The molecular basis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in compound heterozygous patients: is there correlation between genotype and phenotype?
The molecular basis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in compound heterozygous patients: is there correlation between genotype and phenotype?Brage Storstein Andresen1,*, Peter Bross1, Szabolcs Udvari1,2, Jean Kirk3, George Gray4, Stanislav Kmoch5, Nestor Chamoles6, Inga Knudsen1, Vibeke Winter1, Bridget Wilcken7, Ichiro Yokota8, Kimberly Hart9, Seymour Packman9, Jean Paul Harpey10, Jean Marie Saudubray11, Daniel E. Hale12, Lars Bolund13, Steen Kølvraa13 and Niels Gregersen1
1Center for Medical Molecular Biology, Åarhus University Hospital and Faculty of Health Science, Åarhus, Denmark, 2Department of Biochemistry, University College Dublin, Dublin, Ireland, 3Department of Paediatric Biochemistry, Royal Hospital for Sick Children, Edinburgh, UK, 4West Midlands Regional Laboratory for Neonatal Screening and Inherited Metabolic Disorders, Children's Hospital, Birmingham, UK, 5Institute for Inherited Metabolic Diseases, General Faculty Hospital and Charles University 1st School of Medicine, Prague, Czech Republic, 6Fundacion Para El Estudio De Las Infermedades Neurometabolicas, Uriarte, Buenos Aires, Argentina, 7NSW Biochemical Genetics & Newborn Screening Services, The New Children's Hospital, Parramatta, Australia, 8Yale University School of Medicine, Department of Genetics, New Haven, CT, USA, 9Division of Medical Genetics, University of California, San Francisco, CA, USA, 10Clinique de Pediatrie et Genetique Medicale, Groupe Hospitalier Pitié-Salpétrière, Paris, France, 11Departement de Pediatrie, Clinique de Genetique Medicale, Groupe Hospitalier Necker-Enfants Malades, Paris, France, 12Department of Pediatrics, The University of Texas Health Care Center at San Antonio, San Antonio, TX, USA and 13Institute of Human Genetics, University of Åarhus, Åarhus, Denmark
Received December 3, 1996;Revised and Accepted February 12, 1997
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of mitochondrial [beta]-oxidation. It is potentially fatal, but shows a wide clinical spectrum. The aim of the present study was to investigate whether any correlation exists between MCAD genotype and disease phenotype. We determined the prevalence of the 14 known and seven previously unknown non-G985 mutations in 52 families with MCAD deficiency not caused by homozygosity for the prevalent G985 mutation. This showed that none of the non-G985 mutations are prevalent, and led to the identification of both disease-causing mutations in 14 families in whom both mutations had not previously been reported. We then evaluated the severity of the mutations identified in these 14 families. Using expression of mutant MCAD in Escherichia coli with or without co-overexpression of the molecular chaperonins GroESL we showed that five of the missense mutations affect the folding and/or stability of the protein, and that the residual enzyme activity of some of them could be modulated to a different extent depending on the amounts of available chaperonins. Thus, some of the missense mutations may result in relatively high levels of residual enzyme activity, whereas the mutations leading to premature stop codons will result in no residual enzyme activity. By correlating the observed types of mutations identified to the clinical/biochemical data in the 14 patients in whom we identified both disease-causing mutations, we show that a genotype/phenotype correlation in MCAD deficiency is not straightforward. Different mutations may contribute with different susceptibilities for disease precipitation, when the patient is subjected to metabolic stress, but other genetic and environmental factors may play an equally important role.
Medium-chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3) is one of four chain length specific, straight-chain acyl-CoA dehydrogenases which initiate the mitochondrial [beta]-oxidation of straight-chain acyl-CoA esters (1 ,2 ). The human MCAD gene consists of 12 exons that span more than 44 kb encoding a precursor protein of 421 amino acids (3 ,4 ). The first 25 amino acids in the human enzyme are suggested to make up the leader peptide (5 ). On import into mitochondria, the leader peptide is cleaved off, producing the mature MCAD monomer (42.5 kDa). In the mitochondria, folding of the MCAD monomer is assisted by the chaperonin, mitochondrial heat shock protein 60 (Hsp60) (6 ). Subsequently the monomers are assembled to the functional homotetrameric MCAD enzyme, with one molecule of flavin adenine dinucleotide (FAD) per subunit (7 ,8 ). The three dimensional structure of the MCAD enzyme from pig liver has been characterized by X-ray crystallography (9 ).
MCAD deficiency is the most common defect of mitochondrial [beta]-oxidation in humans (10 ,11 ). It is a potentially fatal, autosomal recessively inherited defect, which most often presents in the first years of life (11 -13 ).
The clinical manifestations of MCAD deficiency are diverse, but usually they include fasting induced non-ketotic hypoglycemia with lethargy which may develop into coma (11 ,13 ). Biochemically the disease is characterized by urinary excretion of C6-C10 dicarboxylic acids, acylglycine and acylcarnitine conjugates [hexanoylglycine, phenylpropionylglycine, suberylglycine and octanoylcarnitine (11 ,14 ,15 )]. Between 20 and 25% of patients die suddenly at first presentation of the disease (13 ,16 ), but affected patients, who remain without symptoms for years have also been reported (15 -18 ).
Part of the explanation for this wide clinical spectrum is that fasting stress, often in connection with fever, is usually required to trigger the disease. Differences in MCAD genotype could also be important for the disease manifestation. However, it is well known that the entire clinical spectrum of MCAD deficiency can be observed among patients who are homozygous for the prevalent G985 mutation (13 ,16 ; unpublished results). This clearly shows that the most common MCAD genotype-80% of patients are homozygous for the G985 mutation (19 ,20 )-cannot be correlated with a specific disease phenotype. Despite this, it could still be speculated that patients with other MCAD genotypes exhibit a more distinct correlation between MCAD genotype and clinical phenotype.
Unfortunately, little is known about the prevalence of, and the disease phenotype associated with the different non-G985 mutations in MCAD deficiency. So far 14 non-G985 mutations have been reported (15 ,18 ,21 -24 ), but only a small number of patients have been tested for all of them.
Due to the very variable clinical spectrum of the disease, much effort has been directed towards elucidating the molecular cell pathology in MCAD deficiency. In particular the molecular defect of the mutant protein (K304E) resulting from the prevalent G985 mutation has been investigated (6 ,25 -28 ), but also three other missense mutations have been studied (18 ,25 -27 ,29 ). These experiments showed that the folding and/or assembly of these four mutant MCAD proteins is compromised, and that this to a variable extent can be modulated by increasing the amount of available chaperonins. In addition, recent experiments have shown that the K304E mutant protein resulting from the G985 mutation is temperature sensitive. Both in human lymphoblasts and in expression experiments in Escherichia coli the folding of the K304E mutant protein could be improved by decreasing the temperature (26 ).
On this basis, we have suggested that interindividual variation of endogenous factors, especially those of the mitochondrial folding machinery, may be important in determining the susceptibility to disease precipitation (10 ,26 ,27 ).
