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Short Communication |
From the University Department of Clinical
Neurosciences,*
Royal Free and University College Medical
School, University College London, London; the Metabolic
Unit,
Institute of Child Health, London,
United Kingdom; the Departments of Clinical
Chemistry
and Hepatology,§
Birmingham Children's Hospital, Birmingham; and the University
Department of Clinical Neurology,¶
Institute of Neurology,
London, United Kingdom
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Abstract |
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Introduction |
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Although certain molecular events in the evolution of mtDNA depletion have been defined in cultured cells from patients,8 the primary molecular defect remains to be identified. A nuclear genomic etiology has been defined in two of our families by mtDNA transfer technology,8,9 indicating that the mtDNA depletion is secondary to a nuclear genetic defect. The fact that the affected children are born after uncomplicated pregnancies and are often normal at birth and during the early postnatal period1-6 has led to the suggestion that there may be a defect in a developmentally regulated switch controlling mtDNA copy number.1 Here, we demonstrate mtDNA depletion in amniocytes from an infant who subsequently died from severe mtDNA depletion. These results suggest that the defect of mtDNA replication may be expressed early in embryological development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The female patient was the second child of healthy, unrelated Caucasian parents and was born at term after an uncomplicated pregnancy. She was well at birth but developed hypoglycemia during the first 24 hours. Later, she developed lactic acidemia (blood lactate 5.8 mmol/L) with progressive liver disease. Liver biopsy showed micronodular cirrhosis with proliferation of bile ducts and abundant neutral fat and bile pigment in hepatocytes. The activity of cytochrome-c oxidase was severely depleted in liver. Assay of phosphoenolpyruvate carboxykinase (PEPCK) in liver mitochondria revealed evidence of reduced mitochondrial PEPCK activity but PEPCK activity measured in amniotic fluid cells and fibroblasts was within normal limits. The patient died of progressive liver failure at the age of 6 months. Postmortem examination of skeletal muscle showed relatively uniform fibers with no evidence of ragged-red fibers with Gömöri trichrome staining. There was an excess of coarse lipid droplets in many muscle fibers. Cytochrome-c oxidase activity was normal and present in all fibers.
This patient's elder brother10 had been delivered at 42 weeks of gestation. He was well at birth but very soon developed symptoms compatible with liver failure. Blood lactate was variably increased (2.1–5.0 mmol/L). Assay of fibroblast PEPCK activity revealed marked deficiency. The child died, aged 7 weeks, of progressive liver failure.
Pediatric control skeletal muscle tissue was obtained, with informed parental consent, from children having orthopedic surgery, diagnostic biopsies, or autopsies, in whom no evidence of muscle or metabolic disease was found. Pediatric control liver specimens were derived from autopsies. Approval for study of this material was obtained from the ethical committees for Great Ormond Street Hospital and University College Hospital, both of London. Pediatric control fibroblasts were cultured from diagnostic skin biopsies of infants in whom no evidence of mitochondrial disease was found. Control amniotic fluid cell cultures were obtained from nine pregnancies with no family history of mitochondrial disease.
Southern Blot Analysis
Total cellular DNA was extracted from tissue samples by proteinase K digestion and subsequent phenol:chloroform extraction and ethanol precipitation.11 For Southern blot analysis of cultured fibroblasts, cells were harvested by trypsinization and washed twice in phosphate-buffered saline (PBS). Total cellular DNA was extracted with the Nucleon I kit (Scotlab, Strathclyde, UK), followed by ethanol precipitation.
Similar amounts (~3 µg) of DNA were digested with the restriction
enzyme PvuII, resolved on a 0.8% agarose gel by
electrophoresis, denatured, and blotted onto Hybond-N membrane
(Amersham, Buckinghamshire, UK) as recommended by the supplier. Blots
were hybridized according to standard conditions11
with
two probes simultaneously: a cloned 5.8-kb EcoRI fragment of
the gene encoding 18S rRNA12
and the entire mtDNA
obtained by long-range polymerase chain reaction.13
Probes were labeled with [
-32P]dCTP using
the Rediprime random prime labeling system (Amersham).
Signals on the blot were quantified by volume integration and corrected
for background noise on a PhosphorImager using ImageQuant software
(Molecular Dynamics, Sunnyvale, CA).
