Methods of assessing the risk of clinical signs of hypersensitivity reaction to nucleoside antiviral compounds, including abacavir, are described. The methods include genotyping subjects for polymorphisms in the TNF.alpha. gene, the class 1 HLA genes, or a combination of both the TNF.alpha. and HLA genes.

Description of the Invention

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of identifying genotypes that confer a increased or decreased risk for a hypersensitivity reaction to abacavir in human subjects. In a population of test subjects, each subject is genotyped for polymorphisms in a candidate gene, such as the TNFalpha (TNF.alpha.) gene, MICA, MICB, and/or HLA genes. A therapeutic regime of abacavir is administered to each subject (either prior to, concomitant with, or after genotyping of the subject), and test subjects that exhibit (or exhibited) clinical signs of a hypersensitivity reaction to abacavir are identified. The genotypes of the test subjects at polymorphic sites in the candidate genes are correlated with the occurrence of clinical signs of hypersensitivity reaction, to determine which genotypes are associated with an increased or decreased risk of hypersensitivity reaction (compared to other genotypes or to a general population that has not been stratified by genotype).

A further aspect of the present invention is a method of determining whether an individual is at increased risk of experiencing a hypersensitivity reaction to abacavir, by determining whether the individual has a genotype that is associated with an increased risk of hypersensitivity reaction, compared to the risk in subjects with alternate genotypes.

A further aspect of the present invention is a method of determining whether an individual is at decreased risk of experiencing a hypersensitivity reaction to abacavir, by determining whether the individual has a genotype that is associated with a decreased risk of hypersensitivity reaction, compared to the risk in subjects with alternate genotypes.

A further aspect of the present invention is a method of screening a human subject as an aid in assessing suitability to abacavir administration, by determining whether the subject has a TNF.alpha. genotype that has been associated with an increased risk of hypersensitivity reaction to abacavir compared to the risk in subjects with alternate TNF.alpha. genotypes. The presence of such a TNF.alpha. genotype indicates the subject is at increased risk for a hypersensitivity reaction to abacavir.

A further aspect of the present invention is a method of screening a human subject as an aid in assessing suitability to abacavir administration, by determining whether the subject has an HLA genotype that has been associated with an increased risk of hypersensitivity reaction to abacavir compared to the risk in subjects with alternate HLA genotypes. The presence of such an HLA genotype indicates the subject is at increased risk for hypersensitivity reaction to abacavir.

A further aspect of the present invention is a method of treating a human subject with abacavir, by first genotyping the subject to detect the presence or absence of the HLA-B57 allele, and then administering abacavir if the HLA-B57 allele is not detected.

A further aspect of the present invention is a method of screening a human subject as an aid in predicting the subject's risk of experiencing a hypersensitivity reaction to a therapeutic regime of abacavir, by genotyping a sample of DNA from the subject to determine the presence of a polymorphism in the TNF.alpha. gene, where the polymorphism has previously been associated with an increased risk of abacavir HSR compared to the risk of HSR associated with alternate TNF.alpha. polymorphisms. Detecting the presence of a TNF.alpha. genotype that has been associated with an increased incidence of hypersensitivity reaction to abacavir (compared to the incidence of abacavir HSR associated with other TNF.alpha. genotypes) indicates that the subject is at an increased risk of a hypersensitivity reaction to abacavir.

A further aspect of the present invention is a method of screening a human subject as an aid in predicting the subject's risk of experiencing a hypersensitivity reaction to a therapeutic regime of abacavir, by genotyping a sample of DNA from the subject to determine the presence of a polymorphism in an HLA gene, where the polymorphism has previously been associated with an increased risk of abacavir HSR compared to the risk of HSR associated with alternate polymorphisms. The presence of an HLA genotype that has been associated with an increased incidence of hypersensitivity reaction to abacavir (compared to the incidence of abacavir HSR associated with other HLA genotypes) indicates that the subject is at an increased risk of a hypersensitivity reaction to abacavir.

