Mutations in SYNE1 lead to a newly discovered formof autosomal recessive cerebellar ataxiaFrancois Gros-Louis1, Nicolas Dupre1,2, Patrick Dion1, Michael A Fox3, Sandra Laurent1, Steve Verreault2,Joshua R Sanes3, Jean-Pierre Bouchard2 & Guy A Rouleau1

The past decade has seen great advances in unraveling thebiological basis of hereditary ataxias. Molecular studies ofspinocerebellar ataxias (SCA) have extended our understandingof dominant ataxias1. Causative genes have been identified fora few autosomal recessive ataxias: Friedreich’s ataxia2, ataxiawith vitamin E deficiency3, ataxia telangiectasia4, recessivespastic ataxia of Charlevoix-Saguenay5 and ataxia withoculomotor apraxia type 1 (refs. 6,7) and type 2 (ref. 8).Nonetheless, genes remain unidentified for most recessiveataxias. Additionally, pure cerebellar ataxias, which representup to 20% of all ataxias, remain poorly studied with only twocausative dominant genes being described: CACNA1A (ref. 9)and SPTBN2 (ref. 10). Here, we report a newly discoveredform of recessive ataxia in a French-Canadian cohort and showthat SYNE1 mutations are causative in all of our kindreds,making SYNE1 the first identified gene responsible for arecessively inherited pure cerebellar ataxia.

Hereditary ataxias are classified as autosomal dominant, auto-somal recessive, X-linked and mitochondrial. Despite these differences,all ataxias share the prototypic feature of impaired walking with alack of coordination of gait and limbs. Many affected individualsalso have additional neurological symptoms such as pyramidalfeatures, peripheral neuropathy, extrapyramidal signs, cognitive lossor retinopathy.

We identified a geographically defined group of 26 French-Canadian families, including 53 affected family members, most ofwhich originate from the Beauce and Bas-St-Laurent regions of theprovince of Quebec. All of the affected family members have a similarphenotype, which consists of late-onset cerebellar ataxia with slowprogression accompanied by dysarthria, with few associated featuresother than dysmetria, occasional brisk lower-extremity tendon reflexesand minor abnormalities in saccades and smooth pursuit (Table 1 andSupplementary Fig. 1 online). We named this phenotype autosomalrecessive cerebellar ataxia type 1 (ARCA1), also known as recessiveataxia of Beauce.

Genome-wide linkage analysis of selected families (SupplementaryFig. 2 online) showed only one marker, D6S476, with a maximum lodscore above 3.0 (Supplementary Table 1 online). We genotyped 21additional flanking markers surrounding D6S476 and established twodifferent disease haplotypes that segregated with the disease in selectedfamilies. Fine-mapping established a minimum candidate interval ofabout 0.5 Mb on chromosome 6q, between markers D6S420 and

Table 1 Clinical features of individuals with ARCA1

Clinical feature Frequency (%)

Male sex 31/53 (58)

Nystagmus, gaze-evoked 7/53 (13)

Abnormal saccades 16/51 (31)

Slow/jerky pursuit 23/51 (45)

Cerebellar dysarthria 53/53 (100)

Brisk lower limb reflexes 14/53 (26)

Limb ataxia 52/53 (98)

Cerebellar gait ataxia 52/53 (98)

Cerebellar atrophy on MRI or CT 34/34 (100)

Mean age at onset (range) 30.4 (17–46)

Mean age at assessment (range) 42.9 (24–69)

