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WO2002038586A2 - Haplotypes du gene il6 - Google Patents

Haplotypes du gene il6 Download PDF

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Publication number
WO2002038586A2
WO2002038586A2 PCT/US2001/047077 US0147077W WO0238586A2 WO 2002038586 A2 WO2002038586 A2 WO 2002038586A2 US 0147077 W US0147077 W US 0147077W WO 0238586 A2 WO0238586 A2 WO 0238586A2
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WIPO (PCT)
Prior art keywords
haplotype
seq
gene
individual
polymoφhic
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PCT/US2001/047077
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English (en)
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WO2002038586A3 (fr
Inventor
Steven C. Bentivegna
Karyn M. Bieglecki
Anne Chew
R. Rex Denton
Michael Lachowicz
Krishnan Nandabalan
Katie E. Parks
Elizabeth Ann Sausker
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Cogenics Inc
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Genaissance Pharmaceuticals Inc
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Priority to AU2002228871A priority Critical patent/AU2002228871A1/en
Publication of WO2002038586A2 publication Critical patent/WO2002038586A2/fr
Publication of WO2002038586A3 publication Critical patent/WO2002038586A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to variation in genes that encode pharmaceutically-important proteins.
  • this invention provides genetic variants of the human Interleukin-6 (TL6) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.
  • TL6 human Interleukin-6
  • Cunent methods for identifying pharmaceuticals to treat disease often start by identifying, cloning, and expressing an important 'target protein related to the disease. A determination of whether an agonist or antagonist is needed to produce an effect that may benefit a patient with the disease is then made. Then, vast numbers of compounds are screened against the target protein to find new potential drags. The desired outcome of this process is a lead compound that is specific for the target, thereby reducing the incidence of the undesired side effects usually caused by activity at non-intended targets. The lead compound identified in this screening process then undergoes further in vitro and in vivo testing to determine its absorption, disposition, metabolism and toxicological profiles. Typically, this testing involves use of cell lines and animal models with limited, if any, genetic diversity.
  • Tyrosine kinases belonging to the Janus kinase (JAK) family are activated by the binding of IL6 to their receptors. This results in the activation of members of the signal transducers and activators of transcription (STAT) family (Berenson et al, Semin. Cancer Biol 2000; 10:383-391).
  • STAT3 is implicated in prevention of apoptosis in myeloma cells. Multiple myeloma results from the accumulation of terminally differentiated B cells in the manow, which ultimately leads to bone destruction, anemia, and renal failure.
  • IL6 activated signaling triggers binding of STAT3 to the BCL2L1 promoter, resulting in increased expression of this anti-apoptotic protein (Berenson et al., supra). IL6 inhibits bone formation and is capable of enhancing osteoclast differentiation, providing evidence of this cytokine 's involvement in bone disease. Furthermore, monoclonal anti-IL6 antibodies in myeloma patients have been shown to reduce hypercalcemia and bone disease (Bataille et al., Blood 1995; 86:685-691).
  • CAD coronary artery disease
  • CAD CAD Relative to controls, individuals with CAD have elevated levels of the inflammatory markers fibrinogen and C-reactive protein (Woods et al., Eur Heart J 2000; 21:1574-1583).
  • the systemic inflammatory response refers to a collection of pathophysiological pathways, amongst which, the acute phase response is most relevant to CAD.
  • Various cytokines are involved in the acute phase response, however, only IL6 is capable of stimulating all of the proteins involved in this phase: C- reactive protein, serum amyloid A, fibrinogen, al-chymotrypsin and haptoglobin (Castell et al., FEBS Lett.
  • this cytokine also contributes to the onset of CAD by increasing aggregability of platelets and inducing the expression of adhesion molecules in the endothelium (Woods et al., supra).
  • IL6 levels are conelated with cardiovascular disease, as well as being predictive of future heart disease (Woods et al., supra).
  • cardiovascular risk factors such as strenuous exercise and psychological stress are believed to contribute to CAD by increasing IL6 levels.
  • these data implicate IL6 as a central component of the inflammatory processes that lead to the development of CAD.
  • Inflammatory cytokines such as IL6, also promote the development of Kaposi sarcoma (KS), an angioproliferative inflammatory condition that occurs commonly in patients infected with HIV.
  • KS Kaposi sarcoma
  • the polymorphism of guanine or cytosine at nucleotide 765 (position - 174 relative to the start codon) in IL6 is associated with altered levels of IL6 expression.
  • the homozygous G allele of this polymorphism is associated with an increased level of IL6 production, and patients with this genotype are more likely to develop KS (Foster et al., Blood 2000; 96:2562- 2567).
  • polymorphic sites conespond to the following nucleotide positions in Figure 1 : 1157 (PS2), 1245 (PS3), 1248 (PS4), 1337 (PS5), 1394 (PS6), 2350 (PS8), 2589 (PS10), 2671 (PS11), 3079 (PS12), 3409 (PS13), 5134 (PS14), 5251 (PS15), 5366 (PS16) and 5391 (PS17).
  • each IL6 haplotype also represents a naturally-occurring isoform (also refened to herein as an "isogene") of the IL6 gene.
  • the frequency of each haplotype and haplotype pair within the total reference population and within each of the four major population groups included in the reference population was also determined.
  • the invention provides a method, composition and kit for genotyping the IL6 gene in an individual.
  • the genotyping method comprises identifying the nucleotide pair that is present at one or more polymorphic sites selected from the group consisting of PS2, PS3, PS4, PS5, PS6, PS8, PS10, PSI 1, PS12, PS13, PS14, PS15, PS16 and PS17 in both copies of the IL6 gene from the individual.
  • a genotyping composition of the invention comprises an oligonucleotide probe or primer which is designed to specifically hybridize to a target region containing, or adjacent to, one of these novel IL6 polymorphic sites.
  • a genotyping kit of the invention comprises a set of oligonucleotides designed to genotype each of these novel IL6 polymorphic sites.
  • the genotyping kit comprises a set of oligonucleotides designed to genotype each of PS1-PS17.
  • the genotyping method, composition, and kit are useful in determining whether an individual has one of the haplotypes in Table 5 below or has one of the haplotype pairs in Table 4 below.
  • the invention also provides a method for haplotyping the IL6 gene in an individual.
  • the haplotyping method comprises determining, for one copy of the IL6 gene, the identity of the nucleotide at one or more polymorphic sites selected from the group consisting of PS2, PS3, PS4, PS5, PS6, PS8, PS10, PSI 1, PS12, PS13, PS14, PS15, PS16 and PS17.
  • the haplotyping method comprises determining whether one copy of the individual's IL6 gene is defined by one of the IL6 haplotypes shown in Table 5, below, or a sub-haplotype thereof.
  • the haplotyping method comprises determining whether both copies of the individual's IL6 gene are defined by one of the IL6 haplotype pairs shown in Table 4 below, or a sub- haplotype pair thereof. Establishing the IL6 haplotype or haplotype pair of an individual is useful for improving the efficiency and reliability of several steps in the discovery and development of drugs for treating diseases associated with IL6 activity, e.g., myeloma, coronary artery disease, arthritis, and Kaposi sarcoma.
  • diseases associated with IL6 activity e.g., myeloma, coronary artery disease, arthritis, and Kaposi sarcoma.
  • variable IL6 activity has little, if any, involvement with that disease.
  • the pharmaceutical research scientist can, without a priori knowledge as to the phenotypic effect of any IL6 haplotype or haplotype pair, apply the information derived from detecting IL6 haplotypes in an individual to decide whether modulating IL6 activity would be useful in treating the disease.
  • Haplotyping the IL6 gene in an individual is also useful in the design of clinical trials of candidate drugs for treating a specific condition or disease predicted to be associated with IL6 activity. For example, instead of randomly assigning patients with the disease of interest to the treatment or control group as is typically done now, determining which of the IL6 haplotype(s) disclosed herein are present in individual patients enables the pharmaceutical scientist to distribute IL6 haplotypes and/or haplotype pairs evenly to treatment and control groups, thereby reducing the potential for bias in the results that could be introduced by a larger frequency of an IL6 haplotype or haplotype pair that is associated with response to the drug being studied in the trial, even if this association was previously unknown. Thus, by practicing the claimed invention, the scientist can more confidently rely on the information learned from the trial, without first determining the phenotypic effect of any IL6 haplotype or haplotype pair.
  • the invention provides a method for identifying an association between a trait and an IL6 genotype, haplotype, or haplotype pair for one or more of the novel polymorphic sites described herein.
  • the method comprises comparing the frequency of the IL6 genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency of the IL6 genotype or haplotype in a reference population.
