Systemic Carnitine Deficiency Gene and Uses Thereof
The gene responsible for systemic carnitine deficiency was found to be the OCTN2 gene involved in the transportation of organic cations. This invention enables tests for this disease by detecting whether or not the OCTN2 gene has a mutation. Furthermore, systemic carnitine deficiency can be treated using the normal OCTN2 gene and its protein.
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This application is a divisional of U.S. application Ser. No. 10/940,500, filed on Sep. 13, 2004, to issue as U.S. Pat. No. 7,413,860 on Aug. 19, 2008, which is a divisional of U.S. application Ser. No. 09/798,743, filed on Mar. 2, 2001, now U.S. Pat. No. 6,790,831, which is a continuation of PCT/JP99/04853, filed on Sep. 7, 1999, and claims priority from Japanese Patent Application No. 10/252,683, filed on Sep. 7, 1998. The contents of each of these applications are incorporated in their entirety by reference herein.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING FILED ELECTRONICALLYAn electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The computer-readable file, created on Aug. 15, 2008, is 64.0 kilobytes in size and titled 14875073003SeqList.txt.
1. Technical Field
This invention relates to molecules used in the testing and treatment of systemic carnitine deficiency, as well as methods for testing the disease.
2. Background of the Invention
Systemic Carnitine Deficiency (SCD) is a human genetic disease inherited through autosomal recessive inheritance, the main symptoms being skeletal or cardiac muscle disorders (NIM 212140) (Roe, C. R. and Coates, P. M., Mitochondrial fatty acid oxidation disorder, The metabolic and molecular bases of inherited diseases 7th ed., edited by Scriver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D., McGraw-Hill, New York, 1995, 1508-1509; Karpati, G. et al., The syndrome of systemic carnitine deficiency: clinical, morphologic, biochemical, and pathophysiologic features, Neurology 1975, 25:16-24). Serum carnitine levels and intra-tissue carnitine levels are known to be extremely low in these patients compared to healthy individuals. Carnitine is an indispensable co-factor in the long-chain fatty acid metabolism. A carnitine-mediated mechanism enables intracellular fatty acids to permeate mitochondrial outer and inner membranes, and energy is produced when these fatty acids undergo β-oxidation within the mitochondria (Walter, J. H., L-Carnitine, Arch Dis Child, 1996, 74:475-478; Bremer, J., Carnitine metabolism and functions, Physiol Rev, 1983, 1420-1480). The abnormal decrease of carnitine concentration in systemic carnitine deficiency patients is thought to be the direct cause of diseases in tissues such as muscles that require a large amount of energy. Membrane physiological studies done using fibroblasts from systemic carnitine deficiency patients have shown that these cells lack the mechanism to transport carnitine from the outside of the cell to the inside. A gene that encodes a protein involved in this mechanism is presumed to be the gene responsible for this disease (Tein, I. et al., Impaired skin fibroblast carnitine uptake in primary systemic carnitine deficiency manifested by childhood carnitine-responsive cardiomyopathy, Pediatr Res, 1990, 28:247-255). However, the gene responsible for systemic carnitine deficiency is yet to be isolated.
SUMMARY OF THE INVENTIONAn objective of the present invention is to provide the gene responsible for systemic carnitine deficiency. Moreover, this invention aims to provide a molecule used in the testing and treatment of systemic carnitine deficiency, as well as a method for testing the disease.
The Inventors isolated several genes encoding proteins involved in the transport of organic cations. Among these, the Inventors discovered the human gene (human OCTN2 gene) having an activity to transport carnitine in a sodium ion dependent manner, and the corresponding mouse gene (mouse OCTN2 gene) (Japanese Patent Application Hei 9-260972, Japanese Patent Application Hei 10-156660). The Inventors thought that the isolated OCTN2 gene might be the gene responsible for systemic carnitine deficiency, and evaluated this possibility.
Specifically, the nucleotide sequence of the OCTN2 gene of the systemic carnitine deficiency mouse model and systemic carnitine deficiency patients were analyzed. As a result, the Inventors discovered the presence of various mutations in the OCTN2 gene of both the mouse model and systemic carnitine deficiency patients. In other words, for the first time in the world, the Inventors succeeded in revealing that systemic carnitine deficiency is caused by mutations in the OCTN2 gene.
Moreover, due to the close relationship of OCTN2 gene mutation and systemic carnitine deficiency, the Inventors found that this disease can be tested by examining whether or not there is a mutation in the OCTN2 gene of a patient.
It was also found that systemic carnitine deficiency could be treated by using the normal OCTN2 gene and its protein, to complete the invention.