A further aspect of the present invention is a method of identifying human genotypes associated with an increased risk for a hypersensitivity reaction to abacavir, by genotyping each member of a population of test subjects for at least one polymorphism in the TNF.alpha. gene, administering a therapeutic regime of abacavir to each test subject, and identifying test subjects that exhibit clinical signs of a hypersensitivity reaction to abacavir. Correlating TNF.alpha. genotypes with the occurrence of clinical signs of hypersensitivity reaction, will determine which genotypes are associated with an increased risk of hypersensitivity reaction to abacavir (compared to the other detected genotypes).

A further aspect of the present invention is a method of identifying human genotypes associated with an increased risk for a hypersensitivity reaction to abacavir, by genotyping each member of a population of test subjects for at least one polymorphism in an HLA gene, administering a therapeutic regime of abacavir to each test subject, and identifying test subjects that exhibit clinical signs of a hypersensitivity reaction to abacavir. Correlating HLA genotypes with the occurrence of clinical signs of hypersensitivity reaction, will determine which genotypes are associated with an increased risk of hypersensitivity reaction to abacavir (compared to the other detected genotypes).

A further aspect of the present invention is a method of administering or prescribing abacavir to reduce the incidence of abacavir hypersensitivity reaction. The method comprises selecting, based on genotype status, a treatment population from a larger starting population of subjects who have a condition suitable for treatment with abacavir. The treatment population is selected to increase the percentage of subjects in the treatment population who have a genotype that has been associated with reduced risk of abacavir hypersensitivity reaction (the increased percentage of subjects in the treatment population is relative to the percentage of subjects in the starting population). Alternatively, the treatment population is selected to decrease the percentage of subjects in the treatment population who have a genotype that has been associated with increased risk of abacavir hypersensitivity reaction. Abacavir is then administered to the selected treatment population, thereby reducing the incidence of abacavir HSR in the treated population compared to the incidence that would have been expected to occur had abacavir been administered to the larger starting population. The `selection` may occur by any suitable process as will be apparent to those skilled in the art. Examples of suitable selection methods include genetically screening starting population subjects, or otherwise classifying subjects by genotype (e.g., where a subject's genotype is known, genetic testing need not be repeated); or otherwise regulating access to abacavir to decrease the number of subjects in the treatment population who have genotypes that have been associated with an increased risk of abacavir HSR. One such genotype is the HLA-B57 allele, where the treatment population would be selected to minimize the occurrence of the HLA-B57 allele in the treatment population. Alternatively, the genotype of interest may be the TNF G(-237)A polymorphism, where the treatment population is selected to minimize the occurrence of the A allele.

DETAILED DISCUSSION

Anti-retroviral therapy in HIV-infected patients often comprises the use of multiple types of antiretroviral agents, including protease inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTI) and nucleoside reverse transcriptase inhibitors (NRTI). Abacavir is a synthetic purine nucleoside analogue that is commercially available as abacavir sulfate (ZIAGEN.RTM.; GlaxoSmithKline), and that is used in combination with other antiretroviral agents to treat HIV-infected subjects. Abacavir is an inhibitor (NRTI) of the HIV-1 reverse transcriptase that contains an unsaturated cyclopentene ring in place of the 2'deoxyriboside of natural deoxynucleosides, and contains a cyclopropylamino group. The chemical name of abacavir sulfate is (cis)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-met- hanol sulfate (salt) (2:1).

Hypersensitivity reactions are idiosyncratic events of a presumed immunologic nature that occur with a broad range of pharmacological compounds. In the context of abacavir administration, hypersensitivity reactions to abacavir can be serious and progress to become life-threatening (Clay et al., Ann Pharmacotherapy 34(2):247 (2000); Staszewski et al., AIDS 12:F197 (1998)). In clinical trials, hypersensitivity to abacavir has occurred among approximately 5% of subjects.