  • a higher frequency of the IL6 genotype, haplotype, or haplotype pair in the trait population than in the reference population indicates the trait is associated with the IL6 genotype, haplotype, or haplotype pair.
  • the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymorphic variant of a reference sequence for the IL6 gene or a fragment thereof.
  • the reference sequence comprises the contiguous sequences shown in Figure 1 and the polymo ⁇ hic variant comprises at least one polymo ⁇ hism selected from the group consisting of thymine at PS2, adenine at PS3, thymine at PS4, thymine at PS5, adenine at PS6, thymine at PS8, adenine at PS10, cytosine at PSI 1, guanine at PS12, adenine at PS13, adenine at PS14, thymine at PS 15, cytosine at PS 16 and adenine at PS 17.
  • the polymo ⁇ hic variant comprises one or more additional polymo ⁇ hisms selected from the group consisting of cytosine at PSI, thymine at PS7 and thymine at PS9.
  • a particularly prefened polymo ⁇ hic variant is an isogene of the IL6 gene.
  • An IL6 isogene of the invention comprises guanine or cytosine at PSI, guanine or thymine at PS2, cytosine or adenine at PS3, cytosine or thymine at PS4, cytosine or thymine at PS5, guanine or adenine at PS6, cytosine or thymine at PS7, guanine or thymine at PS8, cytosine or thymine at PS9, guanine or adenine at PS 10, guanine or cytosine at PSI 1, thymine or guanine at PS12, guanine or adenine at PS13, thymine or adenine at PS 14, cytosine or thymine at PS 15, thymine or cytosine at PS 16 and guanine or adenine at PS17.
  • the invention also
  • the invention provides a polynucleotide comprising a polymo ⁇ hic variant of a reference sequence for an IL6 cDNA or a fragment thereof.
  • the reference sequence comprises SEQ ID NO:2 (Fig.2) and the polymo ⁇ hic cDNA comprises at least one polymo ⁇ hism selected from the group consisting of adenine at a position conesponding to nucleotide 91, thymine at a position conesponding to nucleotide 94, thymine at a position conesponding to nucleotide 183, adenine at a position conesponding to nucleotide 486 and thymine at a position conesponding to nucleotide 603.
  • a particularly prefened polymo ⁇ hic cDNA variant comprises the coding sequence of an IL6 isogene defined by haplotypes 4, 5, 8, 10 and 17.
  • Polynucleotides complementary to these IL6 genomic and cDNA variants are also provided by the invention. It is believed that polymo ⁇ hic variants of the IL6 gene will be useful in studying the expression and function of IL6, and in expressing IL6 protein for use in screening for candidate drags to treat diseases related to IL6 activity.
  • the invention provides a recombinant expression vector comprising one of the polymo ⁇ hic genomic and cDNA variants operably linked to expression regulatory elements as well as a recombinant host cell transformed or transfected with the expression vector.
  • the recombinant vector and host cell may be used to express IL6 for protein structure analysis and drug binding studies.
  • the invention provides a polypeptide comprising a polymo ⁇ hic variant of a reference amino acid sequence for the IL6 protein.
  • the reference amino acid sequence comprises SEQ ID NO:3 (Fig.3) and the polymo ⁇ hic variant comprises at least one variant amino acid selected from the group consisting of threonine at a position conesponding to amino acid position 31, serine at a position conesponding to amino acid position 32 and glutamic acid at a position conesponding to amino acid position 162.
  • a polymo ⁇ hic variant of IL6 is useful in studying the effect of the variation on the biological activity of IL6 as well as on the binding affinity of candidate drugs targeting IL6 for the treatment of myeloma, coronary artery disease, arthritis, and Kaposi sarcoma.
  • the present invention also provides nonhuman transgenic animals comprising one or more of the IL6 polymo ⁇ hic genomic variants described herein and methods for producing such animals.
  • the transgenic animals are useful for studying expression of the IL6 isogenes in vivo, for in vivo screening and testing of drugs targeted against IL6 protein, and for testing the efficacy of therapeutic agents and compounds for myeloma, coronary artery disease, arthritis, and Kaposi sarcoma in a biological system.
  • the present invention also provides a computer system for storing and displaying polymo ⁇ hism data determined for the IL6 gene.
  • the computer system comprises a computer processing unit; a display; and a database containing the polymo ⁇ hism data.
  • the polymo ⁇ hism data includes one or more of the following: the polymo ⁇ hisms, the genotypes, the haplotypes, and the haplotype pairs identified for the IL6 gene in a reference population.
  • the computer system is capable of producing a display showing IL6 haplotypes organized according to their evolutionary relationships.
  • Figure 1 illustrates a reference sequence for the IL6 gene (Genaissance Reference No. 3868934; contiguous lines), with the start and stop positions of each region of coding sequence indicated with a bracket ([ or ]) and the numerical position below the sequence and the polymo ⁇ hic site(s) and polymo ⁇ hism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymo ⁇ hic site in the sequence.
  • SEQ ID NO: 1 is equivalent to
  • Figure 2 illustrates a reference sequence for the IL6 coding sequence (contiguous lines; SEQ ID NO:2), with the polymo ⁇ hic site(s) and polymo ⁇ hism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymo ⁇ hic site in the sequence.
  • Figure 3 illustrates a reference sequence for the IL6 protein (contiguous lines; SEQ ID NO:3), with the variant amino acid(s) caused by the polymo ⁇ hism(s) of Figure 2 positioned below the polymo ⁇ hic site in the sequence.
  • the IL6 isogenes present in the human reference population are defined by haplotypes for 17 polymo ⁇ hic sites in the IL6 gene, 14 of which are believed to be novel.
  • the IL6 polymo ⁇ hic sites identified by the inventors are refened to as PSI -PS 17 to designate the order in which they are located in the gene (see Table 3 below), with the novel polymo ⁇ hic sites refened to as PS2, PS3, PS4, PS5, PS6, PS8, PS10, PSI 1, PS12, PS13, PS14, PS15, PS16 and PS17.
  • the inventors herein Using the genotypes identified in the Index Repository for PSI -PS 17 and the methodology described in the Examples below, the inventors herein also determined the pair of haplotypes for the IL6 gene present in individual human members of this repository.
  • the human genotypes and haplotypes found in the repository for the IL6 gene include those shown in Tables 4 and 5, respectively.
  • the polymo ⁇ hism and haplotype data disclosed herein are useful for validating whether IL6 is a suitable target for drugs to treat myeloma, coronary artery disease, arthritis, and Kaposi sarcoma, screening for such drugs and reducing bias in clinical trials of such drugs.
  • Allele - A particular form of a genetic locus, distinguished from other forms by its particular nucleotide sequence.
  • Candidate Gene - A gene which is hypothesized to be responsible for a disease, condition, or the response to a treatment, or to be conelated with one of these.
  • Genotype An unphased 5 ' to 3 ' sequence of nucleotide pair(s) found at one or more polymo ⁇ hic sites in a locus on a pair of homologous chromosomes in an individual.
  • genotype includes a full-genotype and/or a sub-genotype as described below.
  • Full-genotype -* The unphased 5' to 3' sequence of nucleotide pairs found at all polymo ⁇ hic sites examined herein in a locus on a pair of homologous chromosomes in a single individual.
  • Sub-genotype - The unphased 5 ' to 3 ' sequence of nucleotides seen at a subset of the polymo ⁇ hic sites examined herein in a locus on a pair of homologous chromosomes in a single individual.
  • Haplotype A 5 ' to 3 ' sequence of nucleotides found at one or more polymo ⁇ hic sites in a locus on a single chromosome from a single individual.
  • haplotype includes a full- haplotype and/or a sub-haplotype as described below.
  • Full-haplotype The 5' to 3 ' sequence of nucleotides found at all polymo ⁇ hic sites examined herein in a locus on a single chromosome from a single individual.
  • Sub-haplotype The 5 ' to 3 ' sequence of nucleotides seen at a subset of the polymo ⁇ hic sites examined herein in a locus on a single chromosome from a single individual.
  • Haplotype pair The two haplotypes found for a locus in a single individual.
  • Haplotyping A process for determining one or more haplotypes in an individual and includes use of family pedigrees, molecular techniques and/or statistical inference.
  • Haplotype data Information concerning one or more of the following for a specific gene: a listing of the haplotype pairs in each individual in a population; a listing of the different haplotypes in a population; frequency of each haplotype in that or other populations, and any known associations between one or more haplotypes and a trait.
