{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T05:37:00Z","timestamp":1793338620321,"version":"4.5.0"},"reference-count":45,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5402","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1999,1,29]]},"abstract":"<jats:p>\n                    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive human disease associated with multiple deletions of skeletal muscle mitochondrial DNA (mtDNA), which have been ascribed to a defect in communication between the nuclear and mitochondrial genomes. Examination of 12 MNGIE probands revealed homozygous or compound-heterozygous mutations in the gene specifying thymidine phosphorylase (TP), located on chromosome 22q13.32-qter. TP activity in leukocytes from MNGIE patients was less than 5 percent of controls, indicating that loss-of-function mutations in\n                    <jats:italic>TP<\/jats:italic>\n                    cause the disease. The pathogenic mechanism may be related to aberrant thymidine metabolism, leading to impaired replication or maintenance of mtDNA, or both.\n                  <\/jats:p>","DOI":"10.1126\/science.283.5402.689","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T05:48:21Z","timestamp":1027748901000},"page":"689-692","source":"Crossref","is-referenced-by-count":719,"title":["Thymidine Phosphorylase Gene Mutations in MNGIE, a Human Mitochondrial Disorder"],"prefix":"10.1126","volume":"283","author":[{"given":"Ichizo","family":"Nishino","sequence":"first","affiliation":[{"name":"Columbia University College of Physicians and Surgeons, Department of Neurology, 630 West 168 Street, P &amp; S 4-443, New York, NY 10032, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antonella","family":"Spinazzola","sequence":"additional","affiliation":[{"name":"Columbia University College of Physicians and Surgeons, Department of Neurology, 630 West 168 Street, P &amp; S 4-443, New York, NY 10032, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michio","family":"Hirano","sequence":"additional","affiliation":[{"name":"Columbia University College of Physicians and Surgeons, Department of Neurology, 630 West 168 Street, P &amp; S 4-443, New York, NY 10032, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1016\/S0005-2728(98)00113-3","volume":"1366","author":"DiMauro S.","year":"1998","unstructured":"DiMauro S., Bonilla E., Davidson M., Hirano M., Schon E. A., Biochim. Biophys. Acta. 1366, 199 (1998).","journal-title":"Biochim. Biophys. Acta."},{"key":"e_1_3_1_3_2","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1038\/ng0295-146","volume":"9","author":"Suomalainen A.","year":"1995","unstructured":"Suomalainen A., et al., Nature Genet. 9, 146 (1995);","journal-title":"Nature Genet."},{"key":"e_1_3_1_3_3","first-page":"763","volume":"58","author":"Kaukonen J. A.","year":"1996","unstructured":"Kaukonen J. A., et al., Am. J. Hum. Genet. 58, 763 (1996).","journal-title":"Am. J. Hum. Genet."},{"key":"e_1_3_1_4_2","doi-asserted-by":"crossref","first-page":"526","DOI":"10.1086\/301979","volume":"63","author":"Hirano M.","year":"1998","unstructured":"Hirano M., et al., Am. J. Hum. Genet. 63, 526 (1998).","journal-title":"Am. J. Hum. Genet."},{"key":"e_1_3_1_5_2","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1007\/BF00688189","volume":"74","author":"Bardosi A.","year":"1987","unstructured":"Bardosi A., et al., Acta Neuropathol. 74, 248 (1987);","journal-title":"Acta Neuropathol."},{"key":"e_1_3_1_5_3","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1212\/WNL.44.4.721","volume":"44","author":"Hirano M.","year":"1994","unstructured":"Hirano M., et al., Neurology 44, 721 (1994).","journal-title":"Neurology"},{"key":"e_1_3_1_6_2","doi-asserted-by":"crossref","first-page":"1086","DOI":"10.1212\/WNL.51.4.1086","volume":"51","author":"Papadimitriou A.","year":"1998","unstructured":"Papadimitriou A., et al., Neurology 51, 1086 (1998);","journal-title":"Neurology"},{"key":"e_1_3_1_6_3","unstructured":". Depletion of mtDNA was first described in patients with infantile mitochondrial myopathy and hepatopathy by C. T. Moraes et al. [ Am. J. Hum. Genet. 