Brain-Derived Neurotrophic Factor GeneticVariants Are Not Susceptibility Factors toAlzheimer’s Disease in ItalySilvia Bagnoli, PhD,1 Benedetta Nacmias, PhD,1

Andrea Tedde, PhD,1 Bianca Maria Guarnieri, MD,2

Elena Cellini, PhD,1 Concetta Petruzzi, MD,2

Antonella Bartoli, MD,2 Luigi Ortenzi, MD,2

and Sandro Sorbi, MD1

Several lines of evidences have suggested an involvement ofthe brain-derived neurotrophic factor (BDNF, 11p13) inthe pathogenesis of neurodegenerative disorders,1,2 in par-ticular, in Alzheimer’s disease (AD). Recently, positive as-sociations between two BDNF polymorphisms (theVal66Met[G196A] and 270C/T) and AD have suggested apossible effect of BDNF genetic variations on the risk ofAD.3,4

In light of these findings, we analyzed the segregation ofthe Val66Met(G196A) and 270C/T BDNF and apolipopro-tein E (ApoE) genes polymorphisms in Italian patients withAD.

We analyzed a sample of 128 Italian patients with spo-radic AD (45 men and 83 women; mean age, 71.1 � 8.5;age at onset, 65.7 � 8.6 mean � SD, with age of onsetdefined as the onset of first cognitive changes) enrolled at theDepartment of Neurology, University of Florence and 97healthy controls (36 men and 61 women; mean age, 72.9 �24 years). All subjects were carefully assessed with a rigorousdiagnostic evaluation to exclude diagnosis of any neurologicaldisorder.

Clinical assessments of patients were done according topublished guidelines and the AD diagnosis fulfilled the Na-tional Institute of Neurological and Communicative Disor-ders and Stroke Alzheimer’s disease and related Disorders As-sociation criteria for probable AD.5

The local ethical committee approved the protocol of thestudy and the written consent for genetic screening was ob-tained from all subjects or, where appropriate, their relativeor legal representative.

All the gene polymorphisms were analyzed by polymerasechain reaction followed by appropriate restriction enzyme di-gestion. Allele frequencies were estimated by gene counting.

Comparisons of genotype and allele frequencies distribu-tion between patients and controls were assessed by �2 test.Both polymorphisms followed a distribution in Hardy–Weinberg equilibrium and did not significantly differ fromthat of controls (p � 0.1) (Table).

In addition, no correlation was observed between age atonset, sex, or any BDNF genotype.

Stratification of data irrespective of the ApoE status(data not shown) did not show an epistatic effect of thetwo genes.

It could be speculated that, in previous studies, the an-alyzed populations were not matched for their ethnic back-ground, a major confounding factor when studying poly-morphisms. Several nonmutually exclusive factors canproduce conflicting results in association studies of com-mon complex diseases including the presence of genetic,clinical, and population heterogeneity. Our data suggestthat BDNF genetic variants are not a susceptibility factorfor AD, nor do they mitigate the effect of ApoE ε4 alleleon AD risk.

1Department of Neurological and Psychiatric Sciences,University of Florence, Florence; 2Casa di Cura “VillaSerena,” Associazione L. Petruzzi, Citta Sant Angelo, Pescara,Italy

This work was supported by the Italian Telethon FoundationONLUS (E.0980, QLK-6-CT-1999-02178) and Tuscany Region(ICS110.1/RA00.53, ICS120.3/RA00.87, ICS120.3/RA00.84).

References1. Murer MG, Yan Q, Raisman-Vosari R. Brain derived neurotro-

phic factor in the control human brain, and in Alzheimer’s dis-ease and Parkinson’s disease. Prog Neurobiol 2001;63:71–124.

2. Hakansson A, Melke J, Westberg L, et al. Lack of associationbetween the BDNF Val66Met polymorphism and Parkinson’sdisease in a Swedish population. Ann Neurol 2003;53:823.

3. Riemenschneider M, Schwarz S, Wagenpfeil S, et al. A polymor-phism of the brain-derived neurotrophic factor (BDNF) is asso-ciated with Alzheimer’s disease in patients lacking the apoli-poprotein E epsilon4 allele. Mol Psychiatry 2002;7:782–785.

