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Clinical and Genetic Study of Autosomal Recessive Cerebellar Ataxia Type 1 Nicolas Dupre ´, MD, MSc, 1,2 Franc ¸ois Gros-Louis, PhD, 2 Nicolas Chrestian, MD, 1 Steve Verreault, MD, MSc, 1 Denis Brunet, MD, 1 Danielle de Verteuil, BSc, 3 Bernard Brais, MD, PhD, 3 Jean-Pierre Bouchard, MD, 1 and Guy A. Rouleau, MD, PhD 2 Objective: Define the phenotype and genotype of a cluster of families with a relatively pure cerebellar ataxia referred to as autosomal recessive cerebellar ataxia type 1 (ARCA-1). Methods: We ascertained 64 probands and affected members of 30 French-Canadian families all showing similar clinical features and originating from the same region of Quebec. After informed consent, we performed detailed clinical history, neurological examination, brain imaging, nerve conduction studies, and SYNE1 mutation detection of all available subjects. Results: Based on the cases examined, ARCA-1 is a cerebellar syndrome characterized by recessive transmission, middle-age onset (mean, 31.60; range, 17– 46 years), slow progression and moderate disability, significant dysarthria, mild oculomotor abnormalities, occasional brisk reflexes in the lower extremities, normal nerve conduction studies, and diffuse cerebellar atrophy on imaging. We identified a total of seven mutations in our population, thereby providing evidence of genotypic heterogeneity. Patients with different mutations did not show significant phenotypic heterogeneity. Interpretation: We identified a cluster of French-Canadian families with a new recessive ataxia of relatively pure cerebellar type caused by mutations in SYNE1. The function of SYNE1 is thus critical in the maintenance of cerebellar structure in humans. We expect that this disease will be a common cause of middle-age-onset recessive ataxia worldwide. Ann Neurol 2007;62:93–98 The hereditary ataxias can be divided based on their mode of inheritance into autosomal dominant, autoso- mal recessive, X-linked, and mitochondrial ataxias. These disease categories share the prototypic feature of impaired walking, although they usually present a va- riety of other neurological symptoms such as pyramidal signs, peripheral neuropathy, extrapyramidal signs, cog- nitive loss, or retinopathy. The autosomal recessive ataxias are also a heterogeneous group of disorders composed mainly of Friedreich’s ataxia, ataxia telangi- ectasia, ataxia with vitamin E deficiency, autosomal re- cessive spastic ataxia of Charlevoix–Saguenay (ARSACS), abetalipoproteinemia, and ataxia with oculomotor apraxia types 1 and 2. 1 Over the past decade, we have identified a large clus- ter of French-Canadian families whose ancestors origi- nate mostly from the same region of the Province of Quebec in Canada. This region, located in southeast- ern Quebec near the US border, is called Beauce. The affected individuals in these families all share similar clinical characteristics that define this new disease en- tity we named ARCA-1, also known as recessive ataxia of Beauce. 2,3 Genome-wide linkage and fine-mapping analysis on selected families with ARCA-1 established a minimum candidate interval of about 0.5Mb on chro- mosome 6q containing a single causal gene (SYNE1). 4 Spanning over 0.5Mb genomic DNA, SYNE1 is one of the biggest genes in the human genome formed of 147 exons that encodes a 27,652kb messenger RNA and an 8,797 amino-acid-long protein. To date, five different truncating mutations within SYNE1 have been de- scribed. 4 In this article, we report two novel mutations, also leading to premature termination of the protein, and define the full clinical and molecular spectrum of ARCA-1. Subjects and Methods Subjects were referred to the study protocol by their treating physician based on a preliminary assessment consistent with the core features of ARCA-1 (ataxia and dysarthria of middle-age onset with cerebellar atrophy) and a recessive family history (parents unaffected). They were informed of the procedures entailed in the protocol and signed, before their participation, a consent form approved by the local eth- From the 1 Faculty of Medicine, Laval University, Department of Neurological Sciences, Centre Hospitalier Affilie ´ Universitaire de Que ´bec–Enfant-Je ´sus, Quebec City; 2 Center for the Study of Brain Diseases, Universite ´ de Montre ´al, Centre Hospitalier de l’Universite ´ de Montre ´al (Notre-Dame); and 3 Laboratory of Neurogenetics of Motion, Center for the Study of Brain Diseases, Universite ´ de Mon- tre ´al, Centre Hospitalier de l’Universite ´ de Montre ´al (Notre-Dame), Montre ´al, Que ´bec, Canada. Received Dec 22, 2006, and in revised form Feb 8, 2007. Accepted for publication Mar 2, 2007. Published online May 14, 2007, in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.21143 Address correspondence to Dr Dupre, Department of Neurological Sciences, CHAUQ–Enfant-Je ´sus, 1401, 18th Street, Quebec City, QC, Canada, G1J 1Z4. E-mail: [email protected] © 2007 American Neurological Association 93 Published by Wiley-Liss, Inc., through Wiley Subscription Services

