Báo cáo khóa học: Genetic defects in fatty acid b-oxidation and acyl-CoA dehydrogenases Molecular pathogenesis and genotype–phenotype relationships

doi:10.1046/j.1432-1033.2003.03949.x

Eur. J. Biochem. 271, 470–482 (2004) (cid:1) FEBS 2004

M I N I R E V I E W

Genetic defects in fatty acid b-oxidation and acyl-CoA dehydrogenases Molecular pathogenesis and genotype–phenotype relationships

Niels Gregersen1, Peter Bross1 and Brage S. Andresen1,2

1Research Unit for Molecular Medicine, Aarhus University Hospital and Faculty of Health Sciences and 2Institute of Human Genetics, Aarhus University, Aarhus, Denmark

ant K304E MCAD protein has been studied intensively, the investigations on biogenesis, stability and kinetic properties for this variant enzyme will be discussed in detail and used as a paradigm for the study of other mis-sense variant proteins. We conclude that the total effect of mis-sense sequence variations may comprise an invariable – sequence variation specific – effect on the catalytic parameters and a conditional effect, which is dependent on cellular, physiological and genetic factors other than the sequence variation itself.

system; molecular

chaperones;

Keywords: fatty acid b-oxidation; acyl-CoA dehydrogenase; VLCAD; MCAD; SCAD; mutation type; protein quality control intracellular proteases; genotype–phenotype.

Mitochondrial fatty acid oxidation deficiencies are due to genetic defects in enzymes of fatty acid b-oxidation and transport proteins. Genetic defects have been identified in most of the genes where nearly all types of sequence vari- ations (mutation types) have been associated with disease. In this paper, we will discuss the effects of the various types of sequence variations encountered and review current know- ledge regarding the genotype–phenotype relationship, espe- cially in patients with acyl-CoA dehydrogenase deficiencies where sufficient material exists for a meaningful discussion. Because mis-sense sequence variations are prevalent in these diseases, we will discuss the implications of these types of sequence variations on the processing and folding of mis-sense variant proteins. As the prevalent mis-sense vari-

Introduction

lic aciduria [medium-chain acyl-CoA dehydrogenase (MCAD) deficiency] in the 1970s [5], defects in many enzymes and transport proteins involved in the oxidation of fatty acids have been discovered (Table 1).

The clinical features in patients with different defects, and among patients with deficiencies of the same transport protein/enzyme, are very diverse but the most prevalent symptoms are always related to heart, liver and/or the neuromuscular systems.

During the last 25 years, the number of known mitochond- rial fatty acid oxidation defects, as well as the number of patients with associated disease states, has been increasing steadily [1,2]. Since the first descriptions of muscle carnitine palmitoyltransferase (carnitine palmitoyl-CoA transferase II; CPTII) deficiency [3]; systemic carnitine (carnitine transporter; CAT) deficiency [4] and nonketotic dicarboxy-

Deficiencies in the transporters and enzymes involved in the oxidation of long-chain fatty acids are generally severe and may cause death and severe morbidity early in life. In contrast, the most common features of disorders of enzymes involved in the metabolism of medium-chain fatty acids are episodic hypoglycaemia and liver-associated disturbances of consciousness, which – if untreated – may lead to coma and death. These severe, acute life-threatening episodes are rarely seen in the defects of short-chain fatty acid oxidation, where the most common symptoms are neuromuscular.

Despite the fact that defects of the long-chain fatty acid metabolism often cause severe fatal disease, it has become evident that the whole range of clinical symptoms, from fatal heart or liver failure to mild muscular disabilities, has been observed in patients with these diseases. An exception is in CPTI deficiency, where liver symptoms predominate. On the other hand, it is unusual to observe heart and liver pathologies in patients with deficiencies of short-chain fatty acid metabolism.

Correspondence to N. Gregersen, Research Unit for Molecular Medicine, Skejby Sygehus, 8200 Aarhus N, Denmark. Fax: + 45 89496018, Tel.: + 45 89495140, E-mail: nig@mmf.au.dk Abbreviations: CPTI (II), carnitine palmitoyl-CoA transferase I (or II); ETF, electron transfer flavoprotein; ETFDH, ETF dehydrogenase; Hsp, heat shock protein; HGMD, Human Gene Mutation Database; MCAD, medium-chain acyl-CoA dehydrogenase; NCBI, National Centre for Biotechnology Information; PKU, phenylketonuria; PTC, premature termination codon; SNP, single nucleotide polymorphism; VLCAD, very-long-chain acyl-CoA dehydrogenase. Definitions: Sequence variation designates all types of gene sequence changes, including conventional disease-causing mutations and null- mutations as well as neutral and susceptibility polymorphisms, as recommended by The Human Genome Variation Society [den Dun- nen, J.T. & Antonarakis, S.E. (2001) Hum. Genet. 109, 121–124]. Where not featured in the abbreviations list, enzyme and transport protein abbreviations are defined in Table 1. Note: A web site is available at http://www.auh.dk/sks/afd/mmf.dk (Received 17 July 2003, revised 13 October 2003, accepted 23 October 2003)

