Produção Nacional
Revisado por pares
Mutations in the KIAA0196 Gene at the SPG8 Locus Cause Hereditary Spastic Paraplegia
2006; Elsevier BV; Volume: 80; Issue: 1 Linguagem: Inglês
10.1086/510782
ISSN1537-6605
AutoresPaul N. Valdmanis, Inge A. Meijer, Annie Reynolds, Adrienne Lei, Patrick MacLeod, David Schlesinger, Mayana Zatz, Evan Reid, Patrick A. Dion, Pierre Drapeau, Guy A. Rouleau,
Tópico(s)Neurological diseases and metabolism
ResumoHereditary 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 individuals. 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 morpholino 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) 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 individuals. 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 morpholino 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 prevalence of 1–18 in 100,0001Silva MC Coutinho P Pinheiro CD Neves JM Serrano P Hereditary ataxias and spastic paraplegias: methodological aspects of a prevalence study in Portugal.J Clin Epidemiol. 1997; 50: 1377-1384Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 2Polo JM Calleja J Combarros O Berciano J Hereditary ataxias and paraplegias in Cantabria, Spain: an epidemiological and clinical study.Brain. 1991; 114: 855-866Crossref PubMed Scopus (145) Google Scholar, 3McMonagle P Webb S Hutchinson M The prevalence of "pure" autosomal dominant hereditary spastic paraparesis in the island of Ireland.J Neurol Neurosurg Psychiatry. 2002; 72: 43-46Crossref PubMed Scopus (50) Google Scholar and is characterized by central-motor-system deficits leading to lower-limb spastic paraperesis.4Fink JK Advances in hereditary spastic paraplegia.Curr Opin Neurol. 1997; 10: 313-318Crossref PubMed Scopus (57) Google Scholar, 5Harding AE Classification of the hereditary ataxias and paraplegias.Lancet. 1983; 1: 1151-1155Abstract PubMed Scopus (707) Google Scholar, 6Harding AE Hereditary spastic paraplegias.Semin Neurol. 1993; 13: 333-336Crossref PubMed Scopus (140) Google Scholar This is due to a "dying back" phenomenon whereby upper motor neurons degenerate progressively, commencing with the longest axons.7Behan WM Maia M Strumpell's familial spastic paraplegia: genetics and neuropathology.J Neurol Neurosurg Psychiatry. 1974; 37: 8-20Crossref PubMed Scopus (158) Google Scholar, 8Deluca GC Ebers GC Esiri MM The extent of axonal loss in the long tracts in hereditary spastic paraplegia.Neuropathol Appl Neurobiol. 2004; 30: 576-584Crossref PubMed Scopus (137) Google Scholar HSP can be classified into pure and complicated forms.5Harding AE Classification of the hereditary ataxias and paraplegias.Lancet. 1983; 1: 1151-1155Abstract PubMed Scopus (707) Google Scholar In pure HSP, lower-limb spasticity is the only major symptom. Alternatively, in complicated HSP, this spasticity can be accompanied by other neurological or nonneurological symptoms, such as ataxia, dementia, mental retardation, deafness, epilepsy, ichthyosis, retinopathy, ocular neuropathy, and extrapyramidal disturbances.5Harding AE Classification of the hereditary ataxias and paraplegias.Lancet. 1983; 1: 1151-1155Abstract PubMed Scopus (707) Google Scholar, 9Soderblom C Blackstone C Traffic accidents: molecular genetic insights into the pathogenesis of the hereditary spastic paraplegias.Pharmacol Ther. 2006; 109: 42-56Crossref PubMed Scopus (63) Google Scholar There is clinical heterogeneity within families, where age at onset and severity can differ markedly; between families that map to the same locus; and certainly between families that map to separate loci. This heterogeneity complicates genotype-phenotype correlations for HSP. HSP is also extremely genetically heterogeneous. From >30 loci mapped (SPG1–33), 11 genes have been identified. This disease can be transmitted in a dominant (13 loci), a recessive (15 loci), or an X-linked manner (4 loci).9Soderblom C Blackstone C Traffic accidents: molecular genetic insights into the pathogenesis of the hereditary spastic paraplegias.Pharmacol Ther. 2006; 109: 42-56Crossref PubMed Scopus (63) Google Scholar, 10Klebe S Azzedine H Durr A Bastien P Bouslam N Elleuch N Forlani S Charon C Koenig M Melki J et al.Autosomal recessive spastic paraplegia (SPG30) with mild ataxia and sensory neuropathy maps to chromosome 2q37.3.Brain. 