Clinical and genetic study of autosomal recessive cerebellar ataxia type 1

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Clinical and Genetic Study of AutosomalRecessive Cerebellar Ataxia Type 1

Nicolas Dupre, MD, MSc,1,2 Francois Gros-Louis, PhD,2 Nicolas Chrestian, MD,1 Steve Verreault, MD, MSc,1

Denis Brunet, MD,1 Danielle de Verteuil, BSc,3 Bernard Brais, MD, PhD,3 Jean-Pierre Bouchard, MD,1

and Guy A. Rouleau, MD, PhD2

Objective: Define the phenotype and genotype of a cluster of families with a relatively pure cerebellar ataxia referred to asautosomal recessive cerebellar ataxia type 1 (ARCA-1).Methods: We ascertained 64 probands and affected members of 30 French-Canadian families all showing similar clinical featuresand originating from the same region of Quebec. After informed consent, we performed detailed clinical history, neurologicalexamination, brain imaging, nerve conduction studies, and SYNE1 mutation detection of all available subjects.Results: Based on the cases examined, ARCA-1 is a cerebellar syndrome characterized by recessive transmission, middle-ageonset (mean, 31.60; range, 17–46 years), slow progression and moderate disability, significant dysarthria, mild oculomotorabnormalities, occasional brisk reflexes in the lower extremities, normal nerve conduction studies, and diffuse cerebellar atrophyon imaging. We identified a total of seven mutations in our population, thereby providing evidence of genotypic heterogeneity.Patients with different mutations did not show significant phenotypic heterogeneity.Interpretation: We identified a cluster of French-Canadian families with a new recessive ataxia of relatively pure cerebellar typecaused by mutations in SYNE1. The function of SYNE1 is thus critical in the maintenance of cerebellar structure in humans.We expect that this disease will be a common cause of middle-age-onset recessive ataxia worldwide.

Ann Neurol 2007;62:93–98

The hereditary ataxias can be divided based on theirmode of inheritance into autosomal dominant, autoso-mal recessive, X-linked, and mitochondrial ataxias.These disease categories share the prototypic feature ofimpaired walking, although they usually present a va-riety of other neurological symptoms such as pyramidalsigns, peripheral neuropathy, extrapyramidal signs, cog-nitive loss, or retinopathy. The autosomal recessiveataxias are also a heterogeneous group of disorderscomposed mainly of Friedreich’s ataxia, ataxia telangi-ectasia, ataxia with vitamin E deficiency, autosomal re-cessive spastic ataxia of Charlevoix–Saguenay (ARSACS),abetalipoproteinemia, and ataxia with oculomotorapraxia types 1 and 2.1

Over the past decade, we have identified a large clus-ter of French-Canadian families whose ancestors origi-nate mostly from the same region of the Province ofQuebec in Canada. This region, located in southeast-ern Quebec near the US border, is called Beauce. Theaffected individuals in these families all share similarclinical characteristics that define this new disease en-tity we named ARCA-1, also known as recessive ataxia

of Beauce.2,3 Genome-wide linkage and fine-mappinganalysis on selected families with ARCA-1 established aminimum candidate interval of about 0.5Mb on chro-mosome 6q containing a single causal gene (SYNE1).4

Spanning over 0.5Mb genomic DNA, SYNE1 is one ofthe biggest genes in the human genome formed of 147exons that encodes a 27,652kb messenger RNA and an8,797 amino-acid-long protein. To date, five differenttruncating mutations within SYNE1 have been de-scribed.4 In this article, we report two novel mutations,also leading to premature termination of the protein,and define the full clinical and molecular spectrum ofARCA-1.

