WO2000003024A2 - Alleles of the human mu opioid receptor, diagnostic methods using said alleles, and methods of treatment based thereon - Google Patents
Alleles of the human mu opioid receptor, diagnostic methods using said alleles, and methods of treatment based thereon Download PDFInfo
- Publication number
- WO2000003024A2 WO2000003024A2 PCT/US1999/015707 US9915707W WO0003024A2 WO 2000003024 A2 WO2000003024 A2 WO 2000003024A2 US 9915707 W US9915707 W US 9915707W WO 0003024 A2 WO0003024 A2 WO 0003024A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- human
- opioid receptor
- variation
- allele
- subject
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
- C12N15/73—Expression systems using phage (lambda) regulatory sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
-
- C—CHEMISTRY; METALLURGY
- 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 generally to alleles of the human mu opioid receptor gene, along with products derived from such alleles. Also included herein are methods of diagnosing various susceptibilities using such alleles and determining treatment for certain diseases based upon the presence of specific alleles of the human mu opioid receptor gene, and various diseases or disorders related to physiological functions regulated by the hypothalamus pituitary adrenal axis (HPA) or the hypothalamus pituitary gonadal axis (HPG).
- HPA hypothalamus pituitary adrenal axis
- HPG hypothalamus pituitary gonadal axis
- Opioid drugs have various effects on perception of pain, consciousness, motor control, mood, autonomic function, and can also induce physical dependence.
- the endogenous opioid system plays an important role in modulating endocrine, cardiovascular, respiratory, gastrointestinal functions, and immune functions.
- Opioids either exogenous or endogenous, exert their actions by binding to specific membrane-associated receptors.
- exogenous opioids examples include, opium, heroin, morphine, codeine, fentanyl, and methadone, to name only a few.
- a family of over 20 endogenous opioid peptides has been identified, wherein the members possess common structural features, including a positive charge juxtaposed with an aromatic ring that is required for interaction with an opioid receptor It has been determined that most, if not all the endogenous opioid peptides are derived from the proteolytic processing of three precursor proteins, 1 e , pro-opiomelanocortm, proenkephahn, and prodynorphin
- a fourth class of endogenous opioids, the endorphms has been identified (the gene encoding these proteins has not yet been cloned) In the processing of the endogenous opioid precursor proteins, initial cleavages are made by membrane-bound proteases that cut next to pairs of positively charged amino acid residues, and then trimming reactions produce the final endogenous opioids secrete
- opioid receptors there are at least three known types of opioid receptors, mu ( ⁇ ), delta ( ⁇ ), and kappa (K), to which morphine, the enkephahns, and the dynorphins can bind
- mu ⁇
- delta ⁇
- K kappa
- the mu receptor is generally regarded as primarily associated with pain relief, and drug or other chemical dependence, I e , addiction and alcoholism
- the human mu opioid receptor which modulates corticotropin releasing hormone, has been isolated and described in PCT Application WO 95/07983 (March 23, 1995) (SEQ ID NO. l) as well as in Chen, Y , Mestek, A , Hurley, J A , & Yu, L (1993) Mol Pharmacol 44, 8-12, and Wang, et al , FEBS letters, (1994)338 217-222 Furthermore, SEQ ID NO 1 can readily be obtained in GENBANK under accession number L25119
- the cDNA therefor contains an open reading frame capable of encoding a protein of 400 amino acid residues with 94 % sequence similarity to the rat mu opioid receptor Hydropathy analysis of the deduced protein indicates the presence of seven hydrophobic domains, typical of G-protem-coupled receptors
- the N-terminus contains five potential N-1 inked glycosylation sites which remain conserved between the human and the rat mu opioid receptor A variant in which Asn-40 is changed to Asp (N40D)
- heroin is hydrolyzed to morphine, which acts at the mu opioid receptor and results in an euphoric effect and confers the reinforcing properties of the drug and contributes to development of addiction Heroin addiction can be managed through treatment, primarily methadone maintenance
- the biological basis of heroin addiction may include diversity of gene structure
- Such genetic diversity of the human mu opioid receptor, and the impact of such diversity on receptor function, could contribute to the success or failure of pharmacological management
- Similar problems with respect to patient response to pharmacological treatment could occur in most, if not all addictive diseases, such as heroin addiction, alcohol addiction, or cocaine addiction to name only a few, or a combination thereof
- addiction to opioid drugs, especially heroin is a major social problem in the United States, and throughout the world
- recent epidemiological assessments sponsored by the NIH-NIDA and other federal agencies have found that around 2 7 million persons in the United States have used heroin at some time
- the numbers of "hardcore" long-term heroin addicts (addiction being defined herein as self administration of a regular, multiple, daily dose use of a short-acting opioid, such as heroin, for one year or more, with the development of tolerance, physical dependence and drug-seeking behavior, a definition codified in the Federal guidelines governing pharmacotherapy using long-acting agents such as methadone or LA AM, and used as the minimal requirement for entry into treatment) are now estimated to be approximately one million persons
- the human mu opioid receptor in the study of addiction, and the epidemic proportions
- hypothalamus pituitary adrenal axis HPA
- HPG hypothalamus pituitary gonadal axis
- the neuroendoc ⁇ ne system involves the integration of the neural and endocrine systems of the body, and is responsible for the coordination of numerous bodily functions
- hypothalamus a specialized portion of the brain involved in receiving and relaying messages from the central nervous system to other parts of the body
- hypothalamus secretes hypothalamic hormones, such as corticotropin releasing factor (CRF) or hormone and gonadotropin releasing hormone or luteinizing hormone releasing hormone
- DNA sequences of heretofore unknown isolated nucleic acid molecules which encode human mu opioid receptors wherein the DNA sequences include a combination of presently known and subsequently discovered polymorphisms of the human mu opioid receptors
- the present invention extends to heretofore unknown polymorphisms of the human mu opioid receptor gene that can serve as genetic markers to map the locus of the human mu opioid receptor gene
- the present invention further extends to DNA sequences of heretofore unknown isolated nucleic acid molecules which encode human mu opioid receptors, wherein the DNA sequences include a combination of presently known polymorphisms and polymorphisms of the human mu opioid receptors discovered by Applicants
- the present invention further extends to the characterization of the binding properties of heretofore unknown human mu opioid receptors produced from the expression of isolated nucleic acid molecules comprising DNA sequences with such heretofore unknown polymorphisms of the human mu opioid receptor gene, or combinations of unknown polymorphisms and known polymorphisms
- the present invention extends to characterizing the activity of such unknown human mu opioid receptors and particularly the increased or decreased ability of mu opioid receptors produced from isolated nucleic acid acids of the present invention to activate G protein-activated inwardly rectifying K + (GIRK) channels via a G protem-mediated mechanism
- the present invention further extends to Applicants' discovery that polymorphisms m an allele comprising a DNA sequence of SEQ ID NO 1 , such as Al 18G and C17T, which are described in further detail infra, are present in the population at a high frequency (greater than 5%)
- the present invention extends to Applicant's discovery of a correlation between polymo ⁇ hisms of the human mu opioid receptor gene, and the increased or decreased susceptibility of a subject to addictive diseases, such as heroin addiction, cocaine addiction, or alcohol addiction, to name only a few
- the present invention further extends to diagnostic methods to determine a subject's increased or decreased susceptibility to addictive diseases With the results of such methods, targeted prevention methods, early therapeutic intervention, and improved chronic treatment to opioid addiction are set forth herein and encompassed by the present invention
- attending medical professionals armed with the results of such diagnostic methods can determine whether administration of opioid analgesics is approp ⁇ ate or whether non-op 10 id derived analgesics should be administered to the subject
- appropriate choice and type of analgesic to treai a subject's pain can be made
- the present invention extends to methods of determining a subject's increased or decreased susceptibility to pam and response to analgesics, and the use of the information in prescribing analgesics to the subject
- the present invention extends to methods of diagnosing a disease or disorder in a subject, wherein the disease or disorder is related to a physiological function regulated by the HPA or HPG axes of the neuroendoc ⁇ ne system
- physiological functions include reproductive or sexual functions, gastrointestinal motihty, immune response, and ability to withstand stress
- the present invention extends to an isolated va ⁇ ant allele of a human mu opioid receptor gene which can serve as a genetic marker, wherein the predominant or most common allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO 1 , and a va ⁇ ant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO 1 , wherein the variation comprises G24A,
- the present invention extends to an isolated variant allele of a human mu opioid receptor gene as set forth above, which is detectably labeled Numerous detectable labels have applications in the present invention, such as radioactive elements, chemicals which fluoresces, or enzymes, to name onlv a few
- the present invention further extends to an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of the human mu opioid receptor gene, wherein the predominant or "most common" allele of a human mu opioid receptor gene found m the population comprises a DNA sequence of SEQ ID NO 1
- a variant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO 1 , wherein the variation comprises
- the present invention extends to an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of the human mu opioid receptor gene, wherein the predominant or 'most common ' allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO 1 , and a variant allele ot the present invention comprises a DNA sequence having a variation in SEQ ID NO 1 , wherein the variation comprises G24A. G779A, or G942A, or combinations thereof, wherein the isolated nucleic acid molecule is detectably labeled Examples of detectable labels that have applications in this embodiment of the present invention are described above
- the present invention extends to an isolated variant allele of a human mu opioid receptor gene, wherein the predominant or "most common" allele of the human mu opioid receptor gene encodes a human mu opioid receptor comprising an amino acid sequence of SEQ ID NO 2, and the variant allele of the human mu opioid receptor gene encodes a va ⁇ ant human mu opioid receptor comprising an ammo acid sequence having a variation in SEQ ID NO 2, wherein the variation comprises Arg260H ⁇ s
- the present invention extends to an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor gene of the present invention, wherein the isolated nucleic acid molecule encodes a va ⁇ ant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO 2, wherein the variation comprises Arg260H ⁇ s
- the present invention extends to a va ⁇ ant human mu opioid receptor comprising an ammo acid sequence having a variation in SEQ ID NO 2, wherein the variation comprises Arg260H ⁇ s
- an antibody having a variant human mu opioid receptor comprising an ammo acid sequence having a variation in SEQ ID NO:2, wherein the variation comprises Arg260H ⁇ s as an lmmunogen
- an antibody of the present invention can be detectably labeled Examples of detectable labels which have applications in this embodiment comprises a radioactive element, a chemical which fluoresces. or an enzyme, to name only a few
- the present invention extends to cloning vectors that can be used to clone copies of a variant alleles of a human mu opioid receptor gene of the present invention
- the present invention extends to a cloning vector comprising an isolated variant allele of a human mu opioid receptor gene and an origin of replication, wherein the predominant or "most common" allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO 1
- a variant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO 1. wherein the variation comprises G24A,
- the present invention extends to a cloning vector comprising an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor gene, and an origin of replication, wherein the predominant or "most common" allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO 1, and a variant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO 1, wherein the variation comprises
- a cloning vector having applications m the present invention includes E coli, bacteriophages such as lambda derivatives, plasmids such as pBR322 derivatives, and pUC plasmid derivatives such as pGEX vectors or pmal-c or pFLAG, to name only a few
- the present invention extends to expression vectors comprising an isolated variant allele a human mu opioid receptor gene operatively associated with a promoter, wherein the predominant or ' most common' allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO 1 , and a variant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO 1 , wherein the variation comprises G24A,
- the present invention extends to an expression vector comprising an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated va ⁇ ant allele a human mu opioid receptor gene, wherein the isolated nucleic acid molecule is operatively associated with a promoter
- the predominant or "most common" allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO 1
- a variant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO 1 , wherein the variation comprises G24A, G779A, or G942A, or combinations thereof
- promoters have applications in an expression vector of the present invention, including but not limited to immediate early promoters of hCMV, early promoters of SV40, early promoters of adenovirus, early promoters of vaccinia, early promoters of polyoma, late promoters of SV40, late promoters of adenovirus.
- late promoters of vaccinia late promoters of vaccinia, late promoters of polyoma, the lac the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage lambda, control regions of fd coat protein, 3- phosphoglycerate kinase promoter, acid phosphatase promoter, or promoters of yeast ⁇ mating factor, to name onlj a few
- the present invention extends to a unicellular host transformed or transfected with an expression vector of the present invention
- hosts which can be transformed or transfected with an expression vector of the present invention, and have applications m the present invention include, but are not limited to, E coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, RI 1. B-W, L-M COS1, COS7, BSC1 , BSC40, BMT10 or Sf9 cells
- the present invention extends to a method ot producing a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO 2, wherein the variation comprises Arg260H ⁇ s
- An example of such a method comprises the steps of culturing a unicellular host transformed or transfected with an expression vector comprising an isolated variant allele a human mu opioid receptor gene, wherein the predominant or "most common" allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO 1 , and a variant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO 1 , wherein the variation comprises G779A, operatively associated with a promoter
- the transformed or transfected unicellular host is then cultured under conditions that provide for expression of the variant allele of the human mu opioid receptor gene
- the variant human mu opioid receptor produced from such induced expression is then recovered from the unicellular host
- Another example comprises the steps of culturing a unicellular host transformed or trans
- the present invention extends to an isolated variant allele of a human mu opioid receptor gene, wherein the predominant or "most common" allele of the human mu opioid receptor gene comprises a DNA sequence of SEQ ID NO 1 , and a variant allele of the present invention comprises a DNA sequence having at least two variations in SEQ ID NO 1 , wherein the variations comprise
- the present invention further extends to an isolated variant allele of a human mu opioid receptor gone comprising a DNA sequence having at least two variations in SEQ ID NO. l , as stated above, which is detectably labeled
- detectable labels having applications in this embodiment include, but are not limited to. a radioactive element, a chemical which fluoresces, or an enzyme
- the present invention further extends to an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor gene, wherein the predominant or most common" allele of the human mu opioid receptor gene comprises a DNA sequence of SEQ ID NO 1 , and a variant allele of the present invention comprises a DNA sequence having at least two variations in SEQ ID NO 1 , wherein the variations comprise A118G,
- the present invention extends to a detectably labeled isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor comprising a DNA sequence having at least two variations in SEQ ID NO 1 , wherein the variations comprise A118G, C17T, G24A,
- detectable labels having applications in this embodiment of the invention include, but are not limited to, a radioactive element, a chemical which fluoresces, or an enzyme
- the present invention extends to an isolated variant allele of a human mu opioid receptor gene comprising a DNA sequence having at least two variations in SEQ ID NO 1 , as set forth above, wherein the predominant or most common allele ot a human mu opioid receptor gene encodes a human mu opioid receptor comprising an ammo acid sequence of SEQ ID NO 2, and a variant allele of the present invention encodes a human mu opioid receptor comprising an amino acid having at least two variations in SEQ ID NO 2, wherein the variations comprise Asn40Asp or conserved variants thereof
- the present invention further extends to an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor gene comprising a DNA sequence having at least two variations in SEQ ID NO l, wherein the variations comprise A118G,
- the isolated nucleic acid molecule encodes a variant human mu opioid receptor comprising an amino acid sequence having at least two variations m SEQ ID NO 2, wherein the variations comprise
- the present invention extends to a va ⁇ ant human mu opioid receptor comprising an ammo acid sequence having at least two variations in SEQ ID NO 2, wherein the variations comprise Asn40Asp or conserved variants thereof,
- the present invention extends to an antibody having as an immunogen a human mu opioid receptor comprising an ammo acid sequence having at least two variations in SEQ ID NO 2, wherein the variations comprise Asn40Asp or conserved variants thereof, Ala ⁇ Val or conserved variants thereof, or Arg260H ⁇ s or conserved variants thereof
- An antibody of the present invention can be a polyclonal antibody, a monoclonal antibody, or a chimeric antibody
- an antibody of the present invention can be detectably labeled Examples of detectable labels having applications in an antibody of the present invention include, but are not limited to, a radioactive element, a chemical which fluo- resces, or an enzyme
- the present invention extends to a cloning vector comprising an isolated variant allele of a human mu opioid receptor gene and an origin of replication, wherein the predominant or "most common" allele of the human mu opioid receptor gene present in the population comprises a DNA sequence of SEQ ID NO 1, and a variant allele of the present invention comprises a DNA sequence having at least two variations m SEQ ID NO 1 , wherein the variations comprise
- the present invention extends to a cloning vector comprising an isolated nucleic acid molecule hybridizable under standard hybridization conditions to a variant allele of a human mu opioid receptor and an origin of replication wherein the va ⁇ ant allele comprises a DNA sequence having at least two variations m SEQ ID NO 1 , wherein the variations comprise A118G,
- vectors include, but are not limited to, E coli, bacteriophages, such as lambda derivatives, plasmids such as pBR322 derivatives, and pUC plasmid derivatives such as pGEX vectors or pmal-c or pFLAG to name only a few
- the present invention extends to an expression vector comprising an isolated variant allele of a human mu opioid receptor gene operatively associated with a promoter, wherein such an isolated va ⁇ ant allele comprises a DNA sequence having at least two variations in SEQ ID NO 1 , wherein the variations comprise
- the present invention extends to an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor gene comprising a DNA sequence having at least two variations in SEQ ID NO 1 wherein the variations comprise
- promoters are available and have applications in an expression vector of the present invention
- examples of promoters having applications include, but are not limited to immediate early promoters of hCMV, early promoters of SV40, early promoters of adenovirus, early promoters of vaccinia, early promoters of polyoma, late promoters of SV40, late promoters of adenovirus.
