WO2000031307A1 - POLYMORPHISMS IN THE 5' LEADER CISTRON OF THE β2-ADRENERGIC RECEPTOR - Google Patents
POLYMORPHISMS IN THE 5' LEADER CISTRON OF THE β2-ADRENERGIC RECEPTOR Download PDFInfo
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- WO2000031307A1 WO2000031307A1 PCT/US1999/027963 US9927963W WO0031307A1 WO 2000031307 A1 WO2000031307 A1 WO 2000031307A1 US 9927963 W US9927963 W US 9927963W WO 0031307 A1 WO0031307 A1 WO 0031307A1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- This invention relates to the fields of pharmacogenomics, diagnostics and gene therapy. More specifically, the present invention relates to methods of screening drugs, which modulate the activity of the ⁇ 2 -adrenergic receptor as well as methods of diagnosing and/or treating diseases involving ⁇ 2 AR or its isoforms.
- One class is composed of polymorphisms that alter receptor function or expression and are the direct cause of a disease. These diseases are typically rare and the polymorphism is not found in healthy (non-affected) individuals. Examples of this class are polymorphisms of the leutinizing hormone receptor that cause constitutive activation and results in familial male precocious puberty (Themmen, A.P.N. et al., J Endocrinol 153:179-183, 1997), the calcium sensing receptor which causes constitutive activation and results in familial hypoparathyroidism (Chattopadhyay, N.
- a second class of genetic variations of G-protein coupled receptors comprises those that alter receptor function or expression but do not appear to be the direct or sole cause of a disease.
- the variation can be common in apparently healthy individuals.
- studies to date indicate that these polymorphisms may act as disease modifiers (Liggett, S.B. 1996.
- ⁇ 2 -adrenergic receptors are G-protein coupled receptors that are activated by endogenous catecholamines. These receptors are widely distributed, and play important roles in regulating cardiac, vascular, pulmonary, and metabolic functions. Studies of such physiologic functions of ⁇ 2 AR in humans have revealed several observations. First, there appears to be substantial interindividual variation in responsiveness, and secondly receptor function appears to be dynamically regulated as indicated by intraindividual variation.
- the Gly 16 receptor was found to undergo enhanced agonist-promoted downregulation of receptor number as compared to the Arg 16 receptor (3).
- the Glu27 receptor was found to undergo very little agonist-promoted downregulation compared to the Gln27 receptor (3).
- These variants are common in the population (1).
- the He 164 receptor which occurs in the heterozygous state in -5% of the population, displays depressed coupling to the stimulatory G protein, G s (4).
- the ⁇ 2 AR is encoded by an intronless gene on chromosome 5q31 (12).
- Receptor transcripts have a 5 ' leader region harboring an open reading frame (ORF) that encodes a 19 amino acid peptide (13).
- ORF open reading frame
- This peptide impedes translation of ⁇ 2 AR mRNA, and thus regulates cellular expression of the receptor (14). Given the importance of this 5' leader cistron in controlling ⁇ 2 AR expression, this region in the human population was examined for genetic variability.
- the present invention is based on the discovery of a common polymorphism that is located in the 5' leader cistron (5'LC) of the ⁇ 2AR gene.
- a polymorphism of cytosine or thymine located 47 bases upstream of the ⁇ 2 AR coding block results in either Arg or Cys being encoded at the terminal amino acid (position 19) of the 5 XC peptide (Argl9Cys) (see Figs. 1-3; SEQ ID NOSJ-3, respectively).
- the inventor herein has also discovered that a substitution of Cys for Arg in the 5'LC peptide results in increased expression of ⁇ 2AR in both recombinant and airway smooth muscle cells, which endogenously express ⁇ 2 AR.
- this ⁇ 2 AR 5' LC polymo ⁇ hic site represents, at least in part, the genetic basis of variable physiologic sympathetic responses, variation in disease phenotypes, and differences in the therapeutic efficacy of ⁇ -agonists and ⁇ -antagonists.
- a 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 the 5'LC polymo ⁇ hic site in the two copies to assign a ⁇ 2AR genotype to the individual.
- 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 the 5 'LC PS and at one or more additional ⁇ 2AR polymo ⁇ hic sites.
- Compositions useful in performing the genotyping and haplotyping methods include oligonucleotide probes and primers designed to specifically hybridize to a target region containing the 5 'LC polymo ⁇ hic site. These genotyping methods and compositions are useful for studying the effect of the 5'LC polymo ⁇ hisms in the etiology of various diseases and efficacy of drugs targeting the ⁇ 2 AR.
- the invention provides a method for detecting which variant(s) of the 5'LC peptide is expressed in an individual.
- the method comprises contacting a biological sample from the individual with a first antibody that specifically recognizes and binds to only one of the ⁇ 2 AR 5 'LC peptide variants and detecting a complex formed with the first antibody.
