Mutations in FGD4 Encoding the Rho GDP/GTP Exchange Factor FRABIN Cause Autosomal Recessive Charcot-Marie-Tooth Type 4H
2007; Elsevier BV; Volume: 81; Issue: 1 Linguagem: Inglês
10.1086/518428
ISSN1537-6605
AutoresValérie Delague, Arnaud Jacquier, Tarik Hamadouche, Yannick Poitelon, Cécile Baudot, Irène Boccaccio, Éliane Chouery, Malika Chaouch, Nora Kassouri, Rosette Jabbour, Djamel Grid, André Mégarbané, Georg Haase, Nicolas Lévy,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoCharcot-Marie-Tooth (CMT) disorders are a clinically and genetically heterogeneous group of hereditary motor and sensory neuropathies characterized by muscle weakness and wasting, foot and hand deformities, and electrophysiological changes. The CMT4H subtype is an autosomal recessive demyelinating form of CMT that was recently mapped to a 15.8-Mb region at chromosome 12p11.21-q13.11, in two consanguineous families of Mediterranean origin, by homozygosity mapping. We report here the identification of mutations in FGD4, encoding FGD4 or FRABIN (FGD1-related F-actin binding protein), in both families. FRABIN is a GDP/GTP nucleotide exchange factor (GEF), specific to Cdc42, a member of the Rho family of small guanosine triphosphate (GTP)–binding proteins (Rho GTPases). Rho GTPases play a key role in regulating signal-transduction pathways in eukaryotes. In particular, they have a pivotal role in mediating actin cytoskeleton changes during cell migration, morphogenesis, polarization, and division. Consistent with these reported functions, expression of truncated FRABIN mutants in rat primary motoneurons and rat Schwann cells induced significantly fewer microspikes than expression of wild-type FRABIN. To our knowledge, this is the first report of mutations in a Rho GEF protein being involved in CMT. Charcot-Marie-Tooth (CMT) disorders are a clinically and genetically heterogeneous group of hereditary motor and sensory neuropathies characterized by muscle weakness and wasting, foot and hand deformities, and electrophysiological changes. The CMT4H subtype is an autosomal recessive demyelinating form of CMT that was recently mapped to a 15.8-Mb region at chromosome 12p11.21-q13.11, in two consanguineous families of Mediterranean origin, by homozygosity mapping. We report here the identification of mutations in FGD4, encoding FGD4 or FRABIN (FGD1-related F-actin binding protein), in both families. FRABIN is a GDP/GTP nucleotide exchange factor (GEF), specific to Cdc42, a member of the Rho family of small guanosine triphosphate (GTP)–binding proteins (Rho GTPases). Rho GTPases play a key role in regulating signal-transduction pathways in eukaryotes. In particular, they have a pivotal role in mediating actin cytoskeleton changes during cell migration, morphogenesis, polarization, and division. Consistent with these reported functions, expression of truncated FRABIN mutants in rat primary motoneurons and rat Schwann cells induced significantly fewer microspikes than expression of wild-type FRABIN. To our knowledge, this is the first report of mutations in a Rho GEF protein being involved in CMT. Hereditary motor and sensory neuropathies (HMSNs), commonly referred to as "Charcot-Marie-Tooth (CMT) disease," are among the most common inherited neurological diseases, with an overall prevalence of about 1–4/10,000.1Skre H Genetic and clinical aspects of Charcot-Marie-Tooth's disease.Clin Genet. 