2006; 129: 1456-1462Crossref PubMed Scopus (52) Google Scholar, 11Zuchner S Kail ME Nance MA Gaskell PC Svenson IK Marchuk DA Pericak-Vance MA Ashley-Koch AE A new locus for dominant hereditary spastic paraplegia maps to chromosome 2p12.Neurogenetics. 2006; 7: 127-129Crossref PubMed Scopus (19) Google Scholar By far, the most common locus for the disease is SPG4 (MIM 604277), with mutations in the microtubule-severing protein spastin accounting for ∼40% of dominant HSP cases.12Hazan J Fonknechten N Mavel D Paternotte C Samson D Artiguenave F Davoine CS Cruaud C Durr A Wincker P et al.Spastin, a new AAA protein, is altered in the most frequent form of autosomal dominant spastic paraplegia.Nat Genet. 1999; 23: 296-303Crossref PubMed Scopus (504) Google Scholar, 13Meijer IA Hand CK Cossette P Figlewicz DA Rouleau GA Spectrum of SPG4 mutations in a large collection of North American families with hereditary spastic paraplegia.Arch Neurol. 2002; 59: 281-286Crossref PubMed Scopus (69) Google Scholar Families that map to SPG8 are considered to have one of the more aggressive subtypes of HSP, with disease onset occurring for patients as early as their 20s or 30s. It was first identified in a white family as a 6.2-cM region between markers D8S1804 and D8S1774.14Hedera P Rainier S Alvarado D Zhao X Williamson J Otterud B Leppert M Fink JK Novel locus for autosomal dominant hereditary spastic paraplegia, on chromosome 8q.Am J Hum Genet. 1999; 64: 563-569Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar The family had 15 patients affected with spasticity, hyperreflexia, extensor plantar reflexes, lower-limb weakness, decreased vibration sensation, and limited muscle wasting. The candidate region was further reduced to 3.4 cM because of a lower recombinant in a second family, which narrows the interval between markers D8S1804 and D8S1179.15Reid E Dearlove AM Whiteford ML Rhodes M Rubinsztein DC Autosomal dominant spastic paraplegia: refined SPG8 locus and additional genetic heterogeneity.Neurology. 1999; 53: 1844-1849Crossref PubMed Google Scholar This family, as well as a third Brazilian family linked to SPG8, also presented with pure adult-onset HSP.16Rocco P Vainzof M Froehner SC Peters MF Marie SK Passos-Bueno MR Zatz M Brazilian family with pure autosomal dominant spastic paraplegia maps to 8q: analysis of muscle beta 1 syntrophin.Am J Med Genet. 2000; 92: 122-127Crossref PubMed Scopus (16) Google Scholar For two of the families, a muscle biopsy was performed14Hedera P Rainier S Alvarado D Zhao X Williamson J Otterud B Leppert M Fink JK Novel locus for autosomal dominant hereditary spastic paraplegia, on chromosome 8q.Am J Hum Genet. 1999; 64: 563-569Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 16Rocco P Vainzof M Froehner SC Peters MF Marie SK Passos-Bueno MR Zatz M Brazilian family with pure autosomal dominant spastic paraplegia maps to 8q: analysis of muscle beta 1 syntrophin.Am J Med Genet. 2000; 92: 122-127Crossref PubMed Scopus (16) Google Scholar; however, no gross histological or histochemical abnormalities were observed. Ragged red fibers have been observed in muscle biopsies of patients with HSP who have paraplegin mutations.17Casari G De Fusco M Ciarmatori S Zeviani M Mora M Fernandez P De Michele G Filla A Cocozza S Marconi R et al.Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a nuclear encoded mitochondrial metalloprotease.Cell. 1998; 93: 973-983Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar In the present study, we identified four additional families that are linked to the SPG8 locus. Genes were screened in an expanded candidate SPG8 locus defined by these four families, along with the British and Brazilian families described above.15Reid E Dearlove AM Whiteford ML Rhodes M Rubinsztein DC Autosomal dominant spastic paraplegia: refined SPG8 locus and additional genetic heterogeneity.Neurology. 