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WO2013006436A1 - Mutations de acsf3 dans des troubles métaboliques - Google Patents

Mutations de acsf3 dans des troubles métaboliques Download PDF

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Publication number
WO2013006436A1
WO2013006436A1 PCT/US2012/044926 US2012044926W WO2013006436A1 WO 2013006436 A1 WO2013006436 A1 WO 2013006436A1 US 2012044926 W US2012044926 W US 2012044926W WO 2013006436 A1 WO2013006436 A1 WO 2013006436A1
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acsf3
subject
sample
cmamma
exon sequence
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Charles P. Venditti
Leslie G. BIESECKER
Jennifer L. SLOAN
Jennifer J. JOHNSTON
Eirini MANOLI
Randy J. CHANDLER
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/385—Heterocyclic compounds having sulfur as a ring hetero atom having two or more sulfur atoms in the same ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/53—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • 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.
  • MMAemias Methylmalonic acidemias
  • MMA methylmalonic acid
  • methylmalonyl-CoA mutase 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 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.
  • MA malonic acid
  • MA malonyl-CoA decarboxylase activity
  • 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.
  • 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.
  • 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.
  • 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, MgCl 2 , 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.
  • 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.
  • the invention provides a method of treating CMAMMA comprising
  • an expression vector comprising ACSF3 (SEQ ID NO: 1) operably linked to a promoter to a subject in need thereof.
  • 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.
  • 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
  • Motif II was aligned independent of the full-length protein to improve the alignment of the ACSF3 and MCS proteins.
  • Figure 3 is a schematic depiction of results of phylogenetic analysis of ACSF3 orthologues and ACS homologues.
  • Figure 4 shows the relative substrate specificity of purified ACSF3 toward malonate, methylmalonate, and acetate.
  • Figure 5 is a schematic depiction of the intracellular cobalamin pathway.
  • 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.
  • 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.
  • 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.
  • the cell sample is obtained from a subject having normal methylmalonyl CoA mutase and intracellular cobalamin function.
  • 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.
  • the metabolic disorder is Combined Malonic and Methylmalonic Aciduria (CMAMMA).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the primer is any of SEQ ID NOS: 2-4, 8-10, 13-18, 22-24, and 27-29.
  • the exome analysis is performed using primers having SEQ ID NOS: 2-29.
  • 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.
  • the ACSF3 alteration detected in exome analysis can be a substitution, insertion, deletion, or a chimeric transcript derived from a chromosomal rearrangement mutation.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • the propionate can be sodium propionate.
  • the proprionate is provided in excess.
  • 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).
  • 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.
  • 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.
  • the complementation assay can be used to confirm that a variant of unknown significance is pathogenic, i.e. is associated with CMAMMA.
  • 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, MgCl 2 , 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.
  • ATP adenosine triphosphate
  • CoA Coenzyme A
  • substrate such as malonate or methylmalonate
  • 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.
  • 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.
  • the biological sample comprises one or more components such as cells, tissues, extracts, and organelles.
  • the biological sample can comprise a tissue sample or a cell sample taken from a subject.
  • 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.
  • organelles such as mitochondria can be further separated for analysis.
  • the ACSF3 can be purified or isolated from the biological sample and employed in the assay.
  • the source of ACSF3 comprises an affinity-tagged ACSF3. It will be understood that a homogenous sample is not necessarily required in such analysis.
  • a cell sample for use in any of the methods of the invention can comprise any suitable type of cells.
  • the cell sample can comprise fibroblasts or lymphocytes.
  • the cell sample comprises lymphocytes that are transformed with Epstein-Barr Virus (EBV).
  • EBV Epstein-Barr Virus
  • Such cells are suitable for use in the cell secretion assay and also can be used as a source of a nucleotide sample.
  • 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.
  • the disorder comprises CMAMMA.
  • 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.
  • 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.
  • 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).
  • 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.
  • This example provides characterization of clinical and biochemical features of individuals diagnosed with CMAMMA.
