Skip to main content

Thank you for visiting nature.com. 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.

  • Article
  • Published:

High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency

Abstract

Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes that are involved. We report a strategy of focused candidate gene prediction, high-throughput sequencing and experimental validation to uncover the molecular basis of mitochondrial complex I disorders. We created seven pools of DNA from a cohort of 103 cases and 42 healthy controls and then performed deep sequencing of 103 candidate genes to identify 151 rare variants that were predicted to affect protein function. We established genetic diagnoses in 13 of 60 previously unsolved cases using confirmatory experiments, including cDNA complementation to show that mutations in NUBPL and FOXRED1 can cause complex I deficiency. Our study illustrates how large-scale sequencing, coupled with functional prediction and experimental validation, can be used to identify causal mutations in individual cases.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1
Figure 2: Definition of 'likely deleterious' variants detected in pooled sequencing screen.
Figure 3: Sixty individuals with complex I deficiency without a previous genetic diagnosis, categorized by type of 'likely deleterious' variants detected per gene.
Figure 4: NUBPL and FOXRED1 cDNA rescue of complex I defects in subject fibroblasts.
Figure 5: Genetic diagnosis of 94 unrelated individuals with definite, isolated complex I deficiency grouped by function of underlying gene and location in the mitochondrial (mtDNA) or nuclear (nDNA) genome.

Similar content being viewed by others

References

  1. Skladal, D., Halliday, J. & Thorburn, D.R. Minimum birth prevalence of mitochondrial respiratory chain disorders in children. Brain 126, 1905–1912 (2003).

    Article  Google Scholar 

  2. Distelmaier, F. et al. Mitochondrial complex I deficiency: from organelle dysfunction to clinical disease. Brain 132, 833–842 (2009).

    Article  Google Scholar 

  3. Janssen, R.J., Nijtmans, L.G., van den Heuvel, L.P. & Smeitink, J.A. Mitochondrial complex I: structure, function and pathology. J. Inherit. Metab. Dis. 29, 499–515 (2006).

    Article  CAS  Google Scholar 

  4. Lazarou, M., Thorburn, D.R., Ryan, M.T. & McKenzie, M. Assembly of mitochondrial complex I and defects in disease. Biochim. Biophys. Acta 1793, 78–88 (2009).

    Article  CAS  Google Scholar 

  5. Bernier, F.P. et al. Diagnostic criteria for respiratory chain disorders in adults and children. Neurology 59, 1406–1411 (2002).

    Article  CAS  Google Scholar 

  6. Morava, E. et al. Mitochondrial disease criteria: diagnostic applications in children. Neurology 67, 1823–1826 (2006).

    Article  CAS  Google Scholar 

  7. McFarland, R. et al. De novo mutations in the mitochondrial ND3 gene as a cause of infantile mitochondrial encephalopathy and complex I deficiency. Ann. Neurol. 55, 58–64 (2004).

    Article  CAS  Google Scholar 

  8. Dimauro, S. & Davidzon, G. Mitochondrial DNA and disease. Ann. Med. 37, 222–232 (2005).

    Article  CAS  Google Scholar 

  9. Fontanesi, F., Soto, I.C., Horn, D. & Barrientos, A. Assembly of mitochondrial cytochrome c-oxidase, a complicated and highly regulated cellular process. Am. J. Physiol. Cell Physiol. 291, C1129–C1147 (2006).

    Article  CAS  Google Scholar 

  10. Bénit, P. et al. Genotyping microsatellite DNA markers at putative disease loci in inbred/multiplex families with respiratory chain complex I deficiency allows rapid identification of a novel nonsense mutation (IVS1nt −1) in the NDUFS4 gene in Leigh syndrome. Hum. Genet. 112, 563–566 (2003).

    PubMed  Google Scholar 

  11. Bugiani, M. et al. Clinical and molecular findings in children with complex I deficiency. Biochim. Biophys. Acta 1659, 136–147 (2004).

    Article  CAS  Google Scholar 

  12. Lebon, S. et al. Recurrent de novo mitochondrial DNA mutations in respiratory chain deficiency. J. Med. Genet. 40, 896–899 (2003).

    Article  CAS  Google Scholar 

  13. Smeitink, J., Sengers, R., Trijbels, F. & van den Heuvel, L. Human NADH:ubiquinone oxidoreductase. J. Bioenerg. Biomembr. 33, 259–266 (2001).

    Article  CAS  Google Scholar 

  14. Pagliarini, D.J. et al. A mitochondrial protein compendium elucidates complex I disease biology. Cell 134, 112–123 (2008).

    Article  CAS  Google Scholar 

  15. Marcotte, E.M., Pellegrini, M., Thompson, M.J., Yeates, T.O. & Eisenberg, D. A combined algorithm for genome-wide prediction of protein function. Nature 402, 83–86 (1999).

    Article  CAS  Google Scholar 

  16. Pellegrini, M., Marcotte, E.M., Thompson, M.J., Eisenberg, D. & Yeates, T.O. Assigning protein functions by comparative genome analysis: protein phylogenetic profiles. Proc. Natl. Acad. Sci. USA 96, 4285–4288 (1999).

