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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/172—Haplotypes

Definitions

• This invention relates to the fields of genomics and pharmacogenetics. More specifically, the present invention relates to polymorphisms and haplotypes of the ⁇ 2 -adrenergic receptor gene and their use as predictors of disease susceptibility and response to ⁇ -agonists.
• ⁇ -adrenergic receptor is a G protein coupled receptor that mediates the actions of catecholamines in multiple tissues and thus plays important roles in regulating cardiac, vascular, pulmonary, and metabolic functions.
• An abnormal level of expression of ⁇ 2 AR is believed to be a risk factor for or to modify the severity of a number of diseases and conditions, including congestive heart failure, arrhythmia, ischemic heart disease, hypertension, migraine, asthma, chronic obstructive pulmonary disease (COPD), anaphylaxis, obesity, diabetes, myasthenia gravis, and premature labor.
• diseases and conditions including congestive heart failure, arrhythmia, ischemic heart disease, hypertension, migraine, asthma, chronic obstructive pulmonary disease (COPD), anaphylaxis, obesity, diabetes, myasthenia gravis, and premature labor.
• COPD chronic obstructive pulmonary disease
• the ⁇ AR is encoded by an intronless gene on chromosome 5q31-32 (Kobilka, B.K. et al., Proc.Natl.Acad.Sci., USA 84:46-50, 1987).
• SNPs single nucleotide polymorphisms
• SNPs are located at nucleotides 46 (A or G), 79 (C or G), and 491 (C or T) of the ⁇ 2 AR coding sequence, and result in variation that occurs in the amino- terminus of the receptor at amino acids 16 (Arg or Gly) and 27 (Gin or Glu) and in the fourth transmembrane spanning domain at amino acid 164 (Thr or He), respectively.
• RNA transcripts of the ⁇ AR gene have a 5' leader region harboring a short open reading frame (ORF), termed the ⁇ 2 AR 5 '-leader cistron (5'LC), that encodes a 19 amino acid peptide (Kobilka, B.K. et al., J.Biol. Chem. : 262:7321-7327, 1996).
• This ⁇ 2 AR upstream peptide (BUP) modulates translation of ⁇ 2 AR mRNA, and thereby regulates cellular expression of the receptor (Parola, A.L. et al., JBiol Chem 269:4497-4505, 1994).
• the polymorphic site located at -47, described above, is in this 5XC and results in either Arg or Cys being encoded at the terminal amino acid (position 19) of the BUP. It was recently reported that the Cys 19 variant of the BUP is associated with greater ⁇ AR expression than the Argl9 BUP variant (McGraw et al., J Clin. Invest. 102:1927-1932, 1998).
• ⁇ 2 AR gene polymorphisms discussed above may act as disease modifiers in asthma or may be the basis for the known interindividual variation in the bronchodilating response to ⁇ -agonists (Liggett, S.B. "The genetics of ⁇ 2 - adrenergic receptor polymorphisms: relevance to receptor function and asthmatic phenotypes.” in: Liggett, S.B. & Meyers, D.A., The Genetics of Asthma (1996) pp. 455-478). Indeed, two groups have reported that individuals homozygous or heterozygous for the Argl6 variant are more likely to respond to albuterol than individuals homozygous for the Glyl6 variant.
• polymorphic sites correspond to the nucleotide positions 879 and 1182 in the promoter region of the ⁇ 2 AR gene (see Fig. 1) and are designated PS2 and PS5, respectively, to reflect their order in the ⁇ 2 AR gene relative to the other 11 polymorphic sites (Table 3).
• the polymorphisms at these sites are cytosine or adenine at PS2 and cytosine or thymine at PS5. It is believed that ⁇ 2 AR-encoding polynucleotides containing one or more of these novel polymorphic sites will be useful in studying the expression and biological function of ⁇ 2 AR, as well as in developing drugs targeting this receptor.
• the inventors have determined the identity of the alternative nucleotides present at these sites, and at the previously identified 11 polymorphic sites described above (see Table 3), in a human reference population of apparently normal unrelated individuals representing four major population groups and in a cohort of asthma patients.
• the inventors herein have also identified how the polymorphisms at these 13 polymo ⁇ hic sites in the ⁇ 2 AR gene (see Figure 1) are combined into haplotypes in the reference and patient populations (see Tables 4 and 5) and discovered that certain pairs of these haplotypes, designated 2/2, 2/4, 2/6, 4/4 and 4/6 are predictive of bronchodilator response to albuterol.
• the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymo ⁇ hic variant of a reference sequence for the ⁇ 2 AR gene or a fragment thereof.
• the reference sequence comprises SEQ ID NO:l and the polymo ⁇ hic variant comprises at least one polymo ⁇ hism selected from the group consisting of adenine at PS2 and thymine at PS 5.
• a particularly preferred polymo ⁇ hic variant is a naturally-occurring isoform (also referred to herein as an "isogene") of the ⁇ 2 AR gene.
• a ⁇ 2 AR isogene of the invention comprises guanine or adenine at PSl, cytosine or adenine at PS2, guanine or adenine at PS3, guanine or cytosine at PS4, cytosine or thymine at PS5, cytosine or thymine at PS6, cytosine or thymine at PS7, thymine or cytosine at PS8, adenine or guanine at PS9, cytosine or guanine at PS 10, guanine or adenine at PSl 1, cytosine or thymine at PS 12, and cytosine or adenine at PS 13.
• the invention also provides a collection of ⁇ 2 AR isogenes, referred to herein as a ⁇ 2 AR genome anthology.
• a ⁇ 2 AR isogene may be defined by the combination and order of these polymo ⁇ hisms in the isogene, which is referred to herein as a ⁇ 2 AR haplotype.
• the invention also provides data on the number of different ⁇ 2 AR haplotypes found in the above four population groups. This haplotype data is useful in methods for deriving a ⁇ 2 AR haplotype from an individual's genotype for the ⁇ 2 AR gene and for determining an association between a ⁇ 2 AR haplotype and a particular trait.
• the invention provides a recombinant expression vector comprising one of the polymo ⁇ hic ⁇ 2 AR genomic 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 ⁇ AR for protein structure analysis and drug binding studies.
• the invention also provides methods, compositions, and kits for haplotyping and/or genotyping the ⁇ 2 AR gene in an individual.
• the genotyping method comprises isolating from the individual a nucleic acid mixture comprising the two copies of the ⁇ 2 AR gene present in the individual and determining the identity of the nucleotide pair at one or both of PS2 and PS5 in the two copies to assign a ⁇ 2 AR genotype to the individual.
