by epilepsy and rapidly developing neurological impairment. Atypical LD was associated with mutations in exon 1 of EPM2A gene. A number of reports suggest that the course of the disease is longer in patients with EPM2B mutations compared to patients with EPM2A mutations, implying that patients with EPM2B-associated LD tend to have a slightly milder course and slower progression (Guerrero et al., 2011; Ganesh et al., 2006; Baykan et al., 2005; Franceschetti et al., 2006; Singh et al., 2006).

In this study, we report four novel EPM2A gene mutations in four index cases with LD. This includes one family with four affected siblings who all show EPM2A mutations but have an unusual later onset in adulthood and slower progression of the disease.

Material and methods Patients Four unrelated patients with LD were included in the genetic screening at the National Institute of Neurology and Neurosurgery (NINN), Mexico City. Patients in two unrelated families had classic LD. The third family consisted of two affected brothers (one with classic LD and the other with atypical LD). The fourth family with four affected sisters had later onset in adulthood and slow progression. Medical and neurological exam, electroencephalogram (EEG) and/or video-EEG, brain magnetic resonance image (MRI), somatosensorial evoked potentials (SSEP) and skin or muscle biopsy were performed in all patients. Medical, neurological exam and EEGs were also performed in some of the patients’ relatives. Informed consent was obtained from all patients or from the patients’ legal guardian, and from the participating relatives. We included 100 healthy Mexican mestizo controls who did not have a family history of epilepsy. This study was approved by the Bioethics Committee of the NINN.

Mutation analysis Genomic DNA was extracted from peripheral venous blood using the QIAmp DNA blood Mini Kit (QIAGEN, Valencia, CA, USA). The four exons of EPM2A were amplified using the primers and polymerase chain reaction (PCR) conditions described by Lesca et al. (2010). The PCR products were purified and sequenced using the Big Dye Terminator sequencing reactions in an automated DNA sequencer (ABI PRISM 3130 Applied Biosystems, Foster City, CA). The variants identified were sequenced at least twice in both directions (forward and reverse). Protein targeting to glycogen (PTG) is encoded by PPP1R3C. PTG interacts with laforin and is believed to condition the symptoms of LD to a less severe phenotype (Guerrero et al., 2011). Therefore, we also screened for mutations in PPP1R3C gene in Patient 4 and her affected siblings (4-S1, 4-S2 and 4-S3) who are all LD affected with later onset in adulthood.

Late onset Lafora disease and novel EPM2A mutations

In silico analysis An in silico analysis was carried out to evaluate the potential effect of the nucleotide change or the amino acid substitution on protein structure and function, using PolyPhen-2 program (Polymorphism Phenotyping v2), a tool which predicts the possible impact of an amino acid substitution on the structure and function of a human protein (http://genetics.bwh.harvard.edu/pph2/). Clustal Omega was used to identify whether mutations were present in regions conserved during evolution (http://www.ebi.ac.uk/Tools/msa/clustalo/).

Results

1503 spike-wave and polyspike-wave complexes. Her MRI was normal and she had abnormal giant SSEP. When genotyping the family, the 33-year old eldest sister (4-S2) of Patient 4 reported suffering from myoclonic seizures and occasional perioral myoclonias during menses. Myoclonic seizures started at 29 years of age. She denied having absence or GTC seizures. No further studies were performed on her and she had never taken any antiepileptic drug (AED). We could not perform a clinical exam on the other sister (4-S3) of Patient 4 because she lives abroad. However, she had had an EEG performed in the past, and her family had an EEG study which was abnormal, showing generalized slow background with intermittent bursts of 2—3 Hz spike and slow waves (Table 2). A formal neuropsychological/cognitive assessment could not be performed in Patient 4 and her siblings with mutations since they live in a distant, rural area with limited access.