    Article  CAS  Google Scholar 

  17. Ogilvie, I., Kennaway, N.G. & Shoubridge, E.A. A molecular chaperone for mitochondrial complex I assembly is mutated in a progressive encephalopathy. J. Clin. Invest. 115, 2784–2792 (2005).

    Article  CAS  Google Scholar 

  18. Saada, A. et al. Mutations in NDUFAF3 (C3ORF60), encoding an NDUFAF4 (C6ORF66)-interacting complex I assembly protein, cause fatal neonatal mitochondrial disease. Am. J. Hum. Genet. 84, 718–727 (2009).

    Article  CAS  Google Scholar 

  19. Sugiana, C. et al. Mutation of C20orf7 disrupts complex I assembly and causes lethal neonatal mitochondrial disease. Am. J. Hum. Genet. 83, 468–478 (2008).

    Article  CAS  Google Scholar 

  20. Bentley, D.R. et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59 (2008).

    Article  CAS  Google Scholar 

  21. Frazer, K.A. et al. A second generation human haplotype map of over 3.1 million SNPs. Nature 449, 851–861 (2007).

    Article  CAS  Google Scholar 

  22. Sherry, S.T. et al. dbSNP: the NCBI database of genetic variation. Nucleic Acids Res. 29, 308–311 (2001).

    Article  CAS  Google Scholar 

  23. Ingman, M. & Gyllensten, U. mtDB: Human Mitochondrial Genome Database, a resource for population genetics and medical sciences. Nucleic Acids Res. 34, D749–D751 (2006).

    Article  CAS  Google Scholar 

  24. Stenson, P.D. et al. The Human Gene Mutation Database: 2008 update. Genome Med 1, 13 (2009).

    Article  Google Scholar 

  25. Kirby, D.M. et al. NDUFS6 mutations are a novel cause of lethal neonatal mitochondrial complex I deficiency. J. Clin. Invest. 114, 837–845 (2004).

    Article  CAS  Google Scholar 

  26. Valente, L. et al. Identification of novel mutations in five patients with mitochondrial encephalomyopathy. Biochim. Biophys. Acta 1787, 491–501 (2009).

    Article  CAS  Google Scholar 

  27. Budde, S.M. et al. Combined enzymatic complex I and III deficiency associated with mutations in the nuclear encoded NDUFS4 gene. Biochem. Biophys. Res. Commun. 275, 63–68 (2000).

    Article  CAS  Google Scholar 

  28. Leshinsky-Silver, E. et al. NDUFS4 mutations cause Leigh syndrome with predominant brainstem involvement. Mol. Genet. Metab. 97, 185–189 (2009).

    Article  CAS  Google Scholar 

  29. Petruzzella, V. et al. A nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit of complex I abolishes assembly and activity of the complex in a patient with Leigh-like syndrome. Hum. Mol. Genet. 10, 529–535 (2001).

    Article  CAS  Google Scholar 

  30. Anderson, S.L. et al. A novel mutation in NDUFS4 causes Leigh syndrome in an Ashkenazi Jewish family. J. Inherit. Metab. Dis. 32, 121 (2009).

    Google Scholar 

  31. van den Heuvel, L. et al. Demonstration of a new pathogenic mutation in human complex I deficiency: a 5-bp duplication in the nuclear gene encoding the 18-kD (AQDQ) subunit. Am. J. Hum. Genet. 62, 262–268 (1998).

    Article  CAS  Google Scholar 

  32. Schuelke, M. et al. Mutant NDUFV1 subunit of mitochondrial complex I causes leukodystrophy and myoclonic epilepsy. Nat. Genet. 21, 260–261 (1999).

    Article  CAS  Google Scholar 

  33. Loeffen, J. et al. The first nuclear-encoded complex I mutation in a patient with Leigh syndrome. Am. J. Hum. Genet. 63, 1598–1608 (1998).

    Article  CAS  Google Scholar 

  34. McKenzie, M., Lazarou, M., Thorburn, D.R. & Ryan, M.T. Mitochondrial respiratory chain supercomplexes are destabilized in Barth Syndrome patients. J. Mol. Biol. 361, 462–469 (2006).

    Article  CAS  Google Scholar 

  35. Choi, M. et al. Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc. Natl. Acad. Sci. USA 106, 19096–19101 (2009).

    Article  CAS  Google Scholar 

  36. Ng, S.B. et al. Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461, 272–276 (2009).

    Article  CAS  Google Scholar 

  37. Ng, S.B. et al. Exome sequencing identifies the cause of a mendelian disorder. Nat. Genet. 42, 30–35 (2010).

    Article  CAS  Google Scholar 

  38. Sheftel, A.D. et al. Human ind1, an iron-sulfur cluster assembly factor for respiratory complex I. Mol. Cell. Biol. 29, 6059–6073 (2009).

    Article  CAS  Google Scholar 

  39. Bych, K. et al. The iron-sulphur protein Ind1 is required for effective complex I assembly. EMBO J. 27, 1736–1746 (2008).