1974; 6: 98-118Crossref PubMed Scopus (663) Google Scholar Clinically, HMSNs are characterized by progressive muscular and sensory defects starting at the distal extremities, with chronic weakness, pes cavus, and loss of deep tendon reflexes.2Dyck PJ Chance P Lebo R Carney AJ Hereditary motor and sensory neuropathies.in: Dyck PJ Thomas PK Griffin JW Low PA Podulso JF Peripheral neuropathy. WB Saunders, Philadelphia1993: 1094-1136Google Scholar Two main subgroups have been defined on the basis of electrophysiological and histopathological characteristics: the demyelinating form (CMT1) and the axonal form (CMT2). CMT1 can be distinguished from CMT2 by measuring motor nerve–conduction velocities (NCVs): patients affected with CMT1 show reduced NCVs (≤38 m/s), whereas patients affected with CMT2 show NCVs ⩾38 m/s; the normal value is ⩾48 m/s. Recently, a new group of CMT diseases with electrophysiological and histopathological characteristics overlapping CMT1 and CMT2, referred to as "intermediate CMT," has been described.3Villanova M Timmerman V De Jonghe P Malandrini A Rizzuto N Van Broeckhoven C Guazzi G Rossi A Charcot-Marie-Tooth disease: an intermediate form.Neuromuscul Disord. 1998; 8: 392-393Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Genetically, CMT disease is characterized by a great heterogeneity4Shy ME Charcot-Marie-Tooth disease: an update.Curr Opin Neurol. 2004; 17: 579-585Crossref PubMed Scopus (90) Google Scholar (Neuromuscular Disease Center and Inherited Peripheral Neuropathies Mutation Database). All modes of inheritance have been reported. Autosomal recessive demyelinating forms of CMT disease (CMT4) are less frequent, usually of earlier onset, and more severe than the autosomal dominant forms (CMT1), with a fast progression to severe disability leading to higher frequency of wheelchair dependency in the life course.5Berciano J Combarros O Hereditary neuropathies.Curr Opin Neurol. 2003; 16: 613-622Crossref PubMed Scopus (38) Google Scholar Among the 50 loci (30 genes) identified to date, 9 correspond to CMT4, among which 6 genes have already been identified: GDAP1 (MIM 606598)6Cuesta A Pedrola L Sevilla T Garcia-Planells J Chumillas MJ Mayordomo F Leguern E Marin I Vilchez JJ Palau F The gene encoding ganglioside-induced differentiation-associated protein 1 is mutated in axonal Charcot-Marie-Tooth type 4A disease.Nat Genet. 2002; 30: 22-24Crossref PubMed Scopus (296) Google Scholar, 7Baxter RV Ben Othmane K Rochelle JM Stajich J Hulette C Dew-Knight S Hentati F Ben Hamida M Bel S Stenger JE et al.Ganglioside-induced differentiation-associated protein-1 is mutated in Charcot-Marie-Tooth disease type 4A/8q21.Nat Genet. 2002; 30: 21-22Crossref PubMed Scopus (309) Google Scholar at chromosome 8q13-q21.1 (CMT4A [MIM 214400])8Ben Othmane K Hentati F Lennon F Ben Hamida C Blel S Roses AD Pericak-Vance MA Ben Hamida M Vance JM Linkage of a locus (CMT4A) for autosomal recessive Charcot-Marie-Tooth disease to chromosome 8q.Hum Mol Genet. 1993; 2: 1625-1628Crossref PubMed Scopus (184) Google Scholar; MTMR2 (MIM 603557)9Bolino A Muglia M Conforti FL LeGuern E Salih MA Georgiou DM Christodoulou K Hausmanowa-Petrusewicz I Mandich P Schenone A et al.Charcot-Marie-Tooth type 4B is caused by mutations in the gene encoding myotubularin-related protein-2.Nat Genet. 2000; 25: 17-19Crossref PubMed Scopus (407) Google Scholar at chromosome 11q22 (CMT4B1 [MIM 601382])10Bolino A Brancolini V Bono F Bruni A Gambardella A Romeo G Quattrone A Devoto M Localization of a gene responsible for autosomal recessive demyelinating neuropathy with focally folded myelin sheaths to chromosome 11q23 by homozygosity mapping and haplotype sharing.Hum Mol Genet. 1996; 5: 1051-1054Crossref PubMed Scopus (110) Google Scholar; SBF2/MTMR13 (MIM 607697)11Azzedine H Bolino A Taieb T Birouk N Di Duca M Bouhouche A Benamou S Mrabet A Hammadouche T Chkili T et al.Mutations in MTMR13, a new pseudophosphatase homologue of MTMR2 and Sbf1, in two families with an autosomal recessive demyelinating form of Charcot-Marie-Tooth disease associated with early-onset glaucoma.Am J Hum Genet. 