Laforin is a cell membrane and endoplasmic reticulum-associated protein tyrosine phosphatase

Abnormal Activity in theGlobus Pallidus in Off-Period DystoniaTakao Hashimoto, MD, PhD,1

Tsuyoshi Tada, MD, PhD,2 Fumi Nakazato, MD,2

Tetsuhiro Maruyama, MD, PhD,3

Satoshi Katai, MD, PhD,3 Yorimichi Izumi, MD,3

Yuzo Yamada, MD, PhD,4 and Shu-ichi Ikeda, MD, PhD1

Pallidotomy was performed in a parkinsonian patientwith off-period foot dystonia. Dystonia appeared at thebeginning of surgery and disappeared after the first mi-croelectrode penetration of the globus pallidus, perhaps amicropallidotomy effect. Neuronal recording during dys-tonia revealed that the mean firing rates were low in boththe internal and external segments of the globus pallidus,and that firing was irregular in the internal segment ofthe globus pallidus, compared with firing patterns in off-state parkinsonian patients without dystonia. These firingpatterns immediately changed into those of nondystonic,off-state parkinsonism after relief of dystonia. These re-sults suggest that off-period dystonia results from thesame physiological change in the basal ganglia as that inprimary dystonia.

Ann Neurol 2001;49:242–245

Dystonia is characterized by abnormal postures drivenby sustained muscle contractions1 and cocontractionsof antagonist muscle groups during voluntary move-ments.2,3 Long-term levodopa administration may leadto the development of dystonia in patients with Par-kinson’s disease (PD). Changes in levels of levodopa inthe brain cause two types of dystonia in parkinsonianpatients: that is, peak-dose dystonia in on-phase4 andoff-period dystonia in off-phase,5 but the physiologicalmechanisms underlying dopa-related dystonia remainobscure. We report the neuronal change in firing pat-tern in the globus pallidus corresponding to off-perioddystonia during surgery.

Patient and MethodsPatientThe patient was a 54-year-old right-handed man with a 16-year history of Parkinson’s disease. Progression of the diseaseled to gradual increases in levodopa therapy, and peak-doselimb dyskinesia, on-off phenomena, and off-period foot dys-tonia developed. Before the surgery, he took levodopa/dopa-decarboxylase 800 mg, trihexyphenidyl 6 mg, bromocriptine20 mg, pergolide 1.5 mg, and amantadine 200 mg daily.

Right pallidotomy was performed using microelectrodeguidance. Elgiloy, glass-coated microelectrodes with an im-pedance of 0.4 MV at 1000 Hz were used for single-cellrecording. Recording tracks were made in the parasagittalplane proceeding from anterodorsal to posteroventral at anangle of approximately 45° from vertical to the anterior-posterior (AC-PC) commissural line. The lateral distance ofthe electrode tip to the cerebral midline was measured byintraoperative computed tomography, 20.0 mm for the firstrecording track, and 21.0 mm for the second and thirdtracks. No sedation was used in surgery, and antiparkinso-nian medication was withheld overnight and during surgery.The coagulation electrode (1.2 mm in diameter, a 3-mm ex-posed tip) was advanced for lesioning in three tracks, twotracks 2.5 mm apart anteroposteriorly at lateral 21.5 mm,and one track at lateral 19.5 mm. Ten- to 30-second samplesof spontaneous single-cell activity from the GPe and the GPiwere recorded during mapping and stored on digital audiotape. Neuronal signals were digitized at 50 kHz, action po-tentials were discriminated according to the peak amplitudeand window aperture onscreen, and the firing rates of single-cell action potentials were calculated. Student’s t test (two-tailed) was used to determine the level of statistical signifi-cance when comparing mean firing rates in the parkinsonianpatients with and without dystonia.

ResultsCardinal parkinsonian signs in off-phase on the rightside were moderately ameliorated 2 months after sur-gery. Dyskinesia contralateral to the surgery was com-pletely abolished, and off-period foot dystonia was alsoameliorated completely on the right side and partiallyon the left side. Parkinsonian signs gradually deterio-rated and returned to the level before surgery at 1 year,but tremor, dyskinesia, and dystonia on the right sidedid not recur.

The activity of 15 cells was sampled from the palli-dum during pallidotomy; Figure 1A demonstrates thedischarge patterns. The first recording track wentthrough the posterior edge of the GPe and missed theGPi. At the end of the first recording track, bilateralfoot dystonia occurred. The electromyelograms(EMGs) recorded from the pretibial muscles and thegastrocnemius-soleus muscles showed sustained cocon-traction discharges. The temporal changes in EMGsfrom the leg muscles on the right side and in the firingrates of pallidal neurons are illustrated in Figure 2.Along the second track, the frequency of 3 GPe cells

From the 1Department of Medicine (Neurology) and 2Departmentof Neurosurgery, Shinshu University School of Medicine, Matsu-moto, Japan; and the 3Department of Neurology and 4Departmentof Neurosurgery, Kakeyu Rehabilitation Center and Clinic, Ma-ruko, Japan.

Received Apr 13, 2000, and in revised form Sep 13. Accepted forpublication Sep 13, 2000.

Address correspondence to Dr Hashimoto, Department of Medicine(Neurology), Shinshu University School of Medicine, Asahi 3–1-1,Matsumoto 390-8621, Japan.

BRIEF COMMUNICATIONS

242 © 2001 Wiley-Liss, Inc.

(mean frequency, Cell 1, 33.5 Hz; Cell 2, 27.6 Hz;Cell 3, 31.0 Hz; mean 6 standard deviation, 30.7 63.0Hz) was low (p , 0.02) compared with that in par-kinsonian patients without dystonia (29 cells from 10patients, 59.3 6 18.7Hz). The frequency of four GPicells (Cell 4, 27.6 Hz; Cell 5, 7.7 Hz; Cell 6, 9.8 Hz;Cell 7, 12.4 Hz; 14.4 6 9.0 Hz) was remarkably low(p , 0.000001) compared with that in those patientswithout dystonia (51 cells from 10 patients, 87.9 625.8 Hz). Cell 6 showed an increase in the dischargerate with passive ankle dorsiflexion, but it showed lowand irregular firing at rest (Fig 1B), and the dischargedid not correlate with EMGs of dystonia. Cell 6 didnot respond to ankle plantar flexion, nor did it respondto the passive movements in another body part. After

penetration of the second track, foot dystonia on theright side disappeared and tremor and rigidity in theright leg were ameliorated compared with those presentbefore the second track. Along the third track, the fre-quency of four GPe cells (Cell 8, 35.8 Hz; Cell 9, 52.7Hz; Cell 10, 64.5 Hz; Cell 11, 54.7 Hz; 51.9 611.9Hz) returned to the range (p . 0.4) of that in parkin-sonian patients without dystonia, and four GPi cellsshowed the same high frequency firing (Cell 12, 62.1Hz; Cell 13, 108.9 Hz; Cell 14, 75.3 Hz; Cell 15,74.1 Hz; 80.1 6 20.1 Hz) (p . 0.5) as that in pa-tients without dystonia. Cells 13 and 14 did not showa clear response to passive joint movements. Cells 4, 5,7, 12, and 15 could not be recorded long enough tocheck response to sensory stimuli.

Fig 1. Raster display of neural activity along microelectrode Tracks 2 and 3. (A) Spontaneous firing showing reduced firing rates inthe GPi and GPe, an irregular firing pattern in the GPi along Track 2 during dystonia compared with that in parkinsonian pa-tients without dystonia, and an increase in firing rates along Track 3 after relief of dystonia. (B) Cell 6 showed firing increase inresponse to passive dorsiflexion of the dystonic ankle.

Brief Communication: Hashimoto et al: Globus Pallidus in Off-Period Dystonia 243

DiscussionJudging from its occurrence in levodopa off-period andits abolishment along with parkinsonian signs by GPiinactivation, off-period dystonia apparently has apathophysiology similar to that underlying parkinso-nian signs, which are caused by dopa depletion. In pal-lidotomy in the present patient, the relief of off-perioddystonia which occurred after the first microelectrodepenetration of the GPi gave us an opportunity to ob-serve the changes in neuronal activities relating to dys-tonia. The relief of off-period dystonia with some im-provement of parkinsonian symptoms, withoutadministration of antiparkinsonian medication, sug-gests a micropallidotomy effect. In contrast to the highfiring rates of GPi cells in akinetic parkinsonian statewithout dystonia,6,7 firing rates in the GPi were ex-tremely low during off-period dystonia. The observa-tions that the GPi neuron responsive to the dystonicfoot movement showed remarkably low and irregularfiring, and that the firing rates in the GPi increasedafter the patient was not dystonic, suggest that irregularfiring with decreased rates in the GPi is highly corre-lated with off-period dystonia. Although the number ofsampled cells was small, the firing rates in the GPe tendedto be low compared with those in nondystonic off-stateparkinsonism, suggesting that the low rates of GPe neu-rons may also be correlated with off-period dystonia.

Similarly decreased firing rates in the GPi and GPe

have been commonly observed in primary dystonia,7–9

indicating that off-period dystonia shares a commonneural mechanism with primary dystonia. The decreasein firing rates in the GPe and GPi in dystonia may beexplained by either of two possibilities: the increasedactivity in the striatum-GPi and striatum-GPe inhibi-tory pathways or the decreased activity of the subtha-lamic nucleus. The former mechanism is more proba-ble in dystonia cases caused by striatal dysfunction, suchas off-period dystonia. In terms of decrease in the meanfiring rates in the GPi, dystonia may be classified as ahyperkinetic movement disorder, as is hemiballism.7,8,10

Irregular firing in the GPi may have a pathogenic rolein primary dystonia and off-period dystonia.7–9 It wasreported in hemiballism that the firing pattern of GPicells was highly irregular,7,10 and the pauses in neuronalfiring of some GPi cells occurred coincident with thegrouping EMG activity.7 In the motor circuit, the GPiprojection to the thalamus is g-aminobutyric acidergicand inhibitory to the thalamocortical excitatory projec-tion. Therefore, decrease in the mean firing rates in theGPi leads to disinhibition of the thalamocortical projec-tion and to an increase in excitability of the frontal mo-tor areas. The synchronous sudden pauses followingbursts of GPi firing are expected to drive ballistic corticalexcitation leading to hemiballism. In dystonia, on theother hand, the pauses or bursts in such a short perioddo not correlate with sustained EMG activities of dysto-

Fig 2. Temporal change in electromyelograms (EMGs) from the leg muscles on the right side and the firing rates of GPe and GPineurons. The sustained EMG discharges from the pretibial muscles and the gastrocnemius-soleus muscles presenting foot dystoniawere recorded during Track 2. The firing rates were low in both GPe and GPi along Track 2 compared with those in nondys-tonic, off-state parkinsonian patients.

244 Annals of Neurology Vol 49 No 2 February 2001

nia. The markedly low firing rates in the GPi may leadto sustained disinhibition of the thalamocortical projec-tion; however, contradictory to this speculation, reducedfiring rates in a pallidal relay nucleus of the thalamuswere reported in primary dystonia.11

As another neuronal change, widened sensory recep-tive fields of single cells or a higher percentage of sen-sory cells in the GPi compared with hemiballism orPD have been reported in patients with primary dys-tonia.7,9 These features may present disruption of recipro-cal organization in the motor circuit leading to dystoniccocontractions. Altered receptive fields in off-perioddystonia should be fully examined in a future study.

The effect of pallidotomy on dystonia is paradoxical inlight of the change in the amount of GPi activity, becausepallidotomy is expected to inactivate the GPi more andto disinhibit the thalamocortical projection more. Aboli-tion of off-period dystonia by pallidotomy suggests thatthe low-rate, irregular firing of GPi neurons, possiblycombined with altered sensory fields, may send patho-logical commands to the thalamocortical projectionsdriving dystonic contractions. Pallidal ablation mayblock the abnormal-patterned signals in the GPi whichcause dystonia and, consequently, allow the frontal mo-tor areas to recover normal execution of motor control.

References1. Fahn S, Marsden CD, Calne DB. Classification and investiga-

tion of dystonia. In: Marsden CD, Fahn S, editors. Movementdisorders. 2. London: Butterworth, 1987:332–358.

2. Yanagisawa N, Goto A. Dystonia musculorum deformans.Analysis with electromyography. J Neurol Sci 1971;13:39–65.

3. Rothwell JC, Obeso JA, Day BL, et al. Pathophysiology of dys-tonias. In: Desmedt JE, editor. Motor control mechanisms inhealth and disease. New York: Raven Press, 1983:851–863.

4. Marsden CD, Parkes JD, Quinn N. Fluctuations of disability ofParkinson’s disease: clinical aspects. In: Marsden CD, Fahn S,editors. Movement disorders. London: Butterworth, 1982:96–122.

5. McHale DM, Sage JI, Sonsalla PK, et al. Complex dystonia ofParkinson’s disease: clinical features and relation to plasma levo-dopa profile. Clin Neuropharmacol 1990;13:164–170.

6. Filion M, Tremblay L. Abnormal spontaneous activity of glo-bus pallidus neurons in monkeys with MPTP-induced parkin-sonism. Brain Res 1991;547:142–151.

7. Vitek JL, Chockkan V, Zhang J-Y, et al. Neuronal activity inthe basal ganglia in patients with generalized dystonia andhemiballism. Ann Neurol 1999;46:22–35.

8. Lozano AM, Kumar R, Gross RE, et al. Globus pallidus inter-nus pallidotomy for generalized dystonia. Mov Disord 1997;12:865–870.

9. Lenz FA, Suarez JI, Verhagen Metman L, et al. Pallidal activityduring dystonia: somatosensory reorganization and changeswith severity. J Neurol Neurosurg Psychiatry 1998;65:767–770.

10. Suarez JI, Verhagen Metman L, Reich SG, et al. Pallidotomyfor hamiballismus: efficacy and characteristics of neuronal activ-ity. Ann Neurol 1997;42:807–811.

11. Lenz FA, Jaeger CJ, Seike MS, et al. Thalamic single neuronactivity in patients with dystonia: dystonia-related activity andsomatic sensory reorganization. J Neurophysiol 1999;82:2372–2392.

Further Evidence thatNeurofilament Light ChainGene Mutations Can CauseCharcot-Marie-ToothDisease Type 2EPeter De Jonghe, MD, PhD,1,2 Irina Mersivanova, BSc,3

Eva Nelis, PhD,1 Jurgen Del Favero, PhD,1

Jean-Jacques Martin, MD, PhD,2

Christine Van Broeckhoven, PhD,1 Oleg Evgrafov, PhD,1,3

and Vincent Timmerman, PhD1

A missense mutation in the neurofilament light chaingene (NEFL, NF-L) at chromosome 8p21 was recentlyreported in a single Charcot-Marie-Tooth type 2 family(CMT2). This new CMT2 variant is designated CMT2E.The NEFL gene mutation showed co-segregation with thedisease phenotype and is thus most likely the disease-causing mutation. However, the possibility that it is aclosely linked rare polymorphism can not be ruled outwith certainty. We observed a novel NEFL missense mu-tation in a second CMT family, providing supporting ev-idence that CMT2E is caused by NEFL gene mutations

Ann Neurol 2001;49:245–249

The most common inherited peripheral neuropathiesare Charcot-Marie-Tooth disease type 1 (CMT1) andtype 2 (CMT2), which are characterized by progressiveweakness and atrophy, initially of the peroneal musclesand later on of the distal muscles of the arms. CMT1is characterized by de- and remyelination and slownerve conduction velocities (NCV). CMT2 is an ex-onal neuropathy characterized by signs of axonal regen-eration in the absence of overt myelin alterations.NCVs are normal or slightly reduced in CMT2. MostCMT families can be classified as either CMT1 orCMT2 using a cut-off value of 38 m/s for the motormedian nerve. However, in some CMT families pa-tients have very variable NCVs ranging from normal toseverely reduced.1,2

From the 1Flanders Interuniversity Institute for Biotechnology(VIB), Born-Bunge Foundation (BBS), University of Antwerp(UIA), and 2Division of Neurology, University Hospital Antwerpen(UZA), Antwerpen, Belgium; and the 3Research Centre for MedicalGenetics, Moscow, Russia.

Received Jun 27, 2000, and in revised form Sep 3 and Sep 22.Accepted for publication Sep 22 2000.

Address correspondence to Dr De Jonghe, Peripheral NeuropathyGroup, Molecular Genetics Laboratory, Department of Biochemis-try, University of Antwerp, Universiteitsplein 1, B-2610 Antwerpen,Belgium. E-mail: [email protected]

© 2001 Wiley-Liss, Inc. 245

Molecular genetic studies have shown that CMT1and CMT2 are heterogeneous. The majority of CMT1patients have a 1.5 Mb tandem duplication in chromo-some 17p11.2-p12 (CMT1A) harboring the peripheralmyelin protein 22 gene (PMP22).3,4 Mutations in thisgene may also result in CMT1. Mutations in the genesencoding myelin protein zero (MPZ/P0) (CMT1B), gap-junction protein connexin 32 (Cx32/GJB1) (CMT1X)and early growth response element 2 (EGR2) result inCMT1 or the related demyelinating neuropathiesDejerine-Sottas syndrome and congenital hypomyelina-tion.5 Recently, mutations in the gene encoding myo-tubularin related protein-2 (MTMR2) have been ob-served in autosomal recessive demyelinating CMTlinked to chromosome 11q22 (CMT4B),6 and muta-tions in the N-myc downstream-regulated gene 1(NDRG1) underlie hereditary motor and sensory neu-ropathy (HMSN)-Lom.7 Molecular genetic studieshave been less productive in CMT2. Mutations inCx32 and MPZ have been observed in a subset ofCMT2 patients.5 Genetic linkage studies have mappedthree CMT2 loci, ie, CMT2A, CMT2B, and CMT2Dat chromosome 1p35-p36, 3q13-q22, and 7p14 respec-tively.8–10 Very recently, a fourth CMT2 locus(CMT2E) was mapped to 8p21 in a single CMT2 ped-igree from Mordovia, Russia. Subsequently a c.998A.Ctransversion mutation resulting in a Gln333Pro in thefirst exon of the neurofilament light gene (NEFL/NF-L)was found to show complete co-segregation with the dis-ease.11,12 We observed another NEFL mutation in a Bel-gian CMT family. These data confirm that mutations inthe NEFL gene are the cause of CMT2E.

