Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-15
2001-11-13
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S810000, C536S023100, C536S023500, C536S024310, C536S024330, C536S023400
Reexamination Certificate
active
06316196
ABSTRACT:
This invention relates to polymorphisms in the LTC
4
synthase gene. The invention also relates to methods and materials for analysing allelic variation in the LTC
4
synthase gene, and to the use of LTC
4
synthase polymorphism in the diagnosis and treatment of leukotriene mediated diseases such as asthma and allergic rhinitis.
The cysteinyl-leukotrienes, LTC
4
, LTD
4
and LTE
4
, are potent bronchoconstrictors, increase vascular permeability and increase mucus production in airways. They are implicated in the pathophysiology of asthma and allergic rhinitis and are found at elevated levels in bronchoalveolar lavage from asthma patients, particularly after allergen challenge LTD
4
and LTE
4
may also enhance the neurogenic inflammatory response in airways. Compounds which inhibit leukotriene synthesis e.g. the 5-lipoxygenase inhibitor, zileuton, or the leukotriene receptor antagonist, zafirlukast, have been shown to be effective against asthma and rhinitis (Busse W. W, Clin. Exp. Allergy, 26, 868-879, 1996; see particularly FIG. 1 therein which shows the arachidonic acid cascade, indicating the role of LTC
4
synthase in catalysing the formation of LTC
4
).
Leukotrienes are derived from membrane phospholipids. Arachidonic acid is released from the phospholipid by cytosolic phospholipase A2 and converted to LTA
4
by 5-lipoxygenase in the presence of 5-lipoxygenase activating protein, FLAP. Polymorphisms in 5-LO have been reported in international patent application WO 97/42347, Brigham & Women's Hospital. LTA
4
is conjugated with reduced glutathione by LTC
4
synthase to form LTC
4
. The biologically active metabolites, LTD
4
and LTE
4
are formed, following carrier mediated export of LTC
4
, by the sequential action of gamma-glutamyl transpeptidase and dipeptidases.
The LTC
4
synthase gene has been cloned and published as a 4,465 nucleotide genomic sequence comprising 1,446 nucleotides of sequence 5′ to the coding sequence, the 5 exons and intervening introns and 3′ sequence extending 398 nucleotide beyond the poly A signal (Penrose et al., J. Biol. Chem., 271, 11356-11361, 1996; EMBL accession no. U50136).
One approach is to use knowledge of polymorphisms to help identify patients most suited to therapy with particular pharmaceutical agents (this is often termed “pharmacogenetics”). Pharmacogenetics can also be used in pharmaceutical research to assist the drug selection process. Polymorphisms are used in mapping the human genome and to elucidate the genetic component of diseases. The reader is directed to the following references for background details on pharmacogenetics and other uses of polymorphism detection: Linder et al. (1997), Clinical Chemistry, 43, 254; Marshall (1997), Nature Biotechnology, 15, 1249; International Patent Application WO 97/40462, Spectra Biomedical; and Schafer et al. (1998), Nature Biotechnology, 16, 33.
Clinical trials have shown that patient response to treatment with leukotriene antagonists is heterogeneous. Thus there is a need for improved approaches to pharmaceutical agent design and therapy with leukotriene antagonists.
The present invention is based on the discovery of five single nucleotide polymorphisms (SNPs) in the LTC
4
synthase gene. Three SNPs have been found in the 5′ untranslated region of the gene and two in the first intron, located at positions 375, 815, 1003, 2169 and 2742 respectively, based on the numbering of U50136. Before our first filing date, we believe there has been no disclosure of polymorphismlallelic variation in the LTC
4
synthase gene.
According to one aspect of the present invention there is provided a method for the diagnosis of a single nucleotide polymorphism in LTC
4
synthase in a human, which method comprises determining the sequence of the nucleic acid of the human at one or more of positions 375, 815, 1003, 2169 and 2742 in the LTC
4
synthase gene as defined by the positions in SEQ ID NO: 1, and determining the status of the human by reference to polymorphism in the LTC
4
synthase gene.
The term human includes both a human having or suspected of having a leukotriene mediated disease and an a symptomatic human who may be tested for predisposition or susceptibility to leukotriene mediated disease. At each position the human may be homozygous for an allele or the human may be a heterozygote.
In one embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymorphism at position 375 is presence of G and/or A.
In another embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymorphism at position 815 is presence of C and/or A.
In another embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymorphism at position 1003 is presence of A and/or C. Testing for the presence of the C allele at this position is especially preferred because, without wishing to be bound by theoretical considerations, of its association with increased levels of LTC
4
synthase (as explained herein).
In another embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymorphism at position 2169 is presence of C and/or T.
In another embodiment of the invention preferably the method for diagnosis described herein is one in which the single nucleotide polymorphism at position 2742 is presence of C and/or T.
The method for diagnosis is preferably one in which the sequence is determined by a method selected from amplification refractory mutation system and restriction fragment length polymorphism.
In another aspect of the invention we provide a method for the diagnosis of leukotriene mediated disease, which method comprises:
i) obtaining sample nucleic acid from an individual,
ii) detecting the presence or absence of a variant nucleotide at one or more of positions 375, 815, 1003 and 2169 in the LTC
4
synthase gene and
iii) determining the status of the individual by reference to polymorphism in the LTC
4
synthase gene.
The published sequence of the LTC
4
synthase gene, EMBL accession number U50136, is shown in SEQ ID NO: 1 in which the variant sites discovered in the present invention are at positions 375, 815, 1003, 2169 and 2742.
Allelic variation at position 375 consists of a single base substitution from G (the published base), for example to A. Allelic variation at position 815 consists of a single base substitution from C (the published base), for example to A. Allelic variation at position 1003 consists of a single base substitution from A (the published base), for example to C. Allelic variation at position 2169 consists of a single base substitution from C (the published base), for example to T. Allelic variation at position 2742 consists of a single base substitution from C (the published base), for example to T. The status of the individual may be determined by reference to allelic variation at one, two, three, four or all five of the above loci.
Sanak et al. (1998), Lancet, 350, 1599, have reported an increased risk of aspirin induced asthma (AIA) being associated with the polymorphism at position 1003. This work suggests that the presence of the C allele at position 1003 leads to increased levels of LTC
4
synthase (see also Cowburnet al. (1998), J. Clin. Invest., 101, 834). AIA affects about 10% of adult asthmatics. Aspirin and other cyclo-oxygenase inhibitors cause release of LTs into airways, leading to an asthma attack in susceptible individuals. Clinical approaches to deal with AIA include pretreatment with anti-leukotriene drugs (Szczeklik (1997), Allergy, 52, 613-9). Commentators have written approvingly of the clinical utility of detection of LTC
4
polymorphisms (Holgate (1998), Lancet, 351, 1300-1301, see last paragraph in particular). Anti-leukotriene drugs have been reviewed in the following publications: Horwitz et al. (1998), Am J Respir Crit Care Med, 157, 1363 (see particularly Table 1 for a list of drugs); and Tan (199
Myers Carla J.
Pillsbury & Winthrop LLP
Zeneca Limited
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