• a method for predicting an individual's haplotype pair comprises determining the individual's genotype at PS3, PS9 and PS 11.
• the compositions contain oligonucleotide probes or primers designed to specifically hybridize to one or more target regions containing, or that are adjacent to, one or both of PS2 and PS5.
• Kits of the invention comprise a set of oligonucleotides for genotyping at least PS3, PS9 and PSl 1 and may also comprise additional oligonucleotides for genotyping one or more additional polymo ⁇ hic sites selected from the group consisting of PSl, PS2, PS4, PS5, PS6, PS7, PS8, PS10, PS12 and PS13.
• the methods and compositions for genotyping or haplotyping PS2 and PS 5 in the ⁇ 2 AR gene are useful for studying the effect of the alternative nucleotides at PS2 and PS5 in the etiology of various diseases and efficacy of drugs targeting the ⁇ 2 AR.
• Methods and kits for genotyping and haplotyping PS3, PS9 and PSl 1 are useful for predicting an asthmatic patient's bronchodilating response to ⁇ -agonists.
• the invention provides a method for identifying an association between a ⁇ 2 AR haplotype and a trait.
• the trait is susceptibility to a disease, disease severity, the staging of a disease or response to a drug.
• Such methods have applicability in developing diagnostic tests and therapeutic treatments for one or more conditions selected from the group consisting of congestive heart failure, arrhythmia, ischemic heart disease, hypertension, migraine, asthma, chronic obstructive pulmonary disease (COPD), anaphylaxis, obesity, diabetes, myasthenia gravis (MG) and premature labor.
• the drug is an agonist or antagonist of ⁇ 2 AR.
• the present invention also provides genetically modified animals comprising one or more of the novel ⁇ 2 AR genomic polymo ⁇ hic variants described herein and methods for producing such animals. Such animals are useful for studying expression of the ⁇ 2 AR isogenes in vivo, for in vivo screening and testing of drugs targeted against ⁇ AR protein, and for testing the efficacy of therapeutic agents and compounds targeting the ⁇ 2 AR in a biological system.
• the present invention also provides a computer system for storing and displaying polymo ⁇ hism data determined for the ⁇ 2 AR gene.
• the computer system comprises a computer processing unit; a display; and a database containing the polymo ⁇ hism data.
• the polymo ⁇ hism data includes the polymo ⁇ hisms, the genotypes and the haplotypes identified for the ⁇ 2 AR gene in one or both of the reference population and the patient population.
• the computer system is capable of producing a display showing ⁇ AR haplotypes organized according to their evolutionary relationships.
• Another aspect of the invention is based on the discovery of novel information relating to linkage disequilibrium in Caucasians between PSl, PS3, PS4 and PS6 and the ⁇ 2 AR polymo ⁇ hic sites previously reported to be associated with various medical conditions, i.e., PS9 and PS 10.
• the present invention also provides a method for predicting a Caucasian individual's genetic predisposition to any disease or condition known to be associated with one of the alternative alleles at PS9 or PS 10 in the ⁇ 2 AR gene.
• the method comprises determining a first genotype for a first polymo ⁇ hic site in the individual's ⁇ 2 AR gene, wherein the first polymo ⁇ hic site is selected from the group consisting of PSl, PS3, PS4 and PS6, and using the first genotype to predict a second genotype for a second polymo ⁇ hic site in the individual's ⁇ AR gene, wherein if the first polymo ⁇ hic site is PSl, PS4 or PS6, then the second polymo ⁇ hic site is PS 10, and if the first polymo ⁇ hic site is PS3, then the second polymo ⁇ hic site is PS9.
• the disease or condition is selected from the group consisting of atopy, nocturnal asthma, childhood asthma, hypertension, obesity, response to treatment for obesity and MG.
• the present invention further provides methods for predicting an asthma patient's response to ⁇ -agonist therapy.
• the method comprises determining the genotype for the patient at PS3, PS9 and PSl 1, wherein the patient is likely to exhibit a good response to a standard dose of the ⁇ -agonist if the patient is homozygous for guanine at each of PS3, PS9 and PSl 1 or if the patient is heterozygous A/G at each of PS3, PS9 and PSl 1.
• the patient is homozygous A/A/G at PS3, PS9 and PSl 1, respectively, then the patient is likely to not respond to standard dosages of the ⁇ -agonist.
• the ⁇ -agonist is albuterol.
• Figure 1 illustrates the DNA sequence for a reference sequence of the human ⁇ 2 AR gene (GenBank Accession No. M15169.1; SEQ ID NO:l), with the underlines indicating the start and stop codons, shading indicating the reference coding sequence, and bold nucleotides indicating the polymo ⁇ hic sites and polymo ⁇ hisms identified by Applicants in the reference and asthma patient populations.
• Figure 2 illustrates a reference amino acid sequence for ⁇ 2 AR (contiguous lines; SEQ ID NO:2), with the bold amino acids indicating the amino acid variations caused by the polymo ⁇ hisms at nucleotides 1633, 1666 and 2078 in the ⁇ 2 AR gene.
• Figure 3 illustrates the phylogeny of haplotypes of the ⁇ 2 AR gene, with each haplotype represented by a circle, the area of which representing the overall frequency of that haplotype in the reference population, and subdivisions representing the frequency of the haplotype in each of the four population groups. Differences between the haplotypes are indicated by the lines connecting the circles, with solid black lines for single-site differences, gray lines for two-site differences and dashed lines for more than two differences.
• Figure 4 illustrates the linkage disequilibrium between thirteen polymo ⁇ hic sites in the ⁇ 2 AR gene with the degree of linkage indicated by shading.
• Figure 5 shows a bar graph of the in vivo FEVi responses to albuterol exhibited by asthma patients having the indicated haplotype pairs.
• Figure 6 A shows a bar graph illustrating the amount of ⁇ 2 AR protein expression in HEK293 cells co-transfected with a vector containing the indicated ⁇ 2 AR isogene and a luciferase construct.
• Figure 6B shows a bar graph illustrating the amount of ⁇ AR protein expression from Fig. 6A after correction for transfection efficiency as quantitated by luciferase activity.
• Figure 6C shows a bar graph of ⁇ 2 AR mRNA expression in the transfected HEK293 cells of Fig. 6 A.
• the inventors herein have discovered novel variants of the ⁇ 2 AR gene.