Mean duration of symptoms 17.7

None of the subjects showed optic atrophy, auditory loss, sensory abnormalities,autonomic disturbances or extrapyramidal signs. Nerve conduction studies carried out on18 affected individuals were all within normal limits. Imaging findings by computedtomography (CT) or magnetic resonance imaging (MRI) invariably showed diffuse purecerebellar atrophy (Supplementary Fig. 1). Therefore, this disease represents the firstrecessively inherited ’pure’ cerebellar ataxia mapped until now. The closest clinicaldescription to this newly identified form of recessive ataxia, which we refer to asautosomal recessive cerebellar ataxia type 1 (ARCA1), is Holmes’ type of hereditaryataxia, first described in 1908 by Gordon Holmes22. In his report, Holmes described afamily of eight siblings in which four members presented an adult-onset dysarthria andataxia. Inheritance was probably autosomal recessive, as the parents were not affected,and the autopsy of one affected individual affected with Holmes’ ataxia showed a diffusecerebellar atrophy. Other histological features included narrowing and sclerosis of themolecular layer, loss of almost all Purkinje cells and a considerable, although variable,loss of granule cells. To our knowledge, no linkage or gene defect has been reported asbeing responsible for this type of ataxia.

Received 4 July; accepted 23 October; published online 10 December 2006; doi:10.1038/ng1927

1Centre for the Study of Brain Diseases, Centre Hospitalier de l’Universite de Montreal and Centre Hospitalier Universitaire – Ste-Justine, Universite de Montreal,Montreal, Quebec, H2L 4M1, Canada. 2Faculty of Medicine, Laval University, Department of Neurological Sciences, Centre Hospitalier Affilie Universitaire de Quebec –Enfant-Jesus Hospital, Quebec City, Quebec, G1J 1Z4, Canada. 3Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138,USA. Correspondence should be addressed to G.A.-R. (guy.rouleau@umontreal.ca).

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GATA186B06 (Fig. 1). Our candidate interval contained only onegene, SYNE1, which spans over 0.5 Mb of genomic DNA. It com-prises 147 exons and encodes a 27,652-kb mRNA and an 8,797-aminoacid protein.

We screened all of the exons and flanking intronic sequences ofSYNE1 for the presence of mutations by direct sequencing in affectedindividuals from selected families. We identified 115 SNPs, includingtwo disease-segregating SNPs that were not detected among 380 age-and ethnicity-matched control chromosomes. This observation led usto believe that these two variants may be causative mutations forARCA1 (Table 2 and Supplementary Table 2 online). The firstmutation affects the invariant A of the AG splice acceptor site at thejunction of exon 85 and intron 84 (310067A-G), and the secondmutation is located in intron 81, 12 bp upstream of exon 82(306434A-G), creating a new AG cryptic slice acceptor site(Fig. 2). RT-PCR and sequencing analysis showed that the detectedintronic mutations had functional consequences on the proper spli-

cing of the gene and resulted in the prematuretermination of the protein (Fig. 2). Based onthe haplotype reconstructions of affectedindividuals from all of the other families, weidentified three other different disease haplo-types, suggesting that other mutations couldbe associated with the disease (Fig. 1). Asecond mutational screen by direct sequen-cing uncovered three additional mutations(R2906X, 334338–334342delATTTG and

Q7640X) that segregated with their respective haplotypes and wereall predicted to lead to premature termination of the protein (Fig. 2).We did not detect these additional mutations among 380 age- andethnicity-matched control chromosomes (Table 2). These resultsestablish SYNE1 as the causative gene for this newly identified formof recessive ataxia with pure cerebellar atrophy. Because we haveidentified five different mutations in a relatively homogenous popula-tion, we predict that mutations in this gene may be responsible for asubstantial fraction of all adult-onset autosomal recessive ataxiasyndromes with cerebellar atrophy.

