Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-03
2001-10-23
Zitomer, Stephanie W. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024300, C536S024330, C536S023100, C435S091200, C435S091100
Reexamination Certificate
active
06306603
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a CD36 mutant gene and its use, more specifically, a method for diagnosing diseases caused by lipid metabolism abnormality using the CD36 mutant gene.
2. Background Art
The development in the search technology in heart nuclear medicine in recent years has made it possible to clinically study myocardial lipid metabolism and discuss abnormalities in the myocardial lipid metabolism in heart diseases. In particular, a number of cases of abnormal fatty acid accumulation on the myocardium in hypertrophic cardiomyopathy have been reported. However, its mechanism has not been revealed.
The heart, which is a driving device for blood circulation in the body, requires a great amount of energy even in normal state, and the energy requirement further increases during exercise and under stress. The major source of energy supply in the myocardium is long chain fatty acids and 70 to 80% of myocardial energy are deemed to be derived from long chain fatty acids. Accordingly, disorders in long chain fatty acid metabolism in the myocardium are considered to result in serious consequence. In fact, it is known that cardiac diseases including sudden death are caused by disorders in the final stage of the long chain fatty acid metabolism, namely, the incorporation system of long chain fatty acids into mitochondria (carnitine shuttle), or abnormalities in enzymes which belong to &bgr; oxidation system.
Various theories were suggested for the mechanism of the incorporation of long chain fatty acids into cells, but none of them were confirmed. Recently, we identified a gene which associates with the mechanism of the incorporation of myocardial long chain fatty acids and reported that the responsible product is a glycoprotein CD36 which is usually expressed in the platelet membrane (BIO Clinica, 12 (14), 86-90 (1997)).
CD36 mutant genes so far reported include C478T substitution gene in which cytosine at position 478 of the CD36 gene (exon 4) is substituted by thymine (F. K. Schattauer Verlagsgesellschaft mbH (Stuttgart), 69(5), 481-484 (1993)), 539AC deletion gene in which adenine and thymine at positions 539 and 540 of the CD36 gene (exon 5) are deleted (Blood, 83(12), 3545-3552 (1994)), and 1159A insertion gene in which adenine is inserted at position 1159 of the CD36 gene (exon 10) (Arteriosclerosis, Thrombosis, and Vascular Biology, 16(8), 1026-1032).
However, neither relationship between these CD36 mutant genes and diseases caused by lipid metabolism abnormality nor the presence of CD36 mutant genes other than the above have not been confirmed.
SUMMARY OF THE INVENTION
The present inventors studied expression of CD36 protein in platelets and monocytes in 41 test samples deficient in fatty acid incorporation among test samples selectively taken from subjects who have history of cardiac diseases or might have possibilities of having cardiac diseases, using flow cytometry. The results obtained revealed that neither the platelet nor the monocyte expressed the CD36 protein in all test samples.
Further, the present inventors prepared the chromosomal DNA from the 41 test samples and analyzed gene mutations for the entire exon region. The present inventors also prepared the chromosomal DNA from tissues removed using the Batista operation (an operation to excise a part of the myocardium lacking contractibility) on 27 cardiomyopathy patients suffering severe cardiac insufficiency and tried to detect CD36 gene mutations. As a result, the present inventors found new mutations in exon 6, exon 9, exon 12 and exon 13 in addition to known mutations in exon 4, exon 5 and exon 10. Moreover, the present inventors specified an additional new mutation present in exon 5, as well as mutations present in exon 6, exon 9 and exon 13.
An object of the present invention is to provide a CD36 mutant gene and a judgment method and a diagnostic kit for diseases caused by abnormal lipid metabolism.
The present invention provides a CD36 mutant gene which comprises a nucleotide sequence selected from the sequences of SEQ ID NO: 1 through NO: 8.
The present invention also provides a nucleotide fragment which comprises a nucleotide sequence selected from the sequences of SEQ ID NO: 1 through NO: 8, or a mutated portion thereof.
The present invention provides a method of judging diseases caused by abnormal lipid metabolism, which comprises the step of detecting a mutation of the CD36 gene.