• two novel polymo ⁇ hic sites by characterizing an approximate 1.8 kb region of the ⁇ 2 AR gene found in genomic DNAs isolated from an asthmatic cohort (121 Caucasians and 13 African Americans) and an Index Repository that contains immortalized cell lines from 93 human individuals, 76 of which comprised a reference population of unrelated individuals self-identified as belonging to one of four major population groups: Caucasian (23 individuals), African descent (19 individuals), Asian (20 individuals) and Hispanic-Latino (15 individuals).
• the Index Repository contains three families: two three-generation Caucasian families from the CEPH-Utah cohort and one two-generation African- American family.
• the inventors herein Using the ⁇ 2 AR genotypes identified in the Index Repository and patient cohort, in conjunction with the methodology described in the Examples below, the inventors herein also determined the haplotypes found on each chromosome for most individuals in these populations, and determined the frequencies of these haplotypes in the four major population groups.
• the ⁇ 2 AR genotypes and haplotypes found in the Index Repository and patient cohort include those shown in Table 4 and 5, respectively.
• ⁇ 2 AR polymo ⁇ hism and haplotype data disclosed herein are useful for studying population diversity, anthropological lineage, the significance of diversity and lineage at the phenotypic level, paternity testing, forensic applications, and for identifying associations between ⁇ 2 AR genetic variation and a trait such as level of drug response or susceptibility to disease.
• certain pairs of these ⁇ 2 AR haplotypes designated 2/2, 2/4, 2/6, 4/4 and 4/6 are predictive of bronchodilator response to albuterol.
• the present invention is useful in prescribing ⁇ -agonists for treating bronchospasm.
• Allele - A particular form of a genetic locus, distinguished from other forms by its particular nucleotide or amino acid sequence.
• 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.
• Full-genotype The unphased 5 ' to 3 ' sequence of nucleotide pairs found at all known polymo ⁇ hic sites 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 known polymo ⁇ hic sites in a locus on a pair of homologous chromosomes in a single individual.
• Genotyping A process for determining a genotype of an individual.
• Haplotype - A phased 5 ' to 3 ' sequence of nucleotides found at two or more polymo ⁇ hic sites in a locus on a single chromosome from a single individual.
• Haplotyping - A process for determining a haplotype of an individual.
• Haplotype pair The two haplotypes found for a locus in a single individual.
• Full-haplotype The 5 ' to 3 ' sequence of nucleotides found at all known polymo ⁇ hic sites 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 known polymo ⁇ hic sites in a locus on a single chromosome from a single individual.
• 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 or the protein encoded thereby, distinguished from other forms by its particular sequence and/or structure.
• Isogene One of the isoforms of a gene found in a population. An isogene 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 for practical pu ⁇ oses free of other biological molecules such as non-desired 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.
• 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 corresponding strands of the two copies of a chromosome in 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 The sequence variation observed in an individual at a polymo ⁇ hic site.
• Polymo ⁇ hisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.
• Polymorphism Database A collection of polymo ⁇ hism data arranged in a systematic or methodical way and capable of being individually accessed by electronic or other means.
• 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%.
• Single Nucleotide Polymorphism (SNP) - Typically, the specific pair of nucleotides observed at a single polymo ⁇ hic site. In rare cases, three or four nucleotides may be found.
• Subject A human individual whose genotypes or haplotypes or response to treatment or disease state are to be determined.
• Treatment A stimulus administered internally or externally to an individual.
• Unphased As applied to a sequence of nucleotide pairs for two or more polymo ⁇ hic sites in a locus, unphased means the combination of nucleotides present at those polymo ⁇ hic sites on a single copy of the locus is not known.
• the inventors herein have discovered two novel polymo ⁇ hic sites in the ⁇ 2 AR gene, which are referred to as of PS2 and PS 5 to designate the order in which they are located in the gene (see Figure 1 and Table 3 below).
• PSl, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl 1, PS12, and PS13 designate the previously identified polymo ⁇ hic sites located at nucleotides 565, 934, 1120, 1221, 1541, 1568, 1633, 1666, 1839, 2078 and 2110 in Figure 1.
• the invention provides an isolated polynucleotide comprising a polymo ⁇ hic variant of the ⁇ 2 AR 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 ⁇ 2 AR gene is identical to the reference genomic sequence for the region of the gene examined, as described in the Examples below, except that it comprises a different nucleotide at one or both of polymo ⁇ hic sites PS2 and PS5.
• nucleotide sequence of a variant fragment of the ⁇ 2 AR gene is identical to the corresponding portion of the reference sequence except for having a different nucleotide at one or more of these polymo ⁇ hic sites.
• the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence (or other reported ⁇ 2 AR sequences) or to portions of the reference sequence (or other reported ⁇ 2 AR sequences), except for genotyping oligonucleotides as described below.
• the location of a polymo ⁇ hism in a variant gene or fragment is identified by aligning its sequence with SEQ ID NO:l.
• the polymo ⁇ hism is selected from the group consisting of cytosine at PS2 and cytosine at PS 5.
• the polymo ⁇ hic variant comprises a naturally-occurring isogene of the ⁇ 2 AR gene which is defined by any one of haplotypes 1-12 shown in Table 5 below.
• Polymo ⁇ hic variants of the invention may be prepared by isolating a clone containing the ⁇ 2 AR gene from a human genomic library.
• the clone may be sequenced to determine the identity of the nucleotides at the polymo ⁇ hic sites described herein. Any particular variant claimed herein could be prepared from this clone by performing in vitro mutagenesis using procedures well-known in the art.
• a polymo ⁇ hic variant of the ⁇ 2 AR gene may be chemically synthesized.
• ⁇ 2 AR isogenes may be isolated using any method that allows separation of the two "copies" of the ⁇ 2 AR 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 copending U.S. application Serial No. 08/987,966.
• Another method which is described in copending U.S. Application Serial No. 08/987,966, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets.
• Yet other methods are single molecule dilution (SMD) as described in Ruano et al., Proc. Natl. Acad. Sci.
• the invention also provides ⁇ 2 AR genome anthologies, which are collections of ⁇ 2 AR 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 sex population.
• a ⁇ 2 AR genome anthology may comprise individual ⁇ 2 AR 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 ⁇ 2 AR isogenes in the anthology may be stored in separate containers. Individual isogenes or groups of isogenes in a genome anthology may be stored in any convenient and stable form, including but not limited to in buffered solutions, as DNA precipitates, freeze-dried preparations and the like.
• a preferred ⁇ 2 AR 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 ⁇ 2 AR 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, retroviruses, 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 "Current Protocols in Molecular Biology", John Wiley and Sons, New York, New York).
• Host cells which may be used to express the variant ⁇ 2 AR 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.
