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• W2171456276 abstract "Distal myopathy with rimmed vacuoles is an autosomal recessive muscle disease with preferential involvement of the tibialis anterior that spares the quadriceps muscles in young adulthood. In a Japanese patient with distal myopathy with rimmed vacuoles, we identified pathogenic mutations in the gene encoding the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase, which catalyzes the initial two steps in the biosynthesis of sialic acid. In this study, we demonstrated the relationship between the genetic mutations and enzymatic activities using an in vitro expression assay system. Furthermore, we also showed that the levels of sialic acid in muscle and primary cultured cells from DMRV patients were reduced to 60–75% of control. The reactivities to lectins were also variable in some myofibers, suggesting that hyposialylation and abnormal glycosylation in muscles may contribute to the focal accumulations of autophagic vacuoles, amyloid deposits, or both in patient muscle tissue. The addition of ManNAc and NeuAc to primary cultured cells normalized sialylation levels, thus demonstrating the therapeutic potential of these compounds for this disease. Distal myopathy with rimmed vacuoles is an autosomal recessive muscle disease with preferential involvement of the tibialis anterior that spares the quadriceps muscles in young adulthood. In a Japanese patient with distal myopathy with rimmed vacuoles, we identified pathogenic mutations in the gene encoding the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase, which catalyzes the initial two steps in the biosynthesis of sialic acid. In this study, we demonstrated the relationship between the genetic mutations and enzymatic activities using an in vitro expression assay system. Furthermore, we also showed that the levels of sialic acid in muscle and primary cultured cells from DMRV patients were reduced to 60–75% of control. The reactivities to lectins were also variable in some myofibers, suggesting that hyposialylation and abnormal glycosylation in muscles may contribute to the focal accumulations of autophagic vacuoles, amyloid deposits, or both in patient muscle tissue. The addition of ManNAc and NeuAc to primary cultured cells normalized sialylation levels, thus demonstrating the therapeutic potential of these compounds for this disease. Distal myopathy with rimmed vacuoles (DMRV) 1The abbreviations used are: DMRV, distal myopathy with rimmed vacuoles; HIBM, hereditary inclusion body myopathy; MAM, Maackia amurensis lectin; MBS, m-maleimidobenzoyl-N-hydroxysuccinimido ester; SSA, Sambucus sieboldiana agglutinin; SBA, soybean agglutinin; GNE, UDP-GlcNAc 2-epimerase/ManNAc kinase; WGA, wheat germ agglutinin. 1The abbreviations used are: DMRV, distal myopathy with rimmed vacuoles; HIBM, hereditary inclusion body myopathy; MAM, Maackia amurensis lectin; MBS, m-maleimidobenzoyl-N-hydroxysuccinimido ester; SSA, Sambucus sieboldiana agglutinin; SBA, soybean agglutinin; GNE, UDP-GlcNAc 2-epimerase/ManNAc kinase; WGA, wheat germ agglutinin. is an autosomal recessive disorder characterized clinically by the preferential involvement of the tibialis anterior muscle, sparing the quadriceps muscles as originally described in 1981 (1Nonaka I. Sunohara N. Ishiura S. Satoyoshi E. J. Neurol. Sci. 1981; 51: 141-155Abstract Full Text PDF PubMed Scopus (253) Google Scholar, 2Nonaka I. Murakami N. Suzuki Y. Kawai M. Neuromuscul. Disord. 1998; 8: 333-337Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The age at onset is relatively late with a mean of 26 years. Muscle biopsy of this disorder is characterized by many rimmed vacuoles, which are particularly abundant in atrophic fibers. Necrotic and regenerating fibers are rarely seen (3Nonaka I. Sunohara N. Satoyoshi E. Terasawa K. Yonemoto K. Ann. Neurol. 1985; 17: 51-59Crossref PubMed Scopus (97) Google Scholar). The nucleus occasionally contains tubulofilamentous inclusions of 15–20 nm in diameter.Hereditary inclusion body myopathy (HIBM) is an autosomal recessive disorder that presents with adult-onset slowly progressive distal and proximal weakness and has characteristic pathological features in muscle tissue, including rimmed vacuoles and filamentous inclusions, that are similar to those seen in DMRV (4Griggs R.C. Askanas V. DiMauro S. Engel A. Karpati G. Mendell J.R. Rowland L.P. Ann. Neurol. 