Therefore, this invention relates to molecules used in the testing and treatment of systemic carnitine deficiency, as well as methods for testing the disease. More specifically, the present invention relates to:
(1) a DNA for testing systemic carnitine deficiency, wherein the DNA hybridizes to a DNA comprising the nucleotide sequence of SEQ ID NO:5, or the transcription regulatory region thereof, and comprises at least 15 nucleotides;
(2) a molecule as in any one of (a) to (c) below, which is used for the treatment of systemic carnitine deficiency,
(a) a protein comprising the amino acid sequence of SEQ ID NO: 1,
(b) a compound that promotes the activity of the protein comprising the amino acid sequence of SEQ ID NO: 1, or,
(c) a DNA encoding the protein comprising the amino acid sequence of SEQ ID NO:1;
(3) a pharmaceutical composition for treating systemic carnitine deficiency, comprising a molecule of (2) as the active ingredient;
(4) a pharmaceutical composition for treating systemic carnitine deficiency, comprising an antibody binding to the protein comprising the amino acid sequence of SEQ ID NO: 1 as the active ingredient;
(5) a test method for systemic carnitine deficiency comprising the detection of a mutation in the DNA encoding the protein comprising the amino acid sequence of SEQ ID NO: 1, or the transcription regulatory region of said DNA;
(6) the test method for systemic carnitine deficiency of (5) comprising the steps of,
(a) preparing a DNA sample from a patient,
(b) amplifying patient-derived DNA using the DNA of (1) as a primer,
(c) cleaving the amplified DNA,
(d) separating the DNA fragments by their size,
(e) hybridizing the DNA of (1) labeled by a detectable label as a probe to the DNA fragments separated, and,
(f) comparing the size of the DNA fragment detected with a control from a healthy individual,
(7) the test method for systemic carnitine deficiency of (5) comprising the steps of,
(a) preparing an RNA sample from a patient,
(b) separating the prepared RNA by size,
(c) hybridizing the DNA of (1) labeled by a detectable label as a probe to the RNA fragments separated, and,
(d) comparing the size of the RNA fragment detected with a control from a healthy individual,
(8) the test method for systemic carnitine deficiency of (5) comprising the steps of,
(a) preparing a DNA sample from a patient,
(b) amplifying patient-derived DNA using the DNA of (1) as a primer,
(c) dissociating the amplified DNA to single-stranded DNA,
(d) separating the dissociated single-stranded DNA on a non-denaturing gel, and,
(e) comparing the mobility of separated single stranded DNA on the gel with a control from a healthy individual,
(9) the test method for systemic carnitine deficiency of (5) comprising the steps of,
(a) preparing a DNA sample from a patient,
(b) amplifying patient-derived DNA using the DNA of (1) as a primer,
(c) separating the amplified DNA on a gel in which the concentration of the DNA denaturant gradually increases, and,
(d) comparing the mobility of separated DNA on the gel with a control from a healthy individual.
The present invention is based on the finding by the present inventors that systemic carnitine deficiency is caused by a mutation in the gene named “OCTN2”. First and foremost, this invention relates to a molecule used in the testing and treatment of systemic carnitine deficiency, as well as a method for testing the disease.
In the present invention, the genomic DNA region (for example, SEQ ID NO:5) containing OCTN2, or an oligonucleotide (probe and primer) that hybridizes to the nucleotide sequence of the regulatory region (comprising the intron, promoter, and enhancer sequences as well) of OCTN2 is used.
This oligonucleotide preferably hybridizes specifically to the genomic DNA region containing OCTN2, or the regulatory region of OCTN2. Herein, “hybridizes specifically” indicates that cross-hybridization does not significantly occur with DNA encoding other proteins, under normal hybridizing conditions, preferably under stringent conditions (for example, the conditions in Sambrook et al., Molecular Cloning second edition, Cold Spring Harbor Laboratory Press, New York, USA, 1989).
When using as a primer, the oligonucleotide is usually, 15 to 100 bp, preferably, 17 to 30 bp. The primer may be any, as long as it can amplify at least a part of the OCTN2 gene or the region regulating its expression. Such regions comprise, for example, the exon region of OCTN2, the intron region, the promoter region, and enhancer region.
On the other hand, the oligonucleotide used as a probe usually comprises at least 15 bp or more if it is a synthetic oligonucleotide. It is also possible to use a double stranded DNA obtained from a clone incorporated into a vector such as plasmid DNA. The probe may be any, as long as it specifically hybridizes to at least a part of the OCTN2 gene or the region regulating the expression of the gene. Regions to which the probe hybridizes include, for example, the exon region, intron region, promoter region, and enhancer region of the OCTN2 gene. When using as the probe, oligonucleotide or double stranded DNA is suitably labeled. Examples of labeling methods are, phosphorylating the 5′ end of the oligonucleotide by 32p using T4 polynucleotide kinase, and incorporating a substrate nucleotide labeled by an isotope such as 32p, a florescent dye, or biotin, using the random hexamer oligonucleotide as a probe and using DNA polymerase such as the Klenow enzyme (random priming technique).
In the present invention, “a test method for systemic carnitine deficiency” includes not only a test for patients showing symptoms of systemic carnitine deficiency caused by a mutation of the OCTN2 gene, but also a test for detecting a mutation of the OCTN2 gene for determining whether or not the person tested is likely to develop systemic carnitine deficiency arising from a OCTN2 gene mutation. In other words, the risk of developing systemic carnitine deficiency may greatly increase in cases where one of the OCTN2 alleles develops a mutation, even when no symptoms are visible on the outside. Therefore, tests for specifying patients (carriers) having a mutation in an OCTN2 allele are also included in the invention.