- the method further comprises contacting the biological sample with a second antibody that specifically recognizes and binds to the other ⁇ 2AR 5'LC peptide variant and detecting a complex formed with the second antibody.
- This method is useful for investigating the effects of the 5'LC peptide variants on ⁇ 2 AR expression.
- the preferred embodiment is useful to determine the genotype. If both antibodies react with the sample, the individual is heterozygous C/T at the 5 'LC polymo ⁇ hic site.
- Another aspect of the invention is based on the discovery that ⁇ 2 AR alleles carrying the 5 'LC C polymo ⁇ hism are most likely to also have the polymo ⁇ hisms which encode Gin and Arg at amino acids 16 and 27, respectively.
- the genotype for the 5'LC polymo ⁇ hic site may be used to predict the identity of the genotype for one or both of these ⁇ 2AR coding block polymo ⁇ hisms.
- the invention provides a method for identifying an association between a ⁇ 2AR 5 ' LC genotype and a trait.
- the method comprises comparing the frequency of the ⁇ 2 AR 5 'LC genotype in a population exhibiting the trait with the frequency of the genotype in a reference population, wherein a higher frequency of the genotype in the trait population than in the reference population indicates the trait is associated with the genotype.
- diseases including arrhythmia, heart failure, hypertension, vascular disease, migraine, asthma, chronic obstructive pulmonary disease (COPD), anaphylaxis, obesity, diabetes and premature labor.
- COPD chronic obstructive pulmonary disease
- the present invention also provides a method for predicting an individual's genetic predisposition to a disease modified by the ⁇ 2 AR.
- the method comprises determining the individual's genotype for the 5'LC polymo ⁇ hic site. If the individual is homozygous T, increased ⁇ 2 AR expression is likely and the individual has an increased risk for diseases affected by too much ⁇ 2 AR expression. If the individual is homozygous C, decreased ⁇ 2 AR expression is likely and the individual has an increased risk for diseases affected by too little ⁇ 2 AR expression.
- the disease modified by the ⁇ 2 AR is selected from the group consisting of arrhythmia, heart failure, hypertension, vascular disease, migraine, asthma, chronic obstructive pulmonary disease (COPD), anaphylaxis, obesity, diabetes and premature labor.
- arrhythmia heart failure
- hypertension vascular disease
- migraine migraine
- asthma chronic obstructive pulmonary disease
- COPD chronic obstructive pulmonary disease
- anaphylaxis obesity, diabetes and premature labor.
- the present invention further provides methods for predicting a patient's response to ⁇ -agonist therapy for bronchospasm, which typically occurs in asthma, COPD and anaphylaxis.
- the method comprises determining the genotype of the 5'LC polymo ⁇ hic site in the patient's ⁇ 2 AR gene. If the patient is homozygous for the T polymo ⁇ hism, failure to respond to a ⁇ -agonist is likely, while a patient is likely to respond if he or she is homozygous for the C polymo ⁇ hism or is heterozygous T/C at this site.
- knowledge of a patient's ⁇ 2 AR 5 'LC genotype provides a physician with information useful for making determinations as to which drug to administer, drug dosages, and duration of treatment.
- Figure 1 illustrates a partial DNA sequence for one allele of the human ⁇ 2 AR gene (SEQ ID NOJ), with the boxed region indicating the ORF for the 5' leader cistron, the underline indicating the start codon for the ORF encoding the ⁇ 2 AR protein (also referred to herein as the ⁇ 2AR coding block), and the location of the novel polymo ⁇ hic site described herein indicated in bold.
- Figure 2 illustrates the DNA sequence of the ⁇ 2 AR 5' leader cistron containing the thymidine polymo ⁇ hism (SEQ ID NOJ) at nucleotide 55 instead of the cytosine polymo ⁇ hism shown in Figure 1.
- Figure 3 illustrates the amino acid sequence of the Cys variant of the ⁇ 2 AR 5' leader cistron peptide (SEQ ID NOJ).
- Figure 4 illustrates identification of the polymo ⁇ hic site in the 5' leader cistron of the human ⁇ 2 AR gene, with Fig. 4A showing representative antisense sequencing results from two homozygous individuals and Fig. 4B showing representative restriction digests of a 163 bp PCR product digested with MspAlI.
- Figure 5 A shows a graph of the level of ⁇ 2 AR expression in COS-7 cells transfected with one of the 5'LC-Arg 19 or the 5'LC-Cysl9 constructs described in the Examples with ⁇ 2AR levels determined by radioligand binding with [ 125 I]CYP performed in triplicate and presented as fmol receptor/mg membrane protein.
- Figure 5B shows a graph of ⁇ 2AR expression in cells co-transfected with one of the above 5 'LC constructs and a construct encoding firefly luciferase, with ⁇ 2 AR expression presented as fmol receptor/light unit receptor.
- Figure 6 illustrates the results of ribonuclease protection assays, which identify levels of ⁇ 2 AR mRNA and actin mRNA in COS-7 cells transfected with one of the above 5 'LC constructs.
- Figure 7 shows a graph of ⁇ 2AR expression in primary cultures of human airway smooth muscle cells (HASM) homozygous for the 5'LC C allele (Argl9) or homozygous for the 5'LC T allele (Cysl9).
- Figure 8 shows a graph relating genotype for the 5 'LC polymo ⁇ hic site and the percent of patients who responded to the ⁇ -agonist albuterol.
- HASM human airway smooth muscle cells
- the inventor herein has discovered a common polymo ⁇ hism of C or T in the 5 ' leader cistron of the ⁇ 2 AR gene.
- the C allele of this polymo ⁇ hism encodes Arg at position 19 of the 5'LC peptide (MRLPGVRSRPAEPRRGSAR) (SEQ ID NO:4) while the T allele encodes Cys at this position (MRLPGVRSRPAEPRRGSAC) (SEQ ID NOJ).
- MRLPGVRSRPAEPRRGSAR 5'LC peptide
- T allele encodes Cys at this position
- ⁇ 2AR expression is significantly increased in cells homozygous for the T allele as compared to cells homozygous for the C allele.
- ⁇ 2 AR is expressed in virtually every tissue of the body, it is believed an individual's genotype for the 5 'LC polymo ⁇ hism of the present invention has the potential to influence, or be a marker of, his or her genetic predisposition for various diseases, including asthma, hypertension, congestive heart failure, ischemic heart disease, arrhythmia, obesity, diabetes, vascular disease, and premature labor.
- 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.
- 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.
- 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.
- 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.
- Phasing The description of the identity of the nucleotides at two or more polymo ⁇ hic sites in the same linear genetic molecule.
- Polymorphic site (PS) A position within a locus at which at least two alternative sequences are found in a population.
- Polymorphic variant - A gene, mRNA, cDNA, polypeptide or peptide whose nucleotide or amino acid sequence varies from a reference sequence due to the presence of a polymo ⁇ hism in the gene.
- 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.
- 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%.
- SNP Single Nucleotide Polymorphism
- Treatment A stimulus administered internally or externally to an individual.
- Population Group A group of individuals sharing a common ethnogeographic origin.
- the invention provides compositions and methods for detecting the ⁇ 2AR 5 'LC polymo ⁇ hisms described herein.
- compositions comprise oligonucleotide probes and primers capable of hybridizing to a target region containing the 5 'LC polymo ⁇ hic site or defining this target region for amplification.
- 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.
- oligonucleo tides 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 the use of radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags and the like.
- Oligonucleotide probes and primers of the invention must be capable of specifically hybridizing to a target region of a ⁇ 2 AR polynucleotide, i.e., a ⁇ 2 AR gene, cDNA, or mRNA.
- specific hybridization means the oligonucleotide reacts with the target region with sufficient specificity to allow the skilled artisan to discriminate between hybridization to the target region and hybridization to a non- target region.
- the oligonucleotide specifically hybridizes to the target region under conventional high stringency conditions.
- 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.
- oligonucleotide may be designed to specifically hybridize to either strand.
- the invention also includes oligonucleotides which are complementary to the sense strand of the ⁇ 2 AR 5' gene.
- 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 n 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.
- 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 5'LC polymo ⁇ hisms comprises a nucleotide sequence selected from the group consisting of: 5'- GTCCGCCCGCTGAGG -3' (SEQ ID NOJ) or its complement and 5'- GTCCGCCTGCTGAGG -3' (SEQ ID NO:6) or its complement.
- 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 5 'LC polymo ⁇ hisms comprises a nucleotide sequence selected from the group consisting of 5'- CCCCGCCGTGGGTCCGCCCG -3 ' (SEQ ID NOJ) and 5'- CCCCGCCGTGGGTCCGCCTG -3' (SEQ ID NO:8).
- a preferred ASO reverse primer for detecting ⁇ 2 AR 5 'LC polymo ⁇ hisms comprises a nucleotide sequence selected from the group consisting of 5'- GGCTGGGGGCGCCTCAGCGG -3 ' (SEQ ID NO:9) and 5'- GGCTGGGGGCGCCTCAGCAG -3' (SEQ ID NO: 10).
- a composition contains two or more differently labeled allele-specific oligonucleotides for simultaneously probing the identity of nucleotides at the ⁇ 2AR 5 'LC polymo ⁇ hic site and one or more additional polymo ⁇ hic sites in the ⁇ 2 AR gene or other locus. 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 ⁇ 2 AR polymo ⁇ hic site, one of which is the ⁇ 2AR 5'LC polymo ⁇ hic site.
- Allele-specific 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 allele-specific oligonucleotides may be used in a variety of polymo ⁇ hism detection assays, including but not limited to probe hybridization and polymerase extension assays.
