Transfection screening for primary defects in the pyruvate dehydrogenase E1[alpha] subunit gene
Transfection screening for primary defects in the pyruvate dehydrogenase E1 [alpha] subunit geneRuth M. Brown, Lucy J. Otero and Garry K. Brown*
Genetics Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
Received April 7, 1997;Revised and Accepted May 28, 1997
In a significant number of patients with biochemical evidence of a defect in the E1 (pyruvate dehydrogenase) component of the pyruvate dehydrogenase complex, it has not proved possible to identify a mutation in the gene coding regions. To assess the need for more extensive genetic analysis in these patients and to establish a test system in which to study the biochemical consequences of mutations in the E1[alpha] subunit gene (which is responsible for the great majority of defined cases of pyruvate dehydrogenase deficiency), we have developed a method to screen for E1[alpha] gene defects based on complementation of the enzyme deficiency in transformed fibroblast cell lines following transfection and expression of the normal cDNA. Using this system, cell lines from patients with a variety of different defined mutations in the E1[alpha] gene show restoration of enzyme activity. A number of patients have been identified in whom deficient enzyme activity is not restored by expression of the normal cDNA indicating that an alternative explanation for the enzyme defect must be sought.
The multienzyme complex pyruvate dehydrogenase (PDH) plays a key role in aerobic energy metabolism catalysing the oxidative decarboxylation of pyruvate to yield acetyl CoA (1 ). The complex comprises three catalytic components: pyruvate dehydrogenase (E1), dihydrolipoamide S-acetyl transferase (E2) and dihydrolipoamide dehydrogenase (E3), two regulatory components: E1 kinase and phospho-E1-phosphatase together with a sixth component, protein X, which is believed to play a role in transacetylation (2 ). Pyruvate dehydrogenase deficiency is a well defined biochemical defect which is clinically very heterogeneous. It is one of the major defined causes of severe primary lactic acidosis in the newborn period and infancy (3 ) and can also present as a more chronic neurodegenerative disease associated with extensive cerebral atrophy and structural anomalies in the brain (4 ).
The activity of the different enzyme components of the PDH complex can be assessed separately to some extent, so it is possible to define the most likely site of the defect biochemically (5 ). In the great majority of cases of PDH deficiency, biochemical analysis indicates a defect in the E1 enzyme (6 ). This component of PDH is itself a complex structure, a heterotetramer of two [alpha] and two [beta] subunits, and enzyme assays cannot distinguish between defects in either subunit. However, genetic studies in patients with PDH E1 deficiency to date have revealed mutations only in the E1[alpha] subunit gene (6 ). The gene for this subunit is located on the X-chromosome in the region Xp22.1 (7 ) and this may account for both the frequency of E1[alpha] mutations and differences in genetic, clinical and biochemical features between males and females with PDH deficiency (8 ). The majority of female patients are heterozygous for frameshift mutations which lead to complete PDH deficiency whereas males have either missense mutations or frameshift mutations near to the C-terminal end of the protein, both of which result in some residual enzyme activity (9 ).
In a significant number of patients who are suspected of having PDH deficiency clinically and who have biochemical evidence of a defect in the E1 component of the complex, it has not proved possible to identify a causative mutation. In a recent analysis of nine males with fibroblast PDH activity >3SD below the mean of normal control fibroblasts, we identified only four mutations. Selection of female patients for genetic analysis is more complex as there may be substantial residual enzyme activity in the fibroblasts used for the enzyme assays depending upon the X-inactivation pattern. In some cases, this pattern is skewed sufficiently in favour of the normal X chromosome for the PDH activity to be within the normal range (4 ,10 ). In an analysis of 12 female patients with either PDH activity >3SD below the mean of normal control fibroblasts or a higher level of residual activity which was compatible with the X-inactivation pattern, six mutations were detected. Mutation analysis was performed using direct sequencing of the cDNA in the case of males and genomic screening by single strand conformation polymorphism and sequencing in the case of the females (10 -13 and unpublished obervations). In patients in whom no mutation was detected, there was no evidence for significant alterations in PDH E1[alpha] gene transcription or mRNA processing by northern blot analysis (unpublished data).