- late promoters of vaccinia late promoters of polyoma the lac the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage lambda, control regions of fd coat protein, 3-phosphoglycerate kinase promoter, acid phosphatase promoter, or promoters of yeast ⁇ mating factor, to name only a few
- the present invention extends to a unicellular host transformed or transfected with an expression vector of the present invention
- unicellular hosts having applications in an embodiment of the present invention include, but are not limited to, E coli, Pseudonomas, Bacillus, Streptomyces. yeast, WHO, RI 1 , B-W, L-M, COS1 , COS7, BSC1, BSC40, BMT10 or Sf9 cells
- the present invention extends to a method for producing a human mu opioid receptor comprising an ammo acid sequence having at least two variations in SEQ ID NO 2, wherein the variations comprise
- an example of a method for producing such a human mu opioid receptor comprises the steps of culturing a unicellular host transformed or transfected with an expression vector comp ⁇ smg an isolated va ⁇ ant allele of a human mu opioid receptor gene operatively associated with a promoter, wherein the va ⁇ ant allele comprises a DNA sequence having at least two variations m SEQ ID NO 1 , wherein the variations comprise A118G,
- a method for producing a human mu opioid receptor of the present invention comprises the steps of culturing a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor gene comprising a DNA sequence having at least two variations in SEQ ID NO 1 , wherein the variations comprise A118G, C17T, G24A, G779A, or
- ⁇ -endo ⁇ hm an endogenous opioid comprising 31 ammo acid residues, and binds to the human mu opioid receptor, has a binding affinity approximately three times greater for a variant human mu opioid receptor produced from expression of a variant allele of the human mu opioid receptor gene comprising an Al 18G variation in SEQ ID NO 1, than for a human mu opioid receptor produced from expression of the predominant or most common' allele of the human mu opioid receptor gene
- ⁇ -endo ⁇ hm induced activity of a receptor produced from the expression of a variant allele of a human mu opioid receptor gene or an isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions comprising the A118G polymo ⁇ hism effected the receptor s activation of GIRK channels via a G protein-mediated mechanism relative to the activity of a receptor produced from the expression of the predominant or most common allele ⁇ -endo ⁇ hin is present in both the central nervous system (CNS) and the periphery It plays a role
- a variant allele of a human mu opioid receptor gene comprising a variation in SEQ ID NO 1, wherein the variation comprises C17T, is present at a statistically significant greater frequency in the genome of at least one defined subset of addicts suffering from at least one addictive disease, than m the genomes of people not suffering from such a disease
- the presence of such a variant allele of a human mu opioid receptor gene may alter perception of pain, susceptibility to develop opioid addiction following exposure to opioids, and influence the subject's reaction to therapeutic agents designed to treat the at least one addictive disease of the subject
- the C17T variant mu opioid receptor has a ⁇ - endo ⁇ hin binding affinity approximately 0 72 times that of the human mu opioid receptor produced from expression of the predominant or "most common" allele of the human mu opioid receptor gene
- the decreased binding affinity of this variant is in contrast to the increased affinity of the A118G variant mentioned above
- one of the important indices of cellular functioning of the mu opioid receptor is the inhibition of adeny
- the present invention extends to a method for determining a susceptibility m a subject to at least one addictive disease, comprising the steps of removing a bodily sample comprising a first and second allele of a human mu opioid receptor gene from the subject, and determining whether the first allele comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO 1 , wherein the variation comprises A118G, or C17T
- the present of at least one of these variations in the human mu opioid receptor gene of the first allele is expected to be indicative of the subject's susceptibility to at least one addictive disease relative to the susceptibility of a standard to at least one addictive disease, wherein the standard comprises a first allele comprising a human mu opioid receptor gene having a DNA sequence of SEQ ID NO 1
- Another embodiment of the method for determining a susceptibility in the subject to at least one addictive disease comprises the further step of determining whether the second allele of the bodily sample of the subject comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO 1. wherein the variations comprise A118G, or C17T
- the presence of at least one variation the second allele of the bodily sample is expected to be indicative of the subject's susceptibility to at least one addictive disease relative to a standard in which both alleles of a human mu opioid receptor gene comprise a DNA sequence of SEQ ID NO 1
- the presence of an Al 18G variation in the DNA sequence of the human mu opioid receptor gene of the first and/or second alleles in the bodily sample from the subject is expected to be indicative of a decreased susceptibility of the subject to at least one addictive disease relative to the standard
- the presence of a C17T variation in the DNA sequence of the human mu opioid receptor gene of the first and/or second alleles in the bodily sample from the subject is expected to be indicative of an increased susceptibility of the subject to at least one addictive disease relative the susceptibility of the standard to at least one addictive disease, wherein both alleles of the standard comprise a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- at least one addictive disease includes, but is not limited to opioid addiction, cocaine addiction or addiction to other psychostimulants, nicotine addiction, barbiturate or sedative hypnotic addiction, anxiolytic addiction, or alcohol addiction
- the present invention extends to a method for determining a susceptibility to at least one addictive disease m a subject relative to susceptibility to at least one addictive disease in a standard, involving the detection of variations in the human mu opioid receptor itself, and particularly, determining whether a variant human mu opioid receptor is present in a bodily sample from a subject
- a method comprises the steps of removing a bodily sample comprising a human mu opioid receptor from the subject, and determining whether the human mu opioid receptor present in the sample is a variant human mu opioid receptor of the invention, wherein the variant human mu opioid receptor comprises an amino acid sequence having at least one variation in SEQ ID NO 2, wherein the variation comprises Asn40Asp or conserved variants thereof, or
- the presence of at least one variation is expected to be indicative of the subject's susceptibility to at least one addictive disease relative to susceptibility to at least one addictive disease in a standard, wherein the human mu opioid receptor of the standard comprises an ammo acid sequence of SEQ ID NO 2
- a variant human mu opioid receptor present in the sample comprising an amino acid sequence having at least one variation in SEQ ID NO 2, wherein the variation comprises Asn40Asp or conserved variants thereof, is expected to be indicative of a decreased susceptibility to at least one addictive disease in the subject relative susceptibility to the at least one addictive disease in the standard, wherein the human mu opioid receptor of the standard comprises an amino acid sequence of SEQ ID NO 2
- a variant human mu opioid receptor present in a sample from the subject comprising a variation in SEQ ID NO 2, wherein the variation comprises Ala ⁇ Val or conserved variants thereof indicates an increased susceptibility to addictive diseases m the subject relative to a standard having a human mu opioid receptor comprising an amino acid sequence of SEQ ID NO 2
- at least one addictive disease includes, but is not limited to, opioid addiction, cocaine addiction or addiction to other psychostimulants, nicotine addiction, barbiturate or sedative hypnotic addiction, anxiolytic addiction, or alcohol addiction
- the present invention extends to a method for determining a susceptibility to pain in a subject relative to susceptibility to pain in a standard, comprising the steps of removing a bodily sample comprising a first and second allele of a human mu opioid receptor gene from the subject, and determining whether the first allele comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO- 1 , wherein the variation comprises A118G, or C17T
- the presence of at least one variation in the human mu opioid receptor gene of the first allele is expected to be indicative of a decreased oi increased susceptibility to pain in the subject relative to susceptibility to pain in the standard, wherein the first allele of the standard comprises a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- a method for determining a susceptibility to pain in a subject may further comprise the step of determining whether the second allele comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO 1 , wherein the variation comprises A118G, or C17T
- the presence of the at least one variation in the human mu opioid receptor gene of the second allele of the bodily sample from the subject is expected to be indicative of an increased or decreased susceptibility to pain in the subject relative to the susceptibility to pain in the standard
- the second allele in the standard comprises a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- the presence of at least one variation comprising C17T in the human mu opioid receptor gene of the first and/or second allele of the bodily sample from the subject is expected to be indicative of an increased susceptibility to pain in the subject relative to the susceptibility to pain in the standard, wherein the first and/or second allele of the standard comprise a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- the present invention extends to a method for determining a susceptibility to pain in a subject relative to susceptibility to pain in a standard by examining a bodily sample taken from the subject for the presence of a variant human mu opioid receptor
- a method for determining a susceptibility to pain in a subject relative to susceptibility to pain in a standard comprises the steps of removing a bodily sample comprising a human mu opioid receptor from the subject, and determining whether the human mu opioid receptor present in the sample is a variant human mu opioid receptor of the invention, 1 e , comprises an amino acid sequence having at least one variation in SEQ ID NO 2, wherein the variation comprises
- a variant human mu opioid receptor comprising an amino acid sequence having at least one variation in SEQ ID NO 2 wherein the variation comprises Asn40Asp or conserved variants thereof, is expected to be indicative of a decreased susceptibility to pain in the subject relative to susceptibility to pain in the standard, wherein the human mu opioid receptor of the standard comprises an amino acid sequence of SEQ ID NO 2
- a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO 2, wherein the variation comprises Ala ⁇ Val or conserved variants thereof, in a bodily sample taken from a subject is expected to be indicative of an increased susceptibility to pain in the subject relative to susceptibility to pain m the standard, wherein the human mu opioid receptor of the standard comprises an amino acid sequence of SEQ ID NO 2
- a susceptibility to pain in the subject has been determined, it is possible for attending medical professionals treating the subject to administer to an appropriate, or therapeutically effective amount of pam reliever in order to induce analgesia in the subject
- Administration of such an amount is important to the subject because, should an inappropriate amount of pa reliever be administered, the subject may not experience analgesia, and may be exposed to potentially deleterious side effects of the pam reliever, such as induction of addiction to the pain reliever, brain damage, or death
- the present invention extends to a method for determining a therapeuticallv effective amount of pain reliever to administer to a subject m order to induce analgesia in the subject relative to a therapeutically effective amount of the pain reliever to administer to a standard in order to induce analgesia in the standard
- the method comprises determining a susceptibility to pain in the subject relative to susceptibility to pain in the standard
- the susceptibility of pain in the subject is expected to be indicative of the therapeutically effective amount of the pain reliever to administer to the subject to induce analgesia in the subject relative to the amount of the pain reliever to administer to the standard to induce analgesia in the standard
- the present invention extends to a method for determining a therapeutically effective amount of pain reliever to administer to a subject in order to induce analgesia in the subject relative to a therapeutically effective amount of the pain reliever to administer to a standard in order to induce analgesia in the standard
- the method comprises the steps of removing a bodily sample comprising a first and second allele of a human mu opioid receptor gene from the subject, and determining whether the first allele comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO 1 , wherein the variation comprises A118G, or C17T
- the presence of at least one variation m the human mu opioid receptor gene of the first allele from the bodily sample is expected to be indicative of the therapeutically effective amount of pain reliever to administer to the subject to induce analgesia in the subject relative to the therapeutically effective amount of pam reliever to administer to the standard to induce analgesia in the standard
- the standard comprises a first allele comprising a
- the present invention further extends to a method for determining a therapeutically effective amount of pain reliever to administer to a subject in order to induce analgesia in the subject relative to a therapeutically effective amount of pain reliever to administer to a standard to induce analgesia therein, further comprising the steps of removing a bodily sample comprising a first and second allele comprising a human mu opioid receptor gene from the subject, and determining whether the second allele of the bodily sample comprises a human mu opioid receptor gene comprising a DNA sequence comprising at least one variation in SEQ ID NO 1. wherein the variation comprises A118G, or
- the presence of at least one variation in the human mu opioid receptor gene of the first and/or second allele ot the bodily sample is expected to be indicative of the therapeutically effective amount ot pain reliever to administer to the subject to induce analgesia therein relative to the amount ot pain reliever to administer to a standard to induce analgesia therein, wherein the first and second alleles of the standard comprise a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- a variation of the human mu opioid receptor gene of the first and/or second allele from the bodily sample taken from the subject comprising a DNA sequence comprising a variation in SEQ ID NO 1 , wherein the variation comprises A118G, is expected to be indicative of a decreased susceptibility to pain in the subject relative susceptibility of pain in the standard Consequently, the subject requires a decreased therapeutically effective amount of pam reliever in order to induce analgesia therein relative to the therapeutically effective amount of pain reliever needed to induce analgesia in the standard
