WO2013006436A1 - Acsf3 mutations in metabolic disorders - Google Patents

Abstract

The invention provides methods of detecting a metabolic disorder comprising an ACSF3 defect in a subject, as well as methods of treating such disorders and related compositions. The invention further provides methods of measuring ACSF3 activity in a biological sample.

Description

ACSF3 MUTATIONS IN METABOLIC DISORDERS

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

[0001] Incorporated by reference in its entirety herein is a computer-readable

nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 15,039 Byte ASCII (Text) file named "710167ST25.TXT," created on April 11 , 2012.

BACKGROUND OF THE INVENTION

[0002] Methylmalonic acidemias (MMAemias) are heterogeneous metabolic disorders that exhibit elevated methylmalonic acid (MMA) in body fluids. Deficiency of

methylmalonyl-CoA mutase (MUT) or the enzymes (MMAA, MMAB, MMADHC) that synthesize 5'-adenosylcobalamin comprise most disease subtypes. Some patients have atypical forms of MMAemia, e.g., combined malonic and methylmalonic aciduria

(CMAMMA) that lack enzymatic and molecular definition.

[0003] CMAMMA was first reported in a child with immunodeficiency, failure to thrive, seizures, increased urinary MMA compared to malonic acid (MA) and normal malonyl-CoA decarboxylase activity. Gregg et al., J. Inherit. Metab. Dis., 21 : 382-90 (1998). A Labrador retriever with similar biochemical features and neurodegeneration has also been described. Podell et al., Metab. Brain. Dis., 1 1 : 239-47 (1996).

[0004] Further characterization of metabolic disorders such as CMAMMA is needed, as are new and/or improved methods of diagnosing and treating such disorders.

BRIEF SUMMARY OF THE INVENTION

[0005] The invention provides methods and compositions relating to the surprising finding that the gene ACSF3, previously identified as an orphan member of the acyl- coenzyme A synthetase gene family, is associated with the metabolic disorder CMAMMA.

[0006] The invention provides a method of diagnosing a metabolic disorder in a subject, wherein the method comprises (a) obtaining a nucleotide sample from a subject; (b) performing exome analysis to determine an exon sequence of ACSF3 in the sample; and (c) comparing the sample exon sequence with a corresponding control exon sequence, wherein the subject is diagnosed with a metabolic disorder comprising a defect in ACSF3 if an alteration is detected between the sample exon sequence and the control exon sequence.

[0007] The invention also provides a method of detecting a metabolic disorder comprising an ACSF3 defect in a subject, wherein the method comprises (a) obtaining a cell sample comprising cells from a subject having normal methylmalonyl-CoA mutase and intracellular cobalamin metabolism; (b) incubating the cells in a medium comprising a propionate for a predetermined interval; (c) obtaining a sample of the medium; (d) measuring the level of methylmalonic acid present in the medium sample; and (e) comparing the level of methylmalonic acid measured in step (d) with a control level of methylmalonic acid, wherein an elevated level of methylmalonic acid as compared to the control level indicates that the subject has a metabolic disorder comprising a defect in ACSF3.

[0008] The invention additionally provides a method of measuring ACSF3 activity in a biological sample comprising: (a) obtaining a biological sample comprising ACSF3 from a subject; (b) suspending the sample in a reaction solution comprising a buffer, MgCl₂, adenosine triphosphate (ATP), Coenzyme A (CoA), and a substrate such as malonate, or methylmalonate; and (c) measuring the rate of formation of a thioester bond between CoA and the substrate, wherein ACSF3 activity is measured in nmol/min/mg total protein in the biological sample.

[0009] The invention provides a method of treating a metabolic disorder comprising a defect in ACSF3, which method comprises administering a composition comprising ACSF3 and/or lipoic acid and/or octanoic acid and a pharmaceutically acceptable carrier.

[0010] The invention provides a method of treating CMAMMA comprising

administering an expression vector comprising ACSF3 (SEQ ID NO: 1) operably linked to a promoter to a subject in need thereof.

[0011] The invention provides an expression vector comprising ACSF3 (SEQ ID NO: 1) operably linked to a promoter, as well as compositions thereof further comprising a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0012] Figure 1 depicts alignment of the motif regions in ACSF3 orthologues and the malonyl-CoA synthase enzymes in bacteria. The ACSF3 alterations identified in the eight subjects and affected dog are indicated. The asterisk (*) indicates the dog variant

p.Gly430Ser, which is orthologous to position p.Gly480 in human ACSF3. An additional three amino acids amino-terminal to Motif I are depicted. Motif II was aligned independent of the full-length protein to improve the alignment of the ACSF3 and MCS proteins.

[0013] Figure 2A is a bar graph that depicts quantitative MMA production by fibroblasts from CMAMMA Subjects 1 -4 as compared with controls. Error bars are +/- 1 standard deviation (n=3 measurements per cell line).

[0014] Figure 2B is a bar graph that depicts quantitative MMA production by fibroblasts from CMAMMA Subjects 1 , 3, and 4 as compared with controls in the presence of lentiviral complementation with ACSF3 or GFP (negative control). Error bars are +/- 1 standard deviation (n=3 replicates per cell line).

[0015] Figure 3 is a schematic depiction of results of phylogenetic analysis of ACSF3 orthologues and ACS homologues.

[0016] Figure 4 shows the relative substrate specificity of purified ACSF3 toward malonate, methylmalonate, and acetate.

[0017] Figure 5 is a schematic depiction of the intracellular cobalamin pathway.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The ACSF3 gene is an orphan member of the acyl-coenzyme A synthetase gene family, which family includes enzymes that thioesterify substrates into CoA derivatives, and that can weakly activate C24:0 fatty acid (Watkins et al., J. Lipid Res., 48: 2736-50 (2007)). CMAMMA is the first human disorder found to be associated with mutations in a member of the acyl-CoA synthetase family, a diverse group of evolutionarily conserved proteins which includes enzymes that activate fatty acids for intermediary metabolism.

[0019] The invention provides a method of diagnosing a metabolic disorder in a subject. The method comprises (a) obtaining a nucleotide sample from a subject; (b) performing exome analysis to determine an exon sequence of ACSF3 in the sample; and (c) comparing the sample exon sequence with a corresponding control exon sequence. The control exon sequence is an exon sequence of ACSF3 of the same type of subject, e.g., a human, that does not have a metabolic disorder. The subject is diagnosed with a metabolic disorder if an alteration is detected between the sample exon sequence and the control exon sequence. Individuals with mutations in only one of the ACSF3 alleles (which mutations result in a defective ACSF3 polypeptide) typically are not clinically affected.

