Abstract
Vitamin B12 (cobalamin) is essential in animals for metabolism of branched chain amino acids and odd chain fatty acids, and for remethylation of homocysteine to methionine1. In the cblF inborn error of vitamin B12 metabolism, free vitamin accumulates in lysosomes, thus hindering its conversion to cofactors2,3. Using homozygosity mapping in 12 unrelated cblF individuals and microcell-mediated chromosome transfer, we identified a candidate gene on chromosome 6q13, LMBRD1, encoding LMBD1, a lysosomal membrane protein with homology to lipocalin membrane receptor LIMR. We identified five different frameshift mutations in LMBRD1 resulting in loss of LMBD1 function, with 18 of the 24 disease chromosomes carrying the same mutation embedded in a common 1.34-Mb haplotype. Transfection of fibroblasts of individuals with cblF with wild-type LMBD1 rescued cobalamin coenzyme synthesis and function. This work identifies LMBRD1 as the gene underlying the cblF defect of cobalamin metabolism and suggests that LMBD1 is a lysosomal membrane exporter for cobalamin.
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References
Chanarin, I., Deacon, R., Lumb, M., Muir, M. & Perry, J. Coblamin-folate interrelations: a critical review. Blood 66, 479–489 (1985).
Rosenblatt, D.S., Hosack, A., Matiaszuk, N.V., Cooper, B.A. & Laframboise, R. Defect in vitamin B12 release from lysosomes: newly described inborn error of vitamin B12 metabolism. Science 228, 1319–1321 (1985).
Vassiliadis, A., Rosenblatt, D.S., Cooper, B.A. & Bergeron, J.J. Lysosomal cobalamin accumulation in fibroblasts from a patient with an inborn error of cobalamin metabolism (cblF complementation group): visualization by electron microscope radioautography. Exp. Cell Res. 195, 295–302 (1991).
Coelho, D. et al. Gene identification for the cblD defect of vitamin B12 metabolism. N. Engl. J. Med. 358, 1454–1464 (2008).
Suormala, T. et al. The cblD defect causes either isolated or combined deficiency of methylcobalamin and adenosylcobalamin synthesis. J. Biol. Chem. 279, 42742–42749 (2004).
Morral, N. et al. The origin of the major cystic fibrosis mutation (delta F508) in European populations. Nat. Genet. 7, 169–175 (1994).
Varon, R. et al. Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome. Cell 93, 467–476 (1998).
Budde, B.S. et al. tRNA splicing endonuclease mutations cause pontocerebellar hypoplasia. Nat. Genet. 40, 1113–1118 (2008).
Abecasis, G.R. & Wigginton, J.E. Handling marker-marker linkage disequilibrium: pedigree analysis with clustered markers. Am. J. Hum. Genet. 77, 754–767 (2005).
Konrad, M. et al. Mutations in the tight-junction gene claudin 19 (CLDN19) are associated with renal magnesium wasting, renal failure, and severe ocular involvement. Am. J. Hum. Genet. 79, 949–957 (2006).
Crisponi, L. et al. Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1. Am. J. Hum. Genet. 80, 971–981 (2007).
Cuthbert, A.P. et al. Construction and characterization of a highly stable human: rodent monochromosomal hybrid panel for genetic complementation and genome mapping studies. Cytogenet. Cell Genet. 71, 68–76 (1995).
Watkins, D., Matiaszuk, N. & Rosenblatt, D.S. Complementation studies in the cblA class of inborn error of cobalamin metabolism: evidence for interallelic complementation and for a new complementation class (cblH). J. Med. Genet. 37, 510–513 (2000).
Wang, Y.H. et al. Novel nuclear export signal-interacting protein, NESI, critical for the assembly of hepatitis delta virus. J. Virol. 79, 8113–8120 (2005).
Clark, R.M., Marker, P.C. & Kingsley, D.M. A novel candidate gene for mouse and human preaxial polydactyly with altered expression in limbs of Hemimelic extra-toes mutant mice. Genomics 67, 19–27 (2000).
Wojnar, P., Lechner, M., Merschak, P. & Redl, B. Molecular cloning of a novel lipocalin-1 interacting human cell membrane receptor using phage display. J. Biol. Chem. 276, 20206–20212 (2001).
Lettice, L.A. et al. Disruption of a long-range cis-acting regulator for Shh causes preaxial polydactyly. Proc. Natl. Acad. Sci. USA 99, 7548–7553 (2002).
Flower, D.R. The lipocalin protein family: structure and function. Biochem. J. 318, 1–14 (1996).
Fluckinger, M., Merschak, P., Hermann, M., Haertle, T. & Redl, B. Lipocalin-interacting-membrane-receptor (LIMR) mediates cellular internalization of β-lactoglobulin. Biochim. Biophys. Acta 1778, 342–347 (2008).