1999; 53: 1844-1849Crossref PubMed Google Scholar, 16Rocco P Vainzof M Froehner SC Peters MF Marie SK Passos-Bueno MR Zatz M Brazilian family with pure autosomal dominant spastic paraplegia maps to 8q: analysis of muscle beta 1 syntrophin.Am J Med Genet. 2000; 92: 122-127Crossref PubMed Scopus (16) Google Scholar This led to the identification of three point mutations in the KIAA0196 gene encoding the strumpellin protein product. Protocols were approved by the Ethics Committee of the Centre Hospitalier de l'Université de Montréal. Patients gave informed consent, after which patient information and blood was collected. DNA was extracted from peripheral blood through use of standard protocols. PCR-amplified fragments incorporating α-35S–2-deoxyadenosine 5-triphosphate were resolved on 6% denaturing polyacrylamide gels. Alleles were run alongside an M13mp18 sequence ladder and were scored on the basis of allele sizes and frequencies from the Fondation Jean Dausset-CEPH database. LOD-score calculations and multipoint analysis were performed using the MLINK program of the LINKMAP software package.18Cottingham Jr, RW Idury RM Schaffer AA Faster sequential genetic linkage computations.Am J Hum Genet. 1993; 53: 252-263PubMed Google Scholar The 28 exons of KIAA0196 were screened by automated sequencing, including at least 50 bp of each intronic region. Primers were designed using the PrimerSelect program (Lasergene) and were synthesized by Invitrogen Canada. Primer sequences and amplification conditions for each exon are listed in table 1.Table 1Primers and Amplification Conditions for KIAA0196ExonPrimer (5′→3′)KIAA0196×1 (forward)GCCAAGAGTGTTAATCTAGCAAAGTCKIAA0196×1(reverse)TTCATGGTTCCCAGAGAAAACACGKIAA0196×2 (forward)TCTGCTTTAAGTTTGGGATGTCTAKIAA0196×2 (reverse)TTAAGATGACCAGTGCCACAGGTAKIAA0196×3 (forward)AATATCAAACTGTGGCCCTAAATCKIAA0196×3 (reverse)TACACCGAGGAGGCTCATAACTTCKIAA0196×4 (forward)CATCCCAGCCATCTGTCCTGATACKIAA0196×4 (reverse)ACATACACTGCATTTTACCGACAGCKIAA0196×5 (forward)AATGGAATTCTACTTTATTGGACTKIAA0196×5 (reverse)CTCAAAAGGTTTTAAAAGGTTCTACCKIAA0196×6 (forward)TGGGCTTTGGAAAAACTGATGTGTCTKIAA0196×6 (reverse)AAGTTTACCTAAGTGATGTTATGTCCKIAA0196×7 (forward)CAAAAAGCAACGTTAATAGGTGTAAKIAA0196×7 (reverse)ATCATTGCATTAAATTATCTAAGTGKIAA0196×8 (forward)TTAATCACAGCCAGAACTAGGATGTAGKIAA0196×8 (reverse)GACAGGGGAGAGCTTTTCAGGTATGCTKIAA0196×9 (forward)TGGCACTCCATGTCAGATTCAACTGTKIAA0196×9 (reverse)ATGTCTATATTCCCCATTAGGKIAA0196×10 (forward)CAGGGTCAATGTTAATTTATAGTGTAKIAA0196×10 (reverse)AGATGGAGGCCAACTGTGACTCTCKIAA0196×11 (forward)TGCTCCAGGCATTTTTGTCGKIAA0196×11 (reverse)GAACAGACTGCTGGGTGGGTCATAKIAA0196×12 and 13 (forward)ATGAGCACCATAGAGTCCATTCAGGKIAA0196×12 and 13 (reverse)ATTATGCTCTCGTGGAAAAACTGCTAKIAA0196×14 (forward)CTTTTTGAAACAAGAAACAGATATACCKIAA0196×14 (reverse)GGCAAGTAAAAACATCTGTACATCCACKIAA0196×15 (forward)TTTGCAGCATTTTTAGAAGGATTAGCKIAA0196×15 (reverse)TTCCCCTGAGAATACTGAGGCGAACAKIAA0196×16 (forward)GGAGGCCAGGGAAGGGGAGGTTACCKIAA0196×16 (reverse)GGAATGTCAAACAGCCAGATGATGTKIAA0196×17 (forward)ACTTTGCTGAAATAAACAGAGTCCKIAA0196×17 (reverse)GTAAGGTCTTGTTCGCGATAGGTTKIAA0196×18 (forward)AGAACGAATAGTTGACAATCTACTCKIAA0196×18 (reverse)TGAGGTTTGGGATGTGTACTCTAAKIAA0196×19 (forward)AATTATATGGAAAAGGGATAACTAGGTKIAA0196×19 (reverse)TAAAGGGTCAGAATATGAGTTGACAAGKIAA0196×20 (forward)TTGGTGCCGCATGTCCTGTTGAGTCKIAA0196×20 (reverse)AAGTCTTATCTTCCCAAGTTGAAACKIAA0196×21 and 22 (forward)CCCAGCCTCTGTTCTGCATAGCATKIAA0196×21 and 22 (reverse)AAGAACAGATCCAGAAACGGAGATKIAA0196×23 (forward)AAGGCCCAGTGAAGAATTGTCATCKIAA0196×23 (reverse)CTGAAGAAACTGGGGTGCGTAGATKIAA0196×24 (forward)CTGAGGCTTGAAAAGATTACATCACKIAA0196×24 (reverse)CTTCCCCTTTGTCATGAGCTTTCACKIAA0196×25 (forward)TCCCACACTCCCCCTATATTCACCTCKIAA0196×25 (reverse)AGAAAAGATCTCATATCCGACATAGGKIAA0196×26 (forward)GACCCCTGGAATGCCCTACCAATCKIAA0196×26 (reverse)CTGGCAGGGTGACTAAGGATGGACKIAA0196×27 (forward)GATAGATAGCAGGGATCGTGTTGTKIAA0196×27 (reverse)AGGCATCTACTGTGAACGACCTATKIAA0196×28 (forward)AAAGGGGCTGTTTCAAGGAGTCGKIAA0196×28 (reverse)AGTTTTTGAATCATAAGCGAGACGNote.