2003; 72: 1141-1153Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 12Senderek J Bergmann C Weber S Ketelsen UP Schorle H Rudnik-Schoneborn S Buttner R Buchheim E Zerres K Mutation of the SBF2 gene, encoding a novel member of the myotubularin family, in Charcot-Marie-Tooth neuropathy type 4B2/11p15.Hum Mol Genet. 2003; 12: 349-356Crossref PubMed Scopus (215) Google Scholar at chromosome 11p15 (CMT4B2 [MIM 604563 and MIM 607739])13Othmane KB Johnson E Menold M Graham FL Hamida MB Hasegawa O Rogala AD Ohnishi A Pericak-Vance M Hentati F et al.Identification of a new locus for autosomal recessive Charcot-Marie-Tooth disease with focally folded myelin on chromosome 11p15.Genomics. 1999; 62: 344-349Crossref PubMed Scopus (67) Google Scholar; SH3CT2/KIAA1985 (MIM 608206)14Senderek J Bergmann C Stendel C Kirfel J Verpoorten N De Jonghe P Timmerman V Chrast R Verheijen MH Lemke G et al.Mutations in a gene encoding a novel SH3/TPR domain protein cause autosomal recessive Charcot-Marie-Tooth type 4C neuropathy.Am J Hum Genet. 2003; 73: 1106-1119Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar at chromosome 5q23-q33 (CMT4C [MIM 601596])15LeGuern E Guilbot A Kessali M Ravise N Tassin J Maisonobe T Grid D Brice A Homozygosity mapping of an autosomal recessive form of demyelinating Charcot-Marie-Tooth disease to chromosome 5q23-q33.Hum Mol Genet. 1996; 5: 1685-1688Crossref PubMed Scopus (113) Google Scholar; NDRG1 (MIM 605262)16Kalaydjieva L Gresham D Gooding R Heather L Baas F de Jonge R Blechschmidt K Angelicheva D Chandler D Worsley P et al.N-myc downstream-regulated gene 1 is mutated in hereditary motor and sensory neuropathy-Lom.Am J Hum Genet. 2000; 67: 47-58Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar at chromosome 8q24.3 (HMSN-Lom [MIM 601455])17Kalaydjieva L Hallmayer J Chandler D Savov A Nikolova A Angelicheva D King RH Ishpekova B Honeyman K Calafell F et al.Gene mapping in gypsies identifies a novel demyelinating neuropathy on chromosome 8q24.Nat Genet. 1996; 14: 214-217Crossref PubMed Scopus (185) Google Scholar; EGR2 (MIM 129010),18Timmerman V De Jonghe P Ceuterick C De Vriendt E Lofgren A Nelis E Warner LE Lupski JR Martin JJ Van Broeckhoven C Novel missense mutation in the early growth response 2 gene associated with Dejerine-Sottas syndrome phenotype.Neurology. 1999; 52: 1827-1832Crossref PubMed Google ScholarP0 (MIM 159440),19Warner LE Hilz MJ Appel SH Killian JM Kolodry EH Karpati G Carpenter S Watters GV Wheeler C Witt D et al.Clinical phenotypes of different MPZ (P0) mutations may include Charcot-Marie-Tooth type 1B, Dejerine-Sottas, and congenital hypomyelination.Neuron. 1996; 17: 451-460Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 20Ikegami T Nicholson G Ikeda H Ishida A Johnston H Wise G Ouvrier R Hayasaka K A novel homozygous mutation of the myelin P0 gene producing Dejerine-Sottas disease (hereditary motor and sensory neuropathy type III).Biochem Biophys Res Commun. 1996; 222: 107-110Crossref PubMed Scopus (50) Google Scholar and PMP22 (MIM 601097)21Roa BB Dyck PJ Marks HG Chance PF Lupski JR Dejerine-Sottas syndrome associated with point mutation in the peripheral myelin protein 22 (PMP22) gene.Nat Genet. 1993; 5: 269-273Crossref PubMed Scopus (224) Google Scholar (CMT4E [MIM 605523] or Dejerine-Sottas syndrome [MIM 145900]); PRX (MIM 605725)22Guilbot A Williams A Ravise N Verny C Brice A Sherman DL Brophy PJ LeGuern E Delague V Bareil C et al.A mutation in periaxin is responsible for CMT4F, an autosomal recessive form of Charcot-Marie-Tooth disease.Hum Mol Genet. 2001; 10: 415-421Crossref PubMed Scopus (186) Google Scholar, 23Boerkoel C Takashima H Stankiewicz P Garcia C Leber S Rhee-Morris L Lupski J Periaxin mutations cause recessive Dejerine-Sottas neuropathy.Am J Hum Genet. 