Syne-1 is a large protein that is expressed in multiple tissues, includ-ing the central nervous system11. Given that we observed cerebellaratrophy in all of the affected individuals, we examined the expressionof Syne-1 by immunohistochemistry in normal mouse brain cross-sections. We detected the greatest expression in the cell bodies ofPurkinje cells in the cerebellar cortex (Fig. 3a) and in neurons fromthe olivary region of the brain stem (Fig. 3b). The Allen Institute for

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Figure 1 Fine-mapping analysis of seven selected

families with two or more affected family

members diagnosed with ARCA1. Overall, five

different disease haplotypes, named A, B, C, D

and E, have been established from fine-mapping

studies. The upper panel represents disease

haplotypes uncovered from selected families

(Supplementary Figure 2) chosen for the initial

screen. Affected individuals homozygous for

haplotype A or compound heterozygous for

haplotypes A and B all developed the disease,

whereas carriers of one or the other haplotypes

were unaffected. None of the affected individuals

was homozygous for haplotype B (haplotype A:

shaded; haplotype B: black). Stars pinpoint upperand lower key recombinants defining the 0.5-Mb

candidate region defined by genetic markers

D6S420 and GATA186B06. The lower panel

shows the detected haplotypes A, B, C, D and E

of 54 affected individuals. Polymorphic

microsatellite markers within and adjacent to the

candidate region and disease segregating SNPs,

identified by sequencing, were used to

reconstruct haplotypes from all other families.

Affected individuals homozygous for haplotype C

and compound heterozygous for haplotypes A,

and either haplotype C, D and E, all developed

disease, whereas carriers of one or the other

haplotypes were unaffected. None of the affected

individuals was homozygous for haplotype D or E.

The nucleotide position of each haplotype

segregating variant is numbered relative to the A

of the ATG initiation codon from genomic DNA(NM_033071). N is the number of alleles of a

given haplotype for each affected individuals. The

causative mutation for each haplotype is outlined

in shaded boxes.

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Brain Research (http://www.brain-map.org) freely provided in situhybridization data that showed that Syne-1 was expressed predomi-nantly in the cerebellum, which is consistent with our results. We didnot observe any immunostaining in non-neuronal cells, including glialcells (Fig. 3b).

Syne-1 is involved in anchoring specialized myonuclei underneathneuromuscular junctions (NMJ)12. These nuclei are transcriptionallyspecialized and express greater amounts of synaptic components, suchas the acetylcholine receptor (AChR) subunit genes, than nonsynapticnuclei in the same muscle fibers13. Using AChR markers, immuno-histochemical analysis of muscle biopsies from one affected individualand one control showed that in B80% (51/63) of the NMJs examinedin control muscles, smooth myonuclei were directly beneath thepostsynaptic membrane (Fig. 4a). In contrast, nuclei were presentbeneath AChRs in only B40% (12/28) of the NMJs in the affectedindividual (difference between control and affected individual,P o 0.001 by w2 test). Instead, nuclei were often adjacent to AChR-rich areas in the affected individual, as has been seen previously intransgenic mice (Fig. 4a). These observations are consistentwith the idea that the anchoring of specialized myonuclei at synapticsites requires Syne-1. The formation of NMJs seems to benormal, and we did not see any apparent structural defects ordifferences in NMJs in muscle biopsies from control and affectedindividuals (Fig. 4b).

The most outstanding feature of SYNE1 is its size. It encodes aprotein of about 8,797 amino acid residues (41,000 kDa). The proteincontains two N-terminal actin-binding regions that comprise tandempaired calponin-homology-domains, a transmembrane domain, mul-tiple spectrin repeats and a C-terminal klarsicht domain (KASH).There are proteins that are homologous to Syne-1 in Caenorhabditiselegans (ANC-1) and Drosophila melanogaster (MSP-300)14,15. Theseorthologous proteins are very large and contain both the N-terminalactin-binding/calponin domains and the C-terminal KASH domain.Overexpression of the KASH domain in C. elegans results in thedisruption of the proper positioning of the nuclei and mitochondria inthe large syncytial hypodermal cells14. RNA interference (RNAi)-mediated knockdown of ANC-1 in C. elegans and the description ofa mutant MSP-300 strain in D. melanogaster have shown that thedisruption of Syne-1 homologous proteins causes larval lethality16,17.In addition, Syne-1 defects in humans result in less severe adult-onsetphenotypes. These findings suggest that higher vertebrates may useadditional compensatory mechanisms for cellular nuclear migrationand seem to indicate that the Syne proteins may have adapted througha functional evolution in vertebrates to perform a specialized functionin the brain. Notably, although we saw abnormal positioning ofmyonuclei at the NMJ in a muscle biopsy from an affected individual,