- Immobilized ASO's 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.
- the invention provides a kit comprising at least two allele-specific oligonucleotides for detecting ⁇ 2AR 5'LC polymo ⁇ hisms packaged in separate containers.
- the kit may also contain other components such as hybridization buffer (where the oligonucleotides are to be used as probes) packaged in another separate container.
- the kit may also contain, a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase, such as PCR.
- oligonucleotide compositions and kits are useful in methods for genotyping and/or haplotyping the ⁇ 2 AR gene in an individual.
- ⁇ 2 AR genotype means the genotype contains at least the nucleotide pair present at the 5 'LC PS and may optionally include the nucleotide pair(s) present at one or more additional polymo ⁇ hic sites in the ⁇ 2 AR gene.
- ⁇ 2 AR haplotype means the haplotype contains the nucleotide present at the 5 'LC PS as well as the nucleotide(s) present at one or more additional polymo ⁇ hic sites in the ⁇ 2 AR gene.
- the additional polymo ⁇ hic sites in these genotyping and haplotyping may be currently known polymo ⁇ hic sites or sites that are subsequently discovered.
- the additional polymo ⁇ hic sites are located 20 nucleotides upstream of the start codon and coding block polymo ⁇ hic sites located 46, 79, 100 and 491 nucleotides downstream of the coding block ATG site. These sites are also referred to herein as -20 PS, +46 PS, +79 PS, +100 PS, and +491 PS.
- One embodiment of the genotyping method comprises 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 the 5 'LC polymo ⁇ hic site (5 'LC PS) in the two copies in order 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 nucleic acid mixture is isolated from a biological sample taken from the individual, such as a blood sample or tissue sample.
- Suitable 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 ⁇ 2AR gene is expressed.
- the ⁇ 2 AR genotype also comprises the nucleotide pair at one or more additional polymo ⁇ hic sites in the ⁇ 2 AR gene. If a ⁇ 2 AR gene fragment is isolated, it must contain the 5 'LC PS and any other ⁇ 2AR sites 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 the 5'LC PS and at one or more additional ⁇ 2 AR polymo ⁇ hic sites.
- the additional ⁇ 2 AR polymo ⁇ hic site(s) is selected from the group consisting of -20 PS, +46 PS, +79 PS, +100 PS and +491 PS.
- the nucleic acid may be isolated using any method that allows separation of the two copies of the ⁇ 2 AR gene present in an individual.
- any individual clone will provide haplotype information on only one of the two ⁇ 2AR 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 will need to be examined to have more than a 90% probability of haplotyping both copies of the ⁇ 2 AR gene in an individual.
- Another method for separating the two copies of the ⁇ 2 AR gene which is described in U.S. Patent No.
- a ⁇ 2 AR haplotype pair is determined for an individual by identifying the nucleotide at the 5 'LC PS and one or more ⁇ 2 AR polymo ⁇ hic sites.
- the additional polymo ⁇ hic site(s) is selected from the group consisting of -20 PS, +46 PS, +79 PS, +100 PS and +491 PS in both copies of the ⁇ 2AR gene present in the individual.
- the haplotyping method comprises identifying the nucleotide at each of the 5'LC PS, -20 PS, +46 PS, +79 PS, +100 PS and +491 PS in both copies of the ⁇ 2 AR gene present in the individual.
- the identifying step is preferably performed using first and second polynucleotides comprising each copy placed in separate containers.
- first and second polynucleotides 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 polynucleotides 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 polynucleotide while detecting a combination of the second and third dyes would identify the polymo ⁇ hism in the second polynucleotide.
- the 5 'LC thymine allele exhibits linkage disequilibrium with the coding block polymo ⁇ hisms encoding the Argl ⁇ and Gln27 ⁇ AR variants and vice versa, the 5'LC cytosine allele is linked to the coding block polymo ⁇ hisms encoding the Gly 16 and Glu27 ⁇ 2 AR variants.
- This relationship is the basis for another embodiment of the invention, which is a method for predicting an individual's genotype at one or both of +46 PS and +79 PS. The method comprises determining the individual's genotype at the 5'LC PS and assigning a genotype for one or both of +46 PS and +79 PS that is consistent with the 5'LC genotype.
- the genotype at +46 PS is probably homozygous A and the genotype at +79 PS is probably homozygous C
- the genotype at +46 PS is probably homozygous G
- the genotype at +79P S is probably homozygous G. It is also contemplated that the above genotyping and haplotyping methods of the invention may be performed in combination with identifying the genotype(s) and/or haplotype(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 identity of the nucleotide at the 5 ' LC PS may also be determined by digesting an amplified target region containing this site with MspAlI and then analyzing the reaction products by standard size separation techniques such as agarose or polyacrylamide gel electrophoresis. The presence or absence of the enzyme restriction site as determined by this MspAlI restriction fragment length polymo ⁇ hism (RFLP) analyses is used to determine whether the individual being tested is homozygous for the C allele or T allele, respectively.