[0020] The invention further provides a method of detecting an ACSF3 defect in a subject comprising (a) obtaining a cell sample comprising cells from a subject; (b) incubating the cells in a medium comprising a propionate for a predetermined interval; (c) obtaining a sample of the medium; (d) measuring the level of methylmalonic acid present in the medium sample; and (e) comparing the level of methylmalonic acid measured in step (d) with a control level of methylmalonic acid, wherein an elevated level of methylmalonic acid as compared to the control level indicates that the subject has a metabolic disorder comprising a defect in ACSF3. The control level is for the same type of subject, e.g., a human, that does not have an ACSF3 defect. In preferred embodiments, the cell sample is obtained from a subject having normal methylmalonyl CoA mutase and intracellular cobalamin function.

[0021] The two aforementioned methods, which relate to an exome analysis and a cell secretion assay, respectively, can be used alone or in combination to determine whether a subject has a metabolic disorder comprising a defect in ACSF3. In preferred embodiments, the metabolic disorder is Combined Malonic and Methylmalonic Aciduria (CMAMMA).

[0022] In some embodiments, the exome analysis is performed prior to the cell secretion assay. In other embodiments, the cell secretion assay is performed prior to the exome analysis. Of course, the exome analysis and the cell secretion assay can be performed simultaneously.

[0023] If an alteration is detected between the sample exon sequence and the control exon sequence, or if the cell secretion assay indicates that the medium sample exhibits an elevated level of methylmalonic acid as compared to the control level, then the subject is identified as having a metabolic disorder comprising a defect in ACSF3. If an alteration is detected between the sample exon sequence and the control exon sequence, and if the medium sample exhibits an elevated level of methylmalonic acid as compared to the control level, then the subject can be diagnosed with CMAMMA. [0024] In some embodiments, the results of the two assays can appear to be inconsistent. For example, a subject whose cell sample exhibited elevated levels of methylmalonic acid in the cell secretion assay but who did not exhibit an alteration in an ACSF3 exon sequence can be diagnosed with CMAMMA if the subject displays otherwise clinically consistent features, such as methylmalonic acidemia. Such results could occur if the subject has a defect in ACSF3 other than an exon mutation. However, a subject exhibiting an alteration in an exon sequence of ACSF3, such as a variant of unknown significance, but having normal results of the cell secretion assay, can be excluded from having CMAMMA if otherwise clinically appropriate.

[0025] In the methods of the present invention, exome analysis can be executed using any suitable method, such as Sanger sequence analysis. However, one of ordinary skill in the art will understand that other sequencing methods can also be used. The exome analysis can be performed using a suitable primer for the exon to be analyzed, such as any of SEQ ID NOS: 2-29 and a corresponding reverse primer. In preferred embodiments, the primer is any of SEQ ID NOS: 2-4, 8-10, 13-18, 22-24, and 27-29. In a more preferred embodiment, the exome analysis is performed using primers having SEQ ID NOS: 2-29. Preferably, the 1 1 identified exons of ACSF3 are each analyzed in the exome analysis. However, in other embodiments, fewer than all exons (e.g., 10, 9, 8, 7, 6, 5, 4, 3, or 2 exons or a single exon) can be analyzed.

[0026] The ACSF3 alteration detected in exome analysis can be a substitution, insertion, deletion, or a chimeric transcript derived from a chromosomal rearrangement mutation. In some embodiments, the alteration comprises a substitution mutation at a nucleic acid position such as 593, 728, 1073, 1075, 1385, 141 1 , 1412, 1288, 1567, 1672, 1406, or 1470, relative to SEQ ID NO: l (NM_174917.2). In other embodiments, the alteration comprises a deletion of one or more nucleic acids at positions, such as 1394-141 1 , 803, or 1718, relative to SEQ ID NOT (NM_174917.2).

[0027] As noted above, however, it will be understood that a subject can exhibit a defect in ACSF3 as determined by, e.g., a cell secretion assay, but without a detectable exon mutation (e.g., a splice variant; deletion mutation in trans; promoter, enhancer, and regulatory mutations; and other mutations that affect mRNA transcript initiation, elongation, splicing, transport, or polyadenylation). In the methods of the present invention, it is not strictly necessary to determine the nature of a subject's defect in ACSF3 in order to diagnose a metabolic disorder comprising a defect in ACSF3, e.g., CMAMMA.

[0028] As in the exon analysis methods, the cell secretion assay can be executed using any appropriate method known to one of ordinary skill in the art. The priopionate can be of any suitable source. For example, the propionate can be sodium propionate. Preferably, the proprionate is provided in excess. Additionally, the concentration of methylmalonic acid present in the medium sample can be measured by any suitable method such as gas chromatography/mass spectrometry (GC/MS) analysis or liquid chromatography tandem mass spectrometry (LC-MS/MS).

[0029] In some embodiments the cell secretion assay can further comprise a

complementation assay having steps (f) transfecting a portion of the cells of step (a) with an expression vector comprising ACSF3 under control of a promoter; and (g) repeating steps (b)-(e) of the cell secretion assay with the transfected cells, wherein a level of methylmalonic acid that is not elevated as compared to a control confirms that the subject has a defect in ACSF3. Such assay can be particularly useful in embodiments where exon analysis failed to indicate a particular alteration of the ACSF3 exome. In preferred embodiments, the complementation assay can be used to confirm a diagnosis of CMAMMA in a subject displaying increased MMA secretion in the cellular secretion assay, regardless of whether alterations have been detected in an ACSF3 sequence analysis, or whether the subject exhibits clinical symptoms. In more preferred embodiments, the complementation assay can be used to confirm that a variant of unknown significance is pathogenic, i.e. is associated with CMAMMA.

[0030] The invention further provides a method of measuring ACSF3 activity in a biological sample. The method comprises (a) obtaining a biological sample comprising ACSF3 from a subject; (b) suspending the sample in a reaction solution comprising a buffer, MgCl₂, adenosine triphosphate (ATP), Coenzyme A (CoA), and a substrate such as malonate or methylmalonate; and (c) measuring the rate of formation of a thioester bond between CoA and the substrate, wherein ACSF3 activity is measured in nmol/min/mg total protein in the biological sample. In some embodiments, ACSF activity comprises the rate of formation of the thioester bond per unit of enzyme where such activity is typically expressed as nanomoles of methylmalonyl-CoA or malonyl-CoA formed per minute per milligram of protein in the reaction. In clinical applications, ACSF3 is present in the biological sample that in turn provides the protein in the reaction mixture. It will be understood that a substrate of acetate can be evaluated as a negative control. The rate of formation of the thioester bond can measured by spectrophotometry, such as absorbance at 232 nm.

