WO2002092838A2

WO2002092838A2 - Allelic variants of delta opioid receptors associated with disorders of feeding and energy homeostasis

Info

Publication number: WO2002092838A2
Application number: PCT/US2002/014940
Authority: WO
Inventors: Andrew W. Bergen
Original assignee: Bioinvest Ltd.
Priority date: 2001-05-11
Filing date: 2002-05-13
Publication date: 2002-11-21
Also published as: AU2002308686A1; WO2002092838A3

Abstract

The invention relates generally to polymorphisms in delta opioid receptors genes that are associated with eating or energy homeostasis disorders such as of anorexia nervosa, bulimia nervosa, obesity and abnormal body mass.

Description

ALLELIC VARIANTS OF DELTA OPIOID RECEPTORS ASSOCIATED WITH DISORDERS OF FEEDING AND ENERGY HOMEOSTASIS

RELATED APPLICATION: This application claims priority to U.S. Provisional Application 60/290,016, which is herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates generally to polymoφhisms in delta opioid receptor genes that are associated with eating or energy homeostasis disorders such as of anorexia nervosa, bulimia nervosa, obesity and abnormal body mass.

BACKGROUND OF THE INVENTION A. Opioid Receptors

The delta opioid receptor, DOR (Gene locus OPRDl, OMIM#165195, chrlp36.1- p34.3), expressed in brain (Bzdega et al, Proc. Natl. Acad. Sci., 90: 9305-9309 (1993); Mansour et al, Trends Neoroscience, 18: 22-29 (1995)), gastric, intestinal and vascular smooth muscle (Saeed et al, Int. J. Mol. Med., 6: 673-677 (2000)) and in small cell lung cancer cell lines (Schreiber et al, Anticancer Res., 18: 1787-1792 (1998)), is a seven transmembrane G protein-linked receptor that binds met- and leu-enkephalin neuropeptides (Gene locus PENK, 131330, chr8q23-q24). Binding of DOR agonists inhibit adenylate cyclase and stimulates mitogen-activated protein kinases (Tso et al, J. Neurochem., 74: 1685-1693 (2000)). DOR monomers heterodimerize with both opioid receptor KOR (Gene locus OPRK1, OMIM#165196, chr8ql l-ql2) and MOR (Gene locus OPRM1, OMIM#60018, chr6q24-25) monomers to form heterodimers with partially selective opiate agonist ligand-binding properties and that synergistically amplify opiate agonist signaling (Jordan and Devi, (1999), Gomes et al, (2000)). The human (OPRDl) and murine (Oprdl) genes have been cloned (Knapp et al, FASEB J., 9: 516-525 (1995)), expression has been mapped (Simonin et al, Mol. Pharmacol., 46: 1015-1021 (1994); Bzdega et al, Proc. Natl. Acad. Sci., 90: 9305-9309 (1993)), and the gene localized to chromosome 1 in the human (Befort et al, Genomics, 20: 143-145 (1994)).

Murine Oprdl knock-outs (the mouse locus of OPRDl) result in no detectable binding of DOR ligands, and also result in locomoter hyperactivity and increased anxiety and depression (Filliol et al, Nat. Genet., Vol. 25, pp. 195-200 (2000)). Specifically, in two behavioral models of anxiety, male Oprdl-/- mice show decreased time spent in open and lit spaces, interpreted as increased anxiety-like behavior, while in a behavioral model of depression, both male and female Oprdl-/- mice showed increased swim-test immobility over wild type mice, interpreted as increased depression-like behavior (Filliol et al, Nat. Genet., 25: 195-200 (2000)). Oprml-/- mice exhibit opposite behavior in these behavioral models and μ and δ antagonists restore Oprdl-/- and Oprm-/- mouse behavior to that of the wild type, suggesting that μ and δ receptors mediate opioid homeostasis related to anxiety and depression. Met and leu enkephalin peptide neurotransmitters are produced by carboxy- and endo- peptidases and prohormone convertases of preprodynorphin (Berman et al.,

2001), and are expressed in brain and adrenal (Legon et al, Nucleic Acids Res., 10: 7905- 7916 (1982). Penk knock-out mice exhibit increased anxiety- like behavior (hiding under the bedding, frantic running or jumping) in response to moderate noise (Konig et al, Nature, 383: 535-538 (1996)), and increased immobility and decreased time spent in open and lit spaces in behavioral models of fear and anxiety (Konig et al, Nature, 383: 535-538 (1996); Ragnauth et al, Proc. Natl. Acad. Sci., 90: 1958-1963 (2001)). These results from Penk knock-out mice are consistent with the δ and μ opioid receptor-ligand system regulating mood and behavioral responses to stressful stimuli.