- MspAlI restriction fragment length polymo ⁇ hism RFLP
- the forward and reverse primers used to amplify the target region comprise the following sequences 5'-GCTGAATGAGGCTTCCAGGC-3 ' (SEQ ID NO: 13) and 5'- CGCATGGCTTCTATTGGGTG-3' (SEQ ID NO: 14), respectively.
- 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. 4,965,188), ligase chain reaction (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.
- PCR polymerase chain reaction
- LCR ligase chain reaction
- OLA oligonucleotide ligation assay
- the oligonucleotides are between 10 and 35 nucleotides in length and preferably, between 15 and 30 nucleotides in length. Most preferably, 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.
- 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, UN 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.
- the identity of polymo ⁇ hisms may also be determined using a mismatch detection technique, including but not limited to the R ⁇ ase protection method using riboprobes (Winter et al., Proc. Natl. Acad. Sci.
- 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. ⁇ lles, ed., pp 321-340, 1996) or denaturing gradient gel electrophoresis (DGG ⁇ ) (Wartell et al., Nucl Acids Res.
- SSCP single strand conformation polymo ⁇ hism
- 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 No. 5,302,509.
- Another such method is allele-specific PCR (Ruano et al., Nucl Acids Res.
- multiple polymo ⁇ hic sites may be investigated by simultaneously amplifying multiple regions of the nucleic acid using sets of allele-specific primers as described in Wallace et al. (WO89/10414).
- Another aspect of the invention relates to detecting which variant(s) of the 5 'LC peptide is expressed in an individual.
- a biological sample from the individual is contacted with a first antibody that is specifically immunoreactive with only one of the 5 ' LC peptide variants and the formation of a complex with the first antibody is detected.
- the method also comprises contacting the biological sample with a second antibody that is specifically immunoreactive with the other 5 'LC peptide variant and the formation of a complex with the second antibody is detected.
- Complex formation with both first and second antibodies indicate the individual is heterozygous at the ⁇ 2 AR 5 'LC polymo ⁇ hic site.
- Suitable immunoassays for use in this detection method include radioimmunoassay, Western blot assay, immuno fluorescent 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; Current 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.
- 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.,
- Proteins may be isolated from test specimens and biological samples by conventional methods, as described in Current Protocols in Molecular Biology, supra.
- Exemplary antibody molecules for detecting ⁇ 2 AR 5 'LC peptide variants 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 Milstein, 1975, Nature, 256:495-497; Campbell “Monoclonal Antibody Technology, the Production and Characterization of Rodent and Human Hybridomas", 1985, In: “Laboratory Techniques in Biochemistry and Molecular Biology,” Eds.
- 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, 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. 86; 10029).
- 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 5 ' 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 may also comprise the nucleotide pair(s) detected at one or more additional ⁇ 2 AR polymo ⁇ hic sites.
- 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 ⁇ 2AR genotypes or haplotypes in reference and trait populations are useful for identifying an association between a trait and a ⁇ 2 AR 5 'LC polymo ⁇ hism, a ⁇ 2 AR genotype or a ⁇ 2 AR haplotype.
- the trait may be any detectable phenotype, including but not limited to genetic predisposition to a disease or response to a treatment.
- the method comprises obtaining data on the frequency of the ⁇ 2AR 5 'LC polymo ⁇ hism, ⁇ 2AR genotype or ⁇ 2 AR haplotype 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 of the methods 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 5'LC polymo ⁇ hism, ⁇ 2 AR genotype or ⁇ 2 AR haplotype of interest are compared in the reference and trait populations.
- 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 response, no response, and adverse response (i..e, side effects).
- a clinical response to a treatment In order to deduce a correlation between a clinical response to a treatment and a 5 'LC polymo ⁇ hism, ⁇ 2 AR genotype or ⁇ 2 AR 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.
- 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. It is preferred that the individuals included in the clinical population have been assessed for the clinical characteristics of the medical condition of interest. Such clinical characteristics may include symptoms, disease severity, response to therapy and the like. This characterization 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.
- correlations are created between individual response and the presence of a ⁇ 2 AR 5 'LC polymo ⁇ hism, ⁇ 2 AR genotype or ⁇ 2 AR haplotype. Correlations may be produced in several ways. In one embodiment, individuals are grouped by their ⁇ 2 AR genotype or ⁇ 2 AR haplotype and then the averages and standard deviations of clinical responses exhibited by the members of each group are calculated. These results are then analyzed to determine if any observed variation in clinical response between genotype or haplotype groups is statistically significant.
- Another method involves categorizing the response (e.g., none, low, medium, high or other such grades) and then assessing whether a particular genotype is more common in one group of responders compared to another.