1995; 38: 705-713Crossref PubMed Scopus (682) Google Scholar). Gene loci of both diseases have been mapped to chromosome 9 (5Eisenberg I. Avidan N. Potikha T. Hochner H. Chen M. Olender T. Barash M. Shemesh M. Sadeh M. Grabov-Nardini G. Shmilevich I. Friedmann A. Karpati G. Bradley W.G. Baumbach L. Lancet D. Asher E.B. Beckmann J.S. Argov Z. Mitrani-Rosenbaum S. Nat. Genet. 2001; 29: 83-87Crossref PubMed Scopus (437) Google Scholar, 6Ikeuchi T. Asaka T. Saito M. Tanaka H. Higuchi S. Tanaka K. Saida K. Uyama E. Mizusawa H. Fukuhara N. Nonaka I. Takamori M. Tsuji S. Ann. Neurol. 1997; 41: 432-437Crossref PubMed Scopus (84) Google Scholar). HIBM is caused by mutations in the UDP-GlcNAc 2-epimerase/ManNAc kinase gene (GNE gene) (7Eisenberg I. Thiel C. Levi T. Tiram E. Argov Z. Sadeh M. Jackson C.L. Thierfelder L. Mitrani-Rosenbaum S. Genomics. 1999; 55: 43-48Crossref PubMed Scopus (21) Google Scholar). Previously we identified homozygous and compound heterozygous mutations in the GNE gene in 27 DMRV patients (8Nishino I. Noguchi S. Murayama K. Driss A. Sugie K. Oya Y. Nagata T. Chida K. Takahashi T. Takusa Y Ohi T. Nishimiya J. Sunohara N. Ciafaloni E. Kawai M. Aoki M. Nonaka I. Neurology. 2002; 59: 1689-1693Crossref PubMed Scopus (193) Google Scholar), demonstrating that the two diseases are allelic.UDP-GlcNAc 2-epimerase/ManNAc kinase is a dual functional enzyme catalyzing two initial steps in the biosynthesis of sialic acid (9Hinderlich S. Stasche R. Zeitler R. Reutter W. J. Biol. Chem. 1997; 272: 24313-24318Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). This enzyme catalyzes the conversions of UDP-GlcNAc to ManNAc and ManNAc to ManNAc 6-phosphate. Despite the identification of the GNE gene mutations, we still do not fully understand how these mutations contribute to the pathophysiology in DMRV/HIBM. Several questions remain unanswered. 1) What is the status of sialylation activity in the patients with GNE mutations? One would expect sialylation to be impaired but not completely absent in DMRV/HIBM patients, because sialic acid is essential for embryonic development (10Schwarzkopf M. Knobeloch K.P. Rohde E. Hinderlich S. Wiechens N. Lucka L. Horak I. Reutter W. Horstkorte R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5267-5270Crossref PubMed Scopus (282) Google Scholar). In fact, homozygous null mutations have never been identified in patients (8Nishino I. Noguchi S. Murayama K. Driss A. Sugie K. Oya Y. Nagata T. Chida K. Takahashi T. Takusa Y Ohi T. Nishimiya J. Sunohara N. Ciafaloni E. Kawai M. Aoki M. Nonaka I. Neurology. 2002; 59: 1689-1693Crossref PubMed Scopus (193) Google Scholar, 11Eisenberg I. Grabov-Nardini G. Hochner H. Korner M. Sadeh M. Bertorini T. Bushby K. Castellan C. Felice K. Mendell J Merlini L. Shilling C. Wirguin I. Argov Z. Mitrani-Rosenbaum S. Hum. Mutat. 2003; 21: 99Crossref PubMed Scopus (108) Google Scholar). 2) Why are symptoms restricted to the skeletal muscles? GNE transcripts are expressed in various tissues and are especially predominant in the liver (12Lucka L. Krause M. Danker K. Reutter W. Horstkorte R. FEBS Lett. 1999; 454: 341-344Crossref PubMed Scopus (57) Google Scholar). 3) Why do mutant proteins not complement each other in patients who have heterozygous mutations in each of the two domains? The two domains in GNE protein have been reported to catalyze the enzymatic reactions separately and independently (13Effertz K. Hinderlich S. Reutter W. J. Biol. Chem. 1999; 274: 28771-28778Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). To address these questions, we studied the relationships between mutations and enzymatic activity using in vitro expression and enzymatic assay systems. We also determined the levels of sialylation in sera, muscles, and primary cultured cells from DMRV patients and normal individuals.EXPERIMENTAL PROCEDURESMutation and Sialic Acid Analyses of Patients—All of the patients were Japanese. The patients were diagnosed as having DMRV based on both clinical features and muscle pathology. Numbering of the patients followed the protocol presented in our previous report (8Nishino I. Noguchi S. Murayama K. Driss A. Sugie K. Oya Y. Nagata T. Chida K. Takahashi T. Takusa Y Ohi T. Nishimiya J. Sunohara N. Ciafaloni E. Kawai M. Aoki M. Nonaka I. Neurology. 