We have shown previously that a variant allele of the short-chain acyl-CoA dehydrogenase (SCAD) gene, 625G -> A, is present in homozygous form in 7% of control individuals and in 60% of 135 patients with elevated urinary excretion of ethylmalonic acid (EMA). We have now characterized three disease-causing mutations (confirmed by lack of enzyme activity after expression in COS-7 cells) and a new susceptibility variant in the SCAD gene of two patients with SCAD deficiency, and investigated their frequency in patients with elevated EMA excretion. The first SCAD-deficient patient was a compound heterozygote for two mutations, 274G -> T and 529T -> C. These mutations were not present in 98 normal control alleles, but the 529T -> C mutation was found in one allele among 133 patients with elevated EMA excretion. The second patient carried a 1147C -> T mutation and the 625G -> A polymorphism in one allele, and a single point mutation, 511C -> T, in the other. The 1147C -> T mutation was not present in 98 normal alleles, but was detected in three alleles of 133 patients with elevated EMA excretion, consistently as a 625A-1147T allele. On the other hand, the 511C -> T mutation was present in 13 of 130 and 15 of 67 625G alleles, respectively, of normal controls and patients with elevated EMA excretion, and was never associated with the 625A variant allele. This over-representation of the haplotype 511T-625G among the common 625G alleles in patients compared with controls was significant (P<0.02), suggesting that the allele 511T-625G-like 511C-625A-confers susceptibility to ethylmalonic aciduria. Expression of the variant R147W SCAD protein, encoded by the 511T-625G allele, in COS-7 cells showed 45% activity at 37°C in comparison with the wild-type protein, comparable levels of activity at 26°C, and 13% activity when incubated at 41°C. This temperature profile is different from that observed for the variant G185S SCAD protein, encoded by the 511C-625A allele, where higher than normal activity was found at 26 and 37°C, and 58% activity was present at 41°C. These results corroborate the notion that the 511C-625A variant allele is one of the possible underlying causes of ethylmalonic aciduria, and suggest that the 511C -> T mutation represents a second susceptibility variation in the SCAD gene. We conclude that ethylmalonic aciduria, a commonly detected biochemical phenotype, is a complex multifactorial/polygenic condition where, in addition to the emerging role of SCAD susceptibility alleles, other genetic and environmental factors are involved.
The importance of fatty acid [beta]-oxidation to sustain mitochondrial energy metabolism during periods of fasting is underscored by the potentially fatal manifestations of known inborn errors in this pathway (1-3). In particular, acyl-CoA dehydrogenase deficiencies constitute an important group of disorders which have been the focus of extensive investigations at the biochemical and molecular levels over the last decade. Despite this effort, short-chain acyl-CoA dehydrogenase (SCAD) deficiency still remains a poorly defined entity, since <10 verified patients with this disorder have been described (4-10), and only one case has been characterized previously at the molecular level (11).
In sharp contrast to this limited number of patients with established SCAD deficiency, elevation of urinary ethylmalonic acid (EMA), a characteristic biochemical finding of diminished SCAD activity, is detected frequently in patients undergoing a metabolic work-up for a clinical suspicion of a metabolic disorder (12-14). Because urinary EMA elevation most likely reflects a cellular accumulation of butyryl-CoA (15), which is secondary to reduced SCAD catalytic activity, these patients are correctly considered as possibly having SCAD deficiency.
In a previous study (16), we analysed the coding region of the SCAD gene in two patients with SCAD deficiency, whose diagnosis was based on the findings of elevated EMA excretion in urine and low SCAD activity in cultured skin fibroblasts (6). Surprisingly, the main result of the study was not the identification of new severe disease-causing mutations, but the discovery of a polymorphic variation in the SCAD gene, 625G/A. This variation results in a glycine/serine amino acid polymorphism at position 185 in the mature SCAD protein (G/S209 in the pre-SCAD protein) (16,17). In expression studies, the G185S variant enzyme, encoded by the 625A variant allele, was catalytically active, and therefore not a disease-causing mutation in a conventional sense, but the protein was more thermolabile than the wild-type enzyme. In addition, the 625A variant allele was found in homozygous form in 60% of 135 patients with elevated EMA excretion, analysed because of a suspicion of a metabolic disorder, compared with 7% of individuals in the general population (12). Taken together, these data suggest that the 625A variant allele is a susceptibility allele of the SCAD gene, which causes elevation of EMA in urine and, in combination with other genetic and/or environmental factors, may lead to a functional impairment of the enzyme's catalytic activity that, in some cases, is sufficiently severe to justify a diagnosis of SCAD deficiency in vitro.
Isolated ethylmalonic aciduria may thus be a biochemical phenotype that can be subdivided into at least two groups. One group is comprised of patients with conventional SCAD deficiency caused by disruptive mutations in the SCAD gene. The second group consists of patients with a polygenic/multifactorial condition caused by the presence of a number of susceptibility alleles, located either in the SCAD locus, as is the 625A variant allele, or elsewhere. This distinction implies that patients with elevated EMA excretion, who are not homozygous for the 625A susceptibility allele, are the most likely candidates for carrying severe disease-causing mutations in the SCAD gene or, alternatively, other susceptibility polymorphisms.
In the present study, we report the identification of three severe disease-causing SCAD gene mutations (274G -> T, 529T -> C and 1147C -> T) and one new disease-associated susceptibility mutation (511C -> T) in two patients, one of whom was homozygous and the other heterozygous for the common 625G allele. We have determined the frequencies of the mutations in normal control individuals and in a population of patients with elevation of EMA excretion in urine, and we have investigated the mechanism by which the mutations effect the protein function.
Direct sequencing of both strands of the PCR-amplified SCAD cDNA fragments covering the coding region from both patients and a control gave consistent results. Comparing the sequences from the patients with the normal (wild-type) sequence revealed five nucleotide substitutions, leading to amino acid replacements, i.e. 511C -> T, 1147C -> T and 625G -> A in patient 1, and 274G -> T and 529T -> C in patient 2 (Table 1). For patient 1, the assignment of the mutations to the two alleles was done by parental analysis, using PCR assays specific for each mutation. The mother was homozygous for the 625A variant allele and heterozygous for the 1147C -> T mutation, and the father was heterozygous for the 511C -> T mutation and homozygous for the common 625G allele, showing that the 1147C -> T mutation was in the 625A variant allele and the 511C -> T mutation was associated with the common 625G allele. Since DNA samples from the parents of patient 2 were not available, a fragment containing both mutations, 274G -> T and 529T -> C, of the patient's cDNA was amplified by PCR, cloned and sequenced. Eleven clones were examined: four harboured the 274G -> T mutation together with the normal 529T, and four showed the 529T -> C mutation together with the normal 274G, indicating that the mutations are located in separate alleles, both of which harbour the common 625G. The remaining three clones showed a variety of PCR errors.
Table 1.
To define the prevalence of the mutations in the general population and in patients with elevated excretion of EMA, we analysed 49 control individuals and 133 patients with elevated urinary EMA (Table 1). None of the control individuals carried the 274G -> T, 529T -> C or 1147C -> T mutations. In contrast, the 511C -> T variation was present in heterozygous form in seven individuals and in homozygous form in one, giving an allele frequency of 9.2%.
In the group of patients with elevated EMA excretion, the 274G -> T mutation was not present, and the 529T -> C and 1147C -> T mutations were found in heterozygous form in one and three patients, respectively. In contrast, the 511C -> T variation was detected in heterozygous form in 13 and in homozygous form in one patient, giving an allele frequency of 5.6%.
To estimate the frequency of the 511T allele in the general population more precisely and to define which haplotypes are represented by the 511C/T and 625G/A polymorphisms, we searched for these two polymorphisms in a total of 86 control individuals, including the 49 individuals analysed above. The 86 individuals were all independent members of 51 unrelated families (father, mother, child), enabling unambiguous definition of the haplotypes in the total of 172 alleles. Three haplotypes were defined: 42 alleles harboured 511C and 625A (511C-625A), 117 were 511C-625G and 13 were 511T-625G. In conclusion, the allele frequency of the 511T variation is 7.6%, and no allele contained both the 511T and 625A variations.
In the group of 133 patients with elevated EMA excretion, 39 were 625G/625A heterozygotes, 14 were 625G homozygotes and 80 were 625A homozygotes (12). The 511T homozygous and two of the 13 heterozygous 511C/511T patients (see above) were homozygous for the 625G allele, thus assigning the 511T variation to the common 625G allele. Although the remaining 11 511C/511T heterozygous patients were also 625A/625G heterozygotes, this combination is still compatible with the presence of 511T and 625G in the same allele. Therefore, these individuals are likely to be compound heterozygotes with 511T-625G in one allele and 511C-625A in the other, in agreement with the results from the control individuals and from the father of patient 1, which consistently showed that the 511T variation was located in the common 625G allele. In conclusion, the analysis indicated that the total of 266 alleles in patients with elevated EMA excretion contained 199 511C-625A alleles, 52 511C-625G alleles and 15 511T-625G.
Western blot analysis of the steady-state amounts of SCAD protein in cultured fibroblasts from the patients and a control individual is shown in Figure 1. Compared with the amounts of [alpha]/[beta]-electron transfer flavoprotein (ETF), patient 2 produced negligible amounts of SCAD protein (lane 3), whereas patient 1 showed appreciable amounts (lane 2). The additional bands in the gels were a consistent finding, and have no influence on the quantification of the SCAD protein. The amounts of SCAD protein in the fibroblasts are in agreement with the residual SCAD activities observed in vitro (see Materials and Methods).
To evaluate the nature of the mutations further, we overexpressed all variant proteins, including the wild-type and the G185S (625A) variant, in COS-7 cells. Northern blot analysis of all constructed vectors showed similar level of expression in COS-7 cells (results not shown), indicating that transcription and processing of SCAD mRNA is not affected by the mutations. The steady-state levels of SCAD variant proteins and the activity levels of SCAD variant enzymes were analysed in extracts from transfected COS-7 cells cultured at 26, 37 and 41°C. The activity of the SCAD wild-type enzyme at each temperature, calculated as the average of two transfection experiments, was taken as reference (100%) to normalize the results obtained by expression of variant proteins (Fig. 2). The wild-type activities at 26 and 41°C were 68 and 96%, respectively, of that at 37°C. At all three temperatures, immunoreactive protein with a size of 42 kDa corresponding to that of mature SCAD protein (18) was observed (Fig. 2, lane 1). When the R147W (511T) and G185S (625A) variant proteins were expressed at 37°C, the relative SCAD activities were 45 and 136%, respectively. Mature variant SCAD protein was also detected in western blots (lanes 4 and 7). Lowering the temperature to 26°C increased the activity of the R147W and G185S variants to 85 and 183% of wild-type activity, respectively. This increase in activity was also reflected in greater abundance of immunoreactive protein for the two variant proteins.
The goals of this study were to identify new mutations in patients with SCAD deficiency and to investigate the association between SCAD gene mutations and elevated EMA in urine. This condition clearly occurs much more frequently than primary SCAD deficiency, a rare inborn error of fatty acid oxidation, and EMA has emerged as a possible biochemical marker for a group of multifactorial/polygenic conditions.
We identified five missense mutations in two patients with enzymatically proven SCAD deficiency, two of them located in the same allele of one patient (patient 1, Table 1). Expression in COS-7 cells confirmed the pathologic nature of the 274G -> T and 529T -> C mutations found in patient 2, as expected by the previous findings of low SCAD activity (6,8) and undetectable amounts of SCAD protein in fibroblasts from this patient (Fig. 1). When the alleles with the 1147C -> T and 511C -> T mutations were expressed, the first showed no detectable activity and the second revealed considerable residual activity, findings compatible with the high residual SCAD activity (~25% of control) and the presence of significant amounts of SCAD protein in cultured fibroblasts of this patient (Fig. 1). Still, in view of the fact that no other amino acid changing mutations were found in the SCAD cDNA in patient 1, the combination of these two mutations, 1147C -> T and 511C -> T, must be sufficient to cause SCAD deficiency. The additional presence in patient 1 of the 625A variant in the allele harbouring the 1147C -> T mutation was functionally irrelevant, since the 1147C -> T mutation alone will abolish SCAD activity.
