Methods of using a genetic polymorphic variation in the human beta-1 adrenergic receptor gene as a drug response marker are presented. Determining the presence or absence of the A145G genetic variation in the human beta-1 adrenergic receptor gene is useful in predicting an individual's relative response to different antihypertensive drugs; optimizing antihypertensive treatment for an individual; selecting candidate human subjects for participation in clinical trials involving antihypertensive drugs; and, predicting the relative responses among a plurality of individuals to an antihypertensive drug.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that a genetic variant in the human beta-1 adrenergic receptor gene is significantly associated with an individual's response to antihypertensive drugs. The genetic variant according to the present invention is the A145G nucleotide substitution mutation, which has been shown to be associated with an individual's response to antihypertensive drugs. Specifically, it has been discovered that an individual homozygous for the A145G genetic variant responds more favorably to a diuretic antihypertensive class of drugs than to angiotensin converting enzyme inhibitors and beta-blockers. Thus, the present invention provides a genetic basis for predicting an individual's response to antihypertensive drugs.

Accordingly, the present invention provides methods for predicting the relative responses of an individual to different classes of antihypertensive drugs by determining the presence or absence of the A145G nucleotide substitution variant in the beta-1 adrenergic receptor gene in the individual, wherein if the individual is homozygous for this genetic variant, the individual will respond more effectively to diuretic antihypertensive drugs (e.g., hydrochlorothiazide) than to angiotensin converting enzyme inhibitors (e.g., fosinopril) and beta-blockers (e.g., atenolol).

The methods of the present invention are useful in optimizing antihypertensive treatment of patients and in optimizing clinical trials involving antihypertensive drug treatment.

The foregoing and other advantages and features of the invention, and the manner in which the same are accomplished, will become more readily apparent upon consideration of the following detailed description of the invention taken in conjunction with the accompanying examples, which illustrate exemplary embodiments.

DETAILED DESCRIPTION OF THE INVENTION

A cDNA sequence of the beta-1 adrenergic receptor gene is disclosed under GenBank Accession No. J03019. This sequence is used herein as a reference sequence for identifying the polymorphic position of the A145G genetic variant of the present invention. The A145G genetic variant is located at nucleotide position +145 of the beta-1 adrenergic receptor gene mRNA, or cDNA, wherein the A of the ATG of the initiation Met codon is nucleotide +1. The amino acid variant Ser49Gly referred to herein is at position +49 of the protein product of the beta-1 adrenergic receptor mRNA or cDNA, wherein the initiation Met amino acid is amino acid +1.

Thus, in accordance with the present invention, the A145G genetic variant of the human beta-1 adrenergic receptor gene is now, according to the present invention, shown to affect individual response to antihypertensive drug treatment in patients. That is, individuals homozygous for the A145G genetic variant, respond more effectively to antihypertensive drug treatment using diuretic antihypertensive drugs (e.g., hydrochlorothiazide), than to either angiotensin converting enzyme inhibitors (e.g., fosinopril) or beta blockers (e.g., atenolol).

The A145G genetic variant results in the substitution of a non-hydroxyl amino acid for the hydroxy group-containing amino acid Ser49. Thus, an individual lacking a beta-1 adrenergic receptor that has a hydroxy group-containing amino acid (e.g., Ser49 or Thr49) would likely also respond more favorably to antihypertensive drug treatment using diuretic antihypertensive drugs (e.g., hydrochlorothiazide), than to either angiotensin converting enzyme inhibitors (e.g., fosinopril) or beta blockers (e.g., atenolol). Thus, other nucleotide variants at the nucleotide position 145, 146 and 147 of beta-1 adrenergic receptor gene leading to the substitution of a non-hydroxyl amino acid for the hydroxy group-containing amino acid Ser49 should also cause similar effect as A145G.

Accordingly, in one aspect, the present invention provides methods for selecting an antihypertensive treatment for an individual, which include the steps of identifying an individual in need of an antihypertensive treatment, and determining the presence or absence of an A145G nucleotide variant or a nucleotide variant resulting in a Ser49Gly amino acid substitution in a nucleic acid of the individual encoding beta-1 adrenergic receptor, or the presence or absence of a Ser49Gly amino acid variant in the beta-1 adrenergic receptor protein of the individual.

In one embodiment, the methods for selecting an antihypertensive treatment for an individual comprise identifying an individual in need of an antihypertensive treatment, and determining the genotype of the individual at the nucleotide 145 position of the beta-1 adrenergic receptor gene.

In another embodiment, the methods for selecting an antihypertensive treatment for an individual comprise determining the genotype of the individual at the nucleotide 145 position of the beta-1 adrenergic receptor gene, wherein the presence of a homozygous A145G genetic variant would indicate an increased likelihood that said individual will respond more favorably to diuretic antihypertensive drugs than to angiotensin converting enzyme inhibitors and beta-blockers. Preferably, the diuretic antihypertensive drugs are thiazides (e.g., hydrochlorothiazide), the angiotensin converting enzyme inhibitor is fosinopril, and beta-blocker is atenolol. The genotype can be determined by analyzing nucleic acids isolated from said individual.

In another aspect, the present invention provides methods for predicting an individual's relative response to different classes of antihypertensive drugs by determining or detecting in the individual the presence or absence of a homozygous nucleotide variant of A145G or determining whether an individual has nucleotide variant(s) that make the individual devoid of a beta-1 adrenergic receptor that has a hydroxy group-containing amino acid (e.g., Ser49 or Thr49). The presence of such homozygous mutation or such other nucleotide variant(s) would indicate that there is an increased likelihood that said individual would be more responsive to diuretic antihypertensives than to angiotensin converting enzyme inhibitors and beta-blockers.

Thus, in one embodiment, the methods for predicting the relative response of an individual to different classes of antihypertensive drugs include a step of determining, in an individual with hypertension, the presence or absence of an A145G nucleotide variant or a nucleotide variant resulting in a Ser49Gly amino acid substitution in a nucleic acid of the individual encoding beta-1 adrenergic receptor, or the presence or absence of a Ser49Gly amino acid variant in the beta-1 adrenergic receptor protein of the individual. The determination can be accomplished by genotyping the individual with hypertension to determine the genotype at the nucleotide 145 position of the beta-1 adrenergic receptor gene. The presence of a homozygous A145G genetic variant or a homozygous genetic variant resulting in the Ser49Gly amino acid variant would indicate an increased likelihood that said individual will respond favorably to diuretics than to angiotensin converting enzyme inhibitors and beta-blockers. In particular, the presence of a homozygous A145G genetic variant or a homozygous genetic variant resulting in the Ser49Gly amino acid variant would indicate an increased likelihood that said individual will respond more favorably to hydrochlorothiazide than to fosinopril and atenolol.

In yet another aspect of the present invention, a method is provided for selecting candidate human subjects for participation in a clinical trial involving an antihypertensive drug. The method comprises determining the genotype of an individual at the nucleotide 145 position of the beta-1 adrenergic receptor gene, and deciding whether to include said individual in the clinical trial based on the result of the determining step. Thus, in one embodiment, the method includes determining the presence or absence of a homozygous mutation of A145G in a candidate human subject. The presence of such homozygous mutation would indicate that there is an increased likelihood that said candidate human subject would be more responsive to diuretic antihypertensives than to angiotensin converting enzyme inhibitors and beta-blockers. In a second embodiment, the candidate human subject is tested for a Ser49Gly amino acid substitution in the human beta-1 adrenergic receptor protein, or nucleotide variant(s) that make the individual devoid of a beta-1 adrenergic receptor that has a hydroxy group-containing amino acid (e.g., Ser49 or Thr49). The presence of a homozygous Ser49Gly amino acid substitution or the other nucleotide variant(s) would indicate that there is an increased likelihood that said candidate human subject would be more responsive to diuretic antihypertensives than to angiotensin converting enzyme inhibitors and beta-blockers.

In a further aspect, a method for optimizing antihypertensive treatment in an individual is also provided. Optimizing antihypertensive treatment in an individual involves deciding which class of antihypertensive drug to use or the amount of such drug to administer based on the presence or absence of the A145G genetic variant or the Ser49Gly amino acid variant of the beta-1 adrenergic receptor in that individual. The homozygous presence of either the nucleotide (A145G) or amino acid (Ser49Gly) variant in a particular individual would indicate that there is an increased likelihood that individual would be more responsive to diuretic antihypertensives than to angiotensin converting enzyme inhibitors and beta-blockers. With that information in mind, a particular antihypertensive drug and/or dosage thereof may be selected to better suit a particular individual.

In yet another aspect of the present invention, a method is also provided for treating hypertension in an individual. The method includes (a) predicting an individual's relative responses to diuretic antihypertensives, angiotensin converting enzyme inhibitors and beta-blockers, by determining the presence or absence of a homozygous nucleic acid mutation of A145G, wherein the presence of such homozygous mutation would indicate that there is an increased likelihood that said individual would be more responsive to diuretic antihypertensives than to angiotensin converting enzyme inhibitors and beta-blockers, and (b) selecting an antihypertensive drug according to the result of step (a). In a second embodiment, the method includes predicting an individual's relative responses to diuretic antihypertensives, angiotensin converting enzyme inhibitors and beta-blockers, by determining the presence or absence of a homozygous amino acid substitution of Ser49Gly, or nucleotide variant(s) that make the individual devoid of a beta-1 adrenergic receptor that has a hydroxy group-containing amino acid (e.g., Ser49 or Thr49). The presence of a homozygous Ser49Gly amino acid substitution or the other nucleotide variant(s) would indicate that there is an increased likelihood that said candidate human subject would be more responsive to diuretic antihypertensives than to angiotensin converting enzyme inhibitors and beta-blockers.