Furthermore, in patients with very-long-chain acyl-CoA dehydrogenase (VLCAD), CPTII and electron transfer flavoprotein (ETF)/ETF dehydrogenase (ETFDH) defects

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Table 1. Transporter proteins and enzymes involved in the mitochondrial saturated fatty acid oxidation. FATP, fatty acid transport protein; CAT, carnitine transporter; CACT, carnitine/acylcarnitine translocase; CPT I, carnitine palmitoyltransferase I (liver); CPT II, carnitine palmitoyl- transferase II; ETF/ETFDH, electron transport flavoprotein/electron transport flavoprotein dehydrogenase; VLCAD, very-long-chain acyl-CoA dehydrogenase; MTP, mitochondrial trifunctional protein (including long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA dehy- drogenase and long-chain 3-oxoacyl-CoA thiolase); MCAD, medium-chain acyl-CoA dehydrogenase; SCHAD, short-chain 3-hydroxyacyl-CoA dehydrogenase; SCKAT, short-chain 3-oxoacyl-CoA thiolase; SCAD, short-chain acyl-CoA dehydrogenase.

Year disease discovered

Typical organ involvement

Recent updates/references

Transporters

FATP (plasma membrane) CAT (plasma membrane) CACT (mitochondrial membrane)

1998 [69] 1975 [4] 1992 [72]

Liver Liver, heart, muscle Heart, liver, muscle

[70] [32] [35]

Enzymes (mitochondrial membrane)

1981 [75] 1973 [3] 1976 [76] 1993 [77]

Liver Heart, liver, muscle Heart, liver, muscle Heart, liver, muscle

[36] [36] [30,31] [23]

CPT I (liver) CPT II ETF/ETFDH VLCAD MTP

LCHAD LCKAT

1989 [78] 1992 [80]

Liver, heart, muscle Liver, heart, muscle

[80] [80]

Enzymes (mitochondrial matrix)

MCAD SCHAD SCKAT SCAD

[71] [55] – [49]

1976 [5] 1991 [53], 2001 [55] 1997 [73] 1987 [74]

Liver Liver Liver, muscle Muscle, brain

UK), which remains the most comprehensive database containing published disease-associated sequence variations in fatty acid oxidation genes.

there is a clear correlation between the degree of deficiency and the clinical phenotype ([1] and references therein). Severe deficiencies generally result in fatal or severe disabilities, while milder defects are associated with mainly muscular symptoms. Such a correlation is not seen in patients with medium- and short-chain defects. In these diseases mild defects may not be associated with detectable disease.

Lastly, to give the descriptions of genes and sequence variations biological significance, we will review the current knowledge concerning genotype–phenotype relationships in acyl-CoA dehydrogenase deficiencies, which will illuminate considerations and ideas that are applicable to the other fatty acid oxidation deficiencies and many other genetic disorders.

Genomic structures and disease-associated sequence variations in genes encoding enzymes of fatty acid oxidation

The realization of these associations – and lack of connections – between enzymatic phenotypes and clinical phenotypes has emerged through careful studies of many patients over many years. However, the cloning and elucidation of the genes and genomic structures for nearly all clinically relevant enzymes and transport proteins of fatty acid oxidation has stimulated our knowledge considerably, both with respect to the possibility of specific molecular genetic diagnostics – which are insensitive to disturbances in the biochemical and cellular factors – and because this knowledge has made genotype–phenotype investigations possible.

In the following we will summarize the current knowledge regarding the genes that code for clinically relevant trans- port proteins and the enzymes of mitochondrial fatty acid oxidation, as available in publicly accessible databases developed and maintained at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm. nih.gov/genome/guide/human/).

Despite the fact that the annotated genomic structures and cDNAs may not be exact, the information is sufficiently accurate for the purpose of the present discussion and the databases are an extremely valuable resource with links to existing original literature. For the discussion concerning the effects of the various types of sequence variations we have used the information in the Human Gene Mutation Database [6] (HGMD, http://www.hgmd.org/; Cardiff,

The draft sequence of the human genome was published in 2001 [7,8] and the assembly of large contigs and the annotation of genes makes it possible to find gene and genome structures for all genes that encode the enzymes and transport proteins of mitochondrial fatty acid oxida- tion (except for carnitine/acylcarnitine translocase) in the NCBI databases (Table 2). The information extracted includes: chromosome localization; gene length (total sequence) and the number of exons in the gene and nucleotides in the coding region of each gene. In addition, the types of sequence alterations identified in patients with fatty acid oxidation defects, as extracted from the HGMD in Cardiff, are also summarized in Table 2. The sequence variations are categorized into those that probably result in no enzyme protein (null-mutations) and those for which the effect is more unpredictable. This is a little different from the categorization in the database. In Table 2 we have on one hand counted large deletions, small out-of- frame deletions/insertions, stop-codon introductions and

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The first level of analysis of the genotype–phenotype relationship in fatty acid oxidation deficiencies is a discus- sion of the various types of sequence variations identified and associated to the disease in patients. As large deletions – where whole parts of the genes are missing – are rare and because the description in the database is restricted to the cDNA level, we do not discuss this type of gene defect further, but concentrate on the other types.

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These have been encountered in nine of the 12 fatty acid oxidation defects where sequence variations have been identified in the corresponding genes (Table 2). The change in reading frame resulting from this type of sequence variation leads to the introduction of a premature termin- ation codon (PTC) shortly downstream of the deletion/ insertion. A PTC may also be created by changing an amino acid codon to a stop-codon. By means of a number of poorly understood mechanisms, the PTC – if it is present more than 50 nucleotides upstream of the last intron in the gene – will be recognized by a RNA surveillance mechanism [9,10]. This mechanism is mediated by a general mRNA quality control system, which targets mRNA species containing PTCs to the so-called nonsense mediated decay (NMD) pathway. The consequence is that the mRNA is degraded and no polypeptide is synthesized. If small amounts of PTC-containing mRNA should escape the NMD system, it is most probable that the encoded truncated polypeptide will be rapidly degraded by intracel- lular proteases, which are part of the protein quality control system, which will be discussed below. Thus, these types of sequence variations will, as a rule, result in null-mutations, characterized by negligible amounts of variant protein product formed.

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A number of different splice site sequence variations have been encountered in genes resulting in fatty acid oxidation deficiencies. Depending on the position in relation to the intron–exon border the effect may vary. Variations in 100% conserved AG and GT dinucleotides immediately before and after an exon may result in exon skipping, intron retention or activation of cryptic splice sites [11], usually resulting in a change of reading frame and consequently degradation of mRNA. In cases where the reading frame is unchanged, the truncated protein is most probably rapidly degraded due to misfolding (see below).

2 A 5 2 C L S T C A C 5 A 2 2 C L S T A C 2 T P C I I T P C

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Fig. 1. The gene structures of the ACADVL (VLCAD), ACADM (MCAD) and ACADS (SCAD) genes. The number and approximate size of all coding regions are shown and the 5¢-UTR (untranslated region) as well as the 3¢-UTR are indicated. The information used for the constructions are: VLCAD [82,83] and NCBI nucleotide database gi: 3273227; MCAD [84] and NCBI nucleotide database gi: 187432 and SCAD [85] and NCBI nucleotide database gi: 2995253; 2821943. Sequence variations are designated according to the position relative to the first nucleotide in the start- codon ATG, and they are taken from the Human Gene Mutation Database (HGMD) in Cardiff (http://www.hgmd.org/).

variation at the protein level and on the cellular conditions, as discussed below.

Mis-sense sequence variations

Splice site variations located further from the exons, as seen in carnitine/acylcarnitine translocase (CACT), VLCAD and long-chain 3-hydroxy acyl-CoA dehydro- genase (LCHAD) deficiencies, may or may not result in complete abolition of the active enzyme [12]. Thus, the effect may range from severe to mild as discussed for mis-sense variations below.

Small in-frame deletions/insertions

These sequence defects delete or insert one or more amino acid codons in the mRNA. Usually sequence variations of this type have no consequences for the sta- bility and processing of the mRNA and a truncated or elongated polypeptide will be produced. This is the case in several of the fatty acid oxidation deficiencies (Table 2) but the consequences are difficult to predict. However, as small insertions or deletions will affect the structural stability more severely if located in a-helixes or b-sheets than in structural loops, some idea of the effect can be predicted if the crystal structure of the protein in question is known.

About two thirds of all disease-associated sequence varia- tions in patients with fatty acid oxidation deficiencies are of the mis-sense type (Table 2), which changes a codon from one amino acid into another. Usually such sequence variations result in normal mRNA production and pro- cessing and normal translation to the corresponding variant polypeptide. By inspecting the available crystal structures of wild-type protein it is seen that the vast majority of such changes are located distant from the active centres. Only a few seem to be involved in the catalytic mechanism. The rest perturb folding, resulting in either impaired production of a correctly folded active enzyme, or in an unstable active enzyme [13]. Although there have been several attempts, it is only possible to predict the effect of the mutation from the nature and position of the altered amino acid [14–16] in a minority of cases. In certain cases, some rationalization – mostly post hoc – may be possible. However, the general conclusion seems to be that predictions on the severity of a given mis-sense variation are still very uncertain. Despite the fact that a certain correlation exists between the molecular interactions in the structured active protein and the

In general, the polypeptide is synthesized, but it may have difficulties in achieving the correct active structure and will most often be degraded by the protein quality control system, which is dependent on the nature of the sequence

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precipitation of the fatty acid oxidation deficiencies, may be assessed.

Genotype–phenotype relation in fatty acid oxidation deficiencies

interactions that are used during the folding process, the folding pathway and the molecular forces along this (or these) path(s) cannot presently be modelled for molecules of more than 15 kDa [17]. Mis-sense sequence variations may therefore affect the folding of the enzyme protein severely or they may only perturb it slightly. The folding process is monitored by the protein quality control systems, compri- sing molecular chaperones, assisting the folding, and intracellular proteases, which eliminate misfolded proteins [13]. As the efficiency of these systems is dependent on the cellular conditions, e.g. the temperature and energy level, and probably also on genetic differences between individ- uals, the effect of mis-sense sequence variations cannot, in general, be predicted [18]. As will be discussed in the next section, experimental evaluation can and should be performed.

A second level of analysis of the genotype–phenotype relation in fatty acid oxidation deficiencies is the investiga- tion of possible associations between the type of sequence variations and the clinical phenotype. As mentioned in the Introduction, such associations seem to exist in patients with certain of the long-chain defects but not – or at least to a lesser extent – in patients with medium- or short-chain defects. As our research has focussed on the acyl-CoA dehydrogenase deficiencies, we will use VLCAD, MCAD and SCAD deficiencies as examples and try to extrapolate conclusions drawn from these diseases to the other fatty acid oxidation defects.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency

Recently it has been demonstrated that mis-sense sequence variations, in addition to influencing protein biogenesis, also may affect the splicing efficiency by interfering with binding sites for splice modulating factors [19]. Although the effect of mis-sense variations on splicing has not yet been published in relation to fatty acid oxidation defects, it has been identified in relation to isovaleryl CoA dehydrogenase [20] as well as 2-methyl butyryl-CoA dehydrogenase deficiencies [21]. This fascinating phenomenon is in the process of being charac- terized in the MCAD and VLCAD genes (K. B. Nielsen, T. Sinnathamby, T. J. Corydon, L. Cartegni, A. R. Krainer, O. N. Elpeleg, N. Gregersen, J. Kjems & B. S. Andresen, unpublished observation).

The clinical spectrum seen in patients with VLCAD deficiency is a prototype for other long-chain defects. As discussed in more detail elsewhere [23], it is possible to distinguish three phenotypes. The first comprises very young infants who die from cardiac and liver disease within the first year of life. The second group comprises older children who do not have cardiac symptoms but show hypoketotic hypoglycemia and hepatomegaly, symptoms which are (cid:1)MCAD deficiency-like(cid:2) (see below). The third group are composed of adolescents and adults who do not show cardiac and hepatic symptoms but who suffer from muscle weakness, which may develop to degenerative disability [24–27]. A large number of patients with VLCAD deficiency have been genotyped and Table 3 shows the distribution of the null-mutation/null-mutation and poten- tial variable/potential variable genotypes in the three clinical groups.

The most striking result

In conclusion, it is only possible to predict the effect for one third of the disease associated sequence variations in the fatty acid oxidation genes, i.e. for large deletions, stop- codon (PTC) introductions, consensus splice site changes and small out-of-frame deletions/insertions. These are sequence variations preventing formation of functional protein (putative null-mutations). The rest, i.e. in-frame deletions/insertions, nonconsensus splice site changes and mis-sense sequence variations, may show an a priori unpre- dictable effect, which should be studied experimentally. With the exception of the few variations directly affecting the catalytic sites, these types of defects represent sequence variations with potential variable effects. However, even if the effect of the sequence variation can be elucidated in vitro, the in vivo effect may be modulated by cellular and genetic factors. In spite of these reservations concerning the predictive value of knowing the disease associated sequence variations in a given patient, this knowledge has obvious diagnostic implications, which have been discussed in detail elsewhere [1,22]. Furthermore, by performing careful genotype–phenotype studies the relative importance of the genetic predisposition and possible cellular and metabolic the disturbances, which are often determinants

for

is that homozygosity for sequence variations, which lead to mRNA/protein elimin- ation (null-mutations), is exclusively present in the patient group with severe symptoms. There is little doubt that this is a reflection of a severe enzyme deficiency, which is also reflected in the profile of acyl-carnitines in blood and in patient cells metabolizing long-chain fatty acids [27]. Not surprisingly, the severe metabolic block results in profound energy deficiency and corresponding severe clinical symp- toms. To what degree the accumulated long-chain fatty acids and their derivatives, especially the acylcarnitines, may contribute to the clinical phenotype is not known with certainty but these species may disturb membrane function,

Table 3. Distribution of VLCAD genotypes among three clinical subtypes of VLCAD deficiency. Data from [23].

Group1 Number of patients with severe childhood form

Group 2 Number of patients with mild childhood form

Group 3 Number of patients with adult form

8 6

0 14

0 6

Null mutation/null mutation Potential variable genotype/ potential variable genotype

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including ion-channels, and thereby perhaps, promote arrhythmia ([28] and references therein).

It is also noteworthy that the sequence variations with potentially varying effects (potentially variable genotypes) are distributed to all three groups. This is most probably a reflection of the fact that the effect of these sequence variations may be total inactivation of VLCAD function or it may be mild, leaving sufficient residual activity to avoid severe energy deficiency. However, the deficiency is suffi- cient to promote long-term muscle damage [24,25]. Whether this damage is a result of energy deficiency or the long-term effect of toxic long-chain fatty acids and their derivatives is impossible to judge at present.

fatal, through treatable acute symptoms, mild disabilities, to asymptomatic throughout life. The features are, however, rather uniform; episodic attacks of hypoketotic hypogly- caemia accompanied by lethargy and vomiting, that may develop into hepatic coma and death if not treated by administration of carbohydrate. Usually, MCAD-deficient patients do not experience life-threatening heart-related symptoms, such as arrhythmia [28,43]. This indicates that the energy deficiency is milder in MCAD than in VLCAD deficiency or, alternatively, that medium chain-fatty acids and their derivatives are less toxic to cardiac function than long-chain fatty acids and their derivatives. However, it is interesting to note that early investigations of the toxicity of medium-chain fatty acids, such as octanoic acid, showed narcotic properties that may contribute significantly to the lethargy and hepatic coma observed in patients during periods of metabolic decomposition [44,45].

As far as we can determine, the same situation and arguments apply to CPTII [29] and to ETF/ETFDH deficiencies [30,31], where the number of patients and sequence alterations are sufficiently large to perform a similar analysis.

With respect to the energy deficiency, the milder mani- festation is probably caused by a combination of the fact that several cycles of oxidation can proceed before the pathway is blocked and that some enzyme activity may arise from long-chain acyl-CoA dehydrogenase (LCAD) and SCAD because of their overlapping substrate activity with that of MCAD [46].

Although it is known that physiological factors, i.e. metabolic stress in connection with fasting and fevers, are important factors for the expression of the disease, it is still an open question whether there exist other cellular and genetic factors that contribute to the susceptibility in some, but not in all, individuals [18].

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency

In contrast to these diseases, the other diseases related to long-chain fatty acid metabolism may not show any significant association between the severity of the defect/ type of sequence variation and the clinical phenotype, i.e. CAT deficiency [32,33], LCHAD/mitochondrial trifunc- tional protein (MTP) deficiencies [34], carnitine acylcarni- tine translocase (CACT) deficiency [35] and CPTI deficiency [36]. However, despite the fact that the number of patients as well as the number of known disease-associated sequence variations is still too small to provide a clear picture of the genotype–phenotype in these diseases, liver-related patho- logies are most often encountered. That other pathologies, such as cardiac dysrhythmia in CPTI and cardiomyopathy in LCHAD deficiencies, are observed emphasizes the notion that factors other than the gene defect itself may be decisive, as is also the case in MCAD deficiency, as discussed below.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency

the clinical

impact of different

SCAD deficiency remains difficult to analyse. Compared to long-chain and medium-chain defects, there are at least three peculiarities that are noteworthy with respect to the relationship between the type of sequence variations and clinical features in patients. The first is that nearly all sequence variations identified so far are of the mis-sense type, which in all cases may result in production of some variant protein possessing some residual activity. Among the first 13 disease-associated sequence variations identified in SCAD-deficient patients, 12 are mis-sense sequence variations and one is an in-frame deletion of three nucleo- tides (Table 2). This trend has continued in more than 100 unpublished cases (N. Gregersen, A. Kølvraa, J. Vockley, D. Matern, I. Tein, R. Ensenauer, C. Vianey-Saban, M. Kjeldsen, V. S. Winter, C. B. Petersen & S. Kølvraa, unpublished observations). Although about half of the mis- sense sequence variations in the SCAD gene are cytidine to thymine substitutions, which usually arises at CpG dinu- cleotides, the overrepresentation is still striking and could be a reflection of negative selection of germ cells with sequence variations abolishing all enzyme activity. This explanation has been proposed for the similar phenomenon in fumarate hydratase deficiency, where the absence of such sequence variations also is striking [47].

The situation in MCAD deficiency is different from that in VLCAD deficiency as a single prevalent sequence variation (985AfiG), resulting in a mis-sense variant protein (K304E), is present in homozygous form in 80% of all patients diagnosed with MCAD deficiency. Eighteen per- cent of patients are compound heterozygous with 985AfiG on one allele and a rare disease associated sequence variation on the other, and only about 2% carry other (rare) sequence variations on both alleles [37]. Thus, studies types of sequence of variations are hampered by the fact that nearly all patients carry one or two copies of the K304E variant MCAD enzyme. This amino acid change exerts its effect primarily by compromising the folding [38–40], but the variant protein is also unstable [41] and the function is impaired [42]. At least the misfolding and instability are influenced by cellular and probably also by genetic factors, thus, resulting in an effect that is totally unpredictable without experimen- tal approaches, which will be discussed in detail below. Suffice to say that the 985AfiG sequence variation may result in varying effects and may blur an analysis similar to the one described for VLCAD above.

The second point of note is the spectrum of sequence variations observed in patients with SCAD deficiency. In a minority of cases, rare inactivating mis-sense sequence variations are present in homozygous or compound hetero- zygous form, whereas such variations in many cases are

The age of MCAD deficiency at presentation may vary from birth to middle age. Clinically, the severity ranges from

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SCAD deficiency, and include hyperinsulinism. Only time will show the degree of clinical and genetic heterogeneity in this rare disease.

Molecular effect of sequence variations with an a priori unpredictable effect

present in compound heterozygous form together with one of two common susceptibility mis-sense sequence variations, 625GfiA and 511CfiT. These variations are present in homozygous or compound heterozygous form in 14% of the general population [48]. Further, a significant fraction of patients with SCAD deficiency carry this genotype, in the absence of any rare inactivating variations [49].

A third level of analysis of the genotype–phenotype correlation in fatty acid oxidation deficiencies may be the experimental dissection of the molecular effects of sequence variations with a priori unpredictable effects.

The above situation is apparently different from that encountered in MCAD deficiency, where a prevalent sequence variation likewise is present in nearly all patients but where all individuals carrying the prevalent 985Adefi Gua on both chromosomes, or in one chromosome with a rare sequence variation in the other, are at risk of developing disease. Although preliminary results have shown that the spectrum of clinical symptoms in patients who are homo- zygous or compound heterozygous for 625GuafiAde or/and 511CytfiThy, are indistinguishable from patients harbouring rare inactivating sequence variation [50], it must be assumed that only a minority of individuals, who carry the two susceptibility variations, are at risk of developing disease. From this, it follows that there must be other factors, physiological as well as cellular and/or genetic, that are implicated in the expression of the disease.

Traditionally, and long before the genes and protein structures were elucidated, enzymatic diagnosis of most fatty acid oxidation deficiencies was possible and, furthermore, practised in many laboratories. The enzymatic analyses could correctly determine the residual activity in patient cells but the question remained whether the enzyme protein was present with reduced activity, or it was present in diminished amounts. When antibodies against several of the fatty acid oxidation enzyme proteins became available, it was possible to approach this question. It was soon realized that decreased amounts of protein are the rule rather than the exception. However, it was only after gene cloning and synthesis of proteins by recombinant techniques, that it became possible to resolve the molecular pathogenesis of the increasing number of identified disease associated sequence variations. Although it might seem unnecessary to use large resources to investigate molecular mechanisms, particularly as the diagnoses can be made by direct enzyme activity measure- ment in patient cells, there are at least three good reasons for doing so. The first is to corroborate that an identified sequence variation is associated to the clinical phenotype through a functional effect on the variant protein. This is a very practical and important goal that should be achieved every time a new sequence variation is encountered.

This leads to the third point, namely the clinical features in patients with SCAD deficiency. Only a few patients have presented with symptoms related to energy deficiency, such as cardiac symptoms or hypoglycaemia ([49]; N. Gregersen, A. Kølvraa, J. Vockley, D. Matern, I. Tein, R. Ensenauer, C. Vianey-Saban, M. Kjeldsen, V. S. Winter, C. B. Petersen & S. Kølvraa, unpublished observation). The most probable explanation for this is that SCAD deficiency only blocks the last cycle of the pathway and that MCAD activity overlaps with that of SCAD [46], thus, it is probable that near normal amounts of reducing equivalents are generated. A further reason is that butyric acid and its derivatives are neither heart nor liver toxic. On the other hand, butyric acid is known to exert severe cell toxicity by promotion of cell differentiation, inhibition of the cell cycle and induction of apoptosis [51,52]. This may be the reason why the predomi- nant clinical symptoms are neuromuscular.

The second reason is related to the first but extends the purpose to future diagnostic procedures. The ongoing genotype–phenotype studies data will alter the content of sequence variation databases, which, in addition to raw variation data should also contain information about the effects on protein and cellular metabolism. For many diseases, including the fatty acid oxidation deficiencies, it will thus be possible to replace the laborious and expensive enzymatic analysis by gene-based in vitro and in silico methods.

Without going into a detailed discussion about this issue, there are two important remaining questions. First, how is it possible that two genetic variations, which are implicated in severe neuromuscular disease, can achieve such high frequencies in the general population, and second, what are the genetic/cellular/biochemical mechanisms which renders some of the individuals carrying the variations in homozygous or compound heterozygous form at risk of developing clinically relevant disease. The first question can not be answered at this time but we will attempt to answer the second in the next section.

The third reason for elucidating the molecular patho- genesis, at least for some model variant proteins, is that knowledge gained from detailed investigation of such proteins may be generalized to other proteins in other diseases. As a paradigm – with possible implications for future treatment of patients – we will discuss the careful investigation of the biogenesis, stability and function of the disease-associated K304E MCAD enzyme protein, and the application of the gained knowledge and methodological approaches to define the role of the two common suscep- tibility variations in the SCAD gene.

Molecular effects of the 985AfiG MCAD sequence variation

The only other disease affecting the metabolism of short- chain fatty acids, where sequence variations have been associated to an enzyme deficiency, is short-chain 3-hydroxy acyl-CoA dehydrogenase (SCHAD) deficiency. Although the deficiency has been known in a number of patients for more than 10 years [53,54] only one patient with disease- associated sequence variations in both alleles of the SCHAD gene has been published [55]. The clinical symptoms in this patient, who was shown to be homozygous for a mis-sense sequence variation in the SCHAD gene (773CytfiThy; P258L), are quite different from those found in patients with

Surprisingly, at least at the time of discovery, the amino acid lysine, which is replaced by glutamic acid by the

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985AdefiGua sequence variation at position 304 of the MCAD protein, is located far from the active centre. However, Western blot analysis of soluble variant protein after expression in Escherichia coli cells showed diminished amounts of the K304E MCAD protein compared to the result (wild-type) MCAD protein [56]. This normal stimulated further investigations, some of which have provided the basis for the emerging concept that the effects of mis-sense sequence variations are not only dependent on the nature and location of the particular variation but influenced to varying extents by cellular factors related to the protein quality control systems [18,38,41,57].

The studies, which will be summarized below, focused on one hand on the biogenesis and stability of the variant K304E MCAD protein, coded from the 985AdefiGua allele, and on the other hand on the activity and chain-length selectivity of active K304E MCAD enzyme.

Investigations of biogenesis and stability

Fig. 2. An enlarged view of the vicinity of K304 of a monomer of porcine MCAD (PDB accession no. 3MDD or 3MDE). Helices H and I are shown in ribbons and side-chain atoms of K304, D346, Q342, D300 and the main chain carbonyl atoms of Q342 are shown as solid balls. The side chain of R383 of the neighbouring monomer is represented by open ball-and-stick. Distances between polar atoms in A˚ are shown with dotted lines. Reproduced with permission from Journal of Biological Chemistry [41].

give any data relating to either enzyme activity or substrate selectivity.

In the late 1980s and early 1990s it was realized that the expression and folding of many cellular proteins, including mitochondrial proteins, are dependent on assistance by molecular chaperones [58,59]. As the location was distant from the active centre, it was hypothesized that the glutamic acid in position 304 of the K304E MCAD protein distorted the normal folding. This hypothesis was corroborated by experiments performed in K. Tanaka’s group [38,39,60] and in our own [40,41].

Investigations of enzyme kinetics

the folding of

Although the main effect of the 985AdefiGua sequence variation is most probably due to distortion of folding and tetramer assembly/stability, small distortions in the conformation at the active site and substrate binding the pocket could contribute to the pathogenesis of 985AdefiGua MCAD gene variation. Kieweg and coworkers [42] addressed this question by determining the kinetic parameters for purified wild-type and variant MCAD protein from over-expressing E. coli cells. The authors showed that Vmax was similar for wild-type and the variant K304E MCAD proteins (980 vs. 970 lmolÆ min)1), whereas Km was 3–4 times higher for the variant enzyme, indicating a higher saturation concentration for the optimum substrate octanoyl-CoA compared to the wild-type enzyme. This may have consequences for the amounts of available free CoA for other important cellular processes.

Tanaka’s group showed that both wild-type and variant MCAD proteins were assisted in their intramitochondrial folding by, first the chaperone heat shock protein 70 (Hsp70) and, subsequently, by chaperonin Hsp60, and that the K304E protein was retained longer in association with Hsp60 than was the wild-type protein. These elegant experiments, which were performed by using rat liver mitochondria, clearly indicated that MCAD deficiency is at least in part due to compromised folding of the variant enzyme protein. In parallel with these studies, we inves- tigated the effect of co-overexpression of the bacterial groELS (homologous to human Hsp60/10) in E. coli cells, which over-expressed K304E or wild-type MCAD protein. We found that it was possible to increase the yield of active variant enzyme considerably but further studies also showed that it was not possible to rescue more than 40–50% of wild-type activity. These experiments clearly showed that the variant protein is compromised.

Interestingly, the preferred substrate for K304E variant MCAD is dodecanoyl-CoA. At this chain length, both Vmax and Km are similar for wild-type and variant MCAD enzyme.

Taken together, these detailed studies on the molecular pathogenesis of the K304E variant enzyme protein have illuminated a number of important aspects of the effects of mis-sense sequence variations in MCAD deficiency in particular but also in fatty acid oxidation deficiencies in general – which will be discussed for SCAD deficiency below – as well as in other genetic diseases, such as phenylketonuria (PKU) [61,62].

Inspection of the molecular structure surrounding posi- tion 304 in the mature MCAD protein [41] indicated that the lysine at position 304 is in close vicinity to two opposite- charged aspartate residues at positions 300 and 346, respectively (Fig. 2). Second site mutations at, respectively, position 300 and 346, indicated that the presence of lysine at position 304 is important for efficient folding of the monomer and that the charge interaction between lysine 304 and aspartate 346 is important for tetramer assembly and, therefore, for the stability of the assembled enzyme protein [41]. These effects may be decisive for the steady-state level of variant K304E MCAD but these experiments did not

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wild-type activity after co-overexpression of GroELS. This indicated a greater dependence on chaperonin assistance for the variant protein than for the wild-type but also that when the folding capacity is sufficiently high, the biogenesis of the variant enzyme is as effective as the wild-type enzyme.

Fig. 3. Schematic overview of monomeric SCAD with the positions of the 12 published mutations. The figure is based on the coordinates for rat SCAD (PDB acc. no. 1JQI). SCAD protein is shown as a solid ribbon and the Ca atoms at variant residues are represented as balls. FAD and butyryl-CoA are shown as sticks.