Signs and symptoms of a hypersensitivity reaction to abacavir include (but are not limited to) fever, skin rash, fatigue, gastrointestinal symptoms (including nausea, vomiting, diarrhea, abdominal pain), and respiratory symptoms (including pharyngitis, dyspnea and cough). Additional signs and symptoms include malaise, lethargy, myalgia, myolysis, arthralgia, edema, headache and paresthesia. Physical findings include lymphadenopathy, mucous membrane lesions (conjunctivitis and mouth ulcerations). The rash associated with hypersensitivity reaction usually appears maculopapular or urticarial, but the appearance may be variable; up to 30% of hypersensitivity reactions have occurred without rash. Laboratory abnormalities include elevated liver function tests, increased creatine phosphokinase or creatinine, and lymphopenia. See Package Insert, Ziagen (abacavir sulfate), Glaxo Wellcome, Research Triangle Park, N.C. (1998); Clay et al., Management Protocol for Abacavir-related Hypersensitivity Reaction, Ann. Pharmacotherapy 34(2):247 (2000). Clay et al. state that the presence of rash alone does not warrant discontinuation of abacavir unless other systemic symptoms of hypersensitivity reaction occur.

TNFalpha

The immunologic effector molecule Tumor Necrosis Factor alpha (TNF.alpha.) is known to be polymorphic, and a number of polymorphisms have been reported in the TNF.alpha. promoter region. Some reports indicate that such promoter polymorphisms influence immunologic disease (Bouma et al., Scand. J. Immunol. 43:456 (1996); Allen et al., Mol. Immunology 36:1017 (1999)), whereas others suggest that observed associations between TNF.alpha. polymorphisms and disease occurrence are not due to functional effects of TNF.alpha., but due to the linkage disequilibrium of TNF.alpha. with selectable HLA alleles (Uglialoro et al., Tissue Antigens, 52:359 (1998)). A list of TNF.alpha. promoter polymorphisms is provided by Allen et al., Mol. Immunology 36:1017 (1999). The numbering of TNF.alpha. polymorphisms has varied among authors due to the variation in sequences reported for TNF.alpha. promoter region; numbering herein refers to the following consensus sequence provided in Allen et al. (1999) -- see Original Patent.

The transcription start site (+1) is indicated by bold underlined type; the G(-237)A and G(-308)A polymorphisms are indicated by bold, double underlined type. Due to variation in reported sequences and numbering, the G(-237)A polymorphism has also been referred to as G-238A, and the G(-308)A polymorphism is located at the -307 position on the above sequence. A further polymorphism, C(-5,100)G, investigated in the present research was an C/G polymorphism in the 5' untranslated region of TNF.alpha. -- see Original Patent.

Allen et al. (supra) note that a number of the TNF.alpha. promoter polymorphisms observed to date are G/A polymorphisms clustered in the region of -375 to -162 bp; that some of these polymorphisms lie within a common motif; and suggest that the motif could be a consensus binding site for a transcriptional regulator or might influence DNA structure. The G/A polymorphism at -237 has been reported to affect DNA curvature (D'Alfonso et al., Immunogenetics 39:150 (1994)). Huizinga et al. (J. Neuroimmunology 72:149, 1997) reported significantly less TNF.alpha. production by LPS-stimulated cells from individuals heterozygous (G/A) at -237 (compared to G/G individuals); however, a separate study did not observe these effects (Pociot et al., Scand. J. Immunol. 42:501, 1995). The G(-237)A polymorphism has also been reported to affect autoimmune disease (Brinkman et al., Br. J. Rheumatol. 36:516 1997 (rheumatoid arthritis); Huizinga et al., J. Neuroimmunology 72:149 1997 (multiple sclerosis); Vinasco et al., Tissue Antigens, 49:74 1997 (rheumatoid arthritis)) and infectious disease (Hohler et al., Clin. Exp. Immunol. 111:579 1998 (hepatitis B); Hohler et al., J. Med. Virol. 54:173 1998 (hepatitis c)).

As is well known genetics, nucleotide and amino acid sequences obtained from different sources for the same gene may vary both in the numbering scheme and in the precise sequence. Such differences may be due to inherent sequence variability within the gene and/or to sequencing errors. Accordingly, reference herein to a particular polymorphic site by number (e.g., TNF.alpha. G-238A) will be understood by those of skill in the art to include those polymorphic sites that correspond in sequence and location within the gene, even where different numbering/nomenclature schemes are used to describe them.

HLA

The HLA complex of humans (major histocompatibility complex or MHC) is a cluster of linked genes located on chromosome 6. (The TNF.alpha. and HLA B loci are in proximity on chromosome 6). The HLA complex is classically divided into three regions: class I, II, and III regions (Klein J. In: Gotze D, ed. The Major Histocompatibility System in Man and Animals, New York: Springer-Verlag, 1976: 339 378). Class I HLAs comprise the transmembrane protein (heavy chain) and a molecule of beta-2 microglobulin. The class I transmembrane proteins are encoded by the HLA-A, HLA-B and HLA-C loci. The function of class I HLA molecules is to present antigenic peptides (including viral protein antigens) to T cells. Three isoforms of class II MHC molecules, denoted HLA-DR, -DQ, and -DP are recognized. The MHC class II molecules are heterodimers composed of an alpha chain and a beta chain; different alpha- and beta-chains are encoded by subsets of A genes and B genes, respectively. Various HLA-DR haplotypes have been recognized, and differ in the organization and number of DRB genes present on each DR haplotype; multiple DRB genes have been described. Bodmer et al., Eur. J. Immunogenetics 24:105 (1997); Andersson, Frontiers in Bioscience 3:739 (1998).

The MHC exhibits high polymorphism; more than 200 genotypical alleles of HLA-B have been reported. See e.g., Schreuder et al., Human Immunology 60: 1157 1181 (1999); Bodmer et al., European Journal of Immunogenetics 26: 81 116 (1999). Despite the number of alleles at the HLA-A, HLA-B and HLA-C loci, the number of haplotypes observed in populations is smaller than mathematically expected. Certain alleles tend to occur together on the same haplotype, rather than randomly segregating. This is called linkage disequilibrium (LD) and may be quantitated by methods as are known in the art (see, e.g., Devlin and Risch, Genomics 29:311 (1995); BS Weir, Genetic Data Analysis II, Sinauer Associates, Sunderland, Md. (1996)).

The products encoded by the polymorphic HLA loci are commonly typed by serological methods for transplant and transfusion histocompatibility testing, and blood component therapy. Serological typing is based on reactions between characterized sera and the HLA gene products. Known techniques for histocompatibility testing include microlymphocytotoxicity and flow cytometry. Standard microlymphocytotoxicity for HLA antigen typing determines the HLA antigen profile of a subject's lymphocytes, using a panel of well characterized HLA antisera. The HLA-B57 allele is well characterized, and serologic methods of detecting HLA-B57 are known. See e.g., ASHI Laboratory Manual, Fourth Edition, American Society for Histocompatibility and Immunogenetics (2000); Hurley et al., Tissue Antigens 50:401 (1997).

More recently, methods for analysis of HLA polymorphisms at the genetic level have been developed. Non-serological HLA typing methods include the use of DNA restriction fragment length polymorphism (RFLP; see e.g., Erlich U.S. Pat. No. 4,582,788 (1986)), or labelled oligonucleotides, to identify specific HLA DNA sequences. Such methods may detect polymorphisms located in either the coding or noncoding sequence of the genome. See e.g., Bidwell et al, Immunology Today 9:18 (1988), Angelini et al., Proc. Natl. Acad. Sci. USA, 83:4489 (1986); Scharf et al., Science, 233:1076 (1986); Cox et al., Am. J. Hum. Gen., 43:954 (1988); Tiercy et al., Proc. Natl. Acad. Sci. USA 85:198 (1988); and Tiercy et al., Hum. Immunol. 24:1 (1989). The polymerase chain reaction (PCR) process (see U.S. Pat. No. 4,683,202, 1987) allows amplification of genomic DNA and is now used for HLA typing procedures. See Saiki et al., Nature 324:163 (1986); Bugawan et al., J. Immunol. 141:4024 (1988); Gyllensten et al., Proc. Natl. Acad. Sci. USA, 85:7652 (1988). See also e.g., Ennis et al., PNAS USA 87:2833 (1990); Petersdorf et al., Tissue Antigens 46: 77 (1995); Girdlestone et al., Nucleic Acids Research 18:6702 (1990); Marcos et al., Tissue Antigens 50:665 (1997); Steiner et al., Tissue Antigens 57:481 (2001); Madrigal et al., J. Immunology 149:3411 (1992).

As used herein, `genotyping` an HLA locus refers to methods that identify the presence or absence of a particular allele, or nucleic acid or amino acid sequence; sequence variations may be detected directly (by sequencing) or indirectly (e.g., by restriction fragment length polymorphism analysis, or detection of the hybridization of a probe of known sequence, or reference strand conformation polymorphism). HLA alleles may be detected serologically, as is known in the art.

Distinct HLA alleles have been associated with an increased or decreased risk of progression of HIV disease. The HLA-B57 and HLA-B14 alleles have been associated with non-progressive HIV infection, whereas HLA-A29 and HLA-B22 have been associated with rapid progression. Goulder et al., J. Virology 74:5291 (2000); Hendel et al., J. Immunology 162:6942 (1999). Carrington et al., reported that the allele frequency of HLA-B57 in HIV infected patient cohorts is 4.40% in Caucasians and 5.7% in African Americans. Carrington et al., Science, 283:1748 (1999).

MICA and MICB

The MHC (HLA) class I chain-related gene A (MICA) and MHC (HLA) class I chain-related gene B (MICB) belong to a multicopy gene family located in the major histocompatibility complex (MHC) class I region near the HLA-B gene. They are located within a linkage region on chromosome 6p around HLA-B and TNFalpha. The encoded MHC class I molecules are induced by stress factors such as infection and heat shock, and are expressed on gastrointestinal epithelium.

MICA is reported as highly polymorphic. The occurrence of MICA single nucleotide polymorphisms in various ethnic groups is reported by Powell et al., Mutation Research 432:47 (2001). Polymorphisms in MICA have been reported to be associated with various diseases, although in some cases the association was attributable to linkage disequilibrium with HLA genes. See, e.g., Salvarani et al., J Rheumatol 28:1867 (2001); Gonzalez et al., Hum Immunol 62:632 (2001); Seki et al., Tissue Antigens 58:71 (2001).

Various polymorphic forms of MICB have been reported (see, e.g., Visser et al., Tissue Antigens 51:649 (1998); Kimura et al., Hum Immunol 59:500 (1998); Ando et al., Immunogenetics 46:499 (1997); Fischer et al., Eur J Immunogenet 26:399 (1999)).

A partial sequence for homo sapiens MICA gene, including exons 2 and 3, is provided below (GenBank reference AJ295250).

Various MICA polymorphisms were investigated in the present study. The MICA polymorphisms in exon 2 (T/G; rs1063630 in the National Center for Biotechnology Information SNP database (dbSNP)) and exon 3 (A/G; rs1051792) are shown above in bold, double-underlined type. An additional MICA polymorphism investigated in the present study (rs1052416) was located approximately -9,263 bases 5' to the transcription start site -- see Original Patent.

A complete cds for the human MICB gene is provided at SEQ ID NO:4 (GenBank accession U65416). The MICB polymorphisms investigated in the present study included one in exon 2 (rs1065075) and one in exon 3 (rs1051788) -- see Original Patent.

ATP Dependent RNA Helicase p47

The protein encoded by this gene is a member a family of ATP-dependent RNA helicases, and is also known as HLA-B associated transcript 1 (BAT1) (see, e.g., GenBank Accession No. AF029061). A cluster of genes known as BAT1-BAT5 has been localized near the TNF and TNF genes. Various polymorphisms have been identified in ATP dependent RNA Helicase p47, including -- see Original Patent.