Table. BDNF Val66Met(G196A) and 270 C/T Polymorphisms: Genotypes and Corresponding Allele Frequencies inAD and Controls

Samples N

Genotype Allele Frequency

AA (%) AG (%) GG (%) A (%) G (%)

BDNF Val66Met (196 A/G)AD 128 6 (4.7) 60 (46.9) 62 (48.4) 72 (28.1) 184 (71.8)Controls 97 4 (4.2) 38 (39.1) 55 (56.7) 46 (23.7) 148 (76.2)

�2 � 1.52; p � 0.46; df � 2 �2 � 1.11; p � 0.29;df � 1

BDNF 270 C/T CC (%) CT (%) TT (%) C TAD 128 113 (88.3) 14 (11.0) 1 (0.7) 240 (93.7) 16 (0.63)Controls 97 83 (85.6) 14 (14.4) — 180 (92.8) 14 (0.72)

�2 � 1.35; p � 0.51; df � 2 �2 � 0.17; p � 0.68;df � 1

BDNF � brain-derived neurotrophic factor; AD � Alzheimer’s disease.

LETTERS

© 2004 American Neurological Association 447Published by Wiley-Liss, Inc., through Wiley Subscription Services

4. Ventriglia M, Bocchio Chiavetto L, et al. Association betweenthe BDNF 196 A/G polymorphism and sporadic Alzheimer’sdisease. Mol Psychiatry 2002;7:136–137.

5. McKhann G, Drachman D, Folstein M, et al. Clinical diagnosisof Alzheimer’s disease: report of the NINCDS-ADRDA WorkGroup under the auspices of Department of Health and HumanServices Task Force on Alzheimer’s disease. Neurology 1984;34:939–944.

DOI: 10.1002/ana.10842

Progressive Supranuclear Palsy and Parkinson’sDisease in a Family with a New Mutation in thetau GeneGiacomina Rossi, PhD,1 Elisabetta Gasparoli, MD,2

Claudio Pasquali, BSc,1 Giuseppe Di Fede, MD,1

Daniela Testa, MD,1 Alberto Albanese, MD, PhD,1

Fulvio Bracco, MD,2 and Fabrizio Tagliavini, MD1

Mutations in the gene encoding for tau protein are associ-ated with neurodegenerative disorders marked by the pres-ence of abnormally aggregated tau protein, such as fronto-temporal dementia with parkinsonism linked to chromosome17, progressive supranuclear palsy (PSP), and corticobasal de-generation. PSP is clinically characterized by a variable com-bination of postural instability and falls, akinesia, rigidity,axial dystonia, supranuclear gaze palsy, and cognitive or be-havioral changes.1 Most cases appear to be sporadic, with asignificant association with a common haplotype includingthe tau gene and the flanking regions.2 Familial cases showan autosomal dominant pattern of transmission with incom-plete penetrance; genetic analysis of a few cases showed theoccurrence of tau mutations, including a deletion of codon296.3

Here, we report the identification of a novel mutation inthe tau gene, resulting in the deletion of the amino acid 296,in a patient affected by a PSP-like syndrome. The proband isa 39-year-old man who developed antecollis, dysarthria, pos-tural instability with falls, slowing of ocular movements, andincreased deep tendon reflexes at age 36 years. Family historyshowed that (1) a paternal uncle developed an atypical par-kinsonism at age 51 years, characterized by resting tremor,rigidity, bradykinesia, pyramidal signs, cognitive impairment,and unresponsiveness to L-dopa, and died 9 years later; (2) apaternal aunt is currently affected by typical Parkinson’s dis-ease (PD) with onset at age 63 years, and good and sustainedresponse to L-dopa; and (3) a paternal uncle was reported tohave died of amyotrophic lateral sclerosis.

Sequencing of exons 9 to 13 of the tau gene in the pro-band showed an heterozygous deletion of the two last basesof codon 296 and the first base of codon 297, producing anew codon identical to codon 297 and having as a final re-sult the deletion of the highly conserved asparagine 296. Thesame mutation was found in the aunt with PD, in twoasymptomatic sisters of the proband, and in three asymptom-atic daughters of the uncle with atypical parkinsonism.