  • Isoform - A particular form of a gene, mRNA, cDNA, coding sequence or the protein encoded thereby, distinguished from other forms by its particular sequence and/or structure.
  • Isogene - One of the isoforms (e.g., alleles) of a gene found in a population. An isogene (or allele) contains all of the polymo ⁇ hisms present in the particular isoform of the gene.
  • Isolated - As applied to a biological molecule such as RNA, DNA, oligonucleotide, or protein, isolated means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term “isolated” is not intended to refer to a complete absence of such material or to absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the methods of the present invention.
  • Locus - A location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature, where physical features include polymo ⁇ hic sites.
  • Naturally-occurring A term used to designate that the object it is applied to, e.g., naturally- occurring polynucleotide or polypeptide, can be isolated from a source in nature and which has not been intentionally modified by man.
  • Nucleotide pair The nucleotides found at a polymo ⁇ hic site on the two copies of a chromosome from an individual.
  • phased As applied to a sequence of nucleotide pairs for two or more polymo ⁇ hic sites in a locus, phased means the combination of nucleotides present at those polymo ⁇ hic sites on a single copy of the locus is known.
  • PS Polymorphic site
  • Polymorphism data Information concerning one or more of the following for a specific gene: location of polymo ⁇ hic sites; sequence variation at those sites; frequency of polymo ⁇ hisms in one or more populations; the different genotypes and/or haplotypes determined for the gene; fi'equency of one or more of these genotypes and/or haplotypes in one or more populations; any known association(s) between a trait and a genotype or a haplotype for the gene.
  • Polynucleotide - A nucleic acid molecule comprised of single-stranded RNA or DNA or comprised of complementary, double-stranded DNA.
  • Reference Population A group of subjects or individuals who are predicted to be representative of the genetic variation found in the general population.
  • the reference population represents the genetic variation in the population at a certainty level of at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 99%.
  • compositions comprise at least one oligonucleotide for detecting the variant nucleotide or nucleotide pair located at a novel IL6 polymo ⁇ hic site in one copy or two copies of the IL6 gene.
  • oligonucleotides are refened to herein as IL6 haplotyping oligonucleotides or genotyping oligonucleotides, respectively, and collectively as IL6 oligonucleotides.
  • an IL6 haplotyping or genotyping oligonucleotide is a probe or primer capable of hybridizing to a target region that contains, or that is located close to, one of the novel polymo ⁇ hic sites described herein.
  • oligonucleotide refers to a polynucleotide molecule having less than about 100 nucleotides.
  • a prefened oligonucleotide of the invention is 10 to 35 nucleotides long. More preferably, the oligonucleotide is between 15 and 30, and most preferably, between 20 and 25 nucleotides in length. The exact length of the oligonucleotide will depend on many factors that are routinely considered and practiced by the skilled artisan.
  • oligonucleotide may be comprised of any phosphorylation state of ribonucleotides, deoxyribonucleotides, and acyclic nucleotide derivatives " and other functionally equivalent derivatives.
  • oligonucleotides may have a phosphate-free backbone, which may be comprised of linkages such as carboxymethyl, acetamidate, carbamate, polyamide (peptide nucleic acid (PNA)) and the like (Varma, R. in Molecular Biology and Biotechnology, A Comprehensive Desk Reference, Ed. R. Meyers, VCH Publishers, Inc. (1995), pages 617-620).
  • Oligonucleotides of the invention may be prepared by chemical synthesis using any suitable methodology known in the art, or may be derived from a biological sample, for example, by restriction digestion.
  • the oligonucleotides may be labeled, according to any technique known in the art, including use of radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags and the like.
  • Haplotyping or genotyping oligonucleotides of the invention must be capable of specifically hybridizing to a target region of an IL6 polynucleotide.
  • the target region is located in an IL6 isogene.
  • specific hybridization means the oligonucleotide forms an anti-parallel double-stranded structure with the target region under certain hybridizing conditions, while failing to form such a structure when incubated with another region in the IL6 polynucleotide or with a non-IL6 polynucleotide under the same hybridizing conditions.
  • the oligonucleotide specifically hybridizes to the target region under conventional high stringency conditions.
  • the skilled artisan can readily design and test oligonucleotide probes and primers suitable for detecting polymo ⁇ hisms in the IL6 gene using the polymo ⁇ hism information provided herein in conjunction with the known sequence information for the IL6 gene and routine techniques.
  • a nucleic acid molecule such as an oligonucleotide or polynucleotide is said to be a "perfect” or “complete” complement of another nucleic acid molecule if every nucleotide of one of the molecules is complementary to the nucleotide at the conesponding position of the other molecule.
  • a nucleic acid molecule is "substantially complementary” to another molecule if it hybridizes to that molecule with sufficient stability to remain in a duplex form under conventional low-stringency conditions. Conventional hybridization conditions are described, for example, by Sambrook J. et al., in Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and by Haymes, B.D.
  • an oligonucleotide primer may have a non-complementary fragment at its 5 ' end, with the remainder of the primer being complementary to the target region.
  • non-complementary nucleotides may be interspersed into the probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
  • Prefened haplotyping or genotyping oligonucleotides of the invention are allele-specific oligonucleotides.
  • ASO allele-specific oligonucleotide
  • allele-specificity will depend upon a variety of readily optimized stringency conditions, including salt and formamide concentrations, as well as temperatures for both the hybridization and washing steps.
  • Allele-specific oligonucleotides of the invention include ASO probes and ASO primers.
  • ASO probes which usually provide good discrimination between different alleles are those in which a central position of the oligonucleotide probe aligns with the polymo ⁇ hic site in the target region (e.g., approximately the 7 th or 8 th position in a 15mer, the 8 th or 9 th position in a 16mer, and the 10 th or 11 th position in a 20mer).
  • An ASO primer of the invention has a 3 ' terminal nucleotide, or preferably a 3 ' penultimate nucleotide, that is complementary to only one nucleotide of a particular SNP, thereby acting as a primer for polymerase-mediated extension only if the allele containing that nucleotide is present.
  • ASO probes and primers hybridizing to either the coding or noncoding strand are contemplated by the invention.
  • a prefened ASO probe for detecting IL6 gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
  • TGCGCTCKCTCCCCT (SEQ ID NO 4) and its complement, CCCAGTAMCCCCAGG (SEQ ID NO 5) and its complement, AGTACCCYCAGGAGA (SEQ ID NO 6) and its complement, ACATCCTYGACGGCA (SEQ ID NO 7) and its complement, TTGAAGGRCCCGGTG (SEQ ID NO 8) and its complement, TGAAAAGKCCCTCTA (SEQ ID NO 9) and its complement, GCATGCARTCCTGTA (SEQ ID NO 10) and its complement AGGCCAASTTCAAGC (SEQ ID NO 11) and its complement AAAATGGKGCTGTCC (SEQ ID NO 12) and its complement ACTTGGTRTGGGGGA (SEQ ID NO 13) and its complement ATCTAGAWGCAATAA (SEQ ID NO 14) and its complement AGGAGTTYCTGCAGT (SEQ ID NO 15) and its complement GAACTTAYGTTGTTC (SEQ ID NO 16) and its complement and ACTAAAARTATGAGC (SEQ ID NO 17) and
  • a prefened ASO primer for detecting IL6 gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5' to 3', selected from the group consisting of:
  • oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymo ⁇ hic sites identified herein.
  • Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one of the novel polymo ⁇ hisms described herein and therefore such oligonucleotides are refened to herein as "primer-extension oligonucleotides".
  • the 3 '-terminus of a primer- extension oligonucleotide is a deoxynucleotide complementary to the nucleotide located immediately adjacent to the polymo ⁇ hic site.
  • a particularly prefened oligonucleotide primer for detecting IL6 gene polymo ⁇ hisms by primer extension terminates in a nucleotide sequence, listed 5 ' to 3', selected from the group consisting of:
  • GGTACATCCT SEQ ID NO 52 AGATGCCGTC(S ⁇ Q ID NO : 53 )
  • a composition contains two or more differently labeled IL6 oligonucleotides for simultaneously probing the identity of nucleotides or nucleotide pairs at two or more polymo ⁇ hic sites. It is also contemplated that primer compositions may contain two or more sets of allele-specific primer pairs to allow simultaneous targeting and amplification of two or more regions containing a polymo ⁇ hic site.
  • the invention provides a kit comprising at least two IL6 oligonucleotides packaged in separate containers.
  • the kit may also contain other components such as hybridization buffer (where the oligonucleotides are to be used as a probe) packaged in a separate container.
  • the kit may contain, packaged in separate containers, a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase, such as PCR.