DEFINITION:

As used herein, the process of detecting an allele or polymorphism includes but is not limited to serologic and genetic methods. The allele or polymorphism detected may be functionally involved in affecting an individual's phenotype, or it may be an allele or polymorphism that is in linkage disequilibrium with a functional polymorphism/allele. Polymorphisms/alleles are evidenced in the genomic DNA of a subject, but may also be detectable from RNA, cDNA or protein sequences transcribed or translated from this region, as will be apparent to one skilled in the art.

Alleles, polymorphisms or genetic markers that are `associated` with HSR to a NRTI such as abacavir are over-represented in frequency in treated subjects experiencing HSR as compared to treated subjects who do not experience HSR, or as compared to the general population.

According to the present methods, subjects who are being treated with abacavir, or who are considering treatment with abacavir, can be screened as an aid in predicting their risk of experiencing a hypersensitivity reaction to abacavir. Screening comprises obtaining a biological sample from the subject and analyzing it to determine the genotype of the TNF.alpha., and/or HLA genes, i.e., to determine the presence or absence of polymorphisms in one or both of these genes that are associated with an increased risk of abacavir HSR (compared to the risk associated with alternative polymorphisms).

The present inventors have established that a correlation exists between an individual's HLA genotype (particularly class I, and more particularly HLA-B), and/or TNF.alpha. genotype, and the risk of experiencing a hypersensitivity reaction to abacavir administration. Accordingly, a method of assessing an individual's relative risk of an abacavir HSR involves genotyping that individual at the TNF.alpha. gene or the HLA genes to determine whether the individual's genotype places them at increased risk of abacavir HSR. Individuals possessing a TNF.alpha. or HLA genotype that has been previously associated with an increased incidence of abacavir HSR (compared to the incidence of HSR in subjects with alternate genotypes) are at increased risk of HSR.

The present screening methods comprise genotyping a subject at HLA genes, particularly the HLA class I genes, more particularly the HLA-B gene, including to detect the presence or absence of the HLA-B57 allele (as defined herein).

The present screening methods also comprise genotyping a subject at the TNF.alpha. gene, and more particularly, detecting the genotype at the TNF.alpha. G(-237)A polymorphic site (as defined herein), where detection of an A allele indicates increased risk of hypersensitivity reaction, compared to detection of a G/G genotype.

In view of the present disclosure, it will be apparent to one skilled in the art how to determine additional TNF.alpha. and/or HLA genotypes that are associated with an increased risk of abacavir HSR. Various allelic forms of the TNF.alpha. and HLA genes are known, and methods of typing the TNF.alpha. and HLA genes are known in the art. As additional polymorphisms are detected in human TNF.alpha. and HLA genes, typing for such polymorphisms may be based on known methods. Accordingly, one may type a population of subjects who have received abacavir and correlate TNF.alpha. and/or HLA genotype with the occurrence of HSR. In an alternate method, one may genotype only those subjects who have experienced HSR and, where the prevalence of a TNF.alpha. or HLA allele is known in a matched control (non-HSR) population, determine whether the allele is over-represented in the HSR population, indicating that it is associated with HSR. As will be apparent to one skilled in the art, the detection of a TNF.alpha. or HLA allele may be accomplished by typing for genetic markers that are known to be in linkage disequilibrium with the target TNF.alpha. or HLA allele/polymorphism. Preferably such markers are in substantial linkage disequilibrium, more preferably the markers are in complete linkage disequilibrium.

The present invention also provides a method of assessing an individual's relative risk of experiencing HSR to abacavir by determining the genotype at both the TNF.alpha. and HLA genes, to determine whether the individual's genotype places them at increased risk of abacavir HSR. Those individuals possessing a combined TNF.alpha./HLA genotype that is associated with an increased incidence of abacavir HSR (compared to the incidence of HSR in subjects with alternate genotypes) are at increased risk of HSR. In particular, the present methods may comprise detecting the allelic form of the TNF.alpha. G(-237)A polymorphism and the presence or absence of the HLA-B57 allele (and/or markers in linkage disequilibrium with these).