2002; 59: 1689-1693Crossref PubMed Scopus (193) Google Scholar). Gene analyses of DMRV patients were performed as described previously (8Nishino I. Noguchi S. Murayama K. Driss A. Sugie K. Oya Y. Nagata T. Chida K. Takahashi T. Takusa Y Ohi T. Nishimiya J. Sunohara N. Ciafaloni E. Kawai M. Aoki M. Nonaka I. Neurology. 2002; 59: 1689-1693Crossref PubMed Scopus (193) Google Scholar). Primary fibroblasts were obtained from patient 18 and patient 19, whose mutations were reported previously. Primary skeletal myocytes were obtained from patient 5 as reported previously. Sialic acid contents in sera were measured with a SIALIZYME-550 kit (Fujirevio, Tokyo, Japan), and those in muscle and cells were determined by the thiobarbituric acid method. Informed consents were obtained from all subjects using a form approved by the Ethical Review Board at the National Center of Neurology and Psychiatry (Tokyo, Japan).Expression of Recombinant GNE Proteins—The cDNA for wild-type GNE was obtained by reverse transcribed-PCR from normal muscle RNA and cloned into pCR-blunt vector (Invitrogen). The cDNAs for GNE mutants were obtained by reverse transcribed-PCR from skeletal muscle RNA of DMRV patients or by site-directed mutagenesis from wild-type cDNA. All cloned muscle cDNAs were sequenced by ABI cycle-sequencing procedures using an ABI 3100 (Applied Biosystems, Foster City, CA). The sequenced and inserted cDNAs were cut out with EcoRI and blunted, and the purified cDNA fragments were inserted in-frame into the expression vector, pCMV-Myc (Invitrogen). The expression constructs were transiently transfected into COS-7 cells using LipofectAMINE Plus (Invitrogen) according to the manufacturer's protocol. After 24 h, the Myc-tagged wild-type GNE and the mutant proteins were extracted from transfected cells. UDP-GlcNAc 2-epimerase activity was measured as described previously. The ManNAc kinase assay was performed with slight modification according the previous report (13Effertz K. Hinderlich S. Reutter W. J. Biol. Chem. 1999; 274: 28771-28778Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar).Cross-linking of GNE Mutant Proteins—To analyze the oligomer structure of wild-type and mutant GNE, cell lysates were subjected to a reaction with 10 mm MBS for 30 min at room temperature for cross-linking. The Myc-tagged cross-linked products were purified with anti-Myc-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) and eluted by boiling in 2% SDS solution. The products were subjected to SDS-PAGE and Western blot using anti-Myc 9E10 antibody (Santa Cruz Biotechnology).Lectin Staining and Protein Analysis of Skeletal Muscles from Patients—Biotin-labeled Maackia amurensis (MAM), soybean agglutinin (SBA), and Sambucus sieboldiana agglutinin (SSA) (Seikagaku Kogyo, Tokyo, Japan) and fluorescein isothiocyanate-labeled streptavidin (Vector Laboratories, Burlingame, CA) were used for the staining of muscle sections. Unfixed 10-μm-thick muscle sections were blocked in 2% casein/phosphate-buffered saline and stained with lectin solution for 2 h at room temperature. The proteins were extracted from the skeletal muscle sections using 8.5 m urea, 0.5% Nonidet P-40 and analyzed by two-dimensional PAGE. Monoclonal antibodies, H4A3 (for LAMP-1), 43DAG1/8D5 (for β-dystroglycan), and VIA4-I (for α-dystroglycan) were used for Western blotting. Laminin binding to α-dystroglycan was examined as described previously (14Michele D.E. Barresi R. Kanagawa M. Saito F. Cohn R.D. Satz J.S. Dollar J. Nishino I. Kelley R.I. Somer H. Straub V. Mathews K.D. Moore S.A. Campbell K.P. Nature. 