Subjects and MethodsSubjects were referred to the study protocol by their treatingphysician based on a preliminary assessment consistent withthe core features of ARCA-1 (ataxia and dysarthria ofmiddle-age onset with cerebellar atrophy) and a recessivefamily history (parents unaffected). They were informed ofthe procedures entailed in the protocol and signed, beforetheir participation, a consent form approved by the local eth-

From the 1Faculty of Medicine, Laval University, Department ofNeurological Sciences, Centre Hospitalier Affilie Universitaire deQuebec–Enfant-Jesus, Quebec City; 2Center for the Study of BrainDiseases, Universite de Montreal, Centre Hospitalier de l’Universitede Montreal (Notre-Dame); and 3Laboratory of Neurogenetics ofMotion, Center for the Study of Brain Diseases, Universite de Mon-treal, Centre Hospitalier de l’Universite de Montreal (Notre-Dame),Montreal, Quebec, Canada.

Received Dec 22, 2006, and in revised form Feb 8, 2007. Acceptedfor publication Mar 2, 2007.

Published online May 14, 2007, in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/ana.21143

Address correspondence to Dr Dupre, Department of NeurologicalSciences, CHAUQ–Enfant-Jesus, 1401, 18th Street, Quebec City,QC, Canada, G1J 1Z4. E-mail: [email protected]

© 2007 American Neurological Association 93Published by Wiley-Liss, Inc., through Wiley Subscription Services

ics review boards (Centre Hospitalier de l’Universite deMontreal and Centre Hospitalier Affilie Universitaire deQuebec). All 64 available affected and unaffected membersof the 30 recruited families underwent a thorough neurolog-ical examination and were examined independently by atleast two neurologists. We also used an assessment scale5 tograde severity of symptoms: dysarthria (0 � no impairment;1 � mild dysarthria but comprehensible; 2 � moderate dys-arthria with interruption in flow; 3 � severe dysarthria andincomprehensible, very difficult to understand; 4 � com-pletely unintelligible); dysmetria (0 � no impairment; 1 �mild dysmetria but reaches the target; 2 � moderate dysme-tria, reaches target after several attempts; 3 � severe dysme-tria, short of target after many attempts; 4 � cannot usehands); gait (0 � normal; 1 � stance width increased,mildly unstable gait but can walk without support; 2 �moderately unstable gait and needs support for walking; 3 �unable to walk, needs the assistance of two persons; 4 �wheelchair bound). Twenty-two subjects underwent electro-physiological studies including compound motor action po-tentials of median, ulnar, tibial, and peroneal nerves, as wellas sensory nerve action potentials of median, ulnar, radial,and sural nerves, with standard values for filters, stimulusduration, and electrode positioning. Brain imaging withmagnetic resonance imaging was performed on 50 affectedsubjects.

On receipt of informed consent, blood samples were ob-tained from affected individuals in 30 families. DNA wasextracted from peripheral blood by standard methods.6 Formutation screening, a set of 154 polymerase chain reaction(PCR) primer pairs were designed from genomic DNA toamplify each exon of the SYNE1 gene, including the flankingsplice sites and the untranslated regions.4 Products werePCR-amplified, checked on agarose gels, and then sequencedusing the forward primers for all of the amplicons. Each frag-ment containing mutations was PCR-amplified a secondtime and sequenced with the reverse primer to confirm thatthe identified mutations were not due to PCR artifact.