The disease-causing nature of three of the identified variant alleles (274T, 529C or 1147T) is supported by their absence in 98 alleles from normal individuals (Table 1). The allele frequency of the 511T variant, on the contrary, was 9.2%. This variation is therefore a polymorphism, and its potential clinical significance is not as clear as the three other mutations. However, the variable amounts of R147W variant protein, encoded by the 511T variant allele, which were produced in its active form at different expression temperatures suggest that the 511T variant is also a conditional predisposition allele, that may cause SCAD deficiency and ethylmalonic aciduria, depending on the synergetic effect of additional genetic factors and/or cellular conditions. This mechanism is consistent with the one we have proposed previously for the 625A variant allele on the grounds of a highly significant over-representation of this allele in patients with elevated urinary EMA (12). In this population, 90% of subjects carried the variant 625A allele, and 60% were homozygous.
To evaluate this group of patients further at the molecular level, they were tested for the four identified missense mutations found in patients 1 and 2 with SCAD deficiency (Table 1). The 274G -> T mutation was not found, indicating a very low frequency of this mutation in patients with elevated EMA excretion. The 529T -> C and 1147C -> T mutations were found in one and three patients, respectively. Since the single patient harbouring the 529T -> C mutation was homozygous for the common 625G allele, this mutation was obviously located in a 625G allele, as was the case in patient 2. On the other hand, the 1147T variant was associated with homozygosity for the variant 625A allele in all three cases, showing that the presence of the 625A variation does not exclude the presence of other mutations.
Among the 133 patients with elevated excretion of EMA, the variant 511T allele was found with an allele frequency of 5.6% (Table 1), which is not significantly different from the 9.2% found in the 49 control individuals initially tested (Table 1), and the 7.6% found in a larger group of 86 individuals from 51 families, from which the haplotypes represented by the 511C/T and 625G/A polymorphisms could be assigned unambiguously. Interestingly, the 511T variant consistently was located in the common 625G allele in the control population. The same association was very strongly indicated in all patients with elevated EMA excretion carrying the 511T variant allele, as shown in Table 2, where the frequencies of the four possible allelic haplotypes defined by the 511C/T and 625G/A polymorphisms are shown. Based on the qualified assumption that 511T-625A alleles do not exist, the 625A alleles can be excluded for the purpose of statistical analysis of the prevalence of the 511T variation, as shown by the grouping of the data in the lower part of Table 2, where the data were divided into two partially overlapping groups: the alleles harbouring the common 511C, i.e. alleles with haplotypes 511C-625A and 511C-625G, and the alleles harbouring the common 625G, i.e. 511T-625G and 511C-625G. Statistical tests ([chi]2) within these two groups lead to the following conclusions. (i) The alleles with the haplotype 511C-625A are significantly (P<0.01) over-represented in the 511C group of alleles in patients (79.3%) compared with normal controls (26.4%). This is a refinement of our previous results (12), where the over-representation of the variant 625A allele in patients with elevation of urinary EMA was first demonstrated. (ii) The alleles with the 511T-625G haplotype are significantly over-represented (P<0.02) in the 625G group of alleles in patients (22.3%) compared with normal controls (10.0%). The degree of over-representation of the variant 511T allele is much smaller than that calculated for the variant 625A allele (Table 2), but it still indicates a significant association between the variant 511T allele and the occurrence of elevated urinary EMA excretion.
The conclusion that the R147W (511T) and G185S (625A) variant enzymes are disease-predisposing (but not disease-causing in a conventional sense) mutations is thus supported by their over-representation in patients with elevated EMA excretion and by the expression studies, showing that the amounts of these variant proteins and their enzyme activities are temperature-dependent.
Table 2.