In the present invention, a test method for systemic carnitine deficiency using the above oligonucleotides comprises the detection of a mutation in the OCTN2 gene or its transcription regulatory region. One embodiment of this method of testing is the direct determination of the nucleotide sequence of the patient's OCTN2 gene. For example, using the above oligonucleotide as the primer, the whole OCTN2 gene or a part of it is amplified by the Polymerase Chain Reaction (PCR) using as the template a DNA isolated from a patient suspected of having a disease caused by an OCTN2 mutation. By comparing this sequence with that of a healthy individual, it is possible to conduct a test for a disease arising from an OCTN2 gene mutation.
As the testing method of the invention, other than determining the nucleotide sequence of DNA derived directly from the patient, several other methods are also used. One such embodiment comprises the following steps of: (a) preparing a DNA sample from a patient; (b) amplifying the patient-derived DNA using the primer of this invention; (c) dissociating amplified DNA into single-stranded DNA; (d) separating the dissociated single-stranded DNA on a non-denaturing gel; and, (e) comparing the mobility of separated single stranded DNA on the gel with a control from a healthy individual.
An example of such a method is the PCR-single-strand conformation polymorphism (PCR-SSCP) method (Cloning and polymerase chain reaction-single-strand conformation polymorphism analysis of anonymous Alu repeats on chromosome 11, Genomics, 1992 Jan 1, 12(1):139-146; Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products, Oncogene, 1991 August 1, 6(8):1313-1318; Multiple fluorescence-based PCR-SSCP analysis with postlabeling, PCR Methods Appl. 1995 April 1, 4(5):275-282). This method is comparatively easy to handle, and has various advantages such as requiring only a small amount of a sample, and therefore, is suitable for screening a large number of DNA samples. The principle of this method is as follows. When a double stranded DNA fragment is disassociated into single strands, each strand forms an original high-order structure depending on its nucleotide sequence. When these dissociated DNA strands are electrophoresed within a polyacrylamide gel free of denaturants, the single stranded DNAs that are complementary and have the same length, migrate to different positions according to the difference in their high-order structure. This high order structure of the single strands change even by a single nucleotide substitution showing different mobilities in polyacrylamide gel electrophoresis. Therefore, the presence of a mutation in a DNA fragment due to point mutation, deletion, or insertion can be detected by the change in mobility.
Specifically, first, the whole OCTN2 gene or a part of it is amplified by PCR, and such. A length of 200 to 400 bp is usually preferred amplified range. Regions amplified include all the exons and all the introns of the OCTN2 gene, as well as the promoter and enhancer of the OCTN2 gene. PCR can be done, for example, according to conditions described in Example 1. When amplifying the gene fragment by PCR, a primer labeled by an isotope such as 32p, a fluorescent dye, or biotin is used, or the DNA fragment synthesized by PCR after adding a substrate nucleotide labeled by an isotope such as 32p, a fluorescent dye, or biotin, is labeled. Labeling can also be done by adding to the synthesized DNA fragment a substrate nucleotide labeled by an isotope such as 32p, a fluorescent dye, or biotin, using the Klenow enzyme and such after the PCR reaction. The labeled DNA fragment thus obtained is denatured by heating and such, and electrophoresed in a polyacrylamide gel free of denaturants such as urea. Conditions for separating the DNA fragment can be improved by adding a suitable amount (about 5 to 10%) of glycerol to the polyacrylamide gel. Conditions of electrophoresis vary depending on the properties of the DNA fragment, but room temperature (from 20 to 25° C.) is usually used. When a preferable separation cannot be accomplished, the temperature that gives the optimum mobility at 4 to 30° C. is evaluated. Following electrophoresis, the mobility of the DNA fragment is detected by an autoradiography using X-ray films, a scanner that detects fluorescence, and so on, and analyzed. When a band having a difference in mobility is detected, this band is directly excised from the gel, re-amplified by PCR, and is directly sequenced to verify the presence of a mutation. Even when labeled DNA is not used, the band can be detected by staining the gel after electrophoresis with ethidium bromide, silver, and such.
Another embodiment of the test method of the present invention comprises the following steps of: (a) preparing a DNA sample from a patient; (b) amplifying patient-derived DNA using the primer of this invention; (c) cleaving the amplified DNA;
(d) separating the DNA fragments according to their size; (e) hybridizing the probe DNA of the invention labeled with a detectable label to the DNA fragments separated; and
(f) comparing the size of the detected DNA fragment with a control from a healthy individual.
Such methods include those using Restriction Fragment Length Polymorphism (RFLP), PCR-RFLP method, and so on. Restriction enzymes are usually used to cleave DNA. Specifically, compared to a DNA fragment of a healthy individual, the size of one obtained following restriction enzyme treatment changes when a mutation exists at the recognition site of the restriction enzyme, or when nucleotides have been inserted or deleted in the DNA fragment resulting from restriction enzyme treatment. The portion containing the mutation is amplified by PCR, the amplified products are treated with each restriction enzyme and electrophoresed to detect the mutation as the difference of mobility. Alternatively, chromosomal DNA is cleaved with these restriction enzymes, and after electrophoresis, the presence or absence of a mutation can be detected by southern-blotting using the probe DNA of the invention. The restriction enzymes used can be suitably selected according to each mutation. This method can use not only genomic DNA, but also cDNA made by treating RNA prepared from patients with reverse transcriptase, cleaving this cDNA as-it-is with restriction enzymes, and then conducting southern blotting. It is also possible to examine the changes in mobility after amplifying the whole OCTN2 gene, or a part of it, by PCR using the above cDNA as the template, and cleaving the amplified products by restriction enzymes.