ResultsSample CaseThis 42-year-old woman originates from the Beauce re-gion in Quebec. On her initial visit, she mentionedthat around the age of 30 she started noticing that herspeech was slurred. During the same period, she alsonoticed some mild walking impairment. As she ad-vanced in her thirties, the slurred speech and walkingimpairment progressed. Strangers would sometimeswonder if she had been drinking alcohol, and her gaitwas becoming more wide based with a tendency to fallif she did not pay more attention than usual during hermovements. In addition, her hands sometimes feltclumsier. On neurological examination at age 42, shehad normal funduscopy, normal ocular saccades andpursuit, and no nystagmus. She had significant cerebel-lar dysarthria. Strength was normal throughout, withno spasticity. Reflexes were normal in the upper andlower limbs, with down-going toes. Sensory examina-tion was normal to pain, temperature, vibration, pro-

prioception, and light touch. Gait was broad based.There was significant dysmetria on finger-to-nose andheel-to-shin, as well as cerebellar hypotonia (Holmes’sign) in the upper limbs. Magnetic resonance imagingdemonstrated diffuse cerebellar atrophy, and nerveconduction studies were normal. Genetic testing con-firmed that she was homozygous for the g.306434A�Gmutation.

Overall, the ARCA1 phenotype consists of a middle-age onset disease that presents with either dysarthria,cerebellar ataxia, or both coincidentally (Table 1 andFig 1). Over time, all patients develop significant dys-arthria and ataxia, with other associated features suchas dysmetria, brisk lower extremity tendon reflexes, andminor abnormalities in saccade and smooth pursuit.None of the subjects evaluated showed extrapyramidalsigns, cognitive loss, retinopathy, cardiomyopathy, sen-sory abnormalities, or autonomic disturbances. Thedisease progresses slowly and evolves into a moderatedegree of disability. There appears to be no effect onlife expectancy. Nerve conduction studies performedon 22 affected individuals were always normal, show-ing therefore no sign of a peripheral sensory or motorneuropathy in this disease. Single-fiber electromyogra-phy was also normal when performed on one subject,suggesting preserved function at the neuromuscularjunction. Imaging findings on 50 affected individualsinvariably showed marked diffuse cerebellar atrophy(Fig 2). On detailed review of imaging in 38 affectedsubjects, there was no cerebral cortical atrophy; nomidbrain, pontine, or bulbar atrophy; no atrophy ofinferior olives; and no white matter changes.

Genetic analysis demonstrated seven mutations (Ta-ble 2 and Fig 3), including five previously describedmutations4 and two novel truncating mutations(g.409218C�T and g.281100-281101delTG). Themost frequent mutation, g.306434A�G, was presenthomozygously in 20 of 64 subjects (31.2%). This samemutation was present heterozygously with theg.310067A�G mutation in 11 of 64 (17.2%), withthe g.247012A�T mutation in 6 of 64 (9.4%), withthe g.426494C�T mutation in 1 of 64 (1.6%), andwith the g.334338-334342delATTTG mutation in 4of 64 (6.2%). Of these 64 subjects, there was 1 (1.6%)g.310067A�G homozygote, 3 (4.7%) g.247012A�Thomozygotes, and 1 (1.6%) g.247012A�T/g.409218C�T heterozygote. Finally, 6 of 64 (9.4%)carried a known mutation on one chromosome and anunknown mutation on the other chromosome, whereasin 9 (14.0%) the mutation was not found using thetechnique described earlier and in 2 (3.1%) genetictesting could not be performed. The g.306434A�Gmutation was present on 50.8% of chromosomestested, whereas mutations remain to be identified inclose to one-fifth of carrier chromosomes. We per-formed genotype–phenotype correlation studies by seg-

94 Annals of Neurology Vol 62 No 1 July 2007

regating based on the most common genotypes(g.306434A�G homozygotes and g.306434A�G/g.310067A�G heterozygotes) using the following pa-rameters: age of onset (dysarthria, ataxia, overall), dis-ease duration, and eye movement abnormalities. Thisanalysis gave no statistically significant differences. In

addition, when we analyzed the clinical data of patientsbearing the less common genotypes, we were unable todemonstrate that they showed any atypical clinical fea-tures.

DiscussionARCA-1 is thus a new recessive relatively pure cerebel-lar ataxia that is caused by various mutations inSYNE1. ARCA-1 shows relative homogeneity of thephenotype, despite being caused by more than sevendifferent mutations. The age of onset does not varysignificantly in function of given mutations like intrinucleotide repeat disorders, whereas the diseaseshows little associated features accompanying the coresymptoms of dysarthria and dysmetria. SYNE1 encodesa protein of about 8,797 amino acid residues(�1,000kDa).4 The protein contains two N-terminalactin-binding regions that comprise tandem pairedcalponin-homology domains, a transmembrane do-main, multiple spectrin repeats, and a C-terminal Klar-sicht domain. Although SYNE1 is expressed in multi-ple tissues, its greatest level in the central nervoussystem of mice is in the cell bodies of the Purkinje cellsFig 1. Age of onset by decade.

