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

Nature Genetics volume 42, pages 851858 (2010) | Download Citation


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.

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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

Author notes

    • Sarah E Calvo
    • , Elena J Tucker
    •  & Alison G Compton

    These authors contributed equally to this work.


  1. Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Sarah E Calvo
    • , Manuel Rivas
    • , Olga A Goldberger
    • , David Altshuler
    • , Mark J Daly
    •  & Vamsi K Mootha
  2. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.

    • Sarah E Calvo
    • , Olga A Goldberger
    •  & Vamsi K Mootha
  3. Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.

    • Sarah E Calvo
    • , Gabriel Crawford
    • , Noel P Burtt
    • , Manuel Rivas
    • , Candace Guiducci
    • , Michelle C Redman
    • , David Altshuler
    • , Stacey B Gabriel
    • , Mark J Daly
    •  & Vamsi K Mootha
  4. Murdoch Childrens Research Institute and Victorian Clinical Genetics Services, Royal Children's Hospital, Melbourne, Victoria, Australia.

    • Elena J Tucker
    • , Alison G Compton
    • , Denise M Kirby
    • , Damien L Bruno
    •  & David R Thorburn
  5. Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia.

    • Elena J Tucker
    •  & David R Thorburn
  6. Department of Paediatrics and Child Health, University of Otago Wellington, Wellington, New Zealand.

    • Esko Wiltshire
  7. Central Regional Genetics Service, Capital and Coast District Health Board, Wellington, New Zealand.

    • Esko Wiltshire
  8. National Metabolic Service, Starship Children's Hospital, Auckland, New Zealand.

    • Callum J Wilson
  9. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • David Altshuler


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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to David R Thorburn or Vamsi K Mootha.

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    Supplementary Figures 1–10, Supplementary Tables 1, 3–5 and Supplementary Note

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    Supplementary Table 2

    Likely deleterious variants detected and validated in 103 patients with Complex I deficiency

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