ARTICLE |
Correspondence to: Anton K. Raap, Dept. of Cytochemistry and Cytometry, Leiden Univ., Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.
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Summary |
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Pearson's marrow/pancreas syndrome is a disease associated with a large mitochondrial DNA (mtDNA) deletion. The various tissues of a patient contain heteroplasmic populations of wild-type (WT) and deleted mtDNA molecules. The clinical phenotype of Pearson's syndrome is variable and is not correlated with the size and position of the deletion. The histo- and cytological distribution of WT and deleted mtDNA molecules may be factors that correlate with the phenotypical expression of the disease. Here we introduce a new application of two-color FISH to visualize WT and deleted mtDNA simultaneously in a cell population of in vitro cultured skin fibroblasts of two patients with Pearson's syndrome. At the third passage of culturing, fibroblasts showed a remarkable heterogeneity of WT and deleted mtDNA: about 90% of the cells contained almost 100% WT mtDNA, and 10% of the cells contained predominantly deleted mtDNA. At the tenth passage of culturing, fibroblasts showed a reduction of intercellular heteroplasmy from 10% to 1%, while intracellular heteroplasmy was maintained. This new approach enables detailed analysis of distribution patterns of WT and deleted mtDNA molecules at the inter- and intracellular levels in clinical samples, and may contribute to a better understanding of genotype-phenotype relationships in patients with mitochondrial diseases. (J Histochem Cytochem 45:55-61, 1997)
Key Words: Mitochondrial DNA deletion, Fluorescence in situ, hybridization, Heteroplasmy, Pearson's marrow/pancreas, syndrome
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Introduction |
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An average vertebrate cell contains 1000-2000 mitochondria (
Spontaneous or maternally inherited mutations lead to a mixed population of normal and mutant mtDNA molecules, a phenomenon generally called heteroplasmy. However, one can operationally define heteroplasmy at different histological and cytological levels. The most basic level of heteroplasmy can be defined for a single mitochondrion. One mitochondrion contains multiple copies of mtDNA. Therefore, various ratios of mutant-wild type mtDNA molecules can be present within one mitochondrion. This heteroplasmy at the organelle level is referred to as intramitochondrial heteroplasmy. These heteroplasmic mitochondria create in one cell a heteroplasmic situation at the cellular level, i.e., intracellular heteroplasmy. Intracellular heteroplasmy, in turn, leads to heteroplasmy at the cell population or tissue level, a situation referred to as intercellular heteroplasmy.
Pathogenic deletions of the mitochondrial genome are variable in size and are almost always confined to a region delineated by the H-strand and L-strand origins of replication. The most common deletion, found in 30-50% of patients, is flanked by two 13-np direct repeats positioned at np 8470-8482 and 13,447-13,459 [np numbers according to
Pearson's marrow/pancreas syndrome is a disease associated with a large-scale deletion of the mitochondrial genome (
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Materials and Methods |
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Cell Culture
Skin biopsy specimens were taken from two patients diagnosed as having Pearson's marrow/pancreas syndrome. For FISH, patient skin fibroblasts and control (HeLa) fibroblasts were grown on sterilized, uncoated glass microscope slides in Dulbecco's modified Eagle's medium without phenol red, containing 4.5 mg/ml glucose and 110 µg/ml pyruvate (DMEM) supplemented with 10% fetal calf serum (FCS), 0.03% glutamine, 1000 U/ml penicillin/streptomycin in a 5% CO2 atmosphere. After 24-36 hr of culture, cells were washed with PBS and fixed in 4% formaldehyde, 5% acetic acid in PBS for 20 min, followed by three 5-min washes with PBS. Fixed cells were stored in 70% ethanol at 4°C until further use (
Probes
The position of the mtDNA deletions in both patients was determined by PCR and Southern blot analysis (
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All PCR fragments were separated from primers by gel electrophoresis on a 1% agarose gel and purified using an agarose gel extraction kit (Boehringer Mannheim; Mann-heim, Germany) according to manufacturer's instructions. After purification, the four PCR fragments were labeled with digoxigenin-11-dUTP (Boehringer Mannheim) or biotin-11-dUTP (Sigma; St Louis, MO) by nick translation according to standard procedures. After nick translation the PCR probes were ethanol-precipitated and dissolved at a final concentration of 10 ng/µl in 60% deionized formamide, 2 x SSC, 50 mM sodium phosphate buffer, pH 7.0, 10% dex-tran sulfate and 250 ng/µl herring sperm DNA.
In Situ Hybridization
The in situ hybridization was performed as previously described (
Microscopy and Photography
Slides were examined with a Leica DM microscope equipped with single and dual bandpass filters for FITC and Cy3 and with x63 and x100 oil objectives with 1.3 numerical aperture. Photographs were taken with automatic exposure time settings using Scotch 3M 640 ASA color slide films.
