Hepatic disease as the first manifestationof progressive myoclonus epilepsy ofLafora

P. Gomez-Garre, PhD*E. Gutierrez-Delicado,MD*

C. Gomez-Abad, PhDJ. Morales-Corraliza,PhD

V.E. Villanueva, MDS. Rodrıguez de

Cordoba, PhDJ. Larrauri, MD, PhDM. Gutierrez, MD, PhDJ. Berciano, MD, PhDJ.M. Serratosa, MD,PhD

ABSTRACT Background: Lafora disease (LD; progressive myoclonus epilepsy type 2; EPM2) is anautosomal recessive disorder caused by mutations in the EPM2A and EPM2B genes. LD is charac-terized by the presence of strongly PAS-positive intracellular inclusions (Lafora bodies) in severaltissues. Glycogen storage disease type IV (GSD-IV; Andersen disease) is an autosomal recessivedisorder characterized by cirrhosis leading to severe liver failure. GSD-IV has been associatedwith mutations in the glycogen branching enzyme gene (GBE). Histopathologic changes of the liverin both diseases show an identical appearance, although cirrhosis has never been described inpatients with LD. We report a LD family in which the proband presented severe liver failure atonset of the disease. Methods: Clinical histories, physical and neurologic examination, laboratorytests, EEGs, MRI of the brain, and liver or axillary skin biopsies were performed in the two affectedsiblings. The diagnosis was confirmed by molecular genetic analysis of the EPM2A, EPM2B, and GBEgenes and loci. Results: During the first decade of life, abnormalities in liver function tests were de-tected in the two affected siblings. The proband’s liver dysfunction was severe enough to require livertransplantation. Subsequently, both sibs developed LD. Mutation analysis of EPM2A revealed a ho-mozygous Arg241stop mutation in both patients. Conclusions: This is the first description of severehepatic dysfunction as the initial clinical manifestation of LD. The phenotypic differences between thetwo affected siblings suggest that modifier genes must condition clinical expression of the diseaseoutside the CNS. NEUROLOGY 2007;68:1369–1373

Accumulation of polyglucosans represents the morphologic manifestation of several clini-cally heterogeneous disorders such as Lafora disease, glycogen storage disease type IV, adultpolyglucosan body disease, and some cases of phosphofructokinase deficiency (Tauridisease).

Lafora disease or progressive myoclonus epilepsy of the Lafora type (EPM2 [MIN254780])is an autosomal recessive form of progressive myoclonus epilepsy (PME) that initially mani-fests during adolescence and is characterized by epilepsy, myoclonus, and progressive neuro-logic deterioration. The diagnosis is sustained by the presence of periodic acid-Schiff (PAS)positive glycogen-like intracellular inclusion bodies (Lafora bodies). Lafora bodies, first de-scribed by Gonzalo R. Lafora in 1911, consist of an abnormal glucose polymer.1,2 Laforaoriginally called the inclusion bodies “intracellular amyloid bodies” when he observed themin the brain and spinal cord of an adolescent patient who presented a progressive and fatalform of myoclonic epilepsy. In 1955, Harriman and Millar showed that the inclusions werenot limited to the CNS and described similar intracellular inclusions in the heart and liver ofone patient with Lafora disease.3 Lafora disease initially manifests during adolescence, themost common age at onset being between 10 and 17 years. Generalized tonic-clonic seizures,absences, drop attacks, or partial visual seizures are usually the first manifestation, followedsoon after by asymmetric as well as massive myoclonic jerks. As the disease progresses, themyoclonus increases in frequency and becomes constant. A rapidly progressive dementia

*These authors contributed equally to this work.

From Laboratorio y Servicio de Neurologıa (P.G.-G., E.G.-D., C.G.-A., J.M.-C., V.E.V., J.M.S.), Fundacion Jimenez Dıaz, Madrid; Centro deInvestigaciones Biologicas (S.R.C.), Consejo Superior de Investigaciones Cientıficas, Madrid; Departamento de Anatomıa Patologica (J.L., M.G.),Hospital Universitario La Paz, Madrid; and Departamento de Neurologıa (J.B.), Hospital Universitario Marques de Valdecilla (UC), Santander,Spain. V.E.V. is currently affiliated with Unidad de Epilepsia, Servicio de Neurologıa, Hospital La Fe, Valencia, Spain.

