Human Molecular Genetics, 2002, Vol. 11, No. 26 3273-3281
© 2002 Oxford University Press
Cell complementation using Genebridge 4 human:rodent hybrids for physical mapping of novel mitochondrial respiratory chain deficiency genes
1Unité de Recherches sur les Handicaps Génétiques de l'Enfant (INSERM U393), 2Service Informatique, 3Service de Cytogénétique, Hôpital Necker-Enfants Malades, Paris, 4Généthon, Evry and 5Centre National de Séquençage, Evry, France
Received July 29, 2002; Accepted October 19, 2002
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ABSTRACT |
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The mapping and identification of respiratory chain deficiency genes is particularly tedious owing to the large number of genes encoding catalytic subunits and involved in respiratory chain (RC) assembly and maintenance. We have developed a functional complementation approach by: (i) growing the patient's fibroblasts in a highly selective medium; and (ii) transferring human chromosome fragments into RC-deficient fibroblasts by microcell-mediated transfer. In the absence of carbohydrates in the culture medium, the deficient cells rapidly disappeared unless they were rescued by a chromosome fragment carrying the disease gene. Microcells prepared from human:rodent Genebridge 4 panel of whole genome radiation hybrids were fused with fibroblast strains of two patients with complex II or I+IV deficiency and allowed to map the disease-causing genes to small intervals (4 and 12 Mb) on chromosomes 12p13 and 7p21, respectively. These intervals are similar to that obtained by genetic linkage analyses in large informative families. The recovery of normal RC enzyme activity in deficient skin fibroblasts supported the relevance of the transferred chromosome fragment in the disease. This approach makes the physical mapping of the disease genes feasible in some sporadic cases of RC deficiency.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Respiratory chain (RC) deficiency is a group of clinically and genetically heterogeneous conditions, which represents, in our experience, one of the most frequent cause of inherited metabolic diseases in humans (1). Most of the genes involved in mitochondrial functions are nuclearly encoded and only a few of them have been identified as disease-causing genes (2). The candidate gene approach has led to the identification of the disease causing mutation in a flavoprotein subunit of complex II (FP) in two siblings presenting with Leigh syndrome (3). A similar approach has led to the identification of disease mutations in catalytic subunits of complex I (4,5) and in assembly proteins of complex III (BCS1, 6) and complex IV (SCO2, 7). Yet, owing to the large number of possible candidate genes, this approach remains laborious.
A second approach, based on linkage analysis in informative families, resulted in the identification of the disease causing mutations in the thymidine phosphorylase gene in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE, 8), ANT1 gene in autosomal dominant ophthalmoplegia associated with mitochondrial DNA deletions (9), OPA1 gene in dominant optic atrophy (10) and SCO1 and COX10 genes in isolated complex IV deficiency (11,12). However, the scarcity of informative families limits the impact of this approach.
Finally, an elegant approach based on microcell-mediated chromosome transfer led to the identification of SURF1 gene mutations in Leigh syndrome with complex IV deficiency (13,14). This method had previously permitted the identification of a number of metabolic disease genes (15–18) and has been widely used in cancer genetics (19–21). However, apart from SURF1, no RC deficiency gene has been hitherto identified using this approach.
Combining selective culture conditions and microcell-mediated human chromosome fragment transfer using the Genebridge 4 human-rodent hybrids (GB4) previously used for the Human Genome sequencing project, we have devised a novel method of functional complementation in two cases of respiratory enzyme deficiency. This approach combined with computer analysis of complementing GB4 cell lines has allowed the physical mapping of two novel disease genes, for complex II and I+IV deficiency, respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Selective culture medium
We first devised a culture medium highly selective for respiratory competent cells. Both control and RC-deficient skin fibroblasts were found to grow well in RPMI 1640 containing glucose (2 g/l) with 10% fetal calf serum (FCS) supplemented with 2.5 mM sodium pyruvate and 200 µM uridine (which allow an active growth of RC deficient cells, Fig. 1A, (22)). When galactose (2 g/l) that slowly enters the glycolysis, was substituted for glucose in the absence of sodium pyruvate and uridine, control cells grew normally while the patient's cell lines failed to grow and progressively died (Fig. 1B). Withdrawing galactose further decreased glycolytic ATP production by cultured skin fibroblasts (23) and caused cell death in the two deficient cell lines after 2–3 days of culture while control cells grew normally (Fig. 1C). In the absence of carbohydrates, RPMI 1640 was highly selective and the two deficient strains were rapidly counter selected (in less than 3 days). It has been previously shown that cell death in some RC deficiency essentially results from a superoxide-triggered apoptotic process (23). In the cell lines of patients 1 and 2 as well as in five other cell lines showing cell death in selective medium, the increased access of phosphoinositol to the annexin dye suggests that cell death occurred through an apoptotic process, as shown for complex II deficient cells (Fig. 2A–F).
