Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Mutations in ABCD4 cause a new inborn error of vitamin B12 metabolism


Inherited disorders of vitamin B12 (cobalamin) have provided important clues to how this vitamin, which is essential for hematological and neurological function, is transported and metabolized. We describe a new disease that results in failure to release vitamin B12 from lysosomes, which mimics the cblF defect caused by LMBRD1 mutations. Using microcell-mediated chromosome transfer and exome sequencing, we identified causal mutations in ABCD4, a gene that codes for an ABC transporter, which was previously thought to have peroxisomal localization and function. Our results show that ABCD4 colocalizes with the lysosomal proteins LAMP1 and LMBD1, the latter of which is deficient in the cblF defect. Furthermore, we show that mutations altering the putative ATPase domain of ABCD4 affect its function, suggesting that the ATPase activity of ABCD4 may be involved in intracellular processing of vitamin B12.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Figure 1: Distribution of free and protein-bound cobalamin in cell lines from controls and affected individuals.
Figure 2: Somatic complementation analysis confirms a new cobalamin complementation group cblJ.
Figure 3: Expression of wild-type and mutant ABCD4 alleles.
Figure 4: ABCD4 is a membrane protein with ATPase function.
Figure 5: Subcellular localization of ABCD4 detected by fluorescence and confocal microscopy.

Accession codes


NCBI Reference Sequence


  1. Shani, N., Jimenez-Sanchez, G., Steel, G., Dean, M. & Valle, D. Identification of a fourth half ABC transporter in the human peroxisomal membrane. Hum. Mol. Genet. 6, 1925–1931 (1997).

    Article  CAS  Google Scholar 

  2. Coelho, D. et al. Gene identification for the cblD defect of vitamin B12 metabolism. N. Engl. J. Med. 358, 1454–1464 (2008).

    Article  CAS  Google Scholar 

  3. Rutsch, F. et al. Identification of a putative lysosomal cobalamin exporter altered in the cblF defect of vitamin B12 metabolism. Nat. Genet. 41, 234–239 (2009).

    Article  CAS  Google Scholar 

  4. 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).

    Article  CAS  Google Scholar 

  5. Adzhubei, I.A. et al. A method and server for predicting damaging missense mutations. Nat. Methods 7, 248–249 (2010).

    Article  CAS  Google Scholar 

  6. Wanders, R.J., Visser, W.F., van Roermund, C.W., Kemp, S. & Waterham, H.R. The peroxisomal ABC transporter family. Eur. J. Physiol. 453, 719–734 (2007).

    Article  CAS  Google Scholar 

  7. Kemp, S. et al. ABCD1 mutations and the X-linked adrenoleukodystrophy mutation database: role in diagnosis and clinical correlations. Hum. Mutat. 18, 499–515 (2001).

    Article  CAS  Google Scholar 

  8. Matsukawa, T. et al. Identification of novel SNPs of ABCD1, ABCD2, ABCD3, and ABCD4 genes in patients with X-linked adrenoleukodystrophy (ALD) based on comprehensive resequencing and association studies with ALD phenotypes. Neurogenetics 12, 41–50 (2011).

    Article  CAS  Google Scholar 

  9. Kashiwayama, Y. et al. 70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) localizes to endoplasmic reticulum not peroxisomes, and NH2-terminal hydrophobic property determines the subcellular localization of ABC subfamily D proteins. Exp. Cell Res. 315, 190–205 (2009).

    Article  CAS  Google Scholar 

  10. Seeger, M.A. & van Veen, H.W. Molecular basis of multidrug transport by ABC transporters. Biochim. Biophys. Acta 1794, 725–737 (2009).

    Article  CAS  Google Scholar 

  11. Rost, B., Yachdav, G. & Liu, J. The PredictProtein server. Nucleic Acids Res. 32, W321–W326 (2004).

    Article  CAS  Google Scholar 

  12. Kikuchi, M. et al. Proteomic analysis of rat liver peroxisome: presence of peroxisome-specific isozyme of Lon protease. J. Biol. Chem. 279, 421–428 (2004).

    Article  CAS  Google Scholar 

  13. Islinger, M., Lüers, G.H., Li, K.W., Loos, M. & Völkl, A. Rat liver peroxisomes after fibrate treatment. A survey using quantitative mass spectrometry. J. Biol. Chem. 282, 23055–23069 (2007).

    Article  CAS  Google Scholar 

  14. Wiese, S. et al. Proteomics characterization of mouse kidney peroxisomes by tandem mass spectrometry and protein correlation profiling. Mol. Cell. Proteomics 6, 2045–2057 (2007).

    Article  CAS  Google Scholar 

  15. Gloeckner, C.J. et al. Human adrenoleukodystrophy protein and related peroxisomal ABC transporters interact with the peroxisomal assembly protein PEX19p. Biochem. Biophys. Res. Commun. 271, 144–150 (2000).

    Article  CAS  Google Scholar 

  16. Borths, E.L., Poolman, B., Hvorup, R.N., Locher, K.P. & Rees, D.C. In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B12 uptake. Biochemistry 44, 16301–16309 (2005).

    Article  CAS  Google Scholar 

  17. Beedholm-Ebsen, R. et al. Identification of multidrug resistance protein 1 (MRP1/ABCC1) as a molecular gate for cellular export of cobalamin. Blood 115, 1632–1639 (2010).