2002; 418: 417-422Crossref PubMed Scopus (689) Google Scholar).Cell Cultures—COS-7 cells were cultured in 10% fetal bovine serum/Dulbecco's modified Eagle's medium in 5% CO2. Primary fibroblasts and myoblasts from DMRV patients and normal individuals were cultured in 10% fetal bovine serum, Dulbecco's modified Eagle's medium, and Ham's F-12 medium in 5% CO2. The myoblasts were induced to myogenic differentiation by switching the medium to 5% horse serum, Dulbecco's modified Eagle's medium, and Ham's F-12 medium. At 24 h before lectin staining or sialic acid determination, the medium was replaced with serum-free Dulbecco's modified Eagle's medium and Ham's F-12 medium with or without 5 mm GlcNAc, ManNAc, or NeuAc. Cells were fixed and permeabilized as described previously (15Noguchi S. Wakabayashi E. Imamura M. Yoshida M. Ozawa E. Eur. J. Biochem. 2000; 267: 640-648Crossref PubMed Scopus (56) Google Scholar). Biotin-labeled SBA, wheat germ agglutinin (WGA) (Seikagaku Kogyo), an antibody against desmin (ICN Pharmaceuticals, Costa Mesa, CA), and Alexa Fluo 594-labeled secondary antibodies (Molecular Probes, Eugene, OR) were used for staining the cells.RESULTSNovel Mutations in GNE Gene in DMRV Patients—In the previous study, we identified 12 different GNE mutations in either homozygous or compound heterozygous states in 27 DMRV patients (8Nishino I. Noguchi S. Murayama K. Driss A. Sugie K. Oya Y. Nagata T. Chida K. Takahashi T. Takusa Y Ohi T. Nishimiya J. Sunohara N. Ciafaloni E. Kawai M. Aoki M. Nonaka I. Neurology. 2002; 59: 1689-1693Crossref PubMed Scopus (193) Google Scholar). From these results, we concluded that DMRV is allelic to HIBM. Subsequently, we identified six patients harboring five different mutations, of which three were novel mutations: 1622C→T, 89G→C, and 2173G→A (Table I). These novel mutations were absent in 100 control chromosomes from normal Japanese individuals.Table IIdentified mutations The mutations were identified in patients diagnosed as having DMRV based on both clinical features and muscle pathology. Numbering of patients followed that in our previous report (8Nishino I. Noguchi S. Murayama K. Driss A. Sugie K. Oya Y. Nagata T. Chida K. Takahashi T. Takusa Y Ohi T. Nishimiya J. Sunohara N. Ciafaloni E. Kawai M. Aoki M. Nonaka I. Neurology. 2002; 59: 1689-1693Crossref PubMed Scopus (193) Google Scholar). Exon indicates the exon number where mutation was found. Protein domain is predicted ones from the sequence homology as previously (13Effertz K. Hinderlich S. Reutter W. J. Biol. Chem. 1999; 274: 28771-28778Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar).PatientMutationExonPredicted amino acid alterationProtein domain28578A→TE3D176VEpimerase1765G→CE10V572LKinase29578A→TE3D176VEpimerase578A→TE3D176VEpimerase30578A→TE3D176VEpimerase1622C→TE9A524VKinase3189G→CE2C13SEpimerase89G→CE2C13SEpimerase32578A→TE3D176VEpimerase2173G→AE12G708SKinase331765G→CE10V572LKinase1765G→CE10V572LKinase Open table in a new tab Enzymatic Activities of GNE Mutants in DMRV Patients— Site-directed mutagenesis of the GNE protein has shown that the two enzymatic activities are separately and independently catalyzed by two domains, an N-terminal epimerase domain and a C-terminal kinase domain (13Effertz K. Hinderlich S. Reutter W. J. Biol. Chem. 1999; 274: 28771-28778Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Previously, we reported reductions in UDP-GlcNAc 2-epimerase activities in leukocytes from the patients with mutations in the GNE gene (8Nishino I. Noguchi S. Murayama K. Driss A. Sugie K. Oya Y. Nagata T. Chida K. Takahashi T. Takusa Y Ohi T. Nishimiya J. Sunohara N. Ciafaloni E. Kawai M. Aoki M. Nonaka I. Neurology. 2002; 59: 1689-1693Crossref PubMed Scopus (193) Google Scholar). However, the enzyme activity was too weak in leukocytes to clearly demonstrate correlations between gene mutations and the enzymatic activities. To clarify this relationship, we generated recombinant proteins with each of the 13 species of mutations that we identified. All but one of the mutant and wild-type recombinant GNE proteins were expressed in COS cells and migrated at 75 kDa in SDS-PAGE. The single exception was a recombinant protein with a deletion of amino acids 206–256 (Δ206–256) caused by exon 4 skipping. The abnormal protein was degraded in COS cells (Fig. 1A). We determined the specific activities of UDP-GlcNAc 2-epimerase and ManNAc kinase of the mutant proteins relative to wild type (Fig. 1B). The endogenous activities in