The most common specific FAO enzyme deficiency is medium chain acyl-CoA dehydrogenase (ACADM). While ten or more single point mutations have been detected in human ACADM coding sequences, one mutation, G985, accounts for 90- 97% of those with functional disruption. With several methods available and validated to screen for the metabolic defect, numerous states in the U. S. have instituted routine screening oh all newborns within the past 1-2 years. These screening efforts, as well as large epidemiologic studies indicate that the carrier rate for a functional ACADM mutation is 1: 65 to 1: 45. This allelic frequency and epidemiologic studies indicate an estimated 1: 8,000 to 1: 15,000 are ACADM-deficient.

The known consequence of undetected/untreated disease is significant mortality in infants and children. Adult medical problems are unknown and unreported. While rapid dietary intervention at time of crisis may offset some mortality, there needs to be more effective alternatives developed. There is no animal model for this fairly common disorder of lipid beta-oxidation. This limits the ability to examine the mechanisms of acute and potentially chronic tissue effects and impairs the ability to develop acute, longitudinal or corrective interventions.

Description of the Figures Figure 1 shows the nucleotide sequence of wild-type ACADM. The position of the 12 base pair insertion in mACADM is indicated by a downward arrow. The start codon is boxed. The position of the single nucleotide polymorphism is mACADM is underlined (position 1085).

Figure 2 shows the nucleotide sequence of the mutant ACADM, designated mACADM. The 12 base pair insertion in the leader sequence is underlined. The start codon is boxed. The single nucleotide polymorphism at position 1087 (T @ C) is underlined.

Summary of the Invention The invention provides a mutant human medium chain acyl-CoA dehydrogenase, designated"mACADM."The mutant contains a 12 base pair insertion, TGTTCTTTACAG, in the leader sequence and a singie nucleotide polymorphism (SNP) at position 1087 of Figure 2 (or 10985 of Figure 1). The published sequence not containing these changes is shown in Figure 1, from Kelly et al., Proc. Nat'l Acad. Sci., U. S. A. 94: 4068-4072 (1987), incorporated herein by reference for this sequence.

The invention also provides methods for using the mutant for diagnostic, therapeutic, research, and drug screening procedures.

The invention also provides compositions containing the mutant nucleic acid or polypeptide.

Accordingly, the invention is directed to an isolated polynucleotide corresponding to an aberrant sequence of ACADM.

The invention is also directed to a polypeptide encoded by the mACADM polynucleotide sequences. The polypeptide can be isolated.

The invention is also directed to polynucleotide fragments of mACADM that contain the mutations disclosed herein.

The invention is also directed to polypeptide fragments encoded by these polynucleotides.

The invention is also directed to vectors containing the mACADM polynucleotide sequences and to host cells containing or expressing these sequences. These include recombinant vectors and host cells.

The invention is also directed to methods for producing mACADM polynucleotides and polypeptides using these vectors and host cells.

In one embodiment, mACADM polynucleotides are in a transgenic animal and mACADM polypeptides are expressed in the animal.

The invention is also directed to agents that are specifically capable of interacting, e. g., binding, to mACADM polynucleotides or polypeptides.

In one embodiment the agent is an antibody specific for the mutant.

The invention is also directed to agents that can be used to detect the mutations. These include, but are not limited to, antibodies and complementary nucleic acid sequences.

The invention is also directed to methods for detecting the mutation with these or other agents that allow detection of the mutation. Detection can be in vitro, ex vivo, or in vivo, for example.

The invention is also directed to methods for detection in a prognostic or diagnostic procedure, for example, to screen an at-risk individual or population or to screen affects individuals or populations for the presence or expression of the mutation.

The invention is also directed to methods for modulating expression of mACADM polynucleotides or polypeptides in vitro, ex vivo or in vivo, for example.

These methods can be used to treat disorders in which expression of the mutation is relevant to development or progression of the disorder.

The invention is also directed to methods for identifying compounds that modulate expression of the mACADM polynucleotides or polypeptides. Such methods include, but are not limited to, screening for agents that bind to the mACADM polynucleotides or polypeptides. Screening can be done in-vitro, ex vivo, or in vivo, such as in a transgenic animal.