The relevance of codon 296 mutations to the pathogenesisof tauopathies is supported by in vitro experiments,4 showingthat splicing and/or protein function are affected by individ-ual mutations, with decreased microtubule assembly and in-creased tau aggregation. Deletion of codon 296, due to a

distinct mutation, previously was reported in a family withatypical PSP, characterized clinically by cognitive and mem-ory disturbances in addition to oculomotor and gait abnor-malities, and pathologically by PSP-like changes.3,5 The ho-mozygous state produced this severe phenotype, whereasheterozygosity resulted in a mild PD-like condition respon-sive to L-dopa, with reduced penetrance.3 Interestingly, theheterozygous mutation in our family was associated with ei-ther L-dopa–responsive PD (paternal aunt) or PSP-like syn-drome (proband). Because the proband’s father did not showneurological abnormalities until death at age 62 years, in-complete penetrance cannot be excluded. Follow-up ofhealthy relatives carrying the mutation will provide furtherinformation on this issue.

This work was supported by the Italian Ministry of Health, Depart-ment of Social Services (RA 00-48, F.T.).

1National Neurological Institute “Carlo Besta,” Milan; and2Department of Neurological and Psychiatric Sciences,University of Padova, Padova, Italy

References1. Litvan I, Agid Y, Calne D, et al. Clinical research criteria for the

diagnosis of progressive supranuclear palsy (Steele-Richardson-Olszewski syndrome). Neurology 1996;47:1–9.

2. Pastor P, Ezquerra M, Tolosa E, et al. Further extension of theH1 haplotype associated with progressive supranuclear palsy.Mov Dis 2002;17:550–556.

3. Pastor P, Pastor E, Carnero C, et al. Familial atypical progressivesupranuclear palsy associated with homozigosity for the delN296mutation in the tau gene. Ann Neurol 2001;49:263–267.

4. Grover A, DeTure M, Yen SH, et al. Effects on splicing andprotein function of three mutations in codon N296 of tau invitro. Neurosci Lett 2002;323:33–36.

5. Ferrer I, Pastor P, Rey MJ, et al. Tau phosphorylation and ki-nase activation in familial tauopathy linked to delN296 muta-tion. Neuropathol Appl Neurobiol 2003;29:23–34.

DOI: 10.1002/ana.20006

Tau Gene delN296 Mutation, Parkinson’s Disease,and Atypical Supranuclear PalsyRafael Oliva, MD, PhD,1 and Pau Pastor, MD, PhD2

The letter by Rossi and colleagues1 reports a mutation result-ing in the deletion of asparagine 296 in the tau gene in af-fected members with parkinsonism from a family different tothat previously reported.2 Functional studies have shown thatthe delN296 change can decrease the ability of four-repeattau to promote microtubules assembly and increase in theability of recombinant tau protein to aggregate in vitro3,4

with a variable effect on tau exon 10 splicing.3,4

This independent description supports the pathogenicityof this mutation, and therefore it is of outstanding clinicalinterest. The authors also claim a new mutation where thetwo last bases of codon 296 and the first base of codon 297have been deleted, resulting in the deletion of the highlyconserved asparagine 296. However, as compared with thepreviously reported mutation,2 the final result is the same atthe resulting amino acid sequence (deletion of asparagine

448 Annals of Neurology Vol 55 No 3 March 2004

296) as well as in the resulting DNA sequence (GAT ATC).Thus, the mutation reported by Rossi and colleagues1 is notnovel, but it is exactly the same as that previously reported.2

A different issue is to decide for a consensus nomenclatureat the DNA level for this delN296 mutation. The interna-tional recommendations to describe complex mutations es-tablishes that, for variations in tandem repeats, the most 3�copy is arbitrarily assigned to have been changed.5 As shownin the Figure, there are several potential mechanisms result-ing in the deletion of asparagine 296. Thus, we propose fol-lowing these recommendations that the complete nomencla-ture for this mutation1,2 should be arbitrarily “c713-715delATA” at the cDNA level (based on GenBank cDNAaccession number X14474) and delN296 at the protein level.

This tau delN296 mutation initially was reported as caus-ing a severe atypical progressive supranuclear palsy phenotypewhen in an homozygous state and as associated to L-dopa–responsive parkinsonism with low penetrance when in anheterozygous state.2 The report of this tau delN296 muta-tion in heterozygous state in an independent family affectedby parkinsonism1,2 should now prompt its search in otherindependent cases to determine more accurately its pen-etrance. If further detected, the tau delN296 heterozygousvariation will have to be considered as an important risk fac-tor for developing either L-dopa–responsive parkinsonism ora progressive supranuclear palsy–like syndrome.