48 492 (1991)]."},{"key":"e_1_3_1_7_2","doi-asserted-by":"crossref","unstructured":"The diagnosis of MNGIE was based on criteria described previously (3 4). We genotyped families 5 to 7 and confirmed linkage to the MNGIE locus that we reported earlier with families 1 to 4 (3). Patient 10 was previously described by A. B. Threlkeld et al. [ Am. J. Ophthalmol. 114 322 (1992)] and D. R. Johns A. B. Threlkeld N. R. Miller and O. Hurko [ ibid. 115 108 (1993)].","DOI":"10.1016\/S0002-9394(14)73533-0"},{"key":"e_1_3_1_8_2","unstructured":"The Human Chromosome 22 Sequencing Group Sanger Centre Cambridge UK (www.sanger.ac.uk\/HGP\/Chr22) and the Advanced Center for Genome Technology at the University of Oklahoma Norman (www.genome.ou.edu\/maps\/ch22.html). Clone bK384D8 (GenBank accession ) which contains TP was mapped identified and characterized by U.-G. Kim et al. [ Proc. Natl. Acad. Sci. U.S.A. 93 6297 (1996)]."},{"key":"e_1_3_1_9_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1042\/bj3340001","volume":"334","author":"Brown N. S.","year":"1998","unstructured":"Brown N. S., Bicknell R., Biochem. J. 334, 1 (1998);","journal-title":"Biochem. J."},{"key":"e_1_3_1_9_3","doi-asserted-by":"crossref","first-page":"689","DOI":"10.1038\/bjc.1997.447","volume":"76","author":"Griffiths L.","year":"1997","unstructured":"Griffiths L., Stratford I. J., Br. J. Cancer 76, 689 (1997).","journal-title":"Br. J. Cancer"},{"key":"e_1_3_1_10_2","unstructured":"Informed consent for blood samples was obtained from each study participant under a Columbia University Institutional Review Board approval protocol. TP has 10 exons and an open reading frame spanning from exons 2 to 10 (12). We amplified four fragments from genomic DNA which encompassed exons 2 and 3 4 5 and 6 and 7 to 10. All primers were 20 nucleotides in length. Their 5\u2032 positions and direction (F indicates forward and R reverse) are: 267F and 952R for exons 2 and 3; 1283F and 1540R for exon 4; 2258F and 2959R for exons 5 and 6; and 3197F and 4254R for exons 7 to 10. We sequenced the fragments directly using the BigDye Terminator Cycle Sequencing Kit (Perkin-Elmer Foster City CA) and then electrophoresed the samples using an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer)."},{"key":"e_1_3_1_11_2","unstructured":"We used the sequence and numbering system of K. Hagiwara et al. [ Mol. Cell. Biol. 11 2125 (1991)]. However we found that exon 3 ended at nt 878 as illustrated in their figure 1 rather than at nt 833 as in their figure 2."},{"key":"e_1_3_1_12_2","unstructured":"Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr."},{"key":"e_1_3_1_13_2","unstructured":"PROSITE at the Swiss Institute of Bioinformatics. Accession numbers are PS 00647 (thymidine\/pyrimidine-nucleoside phosphorylase consensus) and PS 00029 (leucine zipper pattern). We used the \u201cMOTIF\u201d search program (www.motif.genome.ad.jp\/)."},{"key":"e_1_3_1_14_2","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1006\/jmbi.1998.1941","volume":"281","author":"Pugmire M. J.","year":"1998","unstructured":"Pugmire M. J., Cook W. J., Jasanoff A., Walter M. R., Ealick S. E., J. Mol. Biol. 281, 285 (1998).","journal-title":"J. Mol. Biol."},{"key":"e_1_3_1_15_2","unstructured":"To screen for the mutations G1419A G1443A and A3371C we used mismatch primers that would create new recognition sites for restriction enzymes when the template DNA contained the mutations. For G1419A we first performed PCR with the mismatch primer 5\u2032-TGACCACCAGGTGCCAATGATC T GC-3\u2032 (mismatch sites are underlined) corresponding to nts 1394\u20131418 and the reverse primer corresponding to nts 1540\u20131521. The amplified fragments were then digested overnight with Pst I. The products were subjected to electrophoresis in a 4% agarose gel. Similarly fragments were amplified with the mismatch primers 5\u2032-TAGACTCCAGCTTATCCAAG AA GC-3\u2032 corresponding to nts 1467-1444 and a primer corresponding to nts 1283\u20131302 for G1443A and the mismatch primer 5\u2032-TGCGCCGTCCATGCAGAGCAGCG G C-3\u2032 corresponding to nts 3396\u20133372 and a primer corresponding to nts 3197\u20133216 for A3371C. The PCR products were digested with Hind III and Nar I respectively and subjected to electrophoresis in a 4% agarose gel. The A2744G mutation creates a new recognition site for Hinf III. The flanking region was amplified with primers corresponding to nts 2666\u20132685 and 2950\u20132831 digested with Hinf III and subjected to electrophoresis in a 2% agarose gel."