- a variation of the DNA sequence of the human mu opioid receptor gene of the first and/or second allele from the bodily sample taken from the subject, comprising C17T is expected to be indicative of an increased susceptibility to pain in the subject relative to the susceptibility to pain in the standard
- the therapeutically effective amount of pain reliever to administer to the subject in order to induce analgesia therein is greater than the therapeutically effective amount of pain reliever to administer to the standard to induce analgesia therein
- the present invention extends to determining a therapeutically effective amount of pam reliever to administer to a subject in order to induce analgesia in the subject, by examining a bodily sample from a subject for the presence of a variant human mu opioid receptor comprising an ammo acid sequence having a variation in SEQ ID NO 2 More specifically, the present invention extends to a method for determining a therapeutically effective amount of pam reliever to administer to a subject in order to induce analgesia in the subject, relative to a therapeutically effective amount of pain reliever to administer to a standard in order to induce analgesia in the standard, comprising the steps of removing a bodily sample comprising a human mu opioid receptor from the subject, and determining whether the human mu opioid receptor present m the sample comprises an amino acid sequence having at least one variation in SEQ ID NO 2, wherein the variation comprises Asn40Asp or conserved variants thereof, or
- a variant human mu opioid receptor comprising an ammo acid sequence having a variation in SEQ ID NO 2, wherein the variation comprises Asn40Asp or conserved variants thereof m the sample from the subject, is expected to be indicative of a decreased therapeutically effective amount of pain reliever to administer to the subject to induce analgesia therein relative to the therapeutically effective amount of pam reliever to administer to the standard in order to induce analgesia therein
- the presence of a va ⁇ ant human mu opioid receptor m the sample from the subject, wherein the receptor comprises an amino acid sequence have a variation in SEQ ID NO 2, wherein the variation comprises Ala ⁇ Val or conserved variants thereof is expected to be indicative of an increased therapeutically effective amount of pain reliever to admmis- ter to the subject in order to induce analgesia therein relative to the therapeutically effective amount to administer to the standard to induce analgesia therein
- pa relievers having applications m this embodiment of the present invention include, but are not limited to, mo ⁇ hine, codeine, dihydromo ⁇ hm, mepe ⁇ dme, metha- done, fentanyl and its congeners, buto ⁇ henol, nalbuphme, LAAM, or propoxyphme, to name only a few
- the present invention extends to a method for determining a therapeutically effective amount of a therapeutic agent for treating at least one addictive disease to administer to a subject suffering from at least one addictive disease, relative to a therapeutically effective amount of the therapeutic agent to administer to a standard suffering from the at least one addictive disease
- the dosage of therapeutic agent administered to an addict can be tailored" to the addict's needs based upon the addict's genotype
- An example of such a method comprises the steps of removing a bodily sample from the subject, wherein the bodily sample comprises a first and second allele of the human mu opioid receptor gene, and determining whether the first allele comprises a DNA sequence having at least one variation in SEQ ID NO 1 , wherein the variation comprises A118G, or C17T
- the presence of the at least one variation in the human mu opioid receptor gene of the first allele in the bodily sample from the subject is related to the therapeutically effective amount of therapeutic agent to administer to the subject to treat the subject's at least one addictive disease, relative to the therapeutically effective amount of the therapeutic agent
- a method for determining a therapeutically effective amount of therapeutic agent to administer to a subject suffering from at least one addictive disease may further comprise an additional step of determining whether the second allele of the bodily sample taken from the subject comprises a human mu opioid receptor gene comprises a DNA sequence having at least one variation in SEQ ID NO 1 , wherein the at least one variation comprises
- Such a variation in the first and/or second allele of the bodily sample is expected to be indicative of the therapeutically effective amount of the therapeutic agent to administer to the subject to treat the at least one addictive disease of the subject relative to the therapeutically effective amount of the therapeutic agent to administer to the standard suffering from the at least one addictive disease
- a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO 1 in the first and/or second alleles of the bodily sample taken from the subject, wherein the variation comprises Al 18G is expected to be indicative of a decreased therapeutically effective amount of the therapeutic agent to administer to the subject to treat the at least one addictive disease of the subject, relative to the therapeutically effective amount of the therapeutic agent to administer to the standard suffering from the at least one addictive disease, wherein the two alleles of the standard comprise a human mu opioid receptor gene comprising a DNA sequence ot SEQ ID NO 1
- a human mu opioid receptor gene comprising a DNA sequence having at least one variation of SEQ ID NO 1 in the first and/or second allele of the bodily sample taken from the subject, wherein the variation comprises C17T, is expected to be indicative of an increased therapeutically effective amount of the therapeutic agent to administer to treat the at least one addictive disease of the subject relative to the therapeutically effective amount of the therapeutic agent to administer to the standard suffering from the at least one addictive disease to treat the at least one addictive disease in the standard, wherein the alleles of the standard comprise a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- the present invention extends to determining a therapeutically effective amount of a therapeutic agent for treating at least one addictive disease to administer to a subject suffering from at least one addictive disease by examining a bodily sample from a subject for the presence of a variant human mu opioid receptor comprising an amino acid sequence having at least one variation in SEQ ID NO 2 More specifically, the present invention extends to a method for determining a therapeutically effective amount of therapeutic agent for treating at least one addictive disease to administer to a subject suffering from the at least one addictive disease, relative to a therapeutically effective amount of the therapeutic agent to administer to a standard suffering from the at least one addictive disease, wherein the method comprises the steps of removing a bodily sample comprising a human mu opioid receptor from the subject, and determining whether the human mu opioid receptor present in the sample comprises an amino acid sequence having at least one variation in SEQ ID NO 2, wherein the variation comprises Asn40Asp or conserved variants thereof, or Ala ⁇ Val or conserved variants thereof.
- the variation comprises Asn40Asp or conserved variants
- a variant human mu opioid receptor comprising an amino acid sequence having at least one variation in SEQ ID NO 2 comprising Asn40Asp or conserved variants thereof in the bodily sample of the subject is expected to be indicative of a decreased therapeutically effective amount of the therapeutic agent to administer to the subject to treat the at least one addictive disease in the subject relative to the therapeutically effective amount of the therapeutic agent to administer to the standard to treat the at least one addictive disease therein
- a variant human mu opioid receptor comprising an amino acid sequence having at least one variation in SEQ ID NO 2, wherein the variation comprises Ala ⁇ Val or conserved variants thereof in the bodily sample of the subject is expected to be indicative of an increased therapeutically effective amount of the therapeutic agent to administer to the subject order treat the at least one addictive disease in the subject relative to the therapeutically effective amount of the therapeutic agent to administer to the standard suffering from the at least one addictive disease
- at least one addictive disease includes, but is not limited to opioid addiction, cocaine addiction or addiction to other psychostimulants, nicotine addiction, barbiturate or sedative hypnotic addiction, anxiolytic addiction, or alcohol addiction
- examples of therapeutic agents having applications of the present invention include methadone, LAAM, maltrexone, or bup ⁇ no ⁇ hine, to name only a few
- the present invention extends to a method for diagnosing a disease or disorder related to a physiological function regulated by the HPA or HPG axes of the neuroendoc ⁇ ne system
- the HPA and HPG axes play an important role in regulation of numerous physiological activities such as reproductive and sexual function, gastrointestinal motihty, immune response to an antigen, or an ability to withstand stress
- the HPA and HPG axes exert such regulatory control via the production of endogenous opioids that interact with opioid in many locations of the body
- the mu opioid receptor is centrally involved in tonic regulation of the luteinizmg hormone, particularly in its pulsatile release
- the mu opioid receptor modulates corticotropin releasing factor/hormone (CRF or CRH) in the hypothalamus which m turn modulates production of pro-opiomelanocortin (POMC) in the pituitary which is processed into several active peptides such as ACTH, which stimulates the
- CRF or CRH corticotropin releasing factor/
- the binding affinity of an opioid receptor such as a mu opioid receptor with an endogenous opioid ligand, such as ⁇ -endo ⁇ hin. is expected to modulate such physiological activities
- the binding affinity of variant mu opioid receptors explained above, for endogenous opioid ligands such as ⁇ -endo ⁇ hin is expected to modulate those physiological activities regulated by the HPA and HPG axes relative to those physiological activities in a standard having mu opioid receptors produced from the predominant or "most common" allele of the mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- the present invention extends to a method of diagnosing a disease or disorder related to a physiological function regulated by the HPA or HPG axes
- physiological functions regulated by the HPA and the HPG include, but are not limited to sexual or reproductive functions, gastrointestinal motihty, immune response, or ability to withstand stress
- Such a method comprises the steps of removing a bodilv sample comprising a first and second allele of a
- the presence of at least one variation in the human mu opioid receptor gene of the first allele is expected to be indicative of a disorder related to a physiological function regulated by the HPA or GPA, such as sexual or reproductive functions, gastrointestinal motihty, immune response, and the ability to withstand stress, wherein the first allele of the standard comprises a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- a method for diagnosing a disease or disorder related to a physiological function regulated by the HPA or GPA may further comprise the step of determining whether the second allele of the bodily sample comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO 1 , wherein the variation comprises A118G, or C17T
- the presence of the at least one variation in the human mu opioid receptor gene of the second allele of the bodily sample from the subject may be expected to be indicative of a disease or disorder related to sexual and reproductive functions, gastrointestinal motihty, immune response, or the ability of the subject to withstand stress
- At least one variation m the human mu opioid receptor gene of the first and/or second allele of the bodily sample taken from the subject, wherein the variation comprises A118G is expected to be indicative of decreased HPA and HPG activity, resulting m increased sexual or reproductive functions, increased gastrointestinal motihty, increased immune response, or increased ability to withstand stress relative to the levels of such function observed in a standard
- the presence of at least one variation comprising C17T in the human mu opioid receptor gene of the first and/or second allele of the bodily sample from the subject is expected to be indicative increased HPA or HPG activity, resulting in decreased sexual or reproductive function, decreased gastrointestinal motihty, decreased immune response, or decreased ability to withstand stress relative to the levels of such function observed m a standard
- the present invention extends to a method for diagnosing a disease or disorder related to a physiological function regulated by the HPA or GPA by examining a bodily sample taken from the subject for the presence of a variant human mu opioid receptor
- a method for diagnosing a disease or disorder related to a physiological function regulated by the HPA or GPA comprises the steps of removing a bodily sample comprising a human mu opioid receptor from the subject, and determining whether the human mu opioid receptor present in the sample is a va ⁇ ant human mu opioid receptor of the invention, l e , comprises an ammo acid sequence having at least one variation in SEQ ID NO 2, wherein the variation comprises
- a variant human mu opioid receptor comprising an ammo acid sequence having at least one variation m SEQ ID NO 2 wherein the variation comprises Asn40Asp or conserved variants thereof, is expected to be indicative decreased HPA and HPG activity, resulting in increased sexual or reproductive functions, increased gastrointestinal motility, increased immune response, or increased ability to withstand stress relative to the levels of such function observed in a standard having a mu opioid receptor comprising an amino acid sequence of SEQ ID NO:2.
- a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO:2, wherein the variation comprises Ala ⁇ Val or conserved variants thereof, in a bodily sample taken from a subject is expected to be indicative of increased activity of the HPA and HPG axes, resulting in decreased sexual or reproductive functions, decreased gastrointestinal motility, decreased immune response, or decreased ability to withstand stress relative to the levels of such function observed in a standard having a mu opioid receptor comprising an amino acid sequence of SEQ ID NO:2.
- specific diseases or disorders related to regulation of physiological functions by the HPA or HPG axes include infertility, constipation, diarrhea, decreased immune response to antigens relative to a standard, or decreased of ability to withstand stress relative to a standard.
- the present invention extends to a method for determining an appropriate therapeutic agent to administer to a subject suffering from a disease or disorder related to a physiological function regulated by the HPA or HPG axes, comprising removing a bodily sample from the subject, and determining the presence of at least one variant allele of a mu opioid receptor gene in the bodily sample, wherein the variant allele comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO: l , wherein the variation comprises: A118G; or C17T.
- the present invention further extends to a method for selecting an appropriate therapeutic agent to administer to a subject suffering from a disease or disorder related to a physiologi- cal function regulated by the HPA or HPG axes as set forth above, further comprising determining whether the bodily sample comprises a second variant allele of the mu opioid receptor gene comprising a DNA sequence having a variation in SEQ ID NO 1, wherein the variation comprises A118G, or
- the mu opioid receptors of the subject are expected to have increased function relative to mu opioid receptors of a standard produced from expression of the predominant or "most common" mu opioid receptor allele comprising a DNA sequence of SEQ ID NO 1
- This increased function is expected to result m decreased function of the HPA and HPG axes
- an appropriate therapeutic agent for treating a disease or disorder related to decreased activity of the HPA or HPG axes, such as diarrhea can be selected
- a human mu opioid receptor produced from expression of a variant allele of a mu opioid receptor gene comprising a variation in SEQ ID NO 1 , wherein the variation comprises C17T is expected to have decreased activity relative to a mu opioid receptor produced from expression of the predominant or "most common" allele of the human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- This decreased activity is expected to result in higher activity of the HPA and HPG axes
- a medical professional attending the subject is able to select an appropriate therapeutic agent for treating a disease or disorder related to sexual and reproductive functions, such as infertility, gastrointestinal motihty, such as constipation or diarrhea, decreased immune response towards antigens relative to immune response in a standard, or decreased ability to withstand stress relative to ability to withstand stress in a standard
- the present invention further extends to commercial test kits suitable for use by a medical professional to determine whether either or both alleles of a bodily sample taken from a subject comprise a DNA sequence having at least one variation in SEQ ID NO 1 , wherein the variation comprises A118G, or C17T
- test kits of the present invention have applications in determining susceptibility of pam m the subject relative to a standard Such kits can also be used to determine a subject's increased or decreased susceptibility to at least one addictive disease relative to susceptibility to at least one addictive disease in a standard Also a therapeutically effective amount of pam reliever to administer to the subject in order to induce analgesia in the subject relative to a therapeutically effective amount of pain reliever to administer to a standard to induce analgesia in the standard can be determined Moreover, a test kit of the present invention has applications m determining a therapeutically effective amount of therapeutic agent for treating at least one addictive disease to administer to a subject suffering from the at least one addictive disease, relative to a therapeutically effective amount of therapeutic agent to administer to a standard suffering from at least one addictive disease Furthermore, test kits of the invention have applications in diagnosing a disease or disorder related to a physiological condition regulated by the HPA or HPG axes of the neuroendoc ⁇ ne system, and m selecting an appropriate therapeutic agent for treating such
- a commercial test kit of the present invention can also be used to determine the presence of an isolated variant allele of a human mu opioid receptor gene of the present invention in a bodily sample removed from a subject, which can serve as a genetic marker
- the predominant or ' most common' allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO 1
- a variant allele comprising a DNA sequence having a variation in SEQ ID NO 1. wherein the variation comprises G24A, G779A, or
- an isolated variant allele of a human mu opioid receptor gene detectable with a commercial kit ot the present invention comprises a DNA sequence having at least two variations m SEQ ID NO 1 , wherein the variations comprise
- a commercial test kit may be prepared for determining the presence of at least one variation in a human mu opioid receptor gene of either or both alleles in a bodily sample taken from a subject wherein the commercial test kit comprises a) PCR oligonucleotide primers suitable for detection of an allele comprising a human mu opioid receptor gene having a DNA sequence with a variation in SEQ ID NO 1 , b) other reagents, and c) directions for use of the kit
- the present invention further extends to commercial test kits capable of detecting a variant human mu opioid receptor in a bodily sample taken from a subject
- variant human mu opioid receptors that can be detected with a kit of the present invention comprise a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO 2, wherein the variation comprises Arg260H ⁇ s or conserved variants thereof, or a variant human mu opioid receptor comprising an amino acid sequence having at least two variations in SEQ ID NO 2, wherein the variations comprise Asn40Asp or conserved variants thereof,
- a commercial test kit of the present invention can be used to determine susceptibility to pa in the subject relative to susceptibility to pain in a standard, a therapeutically effective amount of pain reliever to administer to a subject to induce analgesia m the subject relative to a therapeutically effective amount of pain reliever to administer to a standard to induce analgesia in the standard, a therapeutically effective amount of therapeutic agent for treating at least one addictive disease to administer to a subject suffering from at least one addictive disease, relative to a therapeutically effective amount of therapeutic agent to administer to a standard suffering from the at least one addictive disease, diagnosing a disease or disorder related to a physiological condition regulated by the HPA or HPG axes of the neuroendoc ⁇ ne system, or selecting an appropriate therapeutic agent for treating such a disease or disorder, along with a therapeutically effective amount of such agent to administer to the subject
- the present invention extends to a commercial test kit having applications set forth above comprising a predetermined amount of at least one detectably labeled lmmuno- chemically reactive component having affinity for a variant human mu opioid receptor,
- test kit may be prepared and used for the pu ⁇ oses stated above, which operates according to a predetermined protocol (e g "competitive, " “sandwich, double antibody, ' etc ), and comprises
- This insight can be used to determine a therapeutically effective dose of pain reliever to administer to the subject to induce analgesia therein relative to the therapeutically effective amount of pain reliever administered to a standard to induce analgesia therein, wherein the standard comprises two alleles of the human mu opioid receptor gene comp ⁇ s- ing a DNA sequence of SEQ ID NO 1 , or a variant human mu opioid receptor comprising an amino acid sequence of SEQ ID NO 2
- Such information can be used to tailor a regimen for treating a subject suffering from at least one addictive disease, relative to the therapeutically effective amount of therapeutic agent administered to a standard suffering from at least one addictive disease
- the results of such testing can then be used to determine the subject's susceptibility to pain, susceptibility to at least one addictive disease, determining a therapeutically effective amount of pain reliever to administer to the subject m order to induce analgesia, or determining a therapeutically effective amount of therapeutic agent for treating at least one addictive disease to administer to the subject
- the level of activity of the mu opioid receptor effects sexual or reproductive function, gastrointestinal motihtv , immune response, or ability to withstand stress
- Such information can further be used select appropriate therapeutic agents to treat diseases such as infertility, constipation, or diarrhea
- Such information can be used to select appropriate therapeutic agents to increase immune response against an antigen such as a bacterium, a virus or a tumor cell in the subject, and to treat psychiatric diseases or disorders such as obsessive compulsive disorder, schizophrenia, or depression
- the results of such tests can then be used to gain incite into a subject's ability to withstand pain, susceptibility to addiction, to diagnose a disease or disorder related to a physiological function regulated by the HPA or HPG axes such as sexual and reproductive functions, gastrointestinal motihty, immune response, and the ability of the subject to withstand stress
- FIG. 2 Binding of endogenous opioid peptides to the most common (SEQ ID NO 2) and Asn40Asp mu opioid receptors Membrane preparations from cells expressing either the most common (open circles) or the A118G variant (filled squares) receptors were used m binding experiments to displace the [3H]-DAMGO binding Shown are examples of displacement binding for four endogenous peptides Met-enkephahn, dyno ⁇ hm A, ⁇ - endo ⁇ hin. and endo o ⁇ hin- 1
- FIG. 3 Functional comparison of the most common (SEQ ID NO 1) and A118G variant variant human mu opioid receptors in coupling to G protein-activated inwardly rectifying K + (GIRK) channels
- A Example of current trace showing the experimental protocol and calculation method for the agonist-induced response Oocytes were clamped at a holding potential of -80 mV and superfused with different solutions as indicated I max maximum K + currents evoked by DAMGO at a saturating concentration (100 nM) I Tal K + currents evoked by the test dose of agonists
- Figure 4 The nucleic acid sequence of the most common allele of the mu opioid receptor (SEQ ID NO 1) (GENBANK accession number L25119)
- FIG. 5 Ammo acid sequence of the most common human mu opioid receptor (SEQ ID NO 2) referred to hMORl, which is compared to the rat homologs of the mu (rMORl) (SEQ ID NO 3), delta (rDORl) (SEQ ID NO 4) and kappa (rKOR) (SEQ ID NO 5) opioid receptor amino a£ id sequences by the use the program PILEUP Boldface type and shading, transmembrane domain candidates, *, consensus sites for N-hnked glycosylation, italics, amino acid residues different between rat and human mu opioid receptor, @, indicates intron/exon boundary
- the present invention is based upon Applicants' su ⁇ sing and unexpected discovery of heretofore unknown single nucleotide polymo ⁇ hisms (SNPs) m the human mu opioid receptor, along with combinations thereof Furthermore, Applicants have discovered that more than one SNP can be present in either or both alleles of the human mu opioid receptor gene in a subject
- the present invention is based upon Applicants' su ⁇ sing discovery of molecules of heretofore unknown isolated nucleic acid molecules which encode human mu opioid receptors, wherein the DNA sequences include a combination of presently known polymo ⁇ hisms and subsequently of the human mu opioid receptors discovered by Applicants and set forth herein
- the present invention is based upon Applicants' surprising and unexpected discovery that the expression of variant alleles of the human mu opioid gene comprising a DNA sequence having a variation in SEQ ID NO 1 , wherein the variations comprise A118G or C17T, produce a variant mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO.2, wherein the variations comprise Asn40Asp or Ala ⁇ Val, and that these variant receptors exhibit a binding affinity for ⁇ -endo ⁇ hin that is different from tt ⁇ e binding affinity of a mu opioid receptor comprising an amino acid sequence of SEQ ID NO 2, and is encoded by the predominant or "most common" allele of the mu opioid receptor gene comprising a DNA sequence ot SEQ ID NO 1
- the present invention is based upon Applicants' prediction that variant alleles of the mu opioid receptor gene, which comprise a DNA sequence having a variation m SEQ ID NO 1 , wherein the variation comprises A118G or C17T encode variant mu opioid receptors comprising amino acid sequence having a variation in SEQ ID NO 2 wherein the variation comprises Asn40Asp or Ala ⁇ Val, and the variant receptors have an activity in vivo different from the of the predominant or ' most common" mu opioid receptor, the presence of such variant alleles in a bodily sample from a subject is expected to be indicative of the activity of the mu opioid receptors in the subject
- the present invention further extends to heretofore unknown polymo ⁇ hisms of the human mu opioid receptor gene that can serve as genetic markers to map the locus of the human mu opioid receptor gene
- the present invention extends to the characterization of the binding properties of human mu opioid receptors produced from the expression of nucleic acid molecules comprising DNA sequences with such heretofore unknown polymo ⁇ hisms of the human mu opioid receptor gene, or combinations of heretofore unknown polymo ⁇ hisms and known polymo ⁇ hisms
- the human mu opioid receptor is the major pharmacological target for clinically important opioid alkaloids, including mo ⁇ hme, methadone and fentanyl, as well as for endogenous opioid peptides such as ⁇ -endo ⁇ hin, Met- enkephahn-Arg-Phe, the recently identified endomo ⁇ hins [Zadma, J E., Hackler, L , Ge, L J & Kastin, A J.
- a human mu opioid receptor produced from expression of the C17T variant allele of the human mu opioid receptor gene has decreased binding affinity for ⁇ -endo ⁇ hm relative to the binding affinity of a human mu opioid receptor protein produced from the expression of the predominant or ' most common' allele of the human mu opioid receptor gene (SEQ ID NO 1) comprising a DNA sequence of SEQ ID NO 1 Consequently, a variant receptor encoded by a C17T variant allele exhibits decreased activity relative to the predominant or "most common" allele This decreased activity is expected to result in increased activity of HPA and HPG axes Hence, sexual and reproductive functions, gastrointestinal motihty, immune response and/or ability to withstand stress are decreased m the subject relative to the levels of such physiological functions in a standard comprising two alleles of the mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- the present invention extends to characterizing the activity of such heretofore unknown human mu opioid receptors produced from the expression of isolated nucleic acid molecules of the present invention More particularly, the increased or decreased ability of such human mu opioid receptors produced from isolated nucleic acid acids of the present invention to activate G protein-activated inwardly rectifying K + (GIRK) channels via a G protem-mediated mechanism can be determined, and is expected to be indicative of activity
- the present invention further extends to Applicants' discovery that polymo ⁇ hisms such as Al 18G and C17T, are present in the population at a high frequency (greater than 5 %), and that the presence of such polymo ⁇ hisms in the human mu opioid receptor gene of a subject correlates to an increased or decreased susceptibility to addictive diseases, such as heroin addiction, cocaine addiction, or alcohol addiction, to name only a few, and are expected to modulate physiological functions regulated by the HPA and HPG axes, such as sexual and reproductive functions, gastrointestinal motihty, immune response and/or ability to withstand stress, relative to such functions in a standard
- the present invention extends to diagnostic methods to determine a subject's increased or decreased susceptibility to at least one addictive disease With the results of such methods, targeted prevention methods, early therapeutic intervention, and improved chronic treatment to opioid addiction are set forth herein and encompassed by the present invention
- attending medical professionals of subjects armed with the results of such diagnostic methods can determine whether administration of opioid analgesics is approp ⁇ ate or whether non-opioid derived analgesics should be administered to the subject
- appropriate choice and type of analgesic can be made in treating a subject s pain
- the present invention extends to methods of determining a subject's increased or decreased susceptibility to pain and response to analgesics, and using that information when prescribing analgesics to the subject
- the present invention extends to diagnosing a disease or disorder related to a physiological function regulated by the HPA and HPG axes, such as sexual and reproductive functions, gastrointestinal motility, immune response, and the ability to withstand stress
- the present invention further extends to variant alleles of the human mu opioid receptor gene comprising a DNA sequence comprising a heretofore unknown polymo ⁇ hism, such as G24A,
- Applicants' invention extends to variant alleles of the human mu opioid receptor gene comprising a DNA sequence having at least two variations in the predominant or "most common” allele comprising a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1. wherein the variations comprise A118G,
- the present invention is based on Applicants' discovery that su ⁇ smgly and unexpectedly, the C17T va ⁇ ant allele of the human mu opioid receptor is present in a statistically significantly higher frequency in opioid dependent persons than in persons not addicted to opioids
- an initial aspect of the present invention involves isolation of heretofore unknown variant alleles of the human mu opioid receptor gene
- the term "gene” refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids
- a vector' is a replicon such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment
- a 'replicon is any genetic element (e g , plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i e , capable of replication under its own control
- a cassette refers to a segment of DNA that can be inserted into a vector at specific restriction sites
- the segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation
- a cell has been transfected" bv exogenous or heterologous DNA when such DNA has been introduced inside the cell
- a cell has been transformed” by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change
- the transforming DNA should be integrated (covalently linked) into chromosomal DNA making up the genome of the cell
- Heterologous ' DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell
- the heterologous DNA includes a gene foreign to the cell
- a ' nucleic acid molecule refers to the phosphate ester polymeric form of ⁇ bonucleosides (adenosme, guanosine, undine or cytidme, 'RNA molecules") or deoxy ⁇ bonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidme, or deoxycytidme, "DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible
- nucleic acid molecule and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms Thus, this term includes double- stranded DNA found, inter alia, m linear or circular DNA molecules (e g , restriction
- a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al , supra)
- the conditions of temperature and ionic strength determine the "stringency" of the hybridization
- low stringency hybridization conditions corresponding to a T m of 55° , can be used, e g , 5x SSC, 0 1 % SDS, 0 25 % milk, and no formamide, or 30% formamide, 5x SSC 0 5 % SDS)
- Moderate stringency hybridization conditions correspond to a higher T m , e g 40% formamide, with 5x or 6x SSC
- High stringency hybridization conditions correspond to the highest Tberichting e g .
- RNA RNA, DNA RNA, DNA DNA For hybrids of greater than 100 nucleotides in length, equations for calculating T m have been derived (see Sambrook et al , supra, 9.50-0.51).
- a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 20 nucleotides; and more preferably the length is at least about 30 nucleotides; and most preferably 40 nucleotides.
- standard hybridization conditions refers to a T m of 55 °C, and utilizes conditions as set forth above.
- the T m is 60 D C; in a more referred embodiment, the T m is 65°C.
- homologous recombination refers to the insertion of a foreign DNA sequence of a vector in a chromosome
- the vector targets a specific chromosomal site for homologous recombination.
- the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.
- a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
- a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g. , mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
- Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
- polyadenylation signals are control sequences.