It will be apparent to those skilled in the art that, as multiple TNF.alpha. and HLA genotypes exist, the relative risk of abacavir HSR may vary among the multiple genotypes. E.g., in a multilocus screening method where more than two genotypes are found, relative risk may be determined to be highest for one genotype, lowest for another, and intermediate in others. `Increased risk` may be as compared to the risk in a population that has not been stratified by genotype (a general population), or increased as compared to the risk expected in another defined genotype.

The presence of a particular predetermined genotype that is associated with an increased risk of HSR therefore indicates an increased likelihood that the individual will exhibit the associated phenotype (HSR reaction) relative to subjects with alternate genotypes. The genotype will rarely be absolutely predictive, i.e., where a population with a certain genotype displays a high incidence of an associated phenotype, not every individual with that genotype will display the phenotype. Likewise, some individuals with a different genotype may display the same 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 a subject's risk of HSR to treatment with abacavir, and thus assist in treatment decisions. The present methods may further comprise administering abacavir to subjects after screening in subjects where the risk of HSR is deemed acceptable; the final treatment decision will be based on factors in addition to genetic testing (as will be readily apparent to one skilled in the art), including the subject's overall health status and expected treatment outcome.

It will be apparent to those skilled in the art that the present methods are also applicable where hypersensitivity reactions occur in response to synthetic nucleoside analogs other than abacavir, and particularly NRTIs. In particular, such compounds include purine nucleoside analogs, purine nucleoside analogs containing an unsaturated carbon ring in place of the 2'deoxyriboside of natural deoxynucleosides, and purine nucleoside analogs containing an unsaturated cyclopentene ring in place of the 2'deoxyriboside of natural deoxynucleosides. Further, the present methods are applicable where HSR occurs in response to NNRTIs, such as efavirenz (SUSTIVA.TM., Dupont Pharmaceuticals) and nevirapine (VIRAMUNE.RTM., Boerhinger Ingelheim/Roxane).

According to the present methods, a compound (such as an NRTI or NNRTI) may be screened for variation in the incidence of HSR among genetic subpopulations of subjects. Such methods include administering the compound to a population of subjects, obtaining biological samples from the subjects (which may be done either prior to or after administration of the compound), genotyping polymorphic allelic sites in the TNF.alpha. gene and/or the class I HLA genes (particularly the HLA-B gene), and correlating the genotype of the subjects with their phenotypic response (e.g., the absence of hypersensitivity reaction versus the presence of confirmed or suspected hypersensitivity reaction). As will be apparent to those skilled in the art, due to the serious nature of HSR, administration of a pharmaceutical compound may need to be discontinued where a hypersensitivity reaction is suspected due to the presence of rash and/or other symptoms compatible with the clinical syndrome. Correlation of certain genotypes with an increased rate of HSR (where the HSR is either confirmed or clinically suspected), compared to the incidence of HSR in subjects with alternative genotypes, indicates that the incidence of HSR varies among genetic subpopulations.

Stated another way, the methods of the present invention may be used to determine the correlation of a polymorphic allele (such as those in TNF.alpha. and/or HLA alleles), with the incidence of hypersensitivity reaction to a pharmaceutical compound, particularly an NRTI. Subjects are stratified according to genotype and their response to the therapeutic agent is assessed (either prospectively or retrospectively) and compared among the genotypes. In this way, genotypes that are associated with an increased (or decreased) rate of HSR may be identified. The increase or decrease of HSR rates is in comparison to the rates among other genotypes, or to a population as a whole (i.e. the incidence in a population that is not stratified by genotype).