Co-ordinated synthesis, assembly and structural integrity of all of the subunits of the PDH complex are essential for full activity and it is expected that many of these processes will involve other components, both in the cytoplasm and mitochondrion, which are not part of the complex itself. Some indication for the need for such components is provided by the observation of PDH deficiency as part of the general defect in mitochondrial enzymes which results from deficiency of Hsp 60 (14 ) and combined defects of PDH and other genetically unrelated mitochondrial enzymes (15 ). Because of the possibility of secondary defects of PDH E1 activity (which could be due, for example, to abnormalities of import or assembly), it is important to seek other ways to determine whether patients without mutations in the protein coding region, do indeed have a defect in the PDH E1[alpha] subunit as the primary cause of their biochemical abnormality.
One method for demonstrating direct involvement of the PDH E1[alpha] subunit would be to transfect wild-type PDH E1[alpha] cDNA into PDH deficient cells in a suitable expression vector and measure enzyme activity to see if there is complementation of the functional defect. A possible problem with this approach is that PDH is an extremely large mitochondrial enzyme complex and restoration of activity may depend upon precise control of the synthesis of the subunits and their import into the mitochondrion in the correct proportions to allow normal assembly. There may also be problems in patients with residual enzyme activity due to interference between the introduced and endogenous protein products. However, there is some suggestion that these factors may not prove critical in practice from two reports of restoration of [alpha]-ketoacid dehydrogenase activity in deficient cells. In the first, PDH activity was recovered following transfection with the normal PDH E1[alpha] cDNA (16 ), and in the second, activity of the closely related enzyme complex, branched-chain [alpha]-ketoacid dehydrogenase, was restored by retroviral-mediated gene transfer (17 ). We have therefore investigated the feasibility of developing a screening system for identification of primary PDH E1[alpha] gene mutations based on complementation of the enzyme defect by transfection of the normal cDNA.
Transfection studies were performed on fibroblasts from six patients with PDH deficiency, including four who have been reported previously (10 ,11 ,18 ,19 ). Two of the patients (F2 and M1) developed severe lactic acidosis soon after birth and died in the neonatal period (18 ,19 ). Patient M3 also had severe neonatal lactic acidosis but this responded to treatment. However, his subsequent neurological development was delayed and he remained hypotonic. Patients F1, M2 and M4 presented during infancy with delayed development, hypotonia and a variety of other neurological features (10 ,11 ). A cerebral CT scan of patient F1 demonstrated gross cortical atrophy (11 ). All patients had raised blood and/or cerebrospinal fluid lactate concentration, significantly reduced PDH activity in cultured fibroblasts (Table 1 ), reduced E1[alpha] and E1[beta] immunoreactive protein commensurate with the reduction in enzyme activity and no evidence of other mitochondrial enzyme defects. The two female patients showed a mosaic pattern of E1[alpha] positive and negative cells on immunocytochemistry with an affinity purified anti-PDH E1[alpha] antibody. Mutations in the PDH E1[alpha] gene have been defined in the two female patients and two of the male patients (M1 and M2). The female patients, F1 (11 ) and F2 (19 ), had a 20 bp deletion and a 5 bp duplication in exon 10 of the PDH E1[alpha] gene, respectively, the mutation in M1 (18 ) was a 2 bp deletion in the codon for lysine 387 (K387fs) and in M2 (10 ) a G -> A transition in the codon for arginine 378 resulting in replacement by histidine (R378H). The remaining two male patients (M3 and M4) closely resemble the other patients clinically and biochemically. They have enzymatic and immunochemical evidence of a defect in the E1 enzyme only but have no identifiable mutations in either of the PDH E1 gene coding regions.
