Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016:27:1-9.
doi: 10.1007/8904_2015_491. Epub 2015 Sep 25.

Detailed Biochemical and Bioenergetic Characterization of FBXL4-Related Encephalomyopathic Mitochondrial DNA Depletion

Ghadi Antoun  1 , Skye McBride  2 , Jason R Vanstone  2 , Turaya Naas  2   3 , Jean Michaud  4 , Stephanie Redpath  2 , Hugh J McMillan  2 , Jason Brophy  2 , Hussein Daoud  2 , Pranesh Chakraborty  2   3 , David Dyment  2 , Martin Holcik  2 , Mary-Ellen Harper  1 , Matthew A Lines  5
Affiliations

Detailed Biochemical and Bioenergetic Characterization of FBXL4-Related Encephalomyopathic Mitochondrial DNA Depletion

Ghadi Antoun et al. JIMD Rep. 2016.

Abstract

Mutations of FBXL4, which encodes an orphan mitochondrial F-box protein, are a recently identified cause of encephalomyopathic mtDNA depletion. Here, we describe the detailed clinical and biochemical phenotype of a neonate presenting with hyperlactatemia, leukoencephalopathy, arrhythmias, pulmonary hypertension, dysmorphic features, and lymphopenia. Next-generation sequencing in the proband identified a homozygous frameshift, c.1641_1642delTG, in FBXL4, with a surrounding block of SNP marker homozygosity identified by microarray. Muscle biopsy showed a paucity of mitochondria with ultrastructural abnormalities, mitochondrial DNA depletion, and profound deficiency of all respiratory chain complexes. Cell-based mitochondrial phenotyping in fibroblasts showed mitochondrial fragmentation, decreased basal and maximal respiration, absence of ATP-linked respiratory and leak capacity, impaired survival under obligate aerobic respiration, and reduced mitochondrial inner membrane potential, with relative sparing of mitochondrial mass. Cultured fibroblasts from the patient exhibited a more oxidized glutathione ratio, consistent with altered cellular redox poise. High-resolution respirometry of permeabilized muscle fibers showed marked deficiency of oxidative phosphorylation using a variety of mitochondrial energy substrates and inhibitors. This constitutes the fourth and most detailed report of FBXL4 deficiency to date. In light of our patient's clinical findings and genotype (homozygous frameshift), this phenotype likely represents the severe end of the FBXL4 clinical spectrum.

PubMed Disclaimer

Conflict of interest statement

Ghadi Antoun, Skye McBride, Jason R. Vanstone, Turaya Naas, Jean Michaud, Stephanie Redpath, Hugh J. McMillan, Jason Brophy, Hussein Daoud, Pranesh Chakraborty, David Dyment, Martin Holcik, Mary-Ellen Harper, and Matthew A. Lines declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Cranial MRI at five days of age showed (a, b) diffuse white matter edema with bilateral intra- and periventricular cysts, a single right occipital focus of restricted diffusion likely related to focal ischemia (not shown), and (c) a large lactate doublet in the left basal ganglia by MR spectroscopy. Craniofacial morphology (d) was notable for midface hypoplasia, short palpebral fissures, and lightly grooved philtrum. The presenting clinical features were considered to be consistent with a mitochondrial disease and/or pyruvate dehydrogenase deficiency
Fig. 2
Fig. 2
Assessment of respiratory parameters in skin-derived fibroblasts by micro-oximetry. (a–d) Oxygen consumption rate (OCR) in patient and control fibroblasts in the presence of glucose, pyruvate, and glutamine. Values are corrected to non-mitochondrial OCR persisting after addition of antimycin A and rotenone. (a) Basal OCR. (b) Leak OCR measured following inhibition of complex V by oligomycin. (c) Phosphorylation-coupled OCR, estimated as the resulting decrease in OCR after oligomycin. (e, f) Extracellular acidification rate (ECAR), an indirect measure of glycolytic activity in cells. Rates are corrected to non-glycolytic acidification rate as measured prior to the addition of glucose. (e) Glucose-stimulated ECAR. (f) Maximal (oligomycin-stimulated) ECAR. (g) Ratio of basal OCR to glucose-stimulated ECAR. N = 6. Values are presented as mean ± SEM; *p < 0.05, **p < 0.01 analyzed by unpaired, two-tailed Student’s t-test
Fig. 3
Fig. 3
Assessment of mitochondrial content, inner membrane potential, and network branching by cell staining in skin-derived fibroblasts. (a) Spinning disk confocal microscopy of fixed control (left) and patient (right) fibroblasts immunostained for the outer membrane transporter TOMM20. (b) Mitochondrial content (MitoTracker Green) (N = 3) and (c) inner membrane potential (TMRE) (N = 3) were measured by flow cytometry. Values are presented as mean ± SEM; **p < 0.01 analyzed by unpaired, two-tailed Student’s t-test. (d) Time course of patient and control fibroblasts showing increased cell death (as evidenced by YOYO1-positivity) of patient cells versus control cells. (e) This difference is magnified when cells are grown under mandatory respiration in galactose-containing medium after 72 h. N = 3. Values are presented as mean ± SEM; *p < 0.05 analyzed by unpaired, two-tailed Student’s t-test. (f) Levels of glutathione (GSH) and glutathione disulfide (GSSG) from skin-derived fibroblasts of control and subject. Values are corrected to cell number. N = 3. Values are presented as mean ± SEM; *p < 0.05, ***p < 0.001 analyzed by unpaired, two-tailed Student’s t-test
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bonnen PE, Yarham JW, Besse A, et al. Mutations in FBXL4 cause mitochondrial encephalopathy and a disorder of mitochondrial DNA maintenance. Am J Hum Genet. 2013;93:471–481. doi: 10.1016/j.ajhg.2013.07.017. - DOI - PMC - PubMed
    1. Gai X, Ghezzi D, Johnson MA, et al. Mutations in FBXL4, encoding a mitochondrial protein, cause early-onset mitochondrial encephalomyopathy. Am J Hum Genet. 2013;93:482–495. doi: 10.1016/j.ajhg.2013.07.016. - DOI - PMC - PubMed
    1. Gnaiger E. Capacity of oxidative phosphorylation in human skeletal muscle: new perspectives of mitochondrial physiology. Int J Biochem Cell Biol. 2009;41:1837–1845. doi: 10.1016/j.biocel.2009.03.013. - DOI - PubMed
    1. Huemer M, Karall D, Schossig A, et al (2015) Clinical, morphological, biochemical, imaging and outcome parameters in 21 individuals with mitochondrial maintenance defect related to FBXL4 mutations. J Inherit Metab Dis - PMC - PubMed
    1. Invernizzi F, D’Amato I, Jensen P, Ravaglia S, Zeviani M, Tiranti V. Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells. Mitochondrion. 2012;12:328–335. doi: 10.1016/j.mito.2012.01.001. - DOI - PMC - PubMed

LinkOut - more resources