2001; 68: 325-333Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar at chromosome 19q13.3 (CMT4F [MIM 605725]24Delague V Bareil C Tuffery S Bouvagnet P Chouery E Koussa S Maisonobe T Loiselet J Megarbane A Claustres M Mapping of a new locus for autosomal recessive demyelinating Charcot-Marie-Tooth disease to 19q13.1-13.3 in a large consanguineous Lebanese family: exclusion of MAG as a candidate gene.Am J Hum Genet. 2000; 67: 236-243Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar and Dejerine-Sottas syndrome); HMSN-Russe (MIM 601455) at chromosome 10q22-q23,25Rogers T Chandler D Angelicheva D Thomas PK Youl B Tournev I Gergelcheva V Kalaydjieva L A novel locus for autosomal recessive peripheral neuropathy in the EGR2 region on 10q23.Am J Hum Genet. 2000; 67: 664-671Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar for which no corresponding mutated gene has yet been identified; and, finally, CMT4H (MIM 609311), which we recently assigned to chromosome 12p11.21-q13.11.26De Sandre-Giovannoli A Delague V Hamadouche T Chaouch M Krahn M Boccaccio I Maisonobe T Chouery E Jabbour R Atweh S et al.Homozygosity mapping of autosomal recessive demyelinating Charcot-Marie-Tooth neuropathy (CMT4H) to a novel locus on chromosome 12p11.21-q13.11.J Med Genet. 2005; 42: 260-265Crossref PubMed Scopus (36) Google Scholar The roles and function of proteins involved in autosomal recessive CMT (AR-CMT) disease are diverse, including proteins involved in polyposphoinositide signaling27Laporte J Blondeau F Buj-Bello A Mandel J The myotubularin family: from genetic disease to phosphoinositide metabolism.Trends Genet. 2001; 17: 221-228Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar (myotubularins MTMR2 and MTMR13), mitochondrial fission proteins (GDAP1),28Niemann A Ruegg M La Padula V Schenone A Suter U Ganglioside-induced differentiation associated protein 1 is a regulator of the mitochondrial network: new implications for Charcot-Marie-Tooth disease.J Cell Biol. 2005; 170: 1067-1078Crossref PubMed Scopus (316) Google Scholar structural myelin proteins like periaxin (L-PRX), proteins involved in growth arrest and cell differentiation (NDRG1), and proteins with no known function (SH3CT2/KIAA1985). Moreover, several proteins related to GTPase signaling have been identified in CMT disease: mitofusin 2 (MFN2),29Zuchner S Mersiyanova IV Muglia M Bissar-Tadmouri N Rochelle J Dadali EL Zappia M Nelis E Patitucci A Senderek J et al.Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A.Nat Genet. 2004; 36: 449-451Crossref PubMed Scopus (1150) Google Scholar dynamin 2 (DNM2),30Zuchner S Noureddine M Kennerson M Verhoeven K Claeys K De Jonghe P Merory J Oliveira SA Speer MC Stenger JE et al.Mutations in the pleckstrin homology domain of dynamin 2 cause dominant intermediate Charcot-Marie-Tooth disease.Nat Genet. 2005; 37: 289-294Crossref PubMed Scopus (274) Google Scholar RAB7,31Verhoeven K de Jong P Coen K Verpoorten N Auer-Grumbach M Kwon J FitzPatrick D Schmedding E De Vriendt E Jacobs A et al.Mutations in the small GTP-ase late endosomal protein RAB7 cause Charcot-Marie-Tooth type 2B neuropathy.Am J Hum Genet. 2003; 72: 722-727Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar ARHGEF10,32Verhoeven K De Jonghe P Van de Putte T Nelis E Zwijsen A Verpoorten N De Vriendt E Jacobs A Van Gerwen V Francis A et al.Slowed conduction and thin myelination of peripheral nerves associated with mutant Rho guanine-nucleotide exchange factor 10.Am J Hum Genet. 2003; 73: 926-932Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar and SEPT9.33Kuhlenbaumer G Hannibal MC Nelis E Schirmacher A Verpoorten N Meuleman J Watts GD De Vriendt E Young P Stogbauer F et al.Mutations in SEPT9 cause hereditary neuralgic amyotrophy.Nat Genet. 