The Novel Neuronal Ceroid Lipofuscinosis Gene MFSD8 Encodes a Putative Lysosomal Transporter
2007; Elsevier BV; Volume: 81; Issue: 1 Linguagem: Inglês
10.1086/518902
ISSN1537-6605
AutoresEija Siintola, Meral Topçu, Nina Aula, Hannes Lohi, Berge A. Minassian, Andrew D. Paterson, Xiaoqing Liu, Callum Wilson, Ulla Lahtinen, Anna-Kaisa Anttonen, Anna-Elina Lehesjoki,
Tópico(s)Cellular transport and secretion
ResumoThe late-infantile–onset forms are the most genetically heterogeneous group among the autosomal recessively inherited neurodegenerative disorders, the neuronal ceroid lipofuscinoses (NCLs). The Turkish variant was initially considered to be a distinct genetic entity, with clinical presentation similar to that of other forms of late-infantile–onset NCL (LINCL), including age at onset from 2 to 7 years, epileptic seizures, psychomotor deterioration, myoclonus, loss of vision, and premature death. However, Turkish variant LINCL was recently found to be genetically heterogeneous, because mutations in two genes, CLN6 and CLN8, were identified to underlie the disease phenotype in a subset of patients. After a genomewide scan with single-nucleotide–polymorphism markers and homozygosity mapping in nine Turkish families and one Indian family, not linked to any of the known NCL loci, we mapped a novel variant LINCL locus to chromosome 4q28.1-q28.2 in five families. We identified six different mutations in the MFSD8 gene (previously denoted "MGC33302"), which encodes a novel polytopic 518–amino acid membrane protein that belongs to the major facilitator superfamily of transporter proteins. MFSD8 is expressed ubiquitously, with several alternatively spliced variants. Like the majority of the previously identified NCL proteins, MFSD8 localizes mainly to the lysosomal compartment. However, the function of MFSD8 remains to be elucidated. Analysis of the genome-scan data suggests the existence of at least three more genes in the remaining five families, further corroborating the great genetic heterogeneity of LINCLs. The late-infantile–onset forms are the most genetically heterogeneous group among the autosomal recessively inherited neurodegenerative disorders, the neuronal ceroid lipofuscinoses (NCLs). The Turkish variant was initially considered to be a distinct genetic entity, with clinical presentation similar to that of other forms of late-infantile–onset NCL (LINCL), including age at onset from 2 to 7 years, epileptic seizures, psychomotor deterioration, myoclonus, loss of vision, and premature death. However, Turkish variant LINCL was recently found to be genetically heterogeneous, because mutations in two genes, CLN6 and CLN8, were identified to underlie the disease phenotype in a subset of patients. After a genomewide scan with single-nucleotide–polymorphism markers and homozygosity mapping in nine Turkish families and one Indian family, not linked to any of the known NCL loci, we mapped a novel variant LINCL locus to chromosome 4q28.1-q28.2 in five families. We identified six different mutations in the MFSD8 gene (previously denoted "MGC33302"), which encodes a novel polytopic 518–amino acid membrane protein that belongs to the major facilitator superfamily of transporter proteins. MFSD8 is expressed ubiquitously, with several alternatively spliced variants. Like the majority of the previously identified NCL proteins, MFSD8 localizes mainly to the lysosomal compartment. However, the function of MFSD8 remains to be elucidated. Analysis of the genome-scan data suggests the existence of at least three more genes in the remaining five families, further corroborating the great genetic heterogeneity of LINCLs. The neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive neurodegenerative lysosomal storage disorders characterized by the accumulation of autofluorescent storage material in many cell types, including neurons.1Santavuori P Neuronal ceroid-lipofuscinoses in childhood.Brain Dev. 1988; 10: 80-83Abstract Full Text PDF PubMed Scopus (257) Google Scholar Clinically, they present with a variable age at onset, epileptic seizures, progressive psychomotor decline, visual failure, and premature death.2Haltia M The neuronal ceroid-lipofuscinoses.J Neuropathol Exp Neurol. 