we did not see a clinically or electrophysiolo-gically detectable muscle phenotype in theaffected individuals. This suggests that post-synaptic nuclear aggregation abnormalitiesdetected in affected individuals do not havea crucial function in the maturation or main-tenance of NMJs. Instead, Syne-1 seems to beimportant in the development and mainte-nance of cerebellar functions. Given the pre-cise positioning of Purkinje cell nuclei and thefact that Syne-1 is highly expressed in thiscellular type, one might speculate that loss ofSyne-1 function may disrupt cerebellar archi-tecture leading to the ARCA1 phenotype.

The protein product of CPG2, a brain-specific splice variant of SYNE1, is localized to the postsynapticendocytotic zone of excitatory synapses and disrupts glutamatereceptor internalization, suggesting that CPG2 may be necessary forthe rapid cycling of synaptic glutamate receptors18. Because ARCA1involves predominantly central nervous system defects, it is possiblethat interference with the CPG2 isoform of SYNE1 explains ARCA1symptoms, as glutamate toxicity has been implicated in other neuro-degenerative disorders such as Alzheimer disease, Huntington diseaseand amyotrophic lateral sclerosis. The mutations in SYNE1 describedhere, however, are located downstream of the final exon of CPG2 andthus are not predicted to affect its function. Nonetheless, it will beimportant to assess CPG2 expression in brain tissue from individualswith ARCA1.

All spectrin family members, including spectrin itself, dystrophinand utrophin, seem to share a common function: linking the plasmamembrane to the actin cytoskeleton. Syne-1 joins many members ofthe family that are implicated in human diseases. Mutations in thedystrophin and in the beta-III spectrin (SPTBN2) genes cause Duch-enne muscular dystrophy19 and spinocerebellar ataxia type 5 (ref. 10),respectively. In addition, the PLEKHG4 gene, which codes for aprotein with spectrin repeats, is associated with a rare form ofdominant progressive pure cerebellar ataxia20. Finally, the spontaneousoccurrence of recessive mutations in the mouse spectrin beta 4 gene(Spnb4) causes a progressive ataxia with hindlimb paralysis, deafnessand tremor in the quivering mouse21.

Taken together, these results support the idea that spectrinrepeat proteins are important in homeostasis and structural integrity.The identification of additional mutations in SYNE1 in otherfamilies with recessive and pure cerebellar ataxia of different ancestrieswould confirm that this locus is a prominent cause of this typeof disorder. Furthermore, the detection of additional SYNE1mutations will provide insights into the function of this gene andinto the molecular mechanisms involved in ARCA1 and possiblyother neurodegenerative diseases. Ultimately, the complete explana-tion of the function of this gene and of the specific functionalproperties of spectrin repeats in the brain would greatly contributeto the advancement of knowledge in this field and to the developmentof therapies.

METHODSClinical and genetic studies. All available affected and unaffected family

members underwent a standardized neurological examination and were ascer-

tained independently by at least two neurologists. All procedures were in

accordance with the ethical standards of all of the institutional ethic committees

involved in this project, and the Declaration of Helsinki protocols were followed.

After participants gave informed consent, blood samples were obtained from

affected and unaffected individuals in 27 families. DNA was extracted from

Table 2 Details, location, and genomic context of all detected mutations

Varianta Exon or intron Protein change

Control

allele frequency

247012A-T Exon 56 R2906X 0/380

306434A-G Intron 81 Premature stop at position 5244 0/380

310067A-G Intron 84 Premature stop at position 5402 0/380

334338–334342delATTTG Exon 93 Premature stop at position 5880 0/380

426494C-T Exon 126 Q7640X 0/380

Overall, 115 SNPs were detected, including five different mutations identified in 26 French-Canadian families, andascertained based on the common and uniform clinical phenotype. This finding is consistent with the fact that the largesize of the SYNE1 gene makes it a large target for mutation.aVariants were named according to the genomic DNA sequence NM_033071, and nucleotide A from the ATG initiation codon isreferred to as 1.