Cell Culturing Conditions
Fibroblasts and amniotic fluid cells were maintained at 37°C in a humidified atmosphere of 8% CO2 in air. Fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM) that included 25 mmol/L glucose and 4 mmol/L l-glutamine, supplemented with 10% fetal bovine serum, 2 µmol/L uridine, 1 µmol/L sodium pyruvate, 50 U/ml penicillin, and 50 µg/ml streptomycin. Amniotic fluid cells were grown in a mixture of Hams F10 and Chang's medium supplemented with 2 µmol/L uridine, 1 µmol/L sodium pyruvate. Both pyruvate and uridine are essential nutrients for cells deficient in mitochondrial respiratory chain function.14
Immunocytochemical Staining
For immunocytochemical studies, cells were trypsinized and seeded at moderate density on sterile glass coverslips. All cells were grown for a further 24 hours; the culture medium for amniotic fluid cell cultures was changed to DMEM supplemented with 20% fetal bovine serum and 0.2% chick embryo extract in addition to pyruvate and uridine for this period to reduce nonspecific background staining. To stain the mitochondria, cells were cultured for 45 minutes in the presence of 2 µmol/L MitoTracker CM-H2XRos (Molecular Probes, Eugene, OR) followed by culturing for 30 minutes in medium without this fluorescent dye. Cells were subsequently washed in PBS, fixed with 4% paraformaldehyde in PBS for 20 minutes, washed, permeabilized in methanol at -20°C for 15 minutes, and washed again. Protein binding sites were saturated with 10% normal goat serum in PBS for 30 minutes at 37°C in a humidified atmosphere. Next, coverslips were incubated with monoclonal antibodies to the mtDNA-encoded subunit I of cytochrome-c oxidase15 (10 µg IgG of monoclonal 1D6-E1-A8 per ml of PBS) or to the nuclear-encoded flavin protein subunit of succinate dehydrogenase16 (2 µg IgG of monoclonal 2E3-GC12-FB2-AE2 per ml of PBS) for 45 minutes at 37°C in a humidified atmosphere (monoclonals were kindly provided by Dr. R. A. Capaldi). After primary antibody incubation, coverslips were washed and incubated with goat anti-mouse IgG-fluorescein isothiocyanate conjugate (Southern Biotechnology Associates, Birmingham, AL; 100-fold dilution in PBS) for 45 minutes at 37°C in a humidified atmosphere. Finally, coverslips were washed once more in PBS and mounted onto glass slides in Citifluor-glycerol-PBS solution (Agar Scientific Ltd., Stansted, UK) supplemented with 1 µg/ml of 4,6-diamidino-2-phenylindole to reveal the nuclei. Fluorescence was inspected with a Zeiss Axiophot photomicroscope equipped with a 40X Plan-Neofluar lens. Photographs were taken on Kodak Ektachrome EPL 400X film.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Quantification of the signals by
phosphorimaging revealed that mtDNA levels in the patient's liver were
7% of mean control values, whereas mtDNA levels in the patient's
skeletal muscle were 18% of mean control values. Cultured skin
fibroblasts of the infant also exhibited a marked decrease in mtDNA
levels (not shown).
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. Also, patient amniotic fluid cells
negative for subunit I of cytochrome-c oxidase
were readily and consistently identified in all
preparations (Figure 2B)
. Immunostaining for the flavin protein subunit
of succinate dehydrogenase showed, however, that this nuclear-encoded
polypeptide was present in mitochondria of all cultured cells of the
patient (not shown). Control fibroblasts (not shown) and control
amniotic fluid cell cultures (n = 9) showed a
uniform expression of cytochrome-c oxidase subunit I in the
mitochondria of all cells (Figure 2C)
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The elder brother of the
patient died in infancy with acute liver failure originally ascribed to
PEPCK deficiency.10
PEPCK activity in the amniotic fluid
cell culture of the patient was normal and the pregnancy was allowed to
proceed. Following delivery, this second child also developed liver
failure and died at 9 months.
Both fibroblasts and amniotic fluid cells of the second child were
studied with immunocyctochemical techniques. These experiments showed
that all cultured cells contained mitochondria and all mitochondria
contained the nuclear-encoded flavin protein subunit of succinate
dehydrogenase, but the mitochondrially encoded subunit I of
cytochrome-c oxidase was present only in a subset of the
patient fibroblasts and amniotic fluid cells (Figure 2)
. These results
demonstrate that despite the apparently normal fetal development during
pregnancy, fetal amniotic fluid cell cultures expressed mtDNA
depletion.
It is interesting that such early embryological cells as amniocytes express mtDNA depletion. It is possible that mtDNA depletion in the amniocytes developed during culture. Cultured cells in general express fetal isoforms of proteins and so the expression of mtDNA depletion in this patient's amniocytes and fibroblasts, and in the fibroblasts and myoblasts of other patients,8,9 suggests that failure of a developmental switch is unlikely to explain presentation after birth. Analysis of fresh amniotic fluid cells for mtDNA depletion may provide a further clue to the timing of onset of mtDNA depletion during fetal development. The limited quantity of tissue available will require such analysis to be undertaken by immunocytochemical techniques similar to those described here.
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Footnotes |
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Supported by the Research Trust for Metabolic Diseases in Children, the Medical Research Council, and the Wellcome Trust. J. C. B. was an MRC Training Fellow.
Accepted for publication March 31, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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