},{"key":"e_1_3_1_16_2","unstructured":"Total RNA was extracted from fresh blood samples using RNAqueous Kit and RNAqueous Blood Module (Ambion Austin TX) and was reverse-transcribed into cDNA with oligo(dT) 12\u201318 primer using the SuperScript Preamplification System (Life Technologies Gaithersburg MD). Using PCR we amplified the cDNA fragments with primers corresponding to nts 473\u2013492 in exon 2 and nts 3291\u20133272 in exon 7 for the T1504C mutation; and nts 3405\u20133424 in exon 7 and nts 4254\u20134235 in exon 10 for the G3867C mutation. Amplified fragments were gel purified and sequenced (9). RNA from one healthy individual was simultaneously amplified and sequenced to verify all of the exon junctions and confirm that no exon was skipped."},{"key":"e_1_3_1_17_2","doi-asserted-by":"crossref","first-page":"1311","DOI":"10.1016\/S0006-291X(05)80025-7","volume":"184","author":"Usuki K.","year":"1992","unstructured":"Usuki K., et al., Biochem. Biophys. Res. Commun. 184, 1311 (1992).","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"e_1_3_1_18_2","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1016\/0304-4165(90)90160-X","volume":"1034","author":"Yoshimura A.","year":"1990","unstructured":"Yoshimura A., et al., Biochim. Biophys. Acta 1034, 107 (1990);","journal-title":"Biochim. Biophys. Acta"},{"key":"e_1_3_1_18_3","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1016\/0014-5793(92)81428-O","volume":"313","author":"Waltenberger J.","year":"1992","unstructured":"Waltenberger J., Usuki K., Fellstr\u00f6m B., Funa K., Heldin C.-H., FEBS Lett. 313, 129 (1992);","journal-title":"FEBS Lett."},{"key":"e_1_3_1_18_4","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1002\/path.1711760212","volume":"176","author":"Fox S. B.","year":"1995","unstructured":"Fox S. B., et al., J. Pathol. 176, 183 (1995);","journal-title":"J. Pathol."},{"key":"e_1_3_1_18_5","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1016\/0304-3940(95)11622-4","volume":"192","author":"Eccleston P. A.","year":"1995","unstructured":"Eccleston P. A., Funa K., Heldin C.-H., Neurosci. Lett. 192, 137 (1995);","journal-title":"Neurosci. Lett."},{"key":"e_1_3_1_18_6","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1016\/S0167-4889(96)00078-X","volume":"1314","author":"Matsukawa K.","year":"1996","unstructured":"Matsukawa K., Moriyama A., Kawai Y., Asai K., Kato T., Biochim. Biophys. Acta 1314, 71 (1996).","journal-title":"Biochim. Biophys. Acta"},{"key":"e_1_3_1_19_2","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1111\/j.1471-4159.1992.tb08905.x","volume":"59","author":"Asai K.","year":"1992","unstructured":"Asai K., et al., J. Neurochem. 59, 307 (1992);","journal-title":"J. Neurochem."},{"key":"e_1_3_1_19_3","doi-asserted-by":"crossref","first-page":"20311","DOI":"10.1016\/S0021-9258(19)88703-3","volume":"267","author":"Asai K.","year":"1992","unstructured":"Asai K., et al., J. Biol. Chem. 267, 20311 (1992);","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_19_4","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1016\/0006-8993(93)90833-9","volume":"622","author":"Ueki T.","year":"1993","unstructured":"Ueki T., et al., Brain Res. 622, 299 (1993).","journal-title":"Brain Res."},{"key":"e_1_3_1_20_2","unstructured":"Using RT-PCR we amplified TP cDNA from skeletal muscle which indicates that TP is expressed at low levels in this tissue."},{"key":"e_1_3_1_21_2","unstructured":"Preliminary high-performance liquid chromatography experiments have demonstrated markedly elevated thymidine levels with normal thymine levels in the plasma of four patients (about sevenfold) relative to plasma of five controls (A. L. Andreu and A. Naini personal communication)."