On the other hand, Brackett and co-workers (21 ) have stated that compound-heterozygosity with the G985 mutation and one of the non-G985 mutations, A583, gives rise to a particularly severe presentation of the disease. Based on this one might speculate whether in some instances the nature of the non-G985 mutation may in itself be sufficient to explain the phenotypic expression of MCAD deficiency in patients who are not homozygous for the G985 mutation.
In the present study we have investigated if and to what extent a correlation between MCAD genotype and clinical phenotype can be made. We have determined the prevalence of all the 14 known and seven new mutations in members from 52 unrelated families with MCAD deficiency who all harbor one or two non-G985 allele(s), using a combination of mutation-specific PCR/restriction enzyme digestion based assays and direct sequencing of PCR amplified DNA. Then we characterized the molecular consequences of the identified mutations using our E.coli based expression system with or without co-overexpression of the chaperonins GroEL and GroES (25 -27 ). Finally, we related the MCAD genotype to the observed clinical phenotype in the 14 patients in whom we identified non-G985 mutations in the present study.
We used PCR/restriction enzyme digestion based mutation specific assays to test for five of the known non-G985 mutations located outside exon 11, namely T157, [Delta]343-48, A447, C730 and A799 (20 ,24 ,29 ,30 ). The A583 mutation located in exon 7 (21 ), and the seven known non-G985 mutations located in exon 11 (15 ,18 ,20 ,30 ,31 ) were tested for by direct sequencing of amplified genomic DNA.
In 50 of 52 families with non-G985 mutations the index patients were analyzed. In the remaining two families in whom materials from the index patients were not available, parents and siblings were analyzed instead. Using this approach we identified both disease-causing mutations in 14 of the families. In addition we have in our previous studies identified both mutations in seven of the 52 families (15 ,18 ,29 ).
In families where a non-G985 mutation was identified, we also analyzed all available family members for the mutation. Mutations identified by the mutation specific assays were verified in the index patient by direct sequencing of amplified genomic DNA (not shown). Moreover, in those patients in whom we identified non-G985 mutations we investigated whether the two mutations were the only mutations present in the two MCAD alleles. This was done by performing direct sequence analysis of amplified genomic DNA covering the coding region of all exons of the MCAD gene from these patients (see Materials and Methods). In all the index patients from the 14 families described below, and the seven families previously described (15 ,18 ,29 ) the presence of more than one mutation in each allele was excluded in this way. Thus, we were able to ensure that the phenotypes of these patients were not influenced by additional unrecognized mutations in their MCAD gene.
The T157 mutation was only detected in the two families who have previously been described (29 ).
In two unrelated families (Families 1 and 2) of British origin we identified the A447 mutation. In Family 1 the mutation was identified in the index patient and his asymptomatic brother (Fig. 1 A), both of whom are carriers of the G985 mutation. This corresponds well with the fact that the MCAD enzyme defect had also been indicated in the brother, although he had never experienced any symptoms. We also detected the A447 mutation in an unrelated British family (Family 2). In this family the index patient and her three year younger asymptomatic sister, both of whom are carriers of the G985 mutation, are heterozygous for the A447 mutation. This patient is the same as the one described previously by Yokota and co-workers (20 ).
In order to test for the presence of the A583 mutation located in exon 7 (21 ), we PCR amplified and sequenced a 305 bp fragment spanning the entire exon 7 and part of the flanking introns.
The A583 mutation was identified in four unrelated families of Scottish, Irish, Northern-Irish and American origin. In all four families (Families 4-7) the index patient was heterozygous for the G985 mutation.
Analysis of the parents from Family 4 showed that the mother was heterozygous for the A583 mutation and the father was heterozygous for the G985 mutation. In Family 5 analysis of the parents showed that the mother was heterozygous for the A583 mutation and the father was heterozygous for the G985 mutation. Analysis of three healthy siblings, two of whom are heterozygous for the G985 mutation, showed that the A583 mutation was not present in any of them. In Families 6 and 7 there was no material available from family members.
In a French family (Family 8) sequence analysis of the index patient, who is heterozygous for the G985 mutation, showed that this patient was heterozygous for a 474T -> G transition (G474). The G474 mutation has not previously been reported. It changes the second nucleotide of the TAT codon encoding tyrosine133 of the mature MCAD protein to a TAG stop codon. If translated such a protein would be truncated by nearly two thirds (234 amino acids) of the coding region.
In a Czech family (Family 9) sequence analysis of the index patient, who is heterozygous for the G985 mutation, showed her to be heterozygous for a deletion of a T ([Delta]474) corresponding to position 474 or 475 of the cDNA sequence. The [Delta]474 mutation changes the TGT codon encoding cysteine134 to GTG (valine) and leads to a premature stop codon only one codon downstream. If translated such a protein would be truncated by nearly two thirds (232 amino acids) of the coding region. The [Delta]474 mutation has not previously been reported.
In a French family (Family 10) sequence analysis of the index patient showed that he was heterozygous for a 577A -> G transition (G577). We have previously reported that this patient, his father and his healthy sister are heterozygous for a 13 bp insertion mutation ([nabla]999-12) in exon 11 (15 ). The mother was found to be heterozygous for the G577 mutation. The G577 mutation has not previously been reported. It changes the first nucleotide of the ACC codon encoding threonine168 of the mature MCAD protein to GCC for alanine.
We have previously reported the results from sequencing of exon 11 from compound heterozygous patients from 36 unrelated families with MCAD deficiency (18 ). In the present study we sequenced exon 11 from the remaining 16 of our 52 families. In all the investigated persons the results from the G985 assay and the sequencing were in agreement. Only one of the seven known non-G985 mutations located in exon 11 (18 ,22 ) was identified. In an American family (Family 11) we identified the previously described 4 bp deletion ([Delta]1100-03) (30 ,31 ), corresponding to cDNA position 1100-1103 or 1102-1105. The mutation was identified in heterozygous form in the index patient and his father. The index patient is heterozygous for the G985 mutation. Interestingly, his mother is homozygous for the G985 mutation, but she has always been asymptomatic.
The encoded mutant protein would be truncated by 37 amino acids. The deleterious nature of this mutation has previously been verified by expression of recombinant mutant protein in E.coli (30 ).
In an Australian family (Family 12) sequence analysis of the index patient, who is heterozygous for the G985 mutation, showed that this patient was heterozygous for a 1055A -> G transition (G1055). The G1055 mutation has not previously been reported. It changes the second nucleotide of the TAT codon encoding tyrosine327 of the mature MCAD protein to TGT for cysteine.
In a family from Argentina (Family 13) sequence analysis of the index patient, who is heterozygous for the G985 mutation, showed that she was also heterozygous for a 1 bp insertion of a T ([nabla]1189) in exon 11 corresponding to cDNA position 1189 or 1190. The [nabla]1189 mutation will lead to a shifted reading frame from tyrosine362 ending with a premature stop codon only four codons downstream. The encoded mutant protein would be truncated by 31 amino acids. This mutation has not previously been reported.
