Identification of Inheritance Modes of Mitochondrial Diseases by Introduction of Pure Nuclei from mtDNA-less HeLa Cells to Patient-derived Fibroblasts*

(Received for publication, October 17, 1996, and in revised form, February 18, 1997)

Kotoyo Isobe Dagger , Satoshi Kishino Dagger , Kimiko Inoue Dagger , Daisaku Takai Dagger , Hiroko Hirawake §, Kiyoshi Kita §, Shigeaki Miyabayashi and Jun-Ichi Hayashi Dagger par

From the Dagger  Institute of Biological Sciences, University of Tsukuba, Ibaraki 305, the § Department of Parasitology, Institute of Medical Science, University of Tokyo, Tokyo 108, and the  Department of Pediatrics, Tohoku University School of Medicine, Sendai 980, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

A nuclear genome delivery system was developed to deduce the modes of inheritance of the clinical phenotypes observed in patients with mitochondrial diseases by transfer of pure nuclei from normal cells to fibroblasts from the patients. The problem of possible contamination of the nuclei with a small amount of mtDNA was overcome by using mtDNA-less (rho 0) human cells as nuclear donors. In this study, intercellular transfer of pure nuclei was carried out by simple fusion of rho 0 HeLa cells with 533 fibroblasts from a patient with a fatal mitochondrial disease, which were deficient in cytochrome c oxidase and succinate dehydrogenase activities. The results showed that the cytochrome c oxidase and succinate dehydrogenase activities were restored by the introduction of pure HeLa nuclei, suggesting that the observed phenotypes of mitochondrial dysfunction were not due to mtDNA mutations but to nuclear, recessive mutations. Thus, our nuclear transfer system is effective for determining whether a mitochondrial or nuclear genome of a patient is responsible for a disease and whether deficiency of mitochondrial enzymes, including enzymes exclusively encoded by nuclear genomes, is transmitted in a nuclear recessive or nuclear dominant way, providing the parents of the patients with valuable information for genetic counseling on the risk of mitochondrial diseases in their next babies.


INTRODUCTION

Numerous disorders, including mitochondrial encephalomyopathies, cardiomyopathies, and aging, have been reported to be associated with defects of mitochondrial respiratory function, which is under the control of both nuclear and mitochondrial genomes (1-4). For example, nuclear DNA encodes most mitochondrial proteins including factors necessary for replication and expression of the mitochondrial genome. On the other hand, mtDNA encodes 13 polypeptides, which are subunits of respiratory chain complexes and oligomycin-sensitive mitochondrial ATPase, and two rRNAs and 22 tRNAs necessary for their translation in mitochondria. Therefore, mutations in both genomes could contribute to mitochondrial dysfunction and to the pathogenesis of these mitochondrial disorders.

Mitochondrial encephalomyopathies (1) were shown to be closely associated with heteroplasmy of wild-type mtDNA and mutant mtDNA with large scale mtDNA deletions including several tRNA genes (5), with point mutations of the mitochondrial tRNALeu(UUR) gene (6), and with point mutations of the mitochondrial tRNALys gene (7), respectively. The intercellular co-transfer of the mutant mtDNA and mitochondrial dysfunction into human cell lines lacking mtDNA (rho 0 cell lines) provided unambiguous evidence that accumulation of these mutant mtDNA molecules alone without defects in the nuclear genome was sufficient to produce the pathogeneses leading to the diseases (8-11).

In most mitochondrial encephalomyopathies, however, the candidate mutations of mtDNA have not been identified. In these cases, even if a patient's mtDNA and mitochondrial dysfunction are not co-transferred to rho 0 cells, mtDNA mutations could be responsible for the pathogeneses for the following reasons. First, during selection to remove rho 0 cells using medium without pyruvate and uridine, mtDNA-repopulating cells (cytoplasmic hybrids; cybrids) expressing mitochondrial dysfunction due to pathogenic mtDNA mutations might also be preferentially excluded together with rho 0 cells, so that the isolated cybrid clones would be limited to those with normal mitochondrial respiratory function. Furthermore, the possibility that the amount of mtDNA with unidentified pathogenic mutations is not sufficient to induce mitochondrial dysfunction in the cybrids cannot be excluded. Therefore, intercellular transfer of mtDNA from a patient to rho 0 cells is not appropriate for determining the involvement of mtDNA mutations in the pathogenesis, particularly when candidate pathogenic mutations of mtDNA in the patient have not been identified.