Polymorphic alleles may be detected by determining the DNA polynucleotide sequence, or by detecting the corresponding sequence in RNA transcripts from the polymorphic gene, or where the nucleic acid polymorphism results in a change in an encoded protein by detecting such amino acid sequence changes in encoded proteins; using any suitable technique as is known in the art. Polynucleotides utilized for typing are typically genomic DNA, or a polynucleotide fragment derived from a genomic polynucleotide sequence, such as in a library made using genomic material from the individual (e.g. a cDNA library). The polymorphism may be detected in a method that comprises contacting a polynucleotide or protein sample from an 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 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 nucleotides. 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. The binding agent may be a molecule that is structurally similar to polynucleotides that comprises units (such as purine or pyrimidine analogs, peptide nucleic acids, or RNA derivatives such as locked nucleic acids (LNA)) 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, or 100 or more amino acids. The agent may be an antibody (including a fragment of such an antibody that is capable of binding the polymorphism).

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 (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, and the presence of the polymorphism is 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 a 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 that has 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.TM. PCR detection system. 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.

Various other detection techniques suitable for use in the present methods will be apparent to those conversant with methods of detecting, identifying, and/or distinguishing polymorphisms. Such detection techniques include but are not limited to direct sequencing, use of "molecular beacons" (oligonucleotide probes that fluoresce upon hybridization, useful in real-time fluorescence PCR; see e.g., Marras et al., Genet Anal 14:151 (1999)); electrochemical detection (reduction or oxidation of DNA bases or sugars; see U.S. Pat. No. 5,871,918 to Thorp et al.); rolling circle amplification (see, e.g., Gusev et al., Am J Pathol 159:63 (2001)); Third Wave Technologies (Madison Wis.) INVADER.RTM. non-PCR based detection method (see, e.g., Lieder, Advance for Laboratory Managers, 70 (2000))

Accordingly, any suitable detection technique as is known in the art may be utilized in the present methods.

As used herein, "determining" a subject's genotype does not require that a genotyping technique be carried out where a subject has previously been genotyped and the results of the previous genetic test are available; determining a subject's genotype accordingly includes referring to previously completed genetic analyses.

The present invention also provides for a predictive (patient care) test or test kit. Such a test will aid in the therapeutic use of pharmaceutical compounds, including NRTIs, such as abacavir, based on pre-determined associations between genotype and phenotypic response to the therapeutic compound. Such a test may take different formats, including:

(a) a test which analyzes DNA or RNA for the presence of pre-determined alleles and/or polymorphisms. An appropriate test kit may include one or more of the following reagents or instruments: 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), and a gel electrophoresis apparatus. The product may utilise one of the chip technologies as described by the state of the art. The test kit would include printed or machine readable instructions setting forth the correlation between the presence of a specific genotype and the likelihood that a subject treated with a specific pharmaceutical compound will experience a hypersensitivity reaction;

(b) a test which analyses materials derived from the subject's body, such as proteins or metabolites, that indicate the presence of a pre-determined polymorphism or allele. An appropriate test kit may 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). The kit 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 treated with a specific synthetic nucleoside analog will experience a hypersensitivity reaction.

Suitable biological specimens for testing are those which comprise cells and DNA and include, but are not limited to blood or blood components, dried blood spots, urine, buccal swabs and saliva. Suitable samples for HLA serologic testing are well known in the art.
 

Claim 1 of 8 Claims

1. A method of identifying a male Caucasian human subject at increased risk of experiencing a hypersensitivity reaction to a therapeutic regime of abacavir, comprising: (a) performing a genotyping technique on a biological sample from said subject to determine whether the subject's HLA-B genotype includes an allele selected from the HLA-B57 allele and the HLA-B*5701 allele; (b) detecting an HLA-B57 allele or an HLA-B*5701 allele; and (c) correlating the detection of an HLA-B57 or an HLA-B*5701 allele with an increased risk of experiencing a hypersensitivity reaction to a therapeutic regime of abacavir compared to the risk if no HLA-B57 or HLA-B*5701 allele were detected.

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