In a Scottish family (Family 14) direct sequence analysis of the amplified fragment from the index patient showed a normal sequence through exon 11, except that the nucleotides AT corresponding to position 955-956 or 957-958 or alternatively the nucleotides TA corresponding to position 956-957 of the cDNA were deleted. Sequence analysis of her parents revealed that they were both heterozygous for this 2 bp deletion ([Delta]955-56), agreeing well with the fact that they are first cousins, and showing that the index patient is homozygous for the [Delta]955-56 mutation. The [Delta]955-56 deletion changes the reading frame from serine295 and leads to a premature stop codon only four codons downstream. If the mutant MCAD mRNA is translated the resulting protein would be truncated by 99 amino acids.
In order to investigate if the premature stop codon had any effect on the steady state MCAD mRNA amount in patient cells we analyzed MCAD mRNA from the patient by Northern blot analysis (Fig. 2 ). The results from the Northern blot analysis document that the steady state amounts of mutant MCAD mRNA are drastically decreased to a level not detectable by this type of analysis.
In addition to the mutations dealt with above, a deletion covering the entire exon 11 and 12 of the MCAD gene has been reported (23 ). By examination of our total patient material, including both families where the patient is homozygous for the G985 mutation and the 52 families where the patient is not homozygous for the G985 mutation, we excluded the exon 11-12 deletion mutation in 114 families with MCAD deficiency. In 65 families with patients who were diagnosed as being homozygous for the G985 on the basis of results from the G985 assay, hemizygosity, and thus the presence of the deletion, could be ruled out because both parents were found to be heterozygous for the G985 mutation. In 49 non-G985 homozygous patients the deletion could be ruled out either because they were found to be heterozygous for the G985 mutation or because they were found to be heterozygous for the polymorphic silent 1161A -> G mutation in exon 11 (32 ). These results document that the deletion mutation is very rare.
aAll nucleotide numbering is according to Kelly and co-workers (3). bIncludes results from references 15,18 and 29cI/T = Identified alleles/Tested allelesdThe reference for the initial identification of the mutation is given.Nd = Not detected.
The results from the mutation specific assays and sequencing of exon 7 and 11 are listed in Table 1 and 2 . Also the data from the 1991 Workshop report (33 ) have been included, and a compilation of the data from 1991 and the data from the present study is also listed. By comparing patient lists from the laboratories involved, all duplicates have been excluded from the list. Moreover, only one sibling from each family is counted in the tallies.
The 12 different non-G985 mutations identified in patients investigated in the present study (Table 1 ) can be divided into six missense mutations and six mutations that either directly or indirectly results in creation of a premature stop codon (PSC). There can be little doubt that all six of the identified PSC mutations are disease-causing (34 ). In contrast, the disease-causing nature of the identified missense mutations is more complicated to predict.
Therefore they were characterized by expression of recombinant mutant protein in E.coli. A total of 10 missense mutations (including the G985 mutation) have been identified in our sample of MCAD-deficient patients (Table 1 and 2 ). We have previously analyzed four of them (G985, T157, C977 and A1008) using an E.coli based expression system with or without chaperonin co-overexpression and a eukaryotic expression system in COS-7 cells (18 ,25 -27 ,29 ).
Summary of clinical, biochemical and molecular data
DA, dicarboxylic aciduria; Gly, glycine conjugates present in urine; Car, carnitine conjugates present in plasma/urine; mo, months; [dagger], dead.Parents: Y = one mutation identified in each parent. N = material not available.
In the present study we have analyzed five of the remaining six missense mutations present in our patients. Expression of mature wild-type MCAD protein and the mature forms of the mutant MCAD proteins resulting from the A447, G577, A583, A799, G1055 and G985 mutations were performed in E.coli JM109 cells. Transformed JM109 cells harbored expression plasmids encoding the mature form of wild-type or one of the mutant MCAD proteins and the plasmid pGroESL encoding the chaperonins GroEL and GroES or the control plasmid pCaP which lack the GroESL chaperonin genes (see Materials and Methods). The A347 mutation was not tested because it was identified after we had finished testing all the other mutations. Our results from expressing the mutant proteins M124I (A447 mutation), T168A (G577 mutation), G170R (A583 mutation), G242R (A799 mutation) and Y327C (G1055 mutation) without GroESL co-overexpression show that they all exhibit a severely decreased enzyme activity when compared with the wild-type protein (Fig. 3 ). This indicates the disease-causing nature of these mutations. Moreover, our results show that the enzyme activity of all the mutant proteins, except G170R and T168A, to a varying degree can be increased by co-overexpression of the GroESL chaperonins, and thus that compromised folding and/or tetramer formation is part of the molecular defect mechanism.
One of the original goals of the present study was to establish whether any of the known mutations (15 ,18 ,21 ,23 ,24 ,29 ) are prevalent among the non-G985 chromosomes, and thus, if testing with one or a few mutation specific assays would improve the diagnostic efficiency. Our results show that this is not the case. None of the 14 mutations previously described, nor the seven new mutations identified in the present study are prevalent among the non-G985 chromosomes (Table 1 ).
The second aim of this study was to investigate if or to what extent the different MCAD genotypes can be correlated to the variable clinical phenotypes of the disease.
In an attempt to investigate this we evaluated the molecular defect of all mutations identified in the patients of the present study. Firstly, our results from E.coli expression of the mutant proteins document the disease-causing nature of all the missense mutations investigated. It is noteworthy that the disease-causing nature of the A447 and A799 mutations have not previously been verified by expression studies, despite the fact that these mutations were identified already in 1991 (20 ,30 ). Secondly, our results show that the different non-G985 mutations may in fact cause different and variable levels of residual MCAD enzyme activity. Furthermore, the amount of active mutant enzyme is for some of the mutant variants dependent on the level of available chaperonins. In line with the results of our previous reports (18 ,26 ,27 ,29 ) the G985 mutation was shown to cause a milder defect than the other mutations, except for the A447 mutation (characterized herein) and the T157 mutation (characterized in refs 26 ,27 and 29 ).
It is interesting to note that E.coli cells expressing the M124I mutant protein, resulting from the A447 mutation, or the G242R mutant protein, resulting from the A799 mutation, display up to 80 and 40%, respectively, of the MCAD enzyme activity levels measured from cells expressing wild-type MCAD, when the folding defect is alleviated by chaperonin co-overexpression. Thus, the residual MCAD enzyme activity of the G242R mutant protein is similar to the K304E mutant protein and that of M124I is nearly twice as high. It could thus be hypothesized that the seriousness of the A447, A799 and G985 mutations could be influenced by the capacity of the folding machinery in patient cells.
In contrast to this the mutant proteins (T168A, G170R and Y327C) resulting from the G577, A583 and G1055 mutations show very little (Y327C) or no (T168A and G170R) response to chaperonin co-overexpression. This indicates that for these mutations the defect in enzyme biogenesis/stability is so severe that the capacity of the folding machinery in patient cells would have a much smaller (if any) impact on the residual MCAD enzyme activity. It is interesting to note that the G577 mutation is the first mutation in the MCAD protein that directly affects an amino acid (threonine168) known to be functionally important. Threonine168 is located in the [beta]-sheet domain and forms a hydrogen bond with the flavin adenine dinucleotide ring of the bound FAD (9 ). It could be speculated that replacement of this important residue would affect FAD binding and/or perhaps disturb electron transfer. Despite this it appears that the main molecular defect of the G577 mutation is on enzyme biogenesis, like all the other identified missense mutations. Results by Saijo and Tanaka corroborate this observation, as they have shown that FAD cofactor binding is crucial for folding/oligomerization of the MCAD tetramer (35 ). In relation to the possible phenotypic effect of the G577 mutation in the index patient, it should however be noted that we have previously observed that in patient cells the steady state amounts of MCAD mRNA from G577 mutation bearing alleles were for unknown reasons drastically decreased (15 ). The G577 mutation should not by itself interfere with pre-mRNA processing, and sequence analysis of all exon/intron junctions of the MCAD gene from the index patient did not reveal any abnormalities.
Interestingly, glycine170, which is changed to an arginine by the A583 mutation, is located in the same turn in the [beta]-sheet domain close to the FAD molecule (9 ) as threonine168. It may thus seem reasonable to suggest that the drastic substitution of glycine170 with the much larger and charged arginine in this part of the enzyme may disturb FAD-cofactor binding and thus the folding/oligomerization of the MCAD tetramer. The fact that a similar mutation of the homologous glycine residue to a valine in isovaleryl-CoA dehydrogenase has been observed in a patient with IVD deficiency (36 ), corroborates the notion that disturbance of the molecular architecture of this part of the acyl-CoA dehydrogenase enzymes may affect their folding pathway. In this connection it is also interesting to note that a mutation of glutamate365 (E365K) in glutaryl-CoA dehydrogenase (GCD), which is homologous to tyrosine327 (changed to a cysteine by the G1055 mutation) in MCAD, has recently been reported to cause GCD enzyme deficiency, resulting in glutaric-aciduria in patients (37 ). Moreover, we have previously reported that arginine28 (changed to a cysteine by the T157 mutation) in MCAD is homologous to the mutated arginine22 (R22W) in the short-chain acyl-CoA dehydrogenase (SCAD), causing SCAD deficiency (29 ,38 ). Thus, mutations in the homologous amino acids for as many as three of the amino acids changed by the 12 missense mutations identified in MCAD deficiency have been found in other acyl-CoA dehydrogenase deficiencies. This indicates that the mutational pattern in the acyl-CoA dehydrogenase enzymes is similar, and that mutations often affect residues which do not seem to be directly involved in enzyme function, but rather are important for enzyme biogenesis.
Whereas most of the missense mutations may result in residual MCAD activity above background, the identified PSC mutations, which would result in premature termination if translated, would not be expected to give any residual MCAD enzyme activity. Firstly, we have shown that for two of the mutations [[Delta]955-56 in the present study and [nabla]999-12 in our previous study (15 )] where patient cells were available, that they both result in drastically decreased amounts of steady state mutant MCAD mRNA. This is in itself sufficient to explain the enzyme defect, and it is in good agreement with the observation that this type of mutation usually results in severely decreased amounts of mutant mRNA (34 ). Secondly, for those mutations where the truncated enzymes have been tested by expression (1045C -> T, [Delta]1100-03 and 1150G -> T) they were shown to result in very low amounts of immunoreactive protein and no residual enzyme activity, when compared to wild-type MCAD (18 ,30 ). Finally, all eight PSC mutations in the MCAD gene (Table 2 ) encode proteins lacking the active site glutamate376 (39 ,40 ).
We have thus shown that the different non-G985 mutations may cause different levels of residual MCAD enzyme activity. Furthermore, our results from the present and the results from previous studies (15 ,18 ,21 ,29 ) document that the entire clinical spectrum of MCAD deficiency, from remaining without symptoms for several years (Family 11) to patients who die suddenly in the neonatal period (21 ,29 , and Families 9 and 10 of the present study), can be observed in patients who are not homozygous for the G985 mutation (Table 2 ). Based on this it may seem reasonable to expect that differences in MCAD genotype may be important in determining the clinical phenotype. In both Families 1 and 2 there exists a sibling who is also compound heterozygous with the A447 and the G985 mutations, but only the index patients have experienced clinical symptoms (Table 2 ). Moreover, neither of the index patients in these two families has suffered from more than one episode, and there were no dead siblings. This is in good agreement with the observation from our expression experiments that the A447 mutation is rather mild, and the folding of the resulting mutant protein (M124I) may be influenced by the available levels of chaperonins. It could be speculated that in these two families a good capacity of the folding machinery is able to ensure a rather high residual MCAD activity from the two mutant proteins. This is further corroborated by the fact that measurements of the [beta]-oxidation activity towards tritium labeled myristic acid in cultured fibroblasts from the index patient in Family 2 repeatedly have shown the highest activity that we have ever observed among MCAD patients (results not shown). On the other hand, the index patient in Family 11, who is compound heterozygous with the severe [Delta]1100-03 deletion in one allele and the G985 mutation in the other allele, remained without clinical symptoms until 12 years of age (Table 2 ), despite the fact that he suffered from metabolic stress several times during the first two years of life. Moreover, we have recently reported that individuals who harbor the severe [nabla]999-12 insertion mutation or the severe 1045C -> T stop codon mutation in one allele, together with the G985 mutation in the other allele, may remain without symptoms (15 ,18 ). These data clearly show that patients compound heterozygous with the G985 mutation in one of the alleles may remain without clinical symptoms, irrespective of the nature of the second mutation.
Thus the residual activity from the K304E mutant protein expressed from only one G985 mutation bearing allele may, under some circumstances, be sufficient to avoid disease precipitation.
Mismatching nucleotides are indicated by underlining. The cDNA positions of primers located in the coding region are indicated relative to the start codon. The enzymes used for detection of the different mutations in the mutation specific assays are indicated.
In light of this, it is interesting to note that Brackett and co-workers (21 ), based on their observation of sudden neonatal death in two unrelated families, suggested that compound heterozygosity with the A583 and G985 mutations represents a particularly severe genotype (A583/G985) which correlates to a severe, early presentation of the disease. Although our results from E.coli expression of the mutant protein (G170R) resulting from the A583 mutation corroborate the notion that it is one of the most severe missense mutations, the clinical/biochemical data from the four unrelated patients of the present study (Families 4-7, Table 2 ) with the A583/G985 MCAD genotype do not indicate that this genotype is particularly severe. None of the four index patients with the A583/G985 genotype have suffered from an early or particularly severe presentation (Table 2 ). Moreover, Kelly and co-workers (31 ) reported a patient who died suddenly at 22 months of age and had an identical MCAD genotype as the index patient in Family 11, and we have previously reported two unrelated families where the index patients were compound heterozygous for the T157 and G985 mutations, but presented very differently (e.g. one mild episode versus sudden neonatal death) (29 ). These data clearly document the fact that G985 compound heterozygous patients with identical MCAD genotypes may have very different clinical presentations. Taken together with the fact that G985 compound heterozygous patients with the most severe non-G985 mutations may remain asymptomatic for years, and the fact that G985 compound heterozygous patients with the mildest MCAD mutations (i.e. T157) may present with the most severe symptoms i.e. sudden unexpected death at day 3 (29 ) our data show that it is questionable whether it is reasonable, like Brackett and co-workers do, to state that a particular MCAD genotype may explain a severe form of the disease.