• Preferred 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 preferred host cells are mammalian cells.
• nucleic acid molecules containing the ⁇ 2 AR gene may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the corresponding 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 ⁇ AR genomic variants described herein.
• Polynucleotides comprising a polymo ⁇ hic gene variant or fragment 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 ⁇ 2 AR isogene encoding that isoform or may already have at least one copy of that isogene. hi other situations, it may be desirable to decrease or block expression of a particular ⁇ 2 AR isogene.
• Expression of a ⁇ 2 AR 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 for the isogene.
• 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 preferred. Similarly, 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. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994).
• regulatory regions e.g., promoter, introns, enhancers, 3 ' untranslated region
• Oligonucleotides targeting the transcription initiation site e.g., between positions -10 and +10 from the start site are preferred.
• inhibition of transcription can be achieved using oligonu
• Antisense oligonucleotides may also be designed to block translation of ⁇ 2 AR mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of ⁇ 2 AR mRNA transcribed from a particular isogene.
• the 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.
• the oligonucleotides 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. Effect(s) of the novel polymo ⁇ hisms and haplotypes identified herein on expression of ⁇ 2 AR may be investigated by preparing recombinant cells and/or organisms, preferably recombinant animals, containing a polymo ⁇ hic variant of the ⁇ 2 AR 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 into ⁇ AR protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
• the desired ⁇ AR isogene may be introduced into the cell in a vector such that the isogene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location.
• the ⁇ 2 AR isogene is introduced into a cell in such a way that it recombines with the endogenous ⁇ 2 AR gene present in the cell.
• Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired ⁇ 2 AR 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 ⁇ 2 AR isogene may be introduced include, but are not limited to, continuous culture cells, such as COS, NTH/3T3, and primary or culture cells of the relevant tissue type, i.e., they are known to express ⁇ 2 AR, such as the human embryonic kidney cell line HEK293. Such recombinant cells can be used to compare the biological activities of the different protein variants.
• Recombinant organisms i.e., genetically modified animals, expressing a variant ⁇ 2 AR gene are prepared using standard procedures known in the art.
• a construct comprising the variant human gene 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.
• Genetically-modified 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 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.
• a third method involves directly injecting a transgene into the embryo.
• a third method involves the use of embryonic stem cells.
• the genetic modification process results in replacement of the animal's ⁇ 2 AR gene with the human ⁇ 2 AR gene.
• animals into which the human ⁇ 2 AR isogenes may be introduced 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).
• Recombinant nonhuman animals stably expressing a human ⁇ 2 AR isogene and producing human ⁇ 2 AR protein can be used as biological models for studying diseases related to abnormal ⁇ 2 AR 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.
• An additional embodiment of the invention relates to pharmaceutical compositions for treating disorders affected by expression or function of a novel ⁇ 2 AR isogene described herein.
• disorders include congestive heart failure, arrhythmia, ischemic heart disease, hypertension, migraine, asthma, chronic obstructive pulmonary disease (COPD), anaphylaxis, obesity, diabetes, myasthenia gravis, and premature labor.
• COPD chronic obstructive pulmonary disease
• the pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these novel ⁇ 2 AR isogenes; an antisense oligonucleotide directed against one of the novel ⁇ 2 AR isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which activates of inhibits expression of a novel ⁇ 2 AR isogene described herein.
• the composition contains the active ingredient in a therapeutically effective amount.
• therapeutically effective amount is meant that one or more of the symptoms relating to disorders related to the expression or function of a novel ⁇ AR isogene is reduced and/or eliminated.
• the 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).
• determination of the therapeutically effective dose of active ingredient and/or the appropriate route of administration is well within the capability of those skilled in the art.
• 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 drugs being taken by the patient, and tolerance/response to the treatment.
• the invention also provides compositions and methods for detecting the novel ⁇ 2 AR polymo ⁇ hisms and haplotypes identified herein.
• compositions comprise at least one ⁇ 2 AR genotyping oligonucleotide.
• a ⁇ AR genotyping oligonucleotide is a probe or primer capable of hybridizing to a target region that is located close to, or that contains, one of the novel polymo ⁇ hic sites described herein.
• the term "oligonucleotide” refers to a polynucleotide molecule having less than about 100 nucleotides.
• a preferred 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.
• 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.
• Genotyping oligonucleotides of the invention must be capable of specifically hybridizing to a target region of a ⁇ 2 AR polynucleotide, i.e., a ⁇ 2 AR 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 a non-target region or a non- ⁇ 2 AR 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 ⁇ 2 AR gene using the polymo ⁇ hism information provided herein in conjunction with the known sequence information for the ⁇ 2 AR 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 corresponding 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 oligonucleotide probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
• Preferred 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 oligonucleotide 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 15 mer, the 8 th or 9 th position in a 16mer, the 10 th or 11 th position in a 20 mer).
• a preferred ASO probe for detecting ⁇ 2 AR gene porymo ⁇ hisms at PS2 and PS5 comprises a nucleotide sequence selected from the group consisting of:
• An allele-specific oligonucleotide 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. Allele-specific oligonucleotide primers hybridizing to either the coding or noncoding strand are contemplated by the invention.
• a preferred ASO forward primer for detecting ⁇ 2 AR gene polymo ⁇ hisms at PS2 and PS 5 comprises a nucleotide sequence selected from the group consisting of: CGAGTGTGCATGTCG (SEQ ID NO:7); CTCCCAGCTCACCGA (SEQ ID NO:8);
• genotyping 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 ⁇ 2 AR gene polymo ⁇ hisms and thus are referred 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 preferred oligonucleotide primer for detecting ⁇ 2 AR gene polymo ⁇ hisms at PS2 and PS5 by primer extension terminates in a nucleotide sequence selected from the group consisting of:
• GTGTGCATGT SEQ ID NO: 15
• CCAGCTCACC SEQ ID NO: 16
• a composition contains two or more differently labeled genotyping oligonucleotides for simultaneously probing the identity of nucleotides 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. As further described below, the inventors herein have discovered that a patient's bronchodilating response to albuterol may be predicted with high confidence by genotyping only three of the polymo ⁇ hic sites in the ⁇ 2 AR gene: PS3, PS9 and PS 11.
• the invention also provides a diagnostic kit for predicting an individual's response to a ⁇ -agonist.
• the kit comprises a set of genotyping oligonucleotides for genotyping PS3, PS9 and PSl 1 in the ⁇ 2 AR gene packaged in a container.