The OPRDl and OPRMl genes have been investigated as potential candidate genes increasing susceptibility to neuropsychiatric disorders. Sequence variation at the OPRMl locus has been evaluated for association to substance dependence (Bergen et al, Mol. Psychiatry, 2: 490-494 (1997); Berrettini et al, Addiction Biology, 2: 308 (1997); Hoehe et al, Hum. Mol. Genet., 9: 2895-2908 (2000); Kranzler et al, Alcohol Clin. Exp. Res., 22: 1359-1362 (1998); Mayer et al, Neuroreport, 8: 2547-2560 (1997); Town et al, Am. J. Med. Genet., 88: 458-461 (1999)) and idiopathic epilepsy (Sander et al, Epilepsy Res., 39: 57-61 (2000)). Published molecular genetic studies of the OPRDl receptor in the literature (Mayer et al, Neuroreport, 8: 2547-2560 (1997); Franke et al, Am. J. Med. Genet., 88: 462-464 (1999)) provide contradictory evidence that a silent polymorphism (921T>C) at the delta opioid receptor is associated with heroin and alcohol dependence in case control and trio association designs and to alcohol dependence in case control and trio designs.

Identification and genotype determination of this polymorphism and a non-conservative amino acid substitution (80T>G, Phe27Cys) in six populations demonstrates significant allele frequency heterogeneity between continental population groups (Gelernter and Kranzler, Hum. Genet., 107: 86-88 (2000)). No significant linkage disequilibrium was exhibited between the two loci, located in exon 1 and exon 3 of OPRDl and separated by 50,788 base pairs (see http://www.ncbi.nlm.nih.gov/entrez/viewer.cgi?val= NT_004538&db=Nucleotide&dopt=GenBank).

B. Eating Disorders

A variety of life-threatening feeding and energy homeostasis disorders have been recognized in the medical literature. Such disorders include, for example, anorexia nervosa and bulimia nervosa as well as obesity. Only in a few such disorders has the underlying etiology been elucidated at the molecular level. In fact, for some feeding disorders, a questionnaire - rather than molecular probes or monoclonal antibodies specific for a disease-associated marker — is the most probative diagnostic tool available. See, e.g., Walling, Anne D., "A New Screening Tool for Patients with Eating Disorders", American Family Physician" (2000) 7(61): 2186.

Such disorders often are life-threatening and early diagnosis would significantly improve the prognosis and outcome for many patients, particularly the young women who are at greatest risk for certain disorders. For example, anorexia nervosa is associated with a mortality rate of up to 20 percent; and, in addition to weight loss, anorexia nervosa patients also may suffer from cachexia, cardiac dysfunction, leukopenia, osteoporosis and a variety of gastrointestinal and neuropsychiatric conditions. See, e.g., Walling, Anne D., Identifying and Treating Patients with Anorexia Nervosa, American Family Physician (2000) 8(61): 2528. Anorexia nervosa patients also typically have a low self-esteem and are known to have obsessive tendencies in some cases. The etiology of this disorder is obscure. At present, the medical community believes that affected young women use food restriction as an outlet to escape the pressures of home, social group or academic environments. While patients also are tested for depression and other psychological disorders, therapeutics "are not the basis of therapy but may be used to treat concomitant depression." Id. The central therapy is refeeding. Similarly, bulimia nervosa patients typically receive treatment consisting of psychotherapy, antidepressant drugs, or both. The combination of psychotherapy and antidepressants reportedly is more effective than either treatment regimen alone. See, e.g., Kiss, Alexander, "Treatment of chronic bulimic symptoms: new answers, more questions," Lancet ( 2000) 355(9206): 769-770. In clinical practice, serotonin-reuptake inhibitors (or other antidepressants) are the most commonly used drugs because they do not produce many side-effects. However, although the short-term effect of therapy has been documented, the long-term outcome is dismal. For example, 5 to 10 years after initial presentation, there is essentially no difference in recovery rates between treated and untreated individuals. Id.

Psychiatrically defined eating disorders affect ~ 3% of women (Wade et al, Aust. N. Z. J. Psychiatry, 30: 845-851 (1996)), with a significantly increased mortality risk in both anorexia nervosa and bulimia nervosa (Crow et al, Int. J. Eat. Disorder., 26: 97-101