The invention is thus directed to methods to identify agonists and antagonists of mACADM.

The invention is also directed to the mACADM polynucleotide or polypeptide sequences in a computer-readable form.

The invention is also directed to variants of the mutants, for example, to an insertion at the same position but which contains different base changes or an SNP at the same position, but in which A or G is substituted for T.

Disorders that are particularly relevant to the methods and uses described herein include, but are not limited to, diseases of fatty acid oxidation, including, but not limited to, acute liver failure, hypoglycemia, hyperammonemia, acidosis, cardiac fatigue and failure, and striated muscle fatigue and failure, which can lead to respiratory arrest.

Detailed Description of the Invention Defects in fatty acid oxidation are among the more common genetic disorders currently known in man. The consequences of having such defects frequently manifest as acute liver failure in young children who have an acute prodrome that diminishes food intake and sets up a fasting physiology state (Roe, C. R., et al., in The Metabolic Basis of Inherited Disease,"CR Sriver et aL, editors. Pp. 889-914 (1989)). In this state, a key source of energy comes from ketone formation in the liver via fatty acid oxidation of mobilized fatty acids from peripheral storage. Acute fatty liver, hepatic failure, hypoglycemia, hyperammonemia, acidosis and cardiac and striated muscle fatigue result. Death or significant morbidity is common. The disorder appears clinically very similar to Reyes syndrome, and has been reported in a number of retrospective studies of sudden infant death syndrome.

Medium chain acyl-CoA dehydrogenase deficiency is the most common disorder of fatty acid oxidation (Matsubara Y. et al., in"New Developments of Fatty Acid Oxidation"Coates PM era/., editors, pp. 453-462 (1992)). The allelic frequency appears to be in the range of 1: 63-1: 45 in the U. S., and several states are now routinely screening newborns for this defect in hopes of averting medical crises later in childhood. Current therapy is nutritional manipulation at times when children present with the acute syndrome, but this is essentially a logical and empirical therapeutic approach that relies on early recognition of the specific disorder.

ACADM is one of four dehydrogenases involved in fatty acid beta-oxidation.

The others are very long chain, long chain, and short chain dehydrogenases.

ACADM processes fatty acids of 4 to 16 carbons (Finocchiaro G., et al., J. Biol.

Chem., 262: 7892-89 (1987)). All four enzymes are nuclear genome-encoded, are synthesized on cytosylic ribosomes, and contain a leader sequence which targets the propeptide to the mitrochondria. ACADM enters the soluble matrix of the inner mitrochondria) space, the leader peptide (25 aa) is cleaved, the monomer binds flavin adenine dinucleotide (FAD), and then forms a homotetramer which results in the active enzyme.

The most common functional SNP for the ACADM gene is G985A, which allows the entire processing of the proenzyme to proceed normally up through binding FAD. However, the mutation prevents the formation of the homotetramer complex, and the half life of the peptide residence time in the mitochondria is reduced from many hours to a few minutes. The ACADM monomeric peptide is released from the mitochondria and subsequently degraded by the proteosome. Less common mutations interfere with FAD binding, silencing of the catalytic site and other effects.

It is important to note that ninety percent of children with clinical events are G985 homozygotes and an additional 7% are compound heterozygotes for G985 (Workshop on Molecular Aspects of MCAD Deficiency. tn"New Developments of Fatty Acid Oxidation"Coates PM et al., editors. pp. 499-506 (1992)).

The wild-type ACADM cDNA is highly conserved across mouse, rat and human (Tolwani R. J. eraA, Genom/cs23 (1) : 247-9 (1994)). The cDNAs are all the same length (1263 bp) and encode a 421-amino acid precursor protein that includes a 25-amino acid leader sequence for mitochondrial targeting. Of particular interest is the fact that the nucleotides and amino acids involved in the function of ACADM, and disrupted by mutations in humans are identical (12 exons) and exons 2-11 are identical in size (Tolwani, R. J., et aL, Gene 170 (2): 165-71 (1996)). Intron sizes vary, however, with the human locus being about 40kb and the mouse locus being about 25kb.

The invention is based on the discovery of a human ACADM containing mutations at two separate positions, as shown in the Figures. Accordingly, the invention encompasses a mutant, designated herein"mACADM,"containing both mutations as shown.

The mutant cDNA was isolated from a human cell line derived from the normal colon, T84, available from the American Tissue and Cell Culture Repository.

The invention, however, also encompasses fragments of the mutant that contain a mutated sequence. These fragments can be used to construct recombinant mutants containing one or both of the mutations. Fragments can encompass a range of sizes. Thus, any amount of nucleotide or amino acid sequence flanking the mutation on one or both sides is encompassed.

The invention also encompasses variants of the mutants. With respect to the SNP, the variant would contain G or A in place of the T at position 1087 in Figure 2 (1085 in Figure 1).

With respect to the mutation in the leader sequence, the variant could contain any permutation of base pair substitution in the 12 base pair insertion that would provide an in-frame resulting insertion of four amino acids.

Alternatively, the invention also encompasses any permutation of base pair substitution in the insertion sequence that results in in-frame insertions that provide 1- 3 amino acids. In another embodiment, any 3 out of the 12 nucleotides could be deleted to produce a 3 amino acid insertion. The deleted nucleotides could be in order, i. e., as found in Figure 1. In this instance, one to three triplets, as found, could be deleted, i. e., one or more of TGT, TCT, TTA, of CAG. Alternatively, two or more of these triplets could be rearranged in any order. Further, one or more of these triplets could be combined with one or more triplets not found in the insertion in Figure 2.

The invention also encompasses a larger in-frame insertion at. this position, that can be formed by any given combination of triplets (i. e., triplets found in the insertion shown in Figure 2 or other desires triplets).

As used herein, mACADM refers to the polynucleotide and encoded amino acid sequence shown in Figure 2. However, the term also encompasses variants and fragments as described herein. Thus, methods, uses, and compositions encompass these embodiments.

The invention is also directed to isolated or purified mACADM. This means that it is substantially free from cellular components with which it is normally associated. In the case of the polynucleotide, some flanking sequences can be present. Further, mACADM polynucleotide or polypeptide can be joined to non- mACADM sequences, such as in recombinant constructs, and still be considered isolated.

The mACADM can be isolated from cells that express it naturally, such as heart, spleen, kidney, or liver, cell lines that naturally express it, such as T84, or from cells engineered to express it recombinantly or can be chemically synthesized.

In one embodiment, the protein is produced by recombinant DNA techniques.

For example, a nucleic acid molecule encoding the polypeptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.

Functional changes in the mutant ACADM protein caused by one or both mutations are relevant to disorders associated with improper expression (activity) of ACADM.

The mutation in this leader sequence is important for correct subcellular localization of ACADM. The propeptide normally targets the protein to the inner matrix of the mitrochondria where it is then normally cleaved off to form the mature protein. The leader function can be altered by the mutation in a number of ways. For example, the protein could be targeted to another organelle, may be improperly cleaved in the mitochondria (or elsewhere), or not cleaved at all.

Aberrant expression of ACADM has been associated with the disorders described above. Accordingly, any of the uses and methods described herein apply to these disorders. However, other disorders are also included where expression of mACADM is a factor in development or progression of the disorder. Such a disorder can be identified by routine screening for expression of mACADM in cells, tissues, or biological fluids derived from pathological sources. These sources include subjects at risk for the disorder or subjects having the disorder. Screening can be done with any agent that allows detecting in mACADM gene expression, such as are disclosed herein.

The mACADM polynucleotides and polypeptides are useful, therefore, in cell- based and cell-free diagnostic assays. Detectable agents that interact with the mutant (e. g., bind), can be used to detect the mutation. These include, but are not limited to, anti-mACADM antibody to detect the protein and labeled nucleic acid probes, sequence-specific ribozymes, or direct DNA sequencing to detect the polynucleotides.

The mACADM polynucleotides and polypeptides are also useful for drug screening in such assays to identify agents that modulate the level or expression of the mACADM polynucleotides and polypeptides.