[0031] Preferably, the biological sample comprises one or more components such as cells, tissues, extracts, and organelles. For example, the biological sample can comprise a tissue sample or a cell sample taken from a subject. In some embodiments, the cells or tissues can be separated (e.g., by centrifugation) to provide extracts. A tissue sample, such as a liver biopsy, can be homogenized, and one or more fractions extracted for analysis. In some embodiments, organelles such as mitochondria can be further separated for analysis. In some preferred embodiments, the ACSF3 can be purified or isolated from the biological sample and employed in the assay. In particular embodiments, the source of ACSF3 comprises an affinity-tagged ACSF3. It will be understood that a homogenous sample is not necessarily required in such analysis.

[0032] A cell sample for use in any of the methods of the invention can comprise any suitable type of cells. For example, the cell sample can comprise fibroblasts or lymphocytes. In a preferred embodiment, the cell sample comprises lymphocytes that are transformed with Epstein-Barr Virus (EBV). Such cells are suitable for use in the cell secretion assay and also can be used as a source of a nucleotide sample.

[0033] In another embodiment, the invention provides a method of treating a metabolic disorder comprising a defect in ACSF3, which method comprises administering a

composition comprising ACSF3 and a pharmaceutically acceptable carrier. A method of treating a metabolic disorder comprising a defect in ACSF3 can also comprise administering a composition comprising lipoic acid and/or octanoic acid and a pharmaceutically acceptable carrier. In preferred embodiments, the disorder comprises CMAMMA.

[0034] In yet another embodiment, the invention provides an expression vector comprising A CSF3 (SEQ ID NO: 1 ) operably linked to a promoter, as well as a method of treating CMAMMA comprising administering such an expression vector to a subject in need thereof. The expression vector can be any suitable vector for administration to a subject, such as a lentiviral vector. The promoter can be any suitable promoter, such as a CMV promoter. The invention further provides a composition comprising an expression vector ; comprising ACSF3 (SEQ ID NO: 1) operably linked to a promoter and a pharmaceutically acceptable carrier.

[0035] In any of the methods of the present invention, the subject preferably exhibits normal levels of one or more clinical parameters such as Vitamin B12, methylmalonyl-CoA mutase activity, and intracellular cobalamin enzymatic function. Preferably, the subject exhibits at least one clinical symptom associated with CMAMMA such as increased methylmalonic acid compared to malonic acid in the urine and/or blood, seizures, memory loss, neurocognitive decline, frequent urination, coma, ketoacidosis, hypoglycemia, failure to thrive, elevated transaminases, microcephaly, dystonia, axial hypotonia, multiple sclerosis, atypical multiple sclerosis, and developmental delay. Characterization of such symptoms will be understood by one of ordinary skill in the art and are described, for example, in Gregg et al., J. Inherit. Metab. Dis., 21 : 382-90 (1998).

[0036] In any of the methods of the invention, the subject can be any suitable mammal such as a human, a non-human primate, a dog, a cat, a cow, a pig, a horse, a rabbit, a mouse, or a rat. The subject can be an adult or a juvenile.

[0037] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0038] This example provides characterization of clinical and biochemical features of individuals diagnosed with CMAMMA.

[0039] Sixteen individuals with CMAMMA were evaluated. The age of diagnosis and symptoms were variable (Tables 1 A and IB). After uneventful early decades, nine individuals were diagnosed in adulthood with neurological manifestations (seizures, memory problems, psychiatric disease, and/or cognitive decline) without vitamin B12 deficiency. Eight individuals exhibited during childhood symptoms suggestive of an intermediary metabolic disorder (coma, ketoacidosis, hypoglycemia, failure to thrive, elevated

transaminases, microcephaly, dystonia, axial hypotonia, and/or developmental delay). Table 1A

Table IB

[0040] Plasma methylmalonic acid was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) stable isotope dilution analysis, and urine organic acids were measured by gas chromatography-mass spectrometry (GC/MS) (Mayo Medical Laboratories). A GC/MS assay was developed to measure malonic acid (MA) in patient samples. In particular, D₃ methylmalonic and C₂-malonic acid were added to plasma, serum, or urine, adjusted with NaCl, and acidified. An ethyl acetate extraction was performed, and the organic layer was concentrated under N₂ flow. The resulting residue was silylated with BSTFA + 1% TMCS (N,0-bis(trimethylsilyl) trifluoroacetamide + 1% trimethylchlorosilane). The samples were then analyzed by GC/MS in the selected ion monitoring mode. Plasma samples from 19 anonymous controls with normal methylmalonic acid (MMA) levels were used to develop the reference ranges for MA (mean 0.67 μΜ ± 0.14, range 0.38 to 0.89 μΜ).

[0041] Methylmalonic acid and malonic aciduria with a ratio of urinary MMA to MA greater than 5 was present in nine of the fifteen affected subjects (the value for Subject 10 was not determined) (Table 1A).

[0042] In Subjects 1 -9, serum MMA was elevated but serum B12 levels, acylcarnitines, and total homocysteine were normal, as were malonyl-CoA decarboxylase activity, 1 -C¹⁴- propionate incorporation, and sequencing of known MMAemia genes (Table 1 A). Malonyl- CoA decarboxylase (MLYCD) genetic testing also returned no abnormalities in the subjects, except Subject 7, who displayed a heterozygous c.642-5C>T mutation. Conditions that can feature methylmalonic academia (MMAemia), such as those caused by mutations in known genes in the intracellular cobalamin pathway (Figure 5) and methylmalonyl-CoA mutase (LMBDR1, MMACHC, MMADHC, MMAB, MMAA, MMAB, MCEE, MUT, SUCLA2, SUCLG1), or those that cause increased MMA as a secondary manifestation of the disorder, such as malonyl-CoA decarboxylase, were therefore excluded.

[0043] Moreover, although MA and MMA levels for all tested subjects were elevated as compared to controls, urine MM A/MA ratio for all tested subjects was greater than one, and therefore inconsistent with a diagnosis of malonyl-CoA decarboxylase activity. Plasma MA was measured by GC/MS in six subjects and was also markedly elevated (Table 1A). [0044] These results demonstrate characteristic features of subjects with CMAMMA while further demonstrating that the subjects did not display characteristics of other MMemia conditions.