1Genetics Service, Hospital Clınic, and University ofBarcelona, Institut d’Investigacions Biomediques August Pi ISunyer, Barcelona, Spain; and 2Department of Psychiatry,Washington University School of Medicine, St. Louis, MO

References1. Rossi G, Gasparoli E, Pasquali C, et al. Progressive supranuclear

palsy and Parkinson’s disease in a family with a new mutation inthe tau gene. Ann Neurol 2004;55:448.

2. Pastor P, Pastor E, Carnero C, et al. Familial atypical supranu-clear palsy associated with homozigosity for the delN296 muta-tion in the tau gene. Ann Neurol 2001;49:263–267.

3. Grover A, DeTure M, Yen SH, Hutton M. Effects on splicingand protein function of three mutations in codon N296 of tauin vitro. Neurosci Lett 2002;323:33–36.

4. Yoshida H, Crowther RA, Goedert M. Functional effects of taugene mutations deltaN296 and N296H. J Neurochem 2002;80:548–551.

5. Dunnen JT, Antonarakis SE. Mutation nomenclature extensionsand suggestions to describe complex mutations: a discussion.Hum Mutat 2000;15:7–12.

DOI: 10.1002/ana.20025

Deep Brain Stimulation for Parkinson’s Disease:Potential Risk of Tissue Damage Associated withExternal StimulationWassilios Meissner, MD,1,2 Christian E. Gross, PhD,1

Daniel Harnack, MD,2 Bernard Bioulac, MD, PhD,1 andAbdelhamid Benazzouz, PhD1

Deep brain stimulation (DBS) of the subthalamic nucleus(STN) has been shown to alleviate main motor symptoms inlate-stage Parkinson’s disease (PD).1 For this purpose, aquadripolar stimulation electrode is bilaterally placed in theSTN, which then is connected to a subcutaneously placedbattery-operated programmable pulse-generator. Frequency,pulse width, and voltage can be set individually for the pa-tient’s best benefit. In contrast, peroperatively, the clinicaleffectiveness of DBS is tested with an external device. Al-though the implantable pulse-generator delivers a charge bal-anced capacitance-coupled current (Fig, A) in a constantvoltage mode, the external stimulator generates onlymonophasic pulses (see Fig, B). Such a monophasic charge-inbalanced current has been reported to induce extensive tis-sue damage in animal studies.2,3 However, the stimulationparameters in all these studies differed from those used forDBS in humans. Thus, the question remains whether perop-erative external DBS is safe for the integrity of stimulatedbrain tissue. To test this hypothesis, we applied STN-DBS innonhuman primates with the same either implantable (ItrelII; Medtronic, Minneapolis, MN) or external device (Model3625 External Test Stimulator Internal Controls; Medtronic)as used in humans. All experiments were performed in ac-cordance with European Communities Council Directive of24 November 1986 (86/609/EEC) and National Institutes ofHealth Guide for the Care and Use of Laboratory Animals.Animals were housed in a temperature-and humidity-controlled vivarium with a 12-hour light-dark cycle. Foodand water were available ad libitum. In brief, a stimulationelectrode (SNEX 100; RMI, Woodland Hills, CA) was im-planted in the left STN of two Macaca fascicularis (3.8 and2.7kg).4 After recovery from the surgery, animals were ren-dered parkinsonian by repeated injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a cumulative dosing regi-men that leads to the first appearance of parkinsonian signsafter 15 � 1 injections.5 After stabilization of parkinsoniansymptoms, monopolar STN-DBS was applied in a constantvoltage mode. The voltage was set to obtain the best clinicalimprovement (Itrel II: frequency, 130Hz; pulse width, 60microseconds; voltage, 2.5V for 10 days, n � 1, implanted

Fig. Four potential mechanisms resulting in the delN296 taugene mutation.