In yet another embodiment, the method of treating hypertension comprises: (a) determining the genotype of an individual at the nucleotide 145 position of the beta-1 adrenergic receptor gene; (b) selecting an antihypertensive drug for administration to the individual based on the genotype obtained in said determining step; and (c) administering said drug to the individual or instructing the individual to take said drug. In particular, a thiazide is selected if the individual has a homozygous A145G genotype or a homogenous population of beta-1 adrenergic receptor proteins with a Ser49Gly amino acid variant.

Numerous techniques for detecting genetic variants are known in the art and can all be used for the method of this invention. The techniques can be protein-based or DNA-based. In either case, the techniques used must be sufficiently sensitive so as to accurately detect single nucleotide or amino acid variations. Often, a probe is utilized which is labeled with a detectable marker. Unless otherwise specified in a particular technique described below, any suitable marker known in the art can be used, including but not limited to, radioactive isotopes, fluorescent compounds, biotin which is detectable using strepavidin, enzymes (e.g., alkaline phosphatase), substrates of an enzyme, ligands and antibodies, etc. See Jablonski et al., Nucleic Acids Res., 14:6115 6128 (1986); Nguyen et al., Biotechniques, 13:116 123 (1992); Rigby et al., J. Mol. Biol., 113:237 251 (1977).

In a DNA-based detection method, target DNA sample, i.e., a sample containing the beta-1 adrenergic receptor gene sequence must be obtained from the individual to be tested. Any tissue or cell sample containing the beta-1 adrenergic receptor genomic DNA, mRNA, or cDNA or a portion thereof can be used. For this purpose, a tissue sample containing cell nucleus and thus genome DNA can be obtained from the individual. Blood samples can also be useful except that only white blood cells and other lymphocytes have a cell nucleus, while red blood cells are anucleus and contain mRNA. The tissue or cell samples can be analyzed directly without much processing. Alternatively, nucleic acids including the target gene sequence can be extracted, purified, or amplified before they are subject to the various detecting procedures discussed below. Other than tissue or cell samples, cDNAs or genomic DNAs from a cDNA or genomic DNA library constructed using a tissue or cell sample obtained from the individual to be tested are also useful.

To determine the presence of a particular genetic variant, one technique is simply sequencing the target gene sequence, particularly the nucleotide sequence region encompassing the genetic variant locus to be detected. Various sequencing techniques are generally known and widely used in the art including the Sanger method and Gilbert chemical method. The newly developed pyrosequencing method monitors DNA synthesis in real time using a luminometric detection system. Pyrosequencing has been shown to be effective in analyzing genetic polymorphisms such as single-nucleotide polymorphisms and thus can also be used in the present invention. See Nordstrom et al., Biotechnol. Appl. Biochem., 31(2):107 112 (2000); Ahmadian et al., Anal. Biochem., 280:103 110 (2000).

Alternatively, the restriction fragment length polymorphism (RFLP) method may also prove to be a useful technique. In particular, if a genetic variation, e.g., if the SNP in the beta-1 adrenergic receptor gene of the present invention results in the elimination or creation of a restriction enzyme recognition site, then digestion of the target DNA with that particular restriction enzyme will generate a different restriction fragment length pattern. Thus, a detected RFLP will indicate the presence of a particular genetic variant.

Another useful approach is the single-stranded conformation polymorphism assay (SSCA), which is based on the altered mobility of a single-stranded target DNA spanning the genetic variant of interest. A single nucleotide change in the target sequence can result in a different intramolecular base pairing pattern, and thus a different secondary structure of the single-stranded DNA, which can be detected in a non-denaturing gel. See Orita et al., Proc. Natl. Acad. Sci. USA, 86:2776 2770 (1989). Denaturing gel-based techniques such as clamped denaturing gel electrophoresis (CDGE) and denaturing gradient gel electrophoresis (DGGE) detect differences in migration rates of mutant sequences as compared to wild-type sequences in denaturing gel. See Miller et al., Biotechniques, 5:1016 24 (1999); Sheffield et al., Am. J. Hum. Genet., 49:699 706 (1991); Wartell et al., Nucleic Acids Res., 18:2699 2705 (1990); and Sheffield et al., Proc. Natl. Acad. Sci. USA, 86:232 236 (1989). In addition, the double-strand conformation analysis (DSCA) can also be useful in the present invention. See Arguello et al., Nat. Genet., 18:192 194 (1998).