  • the two "copies" of a gene, mRNA or cDNA (or fragment of such IL6 molecules) in an individual may be the same allele or may be different alleles.
  • the identity of the nucleotide pair at one or more of the polymo ⁇ hic sites selected from the group consisting of PSI, PS7 and PS9 is also determined.
  • a genotyping method of the invention comprises determining the identity of the nucleotide pair at each of PS1-PS17.
  • the nucleic acid sample is isolated from a biological sample taken from the individual, such as a blood sample or tissue sample.
  • tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • the nucleic acid sample may be comprised of genomic DNA, mRNA, or cDNA and, in the latter two cases, the biological sample must be obtained from a tissue in which the IL6 gene is expressed.
  • mRNA or cDNA preparations would not be used to detect polymo ⁇ hisms located in introns or in 5 ' and 3 ' untranslated regions if not present in the mRNA or cDNA. If an IL6 gene fragment is isolated, it must contain the polymo ⁇ hic site(s) to be genotyped.
  • the haplotype for the other allele may be infened if the individual has a known genotype for the polymo ⁇ hic sites of interest or if the haplotype frequency or haplotype pair frequency for the individual's population group is known.
  • the IL6 haplotype is assigned to the individual by also identifying the nucleotide at one or more polymo ⁇ hic sites selected from the group consisting of PSI, PS7 and PS9. In a particularly prefened embodiment, the nucleotide at each of PS1-PS17 is identified.
  • an IL6 haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or more polymo ⁇ hic sites selected from the group consisting of PS2, PS3, PS4, PS5, PS6, PS8, PS10, PSI 1, PS12, PS13, PS14, PS15, PS16 and PS17 in each copy of the IL6 gene that is present in the individual.
  • the haplotyping method comprises identifying the phased sequence of nucleotides at each of PS1-PS17 in each copy of the IL6 gene.
  • the identifying step is preferably performed with each copy of the gene being placed in separate containers.
  • the two copies are labeled with different tags, or are otherwise separately distinguishable or identifiable, it could be possible in some cases to perform the method in the same container.
  • first and second copies of the gene are labeled with different first and second fluorescent dyes, respectively, and an allele-specific oligonucleotide labeled with yet a third different fluorescent dye is used to assay the polymo ⁇ hic site(s), then detecting a combination of the first and third dyes would identify the polymo ⁇ hism in the first gene copy while detecting a combination of the second and third dyes would identify the polymo ⁇ hism in the second gene copy.
  • the identity of a nucleotide (or nucleotide pair) at a polymo ⁇ hic site(s) may be determined by amplifying a target region(s) containing the polymo ⁇ hic site(s) directly from one or both copies of the IL6 gene, or a fragment thereof, and the sequence of the amplified region(s) determined by conventional methods. It will be readily appreciated by the skilled artisan that only one nucleotide will be detected at a polymo ⁇ hic site in individuals who are homozygous at that site, while two different nucleotides will be detected if the individual is heterozygous for that site.
  • the polymo ⁇ hism may be identified directly, known as positive-type identification, or by inference, refened to as negative-type identification.
  • a site may be positively determined to be either guanine or cytosine for an individual homozygous at that site, or both guanine and cytosine, if the individual is heterozygous at that site.
  • the site may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanine/guanine).
  • a polymo ⁇ hism in the target region may also be assayed before or after amplification using one of several hybridization-based methods known in the art.
  • allele-specific oligonucleotides are utilized in performing such methods.
  • the allele-specific oligonucleotides may be used as differently labeled probe pairs, with one member of the pair showing a perfect match to one variant of a target sequence and the other member showing a perfect match to a different variant.
  • more than one polymo ⁇ hic site may be detected at once using a set of allele- specific oligonucleotides or oligonucleotide pairs.
  • the members of the set have melting temperatures within 5°C, and more preferably within 2°C, of each other when hybridizing to each of the polymo ⁇ hic sites being detected.
  • Hybridization of an allele-specific oligonucleotide to a target polynucleotide maybe performed with both entities in solution, or such hybridization may be performed when either the oligonucleotide or the target polynucleotide is covalently or noncovalently affixed to a solid support. Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges, hydrophobic interactions, chemical linkages, UV cross-linking baking, etc. Allele-specific oligonucleotides may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis.
  • Solid-supports suitable for use in detection methods of the invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibers, chips, dishes, and beads.
  • the solid support may be treated, coated or derivatized to facilitate the immobilization of the allele-specific oligonucleotide or target nucleic acid.
  • the genotype or haplotype for the IL6 gene of an individual may also be determined by hybridization of a nucleic acid sample containing one or both copies of the gene, mRNA, cDNA or fragment(s) thereof, to nucleic acid anays and subanays such as described in WO 95/11995.
  • the anays would contain a battery of allele-specific oligonucleotides representing each of the polymo ⁇ hic sites to be included in the genotype or haplotype.
  • polymo ⁇ hisms may also be determined using a mismatch detection technique, including but not limited to the RNase protection method using riboprobes (Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230: 1242, 1985) and proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991).
  • riboprobes Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230: 1242, 1985
  • proteins which recognize nucleotide mismatches such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991).
  • variant alleles can be identified by single strand conformation polymo ⁇ hism (SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries et al., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp. 321-340, 1996) or denaturing gradient gel electrophoresis (DGGE) (Wartell et al., Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236, 1989).
  • SSCP single strand conformation polymo ⁇ hism
  • DGGE denaturing gradient gel electrophoresis
  • a polymerase-mediated primer extension method may also be used to identify the polymo ⁇ hism(s).
  • Several such methods have been described in the patent and scientific literature and include the "Genetic Bit Analysis” method (W092/15712) and the ligase/polymerase mediated genetic bit analysis (U.S. Patent 5,679,524.
  • Related methods are disclosed in WO91/02087, WO90/09455, W095/17676, U.S. Patent Nos. 5,302,509, and 5,945,283.
  • Extended primers containing a polymo ⁇ hism may be detected by mass spectrometry as described in U.S. Patent No. 5,605,798.
  • the identity of the allele(s) present at any of the novel polymo ⁇ hic sites described herein may be indirectly determined by haplotyping or genotyping another polymo ⁇ hic site that is in linkage disequilibrium with the polymo ⁇ hic site that is of interest.
  • Polymo ⁇ hic sites in linkage disequilibrium with the presently disclosed polymo ⁇ hic sites may be located in regions of the gene or in other genomic regions not examined herein.
  • Detection of the allele(s) present at a polymo ⁇ hic site in linkage disequilibrium with the novel polymo ⁇ hic sites described herein may be performed by, but is not limited to, any of the above-mentioned methods for detecting the identity of the allele at a polymo ⁇ hic site.
  • an individual's IL6 haplotype pair is predicted from its IL6 genotype using information on haplotype pairs known to exist in a reference population.
  • the haplotyping prediction method comprises identifying an IL6 genotype for the individual at two or more IL6 polymo ⁇ hic sites described herein, accessing data containing IL6 haplotype pairs identified in a reference population, and assigning a haplotype pair to the individual that is consistent with the genotype data.
  • the reference haplotype pairs include the IL6 haplotype pairs shown in Table 4.
  • the IL6 haplotype pair can be assigned by comparing the individual's genotype with the genotypes conesponding to the haplotype pairs known to exist in the general population or in a specific population group, and determining which haplotype pair is consistent with the genotype of the individual.
  • the comparing step may be perfonned by visual inspection (for example, by consulting Table 4).
  • frequency data (such as that presented in Table 7) may be used to determine which of these haplotype pairs is most likely to be present in the individual. This determination may also be performed in some embodiments by visual inspection, for example by consulting Table 7.
  • the comparison may be made by a computer-implemented algorithm with the genotype of the individual and the reference haplotype data stored in computer-readable formats.
  • one computer-implemented algorithm to perform this comparison entails enumerating all possible haplotype pairs which are consistent with the genotype, accessing data containing IL6 haplotype pairs frequency data determined in a reference population to determine a probability that the individual has a possible haplotype pair, and analyzing the determined probabilities to assign a haplotype pair to the individual.
  • the reference population should be composed of randomly-selected individuals representing the major ethnogeographic groups of the world.
  • a prefened reference population allows the detection of any haplotype whose frequency is at least 10% with about 99% certainty and comprises about 20 unrelated individuals from each of the four population groups named above.
  • a particularly prefened reference population includes a 3-generation family representing one or more of the four population groups to serve as controls for checking quality of haplotyping procedures.