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Chromosomal localization of disease loci by microcell-mediated chromosome transfer
In order to identify the chromosome harboring the mutant gene, microcell-mediated transfer of human chromosome into the patient's cell lines was first performed using the Corriell human:rodent monochromosomal hybrid panel. After transfer, the cells were selected in the above-mentioned selective medium. For a period of 1 to 2 weeks, most microcell-fused fibroblasts died and detached and only a few of them were able to proliferate, presumably because they were complemented by a chromosome harboring the defective gene. After 3 to 5 weeks, distinct clones containing condensate and refringent cells could be observed and continued to grow for variable periods of time in selective medium. Some of them reached confluence and after trypsinization were transferred to larger flasks.
Transfer of chromosome 12 rescued patient 1 fibroblasts grown in selective medium, while chromosome 7 rescued fibroblasts of patient 2. No other chromosome restored the ability of cells to grow in the selective medium. These experiments were repeated three times with similar results. Owing to the possible instability of human chromosomes in human:rodent lines, we tested the presence of the human chromosomes in these cell lines. Chromosome painting on normal human metaphases using Alu-LINE-PCR probes derived from a Corriell human:rodent line containing chromosome 12, only revealed chromosomes 12 (Fig. 2G). Then microcells prepared from human:rodent hybrid cells containing intact human chromosome 12 were studied by whole chromosome painting (WCP) FISH and showed that 20% of them contained a chromosome 12 specific signal (Fig. 2I). This result is in apparent contradiction with the low yield of functional complementation, as no more than 6 clones could be obtained after fusion of monochromosomal microcells with 2.106 patient's fibroblasts. Only complemented fibroblasts from patient 1 grew enough in selective medium to yield a suitable amount of cells for enzyme assays. The restoration of complex II activity was confirmed in this strain (normal activity of the succinate cytochrome c reductase when normalized for the glycerol-3-phosphate cytochrome c reductase activity) measured in the same spectrophotometric cuvette, Table 1).
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Chromosomal localization of disease loci using the Genebridge 4 hybrid panel
In order to refine the chromosomal localization of the mutant genes, deficient fibroblasts were complemented using the Genebridge 4 (GB4) human:rodent hybrid panel. The GB4 panel of whole-genome radiation hybrids, previously used for the Human Genome sequencing project (24) includes 93 cell lines, each containing largely one third of the human genome. By comparing the human genetic content of the GB4 cell lines, we selected the following cell lines: for patient 1, lines 2–10, 20, 23, 30, 32–34, 39, 40, 45, 48 and 63 and for patient 2, lines 2, 8–10, 17, 30, 32, 34, 48 and 76. Their combination was respectively representative of chromosomes 12 and 7, previously identified as complementing chromosomes. Each fibroblast strain was fused with each selected GB4 cell line. After fusion, the cells were grown in a selective medium until distinct clones could be observed. As for monochromosome human:rodent hybrid fusion, more than 2.106 RC deficient fibroblasts were fused with the GB4-derived microcells. Five GB4 lines rescued patient 1 fibroblasts grown in the selective medium (lines 2, 8, 9, 10, 20). The disease-causing region, the one shared by these five GB4 lines, is determined by studying all RHdb markers of each chromosome. The presence of a specific marker in a given complementing cell line gives a +1 score, thus chromosomal region harboring several adjacent RHdb markers with maximum score (+5) determined the candidate region. Only positive results were taken into account and negative results were ignored as they can result from artifacts due to occasional loss of genetic material. For patient 1, this approach allowed to define four regions (chromosomes 7p14.3, 7p12.1, 12p13 and 17q25.3). As only chromosome 12 rescued the growth of patient 2 fibroblasts and restored a normal complex II activity, we retained the 12 Mb region on chromosome 12p13 (Fig. 3). Patient 1 and her affected sister showed haploidentity for markers D12S1608, D12S100, D12S1626 and D12S1725 on 12p13 chromosome.
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Similarly, seven GB4 lines rescued patient 2 fibroblasts (lines 2, 9, 10, 32, 33, 34, 76). Analysis of all RHdb markers defined three candidate regions on chromosome 7 (7p21.1, 7p21.2 and 7p13, Fig. 3). The 7p13 region was subsequently excluded, as patient 2, born to consanguineous parents, was heterozygous for various microsatellite markers mapping to this interval (D7S428, D7S478, D7S2427 and D7S2558). Patient 2 was homozygous for markers D7S2553 (7p21.1) and D7S664 and D7S2557 (7p21.2) reducing the physical interval encompassing the disease locus to 4 Mb on chromosome 7p21.
