A form of autosomal recessive spastic ataxia (ARSACS) has been described in the
Charlevoix and Saguenay regions of Quebec. So far a frameshift and a nonsense
mutation have been identified in the SACS gene. The authors report a new mutation
(1859insC), leading to a frameshift with a premature termination of the gene
product sacsin, in two sisters from consanguineous parents. The phenotype is
similar to previously described patients with ARSACS

BANFI S

CRISCUOLO C

DE MICHELE, GIUSEPPE

FILLA, ALESSANDRO

GASPARINI P

MONTICELLI A

ORIO M

PERRETTI, ANNA CARMELA AGNESE

SANTORELLI FM

SANTORO, LUCIO

SCARANO V

English

Archivio della ricerca - Università degli studi di Napoli Federico II

 2004;62;100NeurologyC. Criscuolo, S. Banfi, M. Orio, et al.A novel mutation in SACS gene in a family from southern Italy April 12, 2012This information is current as of    http://www.neurology.org/content/62/1/100.full.htmllocated on the World Wide Web at: The online version of this article, along with updated information and services, is rights reserved. Print ISSN: 0028-3878. Online ISSN: 1526-632X.Allsince 1951, it is now a weekly with 48 issues per year. Copyright © 2004 by AAN Enterprises, Inc. ® is the official journal of the American Academy of Neurology. Published continuouslyNeurology Brief CommunicationsA novel mutation in SACS gene in afamily from southern ItalyC. Criscuolo, MD; S. Banfi, MD; M. Orio, MD; P. Gasparini, MD; A. Monticelli, MD; V. Scarano, MD;F.M. Santorelli, MD; A. Perretti, MD; L. Santoro, MD; G. De Michele, MD; and A. Filla, MDAbstract—A form of autosomal recessive spastic ataxia (ARSACS) has been described in the Charlevoix and Saguenayregions of Quebec. So far a frameshift and a nonsense mutation have been identified in the SACS gene. The authors reporta new mutation (1859insC), leading to a frameshift with a premature termination of the gene product sacsin, in twosisters from consanguineous parents. The phenotype is similar to previously described patients with ARSACS.NEUROLOGY 2004;62:100–102Autosomal recessive spastic ataxia of Charlevoix–Saguenay (ARSACS; MIM 270550) is a neurodegen-erative disorder showing pyramidal, cerebellarprogressive involvement and peripheral neuropathy.More than 300 patients with ARSACS have beendescribed in the Charlevoix–Saguenay region ofnortheastern Quebec, where the estimated carrierfrequency is 1 of every 22 persons.1 Main clinicalfeatures include early-onset progressive ataxia, dys-arthria, spasticity, nystagmus, retinal striation, anddistal amyotrophy. Imaging shows cerebellar vermisatrophy, and peripheral nerve conduction studies re-veal markedly decreased amplitude of the sensorypotentials and reduced motor conduction velocities.The disease is pathologically characterized by atro-phy of the upper cerebellar vermis and loss of Pur-kinje cells.2The gene responsible for ARSACS (SACS) maps tochromosome 13q113 and has a single large exonspanning 12,794 base pairs (bp),4 the largest exon inany vertebrate organism. The SACS product is a pre-dicted 3829 amino acid protein, sacsin, which proba-bly exerts a chaperon-mediated protein-foldingactivity.4 Until now, two causal mutations have beendescribed in this gene in the French-Canadian popu-lation: g.6594 delT, resulting in a frameshift withintroduction of a stop codon, and g.5254 C3T, anonsense mutation.4 Ninety-four percent of the dis-ease alleles carry the g.6594 delT; 3% carry theg.5254 C3T; and the remaining 3% carry an un-known mutation always in the heterozygous statewith g.6594 delT.5 Recently, one Tunisian and twoTurkish families have been linked to 13q11.6-8 Thepresence and the frequency of this disease are notknown in most European countries. In this study, wedescribe a novel single-base insertion mutation inthe SACS gene in two affected sisters from southernItaly.Methods. We recruited 22 index patients with an ARSACS-likephenotype from a series of 85 patients from southern Italy withearly-onset cerebellar ataxia. Consanguinity was present in sixfamilies. The inclusion criteria were sporadic or autosomal reces-sive progressive ataxia, corticospinal signs (at least two amongbrisk tendon reflexes, hypertonus, and Babinski signs), clinical(decreased or absent ankle reflexes and decreased vibration sense)or neurophysiologic signs of peripheral neuropathy, and negativemolecular test for Friedreich ataxia.After informed consent was obtained from all patients,genomic DNA was extracted from peripheral blood samples.Primer pairs were designed to screen for the two French-Canadian mutations in all patients using the web-based version ofthe Primer 3.0 program. PCR products, subjected to electrophore-sis in 1.5% agarose gel, were then analyzed by denaturing high-performance liquid chromatography (DHPLC). DHPLC wasperformed on a WAVE nucleic acid fragment analysis systemHSM (Transgenomic, Crewe, UK). The composition of buffer Awas 0.1 mol/L triethylammonium acetate (TEAA), pH 7.0, 