    Article  CAS  Google Scholar 

  40. Calvo, S. et al. Systematic identification of human mitochondrial disease genes through integrative genomics. Nat. Genet. 38, 576–582 (2006).

    Article  CAS  Google Scholar 

  41. Tarpey, P.S. et al. A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation. Nat. Genet. 41, 535–543 (2009).

    Article  CAS  Google Scholar 

  42. Kirby, D.M. et al. Respiratory chain complex I deficiency: an underdiagnosed energy generation disorder. Neurology 52, 1255–1264 (1999).

    Article  CAS  Google Scholar 

  43. Gnirke, A. et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat. Biotechnol. 27, 182–189 (2009).

    Article  CAS  Google Scholar 

  44. 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 

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

    Article  Google Scholar 

  46. Calvo, S.E., Pagliarini, D.J. & Mootha, V.K. Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. Proc. Natl. Acad. Sci. USA 106, 7507–7512 (2009).

    Article  CAS  Google Scholar 

  47. Karolchik, D., Hinrichs, A.S. & Kent, W.J. The UCSC Genome Browser. Curr. Protoc. Bioinformatics Chapter 1: Unit 1.4 (2009).

  48. Dimmic, M.W., Sunyaev, S. & Bustamante, C.D. Inferring SNP function using evolutionary, structural, and computational methods. Pac. Symp. Biocomput. 382–384 (2005).

  49. Cree, L.M., Samuels, D.C. & Chinnery, P.F. The inheritance of pathogenic mitochondrial DNA mutations. Biochim. Biophys. Acta 1792, 1097–1102 (2009).

    Article  CAS  Google Scholar 

  50. Gabriel, S., Ziaugra, L. & Tabbaa, D. SNP genotyping using the Sequenom MassARRAY iPLEX platform. Curr. Protoc. Hum. Genet. Chapter 2: Unit 2.12 (2009).

  51. Lamandé, S.R. et al. Reduced collagen VI causes Bethlem myopathy: a heterozygous COL6A1 nonsense mutation results in mRNA decay and functional haploinsufficiency. Hum. Mol. Genet. 7, 981–989 (1998).

    Article  Google Scholar 

Download references

Acknowledgements

We thank S. Tregoning, A. Laskowski and S. Smith for assistance with enzyme assays and DNA preparation, M. McKenzie and M. Ryan for the NDUFAF2 antibody, J. Boehm for the lentiviral expression vector, S. Flynn for assistance with human subjects protocols, R. Onofrio for designing PCR primers, K. Ardlie and S. Mahan for assistance in DNA sample preparation, J. Wilkinson and L. Ambrogio for Illumina sequence project management, T. Fennel for sequence alignment, L. Ziaugra for genotyping assistance, M. Cabili for tool evaluation, J. Flannick for assistance with pooled sequence analysis, I. Adzhubei and S. Sunyaev for PolyPhen-2.0 predictions, M. DePristo, E. Banks and A. Sivachenko for advice on sequence data analysis, M. Garber for assistance with evolutionary conservation analyses, J. Pirruccello, R. Do and S. Kathiresan for data and analysis of control data, and the many physicians who referred subjects and assisted with these studies. This work was supported by a grant (436901) and Principal Research Fellowship from the Australian National Health & Medical Research Council awarded to D.R.T., an Australian Postgraduate Award to E.J.T. and a grant from the US National Institutes of Health (GM077465) to V.K.M. The authors wish to dedicate this article to the memory of our co-author Denise Kirby, an outstanding scientist and dear colleague who died during the preparation of this manuscript.

Author information

Authors and Affiliations

Authors

Contributions

This study was conceived and designed by S.E.C., D.R.T. and V.K.M. with input from M.J.D. and S.B.G. Enzyme diagnosis of the cohort was coordinated by D.M.K. E.W. and C.J.W. provided clinical interaction and assisted with sample collection. Samples were collected by D.M.K., E.W. and C.J.W. and prepared by A.G.C. and E.J.T. The pooled sequencing protocol was designed and established at the Broad Institute by D.A., M.J.D. and S.B.G. Project management was performed by S.E.C., N.P.B. and C.G. G.C. performed pooling. M.C.R. and C.G. performed the genotyping. S.E.C. designed and performed the computational analyses, with assistance from E.J.T., A.G.C. and M.R. All experiments were designed and performed by E.J.T., A.G.C. and O.A.G. Affymetrix array-based cytogenetic analysis was performed by D.L.B. Syzygy was developed and run by M.R. and M.J.D. The manuscript was written by S.E.C., E.J.T., A.G.C., D.R.T. and V.K.M. All aspects of the study were supervised by D.R.T. and V.K.M.

Corresponding authors

Correspondence to David R Thorburn or Vamsi K Mootha.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Tables 1, 3–5 and Supplementary Note (PDF 853 kb)

Supplementary Table 2

Likely deleterious variants detected and validated in 103 patients with Complex I deficiency (XLS 103 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Calvo, S., Tucker, E., Compton, A. et al. High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency. Nat Genet 42, 851–858 (2010). https://doi.org/10.1038/ng.659

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.659

This article is cited by

Search

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