A similar detection is also possible using RNA prepared from patients instead of DNA. This method includes the steps of: (a) preparing an RNA sample from a patient; (b) separating the prepared RNA according to their size; (c) hybridizing the probe DNA of the invention labeled by a detectable label to the separated RNA; and (d) comparing the size of the detected RNA with a control from a healthy individual. In a specific example of this method, RNA prepared from a patient is electrophoresed, northern blotting is done using the probe of the invention to detect the mobility change.
Another embodiment of the method of the invention comprises the steps of:
(a) preparing a DNA sample from a patient; (b) amplifying patient-derived DNA using the primer of this invention; (c) separating the amplified DNA on a gel in which the concentration of the DNA denaturant gradually increases; and, (d) comparing mobility of the DNA separated upon the gel with a control from a healthy individual.
An example of such a method is denaturant gradient gel electrophoresis (DGGE). The whole OCTN2 gene or a part of it is amplified by a method such as PCR using the primer of the invention, and the amplified product is electrophoresed in a gel in which the concentration of the DNA denaturant gradually increases, and compared with a control from a healthy individual. In the case of a DNA having a mutation, the DNA fragment will become single stranded at a low denaturant concentration and the moving speed will become extremely slow. The presence or absence of a mutation can be detected by detecting the change in mobility.
Allele Specific Oligonucleotide (ASO) hybridization can be used alternatively when the aim is to detect a mutation at a specific site. When an oligonucleotide comprising a nucleotide sequence thought to have a mutation is prepared and this is hybridized with sample DNA, the hybrid formation efficiency will decrease when there is a mutation. This can be detected by southern blotting and by a method using the property of special fluorescent reagents that quench when intercalated into a hybrid gap. The detection by ribonuclease A mismatch cleavage method can also be used. Specifically, the whole OCTN2 gene, or a part of it, is amplified by a method such as PCR, and the amplified product is hybridized to labeled RNA prepared from OCTN2 cDNA and such incorporated into a plasmid vector, etc. The hybrids will be single stranded in the portion where a mutation exists. This portion is cleaved by ribonuclease A and the existence of a mutation can be detected by autoradiography, and such.
The present invention also relates to a test drug for systemic carnitine deficiency that comprises an antibody binding to the OCTN2 protein as the active ingredient. An antibody binding to the OCTN2 protein can be prepared using methods well known to those skilled in the art. Polyclonal antibodies can be made by, obtaining the serum of small animals such as rabbits immunized with the OCTN2 protein (apart from the natural protein, recombinant OCTN2 proteins expressed in suitable host cells (E. coli, yeasts, mammals, and such), such as recombinant OCTN2 protein expressed in E. coli as a fusion protein with GST) of the present invention, or a partial peptide. The serum is then purified by, for example, ammonium sulfate precipitation, protein A or protein G column chromatography, DEAE ion exchange chromatography, or an affinity chromatography using a column to which the protein of the present invention or synthetic peptide is coupled. Monoclonal antibodies can be made by immunizing small animals such as mice with the OCTN2 protein or a partial peptide thereof, excising the spleen from the mouse, homogenizing it and separating cells, fusing the cells with mouse myeloma cells using a reagent such as polyethylene glycol, and selecting clones that produce an antibody binding to the OCTN2 protein from the fused cells (hybridomas) produced. Next, the obtained hybridomas are transplanted into the abdominal cavity of a mouse, and ascites are extracted from the mouse. The obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column chromatography, DEAE ion exchange chromatography, or an affinity chromatography using a column to which the OCTN2 protein or synthesized peptide is coupled. When using the antibody as a test drug, it is mixed with sterile water, physiological saline, plant oils, surfactants, lipids, solubilizers, stabilizers (BSA, gelatin, etc.), preservatives, and such, according to needs. An example of a test for systemic carnitine deficiency features the staining of tissues collected or cells isolated from a patient by the enzyme-labeled antibody method, fluorescence-labeled antibody method, and test for a deficiency, abnormal accumulation, or abnormal intracellular distribution of the OCTN2 protein. Testing can also be done by preparing a cell-extract of tissues collected or cells isolated from a systemic carnitine deficiency patient, separating the cell-extract by methods such as SDS-PAGE, transferring onto a nitrocellulose membrane, PVDF membrane, and such, and then staining this by a method (western blotting, immunoblotting, etc) using the above-described enzyme-labeled antibody method, etc.
The present invention also relates to a therapeutic drug for systemic carnitine deficiency. One such embodiment has the OCTN2 gene as the active ingredient. When using the OCTN2 gene as a therapeutic drug, it is given to the patient by oral, intravenous, topical administration and such, as the full length OCTN2 chromosomal DNA, a part of it, or by incorporating the OCTN2 DNA into a suitable vector, for example, adenovirus vector, adeno associated virus vector, retro virus vector, or plasmid DNA. The ex vivo method can also be used for administration apart from the in vivo method. The transition and absorption into tissues can be enhanced by enclosing the gene in a liposome prepared by micellization of phospholipids, or by adding a cationic lipid and forming a complex with genomic DNA. Therefore, the method of the invention can replace a patient's mutated OCTN2 gene by a normal gene, and also additionally administer the normal gene, thereby enabling the treatment of systemic carnitine deficiency.
Another embodiment of the invention relating to a therapeutic drug of systemic carnitine deficiency comprises the OCTN2 protein as the active ingredient. The amino acid sequences of human and mouse OCTN2 proteins are shown in SEQ ID NOs: 1 and 3, respectively. The OCTN2 protein can be prepared as a natural protein and also as a recombinant protein. The natural protein can be prepared by a method well known to one skilled in the art, for example, by isolating the OCTN2 protein from tissues or cells that show a high level expression of the protein (e.g. fetal kidney) by affinity chromatography using an antibody against a partial peptide of the OCTN2 protein. On the other hand, a recombinant protein can be prepared by culturing cells transformed by DNA (for example, SEQ ID NO:2) encoding the OCTN2 protein. Cells used for the production of recombinant proteins include mammalian cells such as, COS cells, CHO cells, and NIH3T3 cells, insect cells such as sf9 cells, yeast cells, and E. coli cells. Vectors for expressing the recombinant proteins within cells vary according to the host used, and normally, pcDNA3 (Invitrogen), pEF-BOS (Nucleic Acids Res. 1990, 18(17), 5322) and such are used as vectors for mammalian cells, the “BAC-to-BAC baculovirus expression system” (GIBCO BRL) and such are used for insect cells, “Pichia Expression Kit” (Invitrogen) and such are used for yeast cells, pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen) and such are used for E. coli cells. Vectors are introduced to hosts using, for example, the calcium phosphate method, DEAE dextran method, method using cationic liposome DOTAP (Boehringer Mannheim), and Superfect (Qiagen), electroporation method, calcium chloride method, and such. The recombinant protein can be purified from the transformant obtained usually using methods described in “The Qiaexpressionist handbook, Qiagen, Hilden, Germany”.
When using the obtained OCTN2 protein as a therapeutic drug for treating systemic carnitine deficiency, the OCTN2 protein can be directly administered, or can be given after being formulated into a pharmaceutical composition by a well-known pharmaceutical manufacturing method. For example, the drug can be given after suitably combining with a generally used carrier or medium such as, sterilized water, physiological saline, plant oils, surfactants, lipids, solubilizers, stabilizers, preservatives, and such.
The dosage varies depending on factors such as the patient's body weight, age, healthiness, and method of administration, but a skilled artisan can suitably select the dosage. Usually, it is within the range from 0.01 to 1000 mg/kg. The administration can be done orally, intravenously, intramuscularly, or percutaneously. A skilled artisan can easily replace, add, or delete amino acid(s) in the amino acid sequence of the OCTN2 protein using a well-known method such as the site-specific mutation induction system using PCR (GIBCO-BRL, Gaithersburg, Maryland), site-specific mutagenesis using oligonucleotides (Kramer, W. and Fritz, H J, 1987, Methods in Enzymol, 154:350-367), the Kunkel method (Methods Enzymol., 1988, 85:2763-2766), and such.
Another embodiment of the therapeutic drug for systemic carnitine deficiency uses a compound that enhances the activity of the OCTN2 protein as the active ingredient. Such a compound can be screened as follows. For example, a plasmid expressing the OCTN2 protein is constructed, and this is introduced into HEK293 cells by the calcium phosphate method. Radiolabeled carnitine and a test compound are added to this transformant and the carnitine transporting activity into the cells is determined. A compound that can enhance the carnitine transporting activity is selected by comparing with the activity of the OCTN2 protein in the absence of the test compound. See Japanese Patent Application Hei 9-260972 and Hei 10-156660 for the detailed method.
Similar to the above-mentioned use of the OCTN2 protein as a therapeutic drug, the isolated compound can also be formulated into a pharmaceutical composition using well-known pharmaceutical manufacturing methods. The dose range is usually within 0.01 to 1000 mg/kg.
It is also conceivable to utilize the region regulating OCTN2 gene expression or a factor that binds to this region for the treatment of systemic carnitine deficiency.
The OCTN2 gene comprising the region that regulates OCTN2 gene expression is useful in the above-mentioned gene therapy as it can express the OCTN2 gene under normal expression regulation in vivo by introducing it into patients who lack the OCTN2 gene, or who have a defect in OCTN2 gene expression.
Moreover, if the promoter site is determined from the upstream region of the OCTN2 gene, a compound that regulates OCTN2 gene expression amount can be simply screened by using a reporter gene expression vector having the above promoter site through examining the influence of various compounds on the production of reporter gene products. Such a screening method comprises the following steps of, (a) constructing a vector in which a reporter gene is ligated to the downstream of the promoter site, (b) introducing the vector into a suitable cell, and, (c) detecting the reporter gene activity by contacting or introducing a test compound to the above cell. Examples of the test compound include, proteins, peptides, synthetic compounds, natural compounds, genes, gene products, and such.