To search for the functional basis for the conditional nature of the R147W and G185S variant enzymes, we also considered their localization in the SCAD protein structure. Based on the known crystal structure of bacterial acyl-CoA dehydrogenase, which is homologous to mammalian SCAD (19) (amino acid sequence similarity: 55%), it appears that these mutations are not located in the vicinity of the active sites of the enzyme. It is possible, however, that they compromise either the folding or the tetramerization of the enzyme, as we have observed previously in the case of a number of other SCAD and MCAD variant proteins (20-22) (T.J. Corydon et al., unpublished). The COS-7 cell experiments corroborated this notion, since the R147W variant enzyme showed residual activity only slightly lower than wild-type at low temperature, but has decreased activity at normal temperature and low activity at elevated temperatures (Fig. 2). The temperature profile for the G185S variant protein was different, with a dramatic effect from higher than wild-type activity at 37°C to 58% of wild-type activity at 41°C. Taken together with our previous observation that the thermal stability of the G185S variant enzyme is lower than that of the normal enzyme (12), this indicates that the observed temperature profile for the G185S variant enzyme is caused predominantly by a decreased stability of the tetrameric structure. In contrast, thermal stability experiments with the R147W variant (results not shown) showed that the thermostability of the tetrameric enzyme protein is indistinguishable from that of the normal enzyme. Since this is the behaviour of traditional temperature-sensitive folding mutations (23,24), it is possible that the R147W variation confers instability to intermediates of the folding pathway of the protein (21). Further experiments are in progress to elucidate the exact molecular mechanism underlying this observation, but the present results indicate that the steady-state amounts of the G185S and R147W variant enzymes in cells are dependent on the efficiency of the folding and degradation machinery. Experimental confirmation of this hypothesis will require a full characterization of the mitochondrial protein quality control system, involving chaperones and proteases (25), elements also involved in the handling of other acyl-CoA dehydrogenase mutant proteins (20,21).
The presence of the 625A and 511T variant alleles, either in homozygous (one 511T/511T and 80 625A/625A patients) or in compound heterozygous form (11 511T/625A patients), in 69% of patients with elevated EMA excretion is probably an important reason why this condition was not recognized previously by conventional enzyme assays. Measurements of the SCAD activity in cultured skin fibroblasts of patients with elevated EMA excretion, who are homozygous for the variant 625A allele, have shown inconsistently either normal or variably decreased activities (G. Vockley, M.J. Corydon and N. Gregersen, unpublished). These data indicate that fibroblasts may not represent a sufficiently reliable specimen for diagnostic evaluation of ethylmalonic aciduria/SCAD deficiency.
In spite of these unresolved issues, the present study has substantiated the view that ethylmalonic aciduria is a complex condition, with potential clinical consequences. In many patients, the condition is multifactorial, with the variant 625A allele emerging as the principal predisposing factor. Other susceptibility alleles, such as the 511T allele, and the presence of severe disease-causing mutations, such as 274G -> T, 529T -> C and 1147C -> T, may contribute together with as yet unknown cellular or genetic factors. Such factors include the protein quality control machinery or factors affecting the oxidative phosphorylation pathway, since an association between respiratory chain disorders and elevation of EMA excretion has been reported (26-28). In a small minority of cases with elevated EMA excretion, the disease is classically monogenic and caused by severe mutations such as 274G -> T and 529T -> C in patient 2 or 136C -> T and 319C -> T in the first case with SCAD deficiency to be characterized at the molecular level (11). These two groups, the multifactorial and the monogenic, are, however, not mutually exclusive, since in patients belonging to the monogenic group there may also be additional factors which modify the effect of the mutations, as observed for the variant R147W SCAD enzyme in patient 1 of this study.
In addition to obvious diagnostic benefits, a comprehensive elucidation of the aetiology of ethylmalonic aciduria may also help us to understand better the processes which are important for the biogenesis, processing and degradation of aberrant mitochondrial proteins.
This patient has been reported elsewhere (10): briefly, this Spanish girl presented at 3 months of age with coughing, vomiting and poor feeding. Bronchopneumonia was diagnosed and she recovered in 2 weeks. At 5 months of age, she was readmitted with hypotonia and a low level of consciousness. The urinary organic acid profile showed an increased level of EMA (126 mmol/mol creatinine; controls: <18 mmol/mol creatinine). At 4 years of age, her growth and development were normal. The range of EMA excretion during follow-up was 36-94 mmol/mol creatinine. Specific SCAD activity in cultured fibroblasts, determined by the ETF reduction assay (29), was reduced to 25% of controls, the results being confirmed in two independent laboratories.
This African-American male presented with a shaking-staring spell on the first day of life (6,8). EMA excretion was strikingly abnormal (3900 mmol/mol creatinine), and remained elevated when tested during asymptomatic periods (range: 16-175 mmol/mol creatinine). Specific SCAD activity in fibroblasts showed severe deficiency, with <10% of control activity.