(1999)), though approximately 80% of patient do recover, albeit over a period of 5 - 6 years (Strober et al, Int. J. Eat. Disorder., 22: 339-360 (1997)). The psychiatric criteria for a diagnosis of anorexia nervosa (~0.5% prevalence) include: refusal to maintain weight, fear of gaining weight and a disturbance in the patient's perception of body weight or shape and its effect on self-evaluation, while psychiatric criteria for a diagnosis of bulimia nervosa (~2.5% prevalence) include regular episodes of binge eating and a sense of lack of control during the binge episode, inappropriate compensatory behavior to avoid weight gain and a disturbance in the patient's self-evaluation due to perceived body shape and weight (American Psychiatric Association (1994) Diagnostic and Statistical Manual of Mental Disorders, American Psychiatric Association, Washington, D.C.). Comorbidity of eating disorders and other psychiatric disorders (e.g., depression, OCD, anxiety disorders, bipolar disorder) and extremes of personality traits have been described (Godart et al, Eur. Psychiatry, 15: 38-45 (2000)); Lilenfeld et α/., Arch. Gen. Psychiatry., 55: 603-610 (1998); Simpson et al, J. Nerv. Ment. Dis., 180: 719-722 (1996); Braun et al, Psychol. Med., 24: 859-867 (1994); Klump et al, J. Nerv. Ment. Dis., 188: 559-67 (2000)). The relative risk is approximately 11 for anorexia nervosa and 4 for bulimia nervosa, (Strober et al, Int. J. Eat. Disorder., 22: 339-360 (1997)), and the additive genetic influence on the risk for eating disorders ranges between 50 and 80% (Kendler et al, Am. J. Psychiatry, 148: 1627-1637 (1991); Wade et al, Psychol. Med., 29: 925-934 (1999); Bulik et al, Biol. Psychiatry, 44: 1210-1218 (1998)). These family history and heritability studies provide the required evidence to justify a molecular genetic approach to the study of eating disorder susceptibility factors. SUMMARY OF THE INVENTION

The present invention is based on the discovery nucleotide polymorphisms in a delta opioid receptor gene and the association of these polymorphisms with an eating or energy metabolism disorder such as anorexia nervosa.

In another aspect of the present invention, related composition screening systems and diagnostic and prognostic assays are provided.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 : Molecular Structure of OPRD 1.

Figure 2: Sequences surrounding the single nucleotide polymoφhisms ("SNPs") of the OPRDl gene.

SPECIFIC EMBODIMENTS A. Delta Opioid Receptors

As used herein, the terms "delta opioid receptor" or "delta opioid receptor gene" refer to any mammalian delta opioid receptor gene or protein, and in particular, although not limited to, human delta opioid receptor genes and proteins. As described above, the human delta opioid receptor gene (OPRDl) and the murine receptor gene (Oprdl) have been cloned, expression has been mapped, and the gene localized to chromosome 1 in the human. The terms "delta opioid receptor" or "delta opioid receptor gene," however, are not limited to these specific sequences. For instance, the terms also refer to naturally occurring allelic variants and man-made substitution, insertion or deletion mutants that have a slightly different amino acid sequence than those specifically referred to above. As used herein, the family of proteins related to the human amino acid sequence of the delta opioid receptor refers to proteins that have been isolated from organisms in addition to humans. The methods used to identify and isolate other members of the family of proteins related to these proteins are readily available and known to persons skilled in the molecular biology field, including hybridization and sequence or homology screening methods.

As used herein, the terms "delta opioid receptor variant" and "delta opioid receptor polymoφhism" as well as the gene encoding either the delta opioid receptor variant or polymoφhism refers to the receptor or its encoding gene that is associated with a genetic predisposition to an eating or energy homeostasis disorder, such as anorexia nervosa, bulimia nervosa, obesity or abnormal body mass disorders.

As used herein, the term delta opioid receptor mediated disease refers to a disorder or pathology in which the presence of an "delta opioid receptor variant" or "delta opioid receptor polymoφhism" is associated with or participates in a signaling or other biological pathway in a manner that results in a pathological condition such as those eating and energy metabolism disorders identified above.

The proteins of the present invention are preferably in isolated form. As used herein, a protein is said to be isolated when physical, mechanical or chemical methods are employed to remove the protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain such an isolated protein. Receptor proteins, or peptide fragments thereof may also be covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope).

As used herein, a nucleic acid molecule is said to be "isolated" when the nucleic acid molecule is substantially separated from and relative to contaminant or other nucleic acid molecules encoding other polypeptides with which the nucleic acids of the present invention are customarily associated. Nucleic acid molecules of the invention may be cloned into any available vector for replication and/or expression in suitable host cells. The host cells then may be used to recombinantly produce the encoded protein. Appropriate vectors, host cells and methods of expression are widely available.

B. Methods of Using the Delta Opioid Polymorphisms The invention provides a method for the diagnosis of a delta opioid receptor mediated disease, such as an eating or energy homeostasis disorder comprising the steps of detecting the presence or absence of a variant nucleotide at one or more of positions herein described in a patient sample and determining the status of the individual by reference to polymoφhism in the delta opioid receptor gene. In preferred methods, a polymoφhism is detected at a position corresponding to OPRDl-01, OPRDl-03, OPRD-05 and/or OPRD1- 07 as shown in Table 1. o

o

H U α.

TABLE 1

* AJ=Ashkenazi Jewish (90 individuals); EA= European-American (152 individuals); AA= African-American (69 individuals); B=Bedouin (37 individuals); E=Ethiopian individuals), J=Japanese (89 individuals).