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Results |
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FISH analysis of control fibroblasts revealed a typical mitochondrial staining pattern. When the cells were hybridized with probes for the undeleted region of the mitochondrial genome (further referred to as COMMON 1 and 2), specific fluorescent signals appeared as large, ragged spots lying in a ribbon-like pattern throughout the cytoplasmic volume. Such fluorescent patterns were more abundant around the nucleus, but the ragged distribution could be observed best in the fiber-like extensions of the cells, because there signals from individual mitochondria were not overlapping. When the cells were treated with RNAse the fluorescent pattern remained identical, but the fluorescence intensity was profoundly reduced because of RNA degradation. DNAse treatment resulted in hardly detectable loss of fluorescence.
Double hybridizations to control fibroblasts with COMMON 1 and a probe for the deleted region of the mitochondrial genome of Patient 1 (further referred to as DEL 1) or with COMMON 2 and a probe for the deleted region of the mtDNA of Patient 2 (further referred to as DEL 2) resulted in the same mitochondrial staining pattern. In all cells, the COMMON probe signals visualized in red fluorescence (Figure 2A) and the DEL probe signals detected with a green fluorochrome (Figure 2B) co-localized, resulting in a yellow fluorescent staining pattern (Figure 2C).
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Simultaneous visualization of the COMMON 1 and DEL 1 probes on third-passage fibroblasts of Patient 1 showed, in most of the cells, the same yellow fluorescent pattern as was observed with the control cells (Figure 3A). However, a clear intercellular heteroplasmy was observed in this cell population. About 10% of the cells showed an intense red staining pattern of the COMMON 1 probe (Figure 3A and Figure 3B). Among these red fluorescent signals a small amount of green fluorescent hybridization signals of the DEL 1 probe was clearly present (Figure 3B), indicating the presence of some wild-type mtDNA in these cells. Cells of the tenth passage showed a reduction in the degree of intercellular heteroplasmy from 10% to 1%, whereas the degree of intracellular heteroplasmy was maintained. The cells containing deleted mtDNA appeared in clusters, indicating that these cells still have mitotic activity (Figure 3C) (see also Table 1).
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Essentially the same observations were made with the skin fibroblast cultures from Patient 2: a 10% intercellular heteroplasmy at the third passage and a reduction of intercellular heteroplasmy to 1% at the tenth passage. The intracellular heteroplasmy was less extreme compared to fibroblasts of Patient 1, but still a predominant presence of deleted mtDNA was observed. At the tenth passage this degree of intracellular heteroplasmy was maintained, as was also observed with the fibroblasts of Patient 1 (Table 1).
RNAse-treated cells of both patients showed the same reduction of fluorescent signals due to mtRNA degradation as in the control cells. The staining pattern of the mtDNA appeared as clearly defined spots lying in rows throughout the cytoplasm. The inter- and intracellular heteroplasmy was still evident. DNAse treatment did not result in a detectable loss of fluorescent intensity or of the heterogenous pattern.
Fibroblasts of both patients hybridized with digoxigenin-labeled COMMON probe, detected with fluorescein, and biotinylated DEL probe, visualized with Cy3, showed identical results to those described above, with reversed color patterns.
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Discussion |
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The present findings show that two-color FISH permits the simultaneous detection of deleted and wild-type mtDNA in situ and is therefore well suited to assess levels of intercellular and intracellular heteroplasmy, two factors that are likely to correlate with clinical expression of Pearson's syndrome.
The degree of intercellular heteroplasmy in the third passage of the skin fibroblast cultures from the two patients available for this study was about 10%. The intracellular heteroplasmic cells contained wild-type mtDNA in addition to relative large amounts of deleted mtDNA. These wild-type mtDNAs appeared not to be structurally organized but were randomly scattered throughout the cytoplasmic volume. At the tenth passage of culturing, the intercellular heteroplasmy was reduced to 1%, whereas the degree of intracellular heteroplasmy was maintained. The cells containing predominantly deleted mtDNA were clustered (Figure 3C), indicating that these cells, in spite of high amounts of deleted cytoplasmic mtDNA molecules, still have mitotic activity (
RNAse treatments resulted in considerable reduction of fluorescent signals. However, both inter- and intracellular heteroplasmy were still present, indicating that the deleted mtDNA is transcriptionally active, as has also been observed by in situ hybridization in muscle fibers of muscle biopsies of patients with mitochondrial myopathy (
In both patients the deletion had left the replication initiation sites intact. Because of their shorter sequences, the deleted mtDNA molecules are likely to have a replicative advantage over wild-type mtDNA molecules (
In conclusion, two-color FISH is well suited to assess levels of intercellular and intracellular heteroplasmy in the mitochondrial large-deletion syndromes. Such studies may contribute to a better understanding of the variable clinical expression of Pearson's syndrome. In addition, the technique will be valuable for quantitatively addressing basic questions such as mitotic segregation of mutant mtDNA and drift in inter- and intracellular heteroplasmy.
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Acknowledgments |
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Supported by the Dutch Science Organization, Area Medical Sciences (MW-NWO) project no. 900-543-109, and by a grant from Diabetes Fonds Nederland.
We wish to thank Dr H. Smeets of Nijmegen University Hospital for providing fibroblast cell cultures of Patient 2.
Received for publication May 8, 1996; accepted August 27, 1996.
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