0.025%acetonitrile (v/v). Buffer B contained 0.1 mol/L TEAA, 25% aceto-Additional material related to this article can be found on the NeurologyWeb site. Go to www.neurology.org and scroll down the Table of Con-tents for the December 23 issue to find the title link for this article.See also pages 10, 103, and 107From the Departments of Neurological Sciences (Drs. Criscuolo, Orio, Scarano, Perretti, Santoro, De Michele, and Filla) and Molecular and Cellular Biologyand Pathology (Dr. Monticelli), Federico II University, Naples, Italy; Department of Neurological Sciences (Dr. Santorelli), La Sapienza University, Rome,Italy; and Telethon Institute of Genetics and Medicine (Drs. Banfi and Gasparini), Naples, Italy.Supported in part by grants from Italian Ministry of University and FIRB, and Ministry of Health to A.F.Received July 8, 2003. Accepted in final form September 24, 2003.Address correspondence and reprint requests to Dr. Chiara Criscuolo, Dipartimento di Scienze Neurologiche, Università degli Studi di Napoli Federico II, ViaPansini 5, I-80131, Naples, Italy; e-mail: sky569@libero.it100 Copyright © 2004 by AAN Enterprises, Inc.nitrile (v/v). Analysis was carried out at a flow rate of 0.9 mL/minand buffer B gradient increase of 2% per minute. Start and endconcentrations of buffer B were adjusted according to the size ofthe PCR product. In addition, in the patients from consanguineousmarriages, linkage analysis was performed using polymorphicmarkers spanning the SACS gene (D13S292, D13S787, D13S232,D13S1275) to determine if the disease trait was associated withthe ARSACS locus. Patients homozygous for all tested markerswere considered in possible linkage to ARSACS and were screenedfor mutations in the entire gene. The unique SACS exon, becauseof the large size, was divided in 15 overlapping fragments ofapproximately 800 bp. Oligonucleotide primer pairs and PCR con-ditions are shown in the table available online (see table E-1 onthe Neurology Web site). The PCR reaction was carried out in atotal volume of 25 L containing 100 ng of genomic DNA, 2.5 L10  PCR buffer containing MgCl2 (15 mmol/L), 2 L dNTP (2.5mmol/L), 0.5 L each primer (20 pm/L), and 0.3 L “AmpliTaqGold” (Perkin-Elmer, Foster City, CA). PCR products were puri-fied from agarose gels by the QIAGEN gel extraction kit (Hilden,Germany) and directly sequenced, from the forward and reversestrands, on an automated sequencer (ABI 3100; Applied Biosys-tem, Weiterstadt, Germany) using the ABI-PRISM big-dye termi-nator cycle sequencing ready reaction kit (Applied Biosystem).Peripheral nerve conduction study was performed using needleelectrodes.Results. We did not find any of the previously describedmutations in our patients. Conversely, linkage analysisrevealed that one patient from a consanguineous marriagewas homozygous for all tested markers suggesting linkageto the ARSACS locus. The affected sister showed the samehaplotype (figure 1). Sequencing of the entire gene in bothsisters revealed a novel homozygous 1859insC mutation(figure 2). The screening of this new mutation in the re-maining patients was negative.Clinical and neurophysiologic features of the two pa-tients are summarized in the table. Onset was character-ized by delay in walking, followed by gait unsteadiness andtendency to fall. The patients, now aged in their 40s, needsupport when walking. Mental retardation and hypoacusiaare present in Patient 2. Sural nerve biopsy, performed inPatient 1, showed a severe loss of large myelinated fibers(11 m) with almost normal total number of myelinatedfibers. Neuroimaging showed cerebellar atrophy moremarked in the vermis than in the hemispheres.Discussion. We report a new mutation in theSACS gene in two sisters from consanguineous par-ents in southern Italy. The novel 1859insC mutationresults in a frameshift and the introduction of anearly stop codon at residue 599. The resulting pre-dicted protein lacks 3230 amino acids. The French-Figure 1. Pedigree and haplotypes for the autosomal re-cessive spastic ataxia of Charlevoix–Saguenay (ARSACS)locus.Figure 2. Electropherogram of the SACS normal sequence(A) and the novel single-base insertion [C] mutation in apatient (B). The nucleotide position is based on the tran-script GenBank accession number AF193556.Table Clinical and neurophysiological findingsPatients 1 2Onset age/disease duration, y 2/41 2/36Gait ataxia ~~ ~~Nystagmus  Dysarthria ~~ ~~Dysmetria  Lower limb spasticity ~~ ~~Increased knee jerks ~~ ~~Ankle jerks  Babinski signs  Lower limb amyotrophy  IQ 74 58Median nerveSCV digit III-wrist, m/s 42.2 38.8SAP at wrist, V 0.8 0.7MCV elbow-wrist, m/s 41.6 40.2SSEP N13–N20, m/s 10.7 10.8Presence and severity of features:   mild to moderate;  severe;   absent.SCV  sensory conduction velocity (normal lower limit 51 m/s);SAP  sensory action potential (normal lower limit 6 V);MCV  motor conduction velocity (normal lower limit 54 m/s);SSEP  somatosensory evoked potential (normal upper