• the kit may also contain other components such as hybridization buffer, where the oligonucleotides are to be used as allele-specific probes, or dideoxynucleotide triphosphates (ddNTPs), where the polymo ⁇ hic sites are to be detected by primer extension, hi a preferred embodiment, the set of genotyping oligonucleotides consists of three primer extension oligonucleotides, one for genotyping PS3, one for genotyping PS9 and one for genotyping PS 11.
• the kit may also contain a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase.
• kits may also include detection reagents, such as biotin- or fluorescent-tagged oligonucleotides or ddNTPs and/or an enzyme- labeled antibody and one or more substrates that generate a detectable signal when acted on by the enzyme.
• detection reagents such as biotin- or fluorescent-tagged oligonucleotides or ddNTPs and/or an enzyme- labeled antibody and one or more substrates that generate a detectable signal when acted on by the enzyme.
• each of the genotyping oligonucleotides and all other reagents in the kit have been quality tested for optimal performance in a genotyping assay for each of PS3, PS9 and PSl 1 and the kit also contains instructions for performing the assay and assigning a ⁇ 2 AR haplotype pair from the results.
• ⁇ 2 AR genotyping oligonucleotides of the invention may also be immobilized on or synthesized on a solid surface such as a microchip, bead, or glass slide (see, e.g., WO 98/20020 and WO 98/20019). Such immobilized genotyping oligonucleotides may be used in a variety of polymo ⁇ hism detection assays, including but not limited to probe hybridization and polymerase extension assays.
• Immobilized ⁇ 2 AR genotyping oligonucleotides of the invention may comprise an ordered array of oligonucleotides designed to rapidly screen a DNA sample for polymo ⁇ hisms in multiple genes at the same time.
• ⁇ 2 AR genotype and “ ⁇ 2 AR haplotype” mean the genotype or haplotype contains the nucleotide pair or nucleotide, respectively, that is present at one or both of PS2 and PS5 and may optionally also include the nucleotide pair or nucleotide present at one or more additional polymo ⁇ hic sites in the ⁇ 2 AR gene.
• the additional polymo ⁇ hic sites may be currently known polymorphic sites or sites that are subsequently discovered.
• the additional polymo ⁇ hic sites are selected from the group consisting of PSl, PS3, PS4, PS6, PS7, PS8, PS9, PS10, PS11, PS12 and PS13.
• One embodiment of the genotyping method involves isolating from the individual a nucleic acid mixture comprising the two copies of the ⁇ 2 AR gene, or a fragment thereof, that are present in the individual, and determining the identity of the nucleotide pair at one or more of PS2 and PS5 in the two copies to assign a ⁇ 2 AR genotype to the individual.
• the two "copies" of a gene in an individual may be the same allele or may be different alleles.
• the genotyping method comprises determining the identity of the nucleotide pair at each of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl 1, PS12, and PS13.
• the nucleic acid mixture 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 mixture may be comprised of genomic DNA, mRNA, or cDNA and, in the latter two cases, the biological sample must be obtained from an organ in which the ⁇ 2 AR gene is expressed.
• mRNA or cDNA preparations would not be used to detect polymo ⁇ hisms located in introns or in 5 ' and 3 ' nontranscribed regions. If a ⁇ 2 AR gene fragment is isolated, it must contain the polymo ⁇ hic site(s) to be genotyped.
• One embodiment of the haplotyping method comprises isolating from the individual a nucleic acid molecule containing only one of the two copies of the ⁇ 2 AR gene, or a fragment thereof, that is present in the individual and determining in that copy the identity of the nucleotide at one or both of polymo ⁇ hic sites PS2 and PS5 in that copy to assign a ⁇ 2 AR haplotype to the individual.
• the nucleic acid may be isolated using any method capable of separating the two copies of the ⁇ 2 AR gene or fragment such as one of the methods described above for preparing ⁇ 2 AR isogenes, with targeted in vivo cloning being the preferred approach.
• any individual clone will only provide haplotype information on one of the two ⁇ 2 AR gene copies present in an individual. If haplotype information is desired for the individual's other copy, additional ⁇ 2 AR clones will need to be examined. Typically, at least five clones should be examined to have more than a 90% probability of haplotyping both copies of the ⁇ 2 AR gene in an individual.
• the nucleotide at each of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12 and PS13 is identified.
• a ⁇ 2 AR haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or both of PS2 and PS5 in each copy of the ⁇ 2 AR that is gene present in the individual, h a particularly preferred embodiment, the haplotyping method comprises identifying the phased sequence of nucleotides at each of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl 1, PS12 and PS13 in each copy of the ⁇ 2 AR gene.
• the identifying step is preferably performed with each copy of the gene being placed in separate containers.
• first and second copies of the gene are labeled with different first and second fluorescent dyes, respectively, and an genotyping 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.
• Another aspect of the invention is a method for predicting an individual's bronchodilating response to a ⁇ -agonist, which comprises assigning a ⁇ 2 AR haplotype pair to the individual and using the assigned haplotype pair to make a response prediction selected from the group consisting of: assignment of ⁇ AR haplotype pair 4/6 or 2/2 predicts a good bronchodilating response; assignment of ⁇ 2 AR haplotype pair 2/6 predicts an intermediate bronchodilating response and assignment of ⁇ 2 AR haplotype pair 2/4 or 4/4 predicts no bronchodilating response.
• These haploytpes pairs are set forth in Table 6.
• the ⁇ -agonist is albuterol.
• the assigning step comprises determining a genotype for PS3, PS9 and PSl 1 in the individual's ⁇ 2 AR gene and using the genotype to assign the haplotype pair.
• the genotype and haplotype pair combinations that are predictive of response to ⁇ -agonists are shown in Table 1 below.
• Table 4 Additional genotype and haplotype pair combinations that exist in the population are shown in Table 4 below.
• the ability to assign the ⁇ 2 AR haplotype pair by genotyping only PS3, PS9 and PSl 1 is based on linkage disequilibrium between certain groups of polymo ⁇ hic sites in the ⁇ 2 AR gene (Fig. 4) as well as the frequencies of the individual ⁇ 2 AR haplotypes in the different population groups (Table 5).
• an individual's ⁇ 2 AR haplotype pair is assigned by genotyping one or more additional polymo ⁇ hic sites selected from the group consisting of PSl, PS2, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS12 and PS13.
• the ability to predict a patient's response to a ⁇ -agonist is useful for physicians in making decisions about how to treat an asthma patient for bronchospasm.
• An asthma patient whose haplotype pair indicates the patient will probably respond well to the agonist would be a better candidate for ⁇ -agonist therapy than a patient who is likely to exhibit an intermediate response or no response, and the physician would be able to determine with less trial and error which individuals should receive an alternative form of therapy.