Wojnar, P., Lechner, M. & Redl, B. Antisense down-regulation of lipocalin-interacting membrane receptor expression inhibits cellular internalization of lipocalin-1 in human NT2 cells. J. Biol. Chem. 278, 16209–16215 (2003).
Dufour, E., Marden, M.C. & Haertle, T. β-lactoglobulin binds retinol and protoporphyrin IX at two different binding sites. FEBS Lett. 277, 223–226 (1990).
Schuh, S. et al. Homocystinuria and megaloblastic anemia responsive to vitamin B12 therapy. An inborn error of metabolism due to a defect in cobalamin metabolism. N. Engl. J. Med. 310, 686–690 (1984).
Abecasis, G.R., Cherny, S.S., Cookson, W.O. & Cardon, L.R. GRR: graphical representation of relationship errors. Bioinformatics 17, 742–743 (2001).
Abecasis, G.R., Cherny, S.S., Cookson, W.O. & Cardon, L.R. Merlin–rapid analysis of dense genetic maps using sparse gene flow trees. Nat. Genet. 30, 97–101 (2002).
Thiele, H. & Nürnberg, P. HaploPainter: a tool for drawing pedigrees with complex haplotypes. Bioinformatics 21, 1730–1732 (2005).
Rüschendorf, F. & Nürnberg, P. ALOHOMORA: a tool for linkage analysis using 10K SNP array data. Bioinformatics 21, 2123–2125 (2005).
Litzkas, P., Jha, K.K. & Ozer, H.L. Efficient transfer of cloned DNA into human diploid cells: protoplast fusion in suspension. Mol. Cell. Biol. 4, 2549–2552 (1984).
Tokuyasu, K.T. Immunocytochemistry on ultrathin frozen section. Histochem. J. 12, 381–403 (1980).
Robenek, H. & Severs, N.J. Recent advances in freeze-fracture electron microscopy: the replica immunolabeling technique. Biol. Proced. Online 10, 9–19 (2008).
Robenek, H., Buers, I., Hofnagel, O., Lorkowski, S. & Severs, N.J. GFP-tagged proteins visualized by freeze-fracture immuno-electron microscopy: a new tool in cellular and molecular medicine. J. Cell. Mol. Med. advance online publication, doi:10.1111/j.1582–4934.2008.00407.x (27 June 2008).
Acknowledgements
We thank the following individuals who provided subject samples and/or clinical information: D. Applegarth, J. Bever, S. Cederbaum, G. Davidson, B.S. Dowton, G. Hoganson, R. Laframboise, U. Lichter, M. Lindner, M. Seashore, V. Shih, G.P.A. Smit, J. Vockley and H.E. Wiltse. We thank E. Kirst, C. Kluck and U. Botschen for expert technical assistance. F.R. and S.G. were supported by the fund “Innovative Medical Research” of University of Münster Medical School. B.G. and C.S. were supported by the Centre National de la Recherche Scientifique, the Institut national de la santé et de la recherche médicale and the Agence Nationale de la Recherche - Programme Pluriannuel de Recherche sur les Maladies Rares. I.R.M. was supported by a studentship from the Fonds de la Recherche en Santé du Québec. D.S.R. and I.R.M. were supported by funds from the Canadian Institutes of Health Research and the Hess B. and Diane Finestone Laboratory in memory of Jacob and Jenny Finestone. Financial support was further given by the Swiss National Foundation and the German Federal Ministry of Education and Research through the National Genome Research Network.
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F.R. and P.N. designed the study, analyzed the data and wrote the manuscript. S.G. designed primers, collected data, performed sequencing analysis, LMBD1 expression analysis and biochemical rescue experiments. I.R.M. collected subject information and performed microcell-mediated chromosome transfer. T.S. performed biochemical rescue experiments with the wild-type construct. C.S., I.B., A.S., H.R. and B.G. analyzed the subcellular localization of the tagged construct. M.R.T. performed pyrosequencing of the control chromosomes. G.N. performed the bioinformatic analysis and analyzed the haplotype data. T.W. performed sequencing analysis and edited the manuscript. M.S. and M.B. cloned the construct. C.B. performed the genome scan. T.M., B.G., D.S.R. and B.F. were involved in study design. W.H. and B.G. analyzed the structure and homology of LMBD1. All authors discussed the results and commented on the manuscript.
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Rutsch, F., Gailus, S., Miousse, I. et al. Identification of a putative lysosomal cobalamin exporter altered in the cblF defect of vitamin B12 metabolism. Nat Genet 41, 234–239 (2009). https://doi.org/10.1038/ng.294
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DOI: https://doi.org/10.1038/ng.294
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