Systemic carnitine deficiency gene and use thereof

Details Systemic carnitine deficiency gene and use thereof Systemic carnitine deficiency gene and use thereof

2001-03-02

2004-09-14

Low, Christopher S. F. (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S350000, C530S387200, C536S023500, C435S006120, C435S007100

Reexamination Certificate

active

06790831

ABSTRACT:

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.
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 &bgr;-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 INVENTION
An 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, preferab

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