On the contrary, intercellular transfer of pure nuclei from normal cells to cells from patients should be an effective system for determining which genome, mitochondrial or nuclear, is responsible for pathogenesis. Moreover, with this system it is possible to determine whether a defect is inherited in a nuclear dominant or nuclear recessive way. For example, if mitochondrial dysfunction in cells from a patient is restored by introduction of normal nuclei, the defect can be attributed to nuclear recessive mutations, whereas if it is not, the defect should be due to mtDNA mutations or nuclear dominant mutations. However, the problem with this system is the preparation of pure nuclei completely free from mtDNA, since even a small amount of wild-type mtDNA is known to compensate for the influence of a predominant pathogenic mutant mtDNA and help in maintaining normal mitochondrial translation activity (8-13). This problem can be overcome by using rho 0 cells as nuclear donors; simple fusion of cells from a patient with rho 0 cells results in intercellular transfer of pure normal nuclei uncontaminated by any mtDNA from the nuclear donor cells.

In this study, HeLa nuclei completely free from mtDNA were introduced into 553 fibroblasts from a patient with a fatal mitochondrial disease by fusion of the fibroblasts with rho 0 HeLa cells (14) without using any selective pressure upon mitochondrial respiratory function to remove rho 0 HeLa cells. The 533 fibroblasts showed very low COX1 and SDH activities, and intercellular transfer of nuclei from rho 0 HeLa cells to the 533 fibroblasts restored their reduced COX and SDH activities, suggesting that these clinical phenotypes were not due to mtDNA mutations but to nuclear recessive mutations.


MATERIALS AND METHODS

Cells and Cell Culture

The human skin fibroblast line 553 was isolated from a 3-month-old patient with a fatal mitochondrial disease causing congenital COX and SDH deficiencies.2 A normal human skin fibroblast line TIG3S (15) from a fetus was obtained from Dr. H. Kondo, Department of Biology, Tokyo Metropolitan Institute of Gerontology. The rho 0 HeLa cells are resistant to 20 µM 6-thioguanine and 2 mM ouabain (Oua) (8). The fibroblast lines and rho 0 HeLa cells were grown in normal medium (RPMI 1640 + pyruvate (0.1 mg/ml) + uridine (50 µg/ml) + 10% fetal bovine serum).

Intercellular Transfer of the HeLa Nuclear Genome

Intercellular transfer of HeLa nuclei to TIG3S or 553 fibroblasts was achieved by fusion of the fibroblasts with rho 0 HeLa cells using polyethylene glycol 1500 (Boehringer Mannheim) as described previously (13) with slight modifications. Briefly, cells in the fusion mixtures were cultivated in selective medium (RPMI 1640 + pyruvate (0.1 mg/ml) + uridine (50 µg/ml) + 10% fetal bovine serum + hypoxanthine/aminopterin/thymidine (HAT; Sigma) + 2 mM Oua). On day 10 after fusion, colonies grown in the selection medium were cloned by the cylinder method, and the nuclear hybrid clones were then cultivated in the normal medium.

Southern Blot Analysis of mtDNA

Total DNA (1 µg) extracted from 2 × 105 cells was digested with a single cut restriction enzyme, BamHI, and the restriction fragments were separated by 0.6% agarose gel electrophoresis. After blotting onto a NYTRAN membrane, the DNA fragments were hybridized with [alpha -32P]dATP-labeled HeLa mtDNA. The radioactivity of the fragments was measured with a bioimaging analyzer, Fujix BAS 2000 (Fuji Film, Tokyo).

Northern Blot Analysis of Transcripts of mtDNA

Total cellular RNA was extracted with an ISOGEN RNA isolation kit (Nippon Gene, Toyama, Japan). Total denatured RNA (10 µg) was electrophoresed in 1.1% agarose gel containing formaldehyde and then transferred to a NYTRAN membrane. The membrane was hybridized with the [alpha -32P]dATP-labeled oligonucleotide probes COI (nucleotide positions 5847-7746), ATP6 (8704-9020), and 16 S rRNA (1740-3130) and with [alpha -32P]dATP-labeled cDNA probes of mtTFA and ATPase F1a. The radioactivities of the bands were measured a bioimaging analyzer, Fujix BAS 2000.

Analysis of Mitochondrial Translation Products

Mitochondrial translation products were labeled with [35S]methionine as described (16) with slight modifications. Briefly, semiconfluent cells in a dish were incubated in methionine-free medium containing 10% bovine serum for 1 h at 37 °C. Then the cells were labeled with [35S]methionine for 1 h in the presence of emetine (0.15 mg/ml). The mitochondrial fraction (50 µg/lane) was separated by SDS-urea-polyacrylamide gel electrophoresis. The dried gel was exposed to an imaging plate for 6 h, and the labeled polypeptides were located with a bioimaging analyzer, Fujix BAS 2000.