EXAMPLE 2

[0045] This example demonstrates use of exome and candidate gene sequencing analysis to determine the role of ACSF3 in CMAMMA.

[0046] Whole exome sequencing of a single individual, Subject 1 of Example 1 , with CMAMMA was performed to identify mutations in ACSF3, a putative methylmalonyl-CoA and malonyl-CoA synthetase (MCS). DNA was isolated from whole blood using the salting out method (Qiagen Inc., Valencia, CA) following the manufacturer's instructions. For Subject 1, target-selected libraries were sequenced in the paired-end 101 bp configuration, yielding 1 14,467 variant genotypes. Genetic filters were used for homozygosity or compound heterozygosity. Nonsynonymous, splice, frameshifting, and nonsense variants were included as potential mutations, but dbSNP variants were excluded. Control exome data (Biesecker et al., Genome Res., 19: 1665-74 (2009)) was used to exclude homozygous variants or variants with >10% frequency, as shown in Table 2.

Table 2

[0047] Solution hybridization exome capture was carried out using the SureSelect Human All Exon™ exome sequencing system (Agilent Technologies, Inc., Santa Clara, CA).

Manufacturer's protocol version 1.0 compatible with paired end sequencing (Illumina, Inc., San Diego, CA) was used, with the exception that DNA fragment size and quality was measured using a 2% agarose gel. Flow cell preparation and 101 bp paired-end read sequencing were carried out as per protocol for the GAIIx sequencer (2) (Illumina, Inc., San Diego, CA). A single 101 base pair paired-end lane on a GAIIx flowcell was used per exome sample to generate sufficient reads to generate the aligned sequence. Image analyses and base calling on all lanes of data were performed using Illumina Genome Analyzer Pipeline™ software (GAPipeline versions 1.4.0 or greater, Illumina, Inc., San Diego, CA) with default parameters.

[0048] Reads were aligned to a human reference sequence (UCSC assembly hgl 8, NCBI build 36) using the package called "efficient large-scale alignment of nucleotide databases" (ELAND). Reads that align uniquely were grouped into genomic sequence intervals of about 100 kb, and reads that fail to align were binned with their paired-end mates. Reads in each bin were subjected to a Smith-Waterman-based local alignment algorithm, cross natch using the parameters -minscore 21 and -masklevel 0 to their respective 100 kb genomic sequence. Genotypes were called at all positions where there were high-quality sequence bases (Phred- like Q20 or greater) using a Bayesian algorithm (Most Probable Genotype - MPG). See, e.g., Teer et al, Genome Res., 20: 1420-31 (2010)).

[0049] Filters were applied using criteria that were implemented using the VarSifter (//iubio.bio. indiana.edu/soft/molbio/nhgri/VarSifter/) software program for exome and whole genome data management (Teer et al., unpublished). The filters for homozygosity or compound heterozygosity in the proband were used because most metabolic diseases are autosomal recessive, and those for mutation type (nonsynonymous, splice, frameshift, and nonsense) were selected because they encompass the majority of disease-causing variants. However, these filters would not detect large deletions, regulatory mutations, or non- canonical splice mutations, which can account for several percent of causative mutations. Alleles present in dbSNP were also excluded. A MAF (minor allele frequency) filter of <10% was applied to a cohort of 258 subjects who were sequenced with similar

methodology. Variants that were homozygous in controls and were not present in the patient group were initially filtered out, with the assumption that no member of the control cohort could have CMAMMA.

[0050] The filtering strategy yielded 12 genes. ACSF3, an orphan member of the acyl- CoA synthetase family, was selected for further evaluation based on its putative function and predicted mitochondrial localization. [0051] Sequence analysis of ACSFS was performed using standard methods. Sequencing was performed with a v3.1 BigDye™ terminator cycle sequencing kit (Applied Biosystems, Carlsbad, CA) and the ABI 3130 genetic analyzer (Applied Biosystems, Carlsbad, CA) per the manufacturer's protocol. Sequence data were compared with the published ACSF3 sequence (GenBank reference number NM_174917.2; SEQ ID NO:l) using Sequencher 4.10.1 (Gene Codes Corp., Ann Arbor, MI). Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence. The initiation codon is codon 1.

[0052] As indicated in Table 1 A, three ACSF3 exome variants were detected in Subject 1 (c. l385A>C p.Lys462Thr, c.dell 394_141 1 , p.Gln465_Gly470del, and C.16270T, p.Arg558Trp) and confirmed by Sanger sequencing using primers as provided in Table 3. The variants p.Lys462Thr and p.Gln465_Gly470del were in trans with p.Arg558Trp based on parental genotypes and segregated in two unaffected siblings who, like their parents, had normal serum MM A levels.

Table 3

Gene Exon Forward Primer 5 -3' SEQ Reverse Primer SEQ ID

ID NO: 5 '-3' NO:

Human Exon 3(A) ACGTTTGGATGGG 2 GGATGCTTCC 16 ACSF3 ACAGTTG TGTAGAGGG

G

Exon 3(B) GCAGGCTCTGCGG 3 GACTCCACAG 17 GTGTGTC AAAAGCGAA

TG

Exon 4 AGGTCTGTGTGTGC 4 GAAAGGCGC 18 TGTTGC TTAGGCTGAG

G

Exon 5(A) ATGAGAACGCTGT 5 CTGCTTCCCA 19 GCCTGGAG GAACTTAGTA GG

Exon 5(B) CTACCGAGTGCTTC 6 GAAAGTGGG 20 CTTTCC CTCTTTTCAC

Exon 5(C) GTTCTTAAGTTCTG 7 TTTTCTTCAC 21

AAACG AAACTGCACG

Exon 6(A) GCTAAACCTGCCAC 8 TCGAGACTGG 22 CTTTGC CCCACCTTGG

Exon 6(B) CCTGCCTTTGGTTG 9 GCAGCTGTGG 23 TGCCGCGTAG GAAGTGCTC

Exon 6(C) AGTGCTGGAGAAG 10 CAGAGCCATG 24 TGGAAG . CCGATCTCG Exon 7 TGTGTGCTTCTCTC 11 GATGCACCAG 25

CTCCAG TGTAACCACC

Exon 8 TTTCAGAAAGCACC 12 CAATGAGTTC 26

AATCCC CTGCCTGTCC

Exon 9 AGACCCCACATCAT 13 TCTAAAACTC 27

GGGCACAG AAACATGGA

AGGC

Exon 10 GCCTGTAAGGGTC 14 CGATGCCAAT 28

ACTGAGG ACCTAGGGTG

Exon 1 1 CTGAGTTCCTCCTG 15 CCGTGGTTCT 29

CTGGGC CGGTGTGAAG

Canine Exon 1(B) GAGCGGAGGCATT 30 AGAACAGTG 41 ACSF3 GCTGTCC GCAGCTATGG

Exon 1(A) CGGTGGAACAGGT 31 CGCAAGGAC 42

CTGGTGG CACAGAGCTC

C

Exon 2 ATCTAAGCCCTGAC 32 CCACACCCCA 43

CATGTCC ACTTTCATGC

Exon 3 AGGGCTGTGCCTCT 33 TAAAGGGAG 44

GCTCTTG TGGAATACAC

TGC

Exon 4 CGATACCCTGTTTG 34 TTCTCTCCTG 45

TCATGAAC TCCCCGACTG

G

Exon 5 CAGCCTCAGCCTCA 35 CTGCGTGTCG 46

AGCCTGG GCTATAGACG

Exon 6 AGTTCCAATGTTGA 36 GGGCTCCTGA 47

AAGATGC CCATGATGAC

Exon 7 GGGTAGGGGACCT 37 TGAAATACAC 48

ATGTTCC ATGGAAGCAT G

Exon 8 GGGCTCCACCCAA 38 CTTCCCTGCA 49

AACACAGTG GCCTCAGGAA

TG

Exon 9 ACTTTCACCTTACT 39 TATCTCTAGC 50

GTAGACCG GCTGAGGAGT

GG

Exon 10 CCAGGCTGCCTGTC 40 TCTTGCTTCT 51

CCATGG GTCTGGGTTA

GG

[0053] Follow-up sequencing of the subjects showed that the majority exhibited pathogenic mutations, as shown in Tables 1 A and IB. The identified mutations of Subjects 1-9 included nine missense, one in-frame deletion, and one nonsense mutation (Figure 1 ). One subject had no damaging mutations detected. Four of Subjects 1 -9 were apparently homozygous for ACSF3 mutations. As shown in Figure 1 , most of the variants resided in the C-terminal half of ACSF3. Eight out of nine missense mutations and the in-frame deletion of Subjects 1-9 were located in conserved ACS motifs predicted to be involved in AMP binding (Motif I), conformational change and catalytic function (Motif II), substrate binding (Motifs III, IV), or catalysis (Motif V). See Watkins et al, J. Lipid. Res., 48: 2736-50 (2007);

Hiltunen et al., Prog. Lipid. Res., 49: 27-45 (2010).

[0054] Sequencing was also performed on a canine affected with CMAMMA. DNA was isolated using a commercially available salting out method (Qiagen Inc., Valencia, CA) from a fibroblast cell line.

[0055] As no canine orthologue for ACSF3 was known, the Dog Genome (UCSC browser, May 2005 build) was used to predict the sequence for canine A CSF3, and primers were designed to amplify the exonic regions of the gene. Dog liver cDNA was obtained (Zyagen, San Diego, CA), and primers for the predicted dog cDNA were used to amplify the transcript. The dog ACSF3 partial cDNA sequence has been submitted to GenBank,

Accession Number JF907588.1. The canine ACSF3 orthologue showed a homozygous alteration (c. l288G>A, p.Gly430Ser; orthologous to human p.Gly480) in a conserved residue (Figure 1 , Table IB). This variant was absent in 40 control Labrador DNAs selected for maximum diversity based on American Kennel Club numbers.

[0056] These results provide evidence that mutations in ACSF3 are responsible for the metabolic disorder CMAMMA.

EXAMPLE 3

[0057] This example demonstrates use of exome data analysis of ACSF3 to identify individuals having biochemical features of CMAMMA.

[0058] Exome data was analyzed as described in Example 2 for 401 individuals ascertained for cardiovascular phenotypes (Biesecker et al., Genome Res., 19: 1665-74 (2009)). A 66 year-old female was found to be homozygous for a c.141 1 C>T, p.Arg471Trp ACSF3 variant. She had no previously known metabolic disease symptoms but reported incontinence and mild memory problems. Her laboratory evaluation showed 48 μΜ MMA and 1 1.3 μΜ MA in plasma and 206 mmol/mol Cr MMA and 26.3 mmol/mol Cr MA in urine, and normal serum B12 levels and acylcarnitines. The ratio of urinary MMA to MA was 7.8, a ratio consistent with a diagnosis of CMAMMA. No other mutations of known MMAemia genes were detected in her exome (Table 1A).

[0059] Clinical, biochemical, and mutational analysis indicate a diagnosis of CMAMMA in this individual.

[0060] In one analyzed cohort, an additional four participants were found to be heterozygous for ACSF3 variants (p.Glu359Lys n=T, p.Arg558Trp n=3) also found in subjects with CMAMMA. In another cohort (estimated coverage of 629 genomes), there were six individuals with ACSF3 mutations (p.Glu359Lys n=l, p.Arg558Trp n=5).

Combining these data yields an overall MAF of 0.0058 (95% CI, .0033-.0106).

[0061] Taken together, these results demonstrate that mutation in an individual's ACSF3 exome is predictive of CMAMMA in the individual, and that the estimated disease incidence of CMAMMA is approximately 1/30,000 (95% CI, 1/9,000 - 1/92,000).

[0062] Although individuals heterozygous for ACSF3 variants (i.e., carriers) typically are not biologically affected, elevated plasma MMA levels have been observed (see Table 4).

Table 4

p.Pro268LeufsX13

[0063] The carrier frequency of ACSF3 is believed to be rather high, wherein 1 in 30 or 1 in 40 individuals is heterozygous for ACSF3 variants. Therefore, if a patient exhibits elevated MMA levels, it would be beneficial to screen the individuals for ACSF3 mutations, which could account for the elevated MMA levels and thereby preclude further testing for vitamin B12 deficiency or other disorders.

EXAMPLE 4

[0064] This example provides a method for qualitative and quantitative analysis of ACSF3 expression in individuals with CMAMMA as compared to controls.

[0065] Control fibroblasts and fibroblasts from Subjects 1-4 of Example 1 were incubated in medium containing 5 mM sodium propionate at 37 °C for 72 hours, and the media was removed for GC/MS analysis of MMA.