mock transfected COS cells were determined to correct for the background enzyme activity. UDP-GlcNAc 2-epimerase activities in mutants C13S, H132Q, D176V, D177C, V331A, and D378Y were reduced to less than 20% of the control. In contrast, the I472T and G708S mutants each revealed an ∼50% reduction, and V572L, A630T, and A631V each showed only a 20–30% reduction in activity as compared with wild-type cells. ManNAc kinase activity was retained in the N-terminal mutants C13S, H132Q, D176V, D177C, V331A, and D378Y, whereas the C-terminal mutants I472T, V572L, A630T, A631V, and G703S showed dramatic reductions in activities. These data were essentially compatible with a prior report (13Effertz K. Hinderlich S. Reutter W. J. Biol. Chem. 1999; 274: 28771-28778Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Interestingly, the A524V mutant preferentially affected UDP-GlcNAc 2-epimerase activity, although the mutation is in the kinase domain. None of the DMRV mutants showed complete loss of UDP-GlcNAc 2-epimerase or ManNAc kinase activities.Oligomerization of GNE Mutants—GNE protein forms a homohexamer by oligomerization (13Effertz K. Hinderlich S. Reutter W. J. Biol. Chem. 1999; 274: 28771-28778Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). We examined whether the mutations affect the oligomerization of GNE molecules, because homohexamer structure of GNE protein was reported to be essential for UDP-GlcNAc 2-epimerase activity (13Effertz K. Hinderlich S. Reutter W. J. Biol. Chem. 1999; 274: 28771-28778Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The recombinant mutant proteins were subjected to the cross-linking with MBS. Fig. 1C shows the electrophoretic tracing patterns of cross-linked products of wild-type and mutant GNE proteins. The product of wild-type GNE predominantly migrated at >400 kDa, which corresponds to homohexamer. C13S, V572L, A630T, and A631V gave cross-linked products similar to that of wild-type GNE, whereas the mutants H132Q and D176V mainly generated a 200-kDa product, and D177C, V331A, D378Y, and A524V produced 98-kDa proteins. These data indicate that the hexameric oligomerization is necessary for UDP-GlcNAc 2-epimerase activity and that N-terminal mutants generally fail to form large oligomers although the molecular region responsible for the oligomerization is not clearly related to the predicted oligomerization domains based on the primary structure.Sialic Acid Contents in Skeletal Muscles and Primary Cells from DMRV Patients—GNE gene mutations reduced the enzymatic activity of GNE protein. These results led us to hypothesize that sialylation should be affected in the tissues of DMRV patients. We measured the sialic acid content in sera and muscles from DMRV patients (Fig. 2A). In sera, no difference was detected between patients and normal controls, whereas in skeletal muscle, a 25% reduction of sialic acid was observed in DMRV muscles. We also assessed the status of sialylation in DMRV muscles by lectin staining. We used three lectins: SSA for detection of Siaα2–6Gal/GalNAc, MAM for Siaα2–3Gal, and SBA for GalNAcα1–3Gal (16Shibuya N. Tazaki K. Song Z.W. Tarr G.E. Goldstein I.J. Peumans W.J. J. Biochem. (Tokyo). 1989; 106: 1098-1103Crossref PubMed Scopus (92) Google Scholar, 17Yamamoto K. Konami Y. Irimura T. J. Biochem. (Tokyo). 1997; 121: 756-761Crossref PubMed Scopus (49) Google Scholar, 18Pereira M.E. Kabat E.A. Sharon N. Carbohydr. Res. 1974; 37: 89-102Crossref PubMed Scopus (122) Google Scholar) (Fig. 2B). The results of lectin staining are summarized in Table II. SSA uniformly stained sarcolemma in control muscle, whereas it faintly stained sarcolemma and strongly stained interstitial tissues in DMRV muscle (Fig. 2B, panels d–f). MAM strongly stained sarcolemma and interstitial tissues in both the control and patient muscles. We did not observe any reduction in MAM staining in patients, which may be attributed to the strong intensity in our staining condition (data not shown). In contrast, SBA strongly highlighted the rimmed vacuoles containing fibers and the surrounding atrophic fibers in the patients both in sarcolemma and cytoplasm (Fig. 2B, panels h and i; see arrows), whereas it did not