—PCR was performed using 50 ng DNA, 20 pmol of each primer, 10× buffer, 0.25 nM deoxyribonucleotide triphosphate, and 0.15 μl of Taq (Qiagen). Initial denaturation for 5 min at 94°C was followed by 30 cycles of 30-s denaturation at 94°C, 30 s annealing at 55°C (for all exons except 15 and 26), and 45-s elongation at 72°C. A final extension at 72°C was performed for 7 min. For exon 15, a 50°C annealing temperature was used, and, for exon 26, 10 cycles of a touchdown reaction were performed from 68°C to 63°C, followed by 25 cycles at 63°C. Open table in a new tab Note.— PCR was performed using 50 ng DNA, 20 pmol of each primer, 10× buffer, 0.25 nM deoxyribonucleotide triphosphate, and 0.15 μl of Taq (Qiagen). Initial denaturation for 5 min at 94°C was followed by 30 cycles of 30-s denaturation at 94°C, 30 s annealing at 55°C (for all exons except 15 and 26), and 45-s elongation at 72°C. A final extension at 72°C was performed for 7 min. For exon 15, a 50°C annealing temperature was used, and, for exon 26, 10 cycles of a touchdown reaction were performed from 68°C to 63°C, followed by 25 cycles at 63°C. Variants were first tested in 12 control individuals by sequencing, followed by allele-specific oligomerization (ASO).19Bourgeois S Labuda D Dynamic allele-specific oligonucleotide hybridization on solid support.Anal Biochem. 2004; 324: 309-311Crossref PubMed Scopus (28) Google Scholar, 20Labuda D Krajinovic M Richer C Skoll A Sinnett H Yotova V Sinnett D Rapid detection of CYP1A1, CYP2D6, and NAT variants by multiplex polymerase chain reaction and allele-specific oligonucleotide assay.Anal Biochem. 1999; 275: 84-92Crossref PubMed Scopus (60) Google Scholar In brief, 4 μl of PCR product was hybridized onto Hybond-N+ Nylon membranes (Amersham Biosciences) by use of a dot-blot apparatus. P32-labeled probes specific to the mutation or normal sequence were hybridized and then visualized on autoradiographic film after overnight exposure. ASO primers for exon 11 are 5′-ACTAGAAAACCTTCAAGCT-3′ (normal) and 5′-ACTAGAAGACCTTCAAGCT-3′ (mutated). For exon 14, ASO primers of 5′-GGAGAGTTGGTATC-3′ (normal) and 5′-GGAGAGTTCGTATC-3′ (mutated) were used. Exon 15 ASO primers were 5′-CACTGAAGGTTTTG-3′ (normal) and 5′-CACTGAAGTTTTTG-3′ (mutated). Cluster analysis was performed using the Probcons (v. 1.09) program. Proteins from aligned species included Homo sapiens (Q12768), Canis familiaris (GenBank accession number XP_532327), Pan troglodytes (GenBank accession number XP_519952), Drosophila melanogaster (GenBank accession number CG12272), Caenorhabditis elegans (GenBank accession number CE13235), Xenopus tropicalis (GenBank accession number MGC89323), Rattus norvegicus (GenBank accession number XP_343250), Danio rerio (GenBank accession number BC045490), Gallus gallus (GenBank accession number XP_418441), Dictyostelium discoideum (GenBank accession number EAL63144), and Mus musculus (GenBank accession number NP_705776.2). The size of the strumpellin protein (1,159 aa) made it prohibitive to obtain a template for the entire protein. Instead, 200 aa (amino acids 501–725) around the two mutations were selected and inputted in the Phyre program version 2.0 (Phyre Protein Fold Recognition Server). The template with the highest score was selected—namely, 1dn1b from the Neuronal-Sec1 syntaxin 1a complex. The SwissProt database viewer (v. 3.7)21Guex N Peitsch MC SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling.Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9106) Google Scholar was used to visualize the model, with concentration on the α-helix in which the two mutations lie and on a second α-helix in nearby 3D space. Peptides incorporating one or the other identified point mutation were visualized in the same manner. The KIAA0196 cDNA pBluescript clone was kindly provided by the Kazusa DNA Research Institute. A 1-kb probe specific to the C-terminal region of strumpellin was generated by digesting the KIAA0196 pBluescript vector with XhoI and NotI. Thirty micrograms of total RNA per sample was loaded. RNA was extracted from various regions of the brain of a control individual. An RT-PCR was performed using Moloney murine leukemia virus–reverse transcriptase (Invitrogen). Primers in exons 10 (forward) and 15 (reverse) of KIAA0196 were used. Glyceraldehyde-3-phosphate dehydrogenase cDNA was amplified as a control. Each mutation was introduced into the KIAA0196 pBluescript clone by site-directed mutagenesis through use of the primers 5′-CTGGAGAGTTCGTATCCTATGTG-3′ for the exon 14 variant and 5′-CCTATGTGAGAAAATTTTTGCAGATC-3′ for the exon 15 variant, along with primers of their complementary sequence. Wild-type and mutant KIAA0196 cDNAs were cloned, upstream of Myc and His tags, into a pCS2 vector and were transcribed in vitro by use of the SP6 mMESSAGE mMachine kit (Ambion) for zebrafish studies. The protein expression from each of these constructs was validated after their transient expression in cell (HeLa) culture and subsequent western-blot analysis with an anti-Myc antibody. A band at the appropriate height (∼134 kDa for strumpellin) was observed. Wild-type zebrafish were raised and mated as described elsewhere.22Westerfield M The zebrafish book: a guide for laboratory use of zebrafish (Danio rerio). University of Oregon Press, Eugene1995Google Scholar Antisense morpholinos (AMOs) were purchased from Genetools. The morpholino sequences were designed against the zebrafish strumpellin ortholog BC045490. The oligonucleotide CTCTGCCAGAAAATCAC(CAT)GATG (KIAA MO) binds to the ATG of the KIAA0196 gene, which prevents its translation, and CTCTcCCAcAAAATgAg(CAT)cATG (CTL MO) is a 5-bp mismatch control. AMO injections were performed as described elsewhere, at a concentration of 0.8 mM.23Nasevicius A Ekker SC Effective targeted gene "knockdown" in zebrafish.Nat Genet. 2000; 26: 216-220Crossref PubMed Scopus (2026) Google Scholar The rescue injections were performed as mentioned above, with morpholino and mRNA concentrations of 0.8 mM and 50 ng/μl, respectively. Standard protocols were used for immunohistochemistry.22Westerfield M The zebrafish book: a guide for laboratory use of zebrafish (Danio rerio). University of Oregon Press, Eugene1995Google Scholar In brief, 3-d-old embryos were fixed in 4% paraformaldehyde, were washed, and were blocked at room temperature. Primary antibody (anti-acetylated tubulin; 1:50 [Sigma]) was added overnight. After extensive washing, the embryos were incubated with the fluorescently labeled secondary antibody Alexa 568 (Molecular Probes). Imaging was performed on an UltraView LCI confocal microscope (Perkin Elmer) with use of Methamorph Imaging software (Universal Imaging). The statistical significance between the different conditions was calculated using a χ2 test. Family FSP24 with the SPG8 mutation is from British Columbia. It is composed of 13 members affected with a spastic gait and lower-limb stiffness (fig. 1A); genetic information is available for 10 of them. Symptoms were first observed in individuals between ages 35 and 53 years. Intrafamilial phenotypic heterogeneity exists, as shown by the symptoms presented and the range of disease severity in patients. Deep-tendon reflexes were brisk or increased, and decreased vibration sensation was also noted in three patients. Occasional bladder-control problems were also observed. Walking aids were required for some individuals, whereas one is confined to a wheelchair. Together, these features are consistent with a pure, uncomplicated HSP similar to that described for other families linked to the SPG8 locus. Family FSP29 is of European descent and resides in the United States. There are 31 affected individuals in the family, and genetic information is