Analyses of COX, Oligomycin-sensitive ATPase, and SDH Activities

The activities of the mitochondrial respiratory enzymes COX (complex IV), oligomycin-sensitive mitochondrial ATPase (complex V), and SDH were measured as described in Refs. 17-19, respectively.

Chromosome Analysis

The chromosome compositions of cybrid clones were analyzed immediately after cloning using air-dried chromosome preparations as described previously (20).


RESULTS

The human skin fibroblast line 553 was isolated from a 3-month-old patient with a fatal mitochondrial disease. Since the patient showed progressive COX and SDH deficiencies in skeletal muscles, we examined whether 553 fibroblasts from the patient also expressed these clinical phenotypes. Biochemical studies showed that COX and SDH activities of 553 fibroblasts were about 22 and 10%, respectively, of those of normal skin TIG3S fibroblasts from a fetal subject (Fig. 1, a-b).


Fig. 1. Expressions of clinical phenotypes in fibroblasts from a patient with mitochondrial disease. The mitochondrial respiratory enzyme activities of COX (a), SDH (b), and oligomycin-sensitive mitochondrial ATPase (c) in TIG3S fibroblasts from a normal subject (TIG) and 553 fibroblasts from a patient (553) are compared.
[View Larger Version of this Image (11K GIF file)]

The observed COX deficiency in 553 fibroblasts could be due to reduction of the amount of mtDNA or to down-regulation of transcriptional or translational activity in mitochondria, because the three COX subunits COI, COII, and COIII are encoded by mtDNA. To examine these possibilities, we first compared the total amounts of mtDNA in 553 and TIG3S fibroblasts by Southern blot analysis after digestion of total DNA samples with a single-cut restriction enzyme, XhoI. Results showed that the amounts of mtDNA in these two fibroblast lines were similar (Fig. 2a).


Fig. 2. Comparison of the contents of mtDNA and its transcripts in fibroblast line TIG3S derived from a normal subject (TIG) and 553 from a patient with mitochondrial disease (553). a, Southern blot analysis of a BamHI restriction fragment of mtDNA. Total DNA (5 µg/lane) extracted from fibroblasts was analyzed using [alpha -32P]dATP-labeled HeLa mtDNA as a probe. b, Northern blot analysis. Total RNA (10 µg/lane) extracted from the fibroblasts was separated on agarose/formaldehyde gel and blotted onto filters. Hybridization was carried out with various probes: nuclear DNA-encoded mtTFA, the a subunit of oligomycin-sensitive mitochondrial ATPase, mtDNA-encoded 16 S rRNA, the COI subunit of COX and the ATP6 subunit of oligomycin-sensitive mitochondrial ATPase. The relative contents of the mature mRNAs (indicated by arrowheads) in 553 fibroblasts were 87.0, 105.1, 68.5, 28.2, and 57.9%, respectively.
[View Larger Version of this Image (42K GIF file)]

On the contrary, Northern blot analysis revealed very different transcription profiles of 553 fibroblasts from those of normal TIG3S fibroblasts. Fig. 2b shows that the processings of mitochondrial rRNA and mRNA in 553 fibroblasts were not sufficient, resulting in the productions of increased amounts of unprocessed mitochondrial transcripts and reduced amounts of mature 16 S rRNA (68.5%) and COI mRNA (28.2%), whereas the amounts of mRNAs of nuclear genome-encoded subunits of oligomycin-sensitive ATPase and mtTFA were essentially normal.

Next we examined mitochondrial translation activity using [35S]methionine incorporation in the presence of emetine to determine whether the observed abnormality in processing of mitochondrial transcripts in 553 fibroblasts affected the amounts of newly synthesized polypeptides encoded by mtDNA. Fig. 3 shows that the incorporations of [35S]methionine into most mtDNA-encoded polypeptides, including COI, COII, and COIII, in 553 fibroblasts were about 30% of those in TIG3S fibroblasts. Therefore, the reduced COX activity in 553 fibroblasts might be due at least partly to incomplete processing of mitochondrial transcripts, resulting in reduced translation activity in the mitochondria.