However, in all the patients discussed above the effect of the non-G985 mutant allele may be masked by the simultaneous presence of an allele with the relatively mild G985 mutation. It could be speculated that in these patients interindividual variation in the folding capacity of their cells, by modulating the residual activity of the K304E protein resulting from the G985 mutation, is an equally important factor to consider.
The identification of the index patient in Family 14 is in this connection particularly interesting. She is the first patient identified who is homozygous for a mutation other than the G985 mutation. Despite the fact that she does not produce any functional MCAD, she has only experienced two mild episodes at 20 and 29 months of age. Obviously, the nature of her mutation ([Delta]955-56), for which she is homozygous, implies that her (lack of) MCAD enzyme activity would not be responsive to any factors capable of modulating the biogenesis of the MCAD protein. Thus, she may illustrate that other endogenous factors may also be decisive for the risk of disease precipitation. The overlapping substrate activity (2 ,41 ,42 ) from the other acyl-CoA dehydrogenases is an obvious candidate to provide the enzyme activity sufficient to overcome the `enzyme block' imposed by the lack of MCAD and to ascertain sufficient flow through the [beta]-oxidation in a normal situation. Also, interindividual variations in enzymes involved in the metabolism of potentially toxic intermediates of fatty-acid oxidation accumulating due to the MCAD enzyme defect, could be speculated to be of importance for disease precipitation.
In conclusion, the present study suggests that variation in the MCAD genotype, despite the fact that different MCAD mutations may contribute with different susceptibilities for disease precipitation, is probably not the primary explanation for the observed clinical variation in this potentially severe disease. Finally, it should be emphasized that in the vast majority of patients disease presentation occurs during or following a period of metabolic stress (Table 2 ), for instance after periods of fasting or due to viral infections. Thus, environmental factors are crucial determinants of disease precipitation and the frequency of exposure to metabolic stress and the severity of the metabolic stress are therefore probably the most important determinants of the clinical phenotype, irrespective of the MCAD genotype.
A total of 52 unrelated families with patients in whom MCAD deficiency was not caused by homozygosity for the prevalent G985 mutation were studied. The PCR/restriction enzyme digestion based assay for the prevalent mutation, G985, was performed as described elsewhere (19 ). In 45 of the families the index patients were diagnosed as being heterozygous for the G985 mutation, five index patients did not possess the G985 mutation at all, and in two families material from the index patient was not available for DNA analysis, but investigation of the parents indicated that the index patients should be expected to be heterozygous for the G985 mutation. The number of alleles with non-G985 mutations in the 52 families is therefore 57. In all studied families, the index patient had experienced clinical symptoms of MCAD deficiency, and excreted urine organic acids, including acylglycines, indicative of MCAD deficiency (14 ,15 ). In the majority of the patients, further evidence for the MCAD defect was provided by at least one of the following methods: in vitro enzyme assays (43 -46 ), [beta]-oxidation activity measurements (47 ,48 ) or the phenylpropionic acid loading test (49 ).
In six of the families mutations in exon 11 have previously been reported (15 ,18 ), and in two of the families the T157 mutation has previously been reported (29 ). The case history and the biochemical characterization, as well as the identification of one of the disease-causing mutations ([nabla]999-12 insertion in exon 11) in Family 10 has previously been described (15 ). The index patient in Family 12 has previously been reported as case 4 elsewhere (50 ). A summary of the clinical and biochemical data on all the 14 patients in whom both disease-causing mutations are described for the first time are listed in Table 2 , together with the disease causing mutations identified in each patient. More detailed case histories, and clinical-biochemical data are available upon request.
Genomic DNA was isolated from blood samples and from cultured skin fibroblasts by standard methods (51 ). DNA from blood spots and whole cells were liberated as described elsewhere (19 ,52 ).
PCR amplification and subsequent purification of a 421 bp fragment spanning the entire exon 11 and part of the flanking introns of the MCAD gene was performed as described elsewhere (18 ). Bi-directional solid-phase dideoxy-sequencing was performed using a prism T7 kit (Applied Biosystems) and a semiautomated ABI 373A sequencer (Applied Biosystems). PCR amplification, purification and sequence analysis of a 232 bp fragment spanning the entire exon 5 and part of the flanking introns and of a 305 bp fragment spanning the entire exon 7 and part of the flanking introns was carried out as described for exon 11. PCR, purification and sequence analysis of all exons of the MCAD protein coding region was performed as described for exons 5, 7 and 11. All primers used are listed in Table 3 .
We designed PCR/restriction enzyme digestion based mutation specific assays for the T157, A447, C730 and A799 mutations (20 ,29 ), all four of which are located outside exon 11. These assays are all based on the principle of PCR-based introduction of diagnostic restriction enzyme sites like the G985 assay (19 ). In addition to the diagnostic site, control sites for enzyme cleavage efficiency are introduced in both the variant and control alleles in all assays. The restriction enzymes and primers used for the different assays are listed in Table 3 . It should be noted that the assay for the T157 mutation has been changed from that originally described (29 ), so that the antisense primer is now located in intron 3. In addition, we have substituted the use of SacI with the neoschizomeric enzyme EcoICRI, which is more efficient and less expensive.
The 6 bp deletion mutation ([Delta]343-48) in exon 5 (24 ) was tested for by RsaI digestion of the 232 bp PCR amplified fragment spanning the entire exon 5 and part of the flanking introns. From normals the 232 bp fragment can be cut into fragments of 21, 103 and 108 bp with RsaI. If the [Delta]343-48 deletion is present, RsaI digestion should instead produce two fragments of 21 and 205 bp.
The PCRs for the different mutation specific assays were performed with the following program for 35 cycles: denaturation for 1 min at 94oC, annealing for 2 min at either 50oC (157C -> T assay) or 57oC (447G -> A assay; 730T -> C assay; 799G -> A assay) and chain elongation at 74oC for 3 min. The PCRs were carried out in an automated thermal cycler (Perkin Elmer Cetus, Norwalk, CT) with either genomic DNA, boiled blood spots or boiled whole cells as DNA source in 100 [mu]l with 15 pmol of each primer and 2 U recombinant Taq polymerase (Perkin Elmer Cetus, Norwalk, CT or Northumbria Biologicals, Northumberland, UK). After digestion with the appropriate restriction enzyme, samples were electrophoresed in 12% or 14% polyacrylamide gels together with uncleaved samples. The bands were visualized by staining with ethidium bromide.
Total RNA was prepared from frozen patient fibroblasts using either a RNeasy kit (Qiagen, Hilden, Germany) or a RNAzol kit (WAK-Chemie, Bad Homburg, Germany). Northern blot analysis of 15 [mu]g total RNA from patient and 10 [mu]g total RNA from control fibroblasts and 10 [mu]g total RNA from control placenta was performed as described previously using the MCAD probe (53 ), a VLCAD probe (54 ) and a [beta]-actin probe (Clonetech, Palo Alto, CA).
The mutations A447, G577, A583, A799 and G1055 were all introduced by PCR based site-directed mutagenesis into the previously described expression vector pWt (26 ), which harbors a gene encoding the mature part of human MCAD under the control of a lac promoter. PCR based site-directed mutagenesis was performed according to the method described by Sarkar and Sommer (55 ) using Pfu polymerase (Stratagene, La Jolla, CA) and mutagenic primers for each of the mutations with the pWt plasmid as template. The PCR-products harboring the respective mutations, were digested with restriction enzymes (PstI/EcoRI or EcoRI/HindIII) purified and cloned back into the pWT plasmid, replacing the corresponding fragment with the wild-type sequence. To confirm that no PCR-derived errors were present in the exchanged fragments, all the constructed plasmids were sequenced.
Growth (at 31oC) and disruption of bacterial cells, SDS-PAGE, native PAGE and Western blot analysis was performed as described previously (25 ,26 ). Measurements of the thermal inactivation profiles were performed as previously described for the K304E mutant protein (26 ). The activity measurements were performed by the ferricenium ion based assay (56 ). The concentration of protein in E.coli JM109 cell extracts was determined with a modified Bradford assay kit (BioRad, Richmond, CA). All experiments were performed at least two times, with enzyme activity measurements performed in duplicates each time.
We thank the following physicians/investigators who made the initial diagnosis and/or contributed skin fibroblasts, blood samples or blood spots from their patients: Drs R.A. Chalmers (UK), E. Christensen (Denmark), D. Curtis (UK), P. Divry (France), M. Duran (The Netherlands), B. Francois (Belgium), Y. Gillerot (Belgium), L. Hagenfeldt (Sweden), E. Holme (Sweden), J.B.C. deKlerk (The Netherlands), W.J. Kleijer (The Netherlands), E. Kunert (Germany), W. Lehnert (Germany), J.V. Leonard (UK), S. Lyonnet (France), R. Matalon (Florida), R. Moore (UK), E. Naugthen (Ireland), R. Pollit (UK), W.J. Rhead (Iowa), C.R. Roe (Texas), S. Rosthøj (Denmark), R. Santer (Germany), J. Scholte (The Netherlands), P. Smit (The Netherlands), S. Snodgrass (UK), P. Thornton (Ireland), F. Trefz (Germany), L. Van Maldergem (Belgium), S. Van DerMeer (The Netherlands), C. Vianey-Saban (France), P. Ward (Ireland) and U. Wendel (Germany). This work was supported by grants from The Danish Medical Research Council and The Danish Center for Human Genome Research.
1 Beinert H (1963) Acyl-CoA dehydrogenases. In Boyer, P.D., Lardy, H., Myrback, K. (eds) The Enzymes, Academic Press, New York, Vol.7, pp. 447-476.
2 Aoyama, T., Souri, M., Ushikubo, S., Kamijo, T., Yamaguchi, S., Kelley, R.I., Rhead, W.J. et al. (1995) Purification of human very-long-chain acyl-coenzyme A dehydrogenase and characterization of its deficiency in seven patients. J. Clin. Invest., 95, 2465-2473.MEDLINE Abstract
3 Kelly, D.P., Kim, J.-J.P., Billadello, J.J., Hainline, B.E., Chu, T.W. and Strauss, A.W. (1987) Nucleotide sequence of medium-chain acyl-CoA dehydrogenase mRNA and its expression in enzyme-deficient human tissue. Proc. Natl. Acad. Sci. USA, 84, 4068-4072.MEDLINE Abstract
4 Zhang, Z., Kelly, D.P., Kim, J.-J.P., Zhou, Y., Ogden, M.L., Whelan, A.J. and Strauss, A.W. (1992) Structural organization and regulatory regions of the human medium-chain acyl-CoA dehydrogenase gene. Biochemistry, 31, 81-89.MEDLINE Abstract
5 Matsubara, Y., Kraus, J.P., Ozasa, H., Glassberg, R., Finocchiaro, G., Ikeda, Y., Mole, J. et al. (1987) Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat liver medium-chain acyl coenzyme A dehydrogenase. J. Biol. Chem., 262, 10104-10108.MEDLINE Abstract
6 Saijo, T., Welch, W.J. and Tanaka K (1994) Intramitochondrial folding and assembly of medium-chain acyl-CoA dehydrogenase (MCAD). J. Biol. Chem., 269, 4401-4408.MEDLINE Abstract
7 Hall, C.L. and Kamin, H. (1975) The purification and some properties of electron transfer flavoprotein and general fatty acyl-Coenzyme A dehydrogenase from pig liver mitochondria. J. Biol. Chem., 250, 3476-3486.MEDLINE Abstract
8 Ikeda, Y., Hale, D.E., Keese, S.M., Coates, P.M. and Tanaka, K. (1986) Biosynthesis of variant medium-chain acyl-CoA dehydrogenase in cultured fibroblasts from patients with medium-chain acyl-CoA dehydrogenase deficiency. Ped. Res.,20, 843-847.
9 Kim, J.-J.P., Wang, M. and Paschke, R. (1993) Crystal structures of medium-chain acyl-CoA dehydrogenase from pig liver mitochondria with and without substrate. Proc. Natl. Acad. Sci. USA, 90, 7523-7527.
10 Gregersen, N., Andresen, B.S., Bross, P., Bolund, L. and Kølvraa, S. (1994) Disorders of mitochondrial fatty acid oxidation especially medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In: Farriaux, J.P. and Dhondt, J.L. (eds) New Horizons in Neonatal Screening. Elsevier Science B.V. pp. 247-255.
11 Roe, C.R. and Coates, P.M. (1995) Mitochondrial fatty acid oxidation disorders. In: Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds) The Metabolic and Molecular Basis of Inherited disease. McGraw-Hill, New York, pp. 1501-1533.