Clinical characteristics (see Table 1) Four index cases, three males and one female, with LD were included. Patients’ clinical characteristics are described in Table 1. In all index cases, light periodic acid—Schiff (PAS)—hematoxylin microscopic examination showed PAS+ inclusions in skin and/or muscle biopsies, consistent with LB (Fig. 1). Patients 1 and 2 had a classic progression of LD. Patient 3 also had a classic progression of LD. However, the affected brother (3-1) of Patient 3 exhibited an atypical LD phenotype characterized by earlier onset, learning problems in elementary school. He had his first generalized tonic—clonic (GTC) seizure at 10 years of age. Considering the family history, an EEG was performed in the brother (3-1). The EEG showed frequent bursts of 3—4 Hz spike and polyspike waves, as well as isolated bursts of slow waves. His MRI was normal and his muscle biopsy showed PAS-positive LB. He was then put on valproic acid (VPA) and had rare GTC seizures. Two years later, he developed focal visual seizures and isolated myoclonias. At his last follow-up visit, he was 16 years of age, had no signs of ataxia, and his parents reported only moderate cognitive impairment that had prompted him to leave school. Patient 4 and her three sisters (4-S1, 4-S2 and 4-S3) presented with an unusual adult onset and slow progression for LD. GTC seizures started at 21 years of age in Patient 4. One year later, myoclonic seizures started and involved the arms, legs, shoulders and head. At 25 years of age, absence seizures appeared. Patient 4 was also able to get pregnant as she was not disabled by the seizures. All seizure types were followed by slowly progressive ataxia and mild to moderate intellectual decline. EEG showed slow background with frequent bilaterally-synchronous, spike-wave and polyspikewave complexes. Her MRI was normal and she had abnormal giant SSEP (Fig. 1). Initially we had considered a diagnosis of familial adult myoclonic epilepsy (FAME); however, LBs were seen on skin biopsy and molecular diagnosis confirmed that Patient 4 was affected by LD. The 30 year old sister (4-S1) of Patient 4 started having bilateral myoclonic seizures at 25 years of age. Myoclonic seizures occurred predominantly in right arm and leg, in clusters of three almost every day. Her first GTC seizure appeared at 30 years of age. Mild to moderate intellectual decline followed the GTC seizure. Her EEG showed slow background with occasional bilaterally-synchronous,

Mutation analysis We sequenced EPM2A in four index cases (Patients 1, 2, 3 and 4) and their LD-affected siblings (3-1, 4S-1, 4S-2 and 4S-3). We found four novel mutations and one previously reported mutation in EPM2A (Table 3). Patient 1 showed double heterozygous mutations in exon 4, a novel missense mutation c.794C > T, and a stop mutation c.721C > T. The c.794C > T mutation changed arginine for glycine at position 265 of the protein (p.H265R). The c.721C > T mutation changed arginine by a stop codon in the protein (p.R241X). Patient 2 had a novel nonsense mutation in homozygous state in exon 4 (c.739C > T), causing a change in the protein of a glutamine by a stop codon at position 247 (p.Q247X). Patient 3 and his brother (3-1) showed a novel missense mutation in exon 1 (c.256T > G) in homozygous condition, causing the change of a tyrosine by aspartic acid at position 86 in the protein Laforin (p.Y86D). Both Patient 3 and 31 harbored the mutation in a homozygous state and their parents each carried the mutation in a heterozygous state. Patient 4 and her sisters (4-S1, 4-S2 and 4-S3) were compound heterozygotes for mutations (c.721C > T; p.R241X) and (c.835G > T; p.G279C). We also studied her parents and an asymptomatic brother, and all were heterozygous carriers of only one mutation (Table 2 and Fig. 2). Multiple protein alignment of protein laforin showed that all residues were completely conserved throughout evolution (Fig. 3). In silico analysis by the program Polyphen that predicts possible impact of an amino acid substitution on the structure and function of EPM2A indicated that the missense mutations (H265R, G279C and Y86D) are probably pathogenic. We screened for mutations in the PPP1R3C in Patient 4 and her sisters (4-S1, 4-S2 and 4-S3), but they were all negative. We did not find any of the mutations described above in the 100 healthy Mexican mestizo controls.