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Genomic DNA Complementary DNA

Exon 81N

orm

alH

omoz

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edExon 82

Exon 81 Exon 82

11 extra codingnucleotides

Effect on the proteina

Exon 84 Exon 85

Exon 84 Exon 85

Extra coding nucleotide

Nor

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Exon 126

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C C C CA A AAT T T T T TTG G G C CA ATG

C C C AT T T T T T TG G G

C C C CA AA T T TG G G

C C C CAA T T TG G G G

C C C A AAT TG G G G G

C C CA AT T TG G G G G

C C CA A A A A A AT TG G G

C C CA A A A A A AT TG G G

C CA AT T T T TG G G G G G

C CT T T TG G G N NG G N –

C C CA A A A TG G G G G G G G

C C CA A A A T TG G G G G G

C C C CAA AA AT T T T T TGG G G G

Figure 2 SYNE1 gene mutations and their predicted outcomes on translated proteins. (a) Sequencing results showing the detected intronic 306434A-G

mutation, using genomic DNA and cDNA, in a normal individual and a homozygous affected individual. The normal splicing event, between exon 81 and 82,

is shown in the upper panel. The lower panel shows the abnormal splicing event created by the mutation (in green) followed by the 11 extra coding

nucleotides, along with the resulting eight new amino acids (in red) with a premature stop codon. (b) Sequencing results showing the detected intronic

310067A-G mutation, using genomic DNA and cDNA, in a normal individual and a heterozygote carrier. The upper panel shows the normal splicing event

between exon 84 and 85. The lower panel shows the abnormal splicing event created by the mutation (in green), followed by the extra coding guanine and

the resulting change in the reading frame, with a premature stop codon nine amino acids downstream of the site of the mutation (in red). (c–e) Sequencing

results showing the R2906X, 334338–334342delATTTG and Q7640X mutations, respectively, that detected using genomic DNA. The upper panel shows a

portion of the normal genomic and protein sequences of exons 56, 93 and 126, respectively. The lower panel shows the abnormal effect of each mutation,

highlighted in green.

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peripheral blood by standard methods. Twenty-six samples, including those

from 20 affected individuals and 6 unaffected parents or siblings, were

genotyped with a panel of 388 microsatellite markers (created by the Genome

Quebec Innovation Centre), which spanned all of the chromosomes at approxi-

mately 10-cM intervals. Linkage analysis was then computed with FASTLINK

software (v 4.1) using the following parameter assumptions: all families map to

the same locus, inheritance is autosomal recessive, penetrance is 98%, there are

no phenocopies and gene frequency in the population is 1/1,000. Areas that had

positive linkage scores were further refined using additional microsatellite

markers. For this fine-mapping, we created our own panel of markers, at

approximately 2-cM intervals, within the linkage areas confirmed by the genome

scan. Marker positions were determined using the Marshfield genetic map

(Marshfield Center for Medical Genetics). Primers for each marker were

generated from their respective University of California Santa Cruz (UCSC)

genome browser sequence (UCSC Human Genome Project Working Draft).

Alleles were visualized by incorporating [35S]dATP into PCR products and were

separated on a 6% polyacrylamide gel. The size and frequency of the alleles were

based on values from the Fondation Jean Dausset–Centre d’Etude du Poly-

morphisme Humain (CEPH) database and were compared with an M13mp18

sequence ladder. Two-point and multipoint linkage analyses were carried out

using the MLINK and LINKMAP programs, respectively, of the LINKAGE

software package 11. CEPH allele frequencies for each marker were used to

determine LOD scores, using the parameters described above.

