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

Journal name:
Nature Genetics
Year published:
Published online


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.

At a glance


  1. Schematic overview of the Mito10K project.
    Figure 1: Schematic overview of the Mito10K project.
  2. Definition of 'likely deleterious' variants detected in pooled sequencing screen.
    Figure 2: Definition of 'likely deleterious' variants detected in pooled sequencing screen.

    (a) Barplot of high-confidence and low-confidence variants, categorized by predicted deleterious consequences. (b) Histogram of known disease-associated splice variants, annotated in HGMD24, by position relative to nearest splice donor and splice acceptor exons (black rectangles). Dashed line indicates frequency threshold and asterisk indicates splice positions considered 'likely deleterious'. (c) Histogram of amino acid conservation score (no. species with identical amino acid, out of 44 aligned vertebrate exons) shown for training data: missense variants annotated as disease-associated in HGMD (red curve) or present in dbSNP128 (blue curve). Dashed line indicates minimum conservation required for 'likely deleterious' variants.

  3. Sixty individuals with complex I deficiency without a previous genetic diagnosis, categorized by type of 'likely deleterious' variants detected per gene.
    Figure 3: Sixty individuals with complex I deficiency without a previous genetic diagnosis, categorized by type of 'likely deleterious' variants detected per gene.

    Red indicates individuals with pathogenic variants, blue indicates individuals with variants of uncertain significance (VUS) and gray indicates individuals without 'likely deleterious' variants. Boxes list genes containing 'likely deleterious' variants in each subject. Black arrowheads indicate new experimentally established genetic diagnoses. a,bPairs of affected siblings.

  4. NUBPL and FOXRED1 cDNA rescue of complex I defects in subject fibroblasts.
    Figure 4: NUBPL and FOXRED1 cDNA rescue of complex I defects in subject fibroblasts.

    (a,b) Barplots show complex I activity (CI), normalized by complex IV activity (CIV), measured in control and subject fibroblasts, before and after transduction with wild-type NUBPL-V5 mRNA (a) or wild-type FOXRED1-V5 mRNA (b). Data shown are mean of three biological replicates ± s.e.m. *P < 0.01. Representative dipstick assays shown below.

  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.
    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.

    Red indicates individuals with confirmed genetic diagnosis, and gray indicates absence of genetic diagnosis. Subjects are a representative cohort, selected as all unrelated individuals within the 103 individuals sequenced.


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Author information

  1. These authors contributed equally to this work.

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


  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


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.

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The authors declare no competing financial interests.

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Supplementary information

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

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  1. Supplementary Table 2 (104K)

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

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