Finally, we tested the complementing GB4 lines for the presence of predicted chromosome fragments. Hybridization of a normal human metaphase with an Alu-LINE-PCR probe derived from the GB4 line 10 confirmed the presence of chromosome 12p13 and of other fragments in this GB4 line (Fig. 2J). Interestingly, a clone grown in selective medium after fusion of complex II-deficient fibroblasts (patient 1) with GB4 line 10 yielded enough material to allow enzyme assays (Table 1). The restoration of complex II activity was eventually confirmed in the complemented fibroblasts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Using the Genebridge 4 whole-genome radiation hybrid panel previously used for the Human Genome sequencing project (24), we report here on the mapping of novel loci causing respiratory chain deficiency to chromosome 12p13 (complex II deficiency) and 7p21 (complex I+IV deficiency). The GB4 panel is made up of 93 stable cell lines, each containing one third of the human genome. By combining GB4 lines representative of the chromosomes previously identified by monochromosomal transfer, we were able to reduce the chromosomal regions encompassing the disease genes to only 4 and 12 Mb, respectively. The recovery of normal RC enzyme activity in deficient skin fibroblasts supported the relevance of the transferred chromosome fragment in the disease. To our knowledge, this is the first contribution of this panel to functional complementation studies.
Our data indicate that the human genes carried by these hybrids are functionally expressed, as they rescued RC deficiency in cultured cells. Interestingly, the rodent genome was unable to complement an enzyme defect, as only GB4 cell lines containing a specific human chromosomal fragment could reproducibly rescue the RC deficient human fibroblasts as previously shown with whole genome human:rodent hybrids (14). This feature supports the relative stability of most GB4 lines and suggests that only one specific gene could rescue a given RC deficiency i.e. that no compensatory mechanisms were involved in this rescue (redundant function or protective effect of another gene).
The selection of rescued cells was improved by using a highly selective culture medium causing rapid cell death in RC deficient strains. Indeed, withdrawing glucose, galactose and other carbohydrates hampered anaerobic glycolysis and forced ATP production by the respiratory chain. This approach helped counter-selecting the RC deficient cells. Carbohydrate oxidation is known to control the antioxidant status of the cell (25) and removing carbohydrates made fibroblasts more susceptible to superoxide-induced apoptotic cell death triggered by RC deficiency. This selection considerably improved the use of microcell-mediated chromosome transfer as growth of a given clone required functional complementation i.e. reversion of the phenotype. This approach also avoided the time-consuming antibiotic selection following chromosome transfer for obtaining a sufficient amount of cells for enzymological studies.
However, it should be borne in mind that this approach presents several limitations. First, this approach is applicable to autosomal and X-linked recessive disorders only. Second, the use of a selective medium requires the complete cell death of patient's fibroblasts in a few days. Only a fraction of RC deficient fibroblasts failed to grow in selective medium. We indeed observed cell death in the selective medium devoid of carbohydrates in only 7/15 fibroblast strains and we focused on two strains only. Third, considering that some GB4 lines were unstable, negative results should not be taken into account, thus increasing the number of GB4 lines to be studied. Fourth, we occasionally noted that GB4 line fusions resulted in the rescue of a small number of fibroblasts in selective medium, but in none of them could identifiable clones be eventually amplified. One can hypothesize that in these cases, expression of other genes transiently rescued few cells without allowing emergence of a genuine clone. Despite its limitations and relatively low efficiency, this approach is powerful, as less than 20 different GB4 cell lines were sufficient to map the disease gene in two sporadic cases. This interval is similar to that obtained by linkage analysis in large informative families. The functional approach reported here will hopefully help identifying novel RC deficiency genes, as shown for SURF1, a gene responsible of Leigh disease associated with complex IV deficiency (13,14). Yet, owing to the remarkable diversity of putative disease-causing mechanisms in RC chain deficiency, a candidate gene approach—even when reduced to a short physical interval (18)—will certainly remain particularly tedious.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patients
Patient 1. A girl born to unrelated parents had severe hypotonia, neurological distress and hyperlactatemia at birth. Complex II deficiency was found in her skeletal muscle and cultured skin fibroblasts (Table 2). Her younger sister presented a quite similar clinical course and complex II deficiency. Sequence analysis failed to detect any mutation in the four complex II subunit genes.
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Patient 2. A boy, born to consanguineous parents, presented with neurological distress and hyperlactatemia at birth. A combined complex I and IV deficiency was found in his skeletal muscle (Table 2). Only complex I deficiency was found in his cultured skin fibroblasts.