• the following pairs of ⁇ 2 AR sites exhibit about 100% linkage disequilibrium in Caucasians: PSl and PS10 (amino acid 27); PS3 and PS9 (amino acid 16); PS4 and PS10; and PS6 and PS 10.
• This provides the basis for another aspect of the invention, which is a method for predicting a Caucasian individual's genotype for one or both of PS9 and PS 10, both of which have been linked to various diseases and conditions.
• the method comprises determining a first genotype for one or more of PSl, PS3, PS4 or PS6 in the individual's ⁇ AR gene and using the first genotype to assign a second genotype for one or both of PS9 and PS10 to the individual.
• the first and second genotypes correspond to one of the genotype combinations shown in Table 2 below:
• the first genotype is for PS3 and one or more of PSl, PS4 and PS6, and the second genotype is for both PS9 and PS 10.
• This genotyping prediction method is useful for predicting the individual's predisposition to a disease or condition associated with one of the alternative alleles at PS9 or PS 10 in the ⁇ 2 AR gene.
• a PS9 genotype of G/G means the individual expresses only the ⁇ 2 AR Glyl6 variant and is predisposed to atopy and nocturnal asthma
• a PS9 genotype of A/A indicates the individual expresses only the ⁇ 2 AR Argl6 variant and is predisposed to MG.
• a PS 10 genotype of C/C means the individual expresses only the ⁇ 2 AR Gln27 variant and is predisposed to childhood asthma, while a PS 10 genotype means the individual expresses only the ⁇ 2 AR Glu27 variant and is predisposed to obesity.
• the first genotype is for PS3 and one or more of PSl, PS4, and PS6, the genotypes at PS9 and PS 10 can be predicted, and such genotypes are useful for predicting severity of asthma (G/G at PS9, G/G at PS 10) and BHR.
• the present invention is not limited to the ⁇ 2 AR polymo ⁇ hism or haplotype associations presently known but is applicable to future discoveries of associations between polymo ⁇ hisms or haplotype for PS9 and PS 10 and disease, severity of disease, staging of disease, or any other phenotype.
• genotyping and haplotyping methods of the invention may be performed in combination with identifying the genotype(s) and/or ha ⁇ lotype(s) for other genomic regions.
• the identity of a nucleotide (or nucleotide pair) at a polymo ⁇ hic site may be determined by amplifying a target region(s) containing the polymo ⁇ hic site(s) directly from one or both copies of the ⁇ 2 AR gene present in the individual 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, referred 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).
• the target region(s) may be amplified using any oligonucleotide-directed amplification method, including but not limited to polymerase chain reaction (PCR) (U.S. Patent No.
• PCR polymerase chain reaction
• Oligonucleotide ligation assay LCR (Barany et al., Proc. Natl. Acad. Sci. USA 88:189-193, 1991; WO90/01069), and oligonucleotide ligation assay (OLA) (Landegren et al., Science 241:1077-1080, 1988).
• Oligonucleotides useful as primers or probes in such methods should specifically hybridize to a region of the nucleic acid that contains or is adjacent to the polymo ⁇ hic site. Typically, the oligonucleotides are between 10 and 35 nucleotides in length and preferably, between 15 and 30 nucleotides in length.
• the oligonucleotides are 20 to 25 nucleotides long.
• the exact length of the oligonucleotide will depend on many factors that are routinely considered and practiced by the skilled artisan.
• Other known nucleic acid amplification procedures may be used to amplify the target region including transcription-based amplification systems (U.S. Patent No. 5,130,238; EP 329,822; U.S. Patent No. 5,169,766, WO89/06700) and isothermal methods (Walker et L., Proc. Natl. Acad. Sci. USA 89:392-396, 1992.
• 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 may be 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 one or more polymo ⁇ hic sites in the ⁇ 2 AR gene of an individual may also be determined by hybridization of one or both copies of the gene, or a fragment thereof, to nucleic acid arrays and subarrays such as described in WO 95/11995.
• the arrays 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).
• 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 (WO92/15712) and the ligase/polymerase mediated genetic bit analysis (U.S. Patent 5,679,524.
• Related methods are disclosed in WO91/02087, WO90/09455, WO95/17676, and 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 above described genotyping methods are useful in methods for determining the frequency of a ⁇ 2 AR genotype or haplotype in a population.
• the method comprises determining the genotype or the haplotype pair for the ⁇ 2 AR gene that is present in each member of the population and calculating the frequency any particular ⁇ 2 AR genotype or haplotype is found in the population.
• the ⁇ 2 AR genotype comprises the nucleotide pair(s) detected at each of PS1-PS13.
• the population may be a reference population, a family population, a same sex population, a population group, 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 such ⁇ 2 AR genotypes or haplotypes in reference and trait populations are useful for identifying an association between a trait and any novel ⁇ 2 AR polymo ⁇ hism, genotype or haplotype.
• 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) or haplotype(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 the ⁇ 2 AR gene in each individual in the populations using one of the methods described above.
• the haplotypes for the trait population may be determined directly or, alternatively, by the predictive genotype to haplotype approach 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) or haplotype(s) of interest in the reference and trait populations are compared. In a preferred embodiment, the frequencies of all genotypes and/or haplotypes observed in the populations are compared.
• the trait is predicted to be associated with that ⁇ 2 AR genotype or haplotype.
• the ⁇ 2 AR genotype or haplotype being compared in the trait and reference populations is selected from the full-genotypes and full-haplotypes shown in Tables 4 and 5 respectively, or from sub-genotypes and sub-haplotypes derived from these genotypes and haplotypes.
• the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drug targeting ⁇ 2 AR 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 efficacy of the therapy, no efficacy, and adverse events (i.e., side effects).
• clinical population In order to deduce a correlation between clinical response to a treatment and a ⁇ 2 AR genotype or haplotype, 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 run 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 II and phase III clinical trials. Standard methods are used to define the patient population and to enroll subjects.
• selection of individuals for the clinical population comprises grading such candidate individuals for the existence of the medical condition of interest and then including or excluding individuals based upon the results of this assessment. This is important in cases where the symptom(s) being presented by the patients can be caused by more than one underlying condition, and where treatment of the underlying conditions are not the same. An example of this would be where patients experience breathing difficulties that are due to either asthma or respiratory infections. If both sets were treated with an asthma medication, there would be a spurious group of apparent non-responders that did not actually have asthma. These people would affect the ability to detect any correlation between haplotype and treatment outcome.