- Statistical analysis methods which may be used are described in L.D. Fisher and G. vanBelle, "Biostatistics: A Methodology for the Health Sciences", Wiley-Interscience (New York) 1993.
- the above described genotyping and haplotyping methods are also useful for predicting an individual's predisposition to various diseases modified by the ⁇ 2 AR.
- the ⁇ 2 AR is expressed in a number of tissues in the human body and that the amount of such expression modifies a number of diseases and physiologic processes.
- the ⁇ 2 AR relax airway smooth muscle and open the constricted airways during an asthma attack.
- Congestive heart failure In this syndrome the heart has decreased cardiac output which can be increased by activating ⁇ 2AR which increase heart rate and the force of contraction.
- ⁇ 2AR which increase heart rate and the force of contraction.
- individuals homozygous for the C polymo ⁇ hism at the ⁇ 2 AR 5' LC PS would have an increased risk for decompensation.
- Ischemic Heart Disease Individuals suffering from this condition have blocked coronary arteries and thus a limited capacity to tolerate increased cardiac demand. Thus, an increased cardiac output caused by increased levels of ⁇ 2 AR can lead to myocardial infarction in these patients.
- Cardiac arrhythmias - Activated ⁇ 2AR cause an increase in heart rate which predisposes certain individuals with conduction system defects to arrhythmias.
- the higher ⁇ 2 AR expression expected in individuals homozygous for T at the 5 'LC polymo ⁇ hic site would be a risk factor for arrhythmias.
- Hypertension - Blood pressure is regulated, in part, by systemic vascular resistance which is due to contraction of the smooth muscle surrounding the small resistance arteries known as arterioles. ⁇ 2 AR are expressed on this muscle and thus activation relaxes it and lowers resistance and blood pressure. Because polymo ⁇ hisms leading to lower expression would predispose to hypertension, an individual homozygous for C at the 5 'LC PS would have an increased risk for developing hypertension.
- Vascular disease - Diseases where vascular flow is compromised to a given organ, due to atherosclerosis or abnormal neurogenic control of tone, such as migraine, are amenable to treatment with vasodilators or vasoconstrictors.
- COPD Asthma, COPD, anaphylaxis-
- the airways have smooth muscle which when contracted leads to obstruction of airflow, termed bronchospasm, in asthma, COPD (a syndrome with varying features of emphysema and chronic bronchitis), and anaphylaxis.
- ⁇ 2 AR on airway smooth muscle relax the muscle and open the airways. Therefore, patients homozygous for the C polymo ⁇ hism in the ⁇ 2 AR 5 'LC would be predisposed to bronchospasm.
- Obesity - Fat cells express three ⁇ AR subtypes, including the ⁇ 2 AR. Activation of the ⁇ 2 AR causes breakdown of fat in the cells.
- a polymo ⁇ hism causing reduced ⁇ 2 AR expression would be a risk factor for developing obesity since a lower efficiency of lipolysis in fat cells would be expected in individuals carrying this polymo ⁇ hism. This expectation was recently confirmed by a clinical study, which demonstrated that the 5' LC C polymo ⁇ hism is indeed associated with obesity (29).
- the islet cells of the pancreas are responsible for insulin secretion, and these cells express ⁇ 2 AR, which act to increase insulin secretions.
- ⁇ 2 AR act to increase insulin secretions.
- Skeletal muscle glycogen metabolism is also altered by ⁇ 2 AR activation, and a complex inte ⁇ lay with insulin secretion and lipolysis (see above) can further the potential for the diabetic state.
- this prediction is supported by the results of a recent clinical study, which found significant association between the 5' LC C polymo ⁇ hism and Type 2 diabetes (29).
- Premature labor The uterine muscle expresses ⁇ 2 AR which serve to relax the muscle.
- ⁇ 2 AR agonists such as ritodrine are administered to inhibit contractions of the uterus when premature labor develops.
- Patients with a polymo ⁇ hism that results in decreased ⁇ 2 AR expression would be predisposed to higher uterine muscle tone and at higher risk for early labor.
- An individual's predisposition to these diseases can be predicted by determining the individual's genotype for the ⁇ 2AR 5'LC polymo ⁇ hic site.
- nucleic acid isolated from any tissue from the individual may be used for genotyping
- the typical genotyping procedure comprises drawing a patient's blood and extracting genomic DNA from the cells of the blood (e.g., peripheral blood mononuclear cells, leukocytes, or lymphocytes).
- prenatal diagnosis can be accomplished by testing fetal cells or amniotic fluid.
- the identity of the nucleotide or nucleotide pair at the ⁇ 2 AR 5'LC polymo ⁇ hic site can be determined by any of the means described herein.
- the inventor herein has discovered that asthmatic patients who are homozygous T at the 5 'LC polymo ⁇ hic site are less likely to exhibit a bronchodilating response to the ⁇ -agonist albuterol than asthmatic patients having other 5'LC genotypes.