Blood spots for DNA analysis were collected prospectively in Germany, Denmark, the Czech Republic, Spain and the USA from patients with elevated EMA excretion (18-1185 mmol/mol creatinine). Typically, these patients were referred to a metabolic centre in order to investigate a possible inborn error of metabolism. Many of these patients showed neuromuscular signs such as hypotonia, convulsions and developmental delay, others showed symptoms suggestive of a possible fatty acid oxidation disorder, such as episodes of hypoglycaemia and lethargy. In about half of the cases, the abnormal EMA excretion was documented in more than one urine specimen. In these patients, there was no apparent correlation between the nature and severity of the symptoms and the level of EMA excretion. The samples used in the present study were blood spots from 133 of the 135 patients analysed previously for the SCAD 625G/A polymorphism (12).
DNA was isolated from lymphoblastoid cells of 86 Danish control individuals obtained from the cell bank repository at Panum Institute, Copenhagen, Denmark. They were genetically independent members of 51 unrelated families (father, mother, child), enabling unambiguous assignment of polymorphisms. The use of this control material was approved by the appropriate human investigation committees in Denmark.
RNA and DNA were extracted from cultured fibroblasts of patients, blood cells of the parents of one case, and lymphoblastoid cells of controls. Total RNA was prepared from frozen cells by standard methods (30), and cDNA was synthesized from 1 µg of total RNA using a commercial kit (Clontech, Palo Alto, CA). The cDNA was used immediately for PCR.
cDNA fragments covering the SCAD-coding region were produced by PCR in two steps. The first step was the production of the full-length coding region cDNA, which subsequently was used in the second PCR amplification step to give two overlapping cDNA fragments. Primer sequences are shown in Table 3. The primers for the cDNA amplification were designed from the published SCAD cDNA sequence (18). To obtain efficient amplification of full-length and 5' cDNA fragments it was necessary to locate the 5' primer close to the ATG start codon site at position -20 to -1. Primers located upstream of this region resulted in poor amplification yields. We have later shown that the first nine nucleotides, from -32 to -24, in the published sequence are incorrect. The correct sequence is accessible through EMBL/GenBank/DDBJ databases (31). In the second PCR reaction, each set of primers contained one 5'-biotinylated primer, enabling purification and sequencing of each complementary strand. PCR buffers and conditions were standard, except that the buffer contained 8% dimethylsulfoxide (DMSO) to resolve the GC-rich secondary structure of the cDNA.
Table 3.
Before sequencing, PCR products were purified as single strands by means of strepavidin-coated magnetic beads (DYNA-beads, Dynal, Norway) according to a previously published method (32). Sequencing of the coding region of the SCAD cDNA sequence in both strands was performed by Sequenase and four fluorescence labelled dideoxynucleotides as described in the manufacturer's standard protocol (Perkin Elmer, Norwalk, CA). However, because of the proximity of the PCR primers to the ATG site, the 5' end of the cDNA (six nucleotides in patient 1 and 33 nucleotides in patient 2) could not be sequenced unambiguously with 5' primers. The 5' end of the cDNAs therefore were sequenced repeatedly using different 3' primers. The numbering of the nucleotides in SCAD cDNA starts from the ATG initiation codon for pre-SCAD, and the numbering of the amino acids in the SCAD protein starts at the cleavage site between the leader peptide and the mature protein (18).
A PCR-generated SCAD cDNA fragment covering the sequence from 240 to 774 bp (primers shown in Table 3) was cloned into the pGEM-T vector (Promega, Madison, WI), and the recombinant plasmid DNA was sequenced by cycle sequencing using AmpliTaq DNA polymerase FS and four fluorescence labelled dideoxynucleotides as described by the manufacturer (Perkin Elmer).
Detection of the 274G -> T and 1147C -> T mutations was performed after the introduction of an artificial restriction site by PCR-based mutagenesis, according to procedures described previously (33). To enhance the specificity of the detection of the 274G -> T mutation, this assay was performed in two steps, as indicated by the two sets of primers listed in Table 3.