12 Vianey-Liaud, C., Divry, P., Gregersen, N. and Mathieu, M. (1987) The inborn errors ofmitochondrial fatty acid oxidation. J. Inher. Metab. Dis.,10 (suppl 1), 159-198.MEDLINE Abstract
13 Iafolla, A.K., Thompson, R.J. and Roe, C.R. (1994) Medium-chain acyl-coenzyme A dehydrogenase deficiency: clinical course in 120 affected children. J. Pediatr., 124, 409-415.MEDLINE Abstract
14 Rinaldo, P., O'Shea, J.J., Coates, P.M., Hale, D.E., Stanley, C.A. and Tanaka, K. (1988) Diagnosis of medium-chain acyl-CoA dehydrogenase deficiency by stable isotope dilution analysis of urinary n-hexanoylglycine and 3-phenylpropionylglycine. N. Engl. J. Med., 319, 1308-1313.MEDLINE Abstract
15 Gregersen, N., Winter, V., Lyonnet, S., Saudubray, J.M., Wendel, U., Jensen, T.G., Andresen, B.S. et al. (1994) Molecular genetic characterization and urinary excretion pattern of metabolites in two families with MCAD deficiency due to compound heterozygosity with a 13 basepair insertion in one allele. J. Inher. Metab. Dis., 17, 169-184.MEDLINE Abstract
16 Wilcken, B., Hammond, J. and Silink, M. (1994) Morbidity and mortality in medium chain acyl coenzyme A dehydrogenase deficiency. Arch. Dis. Child., 70, 410-412.MEDLINE Abstract
17 Duran, M., Hofkamp, M., Rhead, W.J., Saudubray, J.M. and Wadman, S.K. (1986) Sudden child death and `healthy' affected family members with medium-chain acyl-CoA dehydrogenase deficiency. Pediatrics,78,1052-57.
18 Andresen, B.S., Jensen, T.G., Bross, P., Knudsen, I., Winter, V., Kølvraa, S., Bolund, L. et al. (1994) Disease-causing mutations in exon 11 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene. Am. J. Hum. Genet.,54, 975-988.MEDLINE Abstract
19 Gregersen, N., Blakemore, A., Winter, V., Andresen, B.S., Kølvraa, S., Bolund, L., Curtis, D. et al. (1991) Specific diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in dried blood spots by a polymerase chain reaction (PCR) assay detecting a point-mutation (G985) in the MCAD gene. Clin. Chim. Acta, 203, 23-34.MEDLINE Abstract
20 Yokota, I., Coates, P., Hale, D.E., Rinaldo, P. and Tanaka, K. (1991) Molecular survey of a prevalent mutation, 985A-to-G transition, and identification of five infrequent mutations in the medium-chain acyl-CoA dehydrogenase (MCAD) gene in 55 patients with MCAD deficiency. Am. J. Hum. Genet., 49, 1280-1291.
21 Brackett, J.C., Sims, H.F., Steiner, R.D., Nunge, M., Zimmerman, E.M., deMartinville, B., Rinaldo, P. et al. (1994) A novel mutation in medium-chain acyl-CoA dehydrogenase causes sudden neonatal death. J. Clin. Invest., 94, 1477-1483.MEDLINE Abstract
22 Tanaka, K., Yokota, I., Coates, P.M., Strauss, A.W., Kelly, D.P., Zhang, Z., Gregersen, N. et al. (1992) Mutations in the medium-chain acyl-CoA dehydrogenase (MCAD) gene. Hum. Mutat.,1, 271-279.MEDLINE Abstract
23 Morris, A.A.M., Taylor, R.W., Lightowlers, R.N., Aynsley-Green, A., Bartlett, K. and Turnbull, D.M. (1995) Medium-chain acyl-CoA dehydrogenase deficiency caused by a deletion of exons 11 and 12. Hum. Mol. Genet., 4, 747-749.
24 Ziadeh, R., Hoffman, E.P., Finegold, D.N., Hoop, R.C., Brackett, J.C., Strauss, A.W. and Naylor, E.W. (1995) Medium-chain acyl-CoA dehydrogenase deficiency in Pennsylvania: Neonatal screening shows high incidence and unexpected mutation frequencies. Pediatr. Res., 37, 675-678.MEDLINE Abstract
25 Bross, P., Andresen, B.S., Winter, V., Kräutle, F., Jensen, T.G., Nandy, A., Kølvraa, S. et al. (1993) Co-overexpression of bacterial GroESL chaperonins partly overcomes non-productive folding and tetramer assembly of E.coli expressed human medium-chain acyl-Coa dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation. Biochim. Biophys. Acta, 1182, 264-274.MEDLINE Abstract
26 Bross, P., Jespersen, C., Jensen, T.G., Andresen, B.S., Kristensen, M.J., Winter, V., Nandy, A. et al. (1995) Effects of two mutations detected in medium-chain acyl-CoA dehydrogenase (MCAD) deficient patients on folding, oligomer assembly, and stability of MCAD enzyme. J. Biol. Chem., 270, 10284-10290.MEDLINE Abstract
27 Jensen, T.G., Bross, P., Andresen, B.S., Lund, T.B., Kristensen, T.J., Jensen, U.B., Winter, V. et al (1995) Comparison between medium-chain acyl-CoA dehydrogenase (MCAD) mutant proteins over-expressed in bacterial and mammalian cells. Hum. Mutat., 6, 226-231.MEDLINE Abstract
28 Yokota, I., Saijo, T., Vockley, J. and Tanaka, K. (1992) Impaired tetramer assembly of variant medium-chain acyl-coenzyme A dehydrogenase with a glutamate or aspartate substitution for lysine 304 causing instability of the protein. J. Biol. Chem., 267, 26004-26010.MEDLINE Abstract
29 Andresen, B.S., Bross, P., Jensen, T.G., Winter, V., Knudsen, I., Kølvraa, S., Jensen, U.B. et al. (1993) A rare disease-associated mutation in the medium-chain acyl-CoA dehydrogenase (MCAD) gene changes a conserved arginine, previously shown to be functionally essential in short-chain acyl-CoA dehydrogenase (SCAD). Am. J. Hum. Genet., 53, 730-739.MEDLINE Abstract
30 Ding, J.H., Bross, P., Yang, B.Z., Iafolla, A.K., Millington, D.S., Roe, C.R., Gregersen, N. et al. (1992) Genetic heterogeneity in MCAD deficiency: frequency of K329E allele and identification three additional mutant alleles. In Coates, P.M. and Tanaka, K. (eds) New Developments in Fatty Acid Oxidation, New York: Wiley-Liss, pp. 479-488.
31 Kelly, D.P., Hale, D.E., Rutledge, S.L., Ogden, M.L., Whelan, A.J., Zhang, Z. and Strauss, A.W. (1992) Molecular basis of inherited medium-chain acyl-CoA dehydrogenase deficiency causing sudden child death. J. Inher. Metab. Dis.,15, 171-180.MEDLINE Abstract
32 Andresen, B.S., Kølvraa, S., Bross, P., Bolund, L., Curtis, D., Eiberg, H., Zhang, Z. et al. (1993) A silent A to G mutation in exon 11 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene. Hum. Mol. Genet., 2, 488.MEDLINE Abstract
33 Workshop on molecular aspects of MCAD deficiency (1992) Mutations causing medium-chain acyl-CoA dehydrogenase deficiency: A collaborative compilation of the data from 172 patients. In Coates, P.M., Tanaka, K. (eds) New Developments in Fatty Acid Oxidation, New York: Wiley-Liss, pp. 499-506.
34 Maquat, L.E. (1995) When cells stop making sense: Effects of nonsense codons on RNA metabolism in vertebrate cells. RNA, 1, 453-465.