- a “promoter sequence” or “promoter” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 ' direction) coding sequence
- the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background
- a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase
- a coding sequence is under the control' of transc ⁇ ptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence
- a coding sequence is operatively associated with" a transcriptional and translational control sequences, such as a promoter for example, when RNA polymerase transcribes the coding sequence into mRNA, which in turn is translated into a protein encoding by the coding sequence
- a “signal sequence ' is included at the beginning of the coding sequence of a protein to be expressed on the surface of a cell This sequence encodes a signal peptide, N-terminal to the mature polypeptide, that directs the host cell to translocate the polypeptide
- translocation signal sequence is used herein to refer to this sort of signal sequence
- An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence
- a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence
- the term "primer” as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, 1 e , in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH
- the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent The exact length of the primer
- the primers herein are selected to be ' substantially' complementary to different strands of a particular target DNA sequence This means that the primers must be sufficiently complementary to hybridize with their respective strands Therefore, the primer sequence need not reflect the exact sequence of the template For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product
- a cell has been ' transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell
- the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell
- the transforming DNA may be maintained on an episomal element such as a plasmid
- a stably transformed cell is one m which the transforming DNA has become integrated into a chromosome so that it is inlierited by daughter cells through chromosome replication This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA
- a "clone” is a population of cells derived from a single cell or common ancestor by mitosis
- a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations
- the phrase "expected to be indicative" is used herein to refer to the correlation between the identity of the allelic va ⁇ at ⁇ on(s) in an individual and the susceptibility of an individual to addictive disease, sensitivity to pam and analgesics, therapeutic effectiveness of analgesics, and other physiological manifestations described herein related to the function of the mu opioid receptor, such as but not limited to the responsiveness to stress, peripheral gastrointestinal function, immune function, and reproductive biology
- the correlations are based on the findings in the present invention of the relationship between the biochemistry and cellular function of the variants of the mu opioid receptor and clinical observations, analyzed statistically, on history of drug dependence, reproductive function, gastrointestinal function, response to stress, and other previous or current conditions
- Expected correlations of mu opioid receptor alleles and susceptibility to various conditions may be increased susceptibility or decreased susceptibility
- DNA sequences encoding variant alleles of a human mu opioid receptor gene of the present invention which comprise at least one variation in the predominant or 'most common" allele of the human mu opioid receptor gene
- the most common allele comprises a DNA sequence of SEQ ID NO 1, and variations in the most common allele comprise
- the present invention comprises DNA sequences encoding variant alleles of a human mu opioid receptor gene, comprising at least two variations in the predominant or "most common" allele of the human mu opioid receptor gene, wherein the most common human mu opioid receptor gene comprises a DNA sequence of SEQ ID NO 1
- Variant alleles of the human mu opioid receptor gene encompassed by the present invention comprise a DNA sequence comprising at least two variations of SEQ ID NO 1 , wherein the variation comprises G24A, G779A, G942A, A118G, or
- variant human mu opioid receptor proteins encoded by variant alleles of the present invention wherein the variant human mu opioid receptors comprise an ammo acid sequence having at least one variation in SEQ ID NO 2, wherein the variations comprise Asn40Asp or conserved variants thereof, Ala ⁇ Val or conserved variants thereof, or Arg260H ⁇ s or conserved variants thereof, or combinations thereof, can be encoded by nucleic acid molecules other than those set forth above "Degenerate nature" refers to the use of different three-letter codons to specify a particular amino acid pursuant to the genetic code It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid
- Vahne GUU or GUC of GUA or GUG Se ⁇ ne (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC
- Threomne Thr or T
- ACU ACC or ACA or ACG
- Tyrosme (Tyr or Y) UAU or UAC Histidine (His or H) CAU or CAC
- Lysme (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAU or GAC
- Arginme CGU or CGC or CGA or CGG or AGA or AGG Gfycme (Gly or G) GGU or GGC or GGA or GGG Tryptophan (T ⁇ or W) UGG
- sequence homology in all its grammatical forms refers to the relationship between proteins that possess a common evolutionary origin," including proteins from superfamihes (e g , the lmmunoglobulin superfamily) and homologous proteins from different species (e g , myosin light chain etc ) (Reeck et al , 1987, Cell 50 667)
- sequence similarity in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or ammo acid sequences of proteins that do not share a common evolutionary origin (see Reeck et al , supra)
- two DNA sequences are "substantially homologous” or “substantially similar” when at least about 50% (preferably at least about 75%, and most preferably at least about 90 or 95 %) of the nucleotides match over the defined length of the DNA sequences
- Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system Defining appropriate hybridization conditions is withm the skill of the art See, e g , Maniatis et al , supra, DNA Cloning, Vols I & II, supra, Nucleic Acid Hybridization, supra
- two ammo acid sequences are "substantially homologous” or “substantially similar” when greater than 30% of the ammo acids are identical, or greater than about 60% are similar (functionally identical)
- the similar or homologous sequences are identified by alignment using, for example
- a variant allele of the human mu opioid receptor gene of the present invention can be isolated from any source, particularly from a human cDNA or genomic library Methods for obtaining an allele of a human mu opioid receptor gene, variants thereof, or the most common, are well known in the art, as described above (see, e g , Sambrook et al , 1989, supra)
- any human cell potentially can serve as the nucleic acid source for the molecular cloning of a variant allele of the human mu opioid receptor gene of the present invention, or a nucleic acid molecule hybridizable to a variant allele of a human mu opioid receptor gene of the present invention
- the DNA may be obtained by standard procedures known in the art from cloned DNA (e g , a DNA "library”) and preferably is obtained from a cDNA library prepared from tissues with high level expression of a human mu opioid receptor protein, by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (See, for example, Sambrook et al , 1989, supra, Glover, D M (ed ), 1985, DNA Cloning A Practical Approach, MRL Press, Ltd , Oxford, U K Vol I, II) Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions, clones derived from cDNA
- identification of the specific DNA fragment containing an allele of a human mu opioid receptor of the present invention may be accomplished in a number of ways For example, if an amount of a portion of an allele of a human mu opioid receptor gene, or its specific RNA, or a fragment thereof, is available and can be purified and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, 1977, Science 196 180, Grunstein and Hogness.
- a set of oligonucleotides corresponding to the partial amino acid sequence information obtained for a human mu opioid receptor protein can be prepared and used as probes for DNA encoding a variant allele of a human mu opioid receptor gene of the present invention, as was done in a specific example, infra, or as primers for cDNA or mRNA (e g , in combination with a poly-T primer for RT-PCR)
- a fragment is selected that is highly unique to a variant allele of the human mu opioid receptor gene of the invention Those DNA fragments with substantial homology to the probe will hybridize As noted above the greater the degree of homology, the more stringent hybridization conditions can be used
- an allele of a human mu opioid receptor gene of the present invention e g , if the allele encodes a variant human mu opioid receptor protein having an lsoelect ⁇ c, electrophoretic, ammo acid composition, or partial amino acid sequence different from that produced from the expression of the most common allele of a human mu opioid receptor gene (SEQ ID NO 1) herein
- SEQ ID NO 1 the most common allele of a human mu opioid receptor gene
- the presence of an allele ot a human mu opioid receptor gene of the present invention may be detected by assays based on the physical, chemical, or immunological properties of its expressed product
- cDNA clones, or DNA clones which hybrid-select the proper mRNAs can be selected which produce a protein that, e g , has different electro- phoretic migration, lsoelect ⁇ c focusing or non-equilibrium pH gel electrophoresis behavior, proteolytic digestion maps, or antigenic properties as known for a human mu opioid receptor produced from
- An allele of a human mu opioid receptor gene of the present invention can also be identified by mRNA selection, i e , by nucleic acid hybridization followed by in vitro translation
- nucleotide fragments are used to isolate complementary mRNAs by hybridization
- Siich DNA fragments may represent available, purified DNA of an allele of a human mu opioid receptor gene of the present invention, or may be synthetic oligonucleotides designed from the partial amino acid sequence information Immunoprecipitation analysis or functional assays of the in vitro translation products of the products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments, that contain the desired sequences
- a labeled cDNA of an alleie of a human mu opioid receptor gene of the present invention, or fragments thereof, or a nucleic acid hybridizable under standard hybridization conditions to an allele of a human mu opioid receptor gene of the present invention can be synthesized using sequences set forth herein
- the radiolabeled mRNA or cDNA may then be used as a probe to identify homologous DNA fragments from among other genomic DNA fragments Suitable labels include enzymes, radioactive isotopes, fluorophores (e g , fluorescene isothiocyanate (FITC), phycoerythrm (PE) Texas red (TR), rhodamine, free or chelated lanthamde series salts especially Eu J , to name a few fluorophores), chromophores, radioisotopes, chelating agents, dyes, colloidal gold, latex particles, ligands (e g , biotin), and chemiluminescent agents When a control
- Direct labels are one example of labels which can be used according to the present invention
- a direct label has been defined as an entity, which in its natural state, is readily visible, either to the naked eye, or with the aid of an optical filter and/or applied stimulation, e g , U V light to promote fluorescence
- colored labels which can be used according to the present invention, include metallic sol particles, for example, gold sol particles such as those described by Leuve ⁇ ng (U S Patent 4,31
- Cloning Vectors The present invention also relates to cloning vectors comprising variant alleles of a human mu opioid receptor gene of the present invention, and an origin of replication
- an "origin of replication refers to those DNA sequences that participate in DNA synthesis
- variant alleles of a human mu opioid receptor gene of the present invention comprise a DNA sequence having at least one variation in the most common allele of a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1 , wherein the variation comprises
- the present invention extends to variant alleles of a human mu opioid receptor gene, comprising a DNA sequence having at least two variations in the DNA sequence of SEQ ID NO 1, wherein the variations comprise G24A, G779A, G942A, A118G, or
- an isolated variant allele of a human mu opioid receptor gene of the present invention can be inserted into an approp ⁇ ate cloning vector m order to produce multiple copies of the variant allele or isolated nucleic acid molecule
- vectors include, but are not limited to, plasmids or modified viruses The vector system used however must be compatible with the host cell used Examples of vectors include having applications herein, but are not limited to E coli, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e g , pGEX vectors, pmal-c, pFLAG, etc
- the insertion into a cloning vector can, for example, be accomplished by hgating a va ⁇ ant allele of the human mu opioid
- an isolated variant allele of a human mu opioid receptor gene of the present invention or an isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach Enrichment for a variant allele, for example, by size fractionation, can be done before insertion into the cloning vector
- the present invention extends to an isolated variant allele of a human mu opioid receptor gene, comprising a DNA sequence having at least one variation in the DNA sequence of the predominant or "most common' allele of the human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1 wherein the variations comprise G24A, G779A, or G942A, or combinations thereof
- the present invention extends to an isolated va ⁇ ant allele of a human mu opioid receptor gene, a DNA sequence having at least two variations m the predominant or "most common" allele of the human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1 wherein the variations comprise
- variant alleles of the present invention can be inserted into an appropriate expression vector, i e , a. vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence
- an appropriate expression vector i e , a. vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence
- a variant allele of the present invention, or an isolated nucleic acid molecule hybridizable to a variant allele of the present invention under standard hybridization conditions is operatively associated with a promoter in an expression vector of the invention
- a DNA sequence is 'operatively associated" to an expression control sequence, such as a promoter, when the expression control sequence controls and regulates the transcription and translation of that DNA sequence
- operatively associated includes having an appropriate start signal (e g , ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence If a variant allele of the present invention, or
- An expression vector also preferably includes a replication origin
- the necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by an allele comprising a human mu opioid receptor gene
- Potential host-vector systems include but are not limited to mammalian cell systems infected with virus (e g , vaccinia virus, adenovirus, etc ), insect cell systems infected with virus (e g , baculovirus), microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA
- virus e g , vaccinia virus, adenovirus, etc
- insect cell systems infected with virus e g , baculovirus
- microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA
- the expression elements of vectors vary in their strengths and specificities Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used
- a variant allele of a human mu opioid receptor gene of the present invention or an isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions may be expressed chromosomally, after integration of the coding sequence by recombination
- any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook et al , 1989, supra)
- a unicellular host transformed or transfected with an expression vector of the present invention is cultured in an appropriate cell culture medium that provides for expression by the unicellular host of the variant allele, or isolated nucleic acid hybridizable thereto under standard hybridization conditions
- any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors of the present invention These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination)
- Expression of a variant allele of a human mu opioid receptor gene of the present invention or an isolated nucleic acid molecule hybridizable to a variant allele of a human mu opioid receptor gene under standard hybridization conditions may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression
- Promoters which may be used to control expression include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, 1981 , Nature 290 304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al , 1980, Cell 22 787-797), the he ⁇ es thymidine kmase promoter (Wagner et al 1981, Proc Natl Acad Sci U S A 78 1441-1445), the regulatory sequences of the metallothionein gene (B ⁇ nster et al , 1982, Nature 296 39-42), prokaryotic expression vectors
- expression vectors comprising a variant allele of a human mu opioid receptor gene of the present invention, or an isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions, can be identified by four general approaches (a) PCR amplification of the desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c) presence or absence of selection marker gene functions, and (d) expression of inserted sequences
- the variant allele or isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions can be amplified by PCR to provide for detection of the amplified product
- the presence of a foreign gene inserted into an expression vector of the present invention can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted marker gene
- the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "selection marker" gene functions (e g , ⁇ -galactosi
- the present invention extends to a method of producing a va ⁇ ant human mu opioid receptor comprising an ammo acid sequence having at least one variation in the amino acid sequence ot SEQ ID NO 2, wherein the variation comprises Arg260H ⁇ s or conserved variants thereof
- An example of such a method comprises the steps of culturing a unicellular host transformed or transfected with an expression vector comprising a variant allele of a human mu opioid receptor gene comprising a DNA sequence having a variation in SEQ ID NO 1, wherein the variation comprises G779A, wherein the variant allele which is operatively associated with a promoter
- the transformed or transfected unicellular host is then cultured under conditions that provide for expression of the variant allele of the human mu opioid receptor gene, and the expression product is recovered from the unicellular host
- Another example involves culturing a unicellular host transformed or transfected with an isolated nucleic acid molecule hybridizable under standard hybridization conditions to a variant allele of a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO 1 , wherein the variation comprises G779A, wherein the isolated nucleic acid molecule is operatively associated with a promotor
- the variant human mu opioid receptor is then recovered from the host
- the present invention extends to a method for producing a va ⁇ ant human mu opioid receptor comprising an amino acid sequence having at least two variations in SEQ ID NO 2, wherein the variations comprise
- Such a method comprises the steps of culturing a unicellular host transformed or transfected with an expression vector comprising a variant allele of a human mu opioid receptor gene of the present invention or an isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions, and operatively associated with a promoter, that provides for expression of the variant allele or the isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions After expression, a variant human mu opioid receptor of the present invention is recovered from the unicellular host
- Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences
- Suitable vectors include derivatives of SV40 and known bacterial plasmids, e g , £ coli plasmids col El, pCRl, pBR322, pMal-C2, pET, pGEX (Smith et al , 1988, Gene 67 31- 40), pMB9 and their derivatives, plasmids such as RP4, phage DNAS, e g , the numerous derivatives of phage ⁇ e g , NM989, and other phage DNA, e g , M13 and filamentous single stranded phage DNA, yeast plasmids such as the 2 ⁇ plasmid or derivatives thereof, vectors useful in eukaryotic cells, such as vectors useful in insect or
- both non-fusion transfer vectors such as but not limited to pVL941 (BamWl cloning site, Summers), pVL1393 (Bam ⁇ l , Smal, Xbal, EcoRI, Notl, Xmalll, Bgtll, and Pstl cloning site, Invitrogen), pVL1392 (Bgl ⁇ , Pstl, Notl, X ⁇ lll, EcoRI, Xbal, Smal, and Bam ⁇ l cloning site, Summers and Invitrogen), and pBlue ⁇ cIII (Bam ⁇ l, BgUl, Pstl, Ncol, and Hindlll cloning site, with blue/white recombinant screening possible, Invitrogen), and fusion transfer vectors, such as but not limited to pAc700 (BamHl and Kp ⁇ l cloning site, in which the BamUl recognition site begins
- Mammalian expression vectors contemplated for use in the invention include vectors with inducible promoters, such as the dihydrofolate reductase (DHFR) promoter, e g , any expression vector with a DHFR expression vector, or a H R/methotrexate co-amplifica- tion vector, such as pED Pstl, SaH, Sbal, Smal, and EcoRI cloning site, with the vector expressing both the cloned gene and DHFR see Kaufman, Current Protocols in Molecular Biology, 16 12 (1991)
- DHFR dihydrofolate reductase
- a glutamine synthetase/methionine sulfoximine co-amplification vector such as pEE14 (Hindlll, Xbal, Smal, Sbal, EcoRI, and Bell cloning site, in which the vector expresses glutamine synthase and the cloned gene, Celltech)
- a vector that directs episomal expression under control of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamHl, Sfil, Xhol, Notl, Nhel, Hindlll, Nhel, Pvull, and Kpnl cloning site, constitutive RSV-LTR promoter, hygromycin selectable marker, Invitrogen), pCEP4 (BamHl , Sfil, Xhol, Notl, Nhel, Hindlll, Nhel Pvull, and Kpnl cloning site, constitutive hCMV immediate early gene, hy
- Yeast expression systems can also be used according to the invention to produce a variant human mu opioid receptor or the present invention.
- the non-fusion pYES2 vector (Xbal, Sphl, Shol, Notl, GstXl, EcoRI, BstXl, BamHl , Sad, Kpnl, and Hindlll cloning sit; Invitrogen) or the fusion pYESHisA, B, C (Xbal, Sphl, Shol, Notl, BstXl, EcoRI, BamHl, Sacl, Kpnl, and Hindlll cloning site, N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen), to mention just two, can be employed according to the invention.
- recombinant expression vectors can be propagated and prepared in quantity.
- the expression vectors which can be used include, but are not limited to the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g. , lambda), and plasmid and cosmid DNA vectors, to name but a few.
- unicellular hosts contemplated by the present invention include, but are not limited to £. coli Pseudonomas, Bacillus, Streptomyces, yeast, CHO, Rl. l, B-W, L-M, COSl , COS7, BSCl, BSC40, BMT10 and Sf9 cells.
- a host cell strain may be chosen which modulates the expression of a variant allele comprising a human mu opioid receptor gene, or an isolated nucleic acid hybridizable thereto under standard hybridization conditions, such that the gene product is modified and processed in the specific fashion desired.
- Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g.
- glycosylation, cleavage [e.g. , of signal sequence]) of proteins.
- Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
- expression in a bacterial system can be used to produce an nonglycosylated core protein product
- a translocation signal sequence of an isolated variant allele of a human mu opioid receptor gene of the present invention, or an isolated nucleic acid hybridizable thereto under standard hybridization conditions, expressed in bacteria may not be properly spliced Expression in yeast can produce a glycosylated product
- Expression in eukaryotic cells can increase the likelihood of "native" glycosylation and folding
- expression m mammalian cells can provide a tool for reconstituting, or constituting activity of the variant human mu opioid receptor gene
- different vector/host expression systems may affect processing reactions, such as proteolytic cleavages, to a different extent
- Vectors are introduced into the desired unicellular hosts by methods known in the art, e g , transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, hpofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e g , Wu et al , 1992, J Biol Chem 267 963-967, Wu and Wu, 1988, J Biol Chem 263 14621-14624, Hartmut et al , Canadian Patent Application No 2,012,311 , filed Marcn 15, 1990)
- an isolated va ⁇ ant human mu opioid receptor of the present invention produced as an integral membrane protein can be isolated and purified by standard methods Generally, the variant human mu opioid receptor can be obtained by lysmg the membrane with detergents, such as but not limited to, sodium dodecyl sulfate (SDS), Triton X-100, nonidet P-40 (NP-40), digoxin, sodium deoxycholate, and the like, including mixtures thereof Solubihzation can be enhanced by somcation of the suspension Soluble forms of an isolated variant of a human mu opioid receptor can be obtained by collecting culture fluid, or solubilizing inclusion bodies, e g , by treatment with detergent, and if desired somcation or other mechanical processes, as described above The solubihzed or soluble protein can be isolated using various techniques, such as polyacrylamide gel electrophoresis (PAGE), lsoelect ⁇ c focusing, 2-d ⁇ mens ⁇ onal gel electrophoresis, chromatography (e g , ion exchange,
- nucleotide coding sequences which encode the variant human mu opioid receptors of the present invention may be used m the practice of the present invention
- these include but are not limited to allelic genes, homologous genes from other species, and nucleotide sequences comprising all or portions of genes which are altered by the substitution of different codons that encode the same ammo acid residue within the sequence, thus producing a silent change
- the conserved variants of human mu opioid receptors of the present invention include, but are not limited to, those containing, as a primary amino acid sequence, substitutions of amino acids in a variant human mu opioid receptor as set forth above, which are functionally equivalent to ammo acids of the variations set forth above, resulting in a conservative ammo acid substitution
- one or more ammo acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration
- Substitutes for an amino acid within the sequence may be
- Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property
- a Cys may be introduced at a potential site for disulfide bridges with another Cys
- a His may be introduced as a particularly "catalytic" site (l e , His can act as an acid or base and is the most common ammo acid in biochemical catalysis)
- Pro may be introduced because of its particularly planar structure, which induces ⁇ -turns m the protein's structure
- variant human mu opioid receptors disclosed herein may be used as an immunogen to generate antibodies that recognize the claimed variant mu opioid receptors
- Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library
- antibodies of the invention may be cross reactive, e g , they may recognize human mu opioid receptors comprising an amino acid sequence of SEQ ID NO 1 , as well as mu opioid receptors from different species
- Polyclonal antibodies have gi eater likelihood of cross reactivity
- an antibody of the invention may be specific for a specific variant allele of a mu opioid receptor
- the variant human mu opioid receptor can be conjugated to an immunogenic carrier, e g , bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH)
- an immunogenic carrier e g , bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH)
- BSA bovine serum albumin
- KLH keyhole limpet hemocyanin
- Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund s (complete and incomplete), mineral gels such as aluminum hydroxide surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides. oil emulsions, keyhole limpet hemocyamns, dimtrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerm) and Corvn
- any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein [Nature 256 495-497 (1975)], as well as the t ⁇ oma technique, the human B-cell hybridoma technique [Kozbor et al , Immunology Today 4 72 1983), Cote et al , Proc Natl Acad Sci U S A 80 2026-2030 (1983)], and the EBV-hyb ⁇ doma technique to produce human monoclonal antibodies [Cole et al , in Monoclonal Antibodies and Cancer Therapy, Alan R Liss, Inc , pp 77-96 (1985)]
- monoclonal antibodies can be produced in germ-free animals utilizing recent technology [PCT/US90/02545]
- Antibody fragments which contain the ldiotype of the antibody molecule can be generated by known techniques
- such fragments include but are not limited to the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule, the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') ⁇ fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent
- antibody binding is detected by detecting a label on the primary antibody
- the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody
- the secondary antibody is labele
- the foregoing antibodies can be used in methods known in the art relating to the localization and activity of a variant human mu opioid receptor, e g , for Western blotting, imaging a variant human mu opioid receptor in situ, measuring levels thereof in appropriate physiological samples etc using any of the detection techniques mentioned above or known
- su ⁇ smgly certain variant human mu opioid receptors have greater affinity for particular opioids than human mu opioid receptors comprising an amino acid sequence of SEQ ID NO 2 More specifically, Applicants have discovered that su ⁇ singly, a variant human mu opioid receptor produced from expression of a va ⁇ ant allele of a human mu opioid receptor comprising a variation in SEQ ID NO 1 , wherein the variation comprises A118G.
- ⁇ -endo ⁇ hin binds three times more tightly to ⁇ -endo ⁇ hm, an endogenous opioid comprising 31 ammo acid residues, than do human mu opioid receptors produced from the expression of the predominant or "most common" allele comprising a DNA sequence of SEQ ID NO 1 Since ⁇ -endo ⁇ hin is believed to play an important role in numerous physiological functions, the presence of a variant comprising A118G, in either or both alleles present m a subject has an impact on such physiological functions Furthermore, ⁇ -endo ⁇ hin induced activity of a receptor produced from the expression of a variant allele of a human mu opioid receptor gene comprising the A118G polymo ⁇ hism effected the receptor's activation of GIRK channels via a G protein-mediated mechanism relative to the activity of a receptor produced from the expression of the predominant or most common” allele Consequently, a subject having an Al 18G variation, in either or both alleles of a human mu opioid receptor gene is expected to have lower susceptibility and greater tolerance to pa
- One such function involves a susceptibility to at least one addictive disease, such as opioid addiction, cocaine addiction or addiction to other psychostimulants, nicotine addiction, barbiturate or sedative hypnotic addiction, anxiolytic addiction, or alcohol addition
- addictive disease such as opioid addiction, cocaine addiction or addiction to other psychostimulants, nicotine addiction, barbiturate or sedative hypnotic addiction, anxiolytic addiction, or alcohol addition
- Applicants have discovered a variant allele of a human mu opioid receptor gene comprising a DNA sequence having at variation in SEQ ID NO 1 , wherein the variation comprises A118G, is present in an Hispanic study population at a statistically significant greater frequency m the genomes of persons not suffering from at least one addictive disease, relative to its presence in the genomes of persons suffering from an addictive disease, such as opioid addiction
- the inventors herein have further discovered that another variant allele of a human mu opioid receptor gene comprising a DNA sequence having a variation m SEQ ID NO 1, wherein the variation comprises C17T, is present at a statistically significant greater frequency in the genomes of persons suffering from at least one addictive disease, relative to its presence in the genome of persons not suffering from at least one addictive disease.
- the C17T variant mu opioid receptor has a ⁇ -endo ⁇ hm binding affinity approximately 0 72 times that of the human mu opioid receptor produced from expression of the predominant or "most common allele of the human mu opioid receptor gene
- the decreased binding affinity of this variant is in contrast to the increased affinity of the A 118G variant mentioned above
- one of the important indices of cellular functioning of the mu opioid receptor is the inhibition of adenylyl cyclase activity following agonist binding to the receptor
- the C17T variant mu opioid receptor has been found to be less sensitive to two endogenous opioids, ⁇ -endo ⁇ hm and
- a variant allele of a human mu opioid receptor gene comprising a DNA sequence having an A118G variation, in SEQ ID NO 1 , has increased affinity for ⁇ -endo ⁇ hm and capability of effecting activation of GIRK channels via a G protein-mediated mechanism provides resistance against susceptibility to addictive diseases
- a variant allele comprising a human mu opioid receptor having a C17T variation in SEQ ID NO 1 has a decreased affinity for ⁇ -endo ⁇ hm and a decreased sensitivity to adenylyl cyclase inhibition by the endogenous ligands ⁇ -endo ⁇ hm and leu-enkephahn, and is indicative of increased susceptibility to at least one addictive disease
- These differences in variant mu opioid receptor function at the biochemical and cellular level and their correlations with physiological manifestations m susceptibility to addictive disease provides a basis for the value of the identity of alleles in identifying individual susceptibility to addictive disease, pain, reaction to therapeutic agents, etc
- the present invention extends to a method for determining a susceptibility of a subject to one addictive disease comprising removing a bodily sample comprising a first and second allele of a human mu opioid receptor gene from the subject, and determining whether either the first or second alleles, or both alleles comprise a DNA sequence having at least one variation in SEQ ID NO 1 , wherein the variation comprises
- the presence of the variation of C17T in either or both alleles of a human mu opioid receptor gene of a sample from the subject indicates the subject has an increased susceptibility to at least one addictive disease relative to a standard having alleles of the human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- the presence of the variation of comprising Al 18G, in either or both alleles of a mu opioid receptor gene in a sample from the subject is expected to indicate the subject as a decreased susceptibility to addictive diseases relative to a standard comprising alleles of a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- the biological sample can be a biological fluid, such as bui not limited to, blood, serum, plasma, interstitial fluid, plural effusions, urine, cerebrospmal fluid, and the like
- variant alleles of a human mu opioid receptor gene as described above, are detected in serum or urine, which are both readily obtained
- variant alleles of a human mu opioid receptor gene indicating increased or decrease susceptibility to addictive diseases in the subject as described above can be detected from cellular sources, such as, but not limited to, brain tissue biopsies, adipocytes, testes, heart, and the like
- cells can be obtained from an individual by biopsy and lysed, e g , by freeze-thaw cycling, or treatment with a mild cy to lytic detergent such as, but not limited to, TRITON X-100 ® , digitonin, NONIDET P (NP)-40 ® , saponin, and the like, or combinations thereof (see, e g ,
- SEQ ID NO 1 wherein the variation comprises C17T
- SEQ ID NO 1 can be used in standard Northern hybridization analysis to detect the presence, and in some instances quantitate the level of transcription of such a variant allele of the present invention
- the presence of this variant allele in a bodily sample from a subject is expected to be indicative of increased susceptibility to at least one addictive disease in the subject
- an optionally detectably labeled isolated nucleic acid molecule hybridizable under standard hybridization conditions to an allele of a human mu opioid receptor gene comprising a DNA sequence having a variation in SEQ ID NO 1, wherein the variation comprises A118G can be used in a standard Northern hybridization analysis to detect the presence of a variant allele in the sample comprising a variation in SEQ ID NO 1, wherein the variation comprises A118G, which is expected to be indicative of a decreased susceptibility to at least one addictive disease relative to the susceptibility of a standard comprising two alleles of a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:
- oligonucleotides of the invention can be used as PCR primers to amplify an allele of a human mu opioid receptor gene of the biological sample e.g. , by reverse transcriptase-PCR, or amplification of the allele itself.
- the amplified mRNA or DNA can then be quantified or sequenced in order to determine the presence of a variant allele, and the susceptibility of the subject to addictive diseases.
- variations in SEQ ID NO: l can be found by creation or deletion of restriction fragment length polymo ⁇ hisms (RFLPs) not found in the predominant or "most common” allele, hybridization with a specific probe engineered to hybridize to variation described above under standard hybridization conditions, (or lack of hybridization with a probe specific for the predominant or “most common” allele), as well as by other techniques.
- RFLPs restriction fragment length polymo ⁇ hisms
- biochemical or immunochemical/biochemical e.g. , immunoprecipitation
- biochemical or immunochemical/biochemical e.g. , immunoprecipitation
- a variant allele of a human mu opioid receptor gene comprising a DNA sequence having a variation in SEQ ID NO: l , wherein the variation comprises:
- the presence of the Al 18G variation in either or both alleles of a human mu opioid receptor gene in a biological sample from the subject indicates a decreased susceptibility to addictive diseases in the subject
- the presence of the C17T variation in either or both alleles of a human mu opioid receptor gene in a biological sample from the subject indicates increased susceptibility to addictive diseases in the subject.
- radioimmunoassay ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioiso- tope labels, for example), western blots, precipitation reactions, agglutination assays (e.g.