Supported by the Spanish Ministry of Science and Education (SAF2004/07151) and Fondo de Investigaciones Sanitarias (PI020536 and C03/06).

Disclosure: The authors report no conflicts of interest.

Address correspondence andreprint requests to Dr. Jose MSerratosa, Servicio deNeurologıa, Fundacion JimenezDıaz, Avda Reyes Catolicos, 2,28040 Madrid, Spainserratosa@telefonica.net

Copyright © 2007 by AAN Enterprises, Inc. 1369

with apraxia, aphasia, and visual loss ensues,leading patients to a vegetative stage anddeath, usually within less than a decade offirst symptoms. Two genes have been associ-ated with Lafora disease: EPM2A (chromo-some 6q24) and EPM2B (chromosome6p22.3). EPM2A encodes a protein tyrosinephosphatase (LAFPTPase or laforin).4,5 Inspite of the remarkable allelic heterogeneityin the EPM2A gene, the R241stop mutationhas been found in approximately 40% ofLafora disease patients with mutations inthis gene.6,7 The EPM2B gene (also calledNHLRC1) encodes an E3 ubiquitin ligase(malin) and the most frequent mutationfound is P69A.8

Glycogen storage disease or glycogenosistype IV, also known as amylopectinosis orAndersen disease (MIN23250), is an autoso-mal recessive disorder caused by a deficiencyof glycogen-branching enzyme (GBE) activ-ity due to mutations in the GBE1 gene.9 Adiagnosis can be made on the basis of thestudy of branching enzyme activity in eryth-rocytes.10 Glycogenosis type IV is highly het-erogeneous in terms of tissue involvement,clinical manifestations, and age at onset. Theclassic form of glycogenosis type IV is char-acterized by rapidly progressive hepato-splenomegaly and liver failure leading toeither liver transplantation or death by theage of 5 years.9 In addition to the classicform, variants such as a milder nonprogres-sive hepatic disease,11 neuromuscular formsvarying in onset and severity (from a fatalneonatal disease12 to a mild adult myop-athy13), a cardiopathic form of childhood,14

or a variant with multisystem involvementincluding liver and muscle15 have been re-ported. GBE belongs to the alpha-amylasefamily and is required for glycogen synthesis.It catalyzes the formation of alpha 1,6-glucosidic branches that play an importantrole increasing the solubility of the moleculeand is most highly expressed in liver andmuscle. Typically, hepatocytes in glycogeno-sis type IV contain glycogen PAS-positive in-clusions resulting in a ground glassappearance. This stored glycogen showsfewer branch points and longer outer chainsresembling amylopectin. For this reason thedisease is also referred to as amylopectinosis.Ultrastructurally, the inclusion bodies ofLafora disease and glycogenosis type IV ap-pear quite similar.16 Liver fibrosis, which canprogress to cirrhosis, is a frequent finding inglycogenosis type IV. However, only mild tomoderate periportal fibrosis has been de-scribed in the liver of patients with Laforadisease.17

Here, we present a clinical and moleculargenetic study of two siblings with Lafora dis-ease, one of whom developed a progressiveliver cirrhosis and failure, requiring livertransplantation.

METHODS Patients and samples. We studied two sib-lings from a Spanish family (figure 1A). Both presented epi-lepsy, myoclonus, rapidly progressive neurologic deterioration,and a slow background activity with polyspike-wave com-plexes in the EEG.18,19 A biopsy of skin from the proband andliver biopsy from the younger brother showed the characteristicPAS-positive Lafora bodies in both. Blood was collected frompatients and their relatives after informed consent.

DNA samples were obtained from peripheral blood lym-phocytes using standard methods. The study was approved bythe Ethics Committee of the Fundacion Jimenez Dıaz.

Microsatellite analysis. Analysis of microsatellite polymor-phisms on chromosomes 6q24 and 3p12 was performed by PCRusing total human genomic DNA. Amplification was per-formed in a total volume of 10 �L containing 40 ng of genomicDNA and isotopic phosphate. Samples were resolved on 6.5%polyacrylamide sequencing gels and bands were visualized us-ing Kodak XAR films for 1 to 14 hours.