Annals of Neurology Vol 54 No 3 March 2004 449

during the same surgery as the electrode; Model 3625 Exter-nal Test Stimulator Internal Controls: frequency, 130Hz;pulse width, 60 microseconds; voltage, 1.5V for 2 hours,n � 1). No abnormal involuntary movements were observedduring STN-DBS. Animals were killed with an overdose ofpentobarbital (150mg/kg IV). DBS was turned off just be-fore brains were removed. STN-DBS for 10 days with theimplantable Itrel II stimulator did not show any morpholog-ical alterations of brain tissue except for a mild gliosis due tothe insertion of the electrode (see Fig, C). In contrast, STN-DBS for 2 hours with the external stimulator resulted in se-vere neuronal tissue damage within a wide region around thetip of the electrode, although duration of DBS and voltagewere lower compared with the implantable device (see Fig,D). Peroperative external STN-DBS usually is applied forminutes. Thus, our data do not prove that short-term stim-ulation for seconds or some minutes may lead to any signif-

icant damage above that incurred by the surgical procedure.However, clinicians should be aware of the potential risk oftissue damage arising from the application of a charge-inbalanced monophasic current. In other words, short-termstimulation with the external device must be restricted to theoperating room for successful placement of the electrodes inthe target and excludes longer postoperative stimulation, thatis, extensive testing of different parameters or continuedtherapeutic DBS before implantation of the chronic device.

We thank S. Dovero and L. Cardoit for their technical assistance.W.M. is a Marie Curie Fellow of the European Community(HPMF-2001-01300). This study was supported by the NationalScientific Research Center (Centre National de Recherche Scienti-fique, CNRS), the University Victor Segalen Bordeaux, and theMedical Research Foundation (Fondation de la Recherche Medicale,INE20020412006/1, A.B.).

1Basal Gang, Laboratoire de Neurophysiologie, CNRS UMR5543, Universite Victor Segalen, Bordeaux Cedex, France,and 2Department of Neurology, Charite Campus Virchow,Humboldt-University, Berlin, Germany

References1. Limousin P, Krack P, Pollak P, et al. Electrical stimulation of the

subthalamic nucleus in advanced Parkinson’s disease. N EnglJ Med 1998;339:1105–1111.

2. Wetzel MC, Howell LG, Bearie KJ. Experimental performanceof steel and platinum electrodes with chronic monophasic stim-ulation of the brain. J Neurosurg 1969;31:658–669.

3. Lilly JC, Hughes JR, Alvord EC, et al. Brief, non-injouriouselectric waveform for stimulation of the brain. Science 1955;121:468–469.

4. Benazzouz A, Gross C, Feger J, et al. Reversal of rigidity andimprovement in motor performance by subthalamic high-frequency stimulation in MPTP-treated monkeys. Eur J Neurosci1993;5:382–389.

5. Bezard E, Dovero S, Prunier C, et al. Relationship between theappearance of symptoms and the level of nigrostriatal degenerationin a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque model of Parkinson’s disease. J Neurosci 2001;21:6853–6861.

DOI: 10.1002/ana.20002

Glutamic Acid Decarboxylase Antibodies inSatoyoshi SyndromeGea Drost, MD,1,2 Aad Verrips, MD, PhD,3

Herbert Hooijkaas, PhD,4 andMachiel Zwarts, MD, PhD1,2

Satoyoshi syndrome is a childhood-onset multisystem disor-der characterized by intermittent painful muscle spasms, mal-absorption, and alopecia. It has a progressive course with anunknown cause, although, mainly because of its associationwith other autoimmune conditions, an autoimmune basis isthought to be likely.1 Here, we report the novel finding ofantibodies against glutamic acid decarboxylase (GAD) in theserum of a child with Satoyoshi syndrome, confirming theautoimmune origin of the disease.

A 6-year-old Turkish girl suffered from progressive painful,mainly exercise-induced muscle contractions and thinning

Fig. Schematic figure of the pulse delivered by the Itrel II (A)and the Model 3625 External Test Stimulator Internal Con-trols (B). Note the difference between the charge-balancedcapacitance-coupled current (A) and the monophasic charge-inbalanced current (B). Cresyl violet staining of subthalamicnucleus sections (C, D). Bars � 680�m. Subthalamic nucleusdeep brain stimulation with the internal stimulator did notshow any morphological alterations of brain tissue except for amild gliosis due to the insertion of the electrode (C). Incontrast, stimulation with the external device resulted in severetissue damage (D). Pathomorphological analysis of cresylviolet–stained sections showed a widespread edema and anelectrolysis-induced cavitation containing necrotic cells andcellular debris around the tip of the electrode.