Table 1. Clinical Results of Patients with ARCA-1 (n � 64)

Sex Male: 37(58%)

Female: 27(42%)

First complaint Dysarthria: 8(12.5%)

Ataxia: 40(62.5%)

Both: 16(25%)

Age of onset (dysarthria), yr Mean: 34.79 SD: 7.62 Range: 17–50

Age of onset (ataxia), yr Mean: 31.60 SD: 7.81 Range: 17–45

Age at evaluation, yr Mean: 45.36 SD: 10.71 Range: 24–69

Duration at evaluation, yr Mean: 14.33 SD: 9.77 Range: 3–40

Assessment scale

Dysarthria Mean: 1.78 Range: 0–3

Dysdiadochokinesis Mean: 1.26 Range: 0–2

Dysmetria Mean: 1.44 Range: 0–3

Gait Mean: 1.48 Range: 0–3

Neurological examination

Dysarthria 64 (100%)

Nystagmus 6 (9.4%)

Slow saccades 20 (31.2%)

Abnormal pursuit 28 (43.8%)

Brisk reflexes in the lower limbs 21 (32.8%)

Ankle clonus and/or Babinski sign 4 (6.2%)

Dysmetria on finger-to-nose 58 (90.6%)

Dysmetria on heal-to-shin 58 (90.6%)

Ataxia 63 (98.4%)

SD � standard deviation.

Dupre et al: SYNE1-Related Cerebellar Ataxia 95

and in neurons of the olivary region of the brainstem,whereas in humans it is also expressed predominantlyin the cerebellum; it is not expressed in glial cells. Inthe peripheral nervous system, SYNE1 is involved inanchoring specialized myonuclei underneath the neuro-muscular junctions.4 It was found in a muscle biopsyof an ARCA-1 patient that fewer myonuclei come tolie beneath the neuromuscular junction, although thishas no consequences clinically, electrophysiologically,or ultrastructurally. SYNE1 is part of the spectrin fam-ily of structural proteins that share a common function

of linking the plasma membrane to the actin cytoskel-eton. This family also includes dystrophin (Duchenne’sand Becker’s muscular dystrophies),7 SPTBN2 (spino-cerebellar ataxia type 5),8 PLEKHG4 (16q-autosomaldominant cerebellar ataxia),9 and Spnb4.10

The closest description of ARCA-1 phenotype in theliterature is Holmes’ hereditary ataxia. Holmes’ type ofhereditary ataxia11 was described a century ago (1907)in a family of eight siblings, with four of them present-ing with middle-age-onset dysarthria, ataxia, and hypo-gonadism (not present in ARCA-1). The inheritancepattern was most likely autosomal recessive, becausethe parents were not affected. Autopsy of one affectedcase showed diffuse cerebellar atrophy with no pontineor olivary involvement. To our knowledge, no linkageor gene defect responsible for this type of ataxia hasbeen reported as yet.

ARSACS is the most common of all autosomal re-cessive ataxias in Quebec with more than 300 affectedindividuals. ARSACS patients exhibit early-onset signsof spasticity in the lower limbs usually observed at gaitinitiation (12–18 months).12 The clinical picture no-ticed by parents from early childhood is always that ofa gait ataxia with a tendency to fall. Nerve conductionstudies demonstrate signs of progressive axonal sensori-motor neuropathy. Ataxia with oculomotor apraxiatype 2 is also present in Quebec, where more than 10families have been described. It is characterized mainlyby cerebellar atrophy, axonal sensorimotor neuropathy,and increased serum �-fetoprotein.13 The main differ-ences clinically between these other recessive ataxiascommon in Quebec and ARCA-1 is the earlier age ofonset, the greater degree of disability, and the associ-ated peripheral neuropathy.

