Mutations in TTC19 cause mitochondrial complex III deficiency and neurological impairment in humans and flies

Journal name:
Nature Genetics
Year published:
Published online

Although mutations in CYTB (cytochrome b) or BCS1L have been reported in isolated defects of mitochondrial respiratory chain complex III (cIII), most cIII-defective individuals remain genetically undefined. We identified a homozygous nonsense mutation in the gene encoding tetratricopeptide 19 (TTC19) in individuals from two families affected by progressive encephalopathy associated with profound cIII deficiency and accumulation of cIII-specific assembly intermediates. We later found a second homozygous nonsense mutation in a fourth affected individual. We demonstrated that TTC19 is embedded in the inner mitochondrial membrane as part of two high–molecularweight complexes, one of which coincides with cIII. We then showed a physical interaction between TTC19 and cIII by coimmunoprecipitation. We also investigated a Drosophila melanogaster knockout model for TTC19 that showed low fertility, adult-onset locomotor impairment and bang sensitivity, associated with cIII deficiency. TTC19 is a putative cIII assembly factor whose disruption is associated with severe neurological abnormalities in humans and flies.

At a glance


  1. Clinical and molecular genetic features of individuals with mutations in TTC19.
    Figure 1: Clinical and molecular genetic features of individuals with mutations in TTC19.

    (a) Brain magnetic resonance image of subject 1. I indicates a transverse T2-weighted image of the cerebellum and medulla oblongata. The arrows indicate bilateral hyperintense signals in the inferior olives. II indicates a transverse T2-weighted image of the supratentorial brain. The arrow shows a hyperintense signal in the right putamen. III indicates a median sagittal T1-weighted image showing atrophy of the cerebral cortex and cerebellar vermis (arrow). IV indicates a coronal T1-weighted image showing brain atrophy with dilation of the lateral ventricles, atrophy of the caudate nuclei and bilateral hyperintensities of the substantia nigra (arrows). (b) Oxygen consumption rate (OCR) of patients fibroblasts (Fb, left panel) and myoblasts (Mb, right panel) before and after (+TTC19) re-expression of wild-type TTC19. Bars indicate the standard deviation for 8–16 technical replicates. *P < 0.05, **P < 0.01. (P values for unpaired, two-tailed Student's t-test). (c) Quantitative real-time PCR of TTC19 mRNA relative to GAPDH mRNA in affected and control (Ct) fibroblasts (Fb), myoblasts (Mb) and muscle biopsies (Ms). P1, P2, P3 and P4 correspond to subjects 1, 2, 3 and 4, respectively. Each value refers to the mean of three independent experiments performed in duplicate. Bars indicate the standard deviation. (d) Immunoblot analysis of total lysates from myoblasts and fibroblasts using α-TTC19, α-Core1 and α-SDHA antibodies. P1, P2 and P3 correspond to subjects 1, 2 and 3. Fb BCS1L indicate fibroblasts of a subject with cIII deficiency due to mutations in BCS1L.

  2. TTC19 subcellular localization.
    Figure 2: TTC19 subcellular localization.

    (a) Confocal immunofluorescence of COS7 cells transfected with TTC19HA. The pattern of the green signal, corresponding to TTCHA, coincides with that of the red signal, corresponding to MitoTracker, a mitochondrial marker, in transfected cells, producing a confocalized image in yellow. Scale bars, 25 μm. (b) Import assay on isolated HeLa cell mitochondria. The in vitro translated TTC protein (lane 1) is partially imported and cleaved in the presence of freshly prepared, fully coupled, energized mitochondria (lane 2). The mature protein species is internalized within the mitochondria, whereas the precursor protein species that remains outside mitochondria is digested by proteinase K (PK) (lane 3). Both mature and precursor TTC19 proteins are digested when the mitochondria are solubilized in Triton-X100 (lane 4). The import is dependent on the integrity of ΔΨ, as no mature protein is formed when ΔΨ is dissipated by valinomycin (lane 5) and is completely digested by PK (lane 6). (c) Immunoblot analysis of HeLa cell fractions. ivT, in vitro translated TTC19; Mt, mitochondria; L, cell lysate; PMF, post-mitochondrial fraction; GAPDH (a cytosolic protein) and Core1 (a mitochondrial protein) were used as controls. (d) Immunoblot analysis of mouse liver mitochondria after treatment with detergent deoxycholate (DOC) or Na2CO3. Mt, mitochondrial fraction; MM, mitochondrial matrix, Mb, mitochondrial membrane fraction. HSP60 (mitochondrial matrix protein) and Core1 (a mitochondrial inner-membrane protein) were used as controls.