450 Annals of Neurology Vol 55 No 3 March 2004

hair, resulting in occipital alopecia. General and neurologicalexamination showed no other abnormalities. Laboratory inves-tigations showed a serum creatine kinase level of 296U/L (ref-erence value �200U/L), the presence of antinuclear antibod-ies, and a transient anemia. Endocrine investigation (thyroidgland, pituitary function, urine steroid profile, serum andurine glucose) and quadriceps muscle biopsy were normal.During muscle spasms, electromyography (EMG) with surfaceelectrodes showed highly synchronized motor unit discharges.Hair root analysis showed a high percentage of catagen folli-cles. A diagnosis of Satoyoshi syndrome was made, and ther-apy was started with prednisone 1mg/kg/day for 3 months.Muscle spasms completely disappeared during therapy.

The abnormal surface EMG findings, and the spasms trig-gered by voluntary contractions in our patient, suggested ab-normal excitability of the motor neuron pool. Such abnor-malities in spinal inhibitory interneuronal circuits also aresuggested to be the underlying cause in another rare autoim-mune disorder, the stiff-person syndrome. Symptoms in stiff-person syndrome are muscle rigidity and episodic spasms in-volving the axial and limb musculature. Various patterns ofpresentation2 and alopecia3 have been described in stiff-person syndrome. Sixty-five percent of patients with stiff-person syndrome may have GAD antibodies.4 We thereforedecided to check for GAD antibodies in the serum of ourpatient, which indeed were present: 1.3U/ml (refence value�0.9U/ml). Low values of anti–GAD antibodies also can befound in patients with type I diabetes or in prediabetic pa-tients; however, our patient had normal glucose tolerancetests. The different clinical expression of the spinal hyperex-citability in Satoyoshi syndrome and stiff-person syndromecould be explained by age-dependent differences in matura-tion of inhibitory circuits.

In conclusion, the finding of GAD antibodies in Satoyo-shi syndrome supports the presumed autoimmune origin ofthe disease. Furthermore, this finding shows that Satoyoshisyndrome in childhood can be a variant of stiff-person syn-drome. We suggest that GAD antibodies should be deter-mined in all patients with Satoyoshi syndrome, especially be-cause stiff-person syndrome is potentially treatable.5

1Department of Clinical Neurophysiology, 2NeuromuscularCentre Nijmegen, Institute of Neurology, University MedicalCentre Nijmegen, 3Department of Neurology, CanisiusWilhelmina Hospital, Nijmegen, 4Department of Immunology,Erasmus MC, University Medical Centre Rotterdam,Rotterdam, The Netherlands

References1. Satoyoshi E. A syndrome of progressive muscle spasm, alopecia,

and diarrhea. Neurology 1978;28:458–471.2. Barker RA, Revesz T, Thom M, et al. Review of 23 patients

affected by the stiff man syndrome: clinical subdivision into stifftrunk (man) syndrome, stiff limb syndrome and progressive en-cephalomyelitis with rigidity. J Neurol Neurosurg Psychiatry1998;65:633–640.

3. Mueller-Schoop JW, Meyer M. Total alopecia, diabetes mellitus,and falls. Lancet 1996;348:1420.

4. Levy LM, Dalakas MC, Floeter MK. The stiff person syndrome:an autoimmune disorder affecting neurotransmission of �-amino-butyric acid. Ann Int Med 1999;131:522–530.

5. Dalakas MC, Fujii M, Li M, et al. High-dose intravenous im-mune globulin for stiff-person syndrome. N Engl J Med 2001;345:1870–1876.

DOI: 10.1002/ana.20007

Neuromuscular Transmission in SCA6H. Jurgen Schelhaas, MD,1

Bart P. C Van de Warrenburg, MD,1

Hubertus P. H. Kremer, MD, PhD,1 andMachiel J. Zwarts, MD, PhD2

Spinocerebellar ataxia type 6 (SCA6) is caused by a smallCAG expansion in the CACNA1A gene encoding the �1A

subunit of the neuronal voltage-dependent P/Q-type Ca2

channel (P/Q-VGCC).1 P/Q-VGCCs are widely expressed,not only in the central nervous system (CNS), but also at theneuromuscular junction.2 The presence of P/Q-VGCCs atmotor nerve terminals, together with the demonstration thatSCA6–related polyglutamine expansions may influencechannel function,3 suggests that neuromuscular transmissionmight be compromised in SCA6. However, muscle weaknessis not a clinical feature of SCA6. Therefore, a possible neu-romuscular defect is likely to be subclinical and might appearas an increased jitter without blockings in single fiber elec-tromyography.