- antibody binding is detected by detecting a label on the primary antibody
- the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody
- the secondary antibody is labeled
- the present invention extends to a method for determining a susceptibility to pam in a subject
- endogenous opioid such as ⁇ -endo ⁇ hin
- ⁇ -endo ⁇ hin induced activity of a receptor produced from the expression of a variant allele of a human mu opioid receptor gene or an isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions comprising the Al 18G polymo ⁇ hism effected the receptor's activation of GIRK channels via a G protein-mediated mechanism relative to the activity of a receptor produced from the
- a variant allele of a human mu opioid receptor gene comprising a DNA sequence having a variation m SEQ ID NO 1 , wherein the variation comprises C17T, is present at a statistically significantly greater frequency in the genomes of opioid addicts relative to its presence in the genomes of persons not addicted to opioids
- the presence of a variant allele of a human mu opioid receptor comprising a DNA sequence having a variation m SEQ ID NO 1, wherein the variation comprises C17T indicates the subject is predicted to have increased susceptibility and decreased tolerance to pam
- a method of determining susceptibility of pain in a subject comprising the steps of removing a bodily sample comprising a first and second allele of a human mu opioid receptor gene from the subject, and determining whether either the first or second alleles, or both alleles, comprise a DNA sequence having at least one variation in SEQ ID NO 1 , wherein the variation comprises
- the presence of at least one variation in either or both alleles of the human mu opioid receptor gene is expected to be indicative of the subject's increased or decreased susceptibility to pain relative to a person homozygous with respect to the predominant or "most common" allele comprising a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- an inappropriate amount of pam reliever is administered to a subject when either the subject is not relieved of pam, or the subject is exposed to potential deleterious side effects of the pain reliever, such as induction of addiction to the pain reliever, brain damage, or death
- the present invention extends to a method for diagnosing a disease or disorder related to a physiological function regulated by the HPA or HPG axes
- physiological functions include sexual or reproductive functions, gastrointestinal motihty, immune response or ability to withstand stress
- diseases or disorders which can be diagnosed with the present invention include infertility, constipation diarrhea, and decreased immune response to name only a few
- endogenous opioid such as ⁇ - endo ⁇ hin
- a variant human mu opioid receptor comprising an amino acid sequence having a m SEQ ID NO 2 wherein the variation comprises Asn40Asp, relative to the binding of ⁇ -endo ⁇ hin to a human mu opioid receptor comprising an ammo acid sequence of SEQ ID NO 2, encoded by the predominant or "most common” allele of a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- ⁇ -endo ⁇ hm induced activity of a receptor produced from the expression of a variant allele of a human mu opioid receptor gene or an isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions comprising the A118G polymo ⁇ hism effected the receptor's activation of GIRK channels via a G protein-mediated mechanism relative to the activity of a receptor produced from the expression of the predominant or "most common” allele Consequently, a subject having an A118G variation
- a human mu opioid receptor produced from expression of the C17T va ⁇ ant allele of the human mu opioid receptor gene is expected to have decreased binding affinity for ⁇ -endo ⁇ hin relative to the binding affinity of a human mu opioid receptor protein produced from the expression of the predominant or 'most common" allele of the human mu opioid receptor gene (SEQ ID NO 1) comprising a DNA sequence of SEQ ID NO 1 Consequently, a variant receptor encoded by a C17T variant allele exhibits decreased activity relative to the predominant or 'most common" allele This decreased activity is expected to result in increased activity of HPA and HPG axes Hence, sexual and reproductive functions, gastrointestinal motihty, immune response and/or ability to withstand stress are expected to be decreased in the subject relative to the levels of such physiological functions in a standard comprising two alleles of the mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- a method of diagnosing a disease or disorder related to a physiological function regulated by the HPA or HPG axes m a subject comprising the steps of removing a bodily sample comprising a first and second allele of a human mu opioid receptor gene from the subject, and determining whether either the first or second alleles or both alleles, comprise a DNA sequence having at least one variation in SEQ ID NO 1 , wherein the variation comprises
- the presence of at least one variation in either or both alleles of the human mu opioid receptor gene is expected to be indicative of a disease or disorder related to a physiological function regulated by the HPA or HPG axes relative to such functions in a person homozygous with respect to the predominant or "most common ' allele comprising a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- physiological functions include sexual or reproductive functions, gastrointestinal moti ty, immune response, or ability to withstand stress
- diseases or disorders which can be diagnosed with the present invention include infertility, constipation, diarrhea, and decreased immune response to name only a few relative to a person homozygous with respect to the predominant or "most common allele comprising a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO 1
- Northern blot hybridization an isolated nucleic acid of the present invention hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor gene comprising a DNA sequence having a variation of SEQ ID NO 1, wherein the variation comprises
- such a method comprises removing a bodily sample from the subject comprising a mu opioid receptor, and determining whether the receptor comprises an ammo acid sequence having a variation in SEQ ID NO 1 , wherein the variation comprises Asn40Asp or conserved variants thereof, or Ala ⁇ Val or conserved variants thereof, such that the presence of at least one variation is expected to be indicative of a disease or disorder related to a physiological function regulated by the HPA or HPG axes, such as sexual function or development, gastric motihty, immune response, or the ability of the subject to withstand stress, relative to regulation of such activities in a standard comprises a human mu opioid receptor having an ammo acid sequence of SEQ ID NO 2
- a variant human mu opioid receptor comprising an amino acid sequence having at least one variation in SEQ ID NO 2 wherein the variation comprises Asn40Asp or conserved variants thereof, is expected to be indicative of increased sexual or reproductive functions increased gastrointestinal motihty, increased immune response, or increased ability to withstand stress relative to the levels of such function observed in a standaid having a mu opioid receptor comprising an ammo acid sequence of SEQ ID NO 2
- a variant human mu opioid receptor comprising an ammo acid sequence having a variation in SEQ ID NO 2, wherein the variation comprises Ala ⁇ Val or conserved variants thereof, in a bodily sample taken from a subject is expected to be indicative of decreased sexual or reproductive functions, decreased gastrointestinal motihty, decreased immune response, or decreased ability to withstand stress relative to the levels of such function observed in a standard having a mu opioid receptor comprising an amino acid sequence of SEQ ID NO 2
- specific diseases or disorders related to regulation of physiological functions regulated by the HPA or HPG axes include infertility, constipation, diarrhea, decreased immune response to antigens or a lack of ability to withstand stress
- a receptor in the bodily sample can be digested into fragments with proteases or CNBr These fragments can then be collected and sequenced using presently known methods Once the sequence of the receptor has been determined, it is a simple matter of comparing it to the amino acid sequence of the predominant or "most common" receptor having an amino acid sequence of SEQ ID NO.2, to determine whether a variation m the amino acid sequence exists
- Other methods involve immune assays described herein using antibodies of the present invention, or a binding assay to determine the binding affinity of the receptor to ⁇ -endo ⁇ hm If its binding to ⁇ - endo ⁇ hin is approximately 3 times greater than the known binding affinity of the predominant or "most common" receptor for ⁇ -endo ⁇ hin, then the receptor is expected to have an ammo acid sequence having a variation in SEQ ID NO 2, wherein the variation comprises Asn40Asp or conserved variants thereof, and is expected to be indicative of a disease or
- the present invention extends to a method for selecting an appropriate therapeutic agent for treating a disease or disorder related to a physiological function regulated by the HPA and HPG axes, wherein such physiological functions include sexual and reproductive functions, gastrointestinal motihty, immune response, and ability to withstand stress
- diseases or disorders related to such functions which can be diagnosed with the present invention include, but are not limited to, infertility, constipation, diarrhea, and decreased immune response, to name only a few
- kits Furthermore, as explained above, the present invention extends to commercial kits having applications in screening a bodily sample taken from a subject tor the presence of a variant allele comprising a human mu opioid receptor comprising a DNA sequence having a variation in SEQ ID NO 1 , wherein the variation comprises A118G, C17T
- an attending health profession can determine whether the subject has an susceptibility to pam relative to a standard, an increased susceptibility to at least one addictive disease relative to the susceptibility of a standard, a therapeutically effective amount of pain reliever to administer to the subject suffering from pam m order to induce analgesia in the subject relative to the therapeutically effective amount of pam reliever to administer to a standard m order to induce analgesia in the standard, or a therapeutically effective amount therapeutic agent to administer to a subject suffering from at least one addictive disease, relative to the therapeutically effective amount of therapeutic agent to administer to standard suffering from at least one addictive disease
- such information can also be used to diagnose a disease or disorder related to a physiological function regulated by the HPA or HPG axes, such as sexual or reproductive functions, gastrointestinal motihty, immune response, or ability to withstand stress, or selecting an appropriate therapeutic agent and a therapeutically effective amount of such an agent to administer to a subject suffering from a
- a test kit of the present invention for determining whether a subject comprises a variant allele of a human mu opioid receptor gene comprising a DNA sequence having a variation m SEQ ID NO 1 comprises means for detecting the presence of a variation m a first and or second allele comprising a human mu opioid receptor in a biological sample from a subject, and optimally packaged with directions for use of the kit
- the means for detecting the presence of a variant allele of a human mu opioid receptor gene comprising a DNA sequence having a variation in SEQ ID NO 1 comprises a specific binding partner of a human mu opioid receptor, such as an antibody, and means for detecting the level of binding of the specific binding partner of the antibody to the particular human mu opioid receptor
- a test kit comprises an oligonucleotide probe for binding to a variant allele of a human mu opioid receptor gene comprising a DNA sequence having a variation in SEQ ID NO 1 , and means for detecting the level of binding of the probe to the
- the sequence of the oligonucleotide probe used m a commercial kit will determine which if any variation is present in an allele comprising a human mu opioid receptor gene Should no binding be detected, it is probable that no such variation exists in either allele of the subject
- a commercial test kit of the present invention comprises a) PCR oligonucleotide primers suitable for detection of a variant allele of a human mu opioid receptor gene comprising a DNA sequence having a variation in SEQ ID NO 1, as set forth above, b) other reagents, and c) directions for use of the kit
- PCR oligonucleotide primer suitable for detection of an allele comprising a human mu opioid receptor gene comprising a DNA sequence having a variation in SEQ ID NO 1 can be readily produced bv a person of ordinary skill in the art with teaching set forth herein, and variations of SEQ ID NO 1 also set forth herein
- the present invention further extends to commercial test kits capable of detecting a variant human mu opioid receptor m a bodily sample taken from a subject
- variant human mu opioid receptors that can be detected with a kit of the present invention com- prise a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO 2, wherein the variation comprises the variation comprises Arg260H ⁇ s or conserved variants thereof, or a variant human mu opioid receptor comprising an amino acid sequence having at least two variations in SEQ ID NO 2, wherein the variations comprise
- a commercial test kit of the present invention can be used to determine a susceptibility to pain in a subject relative to a standard, an increased susceptibility to at least one addictive disease in a subject relative to the susceptibility of a standard, a therapeutically effective amount of pam reliever to administer to the subject suffering from pain in order to induce analgesia in the subject relative to the therapeutically effective amount of pain reliever to administer to a standard in order to induce analgesia in the standard, a therapeutically effective amount of a therapeutic agent to administer to a subject suffering from at least one addictive disease, relative to the therapeutically effective amount of therapeutic agent to administer to standard suffering from at least one addictive disease, a diagnosis of a disease or disorder related to a physiological function regulated by the HPA or HPG axes, such as sexual or reproductive functions, gastrointestinal motility, immune response, or ability to withstand stress, or selecting an appropriate therapeutic agent and a therapeutically effective amount of such an agent to administer to a subject suffering from a disease or disorder related to a physiological function
- the present invention extends to a commercial test kit having applications set forth above, comprising a predetermined amount of at least one detectably labeled immuno- chemically reactive component having affinity for a variant human mu opioid receptor;
- Antibodies of the present invention have readily applications in a commercial test kit of the present invention.
- test kit may be prepared and used for the pu ⁇ oses stated above, which operates according to a predetermined protocol (e.g. "competitive, " “sandwich, “ “double antibody, “ etc.), and comprises:
- the mu opioid receptor is the major target for clinically important opioid alkaloids including mo ⁇ hine, methadone, fentanyl, and other opioid drugs (1,3), as well as for endogenous opioid peptides such ⁇ -endorphin, Met-enkephahn-Arg-Phe, and the recently identified endomo ⁇ hins (5) Furthermore it is the major molecular site of action for heroin (2,6) Rapid activation of the mu opioid receptor, such as occurs in the setting of drug abuse, results in a euphoric effect, thus conferring the reinforcing or rewarding effects of the drug, contributing to the development of addiction Clinical observations have suggested that individuals have varied sensitivity to opioids, suggesting potential variability in the receptor protein and gene
- Inclusion criteria were met by 152 individuals (refer to Methods for details of inclusion/exclusion criteria)
- One hundred thirteen of the study subjects (74 3 %) were opiate dependent with or without previous or current co-dependency for other substances, 39 study subjects (25 7%) had no history of drug dependence Study subjects were well balanced between females and males 69 females (45 4%) and 83 males (56 4%) were included in the study subject pool
- the ethnic breakdown of the study subject populations was as follows African- American 31 , (20 3 %), Caucasian, 52 (34 2%), Hispanic, 67 (44 1 %), Native North- American, 1 , (0 7%) and Other, 1 (0 7%)
- several individuals could be classified into two separate groups if one parent came from one ethnic group and the other parent from another group, including four individuals (2 6%) who reported one parent African- American and one Caucasian, and five individuals (3 3 %) who reported one parent Caucasian and one Hispanic, for the genotype calculations the former were classified as African-American and
- Genotype and allele frequencies for the two most common allelic variants, the A118G and C17T polymo ⁇ hisms, are shown in Table 2
- the following list of individuals with more than one variant form of the receptor were found: (1) Three individuals were double heterozygous for the A118G and C17T variants. The DNA sequencing methodology used cannot determine whether both SNPs are on a single chromosome or the two SNPs are on different chromosomes. These individuals would therefore be predicted either to express both prototype receptors and receptors that have both amino acid substitutions, or, conversely, two variant receptor types, one with the A6V substitution and the other with the N40D substitution. (2) One individual was a double heterozygote for the A118G SNP and G779A SNP.
- This individual would therefore be predicted either to express both prototype receptors and receptors that have both amino acid substitutions, or, conversely, two variant receptor types, one with the N40D substitution and the other with the R260H substitution.
- (3) One individual was homozygous for the C17T SNP and heterozygous for the G924A SNP. This individual is predicted to express variant receptors each of which has the A6V substitution. However the G924A SNP would be useful for ascribing a haplotype in this individual for genetic haplotype analysis.
- the most prevalent genetic polymo ⁇ hism identified is the A118G SNP with a substitution at the nucleotide position 118 with respect to the first base of the initiator codon for methionine (Fig. 1). This allele was observed in 29 of the 152 subjects, with 26 subjects being heterozygous and 3 being homozygous for the variant allele. This gives an allele frequency of 10.5 % in the subject population that we have examined for this study.
- Nucleotide no. 118 is the first base in codon no. 40 of the human mu opioid receptor, and the A118G variant predicts an Asn to Asp change in amino acid residue no. 40 of the receptor (N40D). The Asn residue at amino acid position no.
- the A118G variant would result in the loss of a putative N-glycosylation site.
- the position of amino acid 40 is in the N terminal region of the mu opioid receptor (9) .
- the N terminal region of opioid receptors including that of the mu opioid receptor, is predicted to be in the extracellular space (18).
- position 118 of the most common mu receptor cDNA was mutated by site-directed mutagenesis, and a cDNA clone for the human mu opioid receptor containing the A118G variant was generated. This way, both the most common and the Al 18G variant receptors could be ex- pressed in cells to determine their cellular activity and their binding affinities.