90 90 o o O

Moreover, in another aspect of the invention, the detection of multiple polymoφhisms is also used for the diagnosis of a delta opioid receptor mediated disease, such as an eating or energy homeostasis disorder. Detection of the following two-locus halo type frequencies correspond to an increased risk or genetic predisposition to eating or energy homeostasis disorders, such as Anorexia Nervosa: OPRDl-01-05; OPRDl-01-07; OPRDl-03-07; OPRDl -05-07; and OPRDl -01 -03-05-07. In addition, increased risk or genetic predisposition to either the restricting or purging types of Anorexia Nervosa may be diagnosed based on the linkage disequilibrium of the polymoφhosims. The restricting Anorexia Nervosa subtype is associated with linkage disequilibrium between OPRDl-01 and OPRD 1 -03 and between OPRD 1 -05 and OPRD 1 -07; while the purging Anorexia Nervosa subtype is associated with linkage disequilibrium between OPRDl-01 and OPRDl-03, between OPRDl-03 and both OPRD 1-05 and OPRD 1-07, and between OPRDl-05 and OPRDl-07.

Any sample comprising cells or nucleic acids from the patient or subject to be tested may be used. Preferred samples are those easily obtained from the patient or subject. Such samples include, but are not limited to blood, peripheral lymphocytes, epithelial cell swabs, bronchoalveolar lavage fluid, sputum, or other body fluid or tissue obtained from an individual. It will be appreciated that the test sample may comprise an delta opioid receptor nucleic acid that has been amplified using any convenient technique, e.g. , PCR, before analysis of allelic variation. As described below, any available means of detecting a sequence polymoφhism(s) of the invention may be used in the methods.

In another method of the invention, the diagnostic methods described herein are used in the development of new drug therapies which selectively target one or more allelic variants of an delta opioid receptor gene that are associated with an eating or energy homeostasis disorder. In one format, the diagnostic assays of the invention may be used to stratify patient populations by separating out patients with a genetic predisposition to an eating or homeostasis disorder from the general population. Identification of a link between a particular allelic variant and predisposition to disease development or response to drug therapy may have a significant impact on the design of new drugs by assisting in the analysis of a drugs efficacy or effects on specific populations of patients. For instance, drugs may be designed to regulate the biological activity of variants implicated in the disease process while minimizing effects on other variants. C. Detection of Polymorphisms

As described above, detection of the delta opioid receptor polymoφhisms of the invention generally comprises the step of determining the sequence of a delta opioid receptor gene in a sample, preferably a patient sample, at one or more of the positions herein described.

Any analytical procedure may be used to detect the presence or absence of variant nucleotides at one or more polymoφhic positions of the invention. In general, the detection of allelic variation requires a mutation discrimination technique, optionally an amplification reaction and optionally a signal generation system. Many current methods for the detection of allelic variation are reviewed by Nollau et. al., Clin. Chem., 43: 1114-1120 (1997); and in standard textbooks, for example, Laboratory Protocols for Mutation Detection by U. Landegren, Oxford University Press, 1996 and PCR, 2nd Edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997.

Any means of mutation detection or discrimination may be used. For instance DNA sequencing, scanning methods, hybridization, extension-based methods, incoφoration- based methods, restriction enzyme-based methods and ligation-based methods may be used in the methods of the invention. Sequencing methods include, but are not limited to, direct sequencing and sequencing by hybridization. Scanning methods include, but are not limited to, protein truncation test (PTT), single-strand conformation polymoφhism analysis (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), cleavase, heteroduplex analysis, chemical mismatch cleavage (CMC), and enzymatic mismatch cleavage.

Hybridization-based methods of detection include, but are not limited to, solid phase hybridization such as dot blots, multiple allele specific diagnostic assay (MASDA), and reverse dot blots, oligonucleotide arrays (DNA Chips). Solution phase hybridization methods may also be used, such as Taqman®.

Extension based methods include, but are not limited to, amplification refractory mutation system (ARMS), amplification refractory mutation system linear extension (ALEX), and competitive oligonucleotide priming system (COPS). Incoφoration-based detection methods include, but are not limited to, mini- sequencing and arrayed primer extension (APEX). Restriction enzyme-based detection systems include, but are not limited to, RFLP, and restriction site generating PCR. Lastly, ligation based detection methods include, but are not limited to, oligonucleotide ligation assay (OLA).

Signal generation or detection systems that may be used in the methods of the invention include, but are not limited to, fluorescence methods such as fluorescence resonance energy transfer (FRET), fluorescence quenching, fluorescence polarization as well as other chemiluminescence, electrochemiluminescence, Raman, radioactivity, colorimetric methods, hybridization protection assay and mass spectrometry.

Further amplification methods include, but are not limited to self sustained replication (SSR), nucleic acid sequence based amplification (NASBA), ligase chain reaction (LCR), strand displacement amplification (SDA) and branched DNA (b-DNA).