stain myofibers in the control (Fig. 2B, panel g). These data suggest that sialylation, other glycosylation, or both are at least partly disturbed in some myofibers in DMRV. Furthermore, we examined the expression of glycosylated α-dystroglycan in DMRV muscles using an antibody (VIA4-I) that recognizes a carbohydrate epitope. The α-dystroglycan staining was negative in rimmed vacuoles containing fibers and the surrounding atrophic fibers in one DMRV patient (Fig. 2B, panel l). However, positive staining in another patient demonstrated that α-dystroglycan expression varies among patients (Fig. 2B, panel k). Therefore, we concluded that the loss of α-dystroglycan staining is an extreme down-stream phenomenon in DMRV muscles. We also analyzed muscle sialylated glycoproteins (LAMP-1, and α- and β-dystroglycans) by one- or two-dimensional polyacrylamide gel electrophoresis, but when they were extracted in whole amounts, there was no significant change in the electrophoretic patterns of these proteins between control and patients (data not shown). Furthermore, we analyzed the laminin-binding property of α-dystroglycan from DMRV patients, and the α-dystroglycan showed a strong binding as the control (data not shown).Fig. 2Sialylation of sera and muscles from DMRV patients and controls. A, sialic acid contents of the serum from control (n = 7) and DMRV patients (n = 9, patients 4, 5, 6, 7, 12, 13, 17, 26, and 28) were measured using a SIALIZYME-550 kit. The sialic acid contents of muscles from the control (n = 7) and DMRV patients (n = 4, patients 5, 8, 9, and 13) were measured by the thiobarbituric acid method. *, p < 0.05. B, lectin staining and immunohistochemical staining of α-dystroglycan in skeletal muscles from control (a, d, g, and j) and DMRV patients 5 (b, e, h, and k) and patient 8 (c, f, i, and l). a–c, stained with hematoxylin and eosin; d–f, stained with SSA lectin; g–i, stained with SBA lectin; j–l, stained with an antibody for α-dystroglycan (VIA4-I). Arrows indicate rimmed vacuoles. SBA lectin strongly stained sarcolemma and cytoplasmic areas in the cluster of atrophic or rimmed vacuoles containing myofibers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIThe list of their mutations, GNE activities, and the results of lectin staining of patients used in this study The muscle specimens from patients 5 and 8, the fibroblasts from patients 18 and 19, and the myotubes from patient 5 were used in this study. ND, not determined.PatientMutationPredicted amino acid alterationGNE activityaGNE activity represents UDP-GlcNAc 2-epimerase activityMNK activitybMNK activity represents ManNAc kinase activity as percentage relative to that of wild type GNESSA staining in skeletal muscleSBA staining in skeletal muscleWGA staining in cultured cellSBA staining in cultured cell%%5IVS4+4A→GExon 4 skippingNDNDVariablePositive in atrophic fibersNegative in plasma membranePositive1765G→CV572L68.28.38578A→TD176V18.286.5VariablePositive in atrophic fibersNDND1043T→CV331A16.111418578A→TD176V18.286.5NDNDWeakPositive1466T→CI472T47.54.719578A→TD176V18.286.5NDNDWeakPositive578A→TD176V18.286.5a GNE activity represents UDP-GlcNAc 2-epimerase activityb MNK activity represents ManNAc kinase activity as percentage relative to that of wild type GNE Open table in a new tab Restoration of Sialylation in DMRV Cells by Feeding with ManNAc and NeuAc—Previous studies reported that hyposialylation in GNE-/- embryonic stem cells and GNE-defective cells can be repaired by feeding with the natural sialic acid precursor ManNAc (10Schwarzkopf M. Knobeloch K.P. Rohde E. Hinderlich S. Wiechens N. Lucka L. Horak I. Reutter W. Horstkorte R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5267-5270Crossref PubMed Scopus (282) Google Scholar, 19Keppler O.T. Hinderlich S. Langner J. Schwartz-Albiez R. Reutter W. Pawlita M. Science. 