Pyruvate dehydrogenase activity in primary fibroblasts and derived transformed cell lines
Patient
Primary fibroblasts
Transformed cell lines
F1
0.29
clone 1
ND
F1
clone 2
ND
F1
clone 4
1.01
F1
clone 5
ND
F1
clone 6
ND
F2
0.05
clone 3
ND
F2
clone 5
ND
F2
clone 6
ND
M1
0.09
clone 4
0.03
M1
clone 6
0.02
M2
0.37
0.35
M3
0.17
0.21
M4
0.29
0.46
Enzyme activity is expressed as nmoles/mg protein/min and is the average of duplicate estimations. The range in normal control fibroblasts is 0.7-1.1 and in repeated assays of individual cell cultures, the coefficient of variation is <= 7.5%. ND, No detectable activity.
Fibroblasts from all six patients were transformed using the plasmid T22 as described in Materials and Methods and PDH activity in the starting fibroblast cultures and clonal transformed lines derived from them is summarised in Table 1 . The results demonstrated that four of the five clones derived from patient F1 and all three of the clones from patient F2 were completely deficient. In each case, these deficient cell lines must be expressing the X chromosome carrying the PDH E1[alpha] frameshift mutation which prevents synthesis of any functional PDH E1[alpha] subunit. The results are consistent with the fibroblast X-inactivation patterns in these patients. The starting fibroblast culture from patient F1 has an X-inactivation ratio of 80:20, whereas in the cells from patient F2 the ratio is 95:5. In both cases, the mutant X-chromosome is being predominantly expressed (11 ,19 ). One clone from patient F1 had activity within the normal range for cultured fibroblasts in our laboratory indicating that the normal X chromosome was being expressed. This was confirmed by direct X-inactivation analysis of these cells (results not shown). The PDH activity in the transformed clones derived from the male patients was similar to that found in the original primary fibroblasts.
Northern blot analysis of transformed cell lines showed that the pattern of PDH E1[alpha] mRNA was the same as in primary fibroblasts and this is illustrated by the results obtained with the transformed cell lines from patients F1 and M1 (Fig. 1 ). In the cell line F1-4 which expresses the normal X chromosome only (Fig. 1 A, lane 1), there are two PDH E1[alpha] transcripts of 3.3 and 1.6 kb in length as found in normal human fibroblasts (20 ). The 1.6 kb transcript was not detectable in the cell line F1-2 which is only expressing the mutant X chromosome (Fig. 1 A, lane 2). Transformed fibroblasts from patient M1 showed the normal pattern of E1[alpha] mRNAs (Fig. 1 B, lane 1). The deficient fibroblasts from patient F2 had no detectable 1.6 kb PDH E1[alpha] mRNA, whereas the cell lines from the remaining male patients all had the same pattern as normal control fibroblasts (results not shown).
Complementation of the biochemical defect was studied in cells transiently expressing the pRc/CMV/PDH E1[alpha] construct and in stable transfectants selected by resistance to G418 and the results are summarised in Table 2 . In transient expression experiments, transfection efficiency was monitored by co-expression of pEGFP and was generally in the range 30-50%, although cells from patient M4 attained very high levels of transfection. In the case of the two female patients, PDH activity was readily detectable after transfection of cell lines which were expressing the mutant PDH E1[alpha] gene. As the mutations in these patients result in complete loss of enzyme activity and immunoreactive E1[alpha] protein, this is the simplest case to analyse. In addition to recovery of activity, it was also possible to demonstrate the appearance of immunoreactive protein following transfection of these cells (Fig. 2 ). In the two male patients with defined PDH E1[alpha] mutations, the situation is more complex as both have residual enzyme activity. Nevertheless, there was a significant rise in PDH activity in both cases. Changes in activity were consistent when similar proportions of cells were transfected in separate experiments as shown by the results for patients F1 and M1 and there was some correlation between the restoration of activity and the proportion of cells transfected.
By contrast, there was no increase in enzyme activity following transient transfection of the cell lines from patients M3 and M4 who have reduced enzyme activity but no defined mutation, even though these patients have similar residual activity to patients M1 and M2. The lack of effect is particularly striking in the case of patient M4 where 83% of the cells were co-expressing the pEGFP construct. Again the results were consistent in separate transfection experiments as shown by the results for patient M3.