D. Nucleotide Primers and Probes

The invention further provides nucleotide primers which can detect the polymoφhisms of the invention. In one embodiment of the invention, primers are prepared that are capable of detecting a delta opioid promoter gene polymoφhism at one or more of the positions herein described. Preferred primers allow detection of a delta opioid receptor polymoφhism associated with an eating or energy homeostasis disorder, such as a polymoφhism in an delta opioid receptor gene corresponding to the polymoφhosims designated as OPRDl-01, OPRDl-03, OPRD1-5 and OPRDl-07 in Table 1. Allele specific primers are typically used together with a constant primer, in an amplification reaction such as a PCR reaction, which provides the discrimination between alleles through selective amplification of one allele at a particular sequence position. The allele specific primer is preferably about 10, 12, 15, 17, 19 or up to about 50 or more nucleotides in length, more preferably about 17-35 nucleotides in length, and more preferably about 17-30 nucleotides in length.

The allele specific primer preferably corresponds exactly with the allele to be detected but allele specific primers may be derivatives wherein about 6-8 of the nucleotides at the 3' terminus correspond with the allele to be detected and wherein up to 10, such as up to 8, 6, 4, 2, or 1 of the remaining nucleotides may be varied without significantly affecting the properties of the primer.

Primers may be manufactured using any convenient method of synthesis. Examples of such methods may be found in standard textbooks, for example: Protocols for Oligonucleotides and Analogues; Synthesis and Properties, Methods in Molecular Biology Series; Volume 20; Ed. Sudhir Agrawal, Humana ISBN: 0-89603-247-7; 1993; 1st Edition. If required, the primer(s) may be labeled to facilitate detection.

The invention also provides allele-specific probes that are capable of detecting a delta opioid receptor polymoφhism associated with an eating or energy homeostasis disorder. Preferred probes allow detection of a delta opioid receptor polymoφhism associated with an eating or energy homeostasis disorder, such as a polymoφhism in a delta opioid receptor gene corresponding to the polymoφhisms designated OPRDl-01, OPRDl- 03, OPRD1-5 and OPRDl-07 in Table 1. Such probes are of any convenient length, such as up to about 50 bases or more, up to 40 bases, and more conveniently up to 30 bases in length, such as for example 8-25 or 8- 15 bases in length. In general such probes will comprise base sequences entirely complementary to the corresponding wild type or variant locus in the gene. However, if required, one or more mismatches may be introduced, provided that the discriminatory power of the oligonucleotide probe is not unduly affected. The probes of the invention may carry one or more labels to facilitate detection.

According to another aspect of the present invention there is provided a diagnostic kit comprising an allele specific oligonucleotide probe or primer of the invention and/or an allele-nonspecific primer of the invention. The diagnostic kits may comprise appropriate packaging and instructions for use in the methods of the invention. Such kits may further comprise appropriate buffer(s), nucleotides, and polymerase(s) such as thermostable polymerases, for example Taq polymerase.

The present invention also includes a computer readable medium comprising at least one novel polynucleotide sequence of the invention stored on the medium, such as a nucleotide sequence spanning a polymoφhism in a delta opioid receptor gene as herein described. The computer readable medium may be used, for example, in homology searching, mapping, haplotyping, genotyping or pharmacogenetic analysis or any other bioinformatic analysis.

The polynucleotide sequences of the invention, or parts thereof, particularly those relating to and identifying the single nucleotide polymoφhisms identified herein represent a valuable information source, for example, to characterize individuals in terms of haplotype and other sub-groupings, such as investigating the susceptibility to treatment with particular drugs. These approaches are most easily facilitated by storing the sequence information in a computer readable medium and then using the information in standard bioinformatics programs or to search sequence databases using state of the art searching tools such as "GCC". Thus, the polynucleotide sequences of the invention are particularly useful as components in databases useful for sequence identity and other search analyses. As used herein, storage of the sequence information in a computer readable medium and use in sequence databases in relation to "polynucleotide or polynucleotide sequence of the invention" covers any detectable chemical or physical characteristic of a polynucleotide of the invention that may be reduced to, converted into or stored in a tangible medium, such as a computer disk, preferably in a computer readable form. For example, chromatographic scan data or peak data, photographic scan or peak data, mass spectrographic data, sequence gel (or other) data may be included.

A computer based method is also provided for performing sequence identification, said method comprising the steps of providing a polynucleotide sequence comprising a polymoφhism of the invention in a computer readable medium; and comparing said polymoφhism containing polynucleotide sequence to at least one other polynucleotide or polypeptide sequence to identify identity (homology), i.e., screen for the presence of a polymoφhism.

E. Methods to Identify Agents that Modulate the Expression a Nucleic Acid

Encoding an Delta Opioid Receptor Polymorphism

Another embodiment of the present invention provides methods for identifying agents that modulate the expression of a nucleic acid encoding a delta opioid receptor variant of the invention. Such assays may utilize any available means of monitoring for changes in the expression level of the nucleic acids of the invention. As used herein, an agent is said to modulate the expression of a nucleic acid of the invention if it is capable of up- or down-regulating expression of the nucleic acid in a cell.