Due to the extreme GC-richness of the sequences flanking bases 511 and 529 of the SCAD cDNA, PCR amplification was inefficient from many of the blood spots, resulting in small amounts of product. To overcome this problem, we developed a sensitive SSCP analysis method, using fluorescent phosphoramidite (TET and 6-FAM)-labelled PCR primers (Perkin Elmer) flanking exon 5 of the SCAD gene, which contains bases 511 and 529. The genomic structure of the SCAD is available (34) and accessible from EMBL/GenBank/DDBJ databases (31,35). The primer sequences are shown in Table 3. Determination of the conformers of the 511C, 511T, 529T and 529C alleles was performed on an ABI 377 sequencer (Perkin Elmer) using 6.5% polyacrylamide gels (AT Biochem, Malvern, PA) containing 8% glycerol and electrophoresis at 20°C. This method gave consistent results and was validated by direct sequencing.
Extracts from frozen cultured skin fibroblasts containing ~70 µg of total protein were subjected to polyacrylamide gel electrophoresis using a 12% Tris-glycine gel with 4% stacking gel (Ready Gel) (BioRad, Hercules, CA). Blotting was performed essentially as described by Blake et al. (36). The blots were incubated with polyclonal anti-SCAD (18) or anti-[alpha]/[beta]-ETF antibodies (37) (kindly provided by Kay Tanaka, Yale University, CT), and SCAD and ETF were visualized by chemiluminescence, using alkaline phosphatase-conjugated secondary antibody and the luminescence substrate CSPD (Tropix, Bedford, MA).
For expression of the variant SCAD proteins [G68C (274G -> T), R147W (511C -> T), W153R (529T -> C) and R359C (1147C -> T)] together with the wild-type protein, a series of adeno-associated virus (AAV)-based plasmid vectors were constructed. First, site-directed mutagenesis of the control SCAD cDNA was performed in each case according to the megaprimer PCR procedure (38). Amplified products were first cloned in the pCRII vector (InVitrogen, CA). The insert sequences of the isolated plasmid vectors were checked by sequencing, and cloned into the eucaryotic AAV vector, pMP6 (kindly provided by Ramila Philips, Applied Immune Sci., Santa Clara, CA). For expression of the G185S variant protein, a cDNA insert from a bacterial pCpreA625 expression plasmid containing the 625A variant allele (12) was cloned into pMP6.
COS-7 cells were cultured in RPMI 1640 (Biological Industries, Beit Haemak, Israel) containing 10% fetal calf serum (Gibco BRL), and transfected by the calcium phosphate co-precipitation method as previously described (39). The transfected cells were cultured at 26, 37 and 41°C and harvested 1-2 days post-transfection. Disruption of cells, northern and western blotting were performed as described previously (39), except that the anti-SCAD antibody and the detection system mentioned above for the analyses of cultured fibroblasts were used. The SCAD activity in COS-7 cell extracts was determined by the ferricenium ion-based assay (40). Since the expression of the enzyme protein depends on the transfection efficiency, the transfections with all constructs were done in the same carefully controlled experiment, and two independent transfection experiments were performed, as shown for the activity measurements in Figure 2.
We thank our colleagues from clinical departments in Denmark, Germany, Spain, the Czech Republic and the USA for their valuable contribution to this study by providing data and samples of urine, blood and cultured skin fibroblasts of patients with elevated urinary EMA excretion. The study was supported by the Danish Centre for Human Genome Research, The Danish Medical Research Council, The Institute of Experimental Clinical Research, Aarhus University and Aarhus County Research Initiative.