Stable populations of transformed fibroblasts transfected with the pRc/CMV/PDH E1[alpha] construct and selected with G418 were analysed for both enzyme activity and expression of PDH E1[alpha] mRNA. All of the cell lines from patients with defined PDH E1[alpha] mutations again showed a significant increase in enzyme activity (Table 2 ). Northern blot analysis of mRNA derived from the same population of cells showed very high levels of the 1.6 kb transcript, presumably derived from the pRc/CMV/PDH E1[alpha] plasmid (Fig. 1 A, lane 3 and B, lane 2). Stably transfected cell populations isolated from the two patients with no demonstrable PDH E1 gene mutations, M3 and M4, did not show any significant change in activity (Table 2 ).
Transformed fibroblast lines from patients with PDH deficiency were generated to permit analysis of expression of the normal PDH E1[alpha] cDNA in different genetic and biochemical backgrounds. Generation of transformed fibroblast cell lines provides large amounts of material for analysis and this is a particular advantage for patient cells which grow poorly or are at late passage when the diagnosis is made. Transformed fibroblasts were also necessary to enable selection of populations of stably transfected cells sufficient to allow repeat enzyme assays on several occasions and other investigations, such as northern blots, to be performed on identical populations. Cloning of transformed fibroblast lines from female patients heterozygous for frameshift mutations in exon 10 of the PDH E1[alpha] gene also allowed analysis of the consequences of these mutations in a population of cells expressing only the mutant X chromosome. As well as abolishing enzyme activity, both of these frameshift mutations result in a reduced level of E1[alpha] mRNA. This is a recognised consequence of some nonsense and frameshift mutations and may be due to an increased rate of degradation, although the precise mechanism remains to be elucidated (21 ). Reduction in mRNA levels with nonsense and frameshift mutations appears to be related to the position of the mutation in the coding sequence and is more likely with mutations toward the 5' end (22 ). In contrast to the mutations in the two female patients, the frameshift mutation in patient M1, which is in the codon for the fourth last amino acid, does not affect the level of PDH E1[alpha] mRNA.
The PDH E1[alpha] cDNA was inserted into the mammalian expression vector pRc/CMV (Invitrogen Inc.). In this plasmid, expression in eukaryotic cells is driven by the cytomegalovirus (CMV) promoter. The PDH E1[alpha] cDNA was derived from the plasmid PDH1c (20 ), which has the human liver E1[alpha] coding sequence inserted into the plasmid Bluescribe after the addition of EcoRI linkers. The insert was excised from PDH1c by cleavage with EcoRI and introduced into the NotI site of pRc/CMV after the overhanging ends of both the insert and vector had been filled in using the Klenow fragment of DNA polymerase I. The entire PDH E1[alpha] insert of the pRc/CMV/PDH E1[alpha] construct was sequenced to ensure that no errors had been incorporated during cloning.
Primary fibroblasts were routinely transformed using a plasmid (T22) containing SV40 DNA sequences (kindly supplied by Dr P.H.Gallimore, University of Birmingham). The cells were plated in 25 cm2 flasks and transfected when 60% confluent with 4 [mu]g/ml T22 DNA and 8 [mu]l/ml lipofectin (GIBCO) in a total of 3 ml Optimem I (GIBCO). After 16 h incubation the medium was replaced by Basal Medium Eagle (BME) with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 [mu]g/ml streptomycin. Cells were allowed to recover for 24 h after which cultures were split 1/4 to reduce the risk of overgrowth by non-transformed cells. After 10-14 days, colonies of rapidly dividing cells with a transformed phenotype were visible. Well separated colonies were isolated using cloning cylinders.
pRc/CMV/PDH E1[alpha] DNA was prepared for transfection studies using a Qiagen plasmid maxi-prep kit. Conditions for transient transfection studies were optimised using a luciferase vector, pGL2 (Promega) and measurement of luciferase activity in a luminometer using the Promega Luciferase Assay system. Transfection was achieved by electroporation using an Easyject Plus electroporator (Equibio) and highest activities were obtained using a twin pulse mode and a cell density of ~107 cells/ml. For electroporation with pRc/CMV/PDH E1[alpha], 25 [mu]g of DNA was mixed with 0.8 ml of cell suspension in a 4 mm cuvette. An initial high voltage pulse, with settings of 900 V, 0.5 [mu]F and 99 [Omega] and a time constant of 0.018 ms was followed by a low voltage pulse with settings of 250 V, 1950 [mu]F and 99 [Omega] and a time constant of 26 ms. Cells were immediately diluted into 15 ml culture medium and plated into 80 cm2 flasks. Medium was changed after 24 h and assays were performed ~40 h after transfection.