Discussion To date, at least 64 different mutations in EPM2A gene have been reported in ‘‘The Lafora progressive myoclonus epilepsy mutation and polymorphism database’’ (http://projects.tcag.ca/lafora/). A majority of EPM2A mutations are nonsense — and missense — point mutations

1504 Table 1

A. Jara-Prado et al. Clinical findings of four index patients with Lafora disease and EPM2A mutations.

Clinical characteristics

Patient 1

Patient 2

Patient 3

Patient 4

EPM2A mutations Sex Age at onset (years) Consanguinity/endogamy First seizure type Phenotype Follow-up (years) Onset of cognitive impairment (years of age) Onset of ataxia (years of age) Onset of dementia (years of age) EEG/video-EEG

H265R/R241X Male 12 Absent MS MS, GTC, SP, Abs Three 14

Q246X/Q246X Male 12 Absent GTC GTC, Abs, MS, SP Two 14

Y86D/Y86D Male 14 Present GTC GTC, MS, Abs, SP Seven 13

R241X/G279C Female 21 Present GTC GTC, MS, Abs Three 24

17

14

15

24

14

14

15

25

Abnormal due to severe dysfunction and epileptic activity with multifocal patterns but predominantly generalized. Irregular spike waves and polyspike waves at 3 to 4 Hz with temporal and frontotemporal accentuation, left more than right Absent

Abnormal due to generalized slow background and frequent 2 to 2.5 Hz polyspike and wave complexes consistent with a non-convulsive status epilepticus

Abnormal due to generalized slow background interrupted by frequent 2 to 4 Hz spike and polyspike wave epileptiform complexes that sometimes correlated with generalized myoclonic seizures

Present

Abnormal due to generalized slow background mixed with epileptiform sharps, 1 to 2.5 Hz spke and wave and polyspike and wave complexes consistent with periodic generalized complexes and a non-convulsive status epilepticus Absent

+

+

+

+

CNZ, LEV, VPA At 19, patient was bedridden and discharged to an extended care facility; after that he was lost to follow up

LEV,TPM, VPA 16

CNZ, LEV, VPA 21

CNZ,LEV,LTG, VPA Currently alive Patient is 33 years old

Somato-sensory evoked potentials (SSEP) with giant cortical response Biopsy (skin or muscle) with PAS-positive Lafora inclusion bodies AED treatment Age of death (years of age)

Present

EEG = electronecephalogram, MRI = magentic resonance imaging SSEP = somato-sensory evoked potentials, + = positive = myoclonic seizures, GTC = generalized tonic—clonic seizures, SP = simple partial seizures (visual), Abs = absence seizures, AED = antiepileptic drug, CNZ = clonazepam, LEV = levetiracetam, LTG = lamotrigine, TPM = topiramate, VPA = valproate.

and account for 48% of all mutations in LD. (Serratosa et al., 1999; Chan et al., 2003). EPM2A encodes a protein of 331 amino acids called laforin containing a dual-specificity protein phosphatase catalytic domain in the C-terminus (DSPD) coded by exons 3 and 4, and a carbohydrate binding domain in the N-terminus (CBD4) coded by exon 1 (Delgado-Escueta, 2007). We found five mutations (two nonsense and three missense) in EPM2A, four novel and one already reported. These mutations present in four LD index patients included Q247X, H265R, G279C, Y86D and R241X. The R241X nonsense mutation is common among LD patients from Spain (Minassian et al., 1998, 2000; Goméz-Garre et al., 2000;