Patients and MethodsPatientsWe studied a multigeneration family (CMT-56) in whichCMT segregates as a dominant trait (Fig 1). The proposita,III.5, was first seen at the age of 14 years. She had a steppagegait. Foot extensors were more severely affected than plantarflexor muscles. Hand muscles were slightly weak and atro-phic. Tendon reflexes were absent. Discrete hypoesthesia fortouch, pain, and temperature was present in a glove/stockingdistribution. Clinical examination of family members identi-fied eight additional patients. Patients II.4 and II.13, exam-ined at the age of 56 and 47 years, respectively, showed analmost complete paralysis of distal muscles of the legs and asevere paresis (1–3/5 on the Medical Research Council[MRC] scale) of the intrinsic hand muscles. Examination ofthe youngest generation confirmed a disease onset in the sec-ond decade of life.

Electrophysiological studies in patients II.13, III.5, andIII.9 show severely reduced motor NCVs. The ranges of themotor NCVs are 25 to 39 m/s (n 5 3) for the median and30 to 42 m/s (n 5 2) for the ulnar nerve. Amplitudes ofcompound muscle action potentials (CMAP) in the upperlimbs were severely reduced and ranged from 1.4 to 3.8 mV(normal: higher than 6.0 mV). Sensory nerve action poten-tials were usually absent, and only one NCV measurement of17 m/s for a median nerve was obtained.

Molecular Genetic AnalysisDenaturing high-performance liquid chromatography(DHPLC) was performed on the WAVE automated instru-ment according to the manufacturer’s recommendations(Transgenomics, Santa-Clara, CA). Direct sequencing ofNEFL exon 1 – part 1 was performed on polymerase chainreaction products with primers NEFL1F1 (59-GCACAC-

Fig 1. Pedigree of CMT-56. Symbols: filled 5 affected; open 5 unaffected; square 5 male; circle 5 female; slashed 5 deceased;* 5 DNA sample available; arrow 5 proband.


AGCCATCCATCCTCCC-39)andNEFL1R1(59-GATCCA-GAGCTGGAGGAGTAGC-39)11 and the BigDye TerminatorCycle Sequencing kit with AmpliTaq DNA polymerase FS(ABI PRISM, Applied Biosystems Inc, Foster City, CA).Sequence reactions were run on the ABI automated DNAsequencer 3700 (Applied Biosystems Inc). Data were collectedand analysed using the ABI DNA sequencing analysis soft-ware, version 3.6.

ResultsWe screened 40 unrelated patients diagnosed as CMT2or intermediate CMT for the presence of mutations inthe NEFL gene (Genbank no. NM_006158 for cDNA).Via DHPLC we detected a heteroduplex pattern in PCRfragment NEFL1.1 in the proband of family CMT-56(Fig 2A). DHPLC analysis demonstrated the same het-eroduplex pattern in all patients from family CMT-56but not in unaffected relatives. We sequenced DNAsamples of 3 patients and found a double missense mu-tation at positions 22 and 23 from CC to AG in thefirst exon of NEFL (c.22C.A123C.G) (Fig 2B, nu-cleotide numbering according to the cDNA sequence).This mutation creates an amino-acid change from Proto Arg at codon 8 (P8R). Complete co-segregation ofthe heterozygous mutation with the disease was con-

firmed by DHPLC. This mutation was absent in 160control chromosomes, indicating that this sequencevariation is not a rare polymorphism. Linkage analysiswith the Pro8Arg mutation resulted in a two-point logof the odds (LOD) score of 3.61 in the absence ofrecombinants.

DiscussionNeurofilaments (NFs) form the cytoskeletal compo-nent of the myelinated axon and belong to the mostabundant and widely expressed neuronal intermediatefilament proteins. They are composed of three pro-teins: light (NEFL), mid-sized (NEFM) and heavy(NEFH) chains, encoded by separate genes (reviewedby Julien13). The mouse and human NEFL gene con-tains four coding exons and the 59UTRs are highlyconserved. The NEFL protein contains 543 amino ac-ids with a head, rod and tail domain. The rod domaincontains four coil subdomains separated by three linkermolecules. The tail of the protein has two subdomains:A and B, of which B is acidic.14

Animal models have demonstrated that NFs are in-volved in determining axon diameter. In Japanese quail(Quiverer, quv), a spontaneous recessive mutation in

Fig 2. (A) Denaturing high-performance liquid chromatography analysis of neurofilament light (NEFL)-1 showing a heteroduplexpattern in the proband (III.5) of family CMT-56 (left) and a homoduplex pattern in an unaffected relative (right). The same het-eroduplex pattern at 2.5 minutes retention time was found in all affected individuals and not in their unaffected relatives and 80normal control subjects. (B) DNA sequencing analysis of NEFL1.1 showing the c.22C.A123C.G double mutation in the pro-band (left) and the normal sequence from an unaffected relative (right).

Brief Communication: De Jonghe et al: NEFL Mutation in CMT2E 247

NEFL generates a truncated protein incapable of form-ing NFs.15 Homozygous mutants have no axonal NFsand exhibit mild generalized quivering. Normal radialgrowth of myelinated axons is severely attenuated, re-sulting in a reduction of axonal conduction velocity.Knockout mice lacking axonal NFs, owing to a tar-geted disruption of the NEFL gene, have diminishedaxon caliber and delayed regeneration of myelinatedaxons following crush injury of peripheral nerve.16

When NEFL is lacking, NEFM and NEFH can notform functional 10 nm NFs. Homozygous and het-erozygous NEFL knockouts develop normally, are notlethal, and do not exhibit a clinical phenotype. How-ever, the transgenic mouse mutant NEFL Leu394Prohas massive degeneration of spinal motor neurons withabnormal neurofilament accumulation and severe neu-rogenic atrophy of skeletal muscles. At postnatal day18, this mutant shows an abnormal gait with reducedactivity and weakness of upper and lower limbs.17

These observations suggest that NF abnormalitiesmay contribute to the pathology of human neurode-generative diseases.18 However, so far only alterationsin the NEFH gene had been linked with a human dis-order. A few alterations in the NEFH gene were re-ported in sporadic patients with amyotrophic lateralsclerosis (ALS). Most patients had a deletion in theLys-Ser-Pro repeat region, which is a highly conservedrepetitive region of the NEFH gene.19 Another ALSpatient had a novel 84 bp insertion, leading to an extrafour Lys-Ser-Pro repeats.20

Recently, a novel CMT2 locus (CMT2E) wasmapped to chromosome 8p21 in a single large Russianfamily, obtaining a two-point LOD score of 5.93 witha short tandem repeat marker from the 59UTR regionof the NEFL gene. Mutation analysis of NEFL de-monstrated a c.998A.C mutation at codon 333(Gln333Pro) that showed complete co-segregation withthe disease.11 These findings and the high degree ofconservation of Gln333 between distinct species lendsupport to the hypothesis that the NEFL mutation isindeed the disease causing mutation in this CMT2Efamily.

We identified a dominant double missense mutation(c.22C.A123C.G) at codon 8 in the NEFL gene, re-sulting in a Pro to Arg substitution in a Belgian CMTfamily. This mutation shows perfect co-segregation withthe disease. The Pro8Arg missense mutation most likelydestabilizes the head domain of NEFL. Cumulative ev-idence from the Russian11 and Belgian families providesubstantive proof that NEFL is the CMT2E gene. Bothmutations occur at amino acids that are conserved in allsequenced NEFL genes, ie, from Xenopus to human. Pa-tients in the Belgian family present with a classical, al-though rather severe CMT phenotype with a disease on-set in the second decade of life. The Russian family hadbeen diagnosed as CMT2 based in NCVs ranging from

38 to 52 m/s. In our CMT2E family, however, NCVsare sometimes severely slowed, and patients could beclassified as CMT1 based on NCVs alone. It is impor-tant to note that CMAP amplitudes are always severelyreduced, suggesting that the slowing is, at least partially,due to loss of fast conducting axons. Our data suggestthat patients and families that are diagnosed as CMT1should also be screened for mutations in the NEFL geneonce mutations in the CMT1 genes have been excluded.

This research project was supported by the Fund for ScientificResearch-Flanders (FWO, Belgium), the Geneeskundige StichtingKoningin Elisabeth (GSKE, Belgium), the Association Francaisecontre les Myopathies (AFM, France). Dr Nelis and Dr Timmer-man are postdoctoral fellows of the FWO. Dr Evgrafov is holder ofa research fellowship of the Belgian Office for Scientific, Technicaland Cultural affaires (DWTC).

The authors gratefully acknowledge the cooperation and participa-tion of the family members in this study. We are grateful for theskillful technical assistance of Mrs. E. De Vriendt, Mrs. V. VanGerwen, and Mr. Sam Sluys.

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8. Ben Othmane K, Middleton LT, Loprest LJ, et al. Localizationof a gene (CMT2A) for autosomal dominant Charcot-Marie-Tooth disease type 2 to chromosome 1p and evidence of ge-netic heterogeneity. Genomics 1993;17:370–375.

9. Kwon JM, Elliott JL, Yee WC, et al. Assignment of a secondCharcot-Marie-Tooth type II locus to chromosome 3q. Am JHum Genet 1995;57:853–858.

10. Ionasescu VV, Searby C, Sheffield VC, et al. Autosomal dom-inant Charcot-Marie-Tooth axonal neuropathy mapped onchromosome 7p (CMT2D). Hum Mol Genet 1996;5:1373–1375.

11. Mersiyanova IV, Perepelov AV, Polyakov AV, et al. A new vari-ant of Charcot-Marie-Tooth disease type 2 (CMT2E) is prob-ably the result of a mutation in the neurofilament light gene.Am J Hum Genet 2000;67:37–46.

12. Lupski JR. Axonal Charcot-Marie-Tooth disease and the neu-rofilament light gene (NF-L). Am J Hum Genet 2000;67:8–10.


13. Julien J-P. Neurofilament functions in health and disease. CurrOpin Neurobiol 1999;9:554–560.

14. Julien J-P, Grosveld F, Yazdanbaksh K, et al. The structure of ahuman neurofilament gene (NF-L): a unique exon-intron orga-nization in the intermediate filament gene family. Biochim Bio-phys Acta 1987;909:10–20.

15. Ohara O, Gahara Y, Miyake T, et al. Neurofilament deficiencyin quail caused by nonsense mutation in neurofilament-L gene.J Cell Biol 1993;121:387–395.

16. Zhu Q, Couillard-Despres S, Julien J-P. Delayed maturation ofregenerating myelinated axons in mice lacking neurofilaments.Exp Neurol 1997;148:299–316.

17. Lee MK, Marszalek JR, Cleveland DW. A mutant neurofila-ment subunit causes massive, selective motor neuron death: im-plications for the pathogenesis of human motor neuron disease.Neuron 1994;13:975–988.

18. Julien J-P, Couillard-Despres S, Meier J. Transgenic mice inthe study of ALS: the role of neurofilaments. Brain Pathol1998;8:759–769.

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20. Tomkins J, Usher P, Slade JY, et al. Novel insertion in the KSPregion of the neurofilament heavy gene in amyotrophic lateralsclerosis (ALS). Neuroreport 1998;9:3967–3970.

Absence of EchovirusSequences in Brain andSpinal Cord of AmyotrophicLateral Sclerosis PatientsMichelle Portlance Walker, BS,1 Robert Schlaberg,1

Arthur P. Hays, MD,2 Robert Bowser, PhD,3

and W. Ian Lipkin, MD1

The role of enteroviruses in pathogenesis of amyotrophiclateral sclerosis (ALS) is controversial. A recent study,based on reverse transcription-polymerase chain reaction(RT-PCR) analysis of spinal cord, reported identificationof a novel echovirus in 15 of 17 French subjects withALS and only 1 of 29 subjects with other neurologic dis-eases. We established a real-time RT-PCR method based

on this novel echovirus sequence and used this methodand that previously employed for analysis of the Frenchsubjects to determine the prevalence of echoviral se-quences in spinal cord and motor cortex of sporadic ALSsubjects from the United States. No echoviral sequenceswere found in 20 spinal cord and 10 motor cortex sam-ples from autopsy-confirmed cases of ALS or 13 spinalcord and 5 motor cortex samples from subjects with nomotor neuron disease.

Ann Neurol 2001;49:249–253

Amyotrophic lateral sclerosis (ALS) is a neurodegenera-tive disorder characterized by progressive loss of motorneurons and muscle atrophy. An inherited form causedby mutations in the superoxide dismutase gene hasbeen described1; however, most cases are idiopathic.Proposed mechanisms include abnormalities of neuro-nal metabolism,2 glutamate toxicity,3 neurotoxins,4

neurotrophic factors,5 and neurotropic viruses.6–8

Enteroviral sequences have been reported in cerebro-spinal fluid of patients with ALS by reverse transcrip-tase polymerase chain reaction (RT-PCR)9 and in spi-nal cord samples by RT-PCR,6,7 and in situ RT-PCR.6

Enteroviruses have not been cultivated in samples ob-tained from ALS patients, nor has a specific immuneresponse to enteroviruses been demonstrated. Here wedescribe establishment of a sensitive real-time RT-PCRmethod for detection of echoviral sequences reportedin spinal cord of French ALS patients.6 Application ofthis method and that previously reported to detect echo-viral sequences in ALS spinal cord reveals no evidence ofechoviral infection in spinal cord or motor cortex sam-ples from 20 North American subjects with ALS.

Materials and MethodsPatients and ControlsFive hundred milligram samples of frozen human cervical orlumbar spinal cord and motor cortex from subjects with ALSor other disorders were obtained from postmortem tissuebanks at Columbia University or The University of Pitts-burgh. Details of diagnosis, specimen location, age, gender,postmortem autolysis interval, and race are contained in theTable.

Preparation of SamplesRNA was extracted using TriReagent (Molecular ResearchCenter, Inc., Cincinnati, OH) and quantitated by spectro-photometry.

Sequence Alignments59-untranslated regions of 13 echoviral sequences describedby Berger and colleagues6 were used to probe GenBank forrelated viruses. The search confirmed close similarity toechovirus 7 (Fig A).

From the 1Emerging Diseases Laboratory, Departments of Neurol-ogy, Anatomy and Neurobiology, Microbiology and Molecular Ge-netics, University of California, Irvine, CA; 2Department of Pathol-ogy, Columbia University, New York, NY; and 3Departments ofPathology and Neurobiology, University of Pittsburgh School ofMedicine, Pittsburgh, PA.

Received Jul 7, and in revised form Sep 27. Accepted for publica-tion Sep 27, 2000.