In one assay format, the expression of a nucleic acid encoding a delta opioid receptor variant or polymoφhism of the invention in a cell or tissue sample is monitored directly by hybridization to the nucleic acids of the invention. Cell lines or tissues are exposed to the agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al, (1989) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press). Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared as described above. Hybridization conditions are modified using known methods, such as those described by Sambrook et al. and Ausubel et al. as required for each probe. Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format. For instance, total cellular RNA or RNA enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the sequences of the invention under conditions in which the probe will specifically hybridize. Alternatively, nucleic acid fragments comprising at least one, or part of one of the sequences of the invention can be affixed to a solid support, such as a silicon chip or a porous glass wafer. The chip or wafer can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize to the RNA. By examining for the ability of a given probe to specifically hybridize to an RNA sample from an untreated cell population and from a cell population exposed to the agent, agents which up or down regulate expression are identified.

H. Methods to Identify Agents that Modulate the Levels or at Least One Activity of a Delta Opioid Receptor Polymorphism

Another embodiment of the present invention provides methods for identifying agents that modulate the cellular level or concentration or at least one activity of a variant protein of the invention. Such methods or assays may utilize any means of monitoring or detecting the desired activity.

In one format, the relative amounts of a protein of the invention between a cell population that has been exposed to the agent to be tested compared to an un-exposed control cell population may be assayed. In this format, probes such as specific antibodies are used to monitor the differential expression of the protein in the different cell populations. Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time. Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe.

Antibody probes are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptides, polypeptides or proteins of the invention if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co. (Rockford, IL), may be desirable to provide accessibility to the hapten. The hapten peptides can be extended at either the amino or carboxy terminus with a cysteine residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier. Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation.

While the polyclonal antisera produced in this way may be satisfactory for some applications, for pharmaceutical compositions, use of monoclonal preparations is preferred. Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein (Nature (1975) 256: 495-497) or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known. The immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein. When the appropriate immortalized cell culture secreting the desired antibody is identified, the cells can be cultured either in vitro or by production in ascites fluid.

The desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fab, Fab', of F(ab')₂ fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.

The antibodies or fragments may also be produced, using current technology, by recombinant means. Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras with multiple species origin, such as humanized antibodies.

Agents that are assayed in the above methods can be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of a protein of the invention alone or with its associated substrates, binding partners, etc. An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism. As used herein, an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action. Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up these sites. For example, a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to or a derivative of any functional consensus site.

The agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, nucleic acid molecules such as antisense molecules that specifically recognize a variant delta opioid receptor as well as carbohydrates. Dominant negative proteins, DNAs encoding these proteins, antibodies to these proteins, peptide fragments of these proteins or mimics of these proteins may be introduced into cells to affect function. "Mimic" used herein refers to the modification of a region or several regions of a peptide molecule to provide a structure chemically different from the parent peptide but topographically and functionally similar to the parent peptide (see Grant GA. in: Meyers (ed.) Molecular Biology and Biotechnology (New York, VCH Publishers, 1995), pp. 659-664). A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.

The peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art. In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working example therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example

Identification of delta opioid gene polymorphisms associated with eating and energy homeostasis disorders.

A. Samples

1. Affected sample ascertainment

A multi-center collaborative study was begun with the collection of a large sample of eating disorder affected relative pairs (N=220) ascertained on an anorexic nervosa proband, used a large sample of clinically administered and self-report questionnaires. In addition, a sample of blood was collected from each individual for genetic studies. This multi-center collaborative study has been described (Kaye et al, 2000). The eating disorders affected individuals in this sample are mostly female (94%), and DNA was available from probands fulfilling criteria for DSM-IV anorexia nervosa criteria, which is being utilized to evaluate the association of candidate gene polymoφhisms to DSM-IV AN phenotypes. Several statistical studies of the behavioral, clinical and temperament phenotypes collected by the multi-center collaborative study have been published (Lilenfeld et al, 1998; Halmi et al, 2000; Klump et al, 2000).

2. Control samples

Additional DNA samples were obtained from the Coriell Cell Repositories or other sources. Unrelated Centre Etude Polymoφhism Humaine DNA samples ("CUR") were used for reseqencing, for genotype assay development and for sequence verification of the homozygosity status of individual control DNAs. DNA samples from the Coriell Cell Respositories Human Diversity Panel (Caucasian) samples of N=100 (49 females/ 51 males) ("COR") and a European-American female sample of N=125 ("EAF") were used as samples for genotype and allele frequency comparisons. The EAF sample consisted of

European-American females, who were recruited through advertisements. This sample was screened to exclude obese individuals (>20% ideal weight) and lifetime criteria for Axis I disorders assessed by clinical administration of the Structured Clinical Interview for DSM- III-R (SCTD) and can therefore be considered as a psychiatrically unaffected normal weight sample. B. Molecular Genetic Methods

1. Sequence evaluation and annotation

Evaluation of cDNA and genomic sequence and sequence variation was accomplished using public sequence and variation databases. Alignment and annotation of genomic and cDNA sequences was accomplished using the Sequencher™ sequence evaluation package version 4.0.5 (Gene Codes Coφoration, Ann Arbor, MI). Polymoφhisms (Table 1) at the OPRDl locus (Figure 1) were identified by examination of variation databases including (HGBase, http://hgbase.cgr.ki.se), The SNP Consortium (TSC, http://snp.cshl.org) and resequencing within our laboratory.