2005; 37: 1044-1046Crossref PubMed Scopus (172) Google Scholar Rho GTPases are molecular switches that control a wide variety of signal-transduction pathways in eukaryotic cells, including regulation of the actin and microtubule cytoskeleton and cell polarization, migration, and proliferation.34Jaffe AB Hall A Rho GTPases: biochemistry and biology.Annu Rev Cell Dev Biol. 2005; 21: 247-269Crossref PubMed Scopus (2216) Google Scholar, 35Etienne-Manneville S Hall A Rho GTPases in cell biology.Nature. 2002; 420: 629-635Crossref PubMed Scopus (3654) Google Scholar Along with GTPase-activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs), Rho GDP/GTP nucleotide exchange factors (Rho GEFs) are essential regulators of Rho GTPases. About 1% of the human genome encodes proteins that either regulate or are regulated by direct interactions with members of the Rho family small GTPases, and more than 85 Rho GEFs, regulating 22 GTPases, are known to date.34Jaffe AB Hall A Rho GTPases: biochemistry and biology.Annu Rev Cell Dev Biol. 2005; 21: 247-269Crossref PubMed Scopus (2216) Google Scholar In this article, we report the identification of mutations in FGD4 encoding FGD4/FRABIN (FGD1-related F-actin binding protein [accession number NP_640334]) in two families from Algeria and Lebanon with members affected with CMT4H. FRABIN is a GEF specific to Cdc42, a member of the Rho family of small GTP-binding proteins (Rho GTPases).3Villanova M Timmerman V De Jonghe P Malandrini A Rizzuto N Van Broeckhoven C Guazzi G Rossi A Charcot-Marie-Tooth disease: an intermediate form.Neuromuscul Disord. 1998; 8: 392-393Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 37Umikawa M Obaishi H Nakanishi H Satoh-Horikawa K Takahashi K Hotta I Matsuura Y Takai Y Association of Frabin with the actin cytoskeleton is essential for microspike formation through activation of Cdc42 small G protein.J Biol Chem. 1999; 274: 25197-25200Crossref PubMed Scopus (55) Google Scholar Consistent with these reported functions, expression of truncated FRABIN mutants in rat primary motoneurons and rat Schwann cells induced significantly fewer microspikes than expression of wild-type FRABIN. In all, FRABIN is the sixth protein related to GTPase signaling but the first Rho GEF to be identified in CMT disease. We previously published the localization of CMT4H from one Lebanese family and one Algerian family with CMT4H.26De Sandre-Giovannoli A Delague V Hamadouche T Chaouch M Krahn M Boccaccio I Maisonobe T Chouery E Jabbour R Atweh S et al.Homozygosity mapping of autosomal recessive demyelinating Charcot-Marie-Tooth neuropathy (CMT4H) to a novel locus on chromosome 12p11.21-q13.11.J Med Genet. 2005; 42: 260-265Crossref PubMed Scopus (36) Google Scholar Patients are affected with early-onset demyelinating neuropathy. For detailed clinical, electrophysiological, and histological descriptions and data, see the work of De Sandre-Giovannoli.26De Sandre-Giovannoli A Delague V Hamadouche T Chaouch M Krahn M Boccaccio I Maisonobe T Chouery E Jabbour R Atweh S et al.Homozygosity mapping of autosomal recessive demyelinating Charcot-Marie-Tooth neuropathy (CMT4H) to a novel locus on chromosome 12p11.21-q13.11.J Med Genet. 