Identification of the mutations. The gene structure of SYNE1 was obtained

from publicly available databases (http://genome.ucsc.edu/). A set of 154 PCR

primer pairs was designed from genomic DNA to amplify each exon of the

SYNE-1 gene, including the flanking splice sites and the 5¢ and 3¢ UTRs

(Supplementary Table 3 online). Products were PCR amplified (30 cycles

with the following conditions: 94 1C for 30 s, 601C for 30 s and 721C for

30 s), electrophoresed on agarose gels and then sequenced using the forward

primer for all of the amplicons. The PCR fragment sequence variations were

also sequenced on the reverse strands. All sequencing results was generated

using the sequencing platform of the McGill University Genome Quebec

Innovation Centre.

Total RNA was extracted from transformed lymphoblastoid cell lines derived

from affected individuals using a standard protocol with lithium chloride.

cDNA synthesis was done using standard protocols with a mix of oligo-dT,

random hexamer primers and Moloney murine leukemia virus reverse tran-

scriptase. PCR primer pairs were designed to amplify each implicated exon and

the surrounding DNA sequences to confirm that the identified mutations led to

changes in the coding sequences of the gene.

Figure 4 Neuromuscular junctions in ARCA1

affected individual. (a) Synaptic nuclei were

mislocalized at the NMJs of an individual with

ARCA1. Muscle biopsies from the control or the

affected individual were double-stained with

a-BTX (in red) and antibodies to core histones

(in green). In the control samples, synaptic

nuclei were directly beneath BTX-labeled AChRs

(arrows). In the samples from the individual with

ARCA1, however, nuclei seemed to be displaced

to the periphery of the NMJs (scale bar ¼ 10

mm). (b) The morphologies of NMJs in a control

individual and an individual with ARCA1 were

assessed by labeling presynaptic terminals with

anti-synapsin (in green) and labeling postsynaptic

AChRs with a-BTX (in red). As in the controls,

vesicle-rich nerve terminals in the individual with

ARCA1 were directly apposed to the AChR-rich postsynaptic membrane. The individual with ARCA1 did not differ detectably from the control in the size orshape of pre- or postsynaptic specializations or in the degree of their apposition to each other. NMJs seemed normal in ARCA1 skeletal muscle, suggesting

that synapse formation at the NMJ was not impaired in the absence of full length Syne-1 (scale bar ¼ 20 mm). Unfortunately, antibodies specific for the

N-terminal region of Syne-1 protein are not available, and as all detected mutations led to premature termination of the protein, it was impossible to

determine whether these nuclei contained truncated Syne-1 protein.

Cerebellum

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a Brainstemb Figure 3 Syne-1 expression in the mouse cerebellum and brainstem.

(a) Cerebellar cross-sections stained with antibodies to Syne-1 (in red) and

the Purkinje cells marker calbindin (in green) in the presence of the nuclear

dye 4,6-diamidino-2-phenylindole (DAPI) (in blue). The yellow signal in the

merged picture indicates colocalization of Syne-1 and calbindin, thus

confirming that Syne-1 is expressed in Purkinje cells of the cerebellar

cortex. (b) Olivary-region brainstem cross-sections stained with antibodies to

Syne-1 (in red), glial cells GFAP marker (in green) and the nuclear dye DAPI

(in blue) showing that Syne-1 is not expressed in glial cells but that it is

expressed in olivary neurons from the brainstem.

Control

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Affected Control Affecteda b

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Syne-1 expression in mouse cerebellum. Before their dissection, the animals

were perfused with 4% paraformaldehyde in PBS. Tissues were placed in OCT

embedding compound (Electron Microscopy Sciences). For immunohisto-

chemistry analysis, we used 10-mm sections that were thaw mounted on

Superfrost Plus slides. Immunodetection was carried out using rabbit poly-

clonal antibodies to Syne-1 (anti-Syne-1) (ABCAM: ab5250), mouse mono-

clonal anti-calbindin (Sigma: C9848) and mouse monoclonal anti-NFM

(Chemicon: MAB5254). A mixture of Alexa488-conjugated mouse antibody

and alexa555-conjugated rabbit antibody was used as secondary probe. Sections

were mounted in SlowFade (Molecular Probes).