The presence or absence of the A145G genetic variant in the beta-1 adrenergic receptor gene of an individual can also be detected using the amplification refractory mutation system (ARMS) technique. See e.g., European Patent No. 0,332,435; Newton et al., Nucleic Acids Res., 17:2503 2515 (1989); Fox et al., Br. J. Cancer, 77:1267 1274 (1998); Robertson et al., Eur. Respir. J., 12:477 482 (1998). In the ARMS method, a primer is synthesized matching the nucleotide sequence immediately 5' upstream from the locus being tested except that the 3'-end nucleotide which corresponds to the nucleotide at the locus is a predetermined nucleotide. For example, the 3'-end nucleotide can be the same as that in the mutated locus. The primer can be of any suitable length so long as it hybridizes to the target DNA under stringent conditions only when its 3'-end nucleotide matches the nucleotide at the locus being tested. Preferably the primer has at least 12 nucleotides, more preferably from about 18 to 50 nucleotides. If the individual tested has a mutation at the locus and the nucleotide therein matches the 3'-end nucleotide of the primer, then the primer can be further extended upon hybridizing to the target DNA template, and the primer can initiate a PCR amplification reaction in conjunction with another suitable PCR primer. In contrast, if the nucleotide at the locus is of wild type, then primer extension cannot be achieved. Various forms of ARMS techniques developed in the past few years can be used. See e.g., Gibson et al., Clin. Chem. 43:1336 1341 (1997).

Similar to the ARMS technique is the mini sequencing or single nucleotide primer extension method, which is based on the incorporation of a single nucleotide. An oligonucleotide primer matching the nucleotide sequence immediately 5' to the locus being tested is hybridized to the target DNA or mRNA in the presence of labeled dideoxyribonucleotides. A labeled nucleotide is incorporated or linked to the primer only when the dideoxyribonucleotides matches the nucleotide at the SNP locus being detected. Thus, the identity of the nucleotide at the SNP locus can be revealed based on the detection label attached to the incorporated dideoxyribonucleotides. See Syvanen et al., Genomics, 8:684 692 (1990); Shumaker et al., Hum. Mutat., 7:346 354 (1996); Chen et al., Genome Res., 10:549 547 (2000).

Another set of techniques useful in the present invention is the so-called "oligonucleotide ligation assay" (OLA) in which differentiation between a wild-type locus and a mutation is based on the ability of two oligonucleotides to anneal adjacent to each other on the target DNA molecule allowing the two oligonucleotides joined together by a DNA ligase. See Landergren et al., Science, 241:1077 1080 (1988); Chen et al, Genome Res., 8:549 556 (1998); Iannone et al., Cytometry, 39:131 140 (2000). Thus, for example, to detect the A145G genetic variant in the beta-1 receptor gene, two oligonucleotides can be synthesized, one having the beta-1 adrenergic receptor sequence just 5' upstream from the locus with its 3' end nucleotide being identical to the nucleotide in the mutant locus of the beta-1 adrenergic receptor gene, the other having a nucleotide sequence matching the beta-1 adrenergic receptor sequence immediately 3' downstream from the locus in the beta-1 adrenergic receptor gene. The oligonucleotides can be labeled for the purpose of detection. Upon hybridizing to the target beta-1 adrenergic receptor gene under a stringent condition, the two oligonucleotides are subject to ligation in the presence of a suitable ligase. The ligation of the two oligonucleotides would indicate that the target DNA has a nucleotide variant at the locus being detected.

Detection of small genetic variations can also be accomplished by a variety of hybridization-based approaches. Allele-specific oligonucleotides are most useful. See Conner et al., Proc. Natl. Acad. Sci. USA, 80:278 282 (1983); Saiki et al, Proc. Natl. Acad. Sci. USA, 86:6230 6234 (1989). Oligonucleotide probes hybridizing specifically to a beta-1 adrenergic receptor gene allele having a particular gene variant at a particular locus but not to other alleles can be designed by methods known in the art. The probes can have a length of, e.g., from 10 to about 50 nucleotide bases. The target beta-1 adrenergic receptor DNA and the oligonucleotide probe can be contacted with each other under conditions sufficiently stringent such that the genetic variant can be distinguished from the wild-type beta-1 adrenergic receptor gene based on the presence or absence of hybridization. The probe can be labeled to provide detection signals. Alternatively, the allele-specific oligonucleotide probe can be used as a PCR amplification primer in an "allele-specific PCR" and the presence or absence of a PCR product of the expected length would indicate the presence or absence of a particular genetic variant (e.g., the A145G genetic variant of the beta-1 adrenergic receptor gene).

Other useful hybridization-based techniques allow two single-stranded nucleic acids annealed together even in the presence of mismatch due to nucleotide substitution, insertion or deletion. The mismatch can then be detected using various techniques. For example, the annealed duplexes can be subject to electrophoresis. The mismatched duplexes can be detected based on their electrophoretic mobility that is different from the perfectly matched duplexes. See Cariello, Human Genetics, 42:726 (1988). Alternatively, in RNase protection assay, an RNA probe can be prepared spanning the SNP site to be detected and having a detection marker. See Giunta et al., Diagn. Mol. Path., 5:265 270 (1996); Finkelstein et al., Genomics, 7:167 172 (1990); Kinszler et al., Science 251:1366 1370 (1991). The RNA probe can be hybridized to the target DNA or mRNA forming a heteroduplex that is then subject to the ribonuclease RNase A digestion. RNase A digests the RNA probe in the heteroduplex only at the site of mismatch. The digestion can be determined on a denaturing electrophoresis gel based on size variations. In addition, mismatches can also be detected by chemical cleavage methods known in the art. See e.g., Roberts et al., Nucleic Acids Res., 25:3377 3378 (1997).