},{"key":"e_1_3_1_22_2","doi-asserted-by":"crossref","first-page":"2938","DOI":"10.1016\/S0021-9258(17)33481-6","volume":"251","author":"Bogenhagen D.","year":"1976","unstructured":"Bogenhagen D., Clayton D. A., J. Biol. Chem. 251, 2938 (1976);","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_22_3","first-page":"9300","volume":"257","author":"Bestwick R. K.","year":"1982","unstructured":"Bestwick R. K., Moffett G. L., Mathews C. K., ibid. 257, 9300 (1982);","journal-title":"ibid."},{"key":"e_1_3_1_22_4","unstructured":"; R. K. Bestwick and C. K. Mathews ibid. p. 9305."},{"key":"#cr-split#-e_1_3_1_23_2.1","doi-asserted-by":"crossref","unstructured":"A. J. Berk and D. A. Clayton ibid. 248 2722 (1973)","DOI":"10.1016\/S0021-9258(19)44066-0"},{"key":"#cr-split#-e_1_3_1_23_2.2","doi-asserted-by":"crossref","unstructured":"M. Johansson and A. Karlsson ibid. 272 8454 (1997).","DOI":"10.1074\/jbc.272.13.8454"},{"key":"e_1_3_1_24_2","unstructured":"Elevation of thymidine levels have been shown to alter dNTP pools ["},{"key":"e_1_3_1_24_3","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1016\/0926-6550(63)90411-0","volume":"68","author":"Morris N. R.","year":"1963","unstructured":"Morris N. R., Reichard P., Fischer G. A., Biochim. Biophys. Acta. 68, 93 (1963);","journal-title":"Biochim. Biophys. Acta."},{"key":"e_1_3_1_24_4","doi-asserted-by":"crossref","first-page":"7941","DOI":"10.1128\/JVI.72.10.7941-7949.1998","volume":"72","author":"Julias J. G.","year":"1998","unstructured":"Julias J. G., Pathak V. K., J. Virol. 72, 7941 (1998)].","journal-title":"J. Virol."},{"key":"e_1_3_1_25_2","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1212\/WNL.50.1.99","volume":"50","author":"Carrozo R.","year":"1998","unstructured":"Carrozo R., et al., Neurology 50, 99 (1998).","journal-title":"Neurology"},{"key":"e_1_3_1_26_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/26.1.323"},{"key":"e_1_3_1_27_2","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1016\/S0021-9258(18)71264-7","volume":"207","author":"Friedkin M.","year":"1954","unstructured":"Friedkin M., Roberts D., J. Biol. Chem. 207, 245 (1954);","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_27_3","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1172\/JCI104911","volume":"43","author":"Marsh J. C.","year":"1964","unstructured":"Marsh J. C., Perry S., J. Clin. Invest. 43, 267 (1964);","journal-title":"J. Clin. Invest."},{"key":"e_1_3_1_27_4","doi-asserted-by":"crossref","first-page":"5059","DOI":"10.1016\/S0021-9258(18)99476-7","volume":"242","author":"Gallo R. C.","year":"1967","unstructured":"Gallo R. C., Perry S., Breitman T. R., J. Biol. Chem. 242, 5059 (1967).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_28_2","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(76)90527-3"},{"key":"e_1_3_1_29_2","unstructured":"We thank the families for their cooperation; L. P. Rowland J. R. Carlo A. F. Hahn Y. Shapira I. Butler A. N. Bender L. M. Salberg and S. Hammans for clinical samples; S. DiMauro E. A. Schon A. L. Andreu and T. G. Nygaard for critical advice; and S. Tadesse for excellent technical assistance. Supported by the Columbia-Presbyterian Irving Scholars Program (M.H.); the Uehara Memorial Foundation Tokyo (I.N.); the Consiglio Nazionale delle Ricerche Rome (A.S.); NIH grant RO1-HL59657 (M.H.); and a Muscular Dystrophy Association grant (S. DiMauro)."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.283.5402.689","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,12]],"date-time":"2024-01-12T22:15:29Z","timestamp":1705097729000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.283.5402.689"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1999,1,29]]},"references-count":45,"journal-issue":{"issue":"5402","published-print":{"date-parts":[[1999,1,29]]}},"alternative-id":["10.1126\/science.283.5402.689"],"URL":"https:\/\/doi.org\/10.1126\/science.283.5402.689","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1999,1,29]]}}}