[0066] Thirty to forty micrograms of clarified fibroblast extract from Subjects 1-4 and 7 of Example 1 , as well as similar samples from three unaffected control individuals were analyzed by Western blot using a rabbit polyclonal anti-ACSF3 (abl 00860; Abeam,

Cambridge, MA) or mouse monoclonal anti-PDH-E2 (MSP05; MitoSciences, Eugene, OR) at a dilution of 1 : 1,500. Mouse monoclonal anti-P-actin (ab8226, Abeam, Cambridge, MA) was used as a loading control for immunoblotting at a dilution of 1 : 1 ,000. Horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (NA934 or NA931 ; GE Healthcare Life Sciences, Piscataway, NJ) was used as the secondary antibody and was visualized with chemiluminescence detection (Pierce Biotechnology, Rockford, IL).

[0067] GC/MS analysis of cells from Subjects 1-4 showed increased accumulation of MMA in the media, which were 6, 2.4, 5.3, and 2.4 fold elevated compared to the control cell lines (Figure 2A) after chemical stimulation. Western analyses using fibroblasts from Subjects 1-4 and 7 of Example 1 showed the presence of cross-reactive ACSF3.

[0068] These results demonstrate that immunoreactive ACSF3 is expressed in individuals having ACSF3 mutations at levels similar to healthy controls, but the expressed ACSF3 enzyme is enzymatically inactive, i.e., is not capable of metabolizing methylmalonic acid at normal levels.

EXAMPLE 5

[0069] This example demonstrates use of viral complementation to compensate for defects in ACSF3.

[0070] Wild-type ACFS3 cDNA was generated by RT-PCR from total RNA extracted from normal human liver tissue and sequence validated. This gene was cloned into a Gateway™ retroviral expression vector (Invitrogen, Carlsbad, CA), pLenti6/V5-DEST, as recommended by the manufacturer. The viral constructs express ACSF3 or GFP under the control of the CMV promoter; the backbone also has a blasticidin cassette driven by the E7 promoter. Human fibroblast cell lines from Subjects 1 , 3, and 4 of Example 1 , as well as fibroblast cell lines from three healthy control individuals, were transduced with virus containing either the ACSF3 or GFP.

[0071] The cells were transduced and incubated with cell culture medium for 24-48 hours. Then, the medium was removed and replaced with medium containing 10 μg/ml blasticidin for selection. The cells were further incubated for 5 days, after which dead cells were removed. The resulting blastocidin-resistant cells were then passaged and expanded. Six well tissue culture plates were seeded at a density of 2xl0⁵ or 5xl0⁵ per well in high glucose (4 g/L) DMEM supplemented with 10% fetal bovine serum, penicillin streptomycin, L-glutamine, and sodium pyruvate. The next day, the DMEM growth media was removed and replaced with 1 ml of DMEM growth media containing sodium propionate at a concentration of 5 mM. After 72 hours the media was collected for GC/MS analysis of MMA.

[0072] ACSF3 with a C-terminal GFP fusion was cloned into pCMV6 and sequence verified. Control fibroblasts were electroporated with 3 μg of plasmid DNA using an Amaxa nucleofector electroporator (Amaxa GmbH, Walkersville, MD). Transfected fibroblasts were grown for 48 hours before immunofluorescence experiments.

[0073] Viral expression of ACSF3, but not GFP (Figure 2B), restored metabolism of methylmalonic acid to approximately normal levels in fibroblasts expressing mutant ACSF3.

[0074] These results provide validation of ACSF3 function in a cell culture biochemical assay.

EXAMPLE 6

[0075] This example provides in vitro analysis of the function of ACSF3, as well as its subcellular localization properties.

[0076] Purified, GST-tagged ACSF3 was analyzed under modified assay conditions as described in oo et al., Arch. Biochem. Biophys., 378: 167-74 (2000).

[0077] Full-length ACSF3 containing an N-terminal GST fusion, expressed in wheat germ extract, was obtained from Novus Biologicals (Littleton, CO) and used to assay malonyl- and methylmalonyl-CoA synthetase activity with a previously described spectrophotometric method (Koo et al., Arch. Biochem. Biophys., 378: 167-74 (2000)). The reaction mixture contained the following components in a volume of 500 iL: 100 mM potassium phosphate buffer (pH 7.0), 8 mM malonate, methylmalonate, or acetate, 2 mM MgCl₂, 0.4 mM ATP, 0.2 mM CoA, and 1.43 μg of GST-tagged, purified ACSF3. An increase in absorbance at 232 nm was used to measure the formation of the thioester bond (ε²³²=4.5χ10^'3 M^"1 cm^"1) and determine enzyme activity, represented as specific activity (nmol/min/mg) toward the three substrates assayed.

[0078] The enzyme activated malonate and methylmalonate, but not acetate, into the respective coenzyme thioesters (Table 5).

Table 5

Substrate Specific activity Relative Rate

(nmol/min/mg protein) (% of malonate)

Malonate 1334 100

Methylmalonate 799 60

Acetate Not detected 0 [0079] The specific activity of GST-tagged ACSF3 was higher with malonate as a substrate compared to methylmalonate, similar to its prokaryotic homologues.

[0080] Next, because the first 58 amino acids of ACSF3 were predicted to encode a mitochondrial leader sequence (Figure 4), immunostaining was performed on fibroblasts overexpressing ACSF3 and a C-terminal GFP-ACSF3 fusion protein.

[0081] Control fibroblasts transfected with pCMV-ACSF3-GFP and fibroblasts from Subject 4 stably expressing ACSF3 as described above were grown on chamber slides, fixed with 3% paraformaldehyde in IX phosphate buffered saline (PBS), permeabilized with 0.5% Triton X 100 in IX PBS, and blocked in 1% donkey serum, 0.1% saponin, and 100 μΜ glycine in PBS. Fibroblast slides were incubated with rabbit polyclonal ACSF3 antibody (abl 00860; Abeam, Cambridge, MA) and mouse monoclonal mitochondrial MTC02 antibody (ab3298; Abeam, Cambridge, MA) in a solution containing IX PBS, 0.1 % bovine serum albumin (BSA) and 0.1% saponin overnight at 4 °C. The cells were washed and incubated with donkey anti-rabbit IgG conjugated to Alexa Fluor 555™ and donkey anti- mouse IgG conjugated to Alexa Fluor 488™ or Alexa Fluor 633™ (Invitrogen, Carlsbad, CA) for 1 hour at room temperature. Slides were washed with IX PBS and mounted with VectaShield™ mounting medium (Vector Laboratories, Inc., Burlingame, CA) containing DAPI.