Fig. 3. Analysis of mitochondrial translation products in TIG3S fibroblasts from a normal subject (TIG) and 553 fibroblasts from a patient with mitochondrial disease (553). Protein synthesis in mitochondria was examined after [35S]methionine-labeling of mitochondrial translation products in the presence of emetine (0.2 mg/ml) to protect translation in cytoplasm. Proteins of the mitochondrial fraction (50 mg/lane) were separated by SDS-polyacrylamide gel electrophoresis. ND5, COI, ND4, Cytb (cytochrome b) ND2, ND1, COII, COIII, ATP6, ND6, ND3, ATP8, and ND4L are polypeptides assigned to mtDNA genes. TIG, TIG3S fibroblasts from a normal subject; 553, 553 fibroblasts from a patient.
[View Larger Version of this Image (43K GIF file)]

The profile of mitochondrial translation in 553 fibroblasts also showed another unexpected abnormality, namely the amounts of [35S]methionine incorporation into ATP6 and ATP8 polypeptides were slightly increased over those in control TIG3S fibroblasts (Fig. 3). Moreover, the amounts of their mature mRNAs were also increased correspondingly (Fig. 2b). These observations predicted slight increase of oligomycin-sensitive mitochondrial ATPase activity in 553 fibroblasts, and the results of biochemical analysis shown in Fig. 1c confirmed this prediction.

Since subunits of COX and oligomycin-sensitive mitochondrial ATPase are encoded by both nuclear and mitochondrial genomes, and since mitochondrial protein synthesis is under the control of both genomes, it was necessary to determine which genome was responsible for the reduced COX activity and the elevated ATPase activity in the patient's fibroblasts. Furthermore, although the subunits of SDH are all encoded by the nuclear genome, it was necessary to determine whether the pathogenic mutations causing low SDH activity were recessive or dominant.

For these purposes, pure HeLa cell nuclei were introduced into the 553 fibroblasts and the control TIG3S cells, respectively. As rho 0 HeLa cells were shown to have no mtDNA (8) and to be resistant to both 6-thioguanine and Oua, HeLa nuclei free from mtDNA were introduced into 553 fibroblasts simply by fusion of 553 fibroblasts with rho 0 HeLa cells, and then the cells were cultivated in selective medium with Oua + HAT (Table I; cf. "Materials and Methods") to exclude unfused parental rho 0 HeLa cells and parental fibroblasts by HAT and Oua, respectively, with selective survival of nuclear hybrids (Table I). The possibility that the nuclear hybrid clones we isolated were hybrids between the fibroblasts and contaminating presumptive revertant rho 0 HeLa cells with recovered HeLa mtDNA was excluded by the fact that no HeLa mtDNA was present in these nuclear hybrid clones. This was shown by HhaI digestion of the PCR products (data not shown), a procedure that can distinguish HeLa mtDNA from other human mtDNAs, as described previously (21). Therefore, the nuclear genome of the nuclear hybrids was derived from both parental rho 0 HeLa cells and fibroblasts, while the mitochondrial genome was exclusively from fibroblasts. Thus, pure HeLa nuclei were transferred to 553 fibroblasts from the patient and control TIG3S fibroblasts, respectively (Table I).

Table I. Somatic cell genetic characteristics and chromosome complement of parent cells and their nuclear hybrid clones


Cell line Drug resistancea Selection mtDNAb Chromosome numberc

Parent cell lines
  TIG3S (normal) + 46
  553 (patient) + 46
  rho 0 HeLa(nuclear donor) 6-Thioguaniner, Ouar  - 48
Nuclear hybrid clones
  HTIG-1 (rho 0 HeLa × TIG3S) Oua + HAT + 78
  HTIG-2 (rho 0 HeLa × TIG3S) Oua + HAT + 77
  H553-1 (rho 0 HeLa × 553) Oua + HAT + 80
  H553-2 (rho 0 HeLa × 553) Oua + HAT + 72
  H553-3 (rho 0 HeLa × 553) Oua + HAT + 75

a r, -resistant.
b The absence of HeLa mtDNA in nuclear hybrid clones was confirmed by HhaI digestion of the polymerase chain reaction products.
c Modal chromosome number was obtained from 25 metaphases.