2005; 42: 260-265Crossref PubMed Scopus (36) Google Scholar After informed consent was obtained from all individuals and parents of children included in this study, EDTA blood samples were collected, and genomic DNA was extracted from lymphocytes with the use of standard methods. In all, 29 DNA samples were collected for the study, including 10 from affected individuals (fig. 1). Biopsies of skin and sural nerve samples were performed in Lebanese patient 500.21 under local anesthesia after informed consent was obtained from his parents. All protocols performed in this study complied with the ethics guidelines of the institutions involved. Exploration of the entire coding sequence of candidate genes (KIF21A [accession number AY368076], RAPGEF3 [accession number U78168], and FGD4 [accession numbers AK057294, BC045552, and AY367054]) was performed in Lebanese patient 500.21 and Algerian patient 295.3 (fig. 1). Intronic primers were designed using Primer3 software. DNA sequences were obtained from the UCSC Genome Browser (May 2004 freeze), by comparison of genomic DNA with cDNA sequences. For FGD4, six additional alternatively spliced exons described in other sequences from the UCSC database were screened for mutations: exons 6a and 16b are present in GenBank sequence BC045552, and exons 12a, 15a, 18, and 19 are present in GenBank sequence AY367054. Primer sequences and annealing temperatures used in PCRs for FGD4 are described in table 1. Those used for KIF21A and RAPGEF3 are available on request. Genomic DNA was amplified for one patient of each family under standard PCR conditions. All PCR-amplified fragments were analyzed by denaturing high-performance liquid chromatography (dHPLC)/WAVE (Transgenomic) and were fluorescently sequenced in both directions for those presenting abnormal elution profiles, with the use of sequencing facilities (MWG Biotech). Conditions for dHPLC analysis are available on request. Chromatograms were compared with reference sequences with the use of Sequencher v4.2 (Gene Codes). The identified c.893T→G and the C.893T→C genomic variants were tested in 216 Lebanese and 108 Algerian control chromosomes by restriction endonuclease digestion with the use of MlnII and MaeII (New England Biolabs), respectively.Table 1.Primer Sequences and Annealing Temperatures for FGD4 Analysis of Coding Sequences at the Genomic and Transcriptional LevelPrimer Sequence (5′–3′)Analysis and AmpliconForwardReverseAmplification Product Size (bp)Optimal Annealing Temperature (°C)Genomic DNA: Exon 1gccttaggagggctggttacgataatgcccccacacaaac29656 Exon 2cctgccctttctttctgaacgtggccatcatttttctggt25856 Exon 3cacccaggacaccagaatttcctgccttctcccattgtaa29254 Exon 4accaatgttttcatgcttcctttgagctaagagtggcagcttt28256 Exon 4bctcagggtgcacagacttgtcagggttttccctgctcat39555 Exon 5gatggctgaaagaaccgagtaaaacaggcccttccttcat32955 Exon 6aaAlternatively spliced exons found in GenBank sequence BC045552.ccactgctgctgaccttttaaaaacaaaaacccaaaccttca25755 Exon 6gagcccactatgtgcctagccagcatgactgccttaaaca27256 Exon 7gccactgcactacagtctggggagtagaatgaattttggctagg37060 Exon 8tcatgcaggtgatggacagtgaaaccataaggctgcttgg33756 Exon 9gcacaggaaggacaaagcatttcataaacattctttttggctca23456 Exon 10tgcctgtatgtgatcttgctgctcccaaagtgctgggatta27760 Exon 11cctgatcagtttcccctatttcgttgcctgattttggaaagc27556 Exon 12agttggaacaacaggaaagcacagacagaggctgcagtgag23156 Exon 12abAlternatively spliced exons found in GenBank sequence AY367054.gcctcttgagtagctgggacttgaaaattaagtgaacctgttctga29155 Exon 13tgagaaactttaatgtgtgctttgagggaaaaggttggagaacaa22856 Exon 14tccgaaagtgctgggattatcgtaattggcaccaaaataaa383… Exon 15caaaaatctgccttctatgcttttgcagtgagctgagatcgt36359 Exon 15abAlternatively spliced exons found in GenBank sequence AY367054.gaggctgaggcaggagaatcccagaacagagcctgaactt596… Exon 16cFor the longer exon 16 in sequence BC045552, we used reverse primer 5′-gaaaggggacagatgatcca-3′ and an annealing temperature of 55°C.tttggatagtccagggaaacaattttgagtccgctgtccac32556 Exon 17ctgtttgggacagtggtaggatcctgagacctccacaccat39856 Exon 18bAlternatively spliced exons found in GenBank sequence AY367054.ggacacacttaagttttggctcagactgctcaagtatgttggaagg39355 Exon 19bAlternatively spliced exons found in GenBank sequence AY367054.tgatttgcaattctgtctttccgaccatgattgacttcagattttt49355RT-PCR: Exon 5–exon 8gcaaactgttggaagaagcaagggaatcaggaggcaattt41255 Fragment 1tcgctttagttccaccttgctccttcatctcatggtgctg82659 Fragment 2ctacaggactccaggcataggtgaagtgttccgagcagcta85060 Fragment 3gattccctggactggaatgagaagaaagctgcaca84759 Fragment 4aaaagagccccaagatggattgcatttatgggcctatatttt76855RTQ-PCR: FGD4 (exon 2)tcagatctcatcagtcgctttgacagcagactctttcttcaaatca74… TBP (reference gene 1)gctggcccatagtgatctttcttcacacgccaagaaacagt60… GUSB (reference gene 2)cgccctgcctatctgtattctccccacagggagtgtgtag95…Mouse RT-PCRdPrimers were designed using GenBank sequence NM_139234.: Exon 4–exon 8atgggattggatacgttggaaggaatgcgctgaataggc49860Note.—Specific primers for RTQ-PCR are also mentioned. All human primers for genomic screening of mutations were selected on the basis of comparison of genomic DNA sequence with GenBank cDNA sequence AK057294. Exon 4 was amplified in two amplicons (4a and 4b).a Alternatively spliced exons found in GenBank sequence BC045552.b Alternatively spliced exons found in GenBank sequence AY367054.c For the longer exon 16 in sequence BC045552, we used reverse primer 5′-gaaaggggacagatgatcca-3′ and an annealing temperature of 55°C.d Primers were designed using GenBank sequence NM_139234. Open table in a new tab Note.— Specific primers for RTQ-PCR are also mentioned. All human primers for genomic screening of mutations were selected on the basis of comparison of genomic DNA sequence with GenBank cDNA sequence AK057294. Exon 4 was amplified in two amplicons (4a and 4b). Total RNA was extracted from freshly isolated lymphocytes with the use of either TRIZOL (Invitrogen Life Technologies), on the basis of protocol derived from the work of Chomozynsky and Sacchi,38Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62307) Google Scholar or silica-gel RNeasy colums (QIAGEN). cDNAs were obtained from total RNA with use of Superscript I Reverse Transcriptase (Invitrogen Life Technologies) and random primers (Invitrogen Life Technologies), in accordance with the recommendations of the supplier. cDNAs were then PCR amplified as described above. Primers and annealing temperatures are described in table 1. RT-PCR experiments, which gave rise to several fragments, were cloned into PGEM Vector (PROMEGA), in accordance with the supplier's instructions, and were transformed by electroporation into electrocompetent Escherichia coli cells. Positive clones were selected and amplified by PCR, and fragments were subsequently sequenced as described above. SYBR Green real-time PCRs (RTQ-PCRs) were realized according to standard protocols in a 20-μl final volume, with the use of primers at a final 500 mM concentration on a Lightcycler 480 (Roche). Of the RNAse H–treated (Invitrogen Life Technologies) cDNAs obtained from reverse transcription of DNAse I–treated total RNA extracted from cultured fibroblasts by use of the High Capacity cDNA Archive Kit (Applied Biosystems), 50 ng were used in each reaction. The expression level of FGD4 was normalized to that of two different genes: TBP (TATA-box binding protein) and GUS-B (β-glucuronidase). Two calibrator samples (normal fibroblast cell lines) were used, and experiments were repeated three times. The FGD4 expression ratio for a sample was calculated as the ratio between the average FGD4 signal in the patient's fibroblast and the signal from calibrators. Primer sequences can be found in table 1. One microgram of each used commercial total RNAs extracted from brain, heart, skeletal muscle, uterus and cerebellum (Ambion-Applied Biosystems), placenta and testis (Clontech) were used in RT-PCRs as described above. Primers located in exons 5 (forward) and 8 (reverse) of FGD4 were used for PCR amplification, which resulted in a 412-bp fragment (see table 1). β-actin was used as a control for