limit7.4 m/s).January (1 of 2) 2004 NEUROLOGY 62 101Canadian g.6594T and g.5254C mutations also causethe premature termination of the predicted protein.The recessive character and the truncating nature ofthe sacsin mutations suggest that the developmentof the disease is associated with a loss of function.Sacsin shows significant sequence similarity to pro-teins and domains involved in chaperon-mediatedprotein folding because it contains a “DnaJ” motifand the N-terminal domain of the Hsp90.4 In addi-tion, sacsin is characterized by the presence of twoleucine zipper and three coiled-coil domains andseven nuclear localization signals.4 The mutation de-scribed in this report is localized at the N-terminalregion before any known domain.The proteins paraplegin and HSPD1, involved intwo forms of hereditary spastic paraplegias (SPG7 and13), show chaperon-like activities.9,10 The corticospinaltract involvement present in these two diseases is aprominent feature in patients with ARSACS.There are no phenotypic differences among thepatients homozygous for the g.6594delT, the com-pound heterozygotes g.6594delT/g.5254C3T, andg.6594delT/unknown mutation.5 Although the newmutation leads to a shorter truncated protein, itsharmful effect is not increased. Our patients hadclinical and neurophysiologic findings similar tothose observed in the French-Canadian group exceptfor the presence of mental retardation in one patient.Intellectual impairment was also observed in one ofthe four Turkish patients linked to ARSACS locus.7A finer genotype and phenotype correlation may bepossible when more mutations are identified.This report confirms that ARSACS is present inpopulations other than those previously describedand should be considered in patients with autosomalrecessive cerebellar ataxia, pyramidal signs, and pe-ripheral neuropathy. Failure to detect g.6594delTand g.5254C3T mutations in our series suggeststhat these two mutations may be a rare cause ofARSACS in southern Italy.References1. Bouchard JP, Richter A, Mathieu J, et al. Autosomal recessive spasticataxia of Charlevoix-Saguenay. Neuromuscul Disord 1998;8:474–479.2. Richter A, Morgan K, Bouchard JP, et al. Clinical and molecular geneticstudies on autosomal recessive spastic ataxia of Charlevoix-Saguenay(ARSACS). Adv Neurol 1993;61:97–103.3. Engert JC, Dore C, Mercier J, et al. Autosomal recessive spastic ataxiaof Charlevoix-Saguenay (ARSACS): high-resolution physical and tran-script map of the candidate region in chromosome region 13q11.Genomics 1999;62:156–164.4. Engert JC, Dore C, Mercier J, et al. ARSACS, a spastic ataxia commonin northeastern Quebec, is caused by mutations in a new gene encodingan 11.5-kb ORF. Nat Genet 2000;24:120–125.5. Mercier J, Prèvost C, Engert JC, et al. Rapid detection of the sacsinmutation causing autosomal recessive spastic ataxia of Charlevoix-Saguenay. Genet Test 2001;5:255–259.6. Mrissa N, Belal S, Ben Hamida C, et al. Linkage to chromosome13q11–12 of an autosomal recessive cerebellar ataxia in a Tunisianfamily. Neurology 2000;54:1408–1414.7. Gucuyener K, Ozgul K, Paternotte C. Autosomal recessive spasticataxia of Charlevoix-Saguenay in two unrelated Turkish families. Neu-ropediatrics 2001;32:142–146.8. Pulst SM, Filla A. Ataxias on the march from Quebec to Tunisia. Neu-rology 2000;54:1400–1401.9. Casari G, De Fusco M, Ciarmatori S, et al. Spastic paraplegia andOXPHOS impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease. Cell 1998;93:973–983.10. Hansen JJ, Durr A, Cournu-Rebeix I, et al. Hereditary spastic paraple-gia SPG13 is associated with a mutation in the gene encoding themitochondrial chaperonin Hsp60. Am J Hum Genet 2002;70:1328–1332.102 NEUROLOGY 62 January (1 of 2) 2004 2004;62;100NeurologyC. Criscuolo, S. Banfi, M. Orio, et al.A novel mutation in SACS gene in a family from southern Italy April 12, 2012This information is current as of   ServicesUpdated Information & http://www.neurology.org/content/62/1/100.full.htmlincluding high resolution figures, can be found at: Supplementary Material C1.htmlhttp://www.neurology.org/content/suppl/2004/01/08/62.1.100.DSupplementary material can be found at: References http://www.neurology.org/content/62/1/100.full.html#ref-list-1This article cites 10 articles, 2 of which can be accessed free at:Citations lshttp://www.neurology.org/content/62/1/100.full.html#related-urThis article has been cited by 6 HighWire-hosted articles:Permissions & Licensing http://www.neurology.org/misc/about.xhtml#permissionstables) or in its entirety can be found online at: Information about reproducing this article in parts (figures, Reprints http://www.neurology.org/misc/addir.xhtml#reprintsusInformation about ordering reprints can be found online:

A novel mutation in SACS gene in a family from southern Italy

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A novel mutation in SACS gene in a family from southern Italy

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