In some experiments the proportion of transfected cells was estimated by co-transfecting with 4 [mu]g of the vector pEGFP which encodes a variant of green fluorescent protein (Clontech). An aliquot of the cells was cultured on coverslips, fixed with 4% paraformaldehyde, counterstained with propidium iodide, and the proportion of green fluorescing cells counted.
For selection of stably transfected cell populations, transformed fibroblasts were harvested and resuspended at a density of between 5 * 106 and 1 * 107 per ml. Cell suspension (0.8 ml) was electroporated with 25 [mu]g plasmid DNA with settings of 250 V, 1500 [mu]F and infinite resistance, giving a time constant of ~24 ms. Cells were immediately diluted with culture medium and allowed to recover for 48 h before plating at 3 * 105 cells/80 cm2 flask. After 12-14 days selection in medium containing 400 [mu]g/ml G418, many discrete colonies appeared. These were trypsinised and expanded as bulk populations in G418 for enzyme assay and preparation of RNA for northern blots.
Overall PDH activity was measured in the fibroblasts as described by Wicking et al. (27 ) after maximal activation of the enzyme complex with dichloroacetate and using [1-14C]pyruvate as substrate. Estimations were performed in duplicate in each assay. For stably transfected cells, variability between assays was determined by repeated measurements of the individual cell lines. In transient transfections, the consistency of the results was demonstrated in separate transfection experiments with the same cell lines.
Total cytoplasmic RNA was extracted from fibroblast cell pellets by lysis of the cells, pelleting of the nuclei and phenol:chloroform extraction (28 ). Total RNA (10 [mu]g) was electrophoresed through 1% agarose gels containing 2% formaldehyde and transferred to Boehringer Mannheim Positively Charged membrane in 10% SSC. The PDH E1[alpha] transcripts were detected by hybridisation with a digoxygenin-labelled riboprobe transcribed from pRc/CMV/PDH E1[alpha] using a DIG RNA labelling kit (Boehringer Mannheim).
As an internal control, a probe to 2-oxoglutarate dehydrogenase (OGDH) was included. This was transcribed from a construct synthesised from a 700 bp fragment of OGDH cDNA which was amplified by PCR using the forward primer 5'-GATCGAATTCAGCGGTTCTTGCAGATGTG-3', which binds between nucleotides (nt) 2449 and 2466 of the cDNA sequence reported by Koike et al. (29 ) and the reverse primer 5'-AGCTTCTAGACCCTAGGCAGCATCTACGAG-3', which binds between nt 3126 and 3145. The forward primer incorporates an EcoRI site and the reverse primer, an XbaI site. As these enzymes do not cut within the amplified fragment, the entire 700 bp fragment could be cloned into the EcoRI and XbaI sites of the riboprobe vector pSPT18 (Boehringer Mannheim).
Detection of digoxygenin-labelled riboprobes hybridised to immobilised RNA was performed using a Chemiluminescent Detection Kit (Boehringer Mannheim). The chemiluminescent signal was visualised by autoradiography, exposing the membrane to Kodak X-OMAT AR film for 3-6 h depending on the strength of the signal.
Transiently transfected cells were plated onto glass coverslips, and cultured for 48-72 h. Cells were rinsed three times with phosphate buffered saline and fixed for 1 min with methanol. Expression of PDH E1[alpha] protein was demonstrated using an affinity purified E1[alpha]-specific antibody as previously described (7 ).
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*To whom correspondence should be addressed. Tel: +44 1865 275214; Fax: +44 1865 275318; Email: gkb@bioch.ox.ac.uk
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