- the invention also provides a method for predicting a patient's bronchodilating response to a ⁇ 2 -agonist, which comprises determining the patient's genotype for the 5 'LC PS.
- bronchodilating response means an increase of greater than 12% in the forced expiratory flow between 25% and 75% of the forced vital capacity (FEF 25- - 75 ) after administration of the standard dose of the ⁇ 2 -agonist.
- the physician can determine whether the patient should be treated with higher doses of the ⁇ 2 -agonist, or with an alternative therapy.
- the ⁇ 2 -agonist is albuterol.
- Example 1 This example illustrates the detection of a polymo ⁇ hic site in the 5 ' leader cistron of the ⁇ 2 AR gene.
- Genomic DNA was derived by the cetylmethyl ammonium bromide method (15) from peripheral blood obtained from 176 healthy Caucasian subjects without histories of chronic disease.
- the first nucleotide of the initiator methionine codon of the ⁇ 2 AR coding block is denoted as nucleotide 1 and the adjacent 5' residue as nucleotide -1.
- the 5' leader cistron is thus localized to nucleotides -102 to -42 (nucleotides 1487-1546 of Fig. 1).
- a polymerase chain reaction was carried out using primers that provided for an amplification product spanning this region (sense: 5 '- AAGGACACCACCTCCAGCTTTAG -3', (SEQ ID NOJ 1); antisense:
- Each reaction contained 1 U of Amplitaq polymerase (Perkin Elmer), 500 ng genomic DNA 250 ⁇ M of each dNTP, and 37.5 pmol of each primer in a final volume of 50 ⁇ l. After an initial denaturation step of 98 °C for 2 minutes, 35 cycles of 98 °C for 30 sec, 56 °C for 30 sec, and 72 °C for 30 sec were carried out, followed by a 7-minute final extension at 72 °C. Ten ⁇ l of the PCR reaction was electrophoresed on a 1% agarose gel and visualized by ethidium bromide staining.
- PCR product was purified with a commercial kit (Promega) and was sequenced by an automated sequencer (ABI 377 Prism) using dye-terminator chemistry ( Figure 4A). After sequencing 46 individuals, a single polymo ⁇ hism of C or T at position -47, which corresponds to nucleotide 55 of the leader cistron ORF (nucleotide 1541 of Fig. 1), was identified. This polymo ⁇ hic site in the 5'LC results in the encoded residue at amino acid 19 being either Arg or Cys.
- Example 2 This example illustrates the effect of the ⁇ 2 AR 5'LC polymo ⁇ hisms on ⁇ 2 AR expression.
- cells were transiently transfected with two receptor constructs. These consisted of a contiguous stretch of 1989 bp exactly as found in the human gene, comprising a 93 bp region upstream of the leader cistron, the leader cistron with its stop codon, an additional intervening 41 bp of 5' sequence, the ⁇ 2 AR coding sequence and 557 bp of 3' untranslated region.
- This cDNA was subcloned into the expression vector pBC12BI at the BamHI site.
- the confluent cells were harvested for determination of receptor expression by radioligand binding with [ 125 I]cyanopindolol ([ 125 I]CYP) using 1 ⁇ M propranolol to define non-specific binding.
- Cells were disrupted by scraping with a rubber policeman in cold 5 mM Tris, 2 mM EDTA, pH 7.4 buffer and the particulates centrifuged at 40,000 xg for 10 minutes and resuspended in 75 mM Tris, 12 mM MgCl 2 , 2 mM EDTA, pH 7.4 buffer.
- Membranes (10 ⁇ g) were incubated in triplicate with a saturating concentration of [ !2 J]CYP (400 pM) without or with propranolol in the aforementioned buffer for 2 hrs at 25 °C. The reaction was stopped by dilution and rapid vacuum filtration over Watman GF/C filters. Radioactivity was measured in a gamma counter at 80%) efficiency. Protein was quantitated by the copper bicinchoninic method (19).
- the 5'LC-Cysl9 construct resulted in 72% higher levels of expression over that obtained with the 5'LC-Argl9 construct (4666 ⁇ 302 vs 2711 ⁇ 294 fmol/mg, PO.01). This is consistent with the aforementioned studies (14), which showed that the Arg 5 'LC peptide inhibited ⁇ 2 AR translation.
- the degree of DNA transfection was indeed the same between the two constructs, and the difference in ⁇ 2AR cellular expression was due to differences in peptide mediated inhibition of translation (rather than transcription or mRNA stability)
- mRNA levels were determined by a quantitative ribonuclease protection assay which included a probe for ⁇ -actin as a control.
- RNA was prepared from transfected cells by an acid guanidinium thiocyanate-phenol-chloroform extraction reagent (Molecular Research, Cincinnati, OH) and ⁇ 2 AR transcripts delineated by ribonuclease protection assays (RPA) in a manner similar to that previously described (18).