The SCAD enigma

Due to the ineffective folding behaviour of SCAD compared to MCAD in E. coli cells, we looked into eukaryotic expression that has been shown to be intrinsic- ally more effective than bacterial expression for a number of MCAD mutant proteins [67]. By varying the culture temperature it was possible to detect differences in biogen- esis between wild-type and the two variant proteins, G185S and R147W SCAD. At physiological temperature, 37 (cid:4)C, the relative SCAD activities in extracts from transfected COS-7 cells for G185S and R147W were 136 and 45%, respectively. At 41 (cid:4)C the relative activities were, respect- ively, 58 and 13% for G185S and R147W SCAD, while they were 183 and 85% at 26 (cid:4)C [49]. These results support the notion that the variant proteins in their biogenesis at physiological temperatures may achieve sufficient activity to sustain normal fatty acid oxidation but that both variant proteins at higher temperatures, as experienced during fevers, may result in insufficient amounts and activity and thus the development of SCAD deficiency. This conclusion is supported by further in vitro studies, where the biogenesis of the two variant SCAD enzyme proteins was shown to be delayed and compromised, especially at higher temperatures [68]. Together with the fact that the stability of the active G185S SCAD protein is decreased compared to that of wild-type SCAD [66], these studies further contribute to the notion that especially the G185S SCAD protein may be disease-associated.

As mentioned earlier in this review, the genetic defect in most patients with SCAD deficiency is not due to rare inactivating sequence variations but rather to the presence of one of two (or both) susceptibility gene variations, which are present in 14% of the general population in configurations also seen in patients with enzymatically proven SCAD deficiency [49,63]. The goal is to delineate the nature of these variations, which may help to explain why only certain individuals carrying these variations develop clinically relevant disease.

Whether other perturbations of the cellular homeostasis in addition to high temperatures, such as alterations of redox state, ATP depletion and pH changes, may show differential effects on the biogenesis and/or stability of the two variant proteins are pressing questions. If this is the case, a number of conditions encountered in other metabolic and endocrine diseases may result in (cid:1)functional SCAD deficiency(cid:2) and add to the clinical features of these other diseases.

With the present knowledge levels we still do not know how many of the 14% of the general population are at risk of developing – perhaps in a mild and unrecognized form – SCAD deficiency. We only know that a small fraction develop clinically relevant disease [50], and we know that this is possible by a combination of high fatty acid oxidation activity and high temperature, which may result in accu- mulation of cytotoxic butyric acid.

The structure of SCAD from rat has been elucidated [64]. From an inspection of the positions of the two variations, G185S and R147W, it is not obvious how amino acid changes at these positions could be pathogenic (Fig. 3). Both positions are at the outer surface of the monomeric structure. In agreement with this location and the fact that severe defects on enzyme function would not be compatible with the high frequency in the general population, the kinetic disturbances were not found to be serious. Purified R147W protein had kinetic properties similar to the wild- type, and the kinetic efficiency of G185S protein was about 50% compared to the wild-type enzyme [65]. This probably reflects the change from glycine to serine distorting the conformation and exact positions of other amino acids involved in the enzyme mechanism. These results – at least concerning the G185S variant enzyme – underscore the predisposing nature and indicate that other factors must be involved.

The challenge is to define further the cellular conditions under which the deficiency occurs and to delineate whether there exist inter-individual genetic differences in susceptibi- lity to develop clinical disease.

Generalization and future aspects

Early biogenesis and stability studies showed that wild- type SCAD is more dependent on the chaperonin system Hsp60/10 (GroELS in E. coli) than MCAD [66]. While wild-type MCAD does not need additional assistance by the chaperonins in E. coli at 31 (cid:4)C to achieve the active conformation [40], the yield of functional wild-type SCAD is increased eightfold by co-overexpression of GroELS at the same temperature. In the same type of experiment, G185S variant SCAD showed about 30% of wild-type activity without co-overexpression of GroELS but achieved

Many elements of the above discussion can be generalized to defects in other fatty acid oxidation enzymes and to variant proteins present in other genetic diseases. To our knowledge only a few other metabolic diseases have been investigated in the same detail as MCAD and SCAD deficiencies, and with PKU as a prominent example [61,62].

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Acknowledgements

16. Terp, B.N., Cooper, D.N., Christensen, I.T., Jorgensen, F.S., Bross, P., Gregersen, N. & Krawczak, M. (2002) Assessing the relative importance of the biophysical properties of amino acid substitutions associated with human genetic disease. Hum. Mutat. 20, 98–109.

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19. Cartegni, L., Chew, S.L. & Krainer, A.R. (2002) Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet. 3, 285–298.

The molecular genetic analyses of the VLCAD, MCAD and SCAD genes have been performed by medical laboratory technologists Vibeke Winter, Inga Knudsen, Margrethe Kjeldsen and Lisbeth Schrøder. The investigations of our own group referred to in this review have been supported by The Danish Medical Research Council; Danish Human Genome Centre; Karen Elise Jensen Foundation; Aarhus County Research Initiative; Institute of Experi- mental Clinical Research, Aarhus University; Institute of Human Genetics, Aarhus University and Aarhus University Hospital. We thank colleagues from all over the world for providing genetic and cell material for the studies and certain of them for inspiring discussions concerning genotype–phenotype interactions in especially the acyl-CoA dehydrogenase deficiencies.

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