35 Saijo, T. and Tanaka, K. (1995) Isoalloxazine ring of FAD is required for the formation of the core in the Hsp60-assisted folding of the medium-chain acyl-CoA dehydrogenase subunit into the assembly competent conformation in mitochondria. J. Biol. Chem., 270, 1899-1907.MEDLINE Abstract
36 Vockley, J., Parimoo, B. and Tanaka K (1991) Molecular characterization of four different classes of mutations in the isovaleryl-CoA dehydrogenase gene responsible for isovaleric acidemia. Am. J. Hum. Genet., 49, 147-157.MEDLINE Abstract
37 Biery, B.J., Stein, D.E., Holmes Morton, D. and Goodman S.I. (1996) Gene structure and mutations in glutaryl-Coenzyme A dehydrogenase: impaired association of enzyme subunits that is due to an A421V substitution causes glutaric acidemia type I in the Amish. Am. J. Hum. Genet., 59, 1006-1011.MEDLINE Abstract
38 Naito, E., Indo, Y. and Tanaka, K. (1990) Identification of two variant short-chain acyl-CoA dehydrogenase alleles, each containing a different point mutation in a patient with short-chain acyl-CoA dehydrogenase deficiency. J. Clin. Invest., 85, 1575-1582.MEDLINE Abstract
39 Powell, P.J. and Thorpe, C. (1988) 2-octynoyl coenzyme A is a mechanism-based inhibitor of pig kidney medium-chain acyl-coenzyme A dehydrogenase: Isolation of the target peptide. Biochemistry, 27, 8022-8028.MEDLINE Abstract
40 Bross, P., Engst, S., Strauss, A..W., Kelly, D.P., Rasched, I. and Ghisla, S. (1990) Characterization of wild-type and an active site mutant of human medium-chain acyl-CoA dehydrogenase after expression in Escherichia coli. J. Biol. Chem., 265, 7116-7119.MEDLINE Abstract
41 Ikeda,Y., Okamura-Ikeda, K. and Tanaka, K. (1985) Purification and characterization of short-chain, medium-chain and long-chain acyl-CoA dehydrogenases from rat liver mitochondria. J. Biol. Chem., 260, 1311-1325.MEDLINE Abstract
42 Finocchiaro, G., Ito, M. and Tanaka, K. (1987) Purification and properties of short-chain acyl-CoA, medium-chain acyl-CoA and isovaleryl-CoA dehydrogenases from human liver. J. Biol. Chem.,262, 7982-7989.MEDLINE Abstract
43 Kølvraa, S., Gregersen, N., Christensen, E. and Hobolth, N. (1982) In vitro fibroblast studies in a patient with C6-C10-dicarboxylic aciduria: evidence for a defect in general acyl-CoA dehydrogenase. Clin. Chim. Acta., 126, 53-67.MEDLINE Abstract
44 Rhead, W.J., Amendt, B.A., Fritchman, K.S. and Felts, S.J. (1983) Dicarboxylic aciduria: deficient [1-14C]-octanoate oxidation and medium-chain acyl-CoA dehydrogenase activity in fibroblasts. Science, 22, 73-75.
45 Stanley, C.A., Hale, D.E., Coates, P., Hall, C.L., Corkey, B.E., Yang, W., Kelley, R.I. et al. (1983) Medium-chain acyl-CoA dehydrogenase deficiency in children with non-ketotic hypoglycemia and low carnitine levels. Pediatr. Res., 17, 877-884.MEDLINE Abstract
46 Frerman, F.E. and Goodman, S.I. (1985) Fluorometric assay of acyl-CoA dehydrogenases in normal and mutant human fibroblasts. Biochem. Med., 33, 38-44.MEDLINE Abstract
47 Saudubray, J.M., Coude, F.X., Demaugre, F., Johnson, C., Gibson, K.M. and Nyhan, W.L. (1982) Oxidation of fatty acids in cultured fibroblasts: a model system for the detection and study of defects in oxidation. Pediatr. Res.,16, 877-887.MEDLINE Abstract
48 Manning, N.J., Olpin, S.E., Pollitt, R.J. and Webley, J. (1990) A comparison of [9,10-3H] palmitic and [9,10-3H]myristic acids for the detection of defects of fatty acid oxidation in intact fibroblasts. J. Inher. Metab. Dis.,13, 58-68.MEDLINE Abstract
49 Seakins, J.W.T. and Rumsby, G. (1988) The use of phenylpropionic acid as a loading test for medium-chain acyl-CoA dehydrogenase deficiency. J. Inher. Metab. Dis.,11 (suppl 2), 221-224.
50 Wilcken, B., Carpenter, K.H. and Hammond, J. (1993) Neonatal symptoms in medium chain acyl coenzyme A dehydrogenase deficiency. Arch. Dis. Child., 69, 292-294.MEDLINE Abstract
51 Gustafson, S., Proper, J.A., Bowie, E.J.W. and Sommer, S.S. (1987) Parameters affecting the yield of DNA from human blood. Biochemistry, 165, 294-299.
52 Andresen, B.S., Knudsen, I., Jensen, P.K.A., Rasmussen, K. and Gregersen, N. (1992) Two novel non-radioactive PCR-based assays in which dried blood spots, Genomic DNA or whole cells are used for fast and reliable detection of the Z and S mutations in the gene for [alpha]-1-antitrypsin. Clin. Chem.,38, 2100-2107.MEDLINE Abstract
53 Gregersen, N., Andresen, B.S., Bross, P., Winter, V., Rüdiger, N., Engst, S., Christensen, E. et al. (1991) Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: Identification of a lys329 to glu mutation in the MCAD gene, and expression of inactive mutant protein in E.coli. Hum. Genet.,86, 545-551.
54 Andresen, B.S., Bross ,P., Vianey-Saban, C., Divry, P., Zabot, M..T., Roe, C.R., Nada, M.A. et al. (1996) Cloning and characterization of human very-long-chain acyl-CoA dehydrogenase cDNA, chromosomal assignment of the gene and identification in four patients of nine different mutations within this gene. Hum. Mol. Genet.,5, 461-472.
55 Sarkar, G. and Sommer, S. (1990) The `Megaprimer' method of site-directed mutagenesis. BioTechniques, 8, 404-407.MEDLINE Abstract
56 Lehman, T.C., Hale, D.E., Bhala, A. and Thorpe, C. (1990) An acyl-coenzyme A dehydrogenase assay utilizing the ferricenium ion. Anal. Biochem.,186, 280-284.MEDLINE Abstract
*To whom correspondence should be addressed at: Center for Medical Molecular Biology, Århus University Hospital, Skejby Sygehus, DK 8200 Århus N, Denmark. Tel: +45 8949 5146; Fax: +45 8949 6018; Email: brage@biobase.dk
-->
This page is maintained by OUP admin. Last updated Fri Apr 11 08:44:24 BST 1997. Part of the OUP Journals World Wide Web service.
Copyright
Oxford University Press, 1996