- RPA ribonuclease protection assays
- a template for the synthesis of riboprobes was prepared by subcloning a cDNA encoding the ⁇ 2 AR ORF into the Hindlll/Xbal sites of plasmid pSP72 (Promega).
- Plasmid linearized with EcoNI was then used for in vitro transcription reactions with T7 polymerase and [ 32 P]UTP to generate a 563 bp antisense riboprobe corresponding to the distal 500 bp of the ⁇ 2 AR ORF.
- a radiolabeled antisense riboprobe for ⁇ -actin was generated from a commercial template (Ambion) using T7 polymerase. RPAs were performed as previously reported (18) by hybridizing 20 ⁇ g of total cellular RNA with both the actin and ⁇ 2 AR riboprobes.
- the hybridized products were digested with RNAse A and TI , after which protected fragments were separated by electrophoresis on 6% polyacrylamide gels containing 8M urea. Radiographic bands corresponding to the protected fragments were visualized with a Phospholmager (Molecular Dynamics) and conventional autoradiography. Band density was quantitated on the phosphorimage with the ImageQuant software package (Molecular Dynamics). To account for minor differences in sample loading and recovery, the measured value for the ⁇ 2 AR protected fragment was normalized to that of ⁇ -actin in the same sample. The results of these ribonuclease protection assays are shown in Figure 6.
- This example illustrates there is linkage disequilibrium between the previously described polymo ⁇ hisms at nucleotides 46 and 79 (amino acids 16 and 27 of the receptor protein) and the 5' leader cistron polymo ⁇ hisms.
- This example illustrates there is an association between TT genotype at the ⁇ 2 AR 5'LC polymo ⁇ hic site and lack of response to albuterol.
- the TT genotype for the 5 'LC PS is a specific marker for lack of responsiveness to albuterol.
- the CC genotype and C/T genotype are markers for responsiveness to albuterol.
- cDNA for the human ⁇ 2 -adrenergic receptor a protein with multiple membrane spanning domains and a chromosomal location shared with PDGF receptor gene. Proc.Natl.Acad.Scl, USA 84:46-50.
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GB0115239A GB2361535B (en) | 1998-11-25 | 1999-11-24 | Polymorphisms in the 5'leader cistron of the beta 2 - adrenergic receptor |
AU17445/00A AU1744500A (en) | 1998-11-25 | 1999-11-24 | Polymorphisms in the 5' leader cistron of the beta2-adrenergic receptor |
US09/856,803 US6861217B1 (en) | 1998-11-25 | 1999-11-24 | Variation in drug response related to polymorphisms in the β2-adrenergic receptor |
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Cited By (4)
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---|---|---|---|---|
US6586183B2 (en) | 2000-04-13 | 2003-07-01 | Genaissance Pharmaceuticals, Inc. | Association of β2-adrenergic receptor haplotypes with drug response |
US6797472B1 (en) | 1999-03-12 | 2004-09-28 | Genaissance Pharmaceuticals, Inc. | Variation in drug response related to polymorphisms in β2-adrenergic receptor |
US7041810B2 (en) | 2000-04-17 | 2006-05-09 | University Of Cincinnati | Alpha-2 adrenergic receptor polymorphisms |
US8080578B2 (en) | 2004-09-14 | 2011-12-20 | The Regents Of The University Of Colorado, A Body Corporate | Methods for treatment with bucindolol based on genetic targeting |
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- 1999-11-24 GB GB0115239A patent/GB2361535B/en not_active Expired - Fee Related
- 1999-11-24 WO PCT/US1999/027963 patent/WO2000031307A1/en active Application Filing
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6797472B1 (en) | 1999-03-12 | 2004-09-28 | Genaissance Pharmaceuticals, Inc. | Variation in drug response related to polymorphisms in β2-adrenergic receptor |
US6586183B2 (en) | 2000-04-13 | 2003-07-01 | Genaissance Pharmaceuticals, Inc. | Association of β2-adrenergic receptor haplotypes with drug response |
US7041810B2 (en) | 2000-04-17 | 2006-05-09 | University Of Cincinnati | Alpha-2 adrenergic receptor polymorphisms |
US7572603B2 (en) | 2000-04-17 | 2009-08-11 | University Of Cincinnati | Alpha-2 adrenergic receptor polymorphisms |
US8080578B2 (en) | 2004-09-14 | 2011-12-20 | The Regents Of The University Of Colorado, A Body Corporate | Methods for treatment with bucindolol based on genetic targeting |
US8093286B2 (en) | 2004-09-14 | 2012-01-10 | The Regents Of The University Of Colorado, A Body Corporate | Methods for treatment with bucindolol based on genetic targeting |
US8916603B2 (en) | 2004-09-14 | 2014-12-23 | The Regents Of The University Of Colorado, A Body Corporate | Methods for treatment with bucindolol based on genetic targeting |
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AU1744500A (en) | 2000-06-13 |
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