  3. Protein characterization in individuals with mutations in TTC19.
    Figure 3: Protein characterization in individuals with mutations in TTC19.

    (a) Immunofluorescence images of muscle from a control (Ctrl), a disease control (Ctrl-tRNA) and subject 1 (P1) using α-Core1 antibody. Scale bars, 25 μm. (b) One dimensional BNGE of muscle homogenates from subject 2 (P2) and control (Ctrl). We used an antibody against the Core1 subunit to detect complex III, an antibody against SDH 70 kDa for complex II and an antibody against subunit COX-IV for complex IV. (c) Two dimensional BNGE of subject 2 (P2) and control (Ctrl) muscle homogenates. We used antibodies against Core1, Core2 and RISP to detect complex III and an antibody against COX-IV for complex IV.

  4. Structural analysis of TTC19 interactions.
    Figure 4: Structural analysis of TTC19 interactions.

    (a) Two dimensional BNGE on mouse liver mitochondria using antibodies against TTC19, Core1 and Core2. Dotted vertical lines indicate the dimeric form of cIII (cIII2) and the supercomplex composed of cIII and cIV (cIII2 + cIV). (b) Coimmunoprecipitation assays on mouse liver mitochondria. The antibody used for immunoprecipitation (IP) is indicated on top and the antibodies used for immunodetection are indicated on the right. Sn, supernatant; 2, 5, 10, materials released from beads after treatment with 2%, 5% and 10% Triton X-100, respectively. (c) Two-dimensional BNGE on 143B, 143 rho° and HeLa cells.

  5. Characterization of TTC19-null flies.
    Figure 5: Characterization of TTC19-null flies.

    (a) Spontaneous locomotor activity in D. melanogaster CantonS control (CTRL) and TTC19-null (TTC19) flies. We measured the number of passages of individual flies across an infrared light beam during 30 min. The flies were divided into males (-M) and females (-F). Bars indicate the standard deviation. (b) Locomotor activity after the bang test. Percentage of flies able to climb to four selected 'end-points', corresponding to 2.8, 5.6, 8.4 and 11.2 cm after vigorous shaking in a test tube by vortexing for 10 s at maximum speed. CS, CantonS control flies; TTC19, TTC19-null flies. (c) Number of correct responses (taken in 10 trials) to the Optomotor test in CantonS control (CTRL) and TTC19 knockout (TTC19) flies at different days of age. The stimulus consisted of a black and white striped drum rotating either clockwise or counterclockwise until a response was obtained from the individual flies. Bars indicate the standard deviation. (d) Specific activities of MRC complexes in controls (CTRL1, CantonS; CTRL2, WTALA) and TTC19-null (TTC19) flies normalized to that of Citrate Synthase (CS). For each genotype, we performed three replicate mitochondrial extractions and for each extraction, we determined enzymatic activities from at least ten replicate reactions. The assays have been previously described22.

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Author information

  1. These authors contributed equally to this work.

    • Daniele Ghezzi &
    • Paola Arzuffi


  1. Unit of Molecular Neurogenetics, The Foundation 'Carlo Besta' Institute of Neurology, Milan, Italy.

    • Daniele Ghezzi,
    • Paola Arzuffi,
    • Costanza Lamperti,
    • Daria Diodato &
    • Massimo Zeviani
  2. Neurogenetics and Behaviour of Drosophila Lab, Department of Biology, University of Padova, Padova, Italy.

    • Mauro Zordan,
    • Caterina Da Re,
    • Clara Benna &
    • Rodolfo Costa
  3. Medical Genetics, Istituto di Ricovero e Cura a Carattere Scientifico, Burlo Garofolo, University of Trieste, Trieste, Italy.

    • Pio D'Adamo
  4. Unit of Laboratory Medicine, The Foundation 'Carlo Besta' Institute of Neurology, Milan, Italy.

    • Caterina Mariotti
  5. Unit of Child Neurology, The Foundation 'Carlo Besta' Institute of Neurology, Milan, Italy.

    • Graziella Uziel
  6. Division of Physical Medicine and Rehabilitation, Public Health Hospital, Bassano del Grappa, Italy.

    • Cristina Smiderle


D.G. and P.A. found TTC19 and characterized the mutations in human cells. C.L. performed the histological analysis of muscle biopsies. M. Zordan, C.D.R., C.B. and R.C. carried out the experiments in flies. C.M., G.U. and C.S. identified the subjects and carried out the clinical workout. P.D'A. performed linkage analysis. D.D. carried out the mutational screening on subjects 3 and 4 and the controls. M. Zeviani conceived the experimental planning and wrote the manuscript.

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