NMJ formation. To investigate the effect of mutant SYNE1 in the proper

localization of synaptic nuclei at the NMJ, we obtained muscle biopsies from

one affected individual and one control and stained them with fluorescently

labeled a-bungarotoxin (BTX), which specifically labels AChRs, and a nuclear

dye. Muscle biopsies from an individual with ARCA1 and a normal control

were provided by the pathological department of the CHAUQ–(Enfant-Jesus).

Biopsies were immediately frozen in liquid nitrogen, mounted in OCT

embedding compound (Electron Microscopy Sciences) and cut into 25-mm

sections on a cryostat. Sections, collected on gelatin-coated slides, were allowed

to air-dry for 15 min before being fixed with 4% (vol/vol) paraformaldehyde in

PBS. Nonspecific interactions were blocked by incubating the sections for

30 min in blocking buffer (2.5% (vol/vol) normal goat serum, 2.5% (vol/vol)

bovine serum albumin and 0.2% (vol/vol) Triton X-100 in PBS). We analyzed

the structure of NMJs in the individual with ARCA1 by staining sections from

the biopsies with BTX and antibodies to proteins that are associated with

synaptic vesicles in motor nerve terminals: synapsin, synaptotagmin and

syaptophysin. Primary antibodies (anti-synapsin (see Acknowledgments),

anti-synaptophysin from Invitrogen, and anti-histone from Chemicon) or

fluorescently labeled a-BTX (Invitrogen) were diluted in blocking buffer

and incubated on the sections for 12 h at 4 1C and then removed, and the

sections were washed several times with PBS. Fluorescently labeled secondary

antibodies (Invitrogen) were diluted in blocking buffer and incubated on the

slides for 1 h at room temperature (22 1C). Sections were washed thoroughly

with PBS, cover-slipped with VectaShield and visualized on an Olympus

FV1000 scanning confocal microscope (Olympus America). For controls,

primary antibody incubation was omitted from the immunostaining protocol

described above.

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTSThe authors thank all family members for their entire cooperation. The authorsalso thank D. Verlaan and P. Cossette for helpful discussion on the linkage study,C. Gaspar for careful review of this manuscript, P. Hince for technical assistancewith the immunohistological experiments, F. Gosselin for collecting blood fromaffected individuals and N. Chrestian for data acquisition and management. Weare grateful for the support of T. Hudson from the McGill University GenomeQuebec Innovation Centre. We thank P. Greengard for antibody to synapsin.F.G.L., N.D. and G.A.R. are supported by the Canadian Institutes of HealthResearch (CIHR). This project was funded by the Canadian Genetic DiseasesNetwork (CGDN), by the US National Ataxia Foundation and by a grant fromthe US National Institutes of Health to J.R.S.

AUTHOR CONTRIBUTIONSF.G.-L. generated the data, conducted the data analysis, wrote the manuscriptand led the project; N.D. conducted neurological evaluation of individuals withARCA1, described the ARCA1 phenotype and reviewed the manuscript;P.D. participated in the data analysis and review of the manuscript; M.A.F.conducted and analyzed in vitro neuromuscular junction experiments andreviewed the manuscript; S.L. provided technical assistance in generating data;

S.V. participated in the neurological evaluation of individuals with ARCA1;J.R.S. supervised and analyzed the in vitro neuromuscular junction experimentsand reviewed the manuscript; J.-P.B. conducted neurological evaluation ofindividuals with ARCA1, described the ARCA1 phenotype and reviewed themanuscript; G.A.R. conducted neurological evaluation of individuals withARCA1, described the ARCA1 phenotype, participated in the data analysis,reviewed the manuscript and supervised the project.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/naturegenetics

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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