In the mutS assay, a probe can be prepared matching the beta-1 adrenergic receptor gene sequence surrounding the locus at which the presence or absence of the A145G mutation is to be detected, except that a predetermined nucleotide is used at the SNP locus. Upon annealing the probe to the target DNA to form a duplex, the E. coli mutS protein is contacted with the duplex. Since the mutS protein binds only to heteroduplex sequences containing a nucleotide mismatch, the binding of the mutS protein will be indicative of the presence of a mutation. See Modrich et al., Ann. Rev. Genet., 25:229 253 (1991).

A great variety of improvements and variations have been developed in the art on the basis of the above-described basic techniques, and can all be useful in detecting the genetic variant of the present invention. For example, the "sunrise probes" or "molecular beacons" utilize the fluorescence resonance energy transfer (FRET) property and give rise to high sensitivity. See Wolf et al., Proc. Nat. Acad. Sci. USA, 85:8790 8794 (1988). Typically, a probe spanning the nucleotide locus to be detected are designed into a hairpin-shaped structure and labeled with a quenching fluorophore at one end and a reporter fluorophore at the other end. In its natural state, the fluorescence from the reporter fluorophore is quenched by the quenching fluorophore due to the proximity of one fluorophore to the other. Upon hybridization of the probe to the target DNA, the 5' end is separated apart from the 3'-end and thus fluorescence signal is regenerated. See Nazarenko et al., Nucleic Acids Res., 25:2516 2521 (1997); Rychlik et al., Nucleic Acids Res., 17:8543 8551 (1989); Sharkey et al., Bio/Technology 12:506 509 (1994); Tyagi et al., Nat. Biotechnol., 14:303 308 (1996); Tyagi et al., Nat. Biotechnol., 16:49 53 (1998). The homo-tag assisted non-dimer system (HANDS) can be used in combination with the molecular beacon methods to suppress primer-dimer accumulation. See Brownie et al., Nucleic Acids Res., 25:3235 3241 (1997).

Dye-labeled oligonucleotide ligation assay is a FRET-based method, which combines the OLA assay and PCR. See Chen et al., Genome Res. 8:549 556 (1998). TaqMan is another FRET-based method for detecting SNPs. A TaqMan probe can be oligonucleotides designed to have the nucleotide sequence of the beta-1 adrenergic receptor gene spanning the SNP locus of interest and to differentially hybridize with different beta-1 adrenergic receptor alleles. The two ends of the probe are labeled with a quenching fluorophore and a reporter fluorophore, respectively. The TaqMan probe is incorporated into a PCR reaction for the amplification of a target beta-1 adrenergic receptor gene region containing the locus of interest using Taq polymerase. As Taq polymerase exhibits 5'-3' exonuclease activity but has no 3'-5' exonuclease activity, if the TaqMan probe is annealed to the target beta-1 adrenergic receptor DNA template, the 5'-end of the TaqMan probe will be degraded by Taq polymerase during the PCR reaction thus separating the reporting fluorophore from the quenching fluorophore and releasing fluorescence signals. See Holland et al., Proc. Natl. Acad. Sci. USA, 88:7276 7280 (1991); Kalinina et al., Nucleic Acids Res., 25:1999 2004 (1997); Whitcombe et al., Clin. Chem., 44:918 923 (1998).

In addition, the detection in the present invention can also employ a chemiluminescence-based technique. For example, an oligonucleotide probe can be designed to hybridize to either the wild-type or a mutated beta-1 adrenergic receptor gene locus but not both. The probe is labeled with a highly chemiluminescent acridinium ester. Hydrolysis of the acridinium ester destroys chemiluminescence. The hybridization of the probe to the target DNA prevents the hydrolysis of the acridinium ester. Therefore, the presence or absence of a particular mutation in the target DNA is determined by measuring chemiluminescence changes. See Nelson et al., Nucleic Acids Res., 24:4998 5003 (1996).

The detection of the A145G genetic variant in the beta-1 adrenergic receptor gene sequence in accordance with the present invention can also be based on the "base excision sequence scanning" (BESS) technique. The BESS method is a PCR-based mutation scanning method. BESS T-Scan and BESS G-Tracker are generated which are analogous to T and G ladders of dideoxy sequencing. Mutations are detected by comparing the sequence of normal and mutant DNA. See, e.g., Hawkins et al., Electrophoresis, 20:1171 1176 (1999).

Another useful technique that is gaining increased popularity is mass spectrometry. See Graber et al., Curr. Opin. Biotechnol., 9:14 18 (1998). For example, in the primer oligo base extension (PROBE.TM.) method, a target nucleic acid is immobilized to a solid-phase support. A primer is annealed to the target immediately 5' upstream from the locus to be analyzed. Primer extension is carried out in the presence of a selected mixture of deoxyribonucelotides and dideoxyribonucleotides. The resulting mixture of newly extended primers is then analyzed by MALDI-TOF. See e.g., Monforte et al., Nat. Med., 3:360 362 (1997).