  • a statistically significant difference between the observed and expected haplotype frequencies could be due to one or more factors including significant inbreeding in the population group, strong selective pressure on the gene, sampling bias, and/or enors in the genotyping process. If large deviations from Hardy- Weinberg equilibrium are observed in an ethnogeographic group, the number of individuals in that group can be increased to. see if the deviation is due to a sampling bias. If a larger sample size does not reduce the difference between observed and expected haplotype pair frequencies, then one may wish to consider haplotyping the individual using a direct haplotyping method such as, for example, CLASPER System TM technology (U.S. Patent No.
  • the individual is preferably haplotyped using a direct molecular haplotyping method such as, for example, CLASP ⁇ R SystemTM technology (U.S. Patent No. 5,866,404), SMD, or allele-specific long-range PCR (Michalotos-Beloin et al, supra).
  • the invention also provides a method for determining the frequency of an IL6 genotype, haplotype, or haplotype pair in a population.
  • the method comprises, for each member of the population, determining the genotype or the haplotype pair for the novel IL6 polymo ⁇ hic sites described herein, and calculating the frequency any particular genotype, haplotype, or haplotype pair is found in the population.
  • the population may be e.g., a reference population, a family population, a same gender population, a population group, or a trait population (e.g., a group of individuals exhibiting a trait of interest such as a medical condition or response to a therapeutic treatment).
  • frequency data for IL6 genotypes, haplotypes, and/or haplotype pairs are determined in a reference population and used in a method for identifying an association between a trait and an IL6 genotype, haplotype, or haplotype pair.
  • the trait may be any detectable phenotype, including but not limited to susceptibility to a disease or response to a treatment.
  • the method involves obtaining data on the frequency of the genotype(s), haplotype(s), or haplotype pair(s) of interest in a reference population as well as in a population exhibiting the trait.
  • Frequency data for one or both of the reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one or more of the methods described above.
  • the haplotypes for the trait population may be determined directly or, alternatively, by a predictive genotype to haplotype approach as described above.
  • the frequency data for the reference and/or trait populations is obtained by accessing previously determined frequency data, which may be in written or electronic form.
  • the frequency data may be present in a database that is accessible by a computer. Once the frequency data is obtained, the frequencies of the genotype(s), haplotype(s), or haplotype pair(s) of interest in the reference and trait populations are compared.
  • the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drug targeting IL6 or response to a therapeutic treatment for a medical condition.
  • medical condition includes but is not limited to any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment is desirable, and includes previously and newly identified diseases and other disorders.
  • clinical response means any or all of the following: a quantitative measure of the response, no response, and/or adverse response (i.e., side effects).
  • clinical population In order to deduce a conelation between clinical response to a treatment and an IL6 genotype, haplotype, or haplotype pair, it is necessary to obtain data on the clinical responses exhibited by a population of individuals who received the treatment, hereinafter the "clinical population".
  • This clinical data may be obtained by analyzing the results of a clinical trial that has already been ran and/or the clinical data may be obtained by designing and carrying out one or more new clinical trials.
  • the term "clinical trial” means any research study designed to collect clinical data on responses to a particular treatment, and includes but is not limited to phase I, phase JJ and phase III clinical trials. Standard methods are used to define the patient population and to enroll subjects.
  • the therapeutic treatment of interest is administered to each individual in the trial population and each individual's response to the treatment is measured using one or more predetermined criteria. It is contemplated that in many cases, the trial population will exhibit a range of responses and that the investigator will choose the number of responder groups (e.g., low, medium, high) made up by the various responses.
  • the IL6 gene for each individual in the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment.
  • conelations between individual response and IL6 genotype or haplotype content are created. Conelations may be produced in several ways. In one method, individuals are grouped by their IL6 genotype or haplotype (or haplotype pair) (also refened to as a polymo ⁇ hism group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymo ⁇ hism group are calculated.
  • a second method for finding conelations between IL6 haplotype content and clinical responses uses predictive models based on enor-minimizing optimization algorithms.
  • One of many possible optimization algorithms is a genetic algorithm (R. Judson, "Genetic Algorithms and Their Uses in Chemistry” in Reviews in Computational Chemistry, Vol. 10, pp. 1-73, K. B. Lipkowitz and D. B. Boyd, eds. (VCH Publishers, New York, 1997).
  • Simulated annealing Press et al., "Numerical Recipes in C: The Art of Scientific Computing", Cambridge University Press (Cambridge) 1992, Ch. 10), neural networks (E. Rich and K.
  • the conelation is found using a genetic algorithm approach as described in WO 01/01218.
  • a mathematical model may be readily constructed by the skilled artisan that predicts clinical response as a function of IL6 genotype or haplotype content.
  • the model is validated in one or more follow-up clinical trials designed to test the model. The identification of an association between a clinical response and a genotype or haplotype
  • the diagnostic method may take one of several forms: for example, a direct DNA test (i.e., genotyping or haplotyping one or more of the polymo ⁇ hic sites in the IL6 gene), a serological test, or a physical exam measurement.
  • a direct DNA test i.e., genotyping or haplotyping one or more of the polymo ⁇ hic sites in the IL6 gene
  • a serological test i.e., a serological test
  • a physical exam measurement i.e., a physical exam measurement. The only requirement is that there be a good conelation between the diagnostic test results and the underlying IL6 genotype or haplotype that is in turn conelated with the clinical response.
  • this diagnostic method uses the predictive haplotyping method described above.
  • the invention provides an isolated polynucleotide comprising a polymo ⁇ hic variant of the IL6 gene or a fragment of the gene which contains at least one of the novel polymo ⁇ hic sites described herein.
  • nucleotide sequence of a variant fragment of the IL6 gene is identical to the conesponding portion of the reference sequence except for having a different nucleotide at one or more of the novel polymo ⁇ hic sites described herein.
  • the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence of the IL6 gene, which is defined by haplotype 12, (or other reported IL6 sequences) or to portions of the reference sequence (or other reported IL6 sequences), except for the haplotyping and genotyping oligonucleotides described above.
  • the location of a polymo ⁇ hism in a variant IL6 gene or fragment is preferably identified by alignmg its sequence against SEQ ID NO: 1.
  • the polymo ⁇ hism is selected from the group consisting of thymine at PS2, adenine at PS3, thymine at PS4, thymine at PS5, adenine at PS6, thymine at PS8, adenine at PS10, cytosine at PSI 1, guanine at PS 12, adenine at PS 13, adenine at PS 14, thymine at PS15, cytosine at PS16 and adenine at PS17.
  • the polymo ⁇ hic variant comprises a naturally-occurring isogene of the IL6 gene which is defined by any one of haplotypes 1- 11 and 13 - 18 shown in Table 5 below.
  • Polymo ⁇ hic variants of the invention may be prepared by isolating a clone containing the IL6 gene from a human genomic library.
  • the clone may be sequenced to determine the identity of the nucleotides at the novel polymo ⁇ hic sites described herein.
  • Any particular variant or fragment thereof, that is claimed herein could be prepared from this clone by performing in vitro mutagenesis using procedures well-known in the art.
  • Any particular IL6 variant or fragment thereof may also be prepared using synthetic or semi-synthetic methods known in the art.
  • IL6 isogenes, or fragments thereof may be isolated using any method that allows separation of the two "copies" of the IL6 gene present in an individual, which, as readily understood by the skilled artisan, may be the same allele or different alleles. Separation methods include targeted in vivo cloning (TIVC) in yeast as described in WO 98/01573, U.S. Patent No. 5,866,404, and U.S. Patent No. 5,972,614. Another method, which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets.
  • TIVC targeted in vivo cloning
  • Another method which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets.
  • the invention also provides IL6 genome anthologies, which are collections of at least two IL6 isogenes found in a given population.
  • the population may be any group of at least two individuals, including but not limited to a reference population, a population group, a family population, a clinical population, and a same gender population.
  • An IL6 genome anthology may comprise individual IL6 isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like. Alternatively, two or more groups of the IL6 isogenes in the anthology may be stored in separate containers.
  • a prefened IL6 genome anthology of the invention comprises a set of isogenes defined by the haplotypes shown in Table 5 below.
  • An isolated polynucleotide containing a polymo ⁇ hic variant nucleotide sequence of the invention may be operably linked to one or more expression regulatory elements in a recombinant expression vector capable of being propagated and expressing the encoded IL6 protein in a prokaryotic or a eukaryotic host cell.
  • expression regulatory elements which may be used include, but are not limited to, the lac system, operator and promoter regions of phage lambda, yeast promoters, and promoters derived from vaccinia virus, adenovirus, retro viruses, or SV40.