Single-strand conformational polymorphism (SSCP)analysis. SSCP analysis20 was performed by PCR, using totalgenomic DNA, using the GenePhor DNA Electrophoresis Sys-tem (Amersham Pharmacia Biotech). Amplification was per-formed in a total volume of 10 �L containing 60 ng of genomicDNA. Samples were resolved on 12.5% non-denaturing poly-acrylamide gels with the GeneGel Excel 12.5/24 kit (Amersham

Figure 1 Spanish Lafora disease family(A) Spanish Lafora diseasefamily. Black symbolsindicate affected individuals.(B) Single-strandconformationalpolymorphism analysis.Individuals LD122-1,LD122-2, and LD122-3 aredenoted as 1, 2, and 3.Individual C1 is an unaffectedcontrol. Individual C2 isaffected with theArg241stop mutation inhomozygosis. Individuals 1and 2 carry the mutation inheterozygosis and Individual3 in homozygosis. (C) DNAsequence of exon 4 of theEPM2A gene showing theC¡T change resulting in thereplacement of arginine 241by a stop codon.

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Pharmacia Biotech) and silver stained using the PlusOne DNASilver Staining Kit (Amersham Pharmacia Biotech).

Sequencing of exons. Exons 1, 2, 3, and 4 of the EPM2Agene,6 exon 1 of the EPM2B gene,8 and exons 1 to 16 of theGBEgene were amplified from genomic DNA with specific primersusing standard methods. The corresponding PCR productswere purified by agarose gel electrophoresis and extracted withthe Qiaquick Gel Extraction Kit (Qiagen). Direct sequencing ofPCR products was performed with a dye-terminator cycle-sequencing kit (Perkin-Elmer) using Taq FS DNA polymerase.Sequences were resolved on an ABI PRISM 377 automatic se-quencer, and the results analyzed with the ABI Analysis soft-ware (version 3.1).

RESULTS Clinical findings. Case 1. The probandpresented growth retardation with height andweight deficiencies since early infancy. At age 7, lab-oratory tests revealed an increase in hepatictransaminase levels (alanine aminotransferase 321,aspartate aminotransferase 380) and abdominal dis-tension, ascites, and increasing splenomegaly due toportal hypertension appeared. Hemorrhagic com-plications occurred subsequently and a severe he-patic failure was detected. A liver biopsy wasperformed at age 9. The histopathologic studyshowed ground-glass appearance of hepatocytesdemonstrating positive Best carmine staining andlack of staining with orcein. Severe periportal stel-late fibrosis was observed suggesting type IV glyco-gen storage disease (figure 2, A and B). He requiredliver transplantation at age 10. No clinical compli-cations occurred during follow-up.

At age 16 he noticed transient light spots during aschool test. He had his first generalized tonic-clonicseizure the following day. Six months later, myo-clonic jerks were noticed. At first, these were asym-metric, segmental myoclonic jerks and occurredoccasionally. During the following year, generalizedtonic-clonic seizures increased in frequency andmassive myoclonus appeared. Seizures were refrac-

tory to antiepileptic drugs. Progressive intellectualimpairment occurred during the next 2 years.

An EEG showed diffuse slow background activ-ity with bilateral paroxysms of spike-wave andpolyspike-wave complexes. Marked photosensitiv-ity was noted. Brain magnetic resonance showed ce-rebral atrophy and typical Lafora bodies were seenin an axillary skin biopsy.

Case 2. The proband’s younger brother presentedan increase of liver transaminases levels in a routineanalysis at age 6 (alanine aminotransferase 112, as-partate aminotransferase 80). Although he re-mained clinically asymptomatic, a liver biopsy wasperformed at age 9. Liver biopsy revealed PAS-positive intracellular inclusion bodies with normallobular architecture. No periportal fibrosis was ob-served. At age 14 he initially noticed occasional epi-sodes consisting of seeing sudden and transientbrilliant spots. These episodes lasted for few sec-onds and were especially frequent when he waswatching television. Massive myoclonic jerks ap-peared 3 months later and he had his first general-ized tonic-clonic seizure 1 year after the onset ofneurologic symptoms. EEG showed a slow back-ground activity, occipital spikes increasing with in-termittent photic stimulation, and paroxysms ofirregular spike-wave activity on both frontotempo-ral regions. During follow-up, myoclonic epilepsyand neurologic deterioration progressively devel-oped but no hepatic dysfunction was detected.