Ganesh et al., 2002). Two of our four index patients had the R241X mutation. Mexicans have indigenous pre-Columbian Native American origin mixed with Iberian Spanish ancestry and, thus, finding R241X in our Mexican populations is not surprising and could support the ‘‘founder effect’’ hypothesis that explains the high prevalence of the R241X mutation in Spain (Goméz-Garre et al., 2000; Ganesh et al., 2002). Ganesh et al. (2002) first described and Annesi et al. (2004) confirmed a rare and atypical childhood form of EPM2A deficient LD that manifested dyslexia and learning disorder at onset followed by seizures, cognitive decline and neurologic deterioration. Atypical LD was associated with homozygous mutations in exon1. This atypical earlier onset

Genotype and phenotype of patient’s 4 family with late onset Lafora disease. Current age (years)

Mutation in EPM2A gen

Predicted effect and [genotype in affected]

Phenotype

Pregnancies EEG

Patient 4

21

28

c.721G > T and c.835G > T

p.R241X (nonsense) and p.G279C (missense) [compound heterozygous]

GTC, MS, Abs ataxia dementia

2

Patient 4S1

25

30

c.721G > T and c.835G > T

p.R241X (nonsense) and p.G279C (missense) [compound heterozygous]

MS and GTC mild cognitive impairment no ataxia

1

Patient 4S2

28

33

c.721G > T and c.835G > T

p.R241X (nonsense) and p.G279C (missense) [compound heterozygous]

MS only and perioral MS no cognitive impairment no ataxia

2

Brain MRI

Normal Abnormal due to generalized slow background interrupted by frequent 2 to 4 Hz spike and polyspike and wave epileptiform complexes that sometimes correlated with generalized myoclonic seizures Abnormal due Normal to 3 to 7 Hz slow background, intermittent bursts of 2 to 2.5 Hz generalized slow waves and spike and polyspike wave complexes ND ND

SSEP with giant cortical response

Biopsy with PAS-positive Lafora inclusion bodies (skin or muscle)

Treatment

Present

Present

CNZ, LEV, LTG, VPA

Present

Present

CNZ, VPA

ND

ND

None

1505

Age at onset (years)

Late onset Lafora disease and novel EPM2A mutations

Table 2

1506

Table 2 (Continued) SSEP with giant cortical response

Biopsy with PAS-positive Lafora inclusion bodies (skin or muscle)

Treatment

EEG performed ND at 28 years of age was abnormal due to 5 to 7 Hz slow background, intermittent bursts of 2.5 to 4 Hz generalized slow waves and spike and slow wave complexes ND —

ND

ND

None







ND









ND









Age at onset (years)

Current age (years)

Mutation in EPM2A gen

Predicted effect and [genotype in affected]

Phenotype

Pregnancies EEG

4S3

UNK

32

c.721G > T and c.835G > T

p.R241X (nonsense) and p.G279C (missense) [compound heterozygous]

UNK

2

Brother





c.835G > T

Asymptomatic —

Mother





c.835G > T

p.G279C missense [heterozygous] p.G279C missense [heterozygous]

Father





c.721G > T

MS = myoclonic seizures, GTC = generalized tonic—clonic seizures, SP = simple partial seizures (visual), Abs = absence seizures, ND = not done, UKN = unknown, VPA = valproate, LEV = levetiracetam, LTG = lamotrigine, CNZ = clonazepam, EEG = electronecephalogram, MRI = magentic resonance imaging SSEP = somato-sensory evoked potentials.