2003; 62: 1-13Crossref PubMed Scopus (217) Google Scholar To date, 10 different forms of human NCLs and seven causative genes (PPT1/CLN1 [MIM 600722], TPP1/CLN2 [MIM 607998], CLN3 [MIM 607042], CLN5 [MIM 608102], CLN6 [MIM 606725], CLN8 [MIM 607837], and CTSD/CLN10 [MIM 116840]) have been identified.3The International Batten Disease Consortium Isolation of a novel gene underlying Batten disease, CLN3.Cell. 1995; 82: 949-957Abstract Full Text PDF PubMed Scopus (561) Google Scholar, 4Vesa J Hellsten E Verkruyse LA Camp LA Rapola J Santavuori P Hofmann SL Peltonen L Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis.Nature. 1995; 376: 584-587Crossref PubMed Scopus (627) Google Scholar, 5Sleat DE Donnelly RJ Lackland H Liu CG Sohar I Pullarkat RK Lobel P Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis.Science. 1997; 277: 1802-1805Crossref PubMed Scopus (505) Google Scholar, 6Savukoski M Klockars T Holmberg V Santavuori P Lander ES Peltonen L CLN5, a novel gene encoding a putative transmembrane protein mutated in Finnish variant late infantile neuronal ceroid lipofuscinosis.Nat Genet. 1998; 19: 286-288Crossref PubMed Scopus (244) Google Scholar, 7Ranta S Zhang Y Ross B Lonka L Takkunen E Messer A Sharp J Wheeler R Kusumi K Mole S et al.The neuronal ceroid lipofuscinoses in human EPMR and mnd mutant mice are associated with mutations in CLN8.Nat Genet. 1999; 23: 233-236Crossref PubMed Scopus (255) Google Scholar, 8Gao H Boustany RM Espinola JA Cotman SL Srinidhi L Antonellis KA Gillis T Qin X Liu S Donahue LR et al.Mutations in a novel CLN6-encoded transmembrane protein cause variant neuronal ceroid lipofuscinosis in man and mouse.Am J Hum Genet. 2002; 70: 324-335Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 9Wheeler RB Sharp JD Schultz RA Joslin JM Williams RE Mole SE The gene mutated in variant late-infantile neuronal ceroid lipofuscinosis (CLN6) and in nclf mutant mice encodes a novel predicted transmembrane protein.Am J Hum Genet. 2002; 70: 537-542Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 10Siintola E Partanen S Stromme P Haapanen A Haltia M Maehlen J Lehesjoki AE Tyynela J Cathepsin D deficiency underlies congenital human neuronal ceroid-lipofuscinosis.Brain. 2006; 129: 1438-1445Crossref PubMed Scopus (294) Google Scholar, 11Steinfeld R Reinhardt K Schreiber K Hillebrand M Kraetzner R Bruck W Saftig P Gartner J Cathepsin D deficiency is associated with a human neurodegenerative disorder.Am J Hum Genet. 2006; 78: 988-998Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar The loci and genes for adult-onset NCL (CLN4 [MIM 204300]),12Berkovic SF Carpenter S Andermann F Andermann E Wolfe LS Kufs' disease: a critical reappraisal.Brain. 1988; 111: 27-62Crossref PubMed Scopus (201) Google Scholar the Turkish variant (CLN7) late-infantile–onset NCL (LINCL),13Wheeler RB Sharp JD Mitchell WA Bate SL Williams RE Lake BD Gardiner RM A new locus for variant late infantile neuronal ceroid lipofuscinosis—CLN7.Mol Genet Metab. 1999; 66: 337-338Crossref PubMed Scopus (44) Google Scholar and the recently identified CLN9-variant LINCL (MIM 609055)14Schulz A Dhar S Rylova S Dbaibo G Alroy J Hagel C Artacho I Kohlschutter A Lin S Boustany RM Impaired cell adhesion and apoptosis in a novel CLN9 Batten disease variant.Ann Neurol. 2004; 56: 342-350Crossref PubMed Scopus (53) Google Scholar have remained undetected. Within the NCLs, the late-infantile–onset group is genetically the most heterogeneous, with causative mutations found in CLN1, CLN2, CLN5, CLN6, and CLN8.5Sleat DE Donnelly RJ Lackland H Liu CG Sohar I Pullarkat RK Lobel P Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis.Science. 1997; 277: 1802-1805Crossref PubMed Scopus (505) Google Scholar, 6Savukoski M Klockars T Holmberg V Santavuori P Lander ES Peltonen L CLN5, a novel gene encoding a putative transmembrane protein mutated in Finnish variant late infantile neuronal ceroid lipofuscinosis.Nat Genet. 1998; 19: 286-288Crossref PubMed Scopus (244) Google Scholar, 8Gao H Boustany RM Espinola JA Cotman SL Srinidhi L Antonellis KA Gillis T Qin X Liu S Donahue LR et al.Mutations in a novel CLN6-encoded transmembrane protein cause variant neuronal ceroid lipofuscinosis in man and mouse.Am J Hum Genet. 