2. Resequencing

Although the publicly-available databases contained a number of SNPs at this locus, most were only preliminary; and no confirmation of some SNPs, including frequency information, was available. Therefore, a 4.18kb portion of the genomic sequence surrounding exon 2 was chosen for resequencing in 32 unrelated CEPH individuals. Primer pairs and optimal annealing temperatures are listed in Table 2. PCR reactions were as follows per reaction: 50ng genomic DNA, 25nM each of the forward and reverse primers, lOmM dNTP, 50mM MgCl₂, IX PCR Buffer, and 0.6U Taq (Bioline, loc). All primer pairs except PF-0081 + PF-0082 and PF-0083 + PF-0084 used the supplied buffer from Bioline; the aforementioned assays used the following buffers respectively: 600mM Tris-SO₄,

180mM (NH₄)₂SO₄, pH = 8.98 (substituting 50mM MgSO₄ for MgCl₂), and 200mM Tris- HC1 500mM KC1, pH = 8.3. General conditions for thermocycling were: 94 °C for lmin, followed by 30 cycles of 94 °C for 15s, T_a for 30s, 72 °C for lmin, after which an additional extension of 72 °C for 5min and 4 °C hold, where T_ais the optimal annealing temperature for the particular primer pair. T_a values are listed in Table 2 for each primer pair. Post- PCR, 50μl of each product was purified. Products were then requantitated (A260) and 0.25μg product was mixed with 25pM primer, 4μl Big Dye Terminators (PE Biosystems, loc) with the volume brought to 20μl with water. Reactions were then subjected to the following sequencing reaction thermocyling protocol: 96 °C for 5 minutes, followed by 35 cycles of 96 °C for 10 sec, 50 °C for 10 sec, and 60 °C for 3.5 min, with a 4 °C hold. PCR primers were also used as sequencing primers for the appropriate amplicon. 3. Genotvping

Five SNPs (see Table 1) distributed throughout the >50kb gene and flanking sequence were chosen for genotyping using the 5'exonuclease assay (Morin et al, 1999) (TaqMan™). Probes and primers were chosen using a Biognosis-customized version of ProbelTY (Celadon Laboratories). OPRDl-01 : Primers: Forward 5 '- TGGCTCACACCTGTAA-3', Reverse 5'-ACAAAGCGAGATCCCA-3'; Probes: FAM- cacctggggtcaAgagtttgag-TAMRA TET-acctggggtcaGgagtttga-T AMRA; OPRD 1 -03 : Primers: Forward 5'-TGCTCACCTCCTGTG-3', Reverse 5'CCAGTCTCCCTCCTAAG- 3 ' ; Probes: (note that both probes were synthesized using propyne T) FAM- tgcggattcaAtgggttat-TAMRA, TET-tgcggattcaGtgggtt-TAMRA; OPRD 1-05: Primers: Forward 5'-AGATTTGGTCACCAGATAG-3', Reverse 5'-TTGCCCCTTGCTAGAA-3'; Probes: (note that both probes were synthesized using propyne T) FAM-tgtggcctcaActttgg- TAMRA, TET-tgtggcctcaTctttgg-TAMRA; OPRDl-07: Primers: Forward 5'- TTCCAGACCAGCCTG-3', Reverse 5'-GACTACAGACGCCCA-3'; Probes: FAM- cctatctttactaaaaAtacaaaaatta-MGB , VIC-ccctatctttactaaaaGtacaaaaatta-MGB . General conditions per reaction for PCR and endpoint-read TaqMan™ were as follows (with exceptions noted per assay): 15μl reactions containing 50ng genomic DNA, lOpM forward primer, lOpM reverse primer, 1.5pM TET or VIC probe, 1.5pM FAM probe, 2X Universal MasterMix (PE, loc) in a 96-well plate format with 6 allele 1 homozygous controls, 6 allele 2 homozygous controls, 6 heterozygous controls, and 6 negative (water) controls. Exceptions to this PCR reaction cocktail should be noted for OPRDl-07, since optimization of this assay determined that 3.0mM each of the 2 probes were used. Thermal cycling conditions are as follows: 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 30 seconds and determined annealing temperature for 1 minute. In the case of these experiments, the optimal annealing temperatures as well as CUR samples to be used as assay controls were determined during assay optimization and are as follows: OPRDl- 01, 60°C annealing temperature, allele 1 control 1346-13, allele 2 control 1400-10, heterozygote control 1362-03; OPRDl-03, 55°C annealing temperature, allele 1 control 1347-13, allele 2 control 1347-15, heterozygote control 1333-14; OPRDl-05, 55°C annealing, allele 1 control 1423-12, allele 2 control 1344-12, heterozygote control 1420-11; OPRD1-07, 62°C annealing, allele 1 control 1332-02, allele 2 control -none found- , heterozygote control 1346-11. Allelic discrimination was conducted manually by a technician in the laboratory.