A. Jara-Prado et al.

p.R241X nonsense [heterozygous]

GTC only, in — remission no ataxia, no dementia no cognitive impairment Asymptomatic —

Brain MRI

Late onset Lafora disease and novel EPM2A mutations

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Figure 1 Electroencephalogram, somato-sensory evoked potentials (SSEP) and axillary biopsy of patient 4. (A) and (B) Interictal EEG with generalized slow background interrupted by 4 Hz polyspike wave complexes paroxysms. (C) Left median nerve SSEP with giant cortical response. (D) Periodic acid—Schiff positive Lafora’s bodies (red arrows) in axillary biopsy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

in childhood differed from most LD patients who have the classical adolescent onset of stimuli sensitive epilepsies, cognitive decline and ataxia that associates with homozygous mutations in exon 4. Both atypical and classical LD progress so rapidly that dementia, urinary and fecal incontinence, relentless myoclonus and convulsions characterize late adolescence. And gastrostomy and respiratory support

Table 3

are needed as the patient turns 20 to 22 years of age. Rarely do EPM2A deficient LD patients survive beyond 25 years of age (Delgado-Escueta, 2007). Theoretically, three factors could influence the phenotypic expression of EPM2A mutations. First, is the exon location of the mutations and their pathogenic influence on the protein’s function. Three mutations found in our

Mutations in EPM2A gene in four patients with Lafora disease.

Patient

Nucleotide substitution

Aminoacid substitution (codon)

Region exon

Mutation

1

c.721C > T c.794C > T

R241X H265R

4 4

2 3

c.739C > T c.256T > G

Q247X Y86D

4 1

4

c.721G > T c.835G > T

R241X G279C

4 4

Associated with disease Probably damaging (Polyphen) Truncate protein Probably damaging (Polyphen) Associated with disease Probably damaging (Polyphen)

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A. Jara-Prado et al.

Figure 2 Pedigree of patient’s 4 family with an unusual late onset and slow progression of Lafor a disease and EPM2A mutations.

patients were located in exon 4 that encodes part of the dual specificity phosphatase domain (DSPD). These would impair glycogen phosphatase dephosphorylating activities and result in a hyperphosphorylated glycogen. Exon 4 mutations would also impair interaction with R5 (PTG), a regulatory subunit of protein phosphatase-1 that binds glycogen. Exon 4 mutations would thus impair glycogen binding to laforin and enhance accumulation of hyperphosphorylated poorly branched insoluble glycogen that constitute the Lafora inclusion bodies (Fernández-Sánchez et al., 2003; Roach, 2011; Tagliabracci et al., 2011). Patient 3 and his brother (3-1) showed homozygous mutations in exon 1 where the carbohydrate-binding domain, R5 (PTG), is located. Exon 1 mutations would, thus, also impair glycogen binding, and also result in accumulation of poorly branched insoluble hyperphosphorylated glycogen that constitute the Lafora inclusion bodies. While actual functional studies have yet to be performed to determine pathogenicity of the four novel mutations described in this paper, analysis of our observed three missense mutations (H265R, G279C and Y86D) using Polyphen software (a tool which predicts possible impact of an amino acid substitution on the structure and function of a human protein) showed that all are probably damaging to the protein’s functions. Furthermore, in silico analysis showed that all mutations are located in evolutionarily highly conserved regions, adding further

Homo sapiens Mus musculus Rattus norvegicus Pan troglodytes Macaca mulatta Canis lupus familiaris Bos taurus Gallus gallus