2002; 70: 324-335Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 9Wheeler RB Sharp JD Schultz RA Joslin JM Williams RE Mole SE The gene mutated in variant late-infantile neuronal ceroid lipofuscinosis (CLN6) and in nclf mutant mice encodes a novel predicted transmembrane protein.Am J Hum Genet. 2002; 70: 537-542Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 15Das AK Becerra CH Yi W Lu JY Siakotos AN Wisniewski KE Hofmann SL Molecular genetics of palmitoyl-protein thioesterase deficiency in the U.S.J Clin Invest. 1998; 102: 361-370Crossref PubMed Scopus (137) Google Scholar, 16Ranta S Topcu M Tegelberg S Tan H Ustubutun A Saatci I Dufke A Enders H Pohl K Alembik Y et al.Variant late infantile neuronal ceroid lipofuscinosis in a subset of Turkish patients is allelic to Northern epilepsy.Hum Mutat. 2004; 23: 300-305Crossref PubMed Scopus (78) Google Scholar, 17Cannelli N Cassandrini D Bertini E Striano P Fusco L Gaggero R Specchio N Biancheri R Vigevano F Bruno C et al.Novel mutations in CLN8 in Italian variant late infantile neuronal ceroid lipofuscinosis: another genetic hit in the Mediterranean.Neurogenetics. 2006; 7: 111-117Crossref PubMed Scopus (39) Google Scholar The phenotype of approximately half of the Turkish patients with NCL is classified as variant LINCL (vLINCL), with clinical presentation similar to the other forms of vLINCL.18Topcu M Tan H Yalnizoglu D Usubutun A Saatci I Aynaci M Anlar B Topaloglu H Turanli G Kose G et al.Evaluation of 36 patients from Turkey with neuronal ceroid lipofuscinosis: clinical, neurophysiological, neuroradiological and histopathologic studies.Turk J Pediatr. 2004; 46: 1-10PubMed Google Scholar The age at onset ranges from 2 to 7 years, and, in most patients, onset has been considered to be somewhat later than in the classical LINCL (CLN2 [MIM 204500]).18Topcu M Tan H Yalnizoglu D Usubutun A Saatci I Aynaci M Anlar B Topaloglu H Turanli G Kose G et al.Evaluation of 36 patients from Turkey with neuronal ceroid lipofuscinosis: clinical, neurophysiological, neuroradiological and histopathologic studies.Turk J Pediatr. 2004; 46: 1-10PubMed Google Scholar The initial symptom is most commonly epileptic seizures, but motor, visual, and speech impairment, as well as developmental regression with ataxia, may also be presenting symptoms. The course of seizures is usually more severe than in classical LINCL. With time, progressive cognitive and motor deterioration, myoclonus, personality changes, and loss of vision develop, leading to premature death.18Topcu M Tan H Yalnizoglu D Usubutun A Saatci I Aynaci M Anlar B Topaloglu H Turanli G Kose G et al.Evaluation of 36 patients from Turkey with neuronal ceroid lipofuscinosis: clinical, neurophysiological, neuroradiological and histopathologic studies.Turk J Pediatr. 2004; 46: 1-10PubMed Google Scholar, 19Williams RE Topcu M Lake BD Mitchell W Mole SE CLN7 Turkish variant late infantile NCL.in: Goebel HH Mole SE Lake BD The neuronal ceroid lipofuscinosis (Batten disease). IOS Press, Amsterdam1999: 114-116Google Scholar On electron microscopic analysis, Turkish patients with vLINCL have shown condensed fingerprint profiles in circulating lymphocytes.18Topcu M Tan H Yalnizoglu D Usubutun A Saatci I Aynaci M Anlar B Topaloglu H Turanli G Kose G et al.Evaluation of 36 patients from Turkey with neuronal ceroid lipofuscinosis: clinical, neurophysiological, neuroradiological and histopathologic studies.Turk J Pediatr. 2004; 46: 1-10PubMed Google Scholar Initially, Turkish vLINCL was considered a distinct clinical and genetic entity,13Wheeler RB Sharp JD Mitchell WA Bate SL Williams RE Lake BD Gardiner RM A new locus for variant late infantile neuronal ceroid lipofuscinosis—CLN7.Mol Genet Metab. 1999; 66: 337-338Crossref PubMed Scopus (44) Google Scholar but it has turned out to be genetically heterogeneous. We recently reported four mutations in the CLN8 gene and two mutations in the CLN6 gene accounting for the disease in a subset of Turkish patients with vLINCL.16Ranta S Topcu M Tegelberg S Tan H Ustubutun A Saatci I Dufke A Enders H Pohl K Alembik Y et al.Variant late infantile neuronal ceroid lipofuscinosis in a subset of Turkish patients is allelic to Northern epilepsy.Hum Mutat. 