normalization with the use of primers 5′-CAATAGTGATGACCTGGCCGT-3′ (forward) and 5′-AGAGGGAAATCGTGCGTGAC-3′ (reverse). FGD4 expression in different mouse parts of the brain and CNS tissues was studied by use of the ORIGENE Mouse Brain Rapid-Scan Panel. cDNAs obtained from polyA+ RNAs immobilized on a 96-well plate were amplified using either the aforementioned FGD4 primers (table 1) or β-actin control primers, with the use of PCR conditions recommended by the supplier. The cDNA for rat wild-type Frabin-GFP was isolated from the pMXII Frabin-GFP plasmid kindly provided by Prof. Takai and was subcloned into an XhoI-restricted pCAGGS expression vector.39Jacquier A Buhler E Schafer MK Bohl D Blanchard S Beclin C Haase G Alsin/Rac1 signaling controls survival and growth of spinal motoneurons.Ann Neurol. 2006; 60: 105-117Crossref PubMed Scopus (56) Google Scholar Truncated forms of Frabin were obtained by PCR (Triple master), with the use of pCAGGS Frabin-GFP as template and forward primer Frabin Fw NheI (5′-AAGGAAACTA GCTAGC ACCATGGAGGAGTCTAATCC-3′) together with reverse primer Frabin Rev 297 Not1 (5′-TTCCTTTTTT GCGGCCGC CTTAAGGAATGGTGCCAAC-3′) or Frabin Rev 249 Not1 (5′-TTCCTTTTTT GCGGCCGC CATCTCTGCAGGAAATGAGCCTC-3′) (underlined parts of sequences correspond to endonuclease restriction sites). PCR products were subcloned into pCAGGS-GFP 5′ of the GFP reading frame. The obtained constructs were checked by sequencing. Spinal motoneurons were prepared from E14.5 Sprague Dawley rat embryos as described elsewhere.40Henderson CE Bloch-Gallego E Camu W Purification and culture of embryonic spinal motoneurons.in: Cohen J Wilkin G Nerve cell culture: a practical approach. Oxford University Press, London1995: 69-81Google Scholar Cell pellets were resuspended in electroporation buffer (125 mM NaCl, 5 mM KCl, 1.5 mM MgCl2, 10 mM glucose, and 20 mM HEPES [pH 7.4]) at a density of 50,000 motoneurons per 50 μl and were transferred to 4-mm gap cuvettes (Eppendorf). The cellular suspensions were then incubated for 15 min at room temperature with 5 μg pCAGGS GFP plasmid or the same molar amount of pCAGGS Frabin expression plasmid and were electroporated using a BTX-ECM830 electroporator (Genetronics). Electroporation conditions were 3 square pulses of 5 ms at 200 volts with intervals of 1 s. Motoneurons were cultured on polyornithin/laminin–coated coverslips in supplemented neurobasal medium (Invitrogen Life Technologies) containing 1 ng/ml GDNF, 1 ng/ml BDNF, and 10 ng/ml CNTF. Primary human fibroblasts and RT4 schwannoma cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Invitrogen Life Technologies) supplemented with 10% fetal calf serum (Invitrogen Life Technologies) and l-glutamine in a 37°C incubator stabilized at 5% CO2. RT4 cell lines are described to be arguably equivalent to primary Schwann cells in vitro.41Hai M Muja N DeVries GH Quarles RH Patel PI Comparative analysis of Schwann cell lines as model systems for myelin gene transcription studies.J Neurosci Res. 2002; 69: 497-508Crossref PubMed Scopus (87) Google Scholar Transduced primary motoneurons and RT4 Schwann cells were fixed at 1 division (DIV) with 4% (vol/vol) formaldehyde, were washed three times with PBS, and were blocked and permeabilized with PBS containing 2% (vol/vol) goat serum, 2% (vol/vol) donkey serum, 0.1% (wt/vol) BSA, 50 mM lysine, and 0.01% (vol/vol) Triton X-100. F-actin was stained for 30 min with Alexa Fluor 546-conjugated phalloidin (diluted 1:80 in blocking buffer [Molecular Probes]). Images of GFP–fluorescent cells were acquired with a Zeiss LSM 510 confocal laser scanning microscope that used a 63× water immersion objective and 3× zoom for motoneurons or 1× zoom for RT4 ce
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