Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-02-07
2004-09-28
Wilder, Cynthia (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06797475
ABSTRACT:
BACKGROUND OF THE INVENTION
5-lipoxygenase (5-LO) is the first committed enzyme in the pathway leading to leukotriene synthesis and is responsible for the conversion of arachidonic acid to LTA4 via the unstable intermediate 5-hydroperoxy eicosotetraenoic acid (Samuelsson et al., (1987)
Science
237:1171; Samuelsson (1983)
Science
220:568). Leukotriene A4 can, in turn, be converted to leukotriene B4, C4, D4, or E4. Leukotrienes are potent local mediators which influence inflammatory and allergic response, including asthma, rheumatoid arthritis, psoriasis, thrombotic disease, ulcerative colitis, bronchitis, sinusitis, allergic and non-allergic rhinitis, and lupus. Leukotrienes (LTs) B4, C4, D4, and E4 have been shown experimentally to mimic the pathologic changes seen in asthma and to play a role in several inflammatory mechanisms that lead to airflow obstruction in asthma. Such mechanisms include bronchoconstriction, mucosal edema, increased secretion of mucus, and an inflammatory-cell infiltrate that is rich in eosinophils. (O'Bryne (1997)
Chest
111:27S-34S). The slow-reacting substance of anaphylaxis is now known to be a mixture of leukotrienes C4, D4, and E4, all of which are potent bronchoconstrictors that exert their biological actions through specific ligand-receptor interactions thereby increasing vascular permeability and constricting smooth muscle (U.S. Pat. No. 5,750,565; Silverman, et al. (1998)
Clin Exp Allergy
28 Suppl 5:164-70).
There have been research efforts to develop specific receptor antagonists or inhibitors of leukotriene biosynthesis, to prevent or minimize pathogenic inflammatory responses mediated by these compounds, including the inhibition of 5-LO. For example, European Patent Application Nos. 90117171.0 and 901170171.0 disclose indole, benzofuran, and benzothiophene lipoxygenase inhibiting compounds (U.S. Pat. No. 5,750,565). It has been established that treating patients with agents that have the capacity to inhibit 5-LO results in improvement in lung function, reduction in asthma symptoms, and decreased need for alternative asthma treatments (Persson et al., (1995)
Anesthesiology
82:969; Israel et al., (1993)
Ann. Int. Med.
119:1059). However, in patients with asthma, there is a heterogeneous response to treatment with 5-LO inhibitors. Mutations in the 5-LO gene sequence may be associated with responsiveness to therapy and/or susceptibility to diseases or disorders, e.g., 5 asthma.
The 5-LO gene has been cloned, both as a cDNA (Matsumoto et al., (1988)
Proc. Natl. Acad. Sci. USA
85:3406; Dixon et al., (1988)
Proc. Natl. Acad. Sci. USA
85:416; Balcarek et al., (1988)
J. Biol. Chem.
263:13937) and as a genomic clone (Hoshiko et al., (1990)
Proc. Natl. Acad. Sci. USA
87:9073; Funk et al., (1989)
Proc. Natl. Acad. Sci. USA
86
:
2587
). The 5-LO gene is approximately 85 kilobases in size and contains 14 exons and 15 introns. The region 88 to 212 base pairs upstream of the 5-LO translation start site contains a number of sequences known to be recognition sites for transcriptional regulators (Hoshiko et al., (1990)
Proc. Natl. Acad. Sci. USA
87:9073, incorporated herein by reference). For example, binding sites for AP1 and Sp1, each of which can act as either a transcriptional activator or a transcriptional repressor, depending on context, are found in this region. With respect to the Sp1 binding site, it comprises 5 tandem Sp1 motifs (GGGCGG) found −147 to −176 base pairs upstream of the ATG translation start site. Deletions of the Sp1 motifs reduce transcription from this 5′ upstream regulatory element (Hoshiko et al. (1990)
Proc. Natl. Acad. Sci. USA
87:9073). Sp1 binding sites comprising such motifs are similarly found in the promoter region of a variety of other genes (Li et al., (1995)
Gene
164:229; Wariishi et al., (1995)
Biochem. Biophys. Res. Commun.
216:729; Tang et al., (1995)
Biochem. Biophys. Res. Commun.
213:673; Khachigian et al., (1995)
J. Bio. Chem.
270:27679).
Certain polymorphisms in the 5-LO gene have been shown to be correlated with patient responsiveness to 5-LO inhibitor therapy (U.S. Pat. No. 6,090,547 by Drazen, et al. (2000)). Drazen et al. disclosed that asthma patients having polymorphisms associated with reduced 5-LO gene expression are less responsive to 5-LO inhibitor therapy than patients having normal 5-LO gene expression. Specifically, Drazen et al. showed that 5-LO promoters comprising a variant Sp1 binding site having 3, 4 or 6 Sp1 motifs were less active than the wild-type 5-LO promoter comprising a Sp1 bind site having 5 Sp1 motifs, and that asthma patients homozygous or heterozygous for 5-LO alleles comprising S-LO promoters having such variant Sp1 binding sites were less responsive to S-LO inhibitor therapy than patient homozygous for the wild-type 5-LO allele. Drazen et al. also showed that the 3 Sp1 motif binding site is in linkage disequilibrium with a G to A polymorphism in exon 2 of the 5-LO gene, and that the 4 Sp1motif binding site is in linkage disequilibrium with a C to T polymorphism in exon 1 of the 5-LO gene.
It would also be desirable to identify additional polymorphisms within the 5-LO gene which are in linkage disequilibrium with a variant Sp1 binding site in the 5-LO promoter. It would further be desirable, to provide prognostic, diagnostic, pharmacogenomic and therapeutic methods utilizing the identified polymorphisms.
SUMMARY OF THE INVENTION
The present invention relates to polymorphisms in the 5-LO gene. The invention is based, in part, on the discovery of a novel 5-LO haplotype which comprises five sequence polymorphisms within the 5-LO gene that are in linkage disequilibrium with each other. The five 5-LO polymorphisms are set forth in Table 1. Three of these, 5loprr1, 5lo01a and 5lo04a, are newly identified polymorphisms. The other two, 5lonrra and 5lonrrb, have been described by In et al. in
J. Clinical invest.
99:1130-1137 (1997). The present invention is based, also in part, on the discovery that the haplotype of the invention is in linkage disequilibrium with any one of three variant Sp1 binding sites in the 5-LO promoter region that were previously reported by Drazen et al. U.S. Pat. No. 6,090,547). Accordingly, any one of the five polymorphisms of the invention is a marker for the other four polymorphisms and a variant Sp1 binding site having 3, 4 or 6 Sp1 motifs in the 5-LO promoter region. Moreover, since Drazen et al. (id.) demonstrated that asthma patients homozygous or heterozygous for a 5-LO allele comprising any one of such variant Sp1 binding sites are less susceptible to effective treatment with 5-LO inhibitor therapy, the haplotype of the invention is also a marker of reduced responsiveness amongst inflammatory or allergy disease patients to 5-LO inhibitor therapy.
The present invention is based further, in part, on the discovery that the 5lo01a polymorphism of the invention is associated with an abnormally low eosinophil count. That is, individuals heterozygous or homozygous for the 5lo01a polymorphism have been found to have significantly lower eosinophil levels compared to individuals not having the polymorphism. Thus, the haplotype of the invention and each of its component polymorphisms are all markers of low eosinophil levels.
The invention provides a method for identifying an inflammatory or allergy disease patient who is less susceptible to effective treatment with a 5-LO inhibitor, comprising determining the presence or absence of a 5-LO allelic variant comprising a sequence selected from the group consisting of those set forth in SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO:6, or the complement of the sequence in a nucleic acid sample from an inflammatory or allergy disease patient, wherein the presence of the 5-LO allelic variant in the sample indicates that the patient is less susceptible to effective treatment with a 5-LO inhibitor. In one embodiment, the 5-LO allelic variant comprises the sequences of those set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or the complement of each of those sequences. In
Barnes Glenn
Meyer Joanne
DiRocco Lisa M.
Lahive & Cockfield LLP
Millennium Pharmaceuticals Inc.
Smith DeAnn F.
Wilder Cynthia
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