argument that the mutations are in functional domains and potentially pathogenic (Ganesh et al., 2004; Singh and Ganesh, 2008). A second factor, aside from the inherent pathogenicity of EPM2A mutations that could influence phenotype expression, is whether mutations are homozygous or heterozygous. Compound heterozygozity of EPM2A mutations did not seem to correlate with the late onset phenotype of LD in Patient 4. As Patient 1 was also doubly heterozygous (c.794C > T and c.721C > T) and had classical adolescent onset LD, heterozygous mutations would seem to have no influence on late onset or adolescent onset phenotypes. A third factor that could influence the phenotype of EPM2A mutations is a presently undefined modifier gene that could be genetic or environmental or both. Two clinical variations in the phenotypes of our patients suggest the possible involvement of a modifier. The first phenotype variation concerns the two siblings who had homozygous Y86D mutations in exon 1. Patient 3-1 had homozygous Y86D in exon 1, and presented with the atypical childhood form of Lafora disease in consonance with the reports of Ganesh et al. (2002) and Annesi et al. (2004). However, his brother (Patient 3-1) had the same homozygous Y86D in exon 1 but had the classic adolescent onset LD. Clearly, the homozygous Y86D in exon 1 was not the only determinant of the phenotypes of these two siblings. There has to be a second if not third factor that modified the phenotypic expression of the homozygous Y86D exon 1 mutation. The second phenotypic variation concerns Patient 4 and her sisters who were compound heterozygotes for mutations c.721C > T and c.835G > T in exon 4. All presented with adult onset and slow progression with survival beyond 30 years of age, a form of EPM2A deficient LD previously undescribed. The slowly progressive form of LD is usually associated with EPM2B (malin/E3 ubiquitin ligase) deficient LD and not with EPM2A (laforin/dual specificity phosphatase) deficient LD (Singh et al., 2006; Gómez-Abad et al., 2005). How then do we interpret our present observations in the context of past and more common experiences with EPM2A deficient LD? What the majority of clinical experience and literature has established is that most EPM2A deficient LD has the classical adolescent onset and rapid deterioration. According to our present observation, EPM2A deficient LD in the rare case of Patient 4 and her sisters, can have an adult disease onset (20 years old or more), and survive beyond 30 years of age, not unlike the EPM2B/malin deficient LD (Singh et al., 2006; Gómez-Abad et al., 2005). Guerrero et al. (2011) identified a new variation in PPP1R3C that might be related to a milder phenotype in LD patients with EPM2B mutations.

Y86D R241X Q247X H265R G279C ...PGRVDTFWYKFLKREPGGEL.....MSTEGRVQMLPQAVCLL...HIVYVHCNAGVGRSTAAVCGWLQYVMGWNL... ...PGRVDTFWYKFLQREPGGEL.....MSTEGRVQMLPQAVCLL...HTVYVHCNAGVGRSTAAVCGWLHYVIGWNL... ...PGRIDTFWYKFLQREPGGEL.....MSTEGRVQMLPQAVCLL...HTVYVHCNAGVGRSTAAVCGWLHYVIGWSL... ...PGRVDTFWYKFLKREPGGEL.....MSTEGRVQMLPQAVCLL...HIVYVHCNAGVGRSTAAVCGWLQYVMGWNL... ...PGRVDTFWYKFLKREPGGEL.....MSTEGRVQMLPQAVCLL...HIVYVHCNAGVGRSTAAVCGWFQYVMGWNL... ...PARVDTFWYKFLKREPGGAL.....MSTEGRVQMLPQAVCLL...HTVYVHCNAGVGRSTAAVCGWLQYVMGWNL... ...PGRVDTFWYKFLKREPGGEL.....MSTEGRVQMLPQAVCLL...HTVYVHCNAGVGRSTAAVCGWLQYVLGWSR... ...---ASPFWYKFLRRE-GGQL.....MSTEGRIQMLPQAVCLL...HTVYVHCNAGVGRSTAAVSGWLKYVMGWSL...

Figure 3 Multiple protein alignment of laforin showing conservation of Tyr86, Arg241, Gln247, His265 and Gly 279 residues across of different orthologous proteins.