In addition, the microchip or microarray technologies are also applicable to the detection method of the present invention. Essentially, in microchips, a large number of different oligonucleotide probes are immobilized in an array on a substrate or carrier, e.g., a silicon chip or glass slide. Target nucleic acid sequences to be analyzed can be contacted with the immobilized oligonucleotide probes on the microchip. See Lipshutz et al., Biotechniques, 19:442 447 (1995); Chee et al., Science, 274:610 614 (1996); Kozal et al., Nat. Med. 2:753 759 (1996); Hacia et al., Nat. Genet., 14:441 447 (1996); Saiki et al., Proc. Natl. Acad. Sci. USA, 86:6230 6234 (1989); Gingeras et al., Genome Res., 8:435 448 (1998). Alternatively, the multiple target nucleic acid sequences to be studied are fixed onto a substrate and an array of probes is contacted with the immobilized target sequences. See Drmanac et al., Nat. Biotechnol., 16:54 58 (1998). Numerous microchip technologies have been developed incorporating one or more of the above described techniques for detecting mutations particularly SNPs. The microchip technologies, combined with computerized analysis tools allow fast screening in a large scale. The adaptation of the microchip technologies to the present invention will be apparent to a person of skill in the art apprised of the present disclosure. See, e.g., U.S. Pat. No. 5,925,525 to Fodor et al; Wilgenbus et al., J. Mol. Med., 77:761 786 (1999); Graber et al., Curr. Opin. Biotechnol., 9:14 18 (1998); Hacia et al., Nat. Genet., 14:441 447 (1996); Shoemaker et al., Nat. Genet., 14:450 456 (1996); DeRisi et al., Nat. Genet., 14:457 460 (1996); Chee et al., Nat. Genet., 14:610 614 (1996); Lockhart et al., Nat. Genet., 14:675 680 (1996); Drobyshev et al., Gene, 188:45 52 (1997).

In yet another technique for detecting single nucleotide variations, the Invader.RTM. assay utilizes a novel linear signal amplification technology that improves upon the long turnaround times required of the typical PCR DNA sequenced-based analysis. See Cooksey et al., Antimicrobial Agents and Chemotherapy 44:1296 1301 (2000). This assay is based on cleavage of a unique secondary structure formed between two overlapping oligonucleotides that hybridize to the target sequence of interest to form a "flap." Each "flap" then generates thousands of signals per hour. Thus, the results of this technique can be easily read, and the methods do not require exponential amplification of the DNA target. The Invader.RTM. system utilizes two short DNA probes, which are hybridized to a DNA target. The structure formed by the hybridization event is recognized by a special cleavase enzyme that cuts one of the probes to release a short DNA "flap." Each released "flap" then binds to a fluorescently-labeled probe to form another cleavage structure. When the cleavase enzyme cuts the labeled probe, the probe emits a detectable fluorescence signal.

Furthermore, the detection of the A145G genetic variant in the beta-1 adrenergic receptor gene sequence in accordance with the present invention can also be based on Sniper.TM., a sensitive, high-throughput SNP scoring system designed for the accurate fluorescent detection of specific SNPs from oligonucleotides, PCR fragments or genomic DNA. See Clark and Pickering Life Science News 6, 2000, Amersham Pharmacia Biotech (2000). Because this method depends on two hybridization events combined with a ligation event, it provides highly accurate allele discrimination. For each SNP, two linear, allele-specific probes are designed that circularize when they anneal to the target sequence. Both allele-specific probes are identical with the exception of the 3'-base, which is varied to complement the polymorphic site. Between the two allele-specific probes is a backbone sequence that encodes binding sites for two rolling circle amplication primers. In the first stage of the assay, target genomic DNA is denatured and then hybridized with a pair of single, allele-specific, open-circle oligonucleotide probes resulting in circularization of the probe. When the 3'-base exactly complements the target DNA, ligation of the probe will preferentially occur. Subsequent detection of the circularized oligonucleotide probes is by rolling circle amplification, whereupon the amplified probe products are detected by fluorescence.

As is apparent from the above survey of the suitable detection techniques, it may or may not be necessary to amplify the target DNA, i.e., the beta-1 adrenergic receptor gene sequence to increase the number of target DNA molecule, depending on the detection techniques used. For example, most PCR-based techniques combine the amplification of a portion of the target and the detection of the mutations. PCR amplification is well known in the art and is disclosed in U.S. Pat. Nos. 4,683,195 and 4,800,159, both of which are incorporated herein by reference. For non-PCR-based detection techniques, if necessary, the amplification can be achieved by, e.g., in vivo plasmid multiplication, or by purifying the target DNA from a large amount of tissue or cell samples. See generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989. However, even with scarce samples, many sensitive techniques have been developed in which small genetic variations such as single-nucleotide substitutions can be detected without having to amplify the target DNA in the sample. For example, techniques have been developed that amplify the signal as opposed to the target DNA by, e.g., employing branched DNA or dendrimers that can hybridize to the target DNA. The branched or dendrimer DNAs provide multiple hybridization sites for hybridization probes to attach thereto thus amplifying the detection signals. See Detmer et al., J. Clin. Microbiol., 34:901 907 (1996); Collins et al., Nucleic Acids Res., 25:2979 2984 (1997); Horn et al., Nucleic Acids Res., 25:4835 4841 (1997); Horn et al., Nucleic Acids Res., 25:4842 4849 (1997); Nilsen et al., J. Theor. Biol., 187:273 284 (1997).