Genetic findings. SSCP and mutational analysis ofexons 1, 2, 3, and 4 of the EPM2A gene revealed ahomozygous Arg241stop mutation in both siblings(figure 1, B and C), confirming the diagnosis ofLafora disease. Exons 1 to 16 of the GBE gene andexon 1 of EPM2B gene were also analyzed in bothsiblings in order to search for mutations. No muta-tions were found.

Figure 2 Histologic study of the liver of Individual LD112-3(A) Histologic study of theliver of Individual LD112-3showing loss of hepaticstructure and confluentbands of collagen formingnodules (Masson’s trichromicstain). (B) Ground-glassappearance of hepatocytesas expression of the Laforabodies (arrow).

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Haplotype analysis of the EPM2A and GBEgenes was also performed and showed that both rel-atives presented identical haplotype for these loci(data not shown).

DISCUSSION Lafora disease has been considered aclinically homogeneous degenerative disorder mani-fested by progressive myoclonic epilepsy. AtypicalLafora disease with an early onset cognitive deficitphenotype has been recently reported by differentauthors in a minority of patients.21-23 However, inno case of Lafora disease caused by mutations inEPM2A or EPM2B genes have initial clinical symp-toms outside the CNS been reported. Here we de-scribe the first Lafora disease family in which theproband presented a severe liver failure resemblingglycogenosis type IV at onset of the disease. Hisyounger brother showed abnormal liver functiontests in a routine analysis although he was clinicallyasymptomatic and no signs of hepatic failure weredetected during his follow-up. Both siblings subse-quently developed the typical picture of progressivemyoclonus epilepsy of Lafora type. PAS-positive in-clusions were observed in an axillary skin biopsyfrom the proband and in a liver biopsy from hisyounger brother. The diagnosis of Lafora diseasewas confirmed by mutation analysis of the EPM2Agene, which revealed an Arg241stop mutation in ho-mozygosis in both siblings. Interestingly, only theproband presented severe liver failure preceding theonset of progressive myoclonus epilepsy.

Histopathologic study of the liver in the probandshowed ground-glass appearance of hepatocyteswith PAS-positive inclusions and cirrhosis, leadingto the diagnosis of glycogenosis type IV. His-topathologic changes of the liver in Lafora diseaseand glycogenosis type IV show an identical appear-ance. The similarity applies especially to the groundglass hepatocytes containing PAS-positive inclu-sions. Histochemical reactions of stored materialare similar. However, patients with glycogenosistype IV typically develop cirrhosis while only mildto moderate periportal fibrosis has been describedin patients with Lafora disease.17 The clinical pic-ture and the histopathologic findings in the probandstrongly suggested the diagnosis of glycogenosistype IV. Mutation analysis of the complete codingregion of theGBE gene did not reveal any mutationsand haplotype analysis of the chromosomal regioncontaining the GBE gene revealed identical haplo-types for both sibs, suggesting that this gene is notresponsible for the liver failure present in theproband.

Similarities between Lafora and glycogenosistype IV diseases suggest that these two conditions

are closely related and may share a common meta-bolic disturbance conditioning failures at differentlevels of the same metabolic pathway. Concomitantenzyme deficiencies in the same individual causing anunusual glycogenosis with extensive neuronal polyglu-cosan storage has been reported.24 This is an extremelyrare condition but it is reasonable to think that a simi-lar situation may account for the unusual clinical find-ings found in the patient here presented. This patientpresents a combination of two different clinical pheno-types included in the group of glycogen metabolicpathway disorders. A homozygous mutation inEPM2A and no mutation in GBE were found. Themost plausible hypothesis is that other unidentifiedmodifier genes or genes involved in glycogen metabo-lism might condition the clinical expression of Laforadisease outside the CNS.

ACKNOWLEDGMENTThe authors thank family members who participated in this study.

Received August 21, 2006. Accepted in final form December 15,2006.

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