available for 10 of them (fig. 1B). Age at onset was quite variable, with symptom onset ranging in patients from their 20s to their 60s. The family was negative for mutations in the spastin gene. Two large families that map to the SPG8 locus were identified. In family FSP24, seven markers spanning the candidate region from markers D8S586 to D8S1128 were genotyped in the 18 individuals studied (fig. 1A). A disease haplotype segregated with the disease in all 10 affected individuals (table 2). A recombination event occurred in one individual (fig. 1A) between markers D8S586 and D8S1804, which defined the proximal border of the locus in this family. A lower recombinant was neither identified nor searched for, since the haplotype extended beyond the limits of the SPG8 locus. The maximum LOD score for this family was 3.43 at θ=0, by use of CEPH allele frequencies for the marker D8S1804, along with a maximum multipoint of 4.20 at marker D8S1799.Table 2Haplotype Comparison between SPG8-Linked FamiliesAlleles Number for FamilyMarkerPosition (Mb)FSP24FSP29FSP34D8S586121.211111D8S1804124.8533D8S1832125.422Not typedD8S1179125.9399D8S1774127.5354D8S1128128.5751Note.—Flanking markers in the candidate region are D8S1832 and D8S1774 for family FSP29. The KIAA0196 L619F mutation was at position 126.1 Mb for all three families. The allele for rs2293890 (126.4 Mb) was G for family FSP24 and was C for both families FSP29 and FSP34. Open table in a new tab Note.— Flanking markers in the candidate region are D8S1832 and D8S1774 for family FSP29. The KIAA0196 L619F mutation was at position 126.1 Mb for all three families. The allele for rs2293890 (126.4 Mb) was G for family FSP24 and was C for both families FSP29 and FSP34. The same seven markers tested in family FSP24 were genotyped for family FSP29. A disease haplotype was established for all 10 studied affected individuals; it included many informative recombination events. The proximal recombinant occurred between markers D8S1799 and D8S1832 in three affected individuals (fig. 1B), and the distal recombinant was between markers D8S1774 and D8S1128 for another affected individual (fig. 1B). This yielded a candidate interval of 3.15 Mb. The maximum LOD score for this family was 5.62 (θ=0) for marker D8S1179 when CEPH allele frequencies were used. Multipoint analysis was also conducted for this family in this region, which yielded a maximum LOD score of 6.73, 0.5 cM centromeric to the D8S1128 marker. The previously published SPG8 locus spanned 3.4 cM (1.04 Mb) between markers D8S1804 and D8S1179 on chromosome 8q23-8q24. We screened nine known genes surrounding this candidate region, as annotated in the University of California–Santa Cruz Genome Browser (UCSC) May 2004 update, along with many clustered ESTs and mRNAs that aligned to the locus, without detecting a mutation. Therefore, we opted to redefine the candidate region on the basis of the critical interval determined by an upper recombinant in our FSP29 family at marker D8S1832, and a lower recombinant at D8S1774 was based on published data (fig. 2A).14Hedera P Rainier S Alvarado D Zhao X Williamson J Otterud B Leppert M Fink JK Novel locus for autosomal dominant hereditary spastic paraplegia, on chromosome 8q.Am J Hum Genet. 1999; 64: 563-569Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar This increased the size of the region to 5.43 cM (3.15 Mb), a region that contains three additional known genes (fig. 2B). These additional genes were screened, and three mutations were identified in the KIAA0196 gene (fig. 2C). A valine→phenylalanine mutation was identified in amino acid 626 for families FSP24 and FSP29 (p.V626F) (fig. 3A). All studied affected individuals from each family were screened and were positive for this mutation. The same mutation was also found to segregate in a British family.15Reid E Dearlove AM Whiteford ML Rhodes M Rubinsztein DC Autosomal dominant spastic paraplegia: refined SPG8 locus and additional genetic heterogeneity.Neurology. 