The present invention provides a kit for diagnosing diseases caused by lipid metabolism abnormality which comprises a reagent for detecting a mutation of the CD36 gene.
DETAILED DESCRIPTION OF THE INVENTION
The term “mutation” as used herein refers to deletions, substitutions and insertions.
The term “gene mutation” as used herein refers to a mutation existing in one allele as well as a mutation existing in both alleles.
The expression “insertion at base No. X” as used herein means that a base is inserted between base No. X-1 and base No. X.
The expression “base No. of the CD36 gene” as used herein means base No. of the CD36 cDNA sequence, i.e., the nucleotide sequence of SEQ ID NO: 9.
The CD 36 mutant genes according to the present invention comprise nucleotide fragments shown in SEQ ID NO: 1 through NO: 8.
Examples of the CD36 mutant genes according to the present invention include:
a CD36 gene sequence in which the nucleotide sequence portion of SEQ ID NO: 38 (including the sequence encoding exon 13 and its adjacent intron portion) is the nucleotide sequence of SEQ ID NO: 1, 2 or 3;
a CD36 gene sequence in which the nucleotide sequence portion of SEQ ID NO: 39 (including the sequence encoding exon 12 and its adjacent intron portion) is the nucleotide sequence of SEQ ID NO: 4;
a CD36 gene sequence in which the nucleotide sequence portion of SEQ ID NO: 41 (including the sequence encoding exon 9 and its adjacent intron portion) is the nucleotide sequence of SEQ ID NO: 5;
a CD36 gene sequence in which the nucleotide sequence portion of SEQ ID NO: 42 (including the sequence encoding exon 6 and its adjacent intron portion) is the nucleotide sequence of SEQ ID NO: 6 or 7;
a CD36 gene sequence in which the nucleotide sequence of SEQ ID NO: 43 (including the sequence encoding exon 5 and its adjacent intron portion) is the nucleotide sequence portion of SEQ ID NO: 8.
In the present invention, a mutated portion of either one of the nucleotide sequences of SEQ ID NO: 1 through NO: 8 and NO: 10 through NO: 12 comprises a nucleotide sequence of at least 12 (for example, 12 to 40) consecutive nucleotides, preferably at least 20 (for example, 20 to 40) consecutive nucleotides including portions having substitutions, deletions or insertions.
The term “CD36” gene, refers to the CD36 gene disclosed in J. Biol. Chem., Vol. 269, No. 29, 18985-18991 (1994). The term “CD36 mutant gene” refers to a CD36 gene having mutations.
The cDNA sequence of the CD36 gene is depicted in SEQ ID NO: 9. Locations of exons in the nucleotide sequence of SEQ ID NO: 9 areas follows: Exon 1: 1-27, exon2: 28-121, exon3: 122-330, exon4: 331-491, exon5: 492-639, exon6: 640-819, exon7: 820-911, exon 8: 912-958, exon 9: 959-1028, exon 10: 1029-1216, exon 11: 1217-1335, exon 12: 1336-1409, exon 13: 1410-1464, and exon 14: 1465 to the region containing the termination codon.
The known mutated portions of the CD36 mutant gene mentioned in “Background Technology” are shown in SEQ ID NOS: 10, 11 and 12.
Nucleotide sequences containing sequences coding for exon 13, exon 12, exon 10, exon 9, exon 6, exon 5 and exon 4 of the normal CD36 gene are depicted in SEQ ID NOS: 38, 39, 40, 41, 42, 43 and 44, respectively.
CD36 mutant genes and their mutated portions are useful for the detection of mutations of the CD36 gene, and further for the diagnosis and judgement of diseases caused by abnormal lipid metabolism.
The term “mutation of the CD36 gene” as used herein can refer to mutations in regions including exon 4, exon 5, exon 6, exon 9, exon 10, exon 12 and exon 13. The expression “mutation of the CD36 gene” means deleti
Oka Takanori
Tanaka Takao
Yamane Akio
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Wakunaga Phaemaceutical Co., Ltd.
Wilder Cynthia B
Zitomer Stephanie W.
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