[0082] Upon imaging of the slides, ACSF3 staining showed a distinct mitochondrial distribution and co-localized with a mitochondrial antibody.

[0083] These results show that that ACSF3 is a mitochondrial methylmalonyl-CoA and malonyl-CoA synthetase (MCS), which catalyzes the first step of intramitochondrial fatty acid synthesis.

EXAMPLE 7

[0084] This example provides phylogenic analysis of ACSF3 as compared to putative orthologues.

[0085] ACSF3 orthologues were identified by BLAST search and through Homologene. Sequence alignment of the ACSF3 orthologues: human NP_001 120686.1 , mouse

NP_659181.2, dog JF907588.1 , cow NP_001030240.1 , rat XP__574249.3, zebrafish

XPJ590782.2, Xenopus NP_001086314.1 , B. japonicum NPJ767149.1 and R. leguminosarum AAC83455.1 , was performed by the Clustal W method in MacVector™ version 9.0.2 (MacVector, Inc., Cary, NC). The phylogenetic tree was created in

MegAlign™ (Lasergene, DNASTAR, Inc., Madison, WI) by the Clustal W method.

Similarity was determined by BLAST-P using the BLOSUM 62 matrix. The mitochondrial leader sequence was predicted using MitoProtll (//ihg.gsf.de/ihg/mitoprot.html).

[0086] In comparing ACSF3 to Bradyrhizobium japonicum malonyl-CoA synthetase (MCS), a well-characterized enzyme, the proteins were determined to be more identical (32%) and similar (50%) to each other than ACSF3 was to the next closest human ACS family member (ACSM3vl, 28% identity). Phylogenetic analyses rooted human ACSF3 with the MCS enzymes versus other ACSs (Figure 3).

[0087] MCS from R. trifolii and B. japonicum activate malonate and methylmalonate as substrates in vitro (An et al., Eur. J. Biochem., 257: 395-402 (1998); Koo et al., Arch.

Biochem. Biophys., 378: 167-74 (2000)) as does ACSF3 (Table 5), suggesting that malfunction of this enzyme causes accretion of the proximal substrates that manifests as methylmalonic and malonic aciduria.

[0088] Site-directed mutagenesis experiments with B. japonicum MCS previously showed that p.Glu308Gln abolishes malonate binding (Koo et al., Arch. Biochem. Biophys., 378: 167-74 (2000)). The corresponding human ACSF3 position is the residue mutated in Subject 3 of Example 1 , p.Glu359Lys in Motif III, and predicts that this mutation is likely to effect the K_m for malonate. Arg471 in motif II is nearly invariant in the ACS family (Watkins et al., J. Lipid Res., 48: 2736-50 (2007)) and essential for acyl-CoA synthetase activity. See, e.g., Black et al., J. Biol. Chem., 272: 4896-903 (1997); Stuible et al., FEBS Lett., 467: 1 17-22 (2000); Zou et al., J. Biol. Chem., 277: 31062-71 (2002). Therefore, an Arg471 alteration in ACSF3, as in Subjects 5 and 6 of Example 1 , likely affects enzymatic function. Other missense alterations (p.Pro243Leu, p.Thr358Ile, p.Gly430Ser (dog), p.Arg558Trp) map to conserved residues in B. japonicum and R. leguminosarum MCS (Figure 1) or to conserved residues in other ACSF3 family members (p.Metl98Arg, p.Lys462Thr) and are likely to be deleterious. EXAMPLE 8

[0089] This example demonstrates a technique for constructing an animal model of CMAMMA.

[0090] A targeting vector, designed to eliminate and/or modify the DNA sequence of the mouse homologue of ACSF3 is prepared, using standard techniques. Typically, this involves cloning the 5' and 3' regions that flank a critical exon or coding sequence in ACSF3. The targeting vector carries a selectable marker so that recombination events can be enriched in recombinant clones by antibiotic selection, such as neomycin resistance.

[0091] Mouse embryonic stem cells are then prepared and expanded in cell culture using standard techniques. The targeting construct is then introduced into the cells by

electroporation. In a subset of cells, homologous recombination introduces DNA from the targeting vector into the ACSF3 locus, thereby producing an ACSF3 that has been modified to reduce or eliminate function and creating an ACSF3 mutant allele.

[0092] Antibiotic selection enriches for ACSF3 recombinant clones that are then expanded for DNA and protein analysis. The structure of the recombinant allele is confirmed by Southern analysis and PCR. A karotype is performed on each clone to assess aneuploidy.

[0093] ACSF3 targeted clones with normal chromosomal constitution are injected into the blastula, and resulting chimeric animals are identified by coat color. The chimeras are mated to produce Fl progeny and establish lines that can transmit the ACSF3 targeted locus.

[0094] Carriers of ACSF3 targeted mutation are crossed to generate mice that are homozygous for the ACSF3 targeted mutation. These animals will lack ACSF3 enzyme activity and will provide an animal model of CMAMMA that can be used to study pathophysiology and examine therapeutic interventions such as lipoic acid administration, gene therapy and/or enzyme replacement therapy.

EXAMPLE 9

[0095] This example demonstrates a technique for protein therapy for CMAMMA.

[0096] ACSF3 is cloned into a expression vector to direct either prokaryotic or eukaryotic production, preferably using a vector that contains an inducible promoter as is readily available. An affinity tag may be included into the ACSF3 to facilitate purification. [0097] Cells that express the ACSF3 from the recombinant DNA construct are grown in culture, and expression of ACSF3 is induced, preferably by chemical means, for example by exposure to an inducer such as ITPG (bacterial) or tetracycline (eukaryotic).

[0098] The cells are lysed by chemical, enzymatic, or mechanical means, and ACSF3 is purified from the extract. The purified enzyme may require modification, such as

PEGylation.

[0099] Purified enzyme is then administered to cells, mice, or patients with CMAMMA to restore or augment enzyme activity.

[00100] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[00101] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[00102] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A method of diagnosing a metabolic disorder in a subject, the method comprising:

(a) obtaining a nucleotide sample from a subject;

(b) performing exome analysis to determine an exon sequence of ACSF3 in the sample; and

(c) comparing the sample exon sequence with a corresponding control exon sequence, wherein the subject is diagnosed with a metabolic disorder comprising a defect in ACSF3 if an alteration is detected between the sample exon sequence and the control exon sequence.