2004; 23: 300-305Crossref PubMed Scopus (78) Google Scholar, 20Siintola E Topcu M Kohlschutter A Salonen T Joensuu T Anttonen AK Lehesjoki AE Two novel CLN6 mutations in variant late-infantile neuronal ceroid lipofuscinosis patients of Turkish origin.Clin Genet. 2005; 68: 167-173Crossref PubMed Scopus (24) Google Scholar However, in most patients, the phenotype is not linked to any of the known NCL genes. We here describe the identification and initial characterization of a novel gene, major facilitator superfamily (MFS) domain containing 8 (MFSD8), for Turkish vLINCL. The gene was identified after homozygosity mapping with a panel of 10 families, for which we had excluded, by haplotype analysis, all known human NCL loci as well as two loci homologous for genes causing NCL-like phenotypes in animal models (CLCN3 [MIM 600580]21Stobrawa SM Breiderhoff T Takamori S Engel D Schweizer M Zdebik AA Bosl MR Ruether K Jahn H Draguhn A et al.Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus.Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 22Yoshikawa M Uchida S Ezaki J Rai T Hayama A Kobayashi K Kida Y Noda M Koike M Uchiyama Y et al.CLC-3 deficiency leads to phenotypes similar to human neuronal ceroid lipofuscinosis.Genes Cells. 2002; 7: 597-605Crossref PubMed Scopus (119) Google Scholar and CLCN7 [MIM 602727]23Kasper D Planells-Cases R Fuhrmann JC Scheel O Zeitz O Ruether K Schmitt A Poet M Steinfeld R Schweizer M et al.Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration.EMBO J. 2005; 24: 1079-1091Crossref PubMed Scopus (275) Google Scholar). Nine Turkish families with vLINCL (a, b, c, d, e, f, g, h, and l), one family of Turkish-Georgian origin (k), and one family of Indian origin (j), altogether comprising 13 patients, 21 parents, and five unaffected siblings, were analyzed. All families except one (j) were consanguineous. The clinical phenotypes of eight patients were described elsewhere (patients 17, 18, 22, 24, 25, 27, 28, and 29 in the study by Topcu et al.18Topcu M Tan H Yalnizoglu D Usubutun A Saatci I Aynaci M Anlar B Topaloglu H Turanli G Kose G et al.Evaluation of 36 patients from Turkey with neuronal ceroid lipofuscinosis: clinical, neurophysiological, neuroradiological and histopathologic studies.Turk J Pediatr. 2004; 46: 1-10PubMed Google Scholar). The corresponding patient codes in the present study are e3, a3, f3, b3, c3, d4, d3, and g3, respectively. Patient e5 (the brother of e3) presented at age 3.5 years with a history of delayed speech development, ataxia since age 2.5 years, and stereotyped hand movements. Brain magnetic resonance imaging (MRI) showed cerebellar and cerebral atrophy and increased signal intensity in white matter. At age 5 years, he had prominent myoclonic seizures and rare nocturnal seizures that responded well to treatment with levetiracetam and intravenous immunoglobulin. Electroencephalogram (EEG) showed slow background activity and multifocal epileptic discharges. Eye ground examination showed retinopathy and optic atrophy. At age 6 years, he could not walk without support. For the past 6 mo, he has been wheelchair bound and unable to speak. Patient h3 presented at age 5.5 years with a history of delayed speech development, as well as ataxia that had been worsening over the previous 6 mo, frequent falling, and sleep disorders since age 3.5 years. On examination at 5.5 years, she had ataxic gait with abnormal cerebellar tests. She was able to speak slowly. Brain MRI showed cerebellar atrophy. She is taking sodium valproate for myoclonic seizures. She has tested negative for MECP2 (MIM 300005) mutations and for cerebral folate deficiency. No vacuoles were found on analysis of peripheral blood smear. Patient j3, who was of Indian origin, presented at age 3 years with a history of mild generalized developmental delay. Over the previous 3 mo, her gait had become increasingly ataxic, and, on examination, she walked with a wide-based gait. At age 4 years, she developed frequent clonic seizures that responded well to treatment with sodium valproate. A wide range of metabolic investigations, including enzymologic analysis of PPT1 and TPP1 and molecular analysis of CLN3 and CLN6, were normal. MRI of the brain showed mild cerebellar atrophy and evidence of delayed white matter maturation. A