Of interest, 16q-autosomal dominant cerebellarataxia is characterized by an age of onset older than 55years and sensorineuronal hearing impairment,9 which

Table 2. Known Mutations Causing ARCA-1

Variantsa Exons/Introns Protein Changes Total Carrier ChromosomesTested in the Patient Population(n � 124)

g.306434A�G Intron 81 Premature stop at position 5244 63 (50.8%)

g.310067A�G Intron 84 Premature stop at position 5402 13 (10.5%)

g.247012A�T Exon 56 R2906X 16 (12.9%)

g.426494C�T Exon 126 Q7640X 2 (1.6%)

g.334338-334342delATTTG Exon 93 Premature stop at position 5880 4 (3.2%)

g.409218C�T Exon 118 Q7386X 1 (0.8%)

g.281100-281101delTG Exon 71 4077X 1 (0.8%)

Unknown 24 (19.3%)aVariants were named according to the genomic DNA sequence NM_033071; nucleotide “A” from the ATG initiation codon is referredas 1. Allele frequencies of variants were 0 of 380 French-Canadian control chromosomes.

Fig 2. Magnetic resonance imaging of a 43-year-old autosomalrecessive cerebellar ataxia type 1 (ARCA-1) patient after 5years of disease evolution. Sagittal T1 shows marked diffusecerebellar atrophy with no cerebral cortical atrophy and nomidbrain, pontine, or bulbar atrophy.

96 Annals of Neurology Vol 62 No 1 July 2007

is different from what we found in ARCA-1. However,there are similarities in that it is also a relatively purecerebellar syndrome caused by mutations in PLE-KHG4, also part of the spectrin family of structuralproteins. On pathology, 16q-autosomal dominant cer-ebellar ataxia shows peculiar degeneration of Purkinjecells that undergo shrinkage and are surrounded byamorphous material. Spinocerebellar ataxia type 5 ischaracterized by a slowly progressive cerebellar syn-drome beginning mostly in the third decade.14,15 Themost consistent clinical feature is downbeat nystagmus,whereas other common features included gait, stance,and limb ataxia; dysarthria; intention tremor and rest-ing tremor; impaired smooth pursuit; and gaze-evokednystagmus. Symptom progression is slow, and all pa-tients remain ambulatory despite disease duration of upto 30 years. Magnetic resonance imaging shows atro-phy of the cerebellar vermis and hemispheres. Again,this other ataxia caused by mutations in a spectrin fam-ily protein shows striking similarities with ARCA-1 forthe predominant cerebellar involvement, middle-ageonset, relatively slow progression, and moderate degreeof disability.

In conclusion, ARCA-1, spinocerebellar ataxia type5, and 16q-autosomal dominant cerebellar ataxia taken

together allow us to define a new category of hereditaryataxias related to the spectrin family of structural pro-teins. Despite significant genetic heterogeneity, thiscategory of ataxia shares many common clinical fea-tures. We expect that this new category of inheritedataxias may be more frequent than previously thought,mainly through the contribution of ARCA-1, becausewe have encountered important genetic heterogeneityeven within a homogeneous founder population. Wespeculate that a significant proportion of yet undiag-nosed recessive or “sporadic” ataxias may be due toSYNE1 mutations, which would have great repercus-sions on our ability to diagnose more precisely theseataxia types in specialized clinics worldwide.

This work was supported by the National Ataxia Foundation,(G.A.R.), the Canadian Genetic Disease Network, (G.A.R.), the Ca-nadian Institute of Health Research (F.G.L., N.D.) and the Associ-ation des Ataxies Familiales (D.V.).

F. Gosselin and M. Plante performed blood collection of patientsand obtained their consent. Finally, we also thank the family mem-bers who participated in this study.

Fig 3. SYNE1 identified truncating mutations. (A) Sequence traces of healthy individual (top) and ARCA-1 patients (bottom)showing the novel detected mutations within exons 71 and 118 of SYNE1. (B) Protein structure of SYNE-1. Known ARCA-1disease causing mutations are in black, and newly identified mutations lie within boxes. Light gray region defines areas rich inpredicted spectrin repeats; gray boxes at the N-terminal part of the protein correspond to calponin-homology domains involved inactin binding; dark gray box indicates the C-terminal nuclear envelope binding domains containing sequences homologous to theDrosophila protein Klarsicht.

Dupre et al: SYNE1-Related Cerebellar Ataxia 97

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