A compound regulating OCTN2 gene expression can also be screened by contacting a test sample with the promoter site, and selecting a compound (such as a protein) that binds to the promoter site. For example, a synthetic oligo DNA and such having the nucleotide sequence of the promoter site is prepared, this is bound to a suitable support such as Sepharose, and contacted with a cell-extract, and such. Then, a transcription factor and such that binds to this promoter site and regulates OCTN2 gene expression can be purified by, for example, affinity chromatography.
The invention shall be described in detail below, but it is not to be construed as being limited thereto.
Example 1Proof in mouse and human showing that the gene responsible for systemic carnitine deficiency (SCD) is OCTN2
The Inventors have previously isolated human cDNA encoding a protein having an activity to transport carnitine in a sodium-ion dependent manner, and also the corresponding mouse cDNA (Japanese Patent Application No. Hei 9-260972, Japanese Patent Application No. Hei 10-156660). The nucleotide sequences of the human and mouse OCTN2 cDNA isolated by the Inventors are shown in SEQ ID NO:2 and 4, respectively, and the amino acid sequences of the proteins encoded by these cDNAs are shown in SEQ ID NO: 1 and 3, respectively.
The Inventors drew up a working hypothesis that OCTN2 might be the gene responsible for systemic carnitine deficiency, and conducted experiments to prove this.
(1) OCTN2 Gene Analysis in Juvenile Visceral Steatosis (jvs) Mouse
The juvenile visceral steatosis (jvs) mouse was generated due to a mutation in the C3H.OH mouse. This jvs mouse shows symptoms similar to systemic carnitine deficiency patients, and shows an extremely low carnitine concentration within its blood and tissues. This phenotype is inherited by autosomal inheritance. From the above facts, the jvs mouse is considered to be a mouse model for systemic carnitine deficiency (Hashimoto, N. et al., Gene-dose effect on carnitine transport activity in embryonic fibroblasts of JVS mice as a model of human carnitine transporter deficiency, Biochem Pharmacol, 1998, 55:1729-1732). The Inventors examined the OCTN2 gene arrangement of the jvs mouse. Specifically, whole RNA was extracted from the kidney of a jvs homologous mouse, cDNA was synthesized, jvs mouse OCTN2 cDNA was amplified using this synthesized cDNA as the template by RT-PCR, and the sequence was examined by direct sequencing.
The amplification reaction by PCR was conducted as follows. For the 5′ side fragment, the primers MONB 31 (5′-gataagcttacggtgtccccttattcccatacg-3′/SEQ ID NO:22) and MONB 20 (5′-cccatgccaacaaggacaaaaagc-3′/SEQ ID NO:23) were prepared. Then, amplification was done within a reaction solution (50 μl) containing, cDNA, 5 μl of 10× KOD buffer (Toyobo), 5 μl of 2 mM dNTPs, 2 μl of 25 mM MgCl2, 0.5 μl of KOD DNA polymerase (Toyobo), 1 μl of 20 μM MONB 31 primer, and 1 μl of 20 μM MONB 20 primer at 94° C. for 3 min, 30 cycles of “94° C. for 30 sec, 50° C. for 30 sec, and 74° C. for 1 min”, and 72° C. for 10 min. As for the 3′ side fragment, the primers MONB 6 (5′-tgtttttcgtgggtgtgctgatgg-3′/SEQ ID NO:24) and MONB 26 (5′-acagaacagaaaagccctcagtca-3′/SEQ ID NO:25) were prepared, and amplification was done within a reaction solution (50 μl) containing cDNA, 5 μl of 10× ExTaq buffer (TaKaRa), 4 μl of 2.5 mM dNTPs, 1 μl of a mixture of ExTaq DNA polymerase (TaKaRa) and anti Taq antibody (TaqStart antibody™, CLONTECH), 1 μl of 20 μM MONB 6 primer, and 1 μl of 20 μM MONB 26 primer, at 94° C. for 2 min, 30 cycles of “94° C. for 30 sec, 60° C. for 30 sec, and 74° C. for 2 min”, and 72° C. for 10 min.
Sequencing revealed that the codon encoding the 352nd leucine (CTG) was mutated to a codon encoding arginine (CGG) (
Next, the effect this mutation has on the carnitine transporting activity was examined. Plasmid DNA expressing wild-type mouse OCTN2, and those expressing mutated OCTN2 were separately introduced into BEK293 cells, and then, carnitine transporting ability was measured similar to the assay of human OCTN2 described in Japanese Patent Application Hei 10-156660 (
Thus, the jvs mouse is thought to have developed the disease due to a functional deletion mutation of the OCTN2 gene.
(2) OCTN2 gene analysis in human systemic carnitine deficiency patients
A database search using human OCTN2 cDNA sequence revealed that the human OCTN2 genomic DNA sequence has been decoded by Lawrence Berkeley National Laboratory (LBNL) of the United States as a part of the human genome project. However, it was only recorded as several cosmid clone sequences, therefore, the inventors determined a complete human OCTN2 genomic DNA sequence (SEQ ID NO:5) by comparing with human OCTN2 cDNA sequence and suitably combining the clone sequences. The human OCTN2 gene is an about 26 kb gene comprising ten exons and nine introns. The eight pairs of primers shown below, which can amplify all the exons as eight fragments, were prepared from this gene arrangement.
