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ARTICLE Mutations in the KIAA0196 Gene at the SPG8 Locus Cause Hereditary Spastic Paraplegia

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ARTICLE Mutations in the KIAA0196 Gene at the SPG8 Locus Cause Hereditary Spastic Paraplegia CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector ARTICLE Mutations in the KIAA0196 Gene at the SPG8 Locus Cause Hereditary Spastic Paraplegia Paul N. Valdmanis,* Inge A. Meijer,* Annie Reynolds, Adrienne Lei, Patrick MacLeod, David Schlesinger, Mayana Zatz, Evan Reid, Patrick A. Dion, Pierre Drapeau, and Guy A. Rouleau Hereditary spastic paraplegia (HSP) is a progressive upper-motor neurodegenerative disease. The eighth HSP locus, SPG8, is on chromosome 8p24.13. The three families previously linked to the SPG8 locus present with relatively severe, pure spastic paraplegia. We have identified three mutations in the KIAA0196 gene in six families that map to the SPG8 locus. One mutation, V626F, segregated in three large North American families with European ancestry and in one British family. An L619F mutation was found in a Brazilian family. The third mutation, N471D, was identified in a smaller family of European origin and lies in a spectrin domain. None of these mutations were identified in 500 control indi- viduals. Both the L619 and V626 residues are strictly conserved across species and likely have a notable effect on the structure of the protein product strumpellin. Rescue studies with human mRNA injected in zebrafish treated with mor- pholino oligonucleotides to knock down the endogenous protein showed that mutations at these two residues impaired the normal function of the KIAA0196 gene. However, the function of the 1,159-aa strumpellin protein is relatively unknown. The identification and characterization of the KIAA0196 gene will enable further insight into the pathogenesis of HSP. Hereditary spastic paraplegia (HSP) has a worldwide prev- of the more aggressive subtypes of HSP, with disease onset alence of 1–18 in 100,0001–3 and is characterized by cen- occurring for patients as early as their 20s or 30s. It was tral-motor-system deficits leading to lower-limb spastic first identified in a white family as a 6.2-cM region between paraperesis.4–6 This is due to a “dying back” phenomenon markers D8S1804 and D8S1774.14 The family had 15 pa- whereby upper motor neurons degenerate progressively, tients affected with spasticity, hyperreflexia, extensor plan- commencing with the longest axons.7,8 HSP can be clas- tar reflexes, lower-limb weakness, decreased vibration sen- sified into pure and complicated forms.5 In pure HSP, sation, and limited muscle wasting. The candidate region lower-limb spasticity is the only major symptom. Alter- was further reduced to 3.4 cM because of a lower recom- natively, in complicated HSP, this spasticity can be accom- binant in a second family, which narrows the interval be- 15 panied by other neurological or nonneurological symp- tween markers D8S1804 and D8S1179. This family, as toms, such as ataxia, dementia, mental retardation, deaf- well as a third Brazilian family linked to SPG8, also pre- 16 ness, epilepsy, ichthyosis, retinopathy, ocular neuropathy, sented with pure adult-onset HSP. For two of the families, 14,16 and extrapyramidal disturbances.5,9 There is clinical het- a muscle biopsy was performed ; however, no gross his- tological or histochemical abnormalities were observed. erogeneity within families, where age at onset and severity Ragged red fibers have been observed in muscle biopsies of can differ markedly; between families that map to the patients with HSP who have paraplegin mutations.17 same locus; and certainly between families that map to In the present study, we identified four additional fam- separate loci. This heterogeneity complicates genotype- ilies that are linked to the SPG8 locus. Genes were screened phenotype correlations for HSP. in an expanded candidate SPG8 locus defined by these HSP is also extremely genetically heterogeneous. From four families, along with the British and Brazilian fami- 130 loci mapped (SPG1–33), 11 genes have been identi- lies described above.15,16 This led to the identification of fied. This disease can be transmitted in a dominant (13 three point mutations in the KIAA0196 gene encoding 9– loci), a recessive (15 loci), or an X-linked manner (4 loci). the strumpellin protein product. 11 By far, the most common locus for the disease is SPG4 (MIM 604277), with mutations in the microtubule-sev- Material and Methods ering protein spastin accounting for ∼40% of dominant Subjects 12,13 HSP cases. Protocols were approved by the Ethics Committee of the Centre Families that map to SPG8 are considered to have one Hospitalier de l’Universite´ de Montre´al. Patients gave informed From the Hoˆpital Notre-Dame–Centre Hospitalier de l’Universite´ de Montre´al (P.N.V.; I.A.M.; A.L.; P.A.D.; G.A.R.) and De´partement de Pathologie et Biologie Cellulaire (A.R.; P.D.), Faculte´deMe´decine, Universite´ de Montre´al, and Departments of Human Genetics (P.N.V) and Biology (I.A.M.), McGill University, Montreal; Children’s & Women’s Health Center, Vancouver, Canada (P.M.); Human Genome Research Center, Institute of Biosciences, University of Sa˜o Paulo, Sa˜o Paulo (D.S.; M.Z.); Cambridge Institute for Medical Research and Department of Medical Genetics, University of Cambridge, Cambridge, United Kingdom (E.R.) Received August 7, 2006; accepted for publication November 10, 2006; electronically published December 1, 2006. Address for correspondence and reprints: Dr. Guy A. Rouleau, Faculte´deMe´decine, Universite´ de Montre´al, Hoˆpital Notre-Dame–CHUM, 1560 Sher- brooke E, Room Y-3633, Montreal, Quebec, Canada H2L 4M1. E-mail: [email protected] * These two authors contributed equally to this work. Am. J. Hum. Genet. 