  • regulatory elements include, but are not limited to, appropriate leader sequences, termination codons, polyadenylation signals, and other sequences required for the appropriate transcription and subsequent translation of the nucleic acid sequence in a given host cell.
  • the expression vector contains any additional elements necessary for its transfer to and subsequent replication in the host cell. Examples of such elements include, but are not limited to, origins of replication and selectable markers.
  • Such expression vectors are commercially available or are readily constructed using methods known to those in the art (e.g., F. Ausubel et al., 1987, in "Cunent Protocols in Molecular Biology", John Wiley and Sons, New York, New York).
  • Host cells which may be used to express the variant IL6 sequences of the invention include, but are not limited to, eukaryotic and mammalian cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as E. coli, or algal cells as known in the art.
  • the recombinant expression vector may be introduced into the host cell using any method known to those in the art including, but not limited to, microinjection, electroporation, particle bombardment, transduction, and transfection using DEAE-dextran, lipofection, or calcium phosphate (see e.g., Sambrook et al. (1989) in "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York).
  • eukaryotic expression vectors that function in eukaryotic cells, and preferably mammalian cells, are used.
  • Non-limiting examples of such vectors include vaccinia virus vectors, adenovirus vectors, he ⁇ es virus vectors, and baculovirus transfer vectors.
  • Prefened eukaryotic cell lines include COS cells, CHO cells, HeLa cells, NIH/3T3 cells, and embryonic stem cells (Thomson, J. A. et al., 1998 Science 282: 1145-1147). Particularly prefened host cells are mammalian cells.
  • polymo ⁇ hic variants of the IL6 gene will produce IL6 mRNAs varying from each other at any polymo ⁇ hic site retained in the spliced and processed mRNA molecules.
  • mRNAs can be used for the preparation of an IL6 cDNA comprising a nucleotide sequence which is a polymo ⁇ hic variant of the IL6 reference coding sequence shown in Figure 2.
  • the invention also provides IL6 mRNAs and conesponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ID NO:2 (Fig.
  • a particularly prefened polymo ⁇ hic cDNA variant comprises the coding sequence of an IL6 isogene defined by any one of haplotypes 4, 5, 8, 10 and 17. Fragments of these variant mRNAs and cDNAs are included in the scope of the invention, provided they contain one or more of the novel polymo ⁇ hisms described herein.
  • the invention specifically excludes polynucleotides identical to previously identified IL6 mRNAs or cDNAs, and previously described fragments thereof.
  • Polynucleotides comprising a variant IL6 RNA or DNA sequence may be isolated from a biological sample using well-known molecular biological procedures or may be chemically synthesized.
  • a polymo ⁇ hic variant of an IL6 gene, mRNA or cDNA fragment comprises at least one novel polymo ⁇ hism identified herein and has a length of at least 10 nucleotides and may range up to the full length of the gene.
  • such fragments are between 100 and 3000 nucleotides in length, and more preferably between 200 and 2000 nucleotides in length, and most preferably between 500 and 1000 nucleotides in length.
  • nucleic acid molecules containing the IL6 gene or cDNA may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the conesponding site on the complementary antisense strand.
  • reference may be made to the same polymo ⁇ hic site on either strand and an oligonucleotide may be designed to hybridize specifically to either strand at a target region containing the polymo ⁇ hic site.
  • the invention also includes single-stranded polynucleotides which are complementary to the sense strand of the IL6 genomic, mRNA and cDNA variants described herein.
  • Polynucleotides comprising a polymo ⁇ hic gene variant or fragment of the invention may be useful for therapeutic pu ⁇ oses.
  • an expression vector encoding the isoform may be administered to the patient.
  • the patient may be one who lacks the IL6 isogene encoding that isoform or may already have at least one copy of that isogene. In other situations, it may be desirable to decrease or block expression of a particular IL6 isogene.
  • Expression of an L6 isogene may be turned off by transforming a targeted organ, tissue or cell population with an expression vector that expresses high levels of untranslatable mRNA or antisense RNA for the isogene or fragment thereof.
  • oligonucleotides directed against the regulatory regions (e.g., promoter, introns, enhancers, 3' untranslated region) of the isogene may block transcription. Oligonucleotides targeting the transcription initiation site, e.g., between positions -10 and +10 from the start site are prefened.
  • inhibition of transcription can be achieved using oligonucleotides that base-pair with region(s) of the isogene DNA to form triplex DNA (see e.g., Gee et al. in Huber, B.E. and B.I. Can, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994).
  • Antisense oligonucleotides may also be designed to block translation of IL6 mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of IL6 mRNA transcribed from a particular isogene.
  • the untranslated mRNA, antisense RNA or antisense oligonucleotides may be delivered to a target cell or tissue by expression from a vector introduced into the cell or tissue in vivo or ex vivo. Alternatively, such molecules may be formulated as a pharmaceutical composition for administration to the patient. Oligoribonucleotides and/or oligodeoxynucleotides intended for use as antisense oligonucleotides may be modified to increase stability and half-life.
  • Possible modifications include, but are not limited to phosphorothioate or 2' O-methyl linkages, and the inclusion of nontraditional bases such as inosine and queosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytosine, guanine, thymine, and uracil which are not as easily recognized by endogenous nucleases.
  • the invention also provides an isolated polypeptide comprising a polymo ⁇ hic variant of (a) the reference IL6 amino acid sequence shown in Figure 3 or (b) a fragment of this reference sequence.
  • the location of a variant amino acid in an IL6 polypeptide or fragment of the invention is preferably identified by aligning its sequence against SEQ ID NO:3 (Fig. 3).
  • An IL6 protein variant of the invention comprises an amino acid sequence identical to SEQ ID NO:3 for those regions of SEQ ID NO:3 that are encoded by examined portions of the IL6 gene (as described in the Examples below), except for having one or more variant amino acids selected from the group consisting of threonine at a position conesponding to amino acid position 31, serine at a position conesponding to amino acid position 32 and glutamic acid at a position conesponding to amino acid position 162.
  • an IL6 fragment of the invention also refened to herein as an IL6 peptide variant, is any fragment of an IL6 protein variant that contains one or more of the amino acid variations shown in Table 2.
  • IL6 protein variants included within the invention comprise all amino acid sequences based on SEQ ID NO:3 and having the combination of amino acid variations described in Table 2 below.
  • an IL6 protein variant of the invention is encoded by an isogene defined by one of the observed haplotypes, 4, 5, 8, 10 and 17, shown in Table 5.
  • An IL6 peptide variant of the invention is at least 6 amino acids in length and is preferably any number between 6 and 30 amino acids long, more preferably between 10 and 25, and most preferably between 15 and 20 amino acids long.
  • Such IL6 peptide variants may be useful as antigens to generate antibodies specific for one of the above IL6 isoforms.
  • the IL6 peptide variants may be useful in drug screening assays.
  • an IL6 variant protein or peptide of the invention may be prepared by chemical synthesis or by expressing an appropriate variant IL6 genomic or cDNA sequence described above.
  • the IL6 protein variant may be isolated from a biological sample of an individual having an IL6 isogene which encodes the variant protein. Where the sample contains two different IL6 isoforms (i.e., the individual has different IL6 isogenes), a particular IL6 isoform of the invention can be isolated by immunoaffinity chromatography using an antibody which specifically binds to that particular IL6 isoform but does not bind to the other IL6 isoform.
  • the expressed or isolated 1L6 protein or peptide may be detected by methods known in the art, including Coomassie blue staining, silver staining, and Western blot analysis using antibodies specific for the isoform of the IL6 protein or peptide as discussed further below.
  • IL6 variant proteins and peptides can be purified by standard protein purification procedures known in the art, including differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis, affinity and immunoaffinity chromatography and the like. (Ausubel et. al., 1987, In Cunent Protocols in Molecular Biology John Wiley and Sons, New York, New York). In the case of immunoaffinity chromatography, antibodies specific for a particular polymo ⁇ hic variant may be used.
  • a polymo ⁇ hic variant IL6 gene of the invention may also be fused in frame with a heterologous sequence to encode a chimeric IL6 protein.
  • the non-IL6 portion of the chimeric protein may be recognized by a commercially available antibody.
  • the chimeric protein may also be engineered to contain a cleavage site located between the IL6 and non-IL6 portions so that the IL6 protein may be cleaved and purified away from the non-IL6 portion.
  • An additional embodiment of the invention relates to using a novel IL6 protein isoform, or a fragment thereof, in any of a variety of drag screening assays.
  • screening assays may be performed to identify agents that bind specifically to all known IL6 protein isoforms or to only a subset of one or more of these isoforms.