  • 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).
  • LC-MS/MS liquid chromatography-tandem mass spectrometry
  • GC/MS gas chromatography-mass spectrometry
  • MA malonic acid
  • D 3 methylmalonic and C 2 -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 N 2 flow.
  • 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).
  • MMAemia methylmalonic academia
  • Figure 5 methylmalonyl-CoA mutase
  • LMBDR1 methylmalonyl-CoA mutase
  • MMACHC methylmalonyl-CoA mutase
  • MMADHC methylmalonyl-CoA mutase
  • MMAB methylmalonyl-CoA mutase
  • MCEE methylmalonyl-CoA mutase
  • SUCLA2, SUCLG1 methylmalonyl-CoA decarboxylase
  • 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.
  • ELAND efficient large-scale alignment of nucleotide databases
  • 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)).
  • 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
  • ACSF3 an orphan member of the acyl- CoA synthetase family, was selected for further evaluation based on its putative function and predicted mitochondrial localization.
  • Sequence analysis of ACSFS was performed using standard methods. Sequencing was performed with a v3.1 BigDyeTM 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.
  • AAACG AAACTGCACG
  • 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.
  • Dog Genome 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,
  • 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.
  • 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 ⁇ MMA and 1 1.3 ⁇ 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).
  • 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.
  • This example provides a method for qualitative and quantitative analysis of ACSF3 expression in individuals with CMAMMA as compared to controls.
  • 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.
  • 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).
  • 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.
  • 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 GatewayTM 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.
  • 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 ⁇ g/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 2xl0 5 or 5xl0 5 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.
  • ACSF3 with a C-terminal GFP fusion was cloned into pCMV6 and sequence verified.
  • Control fibroblasts were electroporated with 3 ⁇ g of plasmid DNA using an Amaxa nucleofector electroporator (Amaxa GmbH, Walkersville, MD). Transfected fibroblasts were grown for 48 hours before immunofluorescence experiments.
  • This example provides in vitro analysis of the function of ACSF3, as well as its subcellular localization properties.
  • 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 MgCl 2 , 0.4 mM ATP, 0.2 mM CoA, and 1.43 ⁇ g of GST-tagged, purified ACSF3.
  • 100 mM potassium phosphate buffer (pH 7.0) 100 mM potassium phosphate buffer (pH 7.0), 8 mM malonate, methylmalonate, or acetate, 2 mM MgCl 2 , 0.4 mM ATP, 0.2 mM CoA, and 1.43 ⁇ g of GST-tagged, purified ACSF3.
  • 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 ⁇ glycine in PBS.
  • PBS IX phosphate buffered saline
  • 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 555TM and donkey anti- mouse IgG conjugated to Alexa Fluor 488TM or Alexa Fluor 633TM (Invitrogen, Carlsbad, CA) for 1 hour at room temperature.
  • Slides were washed with IX PBS and mounted with VectaShieldTM mounting medium (Vector Laboratories, Inc., Burlingame, CA) containing DAPI.
  • ACSF3 is a mitochondrial methylmalonyl-CoA and malonyl-CoA synthetase (MCS), which catalyzes the first step of intramitochondrial fatty acid synthesis.
  • This example provides phylogenic analysis of ACSF3 as compared to putative orthologues.
  • ACSF3 orthologues were identified by BLAST search and through Homologene. Sequence alignment of the ACSF3 orthologues: human NP_001 120686.1 , mouse
  • XPJ590782.2, Xenopus NP_001086314.1 , B. japonicum NPJ767149.1 and R. leguminosarum AAC83455.1 was performed by the Clustal W method in MacVectorTM version 9.0.2 (MacVector, Inc., Cary, NC). The phylogenetic tree was created in
  • MegAlignTM (Lasergene, DNASTAR, Inc., Madison, WI) by the Clustal W method.
  • 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.
  • This example demonstrates a technique for constructing an animal model of CMAMMA.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • This example demonstrates a technique for protein therapy for CMAMMA.
  • 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.
  • 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).
  • 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
  • Purified enzyme is then administered to cells, mice, or patients with CMAMMA to restore or augment enzyme activity.

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Abstract

L'invention concerne des procédés de détection d'un trouble métabolique, comprenant un défaut de ACSF3 chez un sujet, ainsi que des méthodes de traitement de tels troubles, et des compositions associées. L'invention concerne en outre des procédés de mesure de l'activité ACSF3 dans un échantillon biologique.
PCT/US2012/044926 2011-07-01 2012-06-29 Mutations de acsf3 dans des troubles métaboliques WO2013006436A1 (fr)

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CN103604763A (zh) * 2013-12-03 2014-02-26 广东中测食品化妆品安全评价中心有限公司 一种保健食品功能成份的检测方法

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103604763A (zh) * 2013-12-03 2014-02-26 广东中测食品化妆品安全评价中心有限公司 一种保健食品功能成份的检测方法

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