Cell lines and culture media
Primary cultures of RC-deficient skin fibroblasts from an early passage (before the sixth passage) were grown at 37°C in a RPMI 1640 medium supplemented with glutamax (446 mg/l), 10% undialyzed fetal calf serum (FCS), 100 µg/ml streptomycin, 100 IU/ml penicillin, plus or minus: (i) glucose (2 g/l); (ii) galactose (2 g/l); (iii) uridine (200 µM) and 2.5 mM sodium pyruvate (26). Cell growth curves were obtained in triplicate by plating 20 000–40 000 cells in 2 cm2 wells and counting the cell number once a day for 5 days.
Transformed rodent cell lines harboring intact human chromosomes (27; Coriell Institute for Medical Research) were grown at 37°C in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% undialyzed fetal calf serum (FCS) and 4.5 g/l glucose. Histidinol, G418, or other selective drugs were added according to the specific resistance gene tagged in the various human chromosomes. Cell lines from the Genebridge 4 (GB4) panel previously obtained by fusing irradiated human fibroblasts with recipient thymidine kinase-deficient A23 hamster cells (24) were grown at 37°C in DMEM medium supplemented with 10% FCS, 4.5 g/l glucose and HAT.
Microcell isolation and fusion with patient's fibroblasts
Confluent rodent cells (about 3.106 cells) were blocked in metaphase by incubation with colcemid (150 ng/ml; 36 h) (28–30). Complete mitotic arrest was then obtained by addition of mitomycin (2 µg/ml; 2 h). The cells were subsequently washed with a phosphate buffer saline (PBS) solution and incubated in DMEM medium supplemented with cytochalasin B (15 µg/ml; 2 h) causing chromosome scattering throughout the cell. After trypsinization, centrifugation of the cells (25 000 g, 1 h, 34°C) resulted in the formation of pelleted microcells containing one or more rodent and/or human chromosomes. This pellet was resuspended in 5 ml of FCS-free DMEM medium and successively filtered through a 11 µm filter (Millipore, Ireland) and a 5 µm filter (Sartorius, Germany). The resulting suspension containing monochromosomal microcells was supplemented with lectin (60 µg/ml) as to favor microcell adhesion to the fibroblast cell surface and was subsequently added to medium-free patient's fibroblasts grown at 80% confluence for 30 min in 24 well plates. Fusion between fibroblasts and microcells was triggered by adding 500 µl of 40% polyethylene glycol (PEG 1000) per well for 1 min. After removing fusion medium, the cells were washed twice with PBS and incubated in glucose-rich RPMI 1640+10% FCS at 37°C under standard conditions. After 24 h, the fused fibroblasts were grown in a selective medium devoid of carbohydrates (RPMI 1640+10% FCS). No antibiotic selection was used. Clones were detected 3 to 5 weeks after fusion.
Representation of human genetic material in each GB4 line
All Radiation Hybrid database (RHdb, http://www.ebi.ac.uk/RHdb/) markers of a given chromosome were tested for all GB4 lines. For each of the 93 GB4 lines, a schematic representation indicating the presence of each marker was used (see Fig. 4, example for chromosome 12, vertical bars). Comparison of the resulting schemes allowed choosing specific GB4 lines for cell complementation. It is worth noting that most human chromosomes could be covered by a combination of GB4 lines. However, small regions were not represented in the GB4 panel (see Fig. 4 for chromosome 12). This made direct use of GB4 cell lines for complementation studies hazardous. For this reason, each deficient cell line was initially ascribed to a given chromosome by using microcell-mediated intact human chromosome transfer before fusion with GB4 cell lines.
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Enzyme studies
Polarographic tests and/or spectrophotometric assays of respiratory chain enzymes were carried out on skeletal muscle mitochondria, muscle homogenate, or cultured skin fibroblasts as previously described (31).
Chromosome painting
DNA was extracted from human:rodent monochromosomal hybrids and from GB4 line 10 by standard phenol/chloroform protocol. Alu-LINE probes were obtained by PCR amplification of DNA from hybrid cell lines using SR1 primer (5'-CCACTGCACTCCAGCCTGGG-3') and L1 primer respectively (5'-CATGGCACATGTATACATATGTAACAAACC-3'). In brief, each PCR product (500 ng), labeled with FITC by standard nick translation, was combined with 50 µg of Cot1 DNA and resuspended in 10 µl of hybridization mixture (50% formamide/1xSSC/10% dextran sulfate). Probe denaturation, hybridization and revelation were performed as described (32).
Microsatellite markers
For genotyping, fluorescent microsatellite markers of the Généthon database were used (33). PCR products were electrophoresed and analysed on an automatic sequencer (ABI377, Applied Biosystems, Foster City, USA).
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ACKNOWLEDGEMENTS |
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This research was supported in part by the Association Française contre les Myopathies (745 AO99).
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FOOTNOTES |
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* To whom correspondence should be addressed at: INSERM U393, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, 75015 Paris, France. Tel: +33 144381584; Fax: +33 147348514; Email: roetig{at}necker.fr
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