1999; 284: 1372-1376Crossref PubMed Scopus (274) Google Scholar). We examined the recovery of sialylation by the addition of NeuAc or ManNAc using primary cultured cells from patients and evaluated the sialylation status by lectin staining (Fig. 3A). We used two lectins, WGA, which specifically recognizes a cluster structure of sialic acids (20Bhavanandan V.P. Katlic A.W. J. Biol. Chem. 1979; 254: 4000-4008Abstract Full Text PDF PubMed Google Scholar), and SBA. WGA strongly stained the perinuclear region in control fibroblasts. Fibroblasts from patient 18, who harbors compound heterozygous missense mutations D176V and I472T, showed weaker staining with WGA compared with normal controls (Fig. 3A; WGA, DMRV, and Control). SBA did not stain the fibroblasts from normal controls but strongly stained cells from DMRV patients. These results were also confirmed in myotubes from another DMRV patient (patient 5) who has compound heterozygous mutations causing exon 4 skipping and V572L (Fig. 3B) and fibroblasts from patient 19 with a homozygous D176V mutation (data not shown). The sialic acid levels in skin fibroblasts and myotubes from DMRV patients were significantly decreased at ∼60–74% of control cells when cells were cultured in serum-free medium (Fig. 3C). By adding ManNAc or NeuAc into the culture medium, sialic acid levels in the fibroblasts and myotubes were restored to normal levels (Fig. 3C, +ManNAc and +NeuAc). Furthermore, WGA staining of cells from patients also increased to normal levels, and particularly strong WGA staining was observed in the plasma membrane of myotubes. In contrast, the SBA staining in DMRV cells disappeared by the addition of either sugar (Fig. 3, A and B; +ManNAc and +NeuAc). The addition of GlcNAc into the medium had no effect on the staining pattern with either lectin (Fig. 3, A and C, +GlcNAc). These results suggest the potential therapeutic use of ManNAc and NeuAc.Fig. 3Recovery of the sialylation in DMRV cells by treatment with ManNAc and NeuAc. A, the fibroblasts from patient 18 (DMRV) and control individuals were stained with WGA and SBA lectins. The patient 18 fibroblasts cultured in the presence of GlcNAc (+GlcNAc), ManNAc (+ManNAc) and NeuAc (+NeuAc) were also stained with both lectins. B, differentiated myotubes from patient 5 cultured without (DMRV) and with ManNAc (+ManNAc) and NeuAc (+NeuAc) were stained by WGA and SBA lectins and desmin antibody. Desmin-positive cells are myotubes. C, sialic acids in myotubes (n = 4 from patient 5, black) and fibroblasts (n = 4 from patients 18 and 19, white) were measured by the thiobarbituric acid method.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONIn this study, we identified six additional DMRV patients with GNE mutations. Combined with prior results, the 1765G→C mutation accounts for 55% (36 of 66) of the abnormal alleles confirming the high frequency of this mutation in Japan. Haplotype analysis suggests that this common mutation is due to a founder effect (8Nishino I. Noguchi S. Murayama K. Driss A. Sugie K. Oya Y. Nagata T. Chida K. Takahashi T. Takusa Y Ohi T. Nishimiya J. Sunohara N. Ciafaloni E. Kawai M. Aoki M. Nonaka I. Neurology. 2002; 59: 1689-1693Crossref PubMed Scopus (193) Google Scholar). All of the mutations identified in DMRV patients caused reduction (but not total loss) of enzymatic activities of either UDP-GlcNAc 2-epimerase or ManNAc kinase. These results strongly suggest that DMRV is caused by partial loss of function of the gene product. Interestingly, we previously identified the compound heterozygous mutations D378Y and A631V in a North American DMRV patient of German and Irish origin; these mutations have also been identified in an Irish HIBM patient (13Effertz K. Hinderlich S. Reutter W. J. Biol. Chem. 1999; 274: 28771-28778Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). D378Y reduced UDP-GlcNAc 2-epimerase activity, and A631V decreased ManNAc kinase activity. Together with clinical and pathological similarities, these biochemical and molecular genetic results suggest that DMRV and HIBM are actually the same disease.Through our study, we obtained information about novel molecular aspects in GNE. The two catalytic domains of the GNE molecule do not always work separately or independently in contrast to a published report (13Effertz K. Hinderlich S. Reutter W. J. Biol. Chem. 1999; 274: 28771-28778Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). For example, the A524V mutation is within the predic" @default.
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