• This grading of potential patients could employ a standard physical exam or one or more lab tests. Alternatively, grading of patients could use haplotyping for situations where there is a strong correlation between haplotype pair and disease susceptibility or severity.
• the therapeutic treatment of interest or the control treatment (active agent or placebo in controlled trials), 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 ⁇ 2 AR gene for each individual in the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment. After both the clinical and polymo ⁇ hism data have been obtained, correlations between individual response and ⁇ 2 AR genotype or haplotype content are created.
• Correlations may be produced in several ways, h one method, individuals are grouped by their ⁇ 2 AR genotype or haplotype (or haplotype pair) (also referred to as a polymo ⁇ hism group), and then the averages and standard deviations of continuous clinical responses exhibited by the members of each polymo ⁇ hism group are calculated.
• the most-common and least common nucleotides at the polymo ⁇ hic site are first defined. Then, for each individual in the trial population, one calculates a "dose" as the number of least-common nucleotides the individual has at the polymo ⁇ hic site of interest. This value can be 0 (homozygous for the least-common nucleotide), 1 (heterozygous), or 2
• a second method for finding correlations between ⁇ 2 AR haplotype content and clinical responses uses predictive models based on error-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.
• C is the measured clinical outcome, i goes over all polymo ⁇ hic sites, ⁇ over all candidate genes, C 0 , w i a and w i ' a are variable weight values, R t a is equal to 1 if site i in gene ⁇ in the first haplotype takes on the most common nucleotide and -1 if it takes on the less common nucleotide.
• L i a is the same as R i a except for the second haplotype.
• the constant term C 0 and the weights w ija and w i ' a are varied by the genetic algorithm during a search process that minimizes the error between the measured value of C and the value calculated from Equation 1.
• Models other than the one given in Equation 1 can be readily inco ⁇ orated by those skilled in the art for analyzing the clinical and polymo ⁇ hism data.
• the genetic algorithm is especially suited for searching not only over the space of weights in a particular model but also over the space of possible models (Judson, supra).
• Correlations may also be analyzed using analysis of variation (ANOVA) techniques to determine how much of the variation in the clinical data is explained by different subsets of the polymo ⁇ hic sites in the ⁇ 2 AR gene.
• ANOVA is used to test hypotheses about whether a response variable is caused by or correlated with one or more traits or variables (in this case, polymo ⁇ hism groups) that can be measured (Fisher and vanBelle, supra, Ch. 10). These traits or variables are called the independent variables.
• the independent variable(s) are measured and individuals are placed into groups based on their values for these variables.
• the independent variable(s) refers to the combination of polymo ⁇ hisms present at a subset of the polymo ⁇ hic sites, and thus, each group contains those individuals with a given genotype or haplotype pair.
• the variation in response within the groups and also the variation between groups is then measured. If the within-group response variation is large (people in a group have a wide range of responses) and the response variation between groups is small (the average responses for all groups are about the same) then it can be concluded that the independent variables used for the grouping are not causing or correlated with the response variable. For instance, if people are grouped by month of birth (which should have nothing to do with their response to a drug) the ANOVA calculation should show a low level of significance. However, if the response variation is larger between groups than within groups, the F-ratio
• the calculated F-ratio is preferably compared with the critical F-distribution value at whatever level of significance is of interest. If the F-ratio is greater than the Critical F-distribution value, then one may be confident that the individual's genotype or haplotype pair for this particular subset of polymo ⁇ hic sites in the ⁇ 2 AR gene is at least partially responsible for, or is at least strongly correlated with the clinical response.
• a mathematical model may be readily constructed by the skilled artisan that predicts clinical response as a function of ⁇ 2 AR 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 (or haplotype pair) for the ⁇ 2 AR gene may be the basis for designing a diagnostic method to determine those individuals who will or will not respond to the treatment, or alternatively, will respond at a lower level and thus may require more treatment, i.e., a greater dose of a drug.
• 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 ⁇ 2 AR 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 ⁇ 2 AR gene
• serological test i.e., a serological test
• a physical exam measurement i.e., a physical exam measurement.
• this diagnostic method uses the predictive haplotyping method described above.
• 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 ⁇ 2 AR 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 ⁇ 2 AR 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.
• This example illustrates examination of the ⁇ 2 AR gene for polymo ⁇ hic sites from about 1100 base pairs upstream of the ATG start site to about 700 base pairs downstream of the ATG start site.
• Overlapping fragments of the ⁇ 2 AR gene were amplified from genomic DNA from the
• sequencing primers were designed to provide for overlapping ⁇ 500 bp reads.
• CDS means coding sequence
• This example illustrates analysis of the ⁇ 2 AR gene polymo ⁇ hisms identified in the Index Repository and asthmatic cohort for genotypes and haplotypes.
• haplotype pairs shown in the Table 4 were estimated from the unphased genotypes using an extension of Clark's algorithm (Clark, A.G. (1990) Mol Bio Evol 7, 111-122), in which 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. In our analysis the list of haplotypes was augmented with haplotypes obtained from three families (two multi-generation Caucasian families, one two-generation African- American family).
• CA Caucasian
• AA African- American
• AS Asian
• HL Hispanic-Latinos
• Haplotype 2 the most frequent in Caucasians (48%) is only seen at frequencies of 6%, 10%, and 27% in samples of African- Americans, Asians, and Hispanic Latinos, respectively. Furthermore, this particular haplotype is by far the most distinctive at the nucleotide level, having unique differences at four sites from all other haplotypes sampled. The distribution of haplotype 1 also indicates population differentiation at this locus with a >20 fold lower frequency in Caucasians compared to the other groups. Also, haplotype 6 is more common in African- Americans and Asians compared to the other two groups. In contrast to the above, the frequency of haplotype 4 is similar in all groups. Assigning haplotypes from unphased genotype data from 200 individuals using the above derivation protocol gave the same results as molecular haplotyping except in a single subject due to a discrepancy at one SNP position (data not shown).
• Example 3 This example illustrates phylo genetic analysis and linkage disequilibrium analysis of the individual ⁇ 2 AR haplotypes shown in Table 5.
• This analysis used a variation of the minimal spanning network algorithm (Excoffier, L., et al., (1992) Genetics 131, 479-491.).
• An advantage of this algorithm over other methods is that it does not force a strictly bifurcating tree as a result.
• actual reticulations in the "tree” such as those arising from evolutionary recombination among the haplotypes, can be visualized and inte ⁇ reted. With this approach every haplotype is connected to the haplotype(s) most similar to itself and the results are shown in Figure 3.