Then we compared the activities of COX, SDH, and oligomycin-sensitive mitochondrial ATPase, and the profiles of mitochondrial protein synthesis in nuclear hybrid clones of rho 0 HeLa cells × 553 fibroblasts and rho 0 HeLa cells × TIG3S fibroblasts. Results showed that all the abnormal clinical phenotypes were reversed in nuclear hybrid clones of rho 0 HeLa cells × 553 fibroblasts to levels comparable to those in control nuclear hybrid clones of rho 0 HeLa cells × TIG3S fibroblasts (Figs. 4 and 5). Moreover, the slight overexpressions of ATP6 and ATP8 polypeptides in 553 fibroblasts (Fig. 3) were no longer observed in their nuclear hybrid clones (Fig. 5). Since rho 0 HeLa cells have no mtDNA, restoration of the normal clinical phenotypes must have been due to cooperation between the imported HeLa nuclear genome and the mitochondrial genome of the patient.


Fig. 4. Recovery of abnormal COX, SDH, and oligomycin-sensitive mitochondrial ATPase activities in 553 fibroblasts by introduction of pure HeLa nuclei. a-c show COX, SDH, and oligomycin-sensitive mitochondrial ATPase activity, respectively. 1, HTIG-1 (a nuclear hybrid clone of rho 0 HeLa cells × TIG3S); 2, H553-1 (a nuclear hybrid clone of rho 0 HeLa cells × 553); 3, H553-2 (a nuclear hybrid clone of rho 0 HeLa cells × 553).
[View Larger Version of this Image (11K GIF file)]


Fig. 5. Recovery of normal mitochondrial translation profile in 553 fibroblasts by introduction of pure HeLa nuclei. Lanes 1-5 are the profiles of the nuclear hybrid clones HTIG-1, HTIG-2, H553-1, H553-2, and H553-3, respectively.
[View Larger Version of this Image (87K GIF file)]

Therefore, mtDNA in the patient was not responsible for the pathogenesis, and all the clinical phenotypes related to mitochondrial dysfunction observed in 553 fibroblasts, i.e. reduced COX and SDH activities, enhanced oligomycin-sensitive mitochondrial ATPase activity, reduced translations of COX subunits (COI, COII, and COIII), and enhanced translations of ATP6 and ATP8 subunits, should be due to nuclear recessive mutations.


DISCUSSION

In this study, we developed a system for delivery of a normal nuclear genome from rho 0 HeLa cells to fibroblasts from a patient to determine whether a mitochondrial or nuclear genome of the patient was responsible for expression of the clinical phenotypes of mitochondrial dysfunction. Intercellular transfer of pure nuclei from rho 0 HeLa cells to 553 fibroblasts from the patient was achieved by simple fusion of rho 0 HeLa cells with the 553 fibroblasts followed by HAT and Oua selection. In these nuclear hybrid clones, the nuclear genome was derived from both parental 553 fibroblasts and rho 0 HeLa cells, whereas the mitochondrial genome was exclusively from the 553 fibroblasts. Therefore, if mitochondrial dysfunction was restored by the introduction of pure rho 0 HeLa nuclei, it should be attributed to nuclear recessive mutations, and if not, it should be due to mtDNA mutations or nuclear dominant mutations.

The 553 fibroblasts showed apparently diverse abnormalities of mitochondrial respiratory function; reduced COX and SDH activities slightly enhanced oligomycin-sensitive mitochondrial ATPase activity, incomplete processing of mitochondrial transcripts, and abnormal mitochondrial translation profiles. Transfer of the pure nuclear genome showed that all the mitochondrial disorders of 553 fibroblasts were reversed completely by the introduction of pure HeLa nuclei, suggesting that the clinical phenotypes were diagnosed to be due to nuclear recessive mutations and not to mtDNA mutations in the 553 fibroblasts. Accordingly, abnormalities of at least two nuclear encoded factors are involved in the expressions of the clinical phenotypes in the patient. One is a factor necessary for the processing of mitochondrial transcripts. Mutation of this factor could be responsible for insufficient processing of mitochondrial transcripts resulting in the productions of abnormal amounts of mitochondrial translation products followed by abnormal COX and ATPase activities. The other is a factor necessary for the expression of SDH activity. Recently, a point mutation in the nuclear encoded flavoprotein subunit gene of SDH, which might be responsible for the pathogenesis, was reported in two SDH-deficient siblings with Leigh's syndrome by Bougeron et al. (22), but it was not present in the patient reported here (data not shown). We are now investigating the regulation of expression of SDH subunits in 553 fibroblasts using Northern blot and Western blot analyses.

In our nuclear genome delivery system, the fate of rho 0 HeLa cell-derived mitochondria in the nuclear hybrids requires consideration, since rho 0 HeLa cells still have these organelles that have no mtDNA or respiratory enzyme activities (14, 23). We recently showed that when HeLa or fibroblast mitochondria were introduced into rho 0 HeLa cells by their fusion with cytoplasts of HeLa cells or fibroblasts, mtDNA from the imported mitochondria rapidly spreads to all the rho 0 HeLa mitochondria (24). Therefore, in our nuclear hybrids, all rho 0 HeLa cell-derived mitochondria come to possess mtDNA, probably by intermitochondrial fusion with the imported mitochondria containing mtDNA (25).