2. The method of claim 1, wherein the exome analysis is Sanger sequence analysis.

3. The method of claim 1 or claim 2, further comprising the steps of:

(d) obtaining a cell sample comprising cells from the subject;

(e) incubating the cells in a medium comprising a propionate for a predetermined interval;

(f) obtaining a sample of the medium;

(g) measuring the level of methylmalonic acid present in the medium sample; and

(h) comparing the level of methylmalonic acid measured in step (d) with a control level of methylmalonic acid, wherein the subject is diagnosed with Combined Malonic and Methylmalonic Aciduria (CMAMMA) if the medium sample exhibits an elevated level of methylmalonic acid as compared to the control level, and an alteration is detected between the sample exon sequence and the control exon sequence.

4. A method of detecting a metabolic disorder comprising an ACSF3 defect in a subject, the method comprising: (a) obtaining a cell sample comprising cells from a subject having normal methylmalonyl CoA mutase and intracellular cobalamin metabolism;

(b) incubating the cells in a medium comprising a propionate for a predetermined interval;

(c) obtaining a sample of the medium;

(d) measuring the level of methylmalonic acid present in the medium sample; and

(e) comparing the level of methylmalonic acid measured in step (d) with a control level of methylmalonic acid, wherein an elevated level of methylmalonic acid as compared to the control level indicates that the subject has a metabolic disorder comprising a defect in ACSF3.

5. The method of claim 3 or 4, wherein the propionate is sodium propionate.

6. The method of claim 3 or 4, wherein the level of methylmalonic acid present in the medium sample is measured using gas chromatography/mass spectrometry (GC/MS) analysis or liquid chromatography tandem mass spectrometry (LC-MS/MS).

7. The method of claim 3 or 4, further comprising:

(f) transfecting a portion of the cells of step (a) with an expression vector comprising ACSF3 under control of a promoter; and

(g) repeating steps (b)-(e) with the transfected cells, wherein a level of methylmalonic acid that is not elevated as compared to a control confirms that the subject has a defect in ACSF3.

8. The method of any one of claims 4-7, further comprising:

(h) obtaining a nucleotide sample from the subject;

(i) performing exome analysis to provide an exon sequence of ACSF3 in the sample;

(J) comparing the sample exon sequence with a corresponding control exon sequence; wherein the subject is confirmed to have a metabolic disorder comprising a defect in ACSF3 if an alteration is detected between the sample exon sequence and the control exon sequence.

9. The method of any one of claims 3-8, wherein the cell sample comprises lymphocytes or fibroblasts.

10. The method of claim 9, wherein the lymphocytes are transformed with Epstein-Barr Virus (EBV).

1 1. A method of treating a metabolic disorder comprising a defect in ACSF3, which method comprises administering a composition comprising lipoic acid and/or octanoic acid and a pharmaceutically acceptable carrier.

12. The method of claim 1 or 8, wherein the exome analysis is performed using a primer selected from the group consisting of SEQ ID NOS: 2-29 and a corresponding reverse primer

13. The method of claim 12, wherein the primer is selected from the group consisting of SEQ ID NOS: 2-4, 8-10, 13-18, 22-24, and 27-29.

14. The method of claim 12, wherein the exome analysis is performed using primers having SEQ ID NOS: 2-29.

15. The method of claim 1 or 8, wherein the alteration is a substitution, insertion, or deletion mutation.

16. The method of claim 15, wherein the alteration comprises a substitution mutation at a nucleic acid position selected from the group consisting of 593, 728, 1073, 1075, 1385, 141 1 , 1412, 1288, 1567, and 1672, relative to SEQ ID NO: l (NM_174917.2).

17. The method of claim 15, wherein the alteration comprises a deletion of one or more nucleic acids at positions selected from the group consisting of 1394-141 1 , relative to SEQ ID NO: l (NM_174917.2).

18. The method of any preceding claim, wherein the subject exhibits normal levels of one or more clinical parameters selected from the group consisting of Vitamin B12, methylmalonyl-CoA mutase, and intracellular cobalamin enzymatic function.

19. The method of any one of claims 1 , 2 or 8-18, wherein the metabolic disorder is Combined Malonic and Methylmalonic Aciduria (CMAMMA).

20. The method of claim 19, wherein the subject exhibits at least one clinical symptom associated with CMAMMA.

21. The method of claim 20, wherein the clinical symptom associated with CMAMMA is selected from the group consisting of: increased methylmalonic

acid/methylamonic urea, seizures, memory loss, neurocognitive decline, frequent urination, coma, ketoacidosis, hypoglycemia, failure to thrive, elevated transaminases, microcephaly, dystonia, axial hypotonia, multiple sclerosis, atypical multiple sclerosis, and developmental delay.

22. A method of measuring ACSF3 activity in a biological sample, the method comprising:

(a) obtaining a biological sample comprising ACSF3 from a subject;

(b) suspending the sample in a reaction solution comprising a buffer, MgCl₂, adenosine triphosphate (ATP), Coenzyme A (CoA), and a substrate selected from the group consisting of malonate and methylmalonate.

(c) measuring the rate of formation of a thioester bond between CoA and the substrate, wherein ACSF3 activity is measured in nmol/min/mg total protein in the biological sample.

23. The method of claim 22, wherein the biological sample comprises one or more components selected from the group consisting of cells, tissues, extracts, and organelles.

24. The method of claim 23, wherein the biological sample comprises liver tissues.

25. The method of claim 22, wherein the organelles comprise mitochondria

26. The method of claim 22, wherein the rate of formation of the thioester bond is measured by spectrophotometry absorbance at 232 nm.

27. A method of treating CMAMMA comprising administering a composition comprising ACSF3 and a pharmaceutically acceptable carrier to a subject in need thereof.

28. A method of treating CMAMMA comprising administering an expression vector comprising ACSF3 (SEQ ID NO: 1) operably linked to a promoter to a subject in need thereof.

29. The method of claim 28, wherein the expression vector is a lentiviral vector.

30. The method of claim 28, wherein the promoter is a CMV promoter.

31. The method of any of claims 1 -30, wherein the subject is a human.

32. The method of any of claims 1-31, wherein the subject is an adult.

33. The method of any of claims 1-31, wherein the subject is a juvenile.

34. An expression vector comprising ACSF3 (SEQ ID NO: 1) operably linked to a promoter.

35. A composition comprising (a) an expression vector comprising ACSF3 (SEQ ID NO: 1) operably linked to a promoter and (b) a pharmaceutically acceptable carrier.

36. A composition comprising ACSF3 and a pharmaceutically acceptable carrier.