C. Informatics infrastructure

A Laboratory Information Management System (LIMS) implemented using an Oracle 8i relational database with Powerbuilder, Visual Basic, Java and SQL facilitated integration and storage of sequence, samples, polymoφhisms and genotype information and analytical results (Ganjei and Bergen, 2001) (http://www.scimag.com/scripts/ShowPR.asp? PUBCODE-030&ACCT=3000039989&ISSUE=0105&RELTYPE-FE&PRODCODE=000 01986&PRODLETT=A&DISPTYPE=). The LIMS and related software was used for tracking samples, primer/probe selection, ABI 7700 data import, Hardy Weinberg evaluation of genotypes, contingency table analysis of genotype and allele frequencies, maximum likelihood haplotype frequency estimation and export of alleles, genotypes and haplotypes data.

1. Statistical Analysis

Quality of control procedures in the laboratory included repeat genotypes in every plate, as well as Hardy- Weinberg disequilibrium tests. The significance (p≤0.05) of an association between poiymorhisms within or close to the OPRDl gene and DSM-IV anorexia nervosa phenotype was analyzed using chi- square (χ²) analysis. In case of low expected cell frequencies, the p-value was based on the Fisher exact test. The magnitude of the association between affected status (i.e., AN versus control group) was evaluated by Spearman rank order correlation test. Haplotype frequency estimation was based on an EM algorithm, programmed by R. Peterson. All analyses, with the exception of the haplotype frequency count estimation were performed using SAS (SAS institute Inc., SAS Procedures Guide 1999 #28).

Linkage disequilibrium was calculated using the program Arlequin using a log likelihood approach where the likelihood ratio statistic (S = -21og (likelihood of the data assuming linkage equilibrium using the product of the allele frequencies/likelihood of the data not assuming linkage equilibrium using Maximum Likelihood estimated allele Linkage disequilibrium was calculated using the program Arlequin using a log likelihood approach where the likelihood ratio statistic (S = -21og (likelihood of the data assuming linkage equilibrium using the product of the allele frequencies/likelihood of the data not assuming linkage equilibrium using Maximum Likelihood estimated allele frequencies) is assumed to follow a chi-square distribution. The significance of the likelihood ratio statistic is estimated by generation of the null distribution of the likelihood ratio statistic, by permutation of alleles between individuals at one locus and re-estimation of the likelihood of the data not assuming linkage equilibrium and therefore of the likelihood ratio statistic.

Transmission disequilibrium analysis was performed using DSM-IV eating disorder phenotypes and the program is available from http://speilman07.med.upenn.edu/WinEd.htm and described in Spielman and Ewens, 1996.

D. RESULTS

1. Sequencing In order to obtain additional SNPs for genotyping in our sample, a portion of the genomic sequence (Table 2) was chosen for resequencing in 32 unrelated CEPH individuals, resulting in the discovery of an unpublished SNP in IVS2 (OPRDl-07, IVS2+898A>G; see Table 1). No other variation was noted.

o relative to the A o ATG intation codon. PCR primers were aso use as sequencing primers in each case.

2. Genotyping

For each assay, 653 AN probands, affected siblings, and other family members were genotyped at OPRDl-01, OPRDl-03, OPRD1-05, OPRDl-07, as well as an additional 244 samples from the CUR, COR, and EAF groups. Additionally, a verification plate consisting of a sample of 72 AN probands and control group samples was genotyped for to assess the repeatability of the genotyping assay. All plates were analyzed using an Applied Biosystems Prism 7700 Sequence Detector (ABI, loc). The average sample dropout rate for all assays was 46.75 individuals (4.8%). A sample of AN probands (N=l 19) and the same sample of CUR, COR and EAF individuals was genotyped for OPRD1 06. Four of the five polymoφhisms typed (In Table of Genotypic and allelic information for the OPRDl polymoφhisms) are informative for association analysis in that the genotype and allele counts in the groups. The fifth polymoφhism, OPRD 1-06/ 80T>C, resulting in F27C, was found at a frequency very similar to the reported frequency in European- Americans (Gelernter and Kranzler, 2000) in a slightly smaller sample of AN probands and in the EAF sample (minor allele frequency of 0.12), and is not included in the following presentation of results.

Table 3. Genotypic and allelic information for the OPRDl polymoφhisms: Number and percentages.

Note: includes the AN 3 cases incorporated in the AN 2 subgroup.

Genotypes Alleles

Polymoφhism Sample N N_n P_π N₁