rectal biopsy showed neuronal curvilinear bodies. She had progressive neurological decline and, by age 6 years, was wheelchair bound and had limited communication, significant visual impairment, and frequent breakthrough seizures. At age 7 years, she developed treatment-resistant status epilepticus. She died just before her 8th birthday. Patient k3, who was of Turkish-Georgian origin, presented at age 8 years with a history of delayed psychomotor development (she walked at age 2.5 years, spoke at age 3 years, and did not learn to read). She showed cognitive deterioration, occasional agitation (with a few hospitalizations in a psychiatric unit), loss of vision, and walking difficulties. At age 22 years, she was agitated, she had axial akinesia, hypertonia, palilalia, and blindness. She developed problems with swallowing. Peripheral blood lymphocytes showed vacuoles, and a skin biopsy revealed vacuoles with fingerprint profiles. Patient l3 presented at age 2 years with a 6-wk history of ataxia and myoclonic and atonic seizures that responded well to treatment with sodium valproate. Psychomotor development, retinal examination, and MRI were normal, whereas EEG showed occipital spikes. An axillary skin biopsy showed fingerprint profiles. Genomic DNA was extracted using standard techniques from EDTA blood samples collected after informed consent was obtained. The control panel for the identified MFSD8 mutations consisted of 212 Turkish and 92 Centre d'Etude du Polymorphisme Humain (CEPH) chromosomes. RNA for the analysis of a splice-site mutation was extracted from peripheral blood samples and for the analysis of the alternative splicing pattern of MFSD8, from a control fibroblast cell line. An institutional review board of the Helsinki University Central Hospital approved the study. The previously known NCL loci (CLN1, CLN2, CLN3, CLN5, CLN6, CLN8, CLN10, CLCN3, and CLCN7) were excluded by microsatellite analysis, as described elsewhere.20Siintola E Topcu M Kohlschutter A Salonen T Joensuu T Anttonen AK Lehesjoki AE Two novel CLN6 mutations in variant late-infantile neuronal ceroid lipofuscinosis patients of Turkish origin.Clin Genet. 2005; 68: 167-173Crossref PubMed Scopus (24) Google Scholar Four microsatellite markers (D4S427, D4S2975, D4S2938, and D4S429) spanning ∼6.5 cM (according to the Marshfield Genetic Map in the Mammalian Genotyping Service) were genotyped around the novel CLN7 locus. Ten families with vLINCL (a, b, c, d, e, f, g, j, l, and k) were included in the genomewide SNP scan. One affected individual was genotyped from each family, except from one pedigree (d) that had four individuals (two affected and two unaffected siblings) genotyped. The SNP scan was performed using Affymetrix's GeneChip Human Mapping 50K Xba 240 arrays in accordance with the manufacturer's instructions. SNPs were merged with the Rutgers map24Kong X Murphy K Raj T He C White PS Matise TC A combined linkage-physical map of the human genome.Am J Hum Genet. 2004; 75: 1143-1148Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar by their physical locations (NCBI build 35). Interval-specific interpolation was performed. From a total of 58,960 SNPs, the ones without chromosome numbers and physical location (466) or with zero heterozygosity values or male heterozygotes on chromosome X (3,904) were removed. Because the sample size in this data set was too small to calculate SNP heterozygosity values, the values from the Affymetrix white sample were used. Since linkage disequilibrium between markers can cause false-positive results when both parents are not genotyped,25Huang Q Shete S Amos CI Ignoring linkage disequilibrium among tightly linked markers induces false-positive evidence of linkage for affected sib pair analysis.Am J Hum Genet. 2004; 75: 1106-1112Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar we selected markers for linkage analysis instead of using all of them. We did this by grouping the remaining 54,590 SNPs into clusters, with at least 0.1 cM between the clusters. One SNP with the largest heterozygosity value was chosen from each cluster, resulting in the use of 8,704 SNPs. The computer program Merlin (version 1.0.1) was used for multipoint homozygosity mapping.26Abecasis GR Cherny SS Cookson WO Cardon LR Merlin—rapid analysis of dense genetic maps using sparse gene flow trees.Nat Genet. 