- Radioligand binding assays were performed with cell lines stably transfected with either the A118G variant or the most common mu receptor, to determine whether the A118G polymo ⁇ hism changes the receptor's ability to bind opioid ligands, especially endogenous opioid peptides, since they are the physiological agonists for the mu opioid receptor.
- the A118G variant and the most common mu receptors yielded similar binding affinity values for most of the opioid ligands tested, including the small endogenous peptide agonists Met- and Leu-enkephalin, each with five amino acid residues; endomo ⁇ hin-1 and -2, each with four residues; the mu-selective synthetic opioid peptide DAMGO, with five amino acid residues; the endogenous ligand for the kappa opioid receptor dyno ⁇ hin A (1-17); as well as the mu-preferring opioid alkaloid agonists mo ⁇ hine, fentanyl, methadone, and the opioid antagonist naloxone (Fig. 2, and data not shown).
- GIRK G protein-activated inwardly rectifying K +
- Agonist stimulation of the Al 18G variant receptor activated a potassium current similar to that seen with the most common mu opioid receptor (9,23).
- the EC 50 values for endomo ⁇ hin-1 are 4.6 nM for the most common receptor and 4.9 nM for the A118G variant receptor (Fig. 3), indicating that endomo ⁇ hin-1 activated both receptors with similar potency.
- the EC 50 values for ⁇ -endo ⁇ hin differed about three fold between the A118G variant and the most common mu opioid receptors (Fig. 3), consistent with the change in the binding affinity (Fig. 2).
- ⁇ - endo ⁇ hin An endogenous opioid with wide distribution in both the CNS and the periphery, ⁇ - endo ⁇ hin has been postulated to play a role in diverse biological functions (25-27). As a neuropeptide, it can modulate neurotransmitter actions in the CNS to mediate antinociception. It is also a mediator in the stress response, of potential importance for the pathophysiology of the addictive diseases (28-36). ⁇ -endorphin can regulate the secretion of both stress and reproductive hormones, thereby influencing a variety of physiological functions. The synthesis and processing of ⁇ -endo ⁇ hin is, in turn, regulated by other factors, including certain neurotransmitters and hormones.
- the A118G polymorphism may change both the binding affinity and functional potency of ⁇ -endorphin.
- affinity and potency values Fig. 2 and 3
- adenylyl cyclase inhibition between the C17T and prototype receptors are compared (Fig. 6).
- the C17T receptor is less sensitive to ⁇ -endo ⁇ hin: the EC50 values differed by a factor of approximately 2.7.
- the receptor was also less sensitive to leu-enkephalin: the EC50 values differed by a factor of approximately 2.8.
- Unrelated healthy volunteer subjects were recruited primarily through posting of notices and newspaper advertisements or referral by physicians or staff at the Rockefeller Univer- sity Hospital Individuals with continuing drug or alcohol abuse or prior extended periods of regular abuse were excluded from this category
- the exclusion criteria were defined as follows for current or continuing abuse, alcohol, at least five (for men) or four (for women) instances of drinking to intoxication during the previous 30 days, opiates, cocaine, amphetamines, or other illicit drugs (excluding cannabis), any use during the previous 30 days Users of nicotine or caffeine were not excluded, nor were individuals who had abused cannabis for up to 12 days during the previous 30 days
- subjects were excluded who had abused illicit drugs, excluding cannabis, for at least three times a week for a period of at least one month All study subjects were rigorously screened to assure appropriate characterization of addictive diseases, status of treatment, and presence or absence of polydrug or alcohol abuse Subjects entering the study were required to be competent to understand the study procedures and understand and sign the Institutional Review Board approved informed consent Patients with schizophrenia or other psychotic mental
- Study subjects were also administered a detailed personal and medical and special addictive disease questionnaire as well as a family history medical and addictive disease questionnaire designed to provide information regarding substance abuse and major mental illness of first and second degree relatives. Study subjects provided detailed information regarding family origin and ethnic background, including country or geographic area of birth. This information was obtained for both the study subjects themselves and their immediate ancestors (parents, grandparents and great- grandparents), to the extent that the information was known by the study subjects. Study subjects were classified into five groups: African- American, Caucasian, Hispanic (Caribbean and Central or South American origin), Native North American, and Other.
- the detailed ancestral information collected by the family origin questionnaire allowed classification of study subjects into defined categories. Following psychiatric and behavioral assessment and informed consent and family history acquisition, venipuncture on the study subject was performed, and a blood specimen was taken. Blood samples were processed for DNA extraction and EBV transformation to create stable cell lines that were stored for future studies. All blood samples were coded; the psychiatrists and nurses who performed psychiatric and psychological assessments were blind to the genotypes of the study subjects, and the identity and categorization of the study subjects was unknown to the laboratory research personnel.
- PCR primers were synthesized for three of the four exons of the gene; the fourth exon was not included in this study because this exon is small (4 or 12 amino acid residues) and alternative splicing in this exon has been shown to occur (40).
- Exon 1 forward primer sequences were based on the 5 '-untranslated region of the receptor (9).
- Exon 1 reverse, exon 2 forward and reverse, and exon 3 forward primer sequences were based on partial intron sequence data obtained from inverse PCR of genomic DNA sequences for the receptor gene (data not shown).
- Exon 3 reverse primers were based on reported intron 3 sequence (40).
- Two sets of primers were designed for each exon to allow for nested PCR reactions to increase amplification specificity. Only one reverse primer was used for exon 1.
- the PCR reactions were performed with 300-400 ng of genomic DNA, PCR products were separated on agarose gels, and the DNA fragments were purified for DNA sequencing . DNA polymo ⁇ hisms were confirmed by both manual and automated sequencing.
- hMOR human mu opioid receptor
- Stable transfection of the A118G SNP plasmid into AV-12 cells was performed as described (41). Individual colonies were then picked, expanded, and tested for expression levels by performing binding assays. Cells were harvested by washing with phosphate-buffered saline (PBS) at room temperature, then they were scraped into homogenization solution (0.3 M sucrose, 25 mM Tris-HCl, pH 7.4, 0.05 % BSA, and protease inhibitor cocktail, including 0.5 mM PMSF, 0.1 ⁇ g/ml leupeptin, and 0.01 % aprotinin), transferred to Dounce homogenizer and homogenized on ice.
- PBS phosphate-buffered saline
- the suspension was centrifuged at 1,000 g for 10 min and the supernatant saved in a clean tube.
- the cell pellet was resuspended in homogenization buffer, homogenized and centrifuged as described above.
- the supernatants from both extractions were combined and centrifuged at 30,000 g for 20 min.
- the pelleted membranes were resuspended in binding buffer (50 mM Tris-HCl, pH 7.4), and binding assays were carried out using membrane protein preparations as described (9).
- Xenopus oocytes were injected with in vitro transcribed mRNAs for the most common or A118G variant mu opioid receptors together with the G protein-activated inwardly rectifying K + channels (GIRKl and GIRK2).
- GIRKl and GIRK2 G protein-activated inwardly rectifying K + channels
- oocytes were voltage-clamped in ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM HEPES, pH 7.6) using a two-electrode voltage-clamp (Axon Instruments).
- Cells were then superfused with a high potassium solution (98 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM HEPES, pH 7.6), and stimulated with opioid ligands to measure the resulting potassium current.
- a high potassium solution 98 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM HEPES, pH 7.6
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU50968/99A AU5096899A (en) | 1998-07-10 | 1999-07-10 | Alleles of the human mu opioid receptor, diagnostic methods using said alleles, and methods of treatment based thereon |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/113,426 | 1998-07-10 | ||
US09/113,426 US6337207B1 (en) | 1998-07-10 | 1998-07-10 | Alleles of the human mu opioid receptor, diagnostic methods using said alleles, and methods of treatment based thereon |
US09/351,198 | 1999-07-09 | ||
US09/351,198 US6335168B1 (en) | 1998-07-10 | 1999-07-09 | Alleles of the human mu opioid receptor and diagnostic methods based thereon |
Publications (3)
Publication Number | Publication Date |
---|---|
WO2000003024A2 true WO2000003024A2 (en) | 2000-01-20 |
WO2000003024A3 WO2000003024A3 (en) | 2000-02-24 |
WO2000003024A9 WO2000003024A9 (en) | 2000-06-02 |
Family
ID=26811041
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1999/015707 WO2000003024A2 (en) | 1998-07-10 | 1999-07-10 | Alleles of the human mu opioid receptor, diagnostic methods using said alleles, and methods of treatment based thereon |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2000003024A2 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005039573A2 (en) * | 2003-10-06 | 2005-05-06 | Novartis Ag | Biomarkers for the prediction of drug-induced diarrhoea |
US7214534B2 (en) | 2002-06-18 | 2007-05-08 | Regents Of The University Of Minnesota | Isolated nucleic acid molecules encoding mutant μ opioid receptors |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995007983A1 (en) * | 1993-09-13 | 1995-03-23 | Indiana University Foundation | Human mu opioid receptor |
WO1995020667A1 (en) * | 1994-01-28 | 1995-08-03 | The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services | cDNA AND GENOMIC CLONES ENCODING HUMAN ν OPIATE RECEPTOR AND THE PURIFIED GENE PRODUCT |
WO1998033937A2 (en) * | 1997-02-03 | 1998-08-06 | Max-Delbrück-Centrum für Molekulare Medizin | GENOMIC SEQUENCE OF THE HUMAN ν-OPIOID RECEPTOR GENE AND THE VARIANTS, POLYMORPHISMS AND MUTATIONS THEREOF |
-
1999
- 1999-07-10 WO PCT/US1999/015707 patent/WO2000003024A2/en active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995007983A1 (en) * | 1993-09-13 | 1995-03-23 | Indiana University Foundation | Human mu opioid receptor |
WO1995020667A1 (en) * | 1994-01-28 | 1995-08-03 | The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services | cDNA AND GENOMIC CLONES ENCODING HUMAN ν OPIATE RECEPTOR AND THE PURIFIED GENE PRODUCT |
WO1998033937A2 (en) * | 1997-02-03 | 1998-08-06 | Max-Delbrück-Centrum für Molekulare Medizin | GENOMIC SEQUENCE OF THE HUMAN ν-OPIOID RECEPTOR GENE AND THE VARIANTS, POLYMORPHISMS AND MUTATIONS THEREOF |
Non-Patent Citations (3)
Title |
---|
BERGEN A W ET AL: "MU OPIOID RECEPTOR GENE VARIANTS: LACK OF ASSOCIATION WITH ALCOHOL DEPENDENCE" MOLECULAR PSYCHIATRY, vol. 2, 1997, pages 490-94, XP002070828 * |
BERRETTINI W ET AL: "Human mu opioid receptor gene polymorphism and vulnerability to substance abuse" ADDICTION BIOLOGY, vol. 2, 1997, pages 303-308, XP000863630 * |
GSCHEIDEL N ET AL: "Association analysis of mu opioid receptor gene variants in cDNA with alcohol dependence by allele specific polymerase chain reaction (AS-PCR)" JOURNAL OF MOLECULAR MEDICINE, vol. 76, no. 6, May 1998 (1998-05), page B33 XP000863490 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7214534B2 (en) | 2002-06-18 | 2007-05-08 | Regents Of The University Of Minnesota | Isolated nucleic acid molecules encoding mutant μ opioid receptors |
WO2005039573A2 (en) * | 2003-10-06 | 2005-05-06 | Novartis Ag | Biomarkers for the prediction of drug-induced diarrhoea |
WO2005039573A3 (en) * | 2003-10-06 | 2006-02-16 | Novartis Ag | Biomarkers for the prediction of drug-induced diarrhoea |
Also Published As
Publication number | Publication date |
---|---|
WO2000003024A3 (en) | 2000-02-24 |
WO2000003024A9 (en) | 2000-06-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6476187B1 (en) | Methods and reagents for discovering and using mammalian melanocortin receptor agonists and antagonists to modulate feeding behavior in animals | |
AU685076B2 (en) | DNA encoding 5-HT4 serotonin receptors and uses thereof | |
GB2292382A (en) | Obesity(OB) polypeptides | |
EP1473367B1 (en) | Means and methods for diagnosing and treating affective disorders | |
JP2007014345A (en) | Polycystic kidney disease 1 gene and uses thereof | |
WO2005007812A2 (en) | Genes as diagnostic tools for autism | |
Tam et al. | Established and emerging strategies to crack the genetic code of obesity | |
JP4208960B2 (en) | Opioid receptor gene | |
US9670544B2 (en) | CC2D2A gene mutations associated with Joubert syndrome and diagnostic methods for identifying the same | |
JP2005124574A (en) | Opioid receptor: composition and method | |
JP3977427B2 (en) | Neuropeptide Y-Y5 receptor | |
US6337207B1 (en) | Alleles of the human mu opioid receptor, diagnostic methods using said alleles, and methods of treatment based thereon | |
US6335168B1 (en) | Alleles of the human mu opioid receptor and diagnostic methods based thereon | |
WO2000003024A2 (en) | Alleles of the human mu opioid receptor, diagnostic methods using said alleles, and methods of treatment based thereon | |
US7071315B2 (en) | Alleles of the human mu opioid receptor, diagnostic methods using said alleles, and methods of treatment based thereon | |
De Maeyer et al. | Alternative splicing and exon duplication generates 10 unique porcine 5-HT4 receptor splice variants including a functional homofusion variant | |
US20040097704A1 (en) | Alleles of the human kappa opioid receptor gene, diagnostic methods using said alleles, and methods of treatment based thereon | |
JP3985007B2 (en) | Body weight modulators, corresponding nucleic acids and proteins, and their diagnostic and therapeutic uses | |
WO2001025431A1 (en) | Primate, particularly human, vomeronasal-like receptor | |
US20020142312A1 (en) | Polymorphism of the human serotonin 1B receptor gene, diagnostic methods and methods of treatment based thereon | |
US20030008289A1 (en) | Alleles of the human orphanin FQ/nociceptin receptor gene, diagnostic methods using said alleles, and methods of treatment based thereon | |
AU772193B2 (en) | Neuropeptide Y-Y5 receptor | |
AU748425B2 (en) | Mammalian melanocortin receptor agonists and antagonists for modulating feeding behavior in animals | |
US20060166207A1 (en) | Hsan II related gene and expression products and uses thereof | |
Class et al. | Patent application title: CC2D2A GENE MUTATIONS ASSOCIATED WITH JOUBERT SYNDROME AND DIAGNOSTIC METHODS FOR IDENTIFYING THE SAME Inventors: John B. Vincent (Toronto, CA) John B. Vincent (Toronto, CA) Muhammad Ayub (Kingston, CA) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
AK | Designated states |
Kind code of ref document: A3 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
AK | Designated states |
Kind code of ref document: C2 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: C2 Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
COP | Corrected version of pamphlet |
Free format text: PAGES 1/6-3/6, 5/6 AND 6/6, DRAWINGS, REPLACED BY NEW PAGES 1/6-3/6, 5/6 AND 6/6; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
122 | Ep: pct application non-entry in european phase |