Specifically, OCN2 43 (5′-GCAGGACCAAGGCGGCGGTGTCAG-3′, SEQ ID NO:6) and OCN2 44 (5′-AGACTAGAGGAAAAACGGGATAGC-3′, SEQ ID NO:7) for exon one; OCN2 25 (5′-AGATTTTTAGGAGCAAGCGTTAGA-3′ SEQ ID NO:8) and OCN2 26 (5′-GAGGCAGACACCGTGGCACTACTA-3′, SEQ ID NO:9) for exon two; OCN2 27 (5′-TTCACACCCACTTACTGGATGGAT-3′ SEQ ID NO: 10) and OCN2 50 (5′-ATTCTGTTTTGTTTTGGCTCTTTT-3′, SEQ ID NO: 11) for exons three and four; OCN2 31 (5′-AGCAGGGCCTGGGCTGACATAGAC-3′, SEQ ID NO: 12) and OCN2 32 (5′-AAAGGACCTGACTCCAAGATGATA-3′, SEQ ID NO: 13) for exon five; OCN2 33 (5′-TCTGACCACCTCTTCTTCCCATAC-3′, SEQ ID NO: 14) and OCN2 34 (5′-GCCTCCTCAGCCACTGTCGGTAAC-3′, SEQ ID NO: 15) for exon six; OCN2 35 (5′-ATGTTGTTCCTTTTGTTATCTTAT-3′, SEQ ID NO: 16) and OCN2 36 (5′-CTTGTTTTCTTGTGTATCGTTATC-3′, SEQ ID NO:17) for exon seven; OCN2 37 (5′-TATGTTTGTTTTGCTCTCAATAGC-3′, SEQ ID NO:18) and OCN2 40 (5′-TCTGTGAGAGGGAGTTTGCGAGTA-3′, SEQ ID NO: 19) for exon eight and nine; and, OCN2 41 (5′-TACGACCGCTTCCTGCCCTACATT-3′, SEQ ID NO:20) and OCN2 42 (5′-TCATTCTGCTCCATCTTCATTACC-3′, SEQ ID NO:21) for exon 10.
Next, human OCTN2 gene mutations in three families that have systemic carnitine deficiency patients, but no blood relationships were examined. The analysis is done by amplifying all the exons using the above primers and genomic DNA prepared from blood cells as the template, and subjecting the amplified products into direct sequencing.
The amplification reaction by PCR was done within a reaction solution (50 μl) containing 100 ng of genomic DNA, 5 μl of 10× ExTaq buffer (TaKaRa), 4 μl of 2.5 mM dNTPs, 1 μl of a mixture of ExTaq DNA polymerase (TaKaRa) and anti Taq antibody (TaqStart antibody™, CLONTECH), and 1 μl of each of the 20 μM primers. The reaction conditions were, 94° C. for 2 min, 36 cycles of “94° C. for 30 sec, 60° C. for 30 sec, and 74° C. for 2 min”, and 72° C. for 10 min. However, in the case of exon one and exon five amplification, a reaction solution (50 μl) containing 100 ng genomic DNA, 25 μl of 2× GC buffer 1 (TaKaRa), 8 μl of 2.5 mM dNTPs, 0.5 μl of LA Taq DNA polymerase (TaKaRa), and 1 μl of each of the 20 μM primers, was used.
In the first family (KR family), a 113 bp deletion was found in first exon of the OCTN2 gene of a systemic carnitine deficiency patient (
In the second family (AK family), the systemic carnitine patients were found to contain two types of mutated OCTN2 genes. One mutation was a cytosine insertion just after the initiation codon, which is thought to cause a frame shift and prevent the proper protein from being produced (
In the third family (TH family), a mutation (AG to AA) was found in the splicing site in the 3′ end of the intron eight of the OCTN2 gene (
The above results revealed that systemic carnitine deficiency is a genetic disease caused by mutations in the OCTN2 gene. Thus, the present invention enables definitive diagnosis, prenatal diagnosis and such, of systemic carnitine deficiency by examining mutations in the OCTN2 gene using analyses described herein, as well as other methods. The present invention also enables treatment of systemic carnitine deficiency by treatments such as gene therapy using the OCTN2 gene.
INDUSTRIAL APPLICABILITYThe present invention revealed that the OCTN2 gene is the gene responsible for systemic carnitine deficiency, thus enabling tests for the disease by detecting mutations in the OCTN2 gene and its protein. Moreover, the present invention facilitates treatment of systemic carnitine deficiency by utilizing the OCTN2 gene and its protein.
Claims
1. A method of screening for a compound that regulates OCTN2 gene expression, the method comprising:
- (a) contacting a test compound with (i) OCTN2 DNA encoding OCTN2; or (ii) a cell comprising said DNA; and
- (b) measuring OCTN2 gene expression, wherein an increase or decrease in gene expression in the presence of the test compound, compared to in the absence of the test compound, is indicative of a compound that regulates OCTN2 gene expression.