Address correspondence to Dr Lipkin, Emerging Diseases Labora-tory, 3101 Gillespie Neuroscience Building, University of Califor-nia, Irvine, CA 92697–4292. E-mail: [email protected]


Construction of pE71c (Positive Control)RNA was extracted from 1 TCID50/0.2 ml echovirus 7,Wallace strain (ATCC, Manassas, VA) using TriReagent LS(Molecular Research Center, Inc.). RNA was precipitatedwith 1 mg glycogen as carrier and resuspended in 10 mlDEPC water. Reverse transcription was performed using spe-cific primer Echo7-L616 (59-AGCTCTATTAGTCACCG-

GATGG-39) with SuperScript II Reverse Transcriptase(Gibco-BRL, Rockville, MD), followed by PCR with prim-ers Echo7-U17 (59-GTCAGCACCCTGGTATCACG-39)and Echo7-L616. The 600-nucleotide product was TAcloned using Promega (Madison, WI) pGEM-T Easy VectorSystem I to create pE71c. Fidelity was confirmed by auto-mated dideoxy sequencing (ABI Prism 377 DNA Sequencer;

Table. Summary of Clinical Diagnosis, Case History, and Results

Diagnosis SpecimenAge(years) Gender

PMI(hr) Race

Real-Time PCR(1.5 mg/5 mg)

RT-PCRa

(2 mg)

ALS CSC 65 M NA W Neg/neg NegALS CSC 56 M NA B Neg/neg NegALS CSC 64 F NA W Neg/neg NegALS CSC 65 M NA W Neg/neg NegALS CSC 66 M NA A Neg/neg NegALS CSC 51 M NA W Neg/neg NegALS CSC 52 F NA A Neg/neg NegALS CSC 66 F NA W Neg/neg NegALS/dementia CSC 63 F NA W Neg/neg NegALS LSC 40 M 6 W Neg/neg NegALS LSC 51 M 4 W Neg/neg NegALS LSC 68 M 4 W Neg/neg NegALS LSC 51 F 7 W Neg/neg NegALS LSC 43 M 6 W Neg/neg NegALS LSC 71 M 8 W Neg/neg NegALS LSC 44 M 3.5 W Neg/neg NegALS LSC 71 F 6 W Neg/neg NegALS LSC 70 F 3 W Neg/neg NegALS/Alzheimer’s disease LSC 75 M NA W Neg/neg NegALS LSC 49 M 10 W Neg/neg NegALS MC 51 F 7 W Neg/neg NegALS MC 43 M 6 W Neg/neg NegALS MC 71 M 8 W Neg/neg NegALS MC 44 M 3.5 W Neg/neg NegALS MC 71 F 6 W Neg/neg NegALS MC 70 F 3 W Neg/neg NegALS MC 49 M 10 W Neg/neg NegALS MC 40 M 6 W Neg/neg NegALS MC 51 M 4 W Neg/neg NegALS MC 68 M 4 W Neg/neg NegAdult acid maltase deficiency LSC 53 M NA B Neg/neg NegAlzheimer’s disease CSC 80 M NA W Neg/neg NegCardiac failure LSC 54 M 6 W Neg/neg NegCardiac failure MC 54 M 6 W Neg/neg NegCardiac failure MC 59 F 6 W Neg/neg NegCIDP LSC 70 F NA W Neg/neg NegCritical illness myopathy CSC 72 M NA W Neg/neg NegEnd-stage liver disease/sepsis MC 65 M 5 W Neg/neg NegEnd-stage renal disease/sepsis MC 50 M 7 W Neg/neg Negb

Hepatic failure, coma LSC 31 F NA W Neg/neg NegHistoplasma meningitis LSC 59 M NA W Neg/neg NegHIV CSC 34 M NA B Neg/neg NegMetastatic breast cancer CSC 39 F NA W Neg/neg NegMultiple system atrophy LSC 66 F NA B Neg/neg NegPulmonary thromboembolus LSC 53 F 4 W Neg/neg NegPulmonary thromboembolus MC 53 F 4 W Neg/neg NegSpinal muscular atrophy CSC 26 F NA W Neg/neg NegStiffman syndrome CSC 66 F NA W Neg/neg Neg

PMI 5 postmortem interval; RT-PCR 5 reverse transcriptase polymerase chain reaction; ALS 5 amyotrophic lateral sclerosis; CSC 5 cervicalspinal cord; LSC 5 lumbar spinal cord; MC 5 motor cortex; M 5 male; F 5 female; W 5 white; B 5 black; CIDP 5 chronic inflammatorydemyelinating polyneuropathy; HIV 5 human immunodeficiency virus; NA 5 not applicable.


Perkin Elmer, Norwalk, CT) on both strands using primersT7 and Sp6 (Fig B).

Establishing a Real-Time PCR Assay for Detection ofEchoviral SequencesPrimers for real-time PCR were designed using the programPrimer Express 1.0 (Perkin Elmer) and sequence alignmentsgiven in Figure A. The specificity of the primers pEforward(59-CAGTGTAGATCAGGTCGATGAGTCA-39), pEreverse(59-TCCTAACTGCGGAGCAGACAC-39), and pEprobe (59-6FAM-TCCGGCCCCTGAATGCGGC-TAMRA-39) was ini-tially assessed in real-time PCR assays (Taqman 7700; PerkinElmer) using Ava II linearized plasmid pE71c. RNA templatesfor calibration of real-time RT-PCR assays were obtained bySp6-primed in vitro transcription of linearized pE71c.

Real-Time PCR Analysis of Human Spinal Cord andMotor Cortex RNATotal RNA from spinal cord or motor cortex (1.5 or 5 mg)was reverse transcribed using Multiscribe RT (Perkin Elmer)and random primers (Perkin Elmer). The resulting cDNAwas subjected to real-time PCR analysis using primerspEforward, pEreverse, and pEprobe. RNA integrity andrandom-primed reverse transcription of clinical samples was

assessed using a GAPDH control primer and JOE (2,7,-dimethoxy-4,5-dichloro-6-carboxyfluorescein)-labeled probeset (Perkin Elmer).

RT-PCR Analysis Using the Method of Berger andColleagues6

Assays were established using synthetic RNA templates ob-tained by Sp6 in vitro transcription of pE71c. Thereafter,analysis of total RNA from spinal cord or motor cortex sam-ples (2 mg) was pursued. Reverse transcription with avianmyeloblastosis virus reverse transcriptase (Promega) wasprimed using oligo-3 (59-ATTGTCACCATAAGCGCCA,nt 584–603)6 for 60 minutes at 42°C. The resulting cDNAwas amplified in two rounds of 30 PCR cycles using primersoligo-2 (59-CAAGCACTTCTGTTTCCCCGG, nt 164–184)6

and oligo-3. PCR conditions were initial denaturation 95°C for5 minutes, annealing at 50°C for 45 seconds, elongation at72°C for 1 minute, and denaturation at 94°C for 30 sec-onds.6,10 Ten microliters of the resulting amplification prod-ucts were size fractionated on 1.5% agarose gels and stainedwith 0.003% ethidium bromide. Amplification productswere isolated from agarose using gel extraction columns(Qiagen, Chatsworth, CA) and sequenced. Resulting se-quences were used to probe databanks for similarity to other

Fig. (A) Sequence alignment for reagents used in real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Thereal-time PCR primers and probe are aligned with echovirus 7 and echoviral sequences proposed for amyotrophic lateral sclerosis(ALS). Positions correspond to proposed ALS echoviral consensus sequence.6 ECHO7, echovirus 7, Wallace strain; 1–9, 11, 13, 15–17, individual echoviral sequences.6 Asterisks represent a deletion in the sequence; base substitutions as listed. (B) pE71c plasmidmap with relative primer positions: a, Echo7-U17; b, pEforward; c, pEprobe; d, oligo-2; e, pEreverse; f, oligo-3; g, Echo7-L616.

Brief Communication: Walker et al: ALS and Echovirus Analysis 251

known sequences using BLAST-NT (NCBI Search). All real-time RT-PCR and RT-PCR assays were performed by inves-tigators blind to diagnosis of clinical materials.

ResultsSensitivities of the real-time RT-PCR method estab-lished for this study and RT-PCR as described by Bergeret al.6 were assessed by limiting dilution analysis of syn-thetic pE71c RNA transcripts. Whereas real-time PCRreproducibly detected between 101 and 102 copies ofpE71c RNA, the threshold for detection by RT-PCRwas 105 copies of pE71c RNA. Amplification productsobtained in assays of pE71c RNA were appropriate insize: real-time RT-PCR, 190 nt; and RT-PCR, 450 nt.

Total RNA extracted from clinical samples was as-sessed for integrity and suitability as template for real-time RT-PCR via comparison to a commercial totalRNA standard (human brain RNA; Research Genetics)using a GAPDH primer set (Perkin Elmer). JOE flu-orescent signal was detected at between 16 and 24 cy-cles with clinical samples vs. 20 cycles with the com-mercial standard. Amplification products were notdetected in real-time RT-PCR assays of clinical samplesusing echoviral primers and either 1.5 mg or 5 mg oftotal RNA (Table). RT-PCR assays of all human sam-ples using oligo-2 and oligo-3 resulted in amplificationproducts of 350 nt rather than the anticipated size(450 nt). One clone from each of 4 different subjects(spinal cord from 3 subjects with ALS and 1 subjectwith multiple system atrophy) was selected for se-quencing. All corresponded to the 59-terminus of a hu-man mitochondrial RNA (GenBank Accession No.NC001807.2) that demonstrates no homology withechoviral sequences. The amplification presumably re-sulted from the use of oligo-3 in both reverse transcrip-tion and two sets of 30 cycle PCR.6 Primer oligo-3bound (capital letters are homologous: 59-caccTaAttgg-AAGCGCCA, nt 9,040–9,058) in the sense orienta-tion and (59-cTcGTgttacatcGCGCCA, nt 9,378–9,396)in antisense orientation. One additional subject, who diedof end-stage liver disease and sepsis, yielded three addi-tional amplification products that represented sequencesderived from Escherichia coli (GenBank Accession No.D90741). Again, homology to echoviruses was not ap-parent. Amplification likely resulted from false primingas follows: sense (59-gAtgGgtggCTGTTTCCCtG, nt3,617–3,636) with antisense (59-TGGCGCTTggGcTGAtAT, nt 3,944–3,962), sense (59-gcaccgCccaAtG-CGCCA, nt 152,086–152,105) with antisense (59-tgcgtAggTggcTTCCtCGG, nt 152,316–152,337), andsense (59-tgaGcCggttTttGCGCCA, nt 2,447–2,465) withantisense (59- caTcctgCtccAgtgGCCA, nt 3,023–3,041).

DiscussionAlthough enteroviruses are clearly established in thepathogenesis of acute motor neuron disease in poliomy-

elitis, efforts to link them to chronic neurologic disor-ders have been less successful. Brahic and colleagues 11

reported in situ hybridization analysis of human spinalcord using probes to poliovirus and Theiler’s virus. Onesubject with ALS and 1 control hybridized; however, 14subjects with ALS, 2 with Guamanian parkinsonian de-mentia, and 5 controls were negative. Woodall and col-leagues7 found enteroviral 59 untranslated region se-quences in RT-PCR analysis of paraffin-embeddedspinal cord sections from 8 of 11 cases of sporadic mo-tor neuron disease, 1 of 2 cases of familial motor neurondisease, and 0 of 6 normal controls. In contrast, Swan-son and colleagues12 were unable to amplify enteroviral59-untranslated region sequences by nested RT-PCR instudies of paraffin-embedded spinal cord from 28 sub-jects with ALS. Recently, a novel enterovirus (echovirus)sequence was identified by RT-PCR analysis of paraffin-embedded spinal cord samples from 13 of 17 Frenchsubjects with ALS and 1 of 29 subjects with other neu-rologic diseases.6 We established a real-time RT-PCR as-say based on this novel echoviral sequence and employedthis assay as well as the RT-PCR method responsible fordetection of the novel echovirus to fresh frozen samplesof spinal cord and motor cortex from 20 subjects withALS and 14 controls. The control group included pa-tients with other neurodegenerative diseases, Alzheimer’sdisease, HIV, and spinal muscular atrophy. Real-timeRT-PCR was three orders of magnitude more sensitivethan RT-PCR for detection of synthetic transcripts.Echoviral sequences were not detected in clinical mate-rials despite integrity of total cellular RNA as measuredwith a GAPDH standard.

Our data do not support the conclusion that echo-viral infection is associated with ALS; however, it isconceivable that differences in results of our study andthat of Berger and colleagues6 reflect differences in ei-ther the sequence or the prevalence of enteroviruses inALS patients in France and the United States. Studies arein progress to address this possibility via domain-specificdifferential display using degenerate enteroviral primers.13

This work was supported by grant NS29425 from the National In-stitutes of Health (M.P.W., R.S., W.I.L.), a research grant from theNational ALS Association (R.B.), the Mario Lemieux Foundation(University of Pittsburgh ALS Tissue Bank), and a center grant toDrs. DiMauro and Rowland (NS11766) from the National Insti-tutes of Health (A.P.H).

We thank Thomas Briese for assistance in designing the real-timePCR assays.

References1. Orrell RW. Amyotrophic lateral sclerosis: copper/zinc superox-

ide dismutase (SOD1) gene mutations. Neuromusc Disord2000;10:63–68.

2. Silani V, Ciammola A, Pizzuti A, Cardin V, Scarlato G. Motorneurone metabolism. J Neurol Sci 1999;169:161–169.


3. Plaitakis A, Constantakakis E, Smith J. The neuroexcitotoxicamino acids glutamate and aspartate are altered in the spinalcord and brain in amyotrophic lateral sclerosis. Ann Neurol1988;3:446–449.

4. Shaw PJ, Ince PG. Glutamate, excitotoxicity and amyotrophiclateral sclerosis. J Neurol 1997;244:3–14.

5. Grundstrom E, Askmark H, Lindeberg J, Nygren I, Ebendal T,Aquilonius SM. Increased expression of glial cell line-derivedneurotrophic factor mRNA in muscle biopsies from patientswith amyotrophic lateral sclerosis. J Neurol Sci 1999;162:169–173.

6. Berger MM, Kopp N, Vital C, Redl B, Aymard M, Lina B.Detection and cellular localization of enterovirus RNA se-quences in spinal cord of patients with ALS. Neurology 2000;54:20–25.

7. Woodall CJ, Riding MH, Graham DI, Clements GB. Se-quences specific for enterovirus detected in spinal cord frompatients with motor neurone disease. Br Med J 1994;308:1541–1543.

8. Brahic M, Smith RA, Gibbs CJ Jr, Garruto RM, TourtellotteWW, Cash E. Detection of picornavirus sequences in nervoustissue of amyotrophic lateral sclerosis and control patients. AnnNeurol 1985;18:337–343.

9. Leparc-Goffart I, Julien J, Fuchs F, Janatova I, Aymard M, Ko-pecka H. Evidence of presence of poliovirus genomic sequencesin cerebrospinal fluid from patients with post-polio syndrome.J Clin Microbiol 1996;34:2023–2026.

10. Leparc I, Aymard M, Fuchs F. Acute, chronic and persistententerovirus and poliovirus infections: detection of viral genomeby seminested PCR amplification in culture-negative samples.Mol Cell Probes 1994;8:487–495.

11. Brahic M, Smith RA, Gibbs CJ Jr, Garruto RM, TourtellotteWW, Cash E. Detection of picornavirus sequences in nervoustissue of amyotrophic lateral sclerosis and control patients. AnnNeurol 1985;18:337–343.

12. Swanson NR, Fox SA, Mastaglia FL. Search for persistent in-fection with poliovirus or other enteroviruses in amyotrophiclateral sclerosis-motor neurone disease. Neuromusc Disord1995;5:457–465.

13. Briese T, Jia XY, Huang C, Grady LJ, Lipkin WI. Identifica-tion of a Kunjin/West Nile-like flavivirus in brains of patientswith New York encephalitis [letter]. Lancet 1999;354:1261–1262.

Anti-Yo Antibodies andCerebellar Degeneration in aMan with Adenocarcinomaof the EsophagusIan J. Sutton, MRCP,1

Christopher J. Fursdon Davis, FRCP,2

Margaret M. Esiri, FRCPath,2 Sharon Hughes, PhD,1

Elisabeth R. Amyes, MSc,2 and Angela Vincent, FRCPath2

Serum antibodies to the Yo antigen are usually associatedwith paraneoplastic cerebellar degeneration arising in fe-male patients with gynecological or breast malignancyand are rarely associated with other tumors. We report amale patient who presented with paraneoplastic cerebel-lar degeneration and anti-Yo antibodies following re-moval of an esophageal adenocarcinoma. This is only thethird report of anti-Yo antibodies occurring in a malepatient. The Yo antigen was expressed by the esophagealtumor but not in a frontal lobe cerebral metastasis iden-tified at postmortem. Interestingly, CD81 T-cell infiltra-tion was also found in the tumor, but not in the metas-tasis, consistent with down-regulation of Yo expressionby the tumor cells leading to evasion from immune-mediated tumor surveillance.

Ann Neurol 2001;49:253–257

A 55-year-old male smoker of 20 cigarettes per daypresented in April 1994, with a 6-month history of in-termittent dysphagia for solids. Endoscopy revealed thepresence of an esophageal tumor arising in an area ofBarretts’ esophagitis, and the patient proceeded to un-dergo an Ivor Lewis esophagogastrectomy. Histopatho-logical examination demonstrated a moderately differ-entiated adenocarcinoma infiltrating through themuscularis propria into the underlying serosa and sur-rounding tissue. There was considerable submucosalspread into the cardia, and a single metastasis was iden-tified in one of eight sampled lymph nodes. Ninemonths later, the patient noted that while driving hehad problems steering his vehicle, resulting in a ten-dency to swerve back and forth across the road. Over aperiod of several weeks, he developed further symp-

From the 1Department of Neurology, Queen Elizabeth Hospital,Birmingham, and the 2Department of Clinical Neurology, Univer-sity of Oxford, Oxford, United Kingdom.

Received Jan 21, 2000, and in revised form Sep 27. Accepted forpublication Sep 29, 2000.

Address correspondence to Dr Sutton, Department of Neurology,Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH,United Kingdom. E-mail: [email protected]


toms of progressive incoordination manifesting as dif-ficulty in writing, slurring of speech, and deterioratingbalance. There was no family history of cerebellar dys-function or excessive alcohol intake.

Neurological examination revealed truncal and gaitataxia in the absence of Rombergism. He had scanningspeech and horizontal nystagmus on lateral gaze.Smooth pursuit and saccadic eye movements were nor-mal. He had dysmetria of all four limbs in the absenceof weakness and sensory loss. His upper limb reflexeswere reduced, lower limb reflexes were normal, andboth plantar responses were flexor. Physical examina-tion was otherwise unremarkable.

Routine haematology and biochemistry were normal.Cranial MRI revealed no cerebellar metastasis, atrophy,or signal change. The cerebrospinal fluid (CSF) con-tained 2 lymphocytes/mm3, 0.77 g/liter protein, and3.7 mmol/liter glucose. CSF cytology was negative, butoligoclonal bands were present in serum and CSF. Im-munohistochemical analysis identified high titers of aserum antibody reactive with a Purkinje cell cytoplas-mic antigen, which was confirmed as anti-Yo antibod-ies by Western blot (see below).

The patient’s cerebellar symptoms continued to de-teriorate despite daily treatment with 20 mg pred-nisolone, two courses of plasma exchange (five 2-literexchanges on each occasion) in May and June, 1995,and a course of intravenous immunoglobulin (0.4 g/kgfor 5 days) in June, 1995. The patient received radio-therapy for a right humeral metastasis diagnosed inNovember, 1995. A postmortem examination in June,1996, revealed an additional right frontal lobe cerebralmetastasis 1 cm in diameter. This lesion was poorlydifferentiated, with no duct formation. Histopathologicexamination of the cerebellum revealed complete ab-sence of Purkinje cells but no cerebellar metastasis,meningeal tumor infiltration, or inflammation.

Materials and MethodsPlasma samples were available from plasma exchanges per-formed in May 1995. Tumor tissue was obtained from par-affin blocks from surgical and postmortem specimens. Im-munohistochemistry and Western blotting of sera against themajor Yo antigen (plasmid kindly provided by Dr. J. Dal-mau, University for Medical Sciences, Arkansas) was per-formed as previously described1 with slight modification(Amyes et al., in press). Controls included sera from normalindividuals and individuals with other autoimmune or neu-rological disorders.

For immunoperoxidase labelling of tumor tissue for ex-pression of Yo antigen, IgG was purified from the patient’splasma and from control pooled plasmas and biotinylated us-ing previously described methods (Binding Site, UnitedKingdom).2 Sections of paraffin-embedded tissue from nor-mal human cerebellum, the esophagogastrectomy, and fron-tal lobe cerebral metastasis specimens were deparaffinized,immersed in 0.01 M citric acid (pH 6.0), and microwaved

for 10 minutes according to the method of Cattoretti et al.3

Following cooling for 30 minutes, tissue sections were incu-bated with 10% normal heat-inactivated goat serum inphosphate-buffered saline for 20 minutes prior to incubationfor 1 hour with serial dilutions between 1 mg/ml and 100

Fig 1. (A) Photomicrograph of patient plasma diluted to1:3,200, reacted with frozen rat cerebellum, and visualizedwith streptavidin-biotin complex and 3-amino-9-ethyl-carbazole(AEC). There is labeling of Purkinje cell (PC) cytoplasm char-acteristic of anti-Yo antibody response. G 5 granular layer,M 5 molecular layer. 3400 before 52% reduction. (B) West-ern blot of recombinant Yo and HuD proteins resolved on 12%SDS-PAGE gel. Antibody reactivity is visualized using a second-ary biotinylated anti-human IgG, streptavidin-biotin complex,and diaminobenzidine (DAB). Lane a: Recombinant Yo 1anti-Yo antibody-positive plasma. Lane b: Recombinant Yo 1patient plasma. Lane c: Recombinant Yo 1 healthy controlserum. Lane d: Recombinant HuD 1 anti-Hu antibody-positive plasma. Lane e: Recombinant HuD 1 patient plasma.Lane f: Recombinant HuD 1 healthy control serum.


mg/ml of biotinylated IgG from the patient. Sections werethen labelled with streptavidin-biotin-peroxidase complexand diaminobenzidine. Biotinylated pooled human IgG wasused as a control. To confirm that reactivity of the tumor wasdue to expression of Yo antigen, a competition assay was per-formed in which sections of tumor were preincubated withserum (1:25) from another anti-Yo antibody-positive patientor a normal individual. The inflammatory infiltrate within thetumor sections was examined using immunohistochemicalmarkers for B cells (CD20) and T cells (CD3 and CD8).

ResultsThe patient’s plasma produced immunohistochemicalstaining of Purkinje cell cytoplasm (titrating to1:6,400; Fig 1A) and reacted in Western blots with the62 kDa Yo fusion protein (Fig 1B) and with a 62 kDaband in neuronal extracts (data not shown). There wasno reactivity with the Hu antigen (see Fig 1B). Biotin-ylated IgG from the patient, at 10mg/ml, reacted withboth Purkinje cells (not shown) and tumor cells (Fig2A). Staining was demonstrated as a general increase inreaction product, particularly evident in the cytoplasmof the columnar epithelium. Reactivity of biotinylatedpatient IgG with the tumor was not uniform, andthere were areas of tumor where Yo expression couldnot be detected. Biotinylated pooled human IgG at thesame concentration did not react with either tumor(Fig 2B) or cerebellum. Specificity of staining for theYo antigen was confirmed by a competitive binding as-say; the binding of biotinylated patient IgG was re-duced when sections were preincubated with serumfrom another patient with anti-Yo antibodies but notwith serum from a normal individual (data not shown).

Neither biotinylated patient nor control IgG reactedagainst the frontal lobe cerebral metastasis (data notshown). An extensive CD81 lymphocytic infiltrate waspresent in the esophageal tumor (Fig 3), but, by con-trast, no CD81 lymphocytes were identified within thecerebral metastasis.

DiscussionWe describe a case of cerebellar ataxia with anti-Yo an-tibodies in a man with esophageal adenocarcinoma andshow that the Yo antigen is expressed in his tumor,although it was not found in a more dedifferentiatedcerebral metastasis. Interestingly, CD81 T cells werealso found in his tumor but not in the metastasis, ap-parently correlating with Yo expression.

Anti-Yo antibodies are normally associated withbreast or gynecological cancers. For instance, among 55female patients with anti-Yo antibody-associated para-neoplastic cerebellar degeneration, malignancy wasidentified in 53.4 Thirty-three cases had an ovarian orother gynecological malignancy, 13 had breast cancer,1 patient had adenocarcinoma of the lung, and 6 caseshad an adenocarcinoma of unknown primary origin.The present case is remarkable in that the patient wasa male with an esophageal tumor. There are only twoother reported males with anti-Yo antibody-associatedparaneopalstic cerebellar degeneration; one patient hadan adenocarcinoma of the parotid gland,5 and the otherhad an adenocarcinoma of unknown primary origin.6 Inthe latter case, examination of tumor sections also con-firmed aberrant expression of the Yo antigen.6 Our pa-

Fig 2. Photomicrograph of esopha-geal tumor sections reacted withbiotinylated patient IgG (A) andcontrol biotinylated IgG (B), visu-alized with streptavidin-biotincomplex and diaminobenzidine(DAB), and counterstained withhematoxylin. Biotinylated patientIgG binds to a cytoplasmic antigenwithin tumor cells and specificityfor the Yo antigen was confirmedby competition experiments; bindingof biotinylated patient IgG wasblocked by preincubation of thetumor with anti-Yo serum fromanother patient but not with serumfrom a normal individual (datanot shown). 3400.

Brief Communication: Sutton et al: Anti-Yo Antibodies and Cerebellar Degeneration 255

tient was a smoker, but antibodies to the Hu antigenassociated with small cell lung cancer were not detected.

The major Yo antigen is a 62 kDa protein with aleucine-zinc DNA binding motif, and expression ofthis antigen is usually confined to the Purkinje cellsand other immune-privileged sites such as the testis.1

However, the Yo antigen is frequently expressed inovarian and breast cancers,7 and Furneaux et al.2 havedemonstrated Yo expression in 10 breast/gynecologicaltumors associated with cerebellar degeneration andanti-Yo antibodies. Aberrant expression of the Yo anti-gen by the tumor appears to be critical in the breakingof immune tolerance, which leads to subsequentimmune-mediated cerebellar dysfunction. Furthermore,it is proposed that there is also immune-mediated re-tardation of tumor growth in patients with paraneo-plastic cerebellar degeneration,8 and the Yo antigen is apossible target for immune recognition of the tumor.Indeed, Albert et al9 identified Yo-specific cytotoxic Tlymphocytes, which can kill tumor cells in vitro, inthree patients with paraneoplastic cerebellar degenera-tion and high titres of anti-Yo antibodies. The markedCD81 T-cell infiltrate and Yo expression within theesophageal tumor, but not in the frontal lobe cerebralmetastasis in this patient, supports the hypothesis thatthere is a cytotoxic T-cell response against the Yo an-tigen, and that down-regulation of Yo expression bytumor cells could lead to evasion from immune-mediated tumor surveillance and subsequent metastasis.

This is the first reported case of paraneoplastic anti-

gen expression in a gastroesophageal tumor and thethird male patient with an adenocarcinoma in whomanti-Yo antibodies have been identified. It is impor-tant, therefore, to appreciate that anti-Yo antibodiescan occur in men, and any male serum staining thePurkinje cell cytoplasm should be tested by Westernblot to exclude anti-Yo antibodies. When this is posi-tive for anti-Yo antibodies, a wide search for an ade-nocarcinoma should be undertaken.

I.J.S. is funded by a Sheldon Research Fellowship and The MidlandNeurosciences Teaching and Research Fund.

We are grateful to John Ridley, Dave Ambler, and Roger Drew-(Binding Site, United Kingdom) and to Dr. Roskell, Department ofPathology, Oxford, for postmortem data and material.

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regulatory mechanism restricts expression of the paraneoplasticcerebellar degeneration antigen cdr2 to immune privileged tis-sues. J Neurosci 1997;17:1406–1415.

2. Furneaux HM, Rosenblum MK, Dalmau J, et al. Selective ex-pression of Purkinje-cell antigens in tumor tissue from patientswith paraneoplastic cerebellar degeneration. N Engl J Med 1990;322:1844–1851.

3. Cattoretti G, Pileri S, Parravicini C, et al. Antigen unmasking onformalin-fixed, paraffin-embedded tissue sections. J Pathol 1993;171:83–98.

4. Peterson K, Rosenblum MK, Kotanides H, Posner JB. Paraneo-plastic cerebellar degeneration. I. A clinical analysis of 55 anti-Yoantibody positive patients. Neurology 1992;42:1931–1937.

5. Felician O, Renard JL, Vega F, et al. Paraneoplastic cerebellar

Fig 3. Photomicrograph of esophageal tumor. Invasive tumor cells (single and tubular formations) within the serosa beneath themuscularis propria surrounded by extensive aggregates of lymphocytes, the majority of which are CD81 on immunohistochemicalstaining. H and E; 3200.


degeneration with anti-Yo antibody in a man. Neurology 1995;45:1226–1227.

6. Krakauer J, Balmaceda C, Torres Gluck J, et al. Anti-Yo-associatedparaneoplastic cerebellar degeneration in a man with adenocarci-noma of unknown origin. Neurology 1996;46:1486–1487.

7. Darnell JC, Albert ML, Darnell RB. Cdr2, a target antigen ofnaturally occurring human tumor immunity, is widely expressedin gynecological tumors. Cancer Res 2000;60:2136–2139.

8. Posner JB. Paraneoplastic syndromes. Curr Opin Neurol 1997;10:471–476.

9. Albert ML, Darnell JC, Bender A, et al. Tumor-specific killercells in paraneoplastic cerebellar degeneration. Nat Med 1998;4:1321–1324.

14-3-3 Protein CerebrospinalFluid Detection in HumanGrowth Hormone–TreatedCreutzfeldt-Jakob DiseasePatientsJean-Philippe Brandel, MD,1,2 Katell Peoc’h, PharmD,3

Patrice Beaudry, MD,3 Arlette Welaratne,1

Corinne Bottos,3 Yves Agid, MD, PhD,1 andJean-Louis Laplanche, PharmD, PhD3

The usefulness of the detection of 14-3-3 protein in thecerebrospinal fluid (CSF) in the diagnosis of Creutzfeldt-Jakob disease transmitted from human growth hormonewas evaluated in 20 French patients. The 14-3-3 proteinwas rarely detectable within the first 3 months of the dis-ease but always positive after 7 months associated withthe aggravation of the disease and the occurrence of de-mentia. 14-3-3 detection was not predictive of the sur-vival time of the patients. The genotype at PRNP codon129 could influence the timing of appearance of the 14-3-3 protein in the CSF.

Ann Neurol 2001;49:257–260

Creutzfeldt-Jakob disease (CJD) is the most frequent ofthe fatal human prion diseases which can present assporadic, genetic, or iatrogenic disorders. The sporadicform of CJD represents more than 80% of all cases. Inmost patients rapidly progressive dementia with neuro-logical dysfunction (myoclonus; cerebellar, visual, pyra-midal and/or extra-pyramidal signs; and akinetic mut-ism) is observed as the main feature.1

In the iatrogenic forms of CJD, two different routesof inoculation have been described. Clinical manifesta-tions following cross-contamination through neurosur-gical instruments, corneal transplants, or dura matergrafts (central route) are similar to those recorded insporadic CJD.2–4 In patients treated with humangrowth hormone (h-GH) (peripheral route), the initialsigns are ataxia, tremor or myoclonus, and oculomotordisorders, with dementia occurring at a later stage.5

Besides these clinical manifestations, the recording ofperiodic discharges on electroencephalogram (EEG) isa useful criterion for the diagnosis of sporadic or iat-rogenic CJD acquired by central route, but it is almostalways absent in patients with h-GH iatrogenic CJD.5,6

An evaluation of biochemical markers for CJD in liv-ing patients has recently led to the proposal that the14-3-3 protein detection in the cerebrospinal fluid(CSF) is a sensitive and specific marker for the diag-nosis of sporadic CJD.7–9 14-3-3 is an abundant pro-tein present in the cells with seven isoforms and acts asa regulatory molecule for several proteins of the cellcycle or apoptosis.10 Its increase in the CSF is thoughtto be due to nerve cell death.

The aim of this study was to evaluate the usefulnessof the 14-3-3 protein detection in the CSF of h-GH–treated CJD patients. This evaluation took into ac-count the time between the first recorded clinical signsand the timing of CSF sampling. Two consecutiveCSF samples were studied when available; data werethen analyzed taking into account the time betweenboth samples and the occurrence of clinical manifesta-tions, particularly dementia. The influence of the ge-notype at PRNP codon 129 on the duration of thedisease and 14-3-3 protein detection was also studied.

Patients and MethodsPatientsTwenty French patients (16 men, 4 women) were includedin the study. The patients were reported to the “Centre Na-tional de Reference de la Maladie de Creutzfeldt-Jakob Iat-rogene” by the physician who suspected the diagnosis of iat-rogenic CJD. All patients had been treated with h-GHduring the high-risk period of contamination in France. Atleast one CSF sample was available for the detection of 14-3-3 protein. For all patients the onset of the disease occurredfrom June 1996 to November 1998. All presented a neuro-logical dysfunction highly suggestive of the diagnosis of iat-rogenic CJD. These manifestations, including dementia,were recorded by the physician who followed the patient andthen analyzed by a qualified neurologist (JPB) who classifiedthe patients according to the criteria used in France (Table1), which are different from those used for sporadic CJD.11

The time of the second CSF sampling in case of an initialnegative 14-3-3 result was decided by the physician.

From the 1Centre National de Reference de la MCJ iatrogene, Hopitalde la Salpetriere; 2INSERM U. 360, Hopital de la Salpetriere; and3Centre de Recherche C. Bernard, IFR 6, Service de Biochimie et Bi-ologie Moleculaire (Pr J-M Launay), Hopital Lariboisiere, Paris, France.

Received Aug 4, 2000, and in revised form Oct 3. Accepted forpublication Oct 3, 2000.

Address correspondence to Dr Brandel, INSERM U. 360, Hopital de laSalpetriere, 47, Boulevard de l’Hopital 75651 Paris Cedex 13, France.


MethodsAfter lumbar puncture, CSF samples were centrifuged andthe supernatant was frozen at 230°C until assessed. 14-3-3proteins were evidenced by Western blotting.7 Briefly, pro-teins from 40 ml of CSF were separated on 12% sodiumdodecyl sulfate–polyacrylamide gel, then electrotransferred toImmobilon-P membrane (Millipore, Bedford, MA). 14-3-3proteins were then revealed by incubating the membranewith a polyclonal antibody at a dilution of 1:750 (sc-629;Santa Cruz Biotech., CA) cross-reacting with all 14-3-3 iso-forms, and chemoluminescent detection (Pierce, Rockford,IL). A positive result showed a band at 30 kD. Molecularweight standards and control CSF from a known CJD casewere run on each gel. After informed consent, genomicDNA was extracted from peripheral blood leukocytes andthe sequence of the PRNP coding region was obtained afterdirect cycle sequencing by the dideoxy chain-terminationmethod (Epicentre Technologies, Madison, WI) and an au-tomated laser fluorescent (ALFexpress) sequencer (AmershamPharmacia Biotech, Uppsala, Sweden).12

ResultsOf 20 patients, 6 were classified as definite, 13 as prob-able, and 1 as possible CJD. The genotype at PRNPcodon 129 was known for 19 of the 20 patients: twovaline/valine (V/V), 11 methionine/methionine (M/M),and six methionine/valine (M/V). This distribution isnot different from that observed in all iatrogenic CJDpatients already studied in France.

Eleven of the 20 patients (55%) had initial positive14-3-3 detection. In those cases, the mean time be-tween the first recorded sign and the CSF samplingwas significantly longer (6 months, range 3 to 12) thanin negative cases (3 months, range 1 to 5) (p , 0.001,Wilcoxon test). The percentage of positive 14-3-3 pro-

tein was 20% in the first quarter, 55% in the second,and 100% later (Fig 1). Cerebellar ataxia was recordedin all patients, whereas oculomotor disorders, tremor,or myoclonus were frequently observed at the time ofsampling. A periodic EEG was recorded in a single pa-tient 14 months after onset. Among the 11 patientswith a positive 14-3-3 CSF detection, dementia waspresent in nine patients (dementia was not clearly men-tioned as present or absent in the remaining two pa-tients). No dementia was recorded in patients withnegative 14-3-3 protein in their CSF.

A second CSF sample was obtained from seven ofthe nine patients having an initial negative 14-3-3 pro-tein detection. The protein became detectable in fourpatients, 6.5 to 9 months after the first clinical signs(Fig 2). All became demented between the two CSFsamplings. The 3 other patients sampled 4.5, 5.5, and6.5 months after onset of the disease remained negativefor 14-3-3. No dementia occurred in these 3 patients.With the second CSF sample, the percentage of positive14-3-3 CSF detection increased from 55% to 75%.

Although based on limited observations, the dura-tion of the disease was significantly longer for heterozy-gous (M/V) patients at PRNP codon 129 (19.8months, range 15 to 28) than for homozygous (M/Mand V/V) cases (12 months, range 7 to 17) (p , 0.001Wilcoxon test). The genotype at codon 129 seemed toinfluence the 14-3-3 protein detection period. All 6M/V patients had a positive CSF 14-3-3 protein detec-tion in the initial course of the disease, whereas all ex-cept one 129 M/M patients were negative (see Fig 2).The 14-3-3 protein was detected long before death inheterozygous patients at PRNP codon 129 (M/V): 7.5,9, 12, 15, and 22 months for the deceased patients and17 months for the living patient. The range was 4 to 9months for homozygous patients (M/M or V/V).

DiscussionAs EEG is not informative in h-GH iatrogenic CJD,the aim of this study was to estimate the diagnosticusefulness of the detection of 14-3-3 protein in theCSF within this specific group of CJD. So far, Western

Table 1. French Diagnostic Criteria in h-GH–Treated CJDPatients

Definite CJDNeuropathological confirmation and/or immunocyto-chemically confirmed PrPSc-positive/Western blot and/orscrapie-associated fibrils (SAF)

Probable CJD1. Treatment with human growth hormone before 19882. No recurrence of the initial cause for treatment3. Severe and progressive aggravation during at least 3

months4. Intellectual impairment with at least three of the fol-

lowing symptoms: cerebellar syndrome, pyramidalsyndrome, rigidity, myoclonus (and/or seizure), oculo-motor disorders, abnormal EEG, abnormal electroreti-nogram (ERG), normal MRI and CSF examinations

Possible CJD1. Treatment with human growth hormone before 19882. Outbreak, after a free interval, of at least one of the

following neurological disorders: cerebellar syndrome,pyramidal syndrome, rigidity, myoclonus (and/or sei-zure), oculomotor disorders, abnormal EEG, abnor-mal electroretinogram (ERG)

Fig 1. Percentage of positive 14-3-3 according to time betweenfirst manifestations and first CSF sampling.


blot is the widely used procedure to detect the 14-3-3protein in patients suspected of CJD.7–9 This tech-nique gave us results consistent with the final diagnosisin 87.6% of neuropathologically proven sporadic CJD(n 5 145) and eliminated the diagnosis of CJD in96% of other neurologic disorders (n 5 687). Theseresults are in good agreement with those already pub-lished.7,8

We observed that in patients with h-GH iatrogenicCJD, the 14-3-3 protein was rarely detectable in theCSF within the first 3 months of the disease but alwayspositive after 7 months. The genotype at PRNP codon129 controls the duration of the disease and its incu-bation time.13 It also seems to influence the timing ofappearance of the 14-3-3 protein in the CSF. As to theclinical course of the disease, the 14-3-3 protein wasnot predictive of the survival time of the patients afterits detection. However, the detection of 14-3-3 proteinin the CSF was often associated with the occurrence ofintellectual impairment, suggesting that the appearance

of the protein in the CSF could reflect diffuse braindamage. In some patients, mainly with the PRNP 129M/V genotype, the 14-3-3 protein was detected severalmonths before death, indicating that brain damage oc-curred relatively early in the long-duration forms of thedisease.

A low rate of 14-3-3 protein detection has been re-ported in new variant CJD (vCJD)14 which sharescommon symptoms with h-GH iatrogenic CJD, nota-bly late-onset dementia. This late dementia could ex-plain the false-negative 14-3-3 results observed invCJD patients.

Our results should be taken into account in the diag-nosis and follow-up of h-GH–treated patients. Becauseof its late detection, the 14-3-3 protein could appear lesshelpful in the diagnosis of CJD in h-GH–treated pa-tients than in sporadic CJD. It should be stressed, how-ever, that in the absence of periodic EEG, the presenceof the 14-3-3 protein in the CSF is the sole indicatorassociated with the clinical signs that strongly supports

Fig 2. 14-3-3 protein detection according to time between first manifestations and CSF sampling, and to the genotype at codon 129.

Brief Communication: Brandel et al: 14-3-3 Protein CSF Detection in CJD Patients 259

the diagnosis of CJD. Serial testing of CSF for at least 7months after the onset of neurological signs in h-GHrecipients may be helpful in diagnosis. The detection ofthe 14-3-3 protein can be considered, along with theneurological manifestations, as a criterion for probableor possible h-GH iatrogenic CJD.

These results could be of importance when testingtreatments, when they become available, in two groupsof patients: h-GH–treated CJD and possibly vCJD pa-tients. Late 14-3-3 protein detection in the group oftreated patients could argue in favor of the efficacy of atherapy.

The authors thank all physicians who referred patients to the “Cen-tre National de Reference de la Maladie de Creutzfeldt-Jakob Iatro-gene” and provided clinical data and CSF samples for the 14-3-3protein detection.

References1. Brown P, Cathala F, Sadowsky D, et al. Creutzfeldt-Jakob dis-

ease in France: II. Clinical characteristics of 124 consecutiveverified cases during the decade 1968–1977. Ann Neurol 1979;6:430–437.

2. Duffy P, Wolf J, Collins G, et al. Possible person-to-persontransmission of Creutzfeldt-Jakob disease. N Engl J Med 1974;290:692–693.

3. Heckmann JG, Lang CJG, Petruch F, et al. Transmission ofCreutzfeldt-Jakob disease via a corneal transplant. J NeurolNeurosurg Psychiatry 1997;63:388–390.

4. Lang CJ, Heckmann JG, Neundorfer B. Creutzfeldt-Jakob dis-ease via dural and corneal transplants. J Neurol Sci 1998;160:128–139.

5. Billette de Villemeur T, Deslys JP, Pradel A, et al. Creutzfeldt-Jakob disease from contaminated growth hormone extracts inFrance. Neurology 1996;47:691–695.

6. Brown P, Preece MA, Will RG. “Friendly fire” in medicine:hormones, homografts, and Creutzfeldt-Jakob disease. Lancet1992;340:24–27.

7. Hsich G, Kenney K, Gibbs C, et al. The 14-3-3 brain proteinin CSF as a marker for transmissible spongiform encephalopa-thies. N Engl J Med 1996;335:924–930.

8. Zerr I, Bodemer M, Gefeller O, et al. Detection of 14-3-3 pro-tein in the cerebrospinal fluid supports the diagnosis ofCreutzfeldt-Jakob disease. Ann Neurol 1998;43:32–40.

9. Beaudry P, Cohen P, Brandel JP, et al. 14-3-3 protein, neuron-specific enolase, and S-100 protein in cerebrospinal fluid of pa-tients with Creutzfeldt-Jakob disease. Dement Geriatr CognDisord 1999;10:40–46.

10. Fu H, Subramanian RR, Masters SC. 14-3-3 proteins: struc-ture, function, and regulation. Annu Rev Pharmacol Toxicol2000;40:617–647.

11. Brandel JP, Delasnerie-Laupretre N, Laplanche JL, et al. Diag-nosis of Creutzfeldt-Jakob disease: effect of clinical criteria onincidence estimates. Neurology 2000;54:1095–1099.

12. Peoc’h K, Manivet P, Beaudry P, et al. Identification of threenovel mutations (E196K, V203I, E211Q) in the prion proteingene (PRNP) in inherited prion diseases with Creutzfeldt-Jakobdisease phenotype. Hum Mutat, 2000;15:482. [Mutation inBrief no. 323, Online: http://journals.wiley.com/1059/7794/pdf/mutation/323.pdf]

13. Huillard d’Aignaux J, Costagliola D, Maccario J, et al. Incuba-

tion period of Creutzfeldt-Jakob disease in human growth hor-mone recipients in France. Neurology 1999;53:1197–1201.

14. Zeidler M, Stewart GE, Barraclough CR, et al. New variantCreutzfeldt-Jakob disease: neurological features and diagnostictests. Lancet 1997;350:903–907.

Propofol in SubanestheticDoses Terminates StatusEpilepticus in a RodentModelMartin Holtkamp, MD,1 Xin Tong, MD,2

and Matthew C. Walker, PhD2

Status epilepticus is commonly refractory to first-linetherapy, and thus better treatments are needed. We haveinvestigated an experimental model of drug-resistant self-sustaining status epilepticus (SSSE) induced by 2 hoursof perforant path stimulation. Propofol in subanestheticdoses administered shortly after the end of stimulationand also after a further 3 hours of SSSE terminated theSSSE without recurrence. This finding calls for a trial ofpropofol in refractory status epilepticus and also raisesthe possibility of using it as first-line therapy.

Ann Neurol 2001;49:260–263

Status epilepticus (SE) is a life-threatening conditionwith an incidence of 25 to 50 per 100,0001 personyears that is terminated in only 50 to 60% of cases byfirst-line antiepileptic treatment.2 Patients with SE re-sistant to first-line therapy usually require general an-esthesia for termination of SE.3 Barbiturates are wellestablished in the treatment of refractory SE, but therehave also been reports of the success of nonbarbiturateanesthetics, such as propofol,4,5 and indeed in onestudy this drug was used for prehospital treatment.6

Propofol has advantageous pharmacokinetics: it has arapid onset of action (2–4 minutes), an eliminationhalf-life of 30 to 60 minutes, and no propensity to ac-

From the 1Klinik fuer Neurologie, Charite, Humboldt-Universitaet,Berlin, Germany; and 2Epilepsy Research Group, University De-partment of Clinical Neurology, Institute of Neurology, London,United Kingdom.

Received Apr 4, 2000, and in revised form Oct 9. Accepted forpublication Oct 10, 2000.

Address correspondence to Dr Walker, Epilepsy Research Group,University Department of Clinical Neurology, Institute of Neurol-ogy, Queen Square, London WC1N 3 BG, United Kingdom.E-mail: [email protected]

260 © 2001 Wiley-Liss, Inc.

cumulate.4 These characteristics make it an ideal drug,not only for prolonged perfusions as an anesthetic inSE, but also as a possible first-line therapy. There have,however, been few studies evaluating the antiepilepticproperties of propofol; concerns about possible procon-vulsant properties are probably unfounded.7 Propofolhas been shown to interrupt SE in an animal model ofgeneralized seizures induced by pentylenetetrazol, butin that model a prolonged infusion had to be given.8

Here we show that subanesthetic boluses of propofolare effective at early and late stages in a drug-resistantrodent model of SE.

MethodsMale Sprague-Dawley rats (300–350 gm; Charles River,Margate, UK) were individually housed under a 12-hourlight-dark cycle with free access to water and to a normallaboratory diet (SDS R and M No. 1 Expanded, ScientificDietary Services, Witham, UK). All animal proceduresstrictly followed Home Office regulations under the Animal(Scientific Procedures) Act of 1986.

The animals were fully anesthetized with 1 to 2% halo-thane in O2. A subcutaneous silver wire was used as the earthelectrode. A 250-mm monopolar recording electrode was im-planted stereotaxically into the hippocampus (coordinates 2.5mm lateral, 4 mm caudal from bregma). Potentials were am-plified and filtered (0.1–500 Hz band pass) via a Neurologamplifier (Digitimer, Welwyn Garden City, UK) onto a stor-age oscilloscope and then via an analogue to digital interfaceonto a computer running Labview (National Instruments,Berkshire, UK; sampling rate 1 kHz). Two twisted 125-mmteflon-coated stainless steel wires were implanted in the righthemisphere and advanced into the angular bundle (coordi-nates 4.4 mm lateral, 8.1 mm caudal from bregma) to stim-ulate the perforant path. Constant current stimuli were de-livered from a Neurolog stimulator (Digitimer). The depthsof the electrodes were adjusted to maximize the slope of thefield potential as previously described.9 The electrodes wereheld in place with dental acrylic and three skull screws. Theanimals were allowed to recover fully from anesthesia.

Seven to 10 days later, in freely moving animals the per-forant path was stimulated with 2 to 3 mA, 50- to 150-msecmonopolar pulses at 20 Hz for 2 hours to induce self-sustaining status epilepticus (SSSE).9 During and, in someanimals, after stimulation, clinical seizures up to stage 4 (clas-sification of Racine)10 occurred. Approximately 1 hour afterthe beginning of stimulation, regular, large-amplitude dis-charges occurred in over 90% of the animals; the experimentwas terminated in animals in which these did not occur.

Phenytoin (50 mg/kg intraperitoneally [IP]; Epanutinready mixed parenteral, Parke-Davis, Hants, UK), propofol(20 or 50 mg/kg IP; Rapinovet, Schering-Plough, Herts,UK), pentobarbital (30 mg/kg IP; Sagatal, Rhone-Poulenc,UK), or diazepam (5 mg/kg IP; Diazemuls, Dumex, Herts)were administered 2 minutes after the end of the stimulusprotocol or after a further 3 hours of SSSE. If SSSE contin-ued for more than 3 hours after drug injection, it was ter-minated with an anesthetic dose of pentobarbital (50–60mg/kg IP).

Epileptic activity was defined electrographically as regulardischarges occurring at a frequency of greater than or equalto 1 Hz. The sedation level of all animals was determined at3, 5, 10, 30, 60, 120, and 180 minutes after drug adminis-tration using an established scale11 (see Fig 2).

ResultsIn all animals completing 2 hours of stimulation, dis-charges continued at a frequency of greater than orequal to 1 Hz after stimulation stopped. We adminis-tered phenytoin (50 mg/kg IP) 2 minutes after the endof stimulation in 5 animals. In all of these animals, theSSSE continued for at least a further 3 hours, confirm-ing the refractory nature of this model. In 5 other an-imals we administered diazepam (5 mg/kg IP) 2 min-utes after the end of stimulation. This terminated SSSEin 2 animals; in the other 3 the discharges continued,albeit at a reduced amplitude (Fig 1a). In a separategroup of 5 animals, administration of propofol (50mg/kg IP) 2 minutes after the end of stimulation rap-idly terminated the SSSE (see Fig 1a). By 3 hours after

Fig 1. Propofol stops self-sustaining status epilepticus givenearly or late. (a) Electrographic recordings from hippocampusof rats in which self-sustaining status epilepticus was treatedwith diazepam 5 mg/kg intraperitoneally (IP) or propofol 50mg/kg IP or remained untreated. (b) Electrographic recordingfrom the hippocampus of 1 rat (the same rat as untreated ina) in which self-sustaining status epilepticus was terminated 3hours after the end of stimulation by propofol 50 mg/kg IP.Note the occurrence of low-frequency discharges by 90 minutes.Each tracing shows 10 seconds of the electroencephalogramrecorded at the time points shown.

Brief Communication: Holtkamp et al: Propofol in Status Epilepticus 261

propofol injection, there was no recrudescence of theepileptic activity (see Fig 1a). We tried a lower, non-sedating dose of propofol (20 mg/kg IP; n 5 4) 2 min-utes after the end of stimulation, but it was ineffective.

Propofol (50 mg/kg IP; n 5 5) or pentobarbital (30mg/kg IP; n 5 3) given 3 hours after the end of stim-ulation stopped SSSE in all animals. Diazepam (5mg/kg IP) at the same stage stopped SSSE in 3 out of4 animals. In all the animals successfully treated withdiazepam and in 4 out of 5 animals treated withpropofol after 3 hours of SSSE, the termination ofSSSE was followed by the emergence of regular dis-charges (1 to 2 every 10 seconds) 90 to 120 minuteslater without clinical accompaniment (Fig 1b). Similardischarges occurred in 2 animals treated with pentobar-bital, 1 of them presenting intermittently with motorseizures. One animal with higher-frequency dischargesduring SSSE that was treated with pentobarbital had arecurrence of SSSE 3 hours after drug injection. Allanimals treated with pentobarbital lost their auditorystartle reflex. Propofol (50 mg/kg) administration re-sulted in minimal sedation; auditory startle reflexeswere maintained in 9 out of 10 animals, and respira-tory function and corneal reflexes were not impaired(Fig 2). The early sedation related to propofol (50 mg/kg) given after 3 hours of SSSE was similar to that ofpropofol (50 mg/kg) given at 2 minutes after the endof stimulation, but the sedation was longer lasting (seeFig 2).

Overall, the sedative effects of propofol (50 mg/kg)were similar to those of diazepam (5 mg/kg). Peak se-dation was not significantly different for propofol anddiazepam (median peak sedation 2.5 for both, Mann-Whitney U test; n 5 19; p . 0.2). Despite this,propofol (50 mg/kg IP) was significantly more effective

than diazepam (5 mg/kg IP). All 10 animals treatedwith propofol (50 mg/kg) had their SSSE stopped,whereas diazepam was only effective in 5 out of 9 an-imals (Fisher’s exact test; p , 0.05).

DiscussionWe have shown that propofol (50 mg/kg IP) termi-nated SSSE in all animals in our model, whether givenearly or late. Diazepam given at a dose that resulted inequivalent sedation terminated SE in only 56% of theanimals. Our data demonstrate the strong antiepilepticproperties of propofol after a single bolus in subanes-thetic doses in a model of SSSE.

Phenytoin at a dose that stopped SE in a similarmodel after 10 minutes of SE activity12 had no effectin our model, demonstrating its refractory nature. Evengiven after 3 hours of SSSE, propofol terminated SEactivity in all 5 animals, although in 4 animals infre-quent, large-amplitude discharges occurred approxi-mately 2 hours after injection. Due to the low fre-quency, these discharges cannot be regarded as SEactivity. The longer-lasting sedative effect of propofoladministered after 3 hours of SSSE compared to im-mediate injection could be due to either an extendedaction of propofol or a prolonged postictal state. Diaz-epam administered after 3 hours of SSSE stopped ac-tivity in 3 out of 4 animals; they also exhibited largeamplitude discharges 2 hours later. Thus, there was noloss of potency of diazepam in our model over time.These findings contrast with those of an SE model in-duced by IP injection of pilocarpine.13,14 The loss ofpotency of diazepam in stimulation models of SE maybe an early phenomenon occurring during stimula-tion,12 explaining why we found no further loss of po-tency from 2 minutes after stimulation to 3 hours afterstimulation. In addition, diazepam has been found tohave varying potencies in various models of SE.15

Pentobarbital was less effective than propofol after 3hours of SSSE, and in 1 of 3 animals SE activity re-curred by 3 hours. Furthermore, the peak sedationlevel after injection of pentobarbital was higher thanthat after injection of propofol.

The therapeutic limitations of benzodiazepines, phe-nytoin, and phenobarbital as first-line drugs to termi-nate SE have been shown in clinical trials2 as well as inanimal models of SE.12 We have not only shown thatpropofol is effective in refractory SE but that it is aneffective first-line therapy at subanesthetic doses. Oneseries has been reported of the successful use of sub-anesthetic doses of propofol in small animals with sei-zures following portosystemic shunts.16 These resultsrequire confirmation in other animal models and even-tually clinical studies, but they do imply that propofolcould be an effective alternative to common first-linedrugs.

Fig 2. Time course of sedation level following administrationof propofol (50 mg/kg intraperitoneally [IP]) given 2 minutesafter the end of stimulation or propofol (50 mg/kg IP) givenafter 3 hours of SSSE.


This study was supported by the Wellcome Trust (M.C.W.).

We thank D.M. Kullmann and K. Chandler for their helpful com-ments.

References1. DeLorenzo RJ, Hauser WA, Towne AR, et al. A prospective,

population-based epidemiologic study of status epilepticus inRichmond, Virginia. Neurology 1996;46:1029–1035.

2. Treiman DM, Meyers PD, Walton NY, et al. A comparison offour treatments for generalized convulsive status epilepticus.N Engl J Med 1998;339:792–798.

3. Walker MC, Smith SJM, Shorvon SD. The intensive care treat-ment of convulsive status epilepticus in the UK. Anaesthesia1995;50:130–135.

4. Shorvon SD. Status epilepticus: its clinical features and treat-ment in children and adults. Cambridge, UK: Cambridge Uni-versity Press, 1994.

5. Stecker MM, Kramer TH, Raps EC, et al. Treatment of refrac-tory status epilepticus with propofol: clinical and pharmacoki-netic findings. Epilepsia 1998;39:18–26.

6. Kuisma M, Roine RO. Propofol in prehospital treatment ofconvulsive status epilepticus. Epilepsia 1995;36:1241–1243.

7. Sneyd JR. Propofol and epilepsy. Br J Anaesth 1999;82:168–169.

8. De Riu PL, Petruzzi V, Testa C, et al. Propofol anticonvulsantactivity in experimental epileptic status. Br J Anaesth 1992;69:177–181.

9. Walker MC, Perry H, Scaravilli F, et al. Halothane as neuro-protectant during constant stimulation of the perforant path.Epilepsia 1999;40:359–364.

10. Racine RJ. Modification of seizure activity by electricalstimulation: II. motor seizure. Electroenceph Clin Neurophysiol1972;32:281–294.

11. Lee VC, Moscicki JC, DiFazio CA. Propofol sedation producesdose-dependent suppression of lidocaine-induced seizures inrats. Anesth Analg 1998;86:652–657.

12. Mazarati AM, Baldwin RA, Sankar R, Wasterlain CG. Time-dependent decrease in the effectiveness of antiepileptic drugsduring the course of self-sustaining status epilepticus. Brain Res1998;814:179–185.

13. Kapur J, Macdonald RL. Rapid seizure-induced reduction ofbenzodiazepine and Zn21 sensitivity of hippocampal dentategranule cell GABAA receptors. J Neurosci 1997;17:7532–7540.

14. Walton NY, Treiman DM. Response of status epilepticus in-duced by lithium and pilocarpine to treatment with diazepam.Exp Neurol 1988;101:267–275.

15. Peterson CJ, Vinayak S, Pazos A, Gale K. A rodent model offocally evoked self-sustaining status epilepticus. Eur J Pharma-col 1992;221:151–155.

16. Heldmann E, Holt DE, Brockman DJ, et al. Use of propofol tomanage seizure activity after surgical treatment of portosystemicshunts. J Small Anim Prac 1999;40:590–594.

Familial Atypical ProgressiveSupranuclear PalsyAssociated withHomozigosity for thedelN296 Mutation in theTau GenePau Pastor, MD,1 Eliana Pastor, MD,2

Cristobal Carnero, MD,3 Rosario Vela, MD,2

Teresa Garcıa, MD, PhD,2 Guillem Amer, MD, PhD,4

Eduardo Tolosa, MD, PhD,1 and Rafael Oliva, MD, PhD5

Heterozygous missense and splice-site mutations in thetau gene have been previously identified in familial fron-totemporal dementia with autosomal dominant inheri-tance. Here we report a Spanish kindred in which twobrothers born from a third-degree consanguineous mar-riage were both affected with atypical progressive su-pranuclear palsy. A homozygous deletion at codon 296(delN296) was identified in one of the affected siblings.Among the heterozygous carriers, two members withprobable Parkinson’s disease were identified, but none ofheterozygotes developed atypical parkinsonism. ThedelN296 mutation lies in the sequence corresponding tothe second tubulin-binding repeat of tau protein and af-fects one asparagine residue absolutely conserved in otherspecies. This finding indicates that homozygous muta-tions in the tau gene may also cause hereditary tauopa-thies.

Ann Neurol 2001;49:263–267

Tau protein is the major component of fibrillar lesionsdescribed in several neurodegenerative disorders re-ferred to as tauopathies.1 “Frontotemporal dementiawith parkinsonism linked to chromosome 17,” a hered-itary form of tauopathy, is due to heterozygous muta-tions in the tau gene.2,3 In familial multisystemtauopathy with presenile dementia, an heterozygous

From the 1 Parkinson’s Disease and Movement Disorders Unit,Neurology Service, Hospital Clınic, Institut d’Investigacions Bio-mediques August Pi i Sunyer (IDIBAPS), Barcelona; 2NeurologyService, Hospital Universitario Virgen de las Nieves, Granada;3Neurology Service, Hospital Torrecardenas, Almerıa; 4NeurologyService, Hospital Son Dureta, Palma de Mallorca; and 5GeneticsService, Hospital Clınic, and Human Genome Research Group,Faculty of Medicine, University of Barcelona, IDIBAPS, Barcelona,Spain.

Received Jun 14, 2000, and in revised form Oct 9. Accepted forpublication Oct 10, 2000.

Address correspondence to Dr Oliva, Genetics Service, HospitalClınic Universitari, Villarroel 170, 08036 Barcelona, Spain.E-mail: [email protected]


splice-donor site mutation of tau gene has also beenidentified, leading to a clinical phenotype and braintau pathologic features reminiscent of progressive su-pranuclear palsy (PSP) and corticobasal degeneration(CBD).4,5 Moreover, missense heterozygous tau muta-tions segregating with autosomal dominant PSP havebeen recently described.6,7 However, no homozygouspathogenic mutations in the tau gene have so far beenreported. We initiated the present work in search ofpotential mutations in the tau gene in a Spanish familywith two members affected by atypical PSP. Figure 1shows the family pedigree.

Subjects and MethodsThe individuals who were genetically tested were formallyexamined by one of the authors (E.P., C.C., G.A., R.V., andP.P.). Generations I, II, III, and IV and the individuals notexamined were ascertained by interviews of family members.

Patient VI:25Patient VI:25 is the index case, who at age 38 years devel-oped forgetfulness, word-finding problems, and slowness. Hewas a smoker and abused cocaine occasionally. He was se-verely dysarthric, with monotonous speech and explosivelaughing and crying. His gaze was spontaneously fixed, andhe had facial dystonia. Ocular movements were severely slowin all directions without limitations. He exhibited imitatoryapraxia with his left hand. Glabellar, palmomental, and bi-lateral grasp reflexes were present. Rigidity and bradykinesiawere seen and were worse in the right hand. Sensory exam-ination revealed intact primary sensory modalities. His gaitwas unsteady, with impairment of postural reflexes. He ab-ducted his right arm from the trunk with dystonic posture inhis right foot. His clinical status is progressively worsening.

Patient VI:23Patient VI:23 is the brother of the index patient and devel-oped altered behavior and parkinsonism at age 39, with ep-isodes of aggressivity, inappropriate laughing, and progressiveclumsiness of his left limbs. He had a history of cocaine use.His facial expression was one of astonishment, with de-creased blink rate, blepharospasm, and marked slowness ofocular saccades. Severe bradyphrenia and bilateral imitatoryapraxia and constructive apraxia were seen. Glabellar reflexwas present. Bilateral limb rigidity was noted, as was severebradykinesia, both worse in the left extremities. Astereognosisin his left hand and impaired positional sensibility in his leftlimbs were observed. His left hand remained lay immobile.His gait was slightly unsteady, with decreased left arm swingand dystonic posture of left leg. The patient was treated withlevodopa, but he continued to worsen. Severe cognitive de-cline and aggressive behavior were added. He was wheel-chair bound and dying from a respiratory infection 3 yearsafter the onset. Magnetic resonance imaging of the 2 broth-ers (VI:23 and VI:25) revealed mild, diffuse cerebral atrophy.

Patient V:8At age of 71, Patient V:8 developed unilateral rest tremor,slowness, and short-steps gait. Rest tremor, moderate asym-metrical rigidity, and bradykinesia were present in the rightlimbs. Treatment with levodopa improved neurologicalsymptoms.

Patient V:9At age 62, Patient V:9 experienced the onset of rest tremorin his right leg, and his left leg was affected 6 months later.He had arterial hypertension and hypercholesterolemia.Later, he presented with an acute left hemiparesis because ofa lacunar infarct, after which he improved moderately. Pro-gressively his slowness worsened, and he developed a freezing

Fig 1. Pedigree of the family in which 2 siblings have developed atypical progressive supranuclear palsy (PSP), one of them beingdelN296 homozygous. An arrowhead indicates the proband of this family. Individuals known to be affected by atypical PSP syn-drome and by typical Parkinson’s disease are indicated by black and gray symbols, respectively. The roman numerals to the left ofthe pedigree denote generations. The numbers below the member symbols denote actual age, age of onset, or age of death. The geno-typic status of the proband’s brothers younger than 34 years is not indicated. cc 5 homozygous for the common allele; mc 5 het-erozygous for the delN296 mutant allele; mm 5 homozygous for the delN296 mutant allele; slash 5 deceased; square 5 male;circle 5 female; diamond 5 sex not declared.


gait. He was treated with anticholinergics because of intoler-ance to levodopa, and his tremor responded slightly. Mentalstatus and ocular movements remained normal in the 2brothers (V:8 and V:9) diagnosed with probable Parkinson’sdisease (PD).

Polymerase Chain Reaction Amplification andMutation DetectionExons of tau gene were amplified by using specific primersderived from the 59 and 39 intronic sequences and the poly-merase chain reaction (PCR) conditions previously described3

and analyzed through single strand conformation polymor-phism (SSCP) (94°C for 5 minutes, 0°C for 1 minute,8–12% polyacrylamide gel at 300–500 V at 4°C for 4–22hours). After electrophoresis, the gel was silver-stained as de-scribed.8 When an abnormal pattern was identified, sequenceanalysis of the PCR products was performed on an auto-mated DNA sequencer (ABI 310; Perkin Elmer/Applied Bio-systems, Foster City, CA).

ResultsSSCP analysis of exon 10 of tau revealed the presenceof a band with an abnormal mobility in Patient VI:25and in some siblings. Additional abnormal bands ob-served after SSCP analysis of the other exons corre-sponded to polymorphisms previously described.9 TheSSCP pattern of exon 10 in Patient VI:25 was differentfrom that seen in other relatives (not shown). Directsequencing analysis identified a trinucleotide (AAT)homozygous deletion at codon 296 (Fig 2A) in PatientVI:25 and a heterozygous (AAT) deletion in both par-ents and in some siblings (see Fig 2A). In addition, wefound that the delN296 mutation creates a new restric-tion site for Eco321 (MBI Fermentas) and generatesone or two bands, depending on whether one or twomutated alleles are present in the siblings (Fig 2B). Tofurther verify that the delN296 exchange represented amutation and not a polymorphism, we analyzed 100control chromosomes from the Spanish population, inwhich the delN296 mutation was absent.

DiscussionThe two affected brothers (VI:23 and VI:25) wereborn from a third-degree consanguineous marriage andbegan in the fourth decade a neurodegenerative disor-

Fig 2. (A) Identification of exon 10 of tau gene mutations infamilial atypical progressive supranuclear palsyPSP syndrome.Sequence chromograms of sense strand for normal control (cc),delN296 heterozygous mutation (mc), and delN296 homozy-gous mutation from the proband (mm). The deleted codonAAT is underlined in the normal control subject and in theheterozygous mutated allele. The nucleotide sequence and thecorresponding amino acid sequence are indicated. (B) Themutation delN296 creates a new Eco 321 restriction site. Thefragment sizes (in base pairs) corresponding to the molecularweight marker (1-kb ladder) are shown in the left lane of theagarose gel. The mutant product containing homozygousdelN296 (mm) generates two 101– and 96–bp fragmentsafter endonuclease digestion that are superimposed in the gel.When the delN296 mutation is heterozygous (mc), an addi-tional fragment of 200 bp appears, corresponding to a normalallele, whereas the nonmutated PCR products (cc) were notdigested, and only one fragment appears (200 bp).

Fig 3. Protein similarity of the second microtubule-bindingrepeat (2R) of the tau protein with the others repeats of thehuman tau protein and with the 2R of distinct microtubule-associated binding proteins (MAPs) in humans and anotherspecies. The shaded regions indicate areas of homology. Theasterisk indicates codon 296.

Brief Communication: Pastor et al: Homozygous Tau Mutation in Atypical PSP 265

der characterized by mild cognitive and memory dis-turbances, frontal lobe signs, oculomotor dysfunction,asymmetrical akinetic-rigid syndrome, and limb dysto-nia. The main clinical characteristic of the two brotherswas the rapidly progressive asymmetrical parkinsonism.Because of the early presence of prominent oculomotordisturbances and gait disequilibrium, the phenotype ofthe two brothers resembled primarily PSP. They alsoexhibited cortical signs, such as apraxia, sensory corticalsigns (Patient VI:23), and language difficulties (PatientVI:25), frequently seen in CBD. The process rapidlyprogressed to death in Patient VI:23. Patient VI:25 washomozygous for the delN296 mutation.

In addition to the atypical PSP patient (VI:25) withthe homozygous mutation, 2 uncles (V:8 and V:9) ofthe patient who developed typical PD (onset at 62 and70) were heterozygous (see Fig 1). These data do notexclude a co-dominant model manifesting in the formof typical PD in heterozygous individuals carryingdelN296 mutation, which would also be more consis-tent with the mechanism of tau mutations in this re-gion of the gene. Thus, it could be possible that thistau mutation gives rise to both parkinsonian pheno-types. In the heterozygous state the mutation wouldhave a reduced penetrance, because many individualsshow no PD phenotype even in advanced ages, andwould give rise to a relatively mild, late-onset, PD-likecondition. In contrast, in the homozygous state thephenotype would be more severe (atypical PSP), withearly onset and complete penetrance, and the conditionis not responsive to levodopa. Indeed, reduced pen-etrance for tau mutations has already been reported.3

Thus, the delN296 mutation in a heterozygous statemay also be a genetic risk factor for developing PD.

The absolute conservation of Asn-296 among theother repeats of human tau10 and the second repeat ofvarious microtubule-associated binding proteins (MAPs)from various species and the absence of the delN296mutation in 100 chromosomes analyzed are findingsthat do not support the suggestion that the delN296mutation may be a rare polymorphism (Fig 3).

A very recent study of mutagenesis identified cis-acting sequences in exon 10 of tau where 9 nucleotidedeletions or combinations of various point mutationsreduced the affinity for a splicing silencer, increasingtau isoforms containing exon 10.11 The delN296 mu-tation reported here lies exactly in this region andtherefore may also increase exon 10 inclusion. The iso-forms containing exon 10 bind microtubules at sitesdifferent from those bound by third-repeat tau.12 Anoverproduction of specific isoforms of tau could resultin a shortage of available microtubule binding sites.13

A disturbance of this balance can result in an excess offree toxic tau susceptible to hyperphosphorylation andassembly into filaments or aggregates seen in familialtauopathies. Overproduction of fourth-repeat isoforms

caused by the delN296 mutation in a heterozygousstate may be insufficient to result in neurodegenerationin most of the individuals, but when two copies thedelN296 mutation are present, it may be sufficient toresult in neurodegeneration. Another possibility is thatthe delN296 mutation affects microtubule binding.

It is interesting to note that the brothers affected byatypical PSP reported here closely resembled some ofthe patients described with heterozygous N279K mu-tation, where early atypical parkinsonism and oculomo-tor disturbances beginning in the fifth decade are thepredominant phenotype.6,14 Familial autosomal reces-sive cases of PSP 15 and CBD16,17 have been described,but there are no reports describing the search for mu-tations in tau in these families. In addition to the ho-mozygous mutation reported here, other cases havebeen reported in which a pathogenic heterozygous anda homozygous mutation of a single gene caused thesame disease.18–19 The identification of a homozygousdeletion in our family indicates that dysfunction of twocopies of the tau protein may also be responsible forsome cases of familial parkinsonism. In addition, thepresent study emphasizes the importance of analyzingadditional members of the families carrying a mutationof the tau gene, particularly in the kindreds with a pos-sible autosomal recessive inheritance.

This work was supported by the “50 Aniversario de la SociedadEspanola de Neurologıa” grant to P.P. and the Generalitat de Cata-lunya grant (1999SGR-00226) to R.O.

We are especially grateful to the members of the family studied fortheir cooperation.

References1. Spillantini MG, Goedert M. Tau protein in neurodegenerative

diseases. Trends Neurosci 1998;21:428–433.2. Foster NL, Wihelmsem K, Sima AAF, et al. Frontotemporal

dementia and parkinsonism linked to chromosome 17: a con-sensus conference. Ann Neurol 1997;41:706–715.

3. Hutton M, Lendon CL, Rizzu P, et al. Association of a mis-sense and 59 splice-site mutations in tau with the inherited de-mentia FTDP-17. Nature 1998;393:702–705.

4. Spillantini MG, Goedert M, Crowther RA, et al. Familial mul-tisystem tauopathy with presenile dementia: a disease withabundant neuronal and glial tau filaments. Proc Natl Acad SciUSA 1997;94:4113–4118.

5. Spillantini MG, Murrell JR, Goedert M, et al. Mutation in thetau gene in familial multisystem tauopathy with presenile de-mentia. Proc Natl Acad Sci USA 1998;95:7737–7741.

6. Delisle MB, Murrell JR, Richardson R, et al. A mutation atcodon 279 (N279K) in exon 10 of the tau gene causes atauopathy with dementia and supranuclear palsy. Acta Neuro-pathol 1999;98:62–77.

7. Stanford PM, Halliday GM, Brooks WS, et al. Progressive su-pranuclear palsy pathology caused by a novel silent mutation inexon 10 of the tau gene: expansion of the disease phenotypecaused gene mutations. Brain 2000;123:880–893.

8. Bassam BJ, Caetano-Anolles G, Gresshoff PM. Fast and sensi-


tive staining of DNA in polyacrylamide gels. Anal Biochem1991;196:80–83.

9. Baker M, Litvan I, Houlden H, et al. Association of an ex-tended haplotype in the tau gene with progressive supranuclearpalsy. Hum Mol Genet 1999;8:711–715.

10. Goedert M, Spillantini MG, Jakes R, et al. Multiple isoforms ofhuman microtubule-associated protein tau: sequences and local-ization in neurofibrillary tangles of Alzheimer’s disease. Neuron1989;3:519–526.

11. D’Souza I, Schellenberg GD. Determinants of 4 repeat tauexpression: coordination between enhancing and inhibitorysplicing sequences for exon 10 inclusion. J Biol Chem 2000;275:17700–17709.

12. Goode BL, Feinstein SC. Identification of a novel microtubulebinding and assembly domain in the developmentally regulatedinter-repeat of tau. J Cell Biol 1994;124:769–782.

13. Heutink P. Untangling tau-related dementia. Hum Mol Genet2000;9:979–986.

14. Yasuda M, Kawamata T, Komure O, et al. A mutation in themicrotubule-associated protein tau in pallido-nigro-luysian de-generation. Neurology 1999;53:864–868.

15. Ohara S, Kondo K, Morita H, et al. Progressive supranuclearpalsy syndrome in two siblings of a consanguineous marriage.Neurology 1992;42:1009–1014.

16. Verin M, Rancurel G, De Marco O, Edan G. First familialcases of corticobasal degeneration. Mov Disord 1997;12(suppl):55.

17. Caselli RJ, Reiman EM, Timmann D, et al. Progressive apraxiain clinically discordant monozygotic twins. Arch Neurol 1995;52:1004–1010.

18. Rosen DR, Siddique T, Patterson D, et al. Mutations in theCu/Zn superoxide dismutase gene are associated with familialamyotrophic lateral sclerosis. Nature 1993;362:59–62.

19. Andersen PM, Nilsson P, Ala-Hurula V, et al. Amyotrophiclateral sclerosis associated with homozygosity for an Aps90Alamutation in CuZn-superoxide dismutase. Nat Genet 1995;10:61–66.

Compound HeterozygousD90A and D96N SOD1Mutations in a RecessiveAmyotrophic LateralSclerosis FamilyCollette K. Hand, PhD,1 Veronique Mayeux-Portas, PhD,2

Jawad Khoris, MD,2,3 Valerie Briolotti,2

Pierre Clavelou, MD, PhD,4 William Camu, MD, PhD,2,3

and Guy A. Rouleau, MD, PhD1

We describe a French amyotrophic lateral sclerosis (ALS)family with two distinct mutations in the Cu/Zn super-oxide dismutase (SOD1) gene. The D90A mutation hasbeen well described and clearly shown to cause recessiveALS. In this family, affected individuals are heterozygousfor the D90A mutation and also carry a single copy of anovel SOD1 mutation, D96N. We propose that in thisfamily both mutations are required for the developmentof disease.

Ann Neurol 2001;49:267–271

Amyotrophic lateral sclerosis (ALS) is a progressiveneurodegenerative disorder, caused by death of motorneurons in the motor cortex, brainstem, and spinalcord.1 The age of disease onset varies, but most indi-viduals present with symptoms in middle adult life anddevelop paralysis leading to death within 3–5 years.2

Approximately 10% of ALS cases are familial, and 20%of these cases have mutations in the Cu/Zn superoxidedismutase (SOD1) gene.3 Presently, 64 SOD1 muta-tions have been described,4 and all are autosomal dom-inant, with the exception of D90A, which has beenobserved in recessive pedigrees, predominantly amongthe Scandinavian population.5 An ALS patient ho-mozygous for a Leu84Phe mutation has been report-ed6; however, there have been no further indicationsthat this is a true recessive mutation.

From the 1Centre for Research in Neuroscience, McGill University,and the Montreal General Hospital Research Institute, Montreal,Quebec, Canada; 2UNCD Molecular Unit, EPI 99–30, Institute ofBiology and 3Department of Neurology, Hopital Gui de Chauliac,Montpellier; and 4Hopital Gabriel Montpied, Clermont-Ferrand,France.

Received Aug 21, 2000, and in revised form Oct 9. Accepted forpublication Oct 10, 2000.

Address correspondence to Dr Rouleau, Montreal General HospitalL7–224,1650 Cedar Avenue, Montreal, H3G 1A4 Canada.E-mail: [email protected]


Haplotype analysis of recessive and dominantD90AALS families indicated that all recessive pedigreeshave a common founder,7 whereas the dominant fam-ilies have a number of different founders. The familywe describe here has one copy of the recessive D90Ahaplotype but no D90A homozygotes. A novel reces-sive SOD1 mutation (D96N) has been identified inaffected and unaffected individuals, and both muta-tions appear to be necessary for development of thedisease in any single individual.

Materials and MethodsPatientsA detailed clinical evaluation of this family has been previ-ously published (see Khoris et al.,8 family 2). Briefly, patientspresent with the characteristic phenotype associated with re-

cessive ALS, i.e., lower limb onset, slow evolution of disease,and age of onset between 30 and 45 years (Table).

Molecular AnalysisBlood was taken from informed, consenting individuals andDNA extracted following standard protocols. SSCP analysisof exon 4 of the SOD1 gene was performed as describedpreviously.6 Electrophoresis using both polyacrylamide(9.5%, with 5% glycerol) and 0.5% mutation detection en-hancement (MDE) gels was followed by autoradiography.Abnormally migrating SSCP bands were sequenced on bothstrands using a thermosequenase 33P cycle sequencing kit(United States Biochemical, Cleveland, OH). Sequenceswere resolved on 6% polyacrylamide gels. A group of normalFrench controls (n 5 160) was examined by SSCP analysis

Table. Clinical Details of Compound Heterozygous ALS Patients

Patient SexAge at Onset(yr)

LMNsigns

UMNsigns

Delay tobulbar onset(yr)

Site ofonset Duration (yr) Atypical signs

V:6 M 42 1 1 4 LL .9 (alive) Electric discharges andcomplaints ofhypoesthesia in LL

V:9 F 44 1 1 4 LL .28 (alive) Severe pain in LL,micturition urgency

V:11 F 30 1 1 6 LL 14 Not known

LMN 5 lower motor neuron; UMN 5 upper motor neuron; LL 5 lower limbs.

Fig 1. Pedigree of ALS family indicating affectedindividuals (solid symbols). SOD1 exon 4 muta-tion status is indicated for the individuals ana-lyzed. In parentheses are the inferred genotypes.The year of birth is displayed when available. Topreserve confidentiality, the sex of individuals inthe last generation (VI) has been disguised andthe year of birth omitted.


of SOD1 exon 4 to determine the prevalence of the D96Nmutation in the population.

ResultsSSCP analysis of individual V:6 from this family (Fig1) yielded abnormally migrating bands in both SSCPgel conditions. Sequencing revealed the D90A muta-tion (GAC3GCC shift) and a novel SOD1 mutation,D96N (GAT3AAT shift). The presence of both mu-tations in a single individual prompted us to analyzethe DNA from all available family members. The twoaffected members for whom DNA was available carrycopies of both mutations. The D90A mutation alonewas found in two family members, whereas D96Nalone was present in four individuals (Figs 1, 2); All ofthese individuals are unaffected. SSCP analysis ofSOD1 exon 4 in 160 control samples did not identifyany mutations.

DiscussionIn this French ALS family (Fig 1), the two affectedindividuals (V:6, V:9) sampled have single copies ofboth D90A and D96N SOD1 mutations. Several rel-atives carry a copy of one or the other mutation butremain unaffected. In addition, based on the segrega-tion of mutations seen in the children of generationVI, we can infer that both mutations were present inthe deceased affected sibling (individual V:11) and thatthe two mutations segregate on different chromosomes.No DNA samples are available from the deceased par-

ents (IV:20 and IV:21), but, insofar as the D96N mu-tation is present in individual IV:24, it is likely to havebeen present in her sister (individual IV:21), and theD90A mutation was possibly introduced by individualIV:20, although we have no background history onother members of his family. There are no individualshomozygous for either mutation.

Because of the late age of onset of ALS, we cannotexclude the possibility of disease onset at a later stagein the last generation (VI) of the family. The dates ofbirth of these individuals range from 1954 to 1972,and all clinical information is current. However, thefact that individual IV:24, born in 1912, remains un-affected, despite the presence of a copy of D96N, sug-gests that this mutation alone is not sufficient to causedisease. Parents IV:21 and IV:20 must each have car-ried one of the mutations, and they both lived to atleast 79 years of age without signs of the disorder. Al-though ALS exhibits incomplete penetrance, this aloneis unlikely to account for all of these unaffected, olderpatients who carry single mutations. It is important tonote that only the individuals carrying both mutationsare affected with ALS, and no unaffected individualwith both mutations has been identified. Within theextended family, there is one more ALS patient. Thisindividual (IV:34) is deceased, but sequencing ofSOD1 exon 4 in three of his children and his brotherdid not detect any mutation. Were he a compoundheterozygote for D90A and D96N mutations, as in the

Fig 2. Sequence of SOD1 exon 4 in the region of interest. Individual V:15 displays the normal sequence; individual VI:7 is aD90A heterozygote and the site of the A3C shift is indicated. Individual IV:24 is a D96N heterozygote, with a G3A shift. Af-fected individual V:6 carries both D90A and D96N mutations.

Brief Communication: Hand et al: SOD1 Compound Heterozygotes in ALS 269

other affected family members, we would expect all ofhis children to carry one mutation. Thus, we suggestthat he represents a sporadic ALS case. Detailed clinicalinformation on this individual is not available.

This is the first description of the D96N SOD1 mu-tation, in which a GAT3AAT shift results in the sub-stitution of aspartic acid (D) by asparagine (N). TheD96N mutation was found in a total of six individualsin this family, but was not seen in 160 controls, whichwould suggest that it is not a polymorphism. This mu-tation has not been described previously, despite thecomprehensive study of the SOD1 gene in ALS pedi-grees. Although this amino acid is not very well-conserved,9 no other mutations have been described atthis codon.4 Amino acids (aa) 96–100 are required forstability of a Greek key.10 No mutations have beenidentified between aa 93 and 100,4 suggesting eitherthat the region is not critically important for the mol-ecule or that only when two mutations interact, as inhomozygotes, or in individuals carrying two distinctmutations, does disease develop.

The D90A mutation is reported to act either as arecessive or as a dominant mutation.5,11 To understandhow the D90A mutation can act in both dominantand recessive fashions, a study examined the origins ofthese mutations by haplotype analysis of both domi-nant and recessive D90A families.7 All families withrecessive inheritance share a common founder, whereasthe dominant kindred appear to have several founders.It was suggested that the D90A mutation normally actsin a dominant fashion but, on one occasion, occurredin a background that modified penetrance, resulting ina recessive allele; all recessive pedigrees have descendedfrom this common founder. The family described herehas one copy of the haplotype associated with recessiveD90A ALS pedigrees (see Al-Chalabi et al.,7 family27), although there are no D90A homozygous individ-uals. This suggests that, alone, the D90A mutation isnot sufficient to result in disease. It appears that in thisfamily copies of both D90A and D96N mutations arerequired to cause ALS. The presence of elderly individ-uals with single mutations and without signs of diseasesupports this hypothesis. At the time of the haplotypestudy, the D96N mutation had not been identified inthis family, probably because the D90A genotypingwas performed by restriction enzyme analysis using ei-ther Cfo17 or Fnu4H1.8 This is a very important pointand emphasizes the necessity to examine abnormalfragments carefully by direct sequencing to confirm themutation and identify any additional mutationspresent. Our findings support the conclusions of thehaplotype study,7 by explaining the occurrence of therecessive D90A haplotype in a family mistakenlythought to segregate a dominant allele.

All ALS patients homozygous for the D90A muta-tion show the same characteristic phenotype of slow

progression of mild disease, with little variation be-tween patients.12–14 In contrast, the D90A heterozy-gous cases show more diverse and aggressive pheno-types.12 This family displays the distinct recessiveclinical phenotype; patients present with lower limbonset, slow evolution of the disease, and onset between30 and 45 years of age.

The D90A mutation has been studied intensively,because it was the only SOD1 mutation described inrecessive pedigrees. Here we present molecular evidencethat D96N is a second recessive SOD1 mutation. Bothmutations are located in the same region of the pro-tein, so it is possible that they act together to destabi-lize the enzyme. However, conclusive evidence thatD96N is indeed acting as a recessive mutation wouldbe the identification of D96N homozygous pedigrees.

In conclusion, despite the large number of dominantSOD1 mutations associated with ALS, we present anovel recessive mutation (D96N). Moreover, this is thefirst report of two distinct SOD1 mutations in a singleALS patient. This finding may have implications forthe interpretation of inheritance patterns in ALS fam-ilies and is of particular importance in the context ofgenetic counseling.

This work was supported by the Muscular Dystrophy Association.G.A.R. is supported by the Medical Research Council of Canada.We also thank the Association pour la Recherche sur la ScleroseLaterale Amyotrophique and the Association Francaise contre lesMyopathies for their financial support.

We thank members of the family for their cooperation.

References1. Siddique T, Deng HX. Genetics of amyotrophic lateral sclero-

sis. Hum Mol Genet 1996;5:1465–1470.2. Brown RH Jr. Amyotrophic lateral sclerosis: recent insights

from genetics and transgenic mice. Cell 1995;80:687–692.3. Siddique T, Nijhawan D, Hentati A. Molecular genetic basis of

familial ALS. Neurology 1996;47:S27–S34.4. Orrell RW. Amyotrophic lateral sclerosis: copper/zinc superox-

ide dismutase (SOD1) gene mutations. Neuromusc Disord2000;10:63–68.

5. Andersen PM, Nilsson P, Ala-Hurula V, et al. Amyotrophic lateralsclerosis associated with homozygosity for an Asp90Ala mutationin CuZn-superoxide dismutase. Nat Genet 1995;10:61–66.

6. Boukaftane Y, Khoris J, Moulard B, et al. Identification of sixnovel SOD1 gene mutations in familial amyotrophic lateralsclerosis. Can J Neurol Sci 1998;25:192–196.

7. Al-Chalabi A, Andersen PM, Chioza B, et al. Recessive amyo-trophic lateral sclerosis families with the D90A SOD1 mutationshare a common founder: evidence for a linked protective fac-tor. Hum Mol Genet 1998;7:2045–2050.

8. Khoris J, Moulard B, Briolotti V, et al. Coexistence of domi-nant and recessive familial amyotrophic lateral sclerosis with theD90A Cu,Zn superoxide dismutase mutation within the samecountry. Eur J Neurol 2000;7:207–211.

9. Rosen DR, Siddique T, Patterson D, et al. Mutations in Cu/Znsuperoxide dismutase gene are associated with familial amyotro-phic lateral sclerosis. Nature 1993;362:59–62.

10. Deng HX, Hentati A, Tainer JA, et al. Amyotrophic lateral


sclerosis and structural defects in Cu,Zn superoxide dismutase.Science 1993;261:1047–1051.

11. Robberecht W, Aguirre T, Van Den Bosch L, et al. D90A het-erozygosity in the SOD1 gene is associated with familial andapparently sporadic amyotrophic lateral sclerosis. Neurology1996;47:1336–1339.

12. Mezei M, Andersen PM, Stewart H, et al. Motor system ab-normalities in heterozygous relatives of a D90A homozygousCuZn-SOD ALS patient of Finnish extraction. J Neurol Sci1999;169:49–55.

13. Andersen PM, Nilsson P, Keranen ML, et al. Phenotypic het-erogeneity in motor neuron disease patients with CuZn-superoxide dismutase mutations in Scandinavia. Brain 1997;120:1723–1737.

14. Andersen PM, Forsgren L, Binzer M, et al. Autosomal recessiveadult-onset amyotrophic lateral sclerosis associated with ho-mozygosity for Asp90Ala CuZn-superoxide dismutase muta-tion. A clinical and genealogical study of 36 patients. Brain1996;119:1153–1172.

Laforin Is a Cell Membraneand Endoplasmic Reticulum–Associated Protein TyrosinePhosphataseBerge A. Minassian, MD,1 Danielle M. Andrade, MD,2

Leonarda Ianzano, PhD,2 Edwin J. Young, BSc,2

Elayne Chan, BSc,2 Cameron A. Ackerley, PhD,3

and Stephen W. Scherer, PhD2

Lafora disease (LD) is the only progressive myoclonusepilepsy with polyglucosan bodies. Among conditionswith polyglucosan bodies, LD is unique for the subcellu-lar location of its polyglucosans in neuronal perikaryaand dendrites and not in axons. Here we report that theprotein encoded by the EPM2A gene, which is mutatedin LD, localizes at the plasma membrane and the endo-plasmic reticulum and that it is a functional protein ty-rosine phosphatase. The significance of these findings inthe epilepsy of LD and in the origin and characteristicsubcellular location of Lafora bodies is discussed.

Ann Neurol 2001;49:271–275

Lafora disease (LD) is a teenage-onset autosomal reces-sive progressive myoclonus epilepsy (PME) with patho-

gnomonic periodic acid–Schiff-positive polyglucosan(PG) inclusions (Lafora bodies; LB).1 Patients with LDhave mutations in the ubiquitously expressed EPM2Agene, which codes for the 331-amino-acid protein, ofunknown function, laforin.2,3

The epilepsy of LD is characterized by worseningmyoclonic, photoconvulsive, and other seizures, includ-ing ictal visual hallucinations.1 Biopsies in patientswith already established intractable epilepsy revealedlittle if any neuronal loss,1,4 implicating factors otherthan neurodegeneration in the epilepsy of LD.

LB are most abundant in the organs with the highestglucose metabolism, brain, heart, liver, and skeletalmuscle, but are also seen in skin and kidneys.1 Theyconsist of aggregates of PG fibrils composed of repeat-ing units of glucose identical to glycogen except for theabsence of a regular branching pattern, with resultantinsolubility.1,4–6 LB have a characteristic subcellulardistribution occupying the perikaryal region.1,4–6 Inneurons, they are present in both perikarya and den-drites, but not axons.1,4–6 To understand better thepathogeneses of the epilepsy of LD and the PG accu-mulations, we investigated the subcellular localizationof laforin and here report it to be at the plasma mem-brane and at the endoplasmic reticulum (ER). In addi-tion, we report experimental confirmation that laforinis a protein tyrosine phosphatase (PTP).

Materials and MethodsSubcellular LocalizationCONSTRUCTION OF MAMMALIAN EXPRESSION VECTORS.To facilitate subcellular localization experiments, heterolo-gous fusion proteins of the Myc epitope and laforin wereconstructed. To rule out any effect of the nine-amino-acidMyc epitope on laforin’s subcellular localization, it wasplaced at either the N or the C terminus of the construct.For brevity, construction of only one of the vectors is de-scribed: For the pcMyc-EPM2A (Myc at the N-terminus),EPM2A was PCR amplified using a 59-primer (59-gccgccgggatccATGcgcttc) including the start codon (uppercase) and tagged (italics) with a BamHI site (primer F) and a39-primer (59-gccgcggccgcctccttagggaaatcagga) tagged with aNotI site. This PCR product was cloned into the corre-sponding sites of the pcDNA3 vector (Invitrogen, La Jolla,CA). Myc was then introduced in-frame after amplifyingfrom a previous vector with 59-KpnI-tagged and 39-BamHI-tagged primers.

EXPRESSION IN COS-1 AND CHO CELLS AND IMMUNOFLU-ORESCENCE LIGHT MICROSCOPY. Each of the above-mentioned vectors was transiently transfected into COS-1cells and stably transfected into CHO cells. Cells were plated(2 3 105/well; Labtek chamber slides; Nalgene-Nunc, Na-

From the 1Division of Neurology, Departments of Paediatrics andGenetics, The Hospital for Sick Children, and Bloorview EpilepsyProgram, The University of Toronto, and Departments of 2Geneticsand 3Pathology, The Hospital for Sick Children, and The Univer-sity of Toronto, Toronto, Ontario, Canada.

Received 12 Sep, 2000, and in revised form Oct 13. Accepted forpublication Oct 13, 2000.

Address correspondence to Dr Scherer, Department of Genetics,The Hospital for Sick Children, and The University of Toronto,

555 University Ave., Toronto, Ontario M5G 1X8, Canada.E-mail: [email protected]


perville, IL), transfected with 1 mg vector usingLipofectamine-Plus (Gibco-BRL, Grand Island, NY), and ex-posed to lipid–DNA complex in DMEM (Sigma, St. Louis,MO) for 5 hours. Forty-eight hours posttransfection, cul-tures were fixed (in methanol for 30 minutes at –20°C) andstained with antibodies against laforin-Myc and organelles.Cultures were blocked for 1 hour (10% BSA/PBS) and in-cubated with antibody against Myc and/or against queriedorganelle (Fig 1) for 45 minutes at room temperature (RT).Slides were washed and incubated with secondary antibody

in blocking solution in the dark for 30 minutes, mounted(Dako Anti-Fade; Carpinteria, CA), and analyzed by confo-cal microscopy using the 363 objective and filters.

IMMUNOGOLD ELECTRON MICROSCOPY. Transfectedcells were fixed in 4% paraformaldehyde/0.1 M phosphatebuffer, pH 7.4, 0.1% glutaraldehyde for 2 hours; lightly cen-trifuged; washed; and stored (PBS 1 10 mM azide). Prior toprocessing, samples were infiltrated with 10% gelatin in PBS

Fig 1. Subcellular localization of laforin to ER by immunofluorescence light microscopy. (A–C) A transfected COS-1 cell (withplasma membrane not preserved) stained with primary mouse monoclonal antibody against Myc (9E10; 0.5 mg/well; Santa CruzBiotechnology, Santa Cruz, CA; SBCT) and primary goat polyclonal antibody against the ER marker GRP94 (C-19; 0.2 mg/well;SCBT); secondary antibodies were FITC-labeled goat anti-mouse (1:400) detectable through the green filter (A) and Texas red-labeled donkey anti-goat (1:200) detectable through the red filter (B; Jackson ImmunoResearch Laboratories, West Grove, PA;JIRL). Exact colocalization of A and B is seen (yellow) when both filters are open (C). (D–F) Same as above except that the pri-mary antiorganelle antibody is against the peroxisome (rabbit polyclonal anti-SKL; 1:100; gift from Dr. R. Rachubinski) with itscorresponding secondary antibody [Texas red-labeled goat anti-rabbit (1:200); JIRL]. (G–I) Same as above except that the primaryantiorganelle antibody is against the golgi apparatus (mouse monoclonal anti-14–3-3; 1 mg/well; StressGen Biotechnology, Victoria,British Columbia, Canada) and the primary anti-Myc antibody is a rabbit polyclonal antibody (A14; 0.5 mg/well; SCBT). Pri-mary antibodies for the remaining organelles tested (data not shown) were rabbit polyclonal anti-b-Gal (lysosome marker; 4 mg/well; gift from Dr. J. Callahan), rabbit polyclonal anti-a-MLRQ (mitochondria marker; 1:1,000 dilution; gift from Dr. B. Robin-son), mouse monoclonal anti-EEA1 (early endosome marker; 10 mg/well; Transduction Labs).


(37°C, 1 hour) and allowed to solidify at 4°C. Cells wereinfused with 2.3 M sucrose overnight, mounted on alumi-num pins, and frozen (liquid nitrogen). Cryosectioning andimmunogold staining are described in Figure 2.

Protein Tyrosine Phosphatase AssayCONSTRUCTION OF BACTERIAL EXPRESSION VECTORS.To produce sufficient quantities of purified wild-type laforinand an inactive mutant, the following glutathione-S-transferase (GST)-EPM2A constructs were generated: ForpgEPM2A (GST-wild-type-laforin), the BamHI/NotI-taggedPCR product used for construct pcMyc-EPM2A was clonedinto vector pGEX-4T1 (Pharmacia, Uppsala, Sweden) in-frame with and 39 to the GST sequence. For pgEPM2AC.S(GST-mutant-laforin), to inactivate laforin’s phosphatase do-main, the main catalytic amino acid, Cys266, was convertedto Ser as follows. A 39-primer (59-agccgcggtggagcggcccacccc-agcgttgGagt) corresponding to nucleotides 294–329 ofEPM2A containing a SacII site and modified to contain therequired mutation (upper case) was used for PCR withprimer F. A second EPM2A SacII site at nucleotide 137 al-lowed replacement of the SacII fragment with the point mu-tation in place of the normal sequence in pgEPM2A.

EXPRESSION, PURIFICATION, AND PHOSPHATASE ASSAYS.Constructs were transformed in BL21(DE3)LysS cells andproteins isolated using the BugBuster/GST-Bind kit (Nova-gen, Madison, WI). Varying amounts of proteins were incu-bated in 100 ml reactions at 37°C for 1 hour withparanitrophenyl-phosphate (pNPP), a PTP substrate that con-verts yellow detectable at 450 nm upon dephosphorylation.7

ResultsSubcellular Localization

IMMUNOFLUORESCENCE LIGHT MICROSCOPY. COS-1and CHO cells transfected with heterologous con-structs tagged at either end were stained with antibod-ies against Myc and against six organelles. Results wereidentical with either cell type and either construct. Mycsignal localized in a reticular fashion in the perikaryalregion (Fig 1A). Costaining with organelle-specific an-tibodies revealed exact colocalization with the ER-specific signal (Fig 1A–C). There was no colocalizationwith antibodies against peroxisomes (Fig 1D–F), golgi(Fig 1G–I), lysosomes, mitochondria, or early endo-

Fig 2. Subcellular localization of laforin to the plasma membrane and ER by immunogold electron microscopy. (A) Immunogold-labeled CHO cell; gold label on the plasma membrane (arrowheads). (B) Cytoplasm of a CHO cell; the majority of the gold labelwas confined to the ER. Grids were prepared as follows: Ultrathin cryosections were sliced using a diamond knife in a cryoultramic-rotome. They were transferred to formvar-coated nickel grids in a drop of molten sucrose. Residual aldehydes were blocked by twowashes in PBS containing 0.15% glycine and 0.5% BSA. Grids were washed in PBS/BSA several times, before a 1 hour incuba-tion in the mouse monoclonal anti-Myc antibody. They were then washed thoroughly in PBS/BSA prior to another incubation for 1hour in the goat anti-mouse IgG bound to 10 nm gold particles. Following multiple rinses, they were stabilized in a thin film ofmethylcellulose containing 0.2% uranyl acetate. As a control, the Myc antibody was omitted from the staining procedure in some ofthe grids. Grids were then examined and photographed in the transmission electron microscope. Scale bars 5 0.2 mm.

Brief Communication: Minassian et al: Laforin Localization and Function 273

somes (not shown). In cells treated with preservedplasma membrane, Myc signal could be seen all alongthe contour of the cell (not shown; see electron micros-copy results below).

IMMUNOGOLD ELECTRON MICROSCOPY. Figure 2 showsthat gold label was found at the plasma membrane (Fig2A) and ER (Fig 2B).

Protein Tyrosine Phosphatase ActivityFigure 3 shows that GST-wild-type-laforin dephospho-rylates the phosphotyrosine substrate pNPP in a dose-dependent manner. GST alone showed no activity.GST-mutant-laforin, which has the active cysteine nu-cleophile2,3,7 in the predicted PTP catalytic domain re-placed by the inert serine, also showed no activity.Similar results were obtained with a phosphoserinesubstrate (data not shown) confirming predictions fromsequence analysis3 that laforin is a dual-specificity PTP.

DiscussionThe observation of laforin at the ER in this study pro-vides the first link between the disease protein and thepathognomonic PG accumulations. As early as themid-1960s, Van Hoof and Hageman-Bal,8 Collins etal.,9 and Toga et al.10 used electron microscopy toshow a direct association of early-stage LB PG withpolyribosomes and the extraluminal surface of roughER (RER). LD PG are found only in cellular compart-ments containing ribosomes and RER; in neurons, thisincludes perikarya and dendrites but not axons.1,4–6,11

PG accumulate in one other inherited neurologicaldisease, adult PG body disease (APBD). APBD is dis-tinct from LD and is characterized by progressive mo-tor and sensory deficits and dementia but no seizuresor myoclonias.12 In contrast to the topography of LB,APBD PG bodies are located exclusively in axons and

not in perikarya or dendrites.6,12 Presence of seizures inLD but not APBD implicates dendritic accumulationsin the epilepsy of LD.

What is the origin of PG, why are APBD PG inaxons, and how could defective laforin result in ER-associated PG? To address these questions, a brief re-view of glycogen metabolism is necessary. Glycogenstrands are elongated by glycogen synthase (GS) andbranched by branching enzyme (BE). They are short-ened by glycogen phosphorylase and debranched bydebranching enzyme.13,14 Branching renders glycogensoluble, and inadequate branching leads strands to co-alesce and form PG bodies.1,5,6,12–14

APBD is caused by BE deficiency.15 Because branch-ing (as well as debranching) occurs in the cytoplasmwith no evidence of ER involvement,13 APBD PG arelikely generated free in the soma and cleared into axonsby axoplasmic flow.6,12 Axonal flow has been invokedas the mode of clearance of excess PG that normallyaccumulate in healthy neurons with aging.6 These PGaggregate in axons in the form of corpora amylacea butnever in neuronal somas or dendrites.6 Deficiencies ofthe other enzymes acting directly on glycogen lead tovarious glycogen storage diseases, with accumulationsof normally or excessively branched soluble glycogenand not PG.13,14

Recently, Raben et al16 showed that overexpressionof GS in muscle also results in PG bodies and pro-posed that GS hyperfunction underlies LD. The nextphase of their work, i.e., systemic or neuronal GS up-regulation, is keenly anticipated, to see whether it re-sults in typical perikaryal and dendritic neuronal LBand LD. GS is an excellent downstream candidate sub-strate for laforin for the following reasons. ER providesthe glucose subunits to be incorporated into glycogenby GS.13,14 GS is tightly bound to the glycogen parti-cle.14 On the other hand, its two regulators, glycogensynthase kinase and glycogen synthase phosphatase(protein phosphatase-1), are associated with ribo-somes17 and ER,18 respectively. These interactions es-tablish a potential link through which laforin coulddown-regulate GS. They might also underlie the asso-ciation of LD PG formed by up-regulated GS to RER.The demonstration that laforin is a PTP is consistentwith a regulatory role for this protein, in that PTPs areregulatory proteins, and many interact closely withcounterpart kinases.2,3,7 Work is in progress to identifylaforin-interacting proteins that may fill the missinglinks between laforin and LD PG. These, as well asknown GS-regulating proteins, are being analyzed ascandidate genes for the ;30% of LD families whosegene locus does not link to EPM2A.19

Finally, laforin was also observed at the plasmamembrane. LD patients commonly have seizures in in-fancy and early childhood many years prior to the on-set of the progressive syndrome.1,3,20 A role for laforin

Fig 3. Laforin functions as a PTP. Dephosphorylation ofpNPP as measured by absorbance of 450 nm light by dephos-phorylated substrate ( y axis) vs increasing concentrations ofindicated protein (x axis). Error bars indicate standard devia-tion of independent trials preformed in triplicate.


in the epilepsy or other aspects of LD separate fromPG metabolism may exist.

Supported by a grant from the Medical Research Council of Canada(MRC) to S.W.S. and by the Quebec and Sweden Lafora diseaseassociations. S.W.S. is a Scholar of the MRC.

We are grateful to Drs. Ameet Sengar, Sean Egan, Sunqu Zhang,Richard Bagshaw, Don Mahuran, Chris Fladd, and Daniela Rotinfor their help and advice.

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