Human Molecular Genetics
Pages
Introduction
Results
Sequence analysis of SCAD cDNA from patients' cultured fibroblasts
Studies of control individuals and patients with elevated EMA excretion
SCAD protein analysis in cultured fibroblasts from the patients
Expression of SCAD variants in COS-7 cells
Discussion
Materials And Methods
Patient 1
Patient 2
Patients with elevated EMA excretion
Control individuals
SCAD cDNA synthesis and sequence analysis
Cloning of cDNA fragments from patient 2
Specific PCR assays for the 274G -> T and 1147C -> T mutations
Single-strand conformation polymorphism (SSCP) analysis for the 511C -> T and 529T -> C mutations
Protein analysis in fibroblasts by western blotting
Expression of SCAD variant enzymes in COS-7 cells
Acknowledgements
References
Patient
625G/625A
Mutation
Frequency
allele
cDNA
Protein
Control
populationPatients with
EMA excretion
1
625G
511C -> T
R147W
9/98
15/266
(9.2%)
(5.6%)
625A
1147C -> T
R359C
0/98
3/266
2
625G
274G -> T
G68C
0/98
0/266
625G
529T -> C
W153R
0/98
1/266
Haplotype
No. of alleles found
Controls
EMA patients
511C-625A
42 (24.4%)
199 (74.8%)
511T-625A
0
0
511C-625G
117 (68.0%)
52 (19.6%)
511T-625G
13 (7.6%)
15 (5.6%)
Total alleles
172 (100%)
266 (100%)
511C-625A/total 511C
42/42 + 117 (26.4%)
199/199 + 52 (79.3%)
511T-625G/total 625G
13/117 + 13 (10.0%)
15/52 + 15 (22.3%)
DNA fragment
Primers
5'-Nucleotide
positionRestriction
enzyme
Full-length
5'-TGGGACTGTGTCTGTCGCCC-3'
cDNA: -20
coding cDNA
5'-CTGGCTCCCGCGCCTTCC-3'
cDNA: 1285
5'-cDNA fragment
5'-TGGGACTGTGTCTGTCGCCC-3'
cDNA: -20
5'-CCCTGGCTCCCCCAGGATGC-3'
cDNA: 774
3'-cDNA fragment
5'-CCCCATGCCAACGCCTGGGC-3'
cDNA: 645
5'-CTGGCTCCCGCGCCTTCC-3'
cDNA: 1285
1147C -> T
5'-GCCGGCAGAGCGGCGGTAC-3'
cDNA: 1128
KpnI
specific assay
5'-CTGGCTCCCGCGCCTTCC-3'
cDNA: 1285
GGTAC/C
274G -> T
I: 5'-GGCCATGGACGTGCCCGAGG-3'
cDNA: 240
specific assay
5'-ACTCCGGTGGAGGCGCAGCC-3'
cDNA: 341
II: 5'-CGAGGAGCTTGGCGCAGCT-3'
cDNA: 255
PvuII
5'-ACTCCGGTGGAGGCGCAGCC-3'
cDNA: 341
CAG/CTG
511C -> T and 529T -> C
5'-[TET]-GTGCGCTGAGCCCTGGGTCT-3'
exon 5: -30
SSCP assay
5'-[6-FAM]-CCGGCTGAACCCCTCTCTGG-3'
exon 5: +30
Cloning of cDNA
5'-GGCCATGGACGTGGCCCGAGG-3'
cDNA: 240
from patient 2
5'-CCCTGGCTCCCCCAGGATGC-3'
cDNA: 774
REFERENCES
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C. B. Pedersen, P. Bross, V. S. Winter, T. J. Corydon, L. Bolund, K. Bartlett, J. Vockley, and N. Gregersen Misfolding, Degradation, and Aggregation of Variant Proteins: THE MOLECULAR PATHOGENESIS OF SHORT CHAIN ACYL-CoA DEHYDROGENASE (SCAD) DEFICIENCY J. Biol. Chem., November 28, 2003; 278(48): 47449 - 47458. [Abstract] [Full Text] [PDF]
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K. P. Battaile, J. Molin-Case, R. Paschke, M. Wang, D. Bennett, J. Vockley, and J.-J. P. Kim Crystal Structure of Rat Short Chain Acyl-CoA Dehydrogenase Complexed with Acetoacetyl-CoA. COMPARISON WITH OTHER ACYL-CoA DEHYDROGENASES J. Biol. Chem., March 29, 2002; 277(14): 12200 - 12207. [Abstract] [Full Text] [PDF]
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T. J. Corydon, P. Bross, T. G. Jensen, M. J. Corydon, T. B. Lund, U. B. Jensen, J.-J. P. Kim, N. Gregersen, and L. Bolund Rapid Degradation of Short-chain Acyl-CoA Dehydrogenase Variants with Temperature-sensitive Folding Defects Occurs after Import into Mitochondria J. Biol. Chem., May 22, 1998; 273(21): 13065 - 13071. [Abstract] [Full Text] [PDF]
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