Late onset Lafora disease and novel EPM2A mutations We searched for mutations in PPP1R3C so as to exclude a potential role of this gene in the slower development of the EPM2A disease in the family of Patient 4, but no mutations were found. Lohi et al. (2007) described a patient with a heterozygous change in EPM2A exon 1, 163C > A (Q55K), which turned out to be rare polymorphism in 7 of 500 healthy controls. Unlike the adult onset and slower progression of LD we observed in the family of Patient 4, Gómez-Abad et al. (2005) described a late onset but rapid decline in EPM2A deficient LD associated with a polymorphism in an Arab family. The patient’s LD started at 20 years of age and progressed rapidly as classical LD does, with death at 28 years of age. Our present observations on interfamilial phenotypic heterogeneity in two brothers with the same (c.256T > G) homozygous mutation in exon 1 also show that the inherent pathogenicity of the mutation and its location in exon 1 are not the only factors that cause the expression of the atypical earlier onset in childhood. Others have reported exon 1 mutations in EPM2A that did not show an early onset of LD symptoms, as we observed in Patient 3 (Franceschetti et al., 2006; Lesca et al., 2010). To our knowledge, however, an earlier onset of LD in childhood has only been reported in exon 1 mutations of EPM2A. Earlier onset in childhood has not been reported in EPM2B mutations. Our present observations again suggest a modifying factor that conditions phenotype expression outside of the defective EPM2A. A second if not third modifying factor was also suggested by Goméz-Garre et al. (2007) when one of two affected siblings developed severe liver failure at onset although both siblings harbored the same homozygous EPM2A mutation. Axillary or muscle biopsies from our patients revealed typical Lafora PAS-positive inclusion bodies. It should be noted that Patient 4 and her sister 4-S1 showed less LB on their axillary and/or muscle biopsies (see Fig. 1). So far, an unequivocal explanation on how mutations in EPM2A gene yield the phenotype of LD and the formation of LB. But a consensus opinion is emerging which consists of the following—–Laforin’s main function as glycogen phosphatase is to correct an error made by glycogen synthase. Glycogen synthase normally adds glucose residues to glycogen which is a branch polymer of glucose. At the same time that glycogen synthase adds glucose residues to glycogen, it mistakenly incorporates the B-phosphate of its substrate UDP glucose to glycogen at a rate of one phosphate per 10,000 glucose residue added as C2 and c3 phosphomonoesters (Tagliabracci et al., 2011; Roach, 2011). The normal function of laforin, a glycogen phosphatase, therefore, is to correct this error and dephosphorylate and remove phosphate residues from glycogen. When a mutation in laforin is present, dephosphorylation does not occur and a hyperphosphorylated poorly branched insoluble glycogen result. These hyperphosphorylated poorly branched insoluble glycogen are the Lafora inclusion bodies. Lafora bodies are ubiquitous despite the fact that the disease manifests mainly in the central nervous system (www. EPM2A—–epilepsy, progressive myoclonus type 2A, Lafora disease (laforin)—–Genetics Home Reference). No studies have correlated the severity of LD with the number of LB noted on biopsies, regardless of whether the biopsy was taken from the skin, muscle or brain. It would be interesting to determine whether the amount of LB correlate with the severity of the disease, as in Patient 4 and her sibling where

1509 we clearly saw fewer bodies compared to the biopsies of our other index patients with mutations.

Conclusions EPM2A mutations appear to be more frequent than EPM2B mutations among patients with LD who are Mexican of indigenous pre-Columbian Native American origin mixed with Iberian Spanish ancestry. We found that an earlier onset in childhood or a classical adolescent onset associate with a mutation in exon 1 of EPM2A. We also found that EPM2A mutations can start late after 20 years of age, progress slowly and survive beyond thirty years of age. It would be important in patients with progressive myoclonic epilepsies of late onset to consider genotyping for EPM2A mutations; thus our findings are different from those previously described for EPM2A, thereby breaking paradigms in the clinical and genetic aspects of LD known to date. Our observations suggest the presence of a modifier, genetic or environmental, that conditioned the phenotypic expression of EPM2A mutations.

Conflict of interest statement None of the authors has any conflict 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.

Acknowledgments This study was supported by grant CONACYT FONSEC 181359. Also supported in part by NIH 5R01NS055057.

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Late onset Lafora disease and novel EPM2A mutations: breaking paradigms.

Lafora disease (LD) is an autosomal recessive progressive myoclonus epilepsy with classic adolescent onset of stimuli sensitive seizures. Patients typ...
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