1999; 53: 1844-1849Crossref PubMed Google Scholar This G→T nucleotide change is at position 1956 of the mRNA (GenBank accession number NM_014846.2). A total of 500 ethnically matched control individuals (400 from North America and 100 from CEPH) were negative for this mutation, on screening by a combination of ASO and sequencing. No unaffected members or spouse control individuals in any family were positive for the mutations. A second mutation was identified in the Brazilian family16Rocco P Vainzof M Froehner SC Peters MF Marie SK Passos-Bueno MR Zatz M Brazilian family with pure autosomal dominant spastic paraplegia maps to 8q: analysis of muscle beta 1 syntrophin.Am J Med Genet. 2000; 92: 122-127Crossref PubMed Scopus (16) Google Scholar in exon 14, a G→C transition at position 1937 of the mRNA (fig. 3B). This leucine→phenylalanine change (p.L619F) is only 7 aa away from the V626F mutation. It was not found, with use of ASO, in 500 controls. The KIAA0196 gene was screened in probands from 24 additional dominant HSP–affected families that are negative for mutations in both spastin and atlastin, resulting in the identification of two more families with missense mutations in the KIAA0196 gene. Thus, the frequency of mutations in our SPG3A- and SPG4-negative autosomal dominant cohort is ∼8% (2 of 24). FSP34 has the same p.V626F change in its three affected studied family members. This family is originally from Great Britain and resides in Canada (fig. 1C). Haplotype analysis of this family with markers D8S1804, D8S1179, D8S1774, and D8S1128 indicated that there is allele sharing between this family and family FSP29, which suggests an ancestral haplotype (table 2). An additional mutation was found in three affected siblings of another North American family of European origin, family FSP91 (fig. 1D). This c.A1491G transition results in an asparagine→aspartate amino acid change (p.N471D) and is not present in the 500 controls tested (fig. 3C). Mutated amino acids at positions 619 and 626 are strictly conserved across all 11 examined species, from human to the social amoeba, D. discoideum (fig. 3D). Indeed, the entire region surrounding these two mutations appears to be functionally relevant for the protein, since 73 consecutive aa (amino acids 576–649) are 100% identical in the human, dog, chicken, mouse, rat, and orangutan. Despite this high level of conservation, this region is an unknown domain, on the basis of searches of the National Center for Biotechnology Information (NCBI) Conserved Domain Database, NCBI BLAST, and the Sanger Institute's Pfam database. Position 471 is conserved across all species except D. melanogaster (with a glutamine residue) and X. tropicalis (with a histidine residue). The exon 15 mutation is in the very first nucleotide of the exon, which leads to the speculation that the splicing of this exon might be compromised in our study families. Splice-site prediction programs, including NetGene2, suggested that the strength of the splice-site acceptor may be reduced by 33% in the mutant form. However, both normal and mutant alleles were observed in cDNA analysis, with use of several pairs of primers, of patient lymphoblasts. The KIAA0196 gene was expressed ubiquitously, including all regions of the brain that were examined by RT-PCR (fig. 3E). There were no alternative splice isoforms detected in control brain samples or patient whole-blood samples by RT-PCR and northern-blot analysis (fig. 3E and 3F). For the full KIAA0196 gene, all spliced ESTs and mRNAs from the UCSC browser, May 2004 draft, were analyzed for potential alternative splice products. One alternative first exon often appears; however, of the 356 entries, only 2 (AK223628 and DA202680) contain exons that are skipped. Thus, overall, the gene is not frequently spliced, and the two spliced entries may represent spurious transcripts. The KIAA0196 gene spans 59.7-kb pairs of ge
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