2. The method of claim 1, wherein the OCTN2 DNA comprises a promoter region or an enhancer region of the OCTN2 gene.
3. The method of claim 1, wherein the OCTN2 DNA comprises the nucleotide sequence of SEQ ID NO:5.
4. The method of claim 1, wherein the test compound is selected from among proteins, peptides, synthetic compounds, natural compounds, genes and gene products.
5. The method of claim 4, wherein the test compound is a protein.
6. The method of claim 5, wherein the protein is a transcription factor.
7. A method of screening for a compound that regulates OCTN2 gene expression, the method comprising:
- (a) providing a vector comprising a reporter gene downstream of a promoter sequence from SEQ ID NO:5;
- (b) contacting a test compound with the vector; and
- (c) detecting expression of the reporter gene, wherein an increase or decrease in expression of the reporter gene in the presence of the test compound, compared to in the absence of the test compound, is indicative of a compound that regulates OCTN2 gene expression.
8. The method of claim 7, wherein the test compound binds to the promoter.
9. A method of screening for a compound that regulates OCTN2 gene expression, the method comprising:
- (a) providing an oligonucleotide DNA, wherein the oligonucleotide DNA comprises a regulatory sequence from SEQ ID NO:5 that is upstream of the OCTN2 coding sequence;
- (b) contacting a test compound with the oligonucleotide DNA; and
- (c) determining whether the test compound binds to the oligo DNA, wherein binding to the oligonucleotide DNA is an indication that the test compound is potentially able to regulate OCTN2 gene expression.
10. The method of claim 9, wherein the regulatory sequence is a promoter region of the OCTN2 gene.
11. A method of screening for a compound that regulates OCTN2 gene expression, the method comprising:
- (a) contacting a test compound with DNA that comprises a promoter region of the OCTN2 gene; and
- (b) measuring binding of the test compound to the promoter, wherein a compound that binds to the promoter is indicative of a compound that regulates OCTN2 gene expression.
12. The method of claim 11, wherein the DNA further comprises (i) OCTN2 genomic DNA (SEQ ID NO:5); (ii) a sequence that regulates transcription of SEQ ID NO:5; or (iii) DNA that encodes an OCTN2 protein comprising the amino acid sequence of SEQ ID NO:1.
13. The method of claim 11, wherein the DNA is bound to a solid support.
14. The method of claim 13, wherein the compound is purified by affinity chromatography.
15. The method of claim 11, wherein the test compound is a protein.
16. The method of claim 15, wherein the protein is a transcription factor.
17. A method of screening for a compound that regulates OCTN2 gene expression, the method comprising:
- (a) providing a cell comprising a reporter gene downstream of DNA that comprises a promoter region of the OCTN2 gene; and
- (b) detecting reporter gene activity in the cell, wherein an increase or decrease in reporter gene expression in the presence of the test compound, compared to in the absence of the test compound, is indicative of a compound that regulates OCTN2 gene expression.
18. The method of claim 17, wherein the test compound is a protein.
19. The method of claim 18, wherein the protein is a transcription factor.
20. A method of screening for a compound that enhances the activity of the OCTN2 protein, the method comprising:
- (a) contacting a test compound with an OCTN2 protein comprising the amino acid sequence of SEQ ID NO: 1; and
- (b) evaluating the activity of the protein, wherein an increase in activity in the presence of the test compound, compared to in the absence of the test compound, is indicative of a compound that enhances the activity of the OCTN2 protein.
21. The method of claim 20, wherein the test compound is introduced into a cell comprising a vector that expresses the OCTN2 protein.
22. The method of claim 21, wherein the activity of the OCTN2 protein is evaluated by measuring carnitine transport into the cell.
23. A pharmaceutical composition comprising an isolated protein comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient, wherein the protein is in an amount effective to increase cellular uptake of carnitine.
24. A method of testing whether an individual's genome carries a mutant OCTN2 allele that, in the homozygous state, may result in systemic carnitine deficiency, the method comprising
- (a) identifying an individual suspected of carrying the mutant allele; and
- (b) analyzing a nucleic acid sample from the individual to determine the presence or absence of a mutation in (i) DNA encoding OCTN2 (SEQ ID NO: 1) or (ii) OCTN2 genomic DNA (SEQ ID NO:5) or (iii) a sequence that regulates expression of SEQ ID NO:5, wherein the presence of the mutation in (i) or (ii) or (iii) indicates that the individual carries a mutant OCTN2 allele that, in the homozygous state, may result in systemic carnitine deficiency.
Type: Application
Filed: Aug 18, 2008
Publication Date: Sep 3, 2009
Applicant: CHUGAI SEIYAKU KABUSHIKI KAISHA (Tokyo)
Inventors: Jun-Ichi Nezu (Shizuoka), Akira Tsuji (Ishikawa), Asuka Ose (Chiba)
Application Number: 12/193,654
International Classification: C12Q 1/68 (20060101); G01N 33/50 (20060101); G01N 33/566 (20060101); G01N 33/68 (20060101); C12Q 1/02 (20060101); C07K 14/47 (20060101);