• haplotype 12 appears to be a recombinant between haplotypes 1 and 2.
• haplotype 8 is best explained as a recombination between the highly frequent haplotypes 4 and 6.
• Example 4 This example illustrates analysis of the ⁇ 2 AR haplotypes in Table 5 for association with asthma patients' response to albuterol.
• the patients in the asthma cohort were enrolled from an outpatient facility as described in detail elsewhere (Yan, L., Galinsky, R.E., Bernstein, J.A., Liggett, S.B. & Weinshilboum, R.M. (1999) Pharmacogenetics in Press). Patients underwent spirometry before and 30 min after inhalation of 180 ⁇ g albuterol delivered by nebulization. Forced expiratory volume in 1 second (FEVi) and forced vital capacity (FVC) were determined in triplicate. The predicted values for these measurements were calculated based on standard algorithms (Morris, J.F., Koski, A. & Johnson, L.C. (1971) Am Rev Respir Dis 103, 57-67).
• FEVi Forced expiratory volume in 1 second
• FVC forced vital capacity
• the change in the % predicted FEV! was considered the primary measure of responsiveness to albuterol (Dales, R.E., Spitzer, W.O., Tousignant, P., Schechter, M. & Suissa, S. (1988) Am Rev Respir Dis 138, 317-320).
• the two rare SNPs at -709 and -406 were not found.
• the haplotypes discussed below continue to list these positions although they were invariant in the Caucasian population group in this cohort.
• No other differences were found in the frequencies of the haplotypes between the Index Repository and the asthmatic population.
• the haplotypes were assembled as pairs, and the eighteen haplotype pairs that were found in the asthmatic cohort are shown in Table 6 below.
• haplotype study discussed herein.
• Two other studies reported limited (2 site) ⁇ 2 AR haplotypes and some association with asthma, neither of which were related to the bronchodilator response to albuterol.
• haplotype encompassing only PS9 and PS 10 and asthma death/near death and severity was examined. Death/near death was not associated with a haplotype, although the more moderate asthmatics were more likely to have the Glyl6/Gln27 haplotype than mild asthmatics.
• the inventors also performed regression calculations to determine the minimal number of polymo ⁇ hisms that predict association between ⁇ 2AR polymo ⁇ hisms and response to albuterol.
• the regression placed individuals in the clinical cohort into groups having 0, 1, or 2 copies of a haplotype or SNP. It then calculated the significance (reported as a p-value) of the regression line connecting number of copies of the haplotype with response (change in % FEV1). Each site was tested independently to find even marginal association (p ⁇ 0.1). Any pair, triple, etc. of sites that showed this nominal association were further combined into haplotypes (sub-haplotypes). A p- value was calculated for the association between each sub-haplotype with response/non-response.
• a haplotype containing n sites was discarded if there was an m-site haplotype with a greater association (smaller p-value) if m ⁇ n. In other words, we favor the simplest association or explanation of the effect, and reject more complex associations. The most significant association was found with the 3 -site haplotype comprised of polymo ⁇ hic sites 3, 9, and 11.
• Example 5 This example illustrates analysis of the amount of in vitro expression of two of the predictive ⁇ 2 AR haplotype pairs.
• transfection studies were carried out in the human embryonic kidney cell line HEK293. These cells were chosen because of their high transfection efficiency, their human origin, and the fact that they express ⁇ 2 AR ( ⁇ 10 fmol/mg) and thus presumably have the relevant transcription factors for expression of the human gene.
• PCR products of the ⁇ 2 AR gene representing haplotypes 2 and 4 were derived from human genomic DNA, subcloned into pCR2.1 and the sequence verified.
• the pCR2.1 vector lacks a eukaryotic responsive promoter and thus the expression of the ⁇ 2 AR gene in mammalian cells is directed by the included ⁇ 2 AR promoter sequence.
• HEK293 cells were transfected using a lipofectamine method (Gene Therapy Systems, San Diego, CA). Cells were transfected with 10 ⁇ g ⁇ AR plasmid, 2 ⁇ g luciferase plasmid, and 50 ⁇ l of lipofectamine reagent. Two days later cells were harvested for radioligand binding, mRNA studies, and luciferase activity as previously described (McGraw, D.W., Forbes, S.L., Kramer, L.A. & Liggett, S.B.
• ⁇ 2 AR density is thus expressed as fmol/mg membrane protein or fmol/light unit (fmol/LU) as previously described (McGraw et al., supra).
• mRNA levels were determined using ribonuclease protection assays with a 563 bp antisense riboprobe corresponding to the most 3' region of the ⁇ 2 AR ORF as described previously (McGraw, D.W., Forbes, S.L., Witte, D.P., former, C.N., Paul, R.j. & Liggett, S.B. (1999) JBiol Chem 274, 32241-32247).
• ⁇ - actin mRNA was simultaneously quantitated confirming the equivalent loading of the samples.
• Comparisons of the sequence of haplotypes 2 and 4 reveal eight differences in the thirteen SNP positions. These include differences in amino acid 19 of the BUP, and amino acids 16 and 27 of the receptor protein. Each of these, studied in isolation, have been shown to alter expression or trafficking of the receptor (Green, S., Turki, J., Innis, M. & Liggett, S.B. (1994) Biochem 33, 9414-9419; McGraw, D.W., Forbes, S.L., Kramer, L.A. & Liggett, S.B. (1998) J Clin Invest 102, 1927-1932), but the effects of the various SNP combinations at these loci have not previously been explored.
• PS4, PS6 and PS8 are located within, or closely flank, a number of potential cz ' s-acting elements.
• PSl flanks potential binding sites for AP-4 and C/EBP; PS3 and PS4 each flank an NF-1 consensus sequence; and PS 6 is within a CP2 consensus sequence.
• the inventors identified thirteen polymo ⁇ hic sites in a contiguous region of the 5' upstream and coding sequence of the ⁇ 2 AR in humans. Twelve distinct haplotypes were represented in a population of four major ethnic groups. Several relatively recent recombination events appear to be responsible for some haplotypes. A striking divergence in ethnic distribution was found for several haplotypes. Five haplotype pairs were common in asthmatics, and there were clear differences in the in vivo response to a ⁇ 2 AR agonist based on haplotype pair. In contrast, no isolated SNP had any predictive utility. The homozygous haplotypes 2/2 and 4/4 with divergent agonist efficacies were shown to have differential effects on ⁇ 2 AR gene and protein expression in vitro, consistent with the direction and magnitude of the in vivo responses.

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