  • the agents may be from chemical compound libraries, peptide libraries and the like.
  • the IL6 protein or peptide variant may be free in solution or affixed to a solid support.
  • high throughput screening of compounds for binding to an IL6 variant may be accomplished using the method described in PCT application WO84/03565, in which large numbers of test compounds are synthesized on a solid substrate, such as plastic pins or some other surface, contacted with the IL6 protein(s) of interest and then washed. Bound IL6 protein(s) are then detected using methods well-known in the art.
  • a novel IL6 protein isoform may be used in assays to measure the binding affinities of one or more candidate drugs targeting the IL6 protein.
  • a particular IL6 haplotype or group of IL6 haplotypes encodes an IL6 protein variant with an amino acid sequence distinct from that of IL6 protein isoforms encoded by other IL6 haplotypes
  • detection of that particular IL6 haplotype or group of IL6 haplotypes may be accomplished by detecting expression of the encoded IL6 protein variant using any of the methods described herein or otherwise commonly known to the skilled artisan.
  • the invention provides antibodies specific for and immunoreactive with one or more of the novel IL6 protein or peptide variants described herein.
  • the antibodies may be either monoclonal or polyclonal in origin.
  • the IL6 protein or peptide variant used to generate the antibodies may be from natural or recombinant sources (in vitro or in vivo) or produced by chemical synthesis or semi-synthetic synthesis using synthesis techniques known in the art. If the IL6 protein or peptide variant is of insufficient size to be antigenic, it may be concatenated or conjugated, complexed, or otherwise covalently linked to a carrier molecule to enhance the antigenicity of the peptide.
  • carrier molecules include, but are not limited to, albumins (e.g., human, bovine, fish, ovine), and keyhole limpet hemocyanin (Basic and Clinical Immunology, 1991, Eds. D.P. Stites, and A.I. Ten, Appleton and Lange, Norwalk Connecticut, San Mateo, California).
  • albumins e.g., human, bovine, fish, ovine
  • keyhole limpet hemocyanin Basic and Clinical Immunology, 1991, Eds. D.P. Stites, and A.I. Ten, Appleton and Lange, Norwalk Connecticut, San Mateo, California.
  • an antibody specifically immunoreactive with one of the novel protein or peptide variants described herein is administered to an individual to neutralize activity of the IL6 isoform expressed by that individual.
  • the antibody may be formulated as a pharmaceutical composition which includes a pharmaceutically acceptable canier.
  • Antibodies specific for and immunoreactive with one of the novel protein isoforms described herein may be used to immunoprecipitate the IL6 protein variant from solution as well as react with IL6 protein isoforms on Western or immunoblots of polyacrylamide gels on membrane supports or substrates.
  • the antibodies will detect IL6 protein isoforms in paraffin or frozen tissue sections, or in cells which have been fixed or unfixed and prepared on slides, coverslips, or the like, for use in im unocytochemical, immunohistochemical, and immunofluorescence techniques.
  • an antibody specifically immunoreactive with one of the novel IL6 protein variants described herein is used in immunoassays to detect this variant in biological samples.
  • an antibody of the present invention is contacted with a biological sample and the formation of a complex between the IL6 protein variant and the antibody is detected.
  • suitable immunoassays include radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme linked immunoassay (ELISA), chemiluminescent assay, immunohistochemical assay, immunocytochemical assay, and the like (see, e.g., Principles and Practice of Immunoassay, 1991, Eds. Christopher P. Price and David J. Neoman, Stockton Press, New York, New York; Cunent Protocols in Molecular Biology, 1987, Eds. Ausubel et al., John Wiley and Sons, New York, New York). Standard techniques known in the art for ELISA are described in Methods in Immunodiagnosis, 2nd Ed., Eds. Rose and Bigazzi, John Wiley and Sons, New York 1980; and
  • Such assays may be direct, indirect, competitive, or noncompetitive as described in the art (see, e.g., Principles and Practice of Immunoassay, 1991, Eds. Christopher P. Price and David J. Neoman, Stockton Pres, NY, NY; and Oellirich, M., 1984, J. Clin. Chem. Clin. Biochem., 22:895-904). Proteins may be isolated from test specimens and biological samples by conventional methods, as described in Cunent Protocols in Molecular Biology, supra.
  • Exemplary antibody molecules for use in the detection and therapy methods of the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, or those portions of immunoglobulin molecules that contain the antigen binding site.
  • Polyclonal or monoclonal antibodies may be produced by methods conventionally known in the art (e.g., Kohler and
  • the antibodies or antigen binding fragments thereof may also be produced by genetic engineering.
  • the technology for expression of both heavy and light chain genes in E. coli is the subject of PCT patent applications, publication number, WO 901443 and WO 9014424 and in Huse et al., 1989, Science, 246:1275-1281.
  • the antibodies may also be humanized (e.g., Queen, C. et al. 1989 Proc. Natl. Acad. Sci.USA 86; 10029).
  • Effect(s) of the polymo ⁇ hisms identified herein on expression of IL6 may be investigated by various means known in the art, such as by in vitro translation of mRNA transcripts of the IL6 gene, ' cDNA or fragment thereof, or by preparing recombinant cells and or nonhuman recombinant organisms, preferably recombinant animals, containing a polymo ⁇ hic variant of the IL6 gene.
  • expression includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA(s) into IL6 protein(s) (including effects of polymo ⁇ hisms on codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
  • the desired IL6 isogene, cDNA or coding sequence may be introduced into the cell in a vector such that the isogene, cDNA or coding sequence remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location.
  • the IL6 isogene, cDNA or coding sequence is introduced into a cell in such a way that it recombines with the endogenous IL6 gene present in the cell. Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired IL6 gene polymo ⁇ hism.
  • Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector or vector construct may be used in the invention. Methods such as electroporation, particle bombardment, calcium phosphate co-precipitation and viral transduction for introducing DNA into cells are known in the art; therefore, the choice of method may lie with the competence and preference of the skilled practitioner.
  • Examples of cells into which the IL6 isogene, cDNA or coding sequence may be introduced include, but are not limited to, continuous culture cells, such as COS, CHO, NIH/3T3, and primary or culture cells of the relevant tissue type, i.e., they express the IL6 isogene, cDNA or coding sequence. Such recombinant cells can be used to compare the biological activities of the different protein variants.
  • Recombinant nonhuman organisms i.e., transgenic animals, expressing a variant EL6 gene, cDNA or coding sequence are prepared using standard procedures known in the art.
  • a construct comprising the variant gene, cDNA or coding sequence is introduced into a nonhuman animal or an ancestor of the animal at an embryonic stage, i.e., the one-cell stage, or generally not later than about the eight-cell stage.
  • Transgenic animals carrying the constructs of the invention can be made by several methods known to those having skill in the art.
  • One method involves transfecting into the embryo a retrovirus constructed to contain one or more insulator elements, a gene or genes (or cDNA or coding sequence) of interest, and other components known to those skilled in the art to provide a complete shuttle vector harboring the insulated gene(s) as a transgene, see e.g., U.S. Patent No. 5,610,053.
  • Another method involves directly injecting a transgene hito the embryo.
  • a third method involves the use of embryonic stem cells.
  • mice examples include, but are not limited to, mice, rats, other rodents, and nonhuman primates (see "The Introduction of Foreign Genes into Mice” and the cited references therein, In: Recombinant DNA, Eds. J.D. Watson, M. Gilman, J. Witkowski, and M. Zoller; W.H. Freeman and Company, New York, pages 254-272).
  • Transgenic animals stably expressing a human IL6 isogene, cDNA or coding sequence and producing the encoded human IL6 protein can be used as biological models for studying diseases related to abnormal IL6 expression and/or activity, and for screening and assaying various candidate drugs, compounds, and treatment regimens to reduce the symptoms or effects of these diseases.
  • compositions for treating disorders affected by expression or function of a novel IL6 isogene described herein.
  • the pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these novel IL6 isogenes (or cDNAs or coding sequences); an antisense oligonucleotide directed against one of the novel IL6 isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel IL6 isogene described herein.
  • the composition contains the active ingredient in a therapeutically effective amount.
  • composition also comprises a pharmaceutically acceptable carrier, examples of which include, but are not limited to, saline, buffered saline, dextrose, and water.
  • a pharmaceutically acceptable carrier examples of which include, but are not limited to, saline, buffered saline, dextrose, and water.
  • Those skilled in the art may employ a formulation most suitable for the active ingredient, whether it is a polynucleotide, oligonucleotide, protein, peptide or small molecule antagonist.
  • the pharmaceutical composition may be administered alone or in combination with at least one other agent, such as a stabilizing compound.
  • Administration of the pharmaceutical composition may be by any number of routes including, but not limited to oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
  • the dose can be estimated initially either in cell culture assays or in animal models.
  • the animal model may also be used to determine the appropriate concentration range and route of administration.
  • Such information can then be used to determine useful doses and routes for administration in humans.
  • the exact dosage will be determined by the practitioner, in light of factors relating to the patient requiring treatment, including but not limited to severity of the disease state, general health, age, weight and gender of the patient, diet, time and frequency of administration, other drags being taken by the patient, and tolerance/response to the treatment.
  • Any or all analytical and mathematical operations involved in practicing the methods of the present invention may be implemented by a computer.
  • the computer may execute a program that generates views (or screens) displayed on a display device " and with which the user can interact to view and analyze large amounts of information relating to the IL6 gene and its genomic variation, including chromosome location, gene structure, and gene family, gene expression data, polymo ⁇ hism data, genetic sequence data, and clinical data population data (e.g., data on ethnogeographic origin, clinical responses, genotypes, and haplotypes for one or more populations).
  • the IL6 polymo ⁇ hism data described herein may be stored as part of a relational database (e.g., an instance of an Oracle database or a set of ASCII flat files).
  • polymo ⁇ hism data may be stored on the computer's hard drive or may, for example, be stored on a CD-ROM or on one or more other storage devices accessible by the computer.
  • the data may be stored on one or more databases in communication with the computer via a network.
  • EXAMPLE 1 This example illustrates examination of various regions of the IL6 gene for polymo ⁇ hic sites.
  • the following target regions were amplified using either the PCR primers represented below or 'tailed' PCR primers, each of which includes a universal sequence forming a noncomplementary 'tail' attached to the 5 ' end of each unique sequence in the PCR primer pairs.
  • the universal 'tail' sequence for the forward PCR primers comprises the sequence 5 '-TGTAAAACGACGGCCAGT-3 ' (SEQ ID NO: 74) and the universal 'tail' sequence for the reverse PCR primers comprises the sequence 5'-AGGAAACAGCTATGACCAT-3' (SEQ ED NO:75).
  • the nucleotide positions of the first and last nucleotide of the forward and reverse primers for each region amplified are presented below and conespond to positions in SEQ ED NO: 1 ( Figure 1).
  • Amplification profile 97°C - 2 min. 1 cycle
  • the PCR products were purified using a Whatman/Polyfiltronics 100 ⁇ l 384 well unifilter plate essentially according to the manufacturers protocol.
  • the purified DNA was eluted in 50 ⁇ l of distilled water.
  • Sequencing reactions were set up using Applied Biosystems Big Dye Terminator chemistry essentially according to the manufacturers protocol.
  • the purified PCR products were sequenced in both directions using either the primer sets represented below with the positions of their first and last nucleotide conesponding to positions in Figure 1, or the appropriate universal 'tail' sequence as a primer. Reaction products were purified by isopropanol precipitation, and ran on an Applied Biosystems 3700 DNA Analyzer.
  • Fragment No. Forward Primer Reverse Primer Fragment 1 831-850 complement of 1350-1331 Fragment 2 832-852 complement of 1151-1131 Fragment 3 Tailed Seq.
  • Fragment 4 1027-1048 complement of 1501-1480 Fragment 5 2278-2297 complement of 2678-2659 Fragment 6 3089-3109 complement of 3571-3551 Fragment 7 4984-5003 complement of 5554-5533
  • Polyld is a unique identifier assigned to each PS by Genaissance Pharmaceuticals, Inc. (R) Reported previously.
  • EXAMPLE 2 This example illustrates analysis of the IL6 polymo ⁇ hisms identified in the Index Repository for human genotypes and haplotypes.
  • the different genotypes containing these polymo ⁇ hisms that were observed in unrelated members of the reference population are shown in Table 4 below, with the haplotype pair indicating the combination of haplotypes determined for the individual using the haplotype derivation protocol described below.
  • Table 4 homozygous positions are indicated by one nucleotide and heterozygous positions are indicated by two nucleotides. Missing nucleotides in any given genotype in Table 4 were infe ⁇ ed based on linkage disequilibrium and/or Mendelian inheritance.
  • haplotype pairs shown in Table 4 were estimated from the unphased genotypes using a computer-implemented extension of Clark's algorithm (Clark, A.G. 1990 Mol Bio Evol 7, 111-122) for assigning haplotypes to unrelated individuals in a population sample, as described in PCT/USOl/12831, filed April 18, 2001.
  • haplotypes are assigned directly from individuals who are homozygous at all sites or heterozygous at no more than one of the variable sites.
  • This list of haplotypes is then used to deconvolute the unphased genotypes in the remaining (multiply heterozygous) individuals.
  • the list of haplotypes was augmented with haplotypes obtained from two families (one three-generation Caucasian family and one two-generation African- American family).
  • IL6 haplotypes shown in Table 5 below.
  • An IL6 isogene defined by a full-haplotype shown in Table 5 below comprises the regions of the SEQ ED NOS indicated in Table 5, with their conesponding set of polymo ⁇ hic locations and identities, which are also set forth in Table 5.
  • Region examined represents the nucleotide positions defining the start and stop positions within SEQ ID NO:l of the regions sequenced;
  • SEQ ID NO : 1 refers to Figure 1 , with the two alternative allelic variants of each polymo ⁇ hic site indicated by the appropriate nucleotide symbol.
  • SEQ ID NO:76 is a modified version of SEQ ED NO:l that shows the context sequence of each of PSI -PS 17 in a uniform format to facilitate electronic searching of the IL6 haplotypes.
  • SEQ ED NO:76 contains a block of 60 bases of the nucleotide sequence encompassing the centrally-located polymo ⁇ hic site at the 30 th position, followed by 60 bases of unspecified sequence to represent that each polymo ⁇ hic site is separated by genomic sequence whose composition is defined elsewhere herein.
  • the size and composition of the Index Repository were chosen to represent the genetic diversity across and within four major population groups comprising the general United States population.
  • this repository contains approximately equal sample sizes of African-descent, Asian- American, European- American, and Hispanic-Latino population groups. Almost all individuals representing each group had all four grandparents with the same ethnogeographic background.
  • the number of unrelated individuals in the Index Repository provides a sample size that is sufficient to detect SNPs and haplotypes that occur in the general population with high statistical certainty. For instance, a haplotype that occurs with a frequency of 5% in the general population has a probability higher than 99.9% of being observed in a sample of 80 individuals from the general population.
  • a haplotype that occurs with a frequency of 10% in a specific population group has a 99% probability of being observed in a sample of 20 individuals from that population group.
  • the size and composition of the Index Repository means that the relative frequencies determined therein for the haplotypes and haplotype pairs of the IL6 gene are likely to be similar to the relative frequencies of these E 6 haplotypes and haplotype pairs in the general U.S. population and in the four population groups represented in the Index Repository. The genetic diversity observed for the three Native Americans is presented because it is of scientific interest, but due to the small sample size it lacks statistical significance.

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Abstract

La présente invention concerne de nouveaux variants génétiques du gène d'interleukine 6 (IL6); divers génotypes, haplotypes, et paires d'haplotypes qui existent dans la population globale des Etats Unis pour le gène IL6; des compositions et des procédés utiles pour haplotyper et/ou génotyper le gène IL6 chez un individu; ainsi que des polynucléotides définis par les haplotypes présentés dans cette invention.
PCT/US2001/047077 2000-11-09 2001-11-09 Haplotypes du gene il6 Ceased WO2002038586A2 (fr)

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US7439019B2 (en) 2003-11-03 2008-10-21 Duke University Methods of identifying individuals at risk of perioperative bleeding, renal dysfunction

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US5521301A (en) * 1988-12-12 1996-05-28 City Of Hope Genotyping of multiple allele systems
US5851762A (en) * 1990-07-11 1998-12-22 Gene Type Ag Genomic mapping method by direct haplotyping using intron sequence analysis

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Publication number Priority date Publication date Assignee Title
US7439019B2 (en) 2003-11-03 2008-10-21 Duke University Methods of identifying individuals at risk of perioperative bleeding, renal dysfunction
US8187807B2 (en) 2003-11-03 2012-05-29 Duke University Methods of identifying individuals at risk of perioperative bleeding, renal dysfunction or stroke
US9556486B2 (en) 2003-11-03 2017-01-31 Duke University Methods of identifying individuals at risk of perioperative bleeding, renal dysfunction or stroke

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