Previously, similar experiments using somatic hybrids isolated by the fusion of HeLa cells with fibroblasts from a patient with Leigh's syndrome were reported by Miranda et al. (26), and their results showed that COX deficiency observed in fibroblasts from a patient was due to nuclear DNA-encoded mutations. To eliminate HeLa cell-derived mtDNA from the hybrids, they used a specific characteristic of HeLa mtDNA, a characteristic which we reported previously, that HeLa mtDNA preferentially segregates from both hybrids and cybrids during prolonged cultivation (27). However, since they did not use rho 0 HeLa cells as nuclear donors, they could not exclude the possible contribution of a slight amount of remaining HeLa mtDNA (about 5%) in maintaining COX activity in the hybrids. In fact, it has been generally thought that a very small amount of wild-type mtDNA (less than 5%) can compensate for the influence of predominant pathogenic mutant mtDNA with tRNALeu(UUR) 3243 (10, 11), 3271 (12), and tRNALys 8344 (9) in cybrids and help in maintaining normal translation activity in mitochondria.

Recently, Tiranti et al. (28) reported the nuclear DNA origin of reduced COX activity in fibroblasts from a patient with Leigh's syndrome based on the absence of cytoplasmic co-transfer of the patient's mtDNA and COX deficiency to rho 0 cells. On the other hand, replacement of mtDNA of fibroblasts from a patient by mtDNA from normal cells did not restore the reduced COX activity. For accomplishment of these experimental processes, however, an SV40-transformed, neomycin-resistant (Neor), and rho 0 fibroblast line must be isolated from the patient's fibroblasts by their SV40-Neor transfection to isolate transformed Neor fibroblast lines followed by long term ethidium bromide treatment to remove their own mtDNA completely. Therefore, this mtDNA transfer system requires complicated experimental processes, and it is possible that SV40-induced transformation and ethidium bromide treatment of fibroblasts from the patient may have side effects on some factors necessary to ensure normal COX activity. Moreover, for isolation of mtDNA-repopulating cells (cybrids), selective medium without uridine and pyruvate must be used for the exclusion of parental rho 0 cells, a medium in which cybrids with deficient COX activity might also be preferentially eliminated together with the rho 0 cells, as reported previously (8, 12, 15), resulting in the possibly misleading conclusion that mtDNA mutations are not involved in the pathogenesis.

In the present study, HeLa nuclei completely free from mtDNA were introduced into fibroblasts from a patient simply by fusion of the fibroblasts with rho 0 HeLa cells followed by HAT selection without using any selective pressure upon mitochondrial respiratory function to remove rho 0 HeLa cells. Therefore, this nuclear genome delivery system using rho 0 HeLa cells can be used to determine the localizations of pathogenic mutations and the modes of inheritance of clinical phenotypes, although possibilities on the loss of a chromosome with dominant allele or increased competition of dominant allele with recessive alleles (1 versus 4 or higher) must be considered carefully before conclusion. This can provide the parents of the patients with valuable information for genetic counseling on the risk of mitochondrial disorders in their next babies.


FOOTNOTES

*   This work was supported in part by grants for a Research Fellowship from the Japan Society for Promotion of Science for Young Scientists (to D. T.), by a University of Tsukuba special research grant (Superior) (to J.-I. H.), by grants from the Naito Foundation (to J.-I. H.), and by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (to J.-I. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be addressed: Institute of Biological Sciences, University of Tsukuba, Ibaraki 305, Japan. Tel.: 298-53-6650; Fax: 298-53-6614; E-mail: jih45{at}sakura.cc.tsukuba.ac.jp.
1   The abbreviations used are: COX, cytochrome c oxidase; SDH, succinate dehydrogenase; Oua, ouabain; HAT, hypoxanthine/aminopterin/thymidine.
2   S. Miyabayashi, H. Hanamizu, K. Haginoya, H. Hirawake, and K. Kita, manuscript in preparation.

ACKNOWLEDGEMENTS

We are grateful to Dr. S. Horai, National Institute of Genetics, Dr. Y.-I. Goto, National Center of Neurology and Psychiatry, and Dr. Y. Kagawa, Jichi Medical School, for providing the probes used in this study.


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