    Article  CAS  Google Scholar 

  18. Verrier, P.J. et al. Plant ABC proteins—a unified nomenclature and updated inventory. Trends Plant Sci. 13, 151–159 (2008).

    Article  CAS  Google Scholar 

  19. Aittoniemi, J. et al. SUR1: a unique ATP-binding cassette protein that functions as an ion channel regulator. Phil. Trans. R. Soc. Lond. B 364, 257–267 (2009).

    Article  CAS  Google Scholar 

  20. 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).

    Article  CAS  Google Scholar 

  21. Watkins, D. Cobalamin metabolism in methionine-dependent human tumour and leukemia cell lines. Clin. Invest. Med. 21, 151–158 (1998).

    CAS  PubMed  Google Scholar 

  22. Yao, J. & Shoubridge, E.A. Expression and functional analysis of SURF1 in Leigh syndrome patients with cytochrome c oxidase deficiency. Hum. Mol. Genet. 8, 2541–2549 (1999).

    Article  CAS  Google Scholar 

  23. 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).

    Article  CAS  Google Scholar 

  24. Alfares, A. et al. Combined malonic and methylmalonic aciduria: exome sequencing reveals mutations in the ACSF3 gene in patients with a non-classic phenotype. J. Med. Genet. 48, 602–605 (2011).

    Article  CAS  Google Scholar 

  25. Li, H., Ruan, J. & Durbin, R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 18, 1851–1858 (2008).

    Article  CAS  Google Scholar 

  26. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  Google Scholar 

  27. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  28. 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).

    Article  CAS  Google Scholar 

  29. Hunt, J.D. Evaluation of phenotypic alteration by microcell-mediated chromosome transfer. Anal. Biochem. 238, 107–116 (1996).

    Article  CAS  Google Scholar 

  30. Fowler, B. & Jakobs, C. Post- and prenatal diagnostic methods for the homocystinurias. Eur. J. Pediatr. 157, S88–S93 (1998).

    Article  Google Scholar 

  31. Miousse, I.R. et al. Clinical and molecular heterogeneity in patients with the cblD inborn error of cobalamin metabolism. J. Pediatr. 154, 551–556 (2009).

    Article  CAS  Google Scholar 

  32. Fowler, B., Whitehouse, C., Wenzel, F. & Wraith, J.E. Methionine and serine formation in control and mutant human cultured fibroblasts: evidence for methyl trapping and characterization of remethylation defects. Pediatr. Res. 41, 145–151 (1997).

    Article  CAS  Google Scholar 

  33. Weraarpachai, W. et al. Mutation in TACO1, encoding a translational activator of COX I, results in cytochrome c oxidase deficiency and late-onset Leigh syndrome. Nat. Genet. 41, 833–837 (2009).

    Article  CAS  Google Scholar 

  34. Lerner-Ellis, J.P. et al. Identification of the gene responsible for methylmalonic aciduria and homocystinuria, cblC type. Nat. Genet. 38, 93–100 (2006).

    Article  CAS  Google Scholar 

  35. Stucki, M. et al. Molecular mechanisms leading to three different phenotypes in the cblD defect of intracellular cobalamin metabolism. Hum. Mol. Genet. 21, 1410–1418 (2012).

    Article  CAS  Google Scholar 

Download references


We thank S. Lutz for technical assistance in carrying out enzyme assays and K. Locher for fruitful discussions. We thank J. Gärtner (University of Göttingen) for providing cell lines with peroxisomal defects and R. Newbold (Brunel University) for providing the mouse-human monochromosomal hybrid cell lines used for microcell-mediated chromosome transfer. This work was supported by the Swiss National Science Foundation (320000_122568 and 31003A_138521) and by the Deutsche Forschungsgemeinschaft (RU816/5-1). D.S.R. is supported by the Canadian Institutes for Health Research (15078).

Author information

Authors and Affiliations



M.R.B., F.R., B.F. and D.S.R. supervised the project. M.R.B., F.R., B.F., D.S.R., J.C.K., T.S. and D.C. designed the study, analyzed the data and wrote the manuscript. T.S., M.d.M. and D.C. performed microcell-mediated chromosome transfer. M.S., D.C. and M.F. designed primers and built constructs. M.d.M., I.B., J.C.K., I.R.M., D.W. and D.C. performed sequencing analysis. J.M., P.N. and H.T. performed whole-exome sequencing. S.F. and E.A.S. performed immortalization and stable transduction of fibroblasts. T.S., P.B., J.C.K. and D.W. performed somatic cell complementation, enzymatic assays, Cbl coenzyme synthesis and expression studies. H.R., I.B. and D.C. analyzed the subcellular localization of ABCD4. D.C. and P.B. performed protein blots. W.H. and D.C. analyzed the structure of ABCD4. N.L., M.P. and E.M. provided clinical information about the affected individuals. All authors discussed the results and reviewed the manuscript.

Corresponding authors

Correspondence to David S Rosenblatt or Brian Fowler.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2, Supplementary Tables 1–7 and Supplementary Note (PDF 126 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Coelho, D., Kim, J., Miousse, I. et al. Mutations in ABCD4 cause a new inborn error of vitamin B12 metabolism. Nat Genet 44, 1152–1155 (2012).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing