The protein encoded by the above is provided at SEQ ID NO:2.

An adenine to cytosine transversion polymorphism (A-444C) is known at the position 444 nucleotides upstream (bold and underlined, above) from the first codon (underlined, above). Accordingly, the "A" allele as defined herein would comprise the sequence CTGGATGGGG ACAGGGAACA (SEQ ID NO:3) (nucleotides 991-1010 of SEQ ID NO:1). In contrast, the "C" allele as defined herein would comprise the sequence CTGGATGGGG ACCGGGAACA (SEQ ID NO:4) (nucleotides corresponding to 991-1010 of SEQ ID NO:1).

The genetic samples were secondly screened for a polymorphism in the region of 176 to 147 base pairs upstream from the ATG start site in 5-lipoxygenase (ALOX5), whereby the presence of 5 tandem repeats of the Sp1 binding motif (GGGCGG; SEQ ID NO:12) is wildtype, and the presence of 3, 4 or 6 such tandem repeats is variant, again using PCR technology. The alleles were labeled as "5" (5 tandem repeats) or "X" (3, 4 or 6 tandem repeats), resulting in three possible genotypes (5,5, 5/X and X/X). Variant ALOX5 alleles have a number of tandem repeats of the Sp1 binding motif in this region that is more than, or fewer than, five repeats. That is, in the region of from about 200 nucleotides to about 125 nucleotides upstream of the ATG start site in ALOX5, there are from one to four, or more than five, repeats of the Sp1 binding motif. In the present studies, variant ALOX5 alleles ("X") had three, four or six tandem repeats in this area.

Screening was also conducted for a G to A transversion polymorphism 1708 bases upstream from the first codon of the ALOX5 gene (G-1708A), and for a single base transversion polymorphism 1728 bases downstream from the first codon (in exon 13) of the ALOX5 gene (A1728G).

The nucleotide sequence of the 5′ region and partial coding sequence of human 5-lipoxygenase (ALOX5) containing five repeats of the Sp1 binding motif, and the G-1708A polymorphism, is provided at GenBank Accession number M38191(SEQ ID NO:5):

The first codon is underlined; the region comprising the repeats of the Sp1 binding motif (GGGCGG) is shown in bold and underlined type; the G-1708A position is also shown in bold and underlined type.

The 5-lipoxygenase gene has been cloned as cDNA (Matsumoto et al., Proc. Natl. Acad. Sci. USA 85:3406 (1988) and as a genomic clone (Hoshiko et al., Proc. Natl. Acad. Sci. USA 87:9073 (1990). The 5-lipoxygenase gene is approximatly 85 kilobases in size, with 14 exons and 15 introns.

Two ALOX5 mRNA sequences are provided in GenBank at Accession numbers NM 000698 and XM 005818. The sequence provided at NM 000698 encodes a protein of 674 amino acids and is shown below:

The 1728 polymorphism site is indicated in underlined and bolded type; the A1728G polymorphism does not change the encoded amino acid (proline at amino acid position 576; SEQ ID NO: 14).

The sequence at XM 005818 is provided below (SEQ ID NO:15) and encodes an amino acid sequence (SEQ ID NO:16) that differs from that encoded by NM000698 in the initial amino acids. The site of the A1728G polymorphism (numbering referenced to NM000698) is shown in underlined bold type.

The present inventors have determined that the C/C genotype at the A-444C site of LTC4S, the X/X Sp1 genotype in ALOX5, and the polymorphism at the A1728G site of ALOX5 are independently associated with diminished response to the Cys LT1 leukotriene receptor antagonist zafirlukast.

According to the present methods, a subject who suffers from asthma that is potentially treatable with an anti-inflammatory inhaled glucocorticoid or a leukotriene antagonist, is genetically screened, to aid in predicting their response to such treatment. Screening comprises obtaining a sample of DNA from the subject and screening the DNA to determine the genotype (presence/absence of polymorphic alleles) at a predetermined polymorphic site in the gene of interest (here ALOX5 and/or LTC4S polymorphisms as described), where different genotypes at that site have previously been associated with different incidences of a phenotypic response to treatment. The presence of a particular genotype therefore indicates an increased likelihood that the individual subject will exhibit the associated phenotype. The genotype will rarely be absolutely predictive, i.e., where a population with a certain genotype displays a high incidence of a particular phenotype, not every individual with that genotype will display the phenotype. However, it will be apparent to those skilled in the art that genotyping a subject as described herein will be an aid in predicting the response a subject will have to treatment with a leukotriene receptor antagonist or a glucocorticoid, and thus assist in the treatment decision.

As used herein, "genotyping a subject (or DNA sample) for a polymorphic allele at a defined genomic locus" or "determining the genotype at a polymorphic allelic site" means detecting which forms of the allele are present in a subject (or a biological sample). As is well known in the art, an individual may be heterozygous or homozygous for a particular allele. More than two forms of an allele may exist, as is the case with microsatellite markers; thus there may be more than three possible genotypes.

As used herein, a subject that is "predisposed to" a particular phenotypic response based on genotyping of a polymorphic allele will be more likely to display that phenotype than an individual with a different genotype at that polymorphic allele. Where the phenotypic response is based on a biallelic polymorphism, the response may differ among the three possible genotypes (Eg. For LTC4S: A,A; A,C and C,C).

As used herein, a "genetic subset" of a population consists of those members of the population having a particular genotype. In the case of a biallelic polymorphism, a population can potentially be divided into three subsets: homozygous for allele 1, heterozygous, and homozygous for allele 2. Where multiple non-wildtype polymorphisms exist, a population can also be divided into three subsets: homozygous wildtype; heterozygous wildtype; and homozygous non-wildtype.

As used herein, asthma treatable with an anti-inflammatory glucocorticoid or treatable with a leukotriene receptor antagonist is a disease in which the administration of such a drug (in an appropriate pharmaceutical formulation, and in a therapeutically effective amount) has been shown to reduce or alleviate symptoms, without causing unacceptable side effects. Such therapeutic effectiveness is typically evidenced by Regulatory Authority (eg FDA, EMEA) approval of the pharmaceutical preparation, or by publication of the results of clinical studies in peer-reviewed medical journals. Therapeutically effective amounts of such compounds can be readily determined by those skilled in the art using, e.g., dose-response studies.

Known leukotriene receptor antagonists include zafirlukast, montelukast, pranlukast or iralukast.

As used herein, a "side effect" is an undesirable response to the administration of a therapeutic compound, e.g., an effect that is not directed to alleviating the symptoms or cause of the disease being treated. Side effects range from minor inconveniences to more serious events.

As used herein, "response" to treatment with a therapeutic compound is a desirable response to the administration of the compound, e.g., alleviation of the symptoms of the disease or of the underlying pathologic causes of the symptoms. Various indicators of a subject's response to therapeutic treatment may be assessed, as will be apparent to one skilled in the art. As an example, the change in Forced Expiratory Volume (FEV; FEV₁=FEV for 1 second duration) may be used as an indicator of response to treatment for asthma, as will be apparent to one skilled in the art.

According to the present methods, a compound with leukotriene receptor antagonism may be screened for variation in its effects among genetic subpopulations of subjects with asthma. Such methods involve administering the compound alone, or in tandem with another anti-asthma compound (such as a glucocorticoid), to a population of subjects suffering from asthma, obtaining DNA samples from the subjects (which may be done either prior to or after administration of the compounds), genotyping a polymorphic allelic site in the gene of interest, and correlating the genotype of the subjects with their phenotypic responses (both favorable and unfavorable) to the treatment. A genotype that is correlated with an increased incidence of a desired therapeutic response, compared to the incidence in subjects with alternative genotypes at the polymorphic allelic site, indicates that the effectiveness of the compound in treating asthma varies among genetic subpopulations.

Stated another way, the methods of the present invention may be used to determine the correlation of a known polymorphic allele with the response of subjects to treatment with a leukotriene receptor antagonist or a glucocorticoid. The population of subjects with the disease of interest is stratified according to genotype for the particular polymorphic allele, and their response to a therapeutic agent is assessed (either prospectively or retrospectively) and compared among the genotypes. The response to the therapeutic agent may include either, or both, desired therapeutic responses (e.g., the alleviation of signs or symptoms) and undesirable side effects. In this way, genotypes that are associated with an increased (or decreased) incidence of therapeutic efficacy, or an increased (or decreased) incidence of a particular side effect, may be identified. The increase or decrease in response is in comparison to the other genotypes, or to a population as a whole.

Polymorphisms are variant sequences within the human genome that may or may not have a functional consequence. These variants can be used in all aspects of genetic investigation including the analysis and diagnosis of genetic disease, forensics, evolutionary and population studies. Two types of genetic analyses are typically performed: linkage and association studies.

A linkage study provides genetic map information with no prior knowledge or assumption about the function of a gene. In a linkage study one uses DNA polymorphisms to identify chromosomal regions that are identical between affected relatives with the expectation that allele sharing frequencies will be higher for a marker (polymorphism) whose chromosomal location is close to that of the disease allele. Physical cloning of a linkage region narrows down the DNA sequence that could harbor the candidate disease gene. While linkage analysis locates the disease locus to a specific chromosome or chromosome region, the region of DNA in which to search for the gene is typically large, on the order of several million base pairs.

In contrast to linkage, association shows the coexistence of a polymorphism and a disease phenotype in a population. Association studies are based upon linkage disequilibrium, a phenomenon that occurs between a marker and a disease loci when the occurrence of two alleles at different loci is larger than the product of the allelic frequencies. Since the marker and disease causing variant are in close proximity, it requires many generations of recombination to separate them in a population. Thus they tend to co-exist together on the same chromosome at a higher than expected frequency. A marker (polymorphism) is said to be associated with a specific phenotype when its frequency is significantly higher among one phenotype group compared to its frequency in another. In general, the closer a marker is to the functionally polymorphic site, the stronger the association.

Association studies offer the opportunity to finely map linkage regions, map loci refractory to linkage analysis and map unknown predisposition loci. Polymorphisms that are in linkage disequilibrium with each other can be spaced over large regions. Linkage disequilibrium has been reported in regions as small as 1 kb or as large as 500 kb. Polymorphisms throughout a gene can be in linkage disequilibrium with each other, such that it is valuable to study the whole genome structure—introns, exons, promoters and transcriptional regulatory regions, and 3′ and 5′ untranslated regions. A marker that is in linkage disequilibrium with a functional polymorphism can be tested for correlation with a phenotype.

As used herein, the term polymorphism includes Single Nucleotide Polymorphisms (SNPs), insertion/deletion polymorphisms; transversion polymorphisms; microsatellite polymorphisms; and variable number of tandem repeat (VNTR) polymorphisms.

Polymorphic alleles are typically detected by directly determining the presence of the polymorphic sequence in a polynucleotide or protein from the subject, using any suitable technique as is known in the art. Such a polynucleotide is typically genomic DNA, or a polynucleotide derived from this polynucleotide, such as in a library made using genomic material from the individual (e.g. a cDNA library). The processing of the polynucleotide or protein before the carrying out of the method of the invention is further discussed below. Typically the presence of the polymorphism is determined in a method that comprises contacting a polynucleotide or protein of the individual with a specific binding agent for the polymorphism and determining whether the agent binds to the polynucleotide or protein, where the binding indicates that the polymorphism is present. The binding agent may also bind to flanking nucleotides and amino acids on one or both sides of the polymorphism, for example at least 2, 5, 10, 15 or more flanking nucleotide or amino acids in total or on each side. In one embodiment the agent is able to bind the corresponding wild-type sequence by binding the nucleotides or amino acids which flank the polymorphism position, although the manner of binding will be different than the binding of a polymorphic polynucleotide or protein, and this difference will be detectable (for example this may occur in sequence specific PCR as discussed below).

In the case where the presence of the polymorphism is being determined in a polynucleotide it may be detected in the double stranded form, but is typically detected in the single stranded form.

The binding agent may be a polynucleotide (single or double stranded) typically with a length of at least 10 nucleotides, for example at least 15, 20, 30, or more polynucleotides. The agent may be a molecule that is a structurally similar polynucleotide that comprises units (such as purines or pyrimidines) able to participate in Watson-Crick base pairing. The agent may be a protein, typically with a length of at least 10 amino acids, such as at least 20, 30, 50, 100 amino acids. The agent may be an antibody (including a fragment of such an antibody that is capable of binding the polymorphism).

A polynucleotide agent which is used in the method will generally bind to the polymorphism of interest, and the flanking sequence, in a sequence specific manner (e.g. hybridize in accordance with Watson-Crick base pairing) and thus typically has a sequence which is fully or partially complementary to the sequence of the polymorphism and flanking region.

In one embodiment of the present methods a binding agent is used as a probe. The probe may be labeled or may be capable of being labeled indirectly. The detection of the label may be used to detect the presence of the probe on (and hence bound to) the polynucleotide or protein of the individual. The binding of the probe to the polynucleotide or protein may be used to immobilize either the probe or the polynucleotide or protein (and thus to separate it from one composition or solution).

In another embodiment of the invention the polynucleotide or protein of the individual is immobilized on a solid support and then contacted with the probe. The presence of the probe immobilized to the solid support (via its binding to the polymorphism) is then detected, either directly by detecting a label on the probe or indirectly by contacting the probe with a moiety that binds the probe. In the case of detecting a polynucleotide polymorphism the solid support is generally made of nitrocellulose or nylon. In the case of a protein polymorphism the method may be based on an ELISA system.

The present methods may be based on an oligonucleotide ligation assay in which two oligonucleotide probes are used. These probes bind to adjacent areas on the polynucleotide which contains the polymorphism, allowing (after binding) the two probes to be ligated together by an appropriate ligase enzyme. However the two probes will only bind (in a manner which allows ligation) to a polynucleotide that contains the polymorphism, and therefore the detection of the ligated product may be used to determine the presence of the polymorphism.

In one embodiment the probe is used in a heteroduplex analysis based system to detect polymorphisms. In such a system when the probe is bound to a polynucleotide sequence containing the polymorphism it forms a heteroduplex at the site where the polymorphism occurs (i.e. it does not form a double strand structure). Such a heteroduplex structure can be detected by the use of an enzyme that is single or double strand specific. Typically the probe is an RNA probe and the enzyme used is RNAse H that cleaves the heteroduplex region, thus allowing the polymorphism to be detected by means of the detection of the cleavage products.

The method may be based on fluorescent chemical cleavage mismatch analysis which is described for example in PCR Methods and Applications 3:268-71 (1994) and Proc. Natl. Acad. Sci. 85:4397-4401 (1998).

In one embodiment the polynucleotide agent is able to act as a primer for a PCR reaction only if it binds a polynucleotide containing the polymorphism (i.e. a sequence—or allele-specific PCR system). Thus a PCR product will only be produced if the polymorphism is present in the polynucleotide of the individual. Thus the presence of the polymorphism may be determined by the detection of the PCR product. Preferably, the region of the primer which is complementary to the polymorphism is at or near the 3′ end the primer. In one embodiment of this system the polynucleotide the agent will bind to the wild-type sequence but will not act as a primer for a PCR reaction.

The method may be an Restriction Fragment Length Polymorphism (RFLP) based system. This can be used if the presence of the polymorphism in the polynucleotide creates or destroys a restriction site that is recognized by a restriction enzyme. Thus treatment of a polynucleotide with such a polymorphism will lead to different products being produced compared to the corresponding wild-type sequence. Thus the detection of the presence of particular restriction digest products can be used to determine the presence of the polymorphism.

The presence of the polymorphism may be determined based on the change that the presence of the polymorphism makes to the mobility of the polynucleotide or protein during gel electrophoresis. In the case of a polynucleotide single-stranded conformation polymorphism (SSCP) analysis may be used. This measures the mobility of the single stranded polynucleotide on a denaturing gel compared to the corresponding wild-type polynucleotide, the detection of a difference in mobility indicating the presence of the polymorphism. Denaturing gradient gel electrophoresis (DGGE) is a similar system where the polynucleotide is electrophoresed through a gel with a denaturing gradient, a difference in mobility compared to the corresponding wild-type polynucleotide indicating the presence of the polymorphism.

The presence of the polymorphism may be determined using a fluorescent dye and quenching agent-based PCR assay such as the Taqman PCR detection system. In brief, this assay uses an allele specific primer comprising the sequence around, and including, the polymorphism. The specific primer is labeled with a fluorescent dye at its 5′ end, a quenching agent at its 3′ end and a 3′ phosphate group preventing the addition of nucleotides to it. Normally the fluorescence of the dye is quenched by the quenching agent present in the same primer. The allele specific primer is used in conjunction with a second primer capable of hybridizing to either allele 5′ of the polymorphism.

In the assay, when the allele comprising the polymorphism is present Taq DNA polymerase adds nucleotides to the nonspecific primer until it reaches the specific primer. It then releases polynucleotides, the fluorescent dye and quenching agent from the specific primer through its endonuclease activity. The fluorescent dye is therefore no longer in proximity to the quenching agent and fluoresces. In the presence of the allele which does not comprise the polymorphism the mismatch between the specific primer and template inhibits the endonuclease activity of Taq and the fluorescent dye in not released from the quenching agent. Therefore by measuring the fluorescence emitted the presence or absence of the polymorphism can be determined.

In another method of detecting the polymorphism a polynucleotide comprising the polymorphic region is sequenced across the region which contains the polymorphism to determine the presence of the polymorphism.

Accordingly, any of the following techniques may be utilized in the present methods for genotyping, as is known in the art.

The present invention also provides for a predictive (patient care) test or test kit. This predictive test could be a product and/or a service which aids in disease management of asthma based on pre-determined associations between genotype and phenotypic response to leukotriene receptor antagonists in treating asthma. Such a test could take two different formats:

A) a molecular test which analyses DNA or RNA for the presence of pre-determined polymorphisms. An appropriate test kit may include one or more of the following reagents or instruments: a means to detect the binding of the agent to the polymorphism, an enzyme able to act on a polynucleotide (typically a polymerase or restriction enzyme), suitable buffers for enzyme reagents, PCR primers which bind to regions flanking the polymorphism, a positive or negative control (or both), a gel electrophoresis apparatus and a means to isolate DNA from a sample. The product may utilise one of the chip technologies as described by the current state of the art. The test kit would include printed or machine readable instructions setting forth the correlation between the presence of a specific polymorphism or genotype and the likelihood that a subject with asthma will respond favorably to therapy with a leukotriene receptor antagonist; or

B) a biochemical test which analyses materials derived from the subject's body, including proteins or metabolites, that indicate the presence of a pre-determined polymorphism. An appropriate test kit would comprise a molecule, aptamer, peptide or antibody (including an antibody fragment) that specifically binds to a predetermined polymorphic region (or a specific region flanking the polymorphism), or a binding agent as defined herein. The product may additionally comprise one or more additional reagents or instruments (as are known in the art). The test kit would also include printed or machine-readable instructions setting forth the correlation between the presence of a specific polymorphism or genotype and the likelihood that a subject with asthma will respond favorably to therapy with a leukotriene receptor antagonist.

The invention provides a method for screening a subject diagnosed with asthma potentially treatable by leukotriene receptor antagonists, to determine the likelihood they will respond in a particular way to treatment with such a drug, more particularly a CysLT1 leukotriene receptor antagonist and most particularly zafirlukast. The method comprises screening the subject for a polymorphism in the ALOX5 gene and/or the LTC4S gene that has previously been associated with a high or low incidence of a particular desirable therapeutic outcome (compared to the incidence in subjects with other genotypes). Subjects are mammalian, and preferably humans.

Treatment of a subject with a leukotriene receptor antagonist comprises administration of an effective amount of the pharmaceutical agent to a subject in need thereof. The dose of agent is determined according to methods known and accepted in the pharmaceutical arts, and can be determined by those skilled in the art. A suitable dosage range for zafirlukast are provided in the disclosure of the Physician's Desk Reference, the entire disclosure of which is hereby incorporated herein by reference.

Genetic testing (also called genetic screening or genotyping) can be defined broadly as analyzing the nucleic acid of a subject to determine if the subject carries mutations (or alleles or polymorphisms) that are either (a) associated with, or causative of, a particular clinical phenotype, or (b) that are in 'linkage disequilibrium' with a mutation, allele or polymorphism that is associated with or causative of a particular clinical phenotype. One such clinical phenotype is the likelihood that the subject will respond favorably to a given therapeutic treatment.

Linkage disequilibrium refers to the tendency of specific alleles to occur together more frequently than would be expected by chance. Alleles at given loci are in equilibrium if the frequency of any particular set of alleles (or haplotype) is the product of their individual population frequencies. Disequilibrium may be due to various forces, including selection for certain allele combinations, or a recent mixing of genetically heterogeneous populations. Where markers link tightley to a disease-causing gene, an association of an allele (or a group of linked alleles) with the disease gene is expected if the disease mutation occurred in the recent historical past, so that sufficient time has not elapsed for equilibrium to be achieved through recombination events in the immediate chromosomal region.
 

Claim 1 of 6 Claims

1. A method of screening a subject suffering from asthma, as an aid in predicting their response to treatment with a leukotriene receptor antagonist ligand, comprising:

a) obtaining a sample of DNA from the subject; and

b) genotyping said DNA sample in the 5′ non-coding region of the LTC₄ Synthase (LTC4S) gene for the presence of SEQ ID NO:3 or SEQ ID NO:4;

wherein homozygosity for an allele comprising SEQ ID NO:4 indicates that the subject is less likely to respond favorably to treatment with a leukotriene receptor antagonist for asthma, compared to a subject having an allele comprising SEQ ID NO:3.

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