Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-10-23
2001-02-06
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S803000, C436S501000, C536S024310, C536S024330
Reexamination Certificate
active
06183963
ABSTRACT:
BACKGROUND OF THE INVENTION
(a) Field of the Invention
This invention relates to a method of detecting genetic variation in individuals by PCR amplification of the locus of interest, transferring the resulting PCR products to a membrane filter, hybridizing with allele-specific-oligonucleotides (ASOs), and visualizing the results. Such a method is in the field of diagnostics, pharmacogenetics, cancer therapeutics, and drug metabolism. In particular, the present invention relates to the detection of genetic polymorphisms in gene encoding xenobiotics metabolizing enzymes CYP1A1, CYP2D6, CYP3A4, and NAT2.
(b) Description of Prior Art
It is recognized that marked interindividual differences exist in enzymes that metabolize xenobiotics and that this variability can be genetically determined. Considerable progress has been made in identifying a number of individual enzymes that are subject to genetic polymorphism. A polymorphism is generally considered to be a stably inherited trait. Genetic variation means differences between individuals in regard to the base sequence of their DNA. Genotyping involves identification of (defined) genetic mutations that, in a particular case, give rise to the specific drug metabolism phenotypes. These mutations include genetic alterations that lead to increased activity, absence of an active protein (null allele) or production of a mutant protein with altered catalytic capacity. A number of types of genetic polymorphism in xenobiotics metabolizing enzymes are relevant to clinical practice. These include those relating to N-ace-tyltransferase 2 (NAT2) and the cytochromes P450 1A1 (CYP1A1), 3A4 (CYP3A4) and 2D6 (CYP2D6). Genetic variation can be detected by means of electrophoresis of DNA fragments, generated by digestion of PCR products with a restriction enzyme the so-called PCR-RFLP approach. This method that has proven useful in screening for genetic mutations associated with altered metabolism of drugs and/or cancer susceptibility. It consists of amplification of a specific region of the gene of interest by PCR followed by digestion of the amplified DNA product with restriction endonucleases. Restriction endonucleases have the capacity to digest DNA with a high degree of nucleotide sequence specificity. Thus, point mutations within the recognition sequence of a specific restriction endonuclease may be detected through determining whether the DNA of interest serves as a substrate for that endonuclease. These studies are routinely carried out by comparing the size of digestion products generated from a DNA substrate amplified from control subject DNA with respect to study subject DNAS. The size of the digestion products is easily evaluated by agarose gel electrophoresis with ethidium bromide staining and UV transillumination. In this case the position of the bands varies between individuals, as a result of gain or loss of the recognition site for the restriction enzyme used, this is restriction fragment length polymorphism (RFLP). However, the PCR-RFLP is not well suited for genotyping a considerable number of samples that are usually analyzed in such investigations.
It would be highly desirable to be provided with a PCR-based assay to detect point mutations in CYP1A1, CYP2D6, CYP3A4 and NAT2.
SUMMARY OF THE INVENTION
The present invention relates to the development of a PCR-based assay to detect point mutations in CYP1A1, CYP2D6, CYP3A4 and NAT2. The presence of these mutant alleles is associated with an altered enzyme activity potentially leading (i) to toxicity when individuals are treated with standard doses of certain prescribed drugs or (ii) to increased susceptibility to cancer following environmental exposures. Detection of DNA common variants at the CYP1A1, CYP2D6, CYP3A4 and NAT2 loci would offer a strategy for identifying individuals <<at risk>> based on their genotype, prior to treatment with potentially toxic doses of drugs or to exposure to environmental toxins. Another approach for detection of specific mutations within a gene of interest is through hybridization of the PCR products with allele-specific oligonucleotide (ASO) probes for the wild type or variant alleles utilized in parallel hybridizations. Only the oligonucleotide that precisely hybridizes to the target sequences produces a signal from a labeled probe. This genotyping method, which require small amounts of nucleated cells derived from a variety of sources, is not affected by the underlying disease or by drugs taken by the patient. It provides results within 24-48 h, allowing for rapid intervention.
One aim of the present invention is to provide a diagnostic test to identify individuals with altered xenobiotics-metabolizing activities based on their genotypes. Such diagnostic test to determine genotype of individuals is quite advantageous because measuring the enzymatic activity has many limitations. To achieve this goal, 11 different tests, 3 to detect mutations in CYP1A1 alleles, 2 for CYP2D6, 1 for CYP3A4 and 5 for NAT2 variants, have been developed. These tests involved PCR-based amplification of these genes where the mutations of interest are found. Following amplification, the amplified fragments are dot blotted on a membrane filter and assayed for the presence or absence of the specific mutation of interest (i.e. at least one of the 11 mutations). Although much of these assays can be done in any molecular biology facilities, procedures and kits are designed that contain all the reagents, primers and solutions for the genotyping test to facilitate the procedure for use in general clinical laboratories, such as those found in a typical hospital, clinic and even private reference laboratories.
In accordance with the present invention there is provided an isolated oligonucleotide molecule comprising a mutant allele of CYP1A1, which contains a point mutation at one position selected from the group consisting of position 4887, 4889, and 6235, such as an adenine at position 4887, a guanine at position 4889 and a cytosine at position 6235. The mutant oligonucleotide molecules have a nucleic acid sequence as set forth in SEQ ID NOS:27, 28 and 31.
1. In accordance with the present invention there is provided an isolated oligonucleotide molecule comprising a wild-type allele of CYP1A1, which contains a normal nucleotide at one position selected from the group consisting of position 4887, 4889, and 6235, such as a cytosine at position 4887, a adenine at position 4889 and a thymine at position 6235. The wild type oligonucleotide molecules have a nucleic acid sequence as set forth in SEQ ID NOS:26, 29 and 30.
In accordance with the present invention there is provided an isolated oligonucleotide molecule comprising a mutant allele of CYP3A4, which contains a point mutation at position −290, such as a guanine. The mutant oligonucleotide molecule has a nucleic acid sequence as set forth in SEQ ID NO:37.
In accordance with the present invention there is provided an isolated oligonucleotide molecule comprising a wild-type allele of CYP3A4, which contains a normal nucleotide at position 290, such as an adenine. The wild type oligonucleotide molecule have a nucleic acid sequence as set forth in SEQ ID NO:36.
In accordance with the present invention there is provided an isolated oligonucleotide molecule comprising a mutant allele of CYP2D6, which contains a point mutation at one position selected from the group consisting of position 1934 and 2637, such as an adenine at position 1934 and a deletion at position 2637. The mutant oligonucleotide molecules have a nucleic acid sequence as set forth in SEQ ID NOS:33 and 35.
In accordance with the present invention there is provided an isolated oligonucleotide molecule comprising a wild-type allele of CYP2D6, which contains a normal nucleotide at one position selected from the group consisting of position 1934 and 2637, such as a guanine at position 1934 and an adenine at position 2637. The wild type oligonucleotide molecules have a nucleic acid sequence as set forth in SEQ ID NOS:32 and 34.
In accordance with the present in
Labuda Damian
Sinnett Daniel
Chakrabarti Arun
Côté France
Fredman Jeffrey
Signalgene
Swabey Ogilvy Renault
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