A number of other techniques that avoid amplification all together include, e.g., surface-enhanced resonance Raman scattering (SERRS), fluorescence correlation spectroscopy, and single-molecule electrophoresis. In SERRS, a chromophore-nucleic acid conjugate is absorbed onto colloidal silver and is irradiated with laser light at a resonant frequency of the chromophore. See. Graham et al., Anal. Chem., 69:4703 4707 (1997). The fluorescence correlation spectroscopy is based on the spatio-temporal correlations among fluctuating light signals and trapping single molecules in an electric field. See Eigen et al., Proc. Natl. Acad. Sci. USA, 91:5740 5747 (1994). In single-molecule electrophoresis, the electrophoretic velocity of a fluorescently tagged nucleic acid is determined by measuring the time required for the molecule to travel a predetermined distance between two laser beams. See Castro et al., Anal. Chem., 67:3181 3186 (1995).

In addition, the allele-specific oligonucleotides (ASO) can also be used in in situ hybridization using tissues or cells as samples. The oligonucleotide probes which can hybridize differentially with the wild-type gene sequence or the gene sequence harboring a mutation may be labeled with radioactive isotopes, fluorescence, or other detectable markers. In situ hybridization techniques are well known in the art and their adaptation to the present invention for detecting the presence or absence of the A145G genetic variant in the beta-1 adrenergic receptor gene of a particular individual should be apparent to a skilled artisan apprised of this disclosure.

Protein-based detection techniques may also prove to be useful, especially in detecting the Ser49Gly amino acid substitution of the present invention. To detect amino acid variations, protein sequencing techniques may be used. For example, a beta-1 adrenergic receptor protein or fragment thereof can be synthesized by recombinant expression using a beta-1 adrenergic receptor DNA fragment isolated from an individual to be tested. Preferably, a beta-1 adrenergic receptor cDNA fragment of no more than 100 to 150 base pairs encompassing the polymorphic locus to be determined is used. The amino acid sequence of the peptide can then be determined by conventional protein sequencing methods. Alternatively, the recently developed HPLC-microscopy tandem mass spectrometry technique can be used for determining the amino acid sequence variations. In this technique, proteolytic digestion is performed on a protein, and the resulting peptide mixture is separated by reversed-phase chromatographic separation. Tandem mass spectrometry is then performed and the data collected therefrom is analyzed. See Gatlin et al., Anal. Chem., 72:757 763 (2000).

Other useful protein-based detection techniques include immunoaffinity assays based on idiotype-specific antibodies, i.e., antibodies specific to mutant beta-1 adrenergic receptor proteins according to the present invention. The method for producing such antibodies is described above in detail. Antibodies can be used to immunoprecipitate specific proteins from solution samples or to immunoblot proteins separated by, e.g., polyacrylamide gels. Immunocytochemical methods can also be used in detecting specific protein polymorphisms in tissues or cells. Other well known antibody-based techniques can also be used including, e.g., enzyme-linked immunosorbent assay (ELISA), radioimmuno-assay (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal or polyclonal antibodies. See e.g., U.S. Pat. Nos. 4,376,110 and 4,486,530, both of which are incorporated herein by reference.

Thus, various techniques can be used in genotyping the human beta-1 adrenergic receptor gene of an individual to determine, in the individual, the presence or absence of the A145G genetic variant or the Ser49Gly amino acid variant. Typically, once the presence or absence of the nucleotide or amino acid variant of the present invention is determined, the result can be cast in a transmittable form that can be communicated to others (including the patient). Such a form can vary and can be tangible or intangible. The result with regard to the presence or absence of a beta-1 adrenergic receptor genetic variant of the present invention in the individual tested can be embodied in descriptive statements, diagrams, photographs, charts, images or any other visual forms. For example, images of gel electrophoresis of PCR products can be used in explaining the results. Diagrams showing where a genetic variant occurs in an individual's beta-1 adrenergic receptor gene are also useful in indicating the testing results. The statements and visual forms can be recorded on a tangible media such as papers, computer readable media such as floppy disks, compact disks, etc., or on an intangible media, e.g., an electronic media in the form of email or website on the Internet or an intranet. In addition, the result with regard to the presence or absence of a beta-1 adrenergic receptor genetic variant of the present invention in the individual tested can also be recorded in a sound form and transmitted through any suitable media, e.g., analog or digital cable lines, fiber optic cables, etc., via telephone, facsimile, wireless mobile phone, internet phone and the like.