2002; 30: 97-101Crossref PubMed Scopus (2780) Google Scholar The disease-allele frequency was set at 0.0001, with penetrances of 0, 0, and 1 for an autosomal recessive model. The gene content of the ∼19.5-Mb genomic region between rs2051785 and rs984369 on chromosome 4q28.1-q28.2 was examined with the NCBI Map Viewer, build 35.1. Positional candidate genes were chosen on the basis of the known or putative functions of the encoded proteins. The exons and the exon-intron boundaries of the candidate genes were screened for mutations by genomic sequencing. PCR conditions and primer sequences are available from the authors on request. Sequencing of the purified PCR products was performed with an ABI 3730 DNA Analyzer (Applied Biosystems). Controls were screened for the identified mutations by genomic sequencing. The tissue expression of MFSD8 was analyzed with Multiple Tissue Northern Blots (Human, Human Brain II, and Human Brain V [BD Biosciences Clontech]) with a 559-bp (c.36_594) probe that was PCR amplified from MFSD8 cDNA (I.M.A.G.E Consortium cDNA clone ID 527266427Lennon G Auffray C Polymeropoulos M Soares MB The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression.Genomics. 1996; 33: 151-152Crossref PubMed Scopus (1089) Google Scholar and RZPD clone IRATp970E0532D). The probe was [32P]-labeled with the Rediprime II Random Prime Labeling System (Amersham Biosciences) and was hybridized in accordance with the manufacturer's protocol (BD Biosciences Clontech). For RT-PCR, RNA isolated from blood or from cultured fibroblasts was reverse transcribed using M-MLV Reverse Transcriptase (Promega). The consequence of the c.754+2T→A mutation was analyzed using primers from exons 6 and 11, and the alternative splicing of MFSD8 in fibroblasts was analyzed using several exonic primer pairs that covered the entire coding region, as well as primers specific for various exon-lacking transcripts. The PCR products were extracted from agarose gels and were sequenced. The PCR-amplified ORF of the human MFSD8 cDNA was cloned in frame into the aminoterminal hemagglutinin (HA) tag containing pAHC expression vector (kindly provided by Prof. T. Mäkelä, University of Helsinki, Finland), a derivative of pCIneo (Promega) (HAMFSD8 construct), and into the pcDNA3.1(+) expression vector (Invitrogen), into which a carboxylterminal HA tag was generated (MFSD8HA construct) by site-directed mutagenesis by use of the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Two nucleotide changes, c.929G→A (p.Gly310Asp) and c.1286G→A (p.Gly429Asp), corresponding to patient missense mutations, were introduced into the HAMFSD8 construct by site-directed mutagenesis. All constructs were verified by sequencing. In vitro translation was performed with the TnT Quick Coupled Transcription/Translation System (Promega) and the Canine Pancreatic Microsomal Membranes (Promega) by use of the HAMFSD8 and MFSD8HA constructs as templates. The [35S]-labeled translation products were analyzed on 12% SDS-PAGE gels and were visualized by autoradiography. COS-1 and HeLa cells (American Type Culture Collection) were cultivated in Dulbecco's modified Eagle's medium (BioWhittaker) supplemented with 10% and 5% fetal calf serum (PromoCell), respectively, penicillin, streptomycin, and 1× GlutaMAX (Gibco). The cells (1.5–2×105 cells) were plated onto 6-well plates on coverslips 1 d before transfection and were transfected with 2 μg of wild-type or missense mutation containing HAMFSD8 or wild-type MFSD8HA constructs per well by use of FuGENE 6 Transfection Reagent (Roche). At 18 h after transfection, cells were incubated with cycloheximide for 2 h, were fixed with 4% paraformaldehyde, and were permeabilized either with 0.1% Triton X-100 in PBS or with 0.2% saponin in PBS supplemented with 0.5% BSA. Overexpressed proteins were detected using mouse monoclonal or rabbit polyclonal anti-HA antibodies (clones 16B12 [Covance Research Products] and Y-11 [Santa Cruz Biotechnology], respectively). Antibodies used as organelle markers were mouse monoclonal anti-early endosome antigen 1 (EEA1 [BD Biosciences]), rabbit polyclonal anti-cathepsin D (CTSD [DAKO]), rabbit polyclonal anti-giantin (BioSite), mouse monoclonal anti–lysosomal–associated membrane protein 1 (Lamp-1, H4A3 [developed by J. T. August and J. E. K. Hildreth and obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences, Iowa City]), mouse monoclonal anti-lysobisphosphatidic acid (LBPA, 6C4 [kindly provided by Professor Jean Gruenberg, University of Geneva, Switzerland]), mouse monoclonal anti–mannose-6-phosphate receptor 46 (MPR46 [kindly provided by Professor Kurt von Figura, Göttingen, Germany]), and mouse monoclonal anti–protein-disulfide isomerase (PDI [Stressgen]). Cy2- or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch) were used to visualize the primary antibodies. The cells were viewed using an Axioplan 2 microscope and were photographed with AxioVision 3.1 (Zeiss). Alternatively spliced transcripts were identified using NCBI AceView. The topology of MFSD8 was predicted using HMMTOP, TMHMM, and TMpred prediction programs. Protein domains were searched for in the Pfam 20.0 database. Proteins homologous and similar to MFSD8 were searched for by NCBI protein-protein BLAST. The peptide sequences were aligned using MAFFT version 5.8. Analysis of genomewide SNP-scan data in 10 families with vLINCL—for which we had excluded all known NCL loci either elsewhere20Siintola E Topcu M Kohlschutter A Salonen T Joensuu T Anttonen AK Lehesjoki AE Two novel CLN6 mutations in variant late-infantile neuronal ceroid lipofuscinosis patients of Turkish origin.Clin Genet. 2005; 68: 167-173Crossref PubMed Scopus (24) Google Scholar or in this study (data not shown) (see the "Material and Methods" section)—revealed three regions, on chromosomes 4, 8, and 15, with heterozygosity LOD (HLOD) scores >2 (see the tab-delimited ASCII file, which can be imported into a spreadsheet, of data set 1). Six families (a, b, c, e, f, and j) contributed to the highest HLOD score of 3.39 at SNP rs348085 on chromosome 4q28.1-q28.2, where an ∼19.5-Mb region (from rs7657655 to rs10518621) of overlapping homozygosity was detected in all but one of these families. In family a, the homozygous region was <1 Mb, which was considered to be too short to reflect a region homozygous by descent, considering the close consanguinity. The haplotypes across the region were different in each family (data not shown). Four families (d, e, g, and l) contributed to the HLOD score of 2.48 on chromosome 15 (at SNP rs2956147). Four families (b, c, e, and l) contributed to the HLOD score of 2.05 on chromosome 8 (at SNP rs2432960). We decided to first focus on the ∼19.5-Mb region on chromosome 4 because it had the strongest evidence for linkage. Genotyping four microsatellite markers (D4S427, D4S2975, D4S2938, and D4S429) in patients in families b, c, e, f, and j and in an additional family (h) did not narrow the region but revealed that the patient in family h also is homozygous for these markers. Within the critical region of ∼19.5 Mb, we identified at least 90 known or putative genes. After excluding two of the genes (TRAM1L1 and TRPC3 [MIM 602345]) by sequencing, we identified six homozygous mutations (table 1 and fig. 1) in MFSD8 (a HUGO Gene Nomenclature Committee–approved symbol; previously denoted "MGC33302"; GenBank accession number NM_152778), by screening the 12 protein-coding exons (exons 2–13) of the gene. All six mutations cosegregated with the disease phenotype in the respective families and were not found in 212 Turkish and 92 CEPH control chromosomes. Patient j3 was homozygous for nonsense mutation c.894T→G in exon 10, which created a premature stop codon (p.Tyr298X) and was predicted to truncate the protein by 221 aa. Patients c3 and b3 were homozygous for missense mutations c.929G→A (p.Gly310Asp) in exon 10 and c.1286G→A (p.Gly429Asp) in exon 12, respectively, which affect amino acids that are conserved across vertebrates (fig. 2). In two patients, we identified homozygous nucleotide changes at the exon-intron junctions that may either change an amino acid or affect the splicing of the transcript: an A→G transition at the second-to-last nucleotide of exon 7 (c.697A→G; p.Arg233Gly) in patient f3, and a transversion of G→C at the last nucleotide of
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