The polynucleotides and polypeptides are also useful to identify agents that increase or decrease their interaction with a target compound, such as substrate, effector molecule, modification enzyme (e. g., glycosylation or phosphorylation), peptidase or other target, such as a disease-associated target (e. g., a target with which the polynucleotides and polypeptides must interact to cause disease).

Agents that modulate expression or affect interaction can be used to treat a disorder associated with expression or function (e. g., interaction) of the mACADM.

In one embodiment the interaction is binding. Accordingly, agents can be identified that increase or decrease binding.

Accordingly, the invention provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate nucleic acid expression. Modulation includes both up-regulation (i. e., activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

The invention also provides method of treatment, with the protein as a target, using a compound identified through drug screening as a gene product modulator to modulate protein expression. Modulation includes both up-regulation (i. e., activation or agonization) or down-regulation (suppression or antagonization) or protein expression.

Agents can also be identified in disease models in vivo, such as in transgenic animals expressing the mACADM.

Candidate agents for the methods above include, but are not limited to, non- peptide organic molecules, inorganic molecules, antibodies, peptides, and proteins.

Peptides/proteins can include the wild-type protein or fragments thereof, especially fragments that compete with the mutant for binding to other cellular components.

Peptides/proteins also include those capable of correcting the defect caused by the mutation, for example, by complementing the defect in activity or processing. These could function to increase or decrease normal subcellular localization (targeting), peptide cleavage, or other post-translational modification, for example.

ACADM is expressed in virtually all cell types. Expression, and also mis- expression, is significant in the liver since this tissue also expresses the other enzymes involved in the FAO pathway.

MCAD deficiency is a naturally occurring disorder that under physiologic stress kills hepatocytes. Therefore, genetic correction of autologous cells should result in a survival advantage to these repaired cells. Under subsequent physiologic stresses, the corrected cells should have the capacity to respond by proliferation in response to uncorrected hepatocyte loss. This response pattern (survival advantage) is different from such models as correction of hepatocyte LDL receptor defects or introduction of a wild-type alpha-1-antitrypsin minigene into A1AT deficient cells.

In one embodiment of the invention, therefore, hepatocytes are a relevant cell type for various purposes. First, the invention pertains to hepatocytes in vitro.

Hepatocytes carrying the mutation, either recombinant or naturally-occurring, can be used as a model to screen for compounds that correct the deficiency cause by the mutation. Such compounds can act directly on the mACADM nucleic acid or protein or can correct the defect by acting on other cellular components that biochemically overcome the defect. In one example, reduced cleavage caused by the mutation in the leader sequence could be compensated by a peptidase having enhanced activity.

In another example, improper subcellular localization of the mACADM could be compensated by a transport component that abnormally targets the mACADM to mitochondria.

Such hepatocytes in vitro could also be used to study protein processing function perse.

Such hepatocytes can also be used as a model of FAO deficiencies, such as acute fatty liver disease and Reyes syndrome.

The uses above also apply to hepatocytes in vivo, such as in transgenic animals and human clinical trials. In transgenic animals, it is further desirable to use inducible promoters to modulate the level of mACADM expression. Moreover, in one embodiment of this aspect, transgenic animals contain both wild-type ACADM and mACADM, both of which can be modulated by external signals. This, in effect, allows the production of functional enzyme and knock-out in the same animal. Alternatively, true knock-out mice for the wild-type enzyme can be created that can express the mACADM. Knock-out mice can also be created that express the mutation in transplanted hepatocytes. Alternatively, animals expressing the mutation can contain normal transplanted hepatocytes.

It is understood, however, that animals naturally expressing the mutation (i. e. non-recombinant) are also useful as a disease model in embodiments described above.

It is also understood that such models pertain to any tissue in which aberrant ACADM expression is relevant. Accordingly, not only inducible, but also tissue- specific promoters are relevant to the methods and compositions of the invention.

A further use involves correcting the mutation ex vivo or in vivo, for example, by gene replacement or other manipulation.) n one embodiment, as briefly discussed above, hepatocytes are manipulated ex vivo to correct the mutation and then returned to the donor.