The present invention also provides a kit for predicting, in an individual, effective response to one of three classes of antihypertensive drugs. The kit may include a carrier for the various components of the kit. The carrier can be a container or support, in the form of, e.g., bag, box, tube, rack, and is optionally compartmentalized. The carrier may define an enclosed confinement for safety purposes during shipment and storage. The kit also includes various components useful in detecting nucleotide or amino acid variants discovered in accordance with the present invention using the above-discussed detection techniques.

In one preferred embodiment, the detection kit includes one or more oligonucleotides useful in detecting the A145G genetic variant in the beta-1 adrenergic receptor gene sequence. Preferably, the oligonucleotides are designed such that they hybridize only to a beta-1 adrenergic receptor gene sequence containing the particular A145G genetic variant discovered in accordance with the present invention, under stringent conditions. Thus, the oligonucleotides can be used in mutation-detecting techniques such as allele-specific oligonucleotides (ASO), allele-specific PCR, TaqMan, chemiluminescence-based techniques, molecular beacons, and improvements or derivatives thereof, e.g., microchip technologies. The oligonucleotides in this embodiment preferably have a nucleotide sequence that matches a nucleotide sequence of the mutant beta-1 adrenergic receptor gene allele containing the A145G genetic variant to be detected. The nucleotide variant preferably is not located at the 5' or 3' end, but in other positions in the oglionucleotides. The length of the oligonucleotides in accordance with this embodiment of the invention can vary depending on its nucleotide sequence and the hybridization conditions employed in the detection procedure. Preferably, the oligonucleotides contain from about 10 nucleotides to about 100 nucleotides, more preferably from about 15 to about 75 nucleotides. Under certain conditions, a length of 18 to 30 may be optimum. In any event, the oligonucleotides should be designed such that it can be used in distinguishing one genetic variant from another at a particular locus under predetermined stringent hybridization conditions. The hybridization of an oligonucleotide with a nucleic acid and the optimization of the length and hybridization conditions should be apparent to a person of skill in the art. See generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989. Notably, the oligonucleotildes in accordance with this embodiment are also useful in mismatch-based detection techniques described above, such as electrophoretic mobility shift assay, RNase protection assay, mutS assay, etc.

In another embodiment of this invention, the kit includes one or more oligonucleotides suitable for use in detecting techniques such as ARMS, oligonucleotide ligation assay (OLA), and the like. The oligonucleotides in this embodiment include a beta-1 adrenergic receptor gene sequence immediately 5' upstream from the A145G genetic variant to be analyzed. The 3' end nucleotide is a nucleotide variant in accordance with this invention.

The oligonucleotides in the detection kit can be labeled with any suitable detection marker including but not limited to, radioactive isotopes, fluorephores, biotin, enzymes (e.g., alkaline phosphatase), enzyme substrates, ligands and antibodies, etc. See Jablonski et al., Nucleic Acids Res., 14:6115 6128 (1986); Nguyen et al., Biotechniques, 13:116 123 (1992); Rigby et al., J. Mol. Biol., 113:237 251 (1977). Alternatively, the oligonucleotides included in the kit are not labeled, and instead, one or more markers are provided in the kit so that users may label the oligonucleotides at the time of use.

In another embodiment of the invention, the detection kit contains one or more idiotype-specific antibodies, i.e., antibodies only recognize certain beta-1 adrenergic receptor proteins or polypeptides containing the A145G nucleotide variant of the present invention. Methods for producing and using such antibodies have been described above in detailed.

Various other components useful in the detection techniques may also be included in the detection kit of this invention. Examples of such components include, but are not limited to, Taq polymerase, deoxyribonucleotides, dideoxyribonucleotides other primers suitable for the amplification of a target DNA sequence, RNase A, mutS protein, and the like. In addition, the detection kit preferably includes instructions on using the kit for detecting the A145G genetic variant of the beta-1 adrenergic receptor gene sequences.

In one embodiment of the present invention, the method is primarily based on binding affinities to screen for compounds capable of interacting with or binding to a beta-1 adrenergic receptor protein containing one or more amino acid variants. Compounds to be screened may be peptides or derivatives or mimetics thereof, or non-peptide small molecules. Conveniently, commercially available combinatorial libraries of compounds or phage display libraries displaying random peptides are used.
 

Claim 1 of 12 Claims

1. A method for selecting an antihypertensive treatment for an individual, comprising: determining the genotype of an individual at the nucleotide 145 position of the beta-1 adrenergic receptor gene, wherein the presence of a homozygous A145G genetic variant would indicate an increased likelihood that said individual will respond more favorably to hydrochlorothiazide than to fosinopril or atenolol; and selecting hydrochlorothiazide as the antihypertensive treatment for said individual if said individual is homozygous for said A145G genetic variant.
 

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