2007;80:152–161. ᭧ 2006 by The American Society of Human Genetics. All rights reserved. 0002-9297/2007/8001-0015$15.00 152 The American Journal of Human Genetics Volume 80 January 2007 www.ajhg.org Table 1. Primers and Amplification the model, with concentration on the a-helix in which the two Conditions for KIAA0196 mutations lie and on a second a-helix in nearby 3D space. Pep- The table is available in its entirety in the online tides incorporating one or the other identified point mutation edition of The American Journal of Human Genetics. were visualized in the same manner. Expression Studies consent, after which patient information and blood was collected. DNA was extracted from peripheral blood through use of standard protocols. Northern-blot and RT-PCR analyses.—The KIAA0196 cDNA pBlue- script clone was kindly provided by the Kazusa DNA Research Genotyping and Locus Exclusion Institute. A 1-kb probe specific to the C-terminal region of strum- pellin was generated by digesting the KIAA0196 pBluescript vec- 35 PCR-amplified fragments incorporating a- S–2-deoxyadenosine tor with XhoI and NotI. Thirty micrograms of total RNA per sam- 5-triphosphate were resolved on 6% denaturing polyacrylamide ple was loaded. RNA was extracted from various regions of the gels. Alleles were run alongside an M13mp18 sequence ladder brain of a control individual. An RT-PCR was performed using and were scored on the basis of allele sizes and frequencies from Moloney murine leukemia virus–reverse transcriptase (Invitro- the Fondation Jean Dausset-CEPH database. LOD-score calcula- gen). Primers in exons 10 (forward) and 15 (reverse) of KIAA0196 tions and multipoint analysis were performed using the MLINK were used. Glyceraldehyde-3-phosphate dehydrogenase cDNA program of the LINKMAP software package.18 was amplified as a control. Mutation Screening Constructs The 28 exons of KIAA0196 were screened by automated sequenc- ing, including at least 50 bp of each intronic region. Primers were Each mutation was introduced into the KIAA0196 pBluescript designed using the PrimerSelect program (Lasergene) and were clone by site-directed mutagenesis through use of the primers 5- synthesized by Invitrogen Canada. Primer sequences and ampli- CTGGAGAGTTCGTATCCTATGTG-3 for the exon 14 variant and fication conditions for each exon are listed in table 1. 5-CCTATGTGAGAAAATTTTTGCAGATC-3 for the exon 15 vari- Variants were first tested in 12 control individuals by sequenc- ant, along with primers of their complementary sequence. Wild- ing, followed by allele-specific oligomerization (ASO).19,20 In brief, type and mutant KIAA0196 cDNAs were cloned, upstream of Myc 4 ml of PCR product was hybridized onto Hybond-Nϩ Nylon membranes (Amersham Biosciences) by use of a dot-blot appa- and His tags, into a pCS2 vector and were transcribed in vitro by ratus. P32-labeled probes specific to the mutation or normal se- use of the SP6 mMESSAGE mMachine kit (Ambion) for zebrafish quence were hybridized and then visualized on autoradiographic studies. The protein expression from each of these constructs was film after overnight exposure. ASO primers for exon 11 are 5- validated after their transient expression in cell (HeLa) culture ACTAGAAAACCTTCAAGCT-3 (normal) and 5-ACTAGAAGACC- and subsequent western-blot analysis with an anti-Myc antibody. TTCAAGCT-3 (mutated). For exon 14, ASO primers of 5-GGAGA- A band at the appropriate height (∼134 kDa for strumpellin) was GTTGGTATC-3 (normal) and 5-GGAGAGTTCGTATC-3 (mu- observed. tated) were used. Exon 15 ASO primers were 5-CACTGAAGGTT- TTG-3 (normal) and 5-CACTGAAGTTTTTG-3 (mutated). Zebrafish Knockdown Studies Protein-Sequence Alignment Morpholino injections.—Wild-type zebrafish were raised and mated 22 Cluster analysis was performed using the Probcons (v. 1.09) pro- as described elsewhere. Antisense morpholinos (AMOs) were pur- gram. Proteins from aligned species included Homo sapiens chased from Genetools. The morpholino sequences were designed (Q12768), Canis familiaris (GenBank accession number XP_ against the zebrafish strumpellin ortholog BC045490. The oli- 532327), Pan troglodytes (GenBank accession number XP_519952), gonucleotide CTCTGCCAGAAAATCAC(CAT)GATG (KIAA MO) Drosophila melanogaster (GenBank accession number CG12272), binds to the ATG of the KIAA0196 gene, which prevents its trans- Caenorhabditis elegans (GenBank accession number CE13235), lation, and CTCTcCCAcAAAATgAg(CAT)cATG (CTL MO) is a 5- Xenopus tropicalis (GenBank accession number MGC89323), Rat- bp mismatch control.
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