Ten SCA6 patients from eight different families partici-pated in the study. In all patients, genetic analysis showed aCAG expansion in the SCA6 gene of 22 repeats. To studyboth proximal and distal muscles, we assessed the variabilityin latency (jitter) of single-fiber action potentials both in theextensor digitorum communis muscle (four patients) and inthe orbicularis oculi muscle (six patients). The data areshown and the results were all normal (Table). Our resultsconfirm a preliminary observation that neuromuscular dys-function is not a feature of SCA6.4 These findings appear tobe inconsistent with the results in patients with episodicataxia type 2 (EA-2),4 a disorder allelic with SCA6, but usu-ally caused by more damaging frameshift mutations or aber-rant splicing that both result in truncated proteins. Cur-rently, the question whether SCA6 is a channelopathy causedby an endogenous dysfunction of the calcium channel in-volved or results from a toxic gain of function attributable tothe expanded polyglutamine tract, as seen in other CAG ex-pansion disorders, remains unanswered. Either way, ourstudy indicates that SCA6 results from CNS-specific diseasepathways. Two possible mechanisms might account for theCNS selectivity. First, the presence of pathogenic spliceforms of the CACNA1A gene products may be confined tothe CNS compartment. Second, interaction with tissue-specific subunits of the channel seems to be important, as aprevious study suggested that interaction with the 4 sub-unit, which is primarily found in Purkinje cells and not inthe neuromuscular junction, is crucial for the developmentof neuronal dysfunction.5

Although some believe that SCA6, EA-2, and familialhemiplegic migraine are representatives of the same diseasewith a large phenotypical variability, the nature of the mu-tation apparently results in a distinctive phenotype at thelevel of neuromuscular transmission.

Annals of Neurology Vol 55 No 3 March 2004 451

Departments of 1Neurology and 2Clinical Neurophysiology,Institute of Neurology, University Medical Center Nijmegen,The Netherlands

References1. Zhuchenko O, Bailey J, Bonnen P, et al. Autosomal dominant

cerebellar ataxia type 6 (SCA6) associated with small polyglu-tamine expansions in the alpha 1A-voltage dependent calciumchannel. Nat Genet 1997;15:62–69.

2. Uchitel OD, Protti DA, Sanchez V, et al. P-type voltage depen-dant calcium channel mediates presynaptic calcium influx andtransmitter release in mammalian synapses. Proc Natl Acad SciUSA 1992;89:3330–3333.

3. Piedras-Renteria ES, Watase K, Harata N, et al. Increased ex-pression of �1A Ca2 channel currents arising from expandedtrinucleotide repeats in spinocerebellar ataxia type 6. J Neurosci2001;21:9185–9193.

4. Jen J, Wan J, Graves M, et al. Loss-of-function EA2 mutationsare associated with impaired neuromuscular transmission. Neu-rology 2001;57:1843–1848.

5. Restituito S, Thompson RM, Eliet J, et al. The polyglutamineexpansion in spinocerebellar ataxia type 6 causes a subunit-specific enhanced activation of P/Q-type calcium channels in Xe-nopus oocytes. J Neurosci 2000;20:6394–6403.

DOI: 10.1002/ana.20007

Table. Single-Fiber Electromyogram in 10 Patients with Spinocerebellar Ataxia Type 6

Age (yr)/Sex

Musculus ExtensorDigitorum Communis M. Orbicularis Oculi

Mean MCD(�sec)

(N � 25)

MCD,Range (�sec)

(N � 40)

Percentage of PulsesBlocked(N � 0)

Mean MCD(�sec)

(N � 20)

MCD,Range (�sec)

(N � 31)

Percentage of PulsesBlocked(N � 0)

62/F 24 12–38 039/F 18 8–32 073/F 23 12–35 034/F 22 13–30 027/M 17 6–29 033/M 20 13–26 056/F 18 10–29 058/M 16 10–23 049/M 16 10–27 069/M 18 9–29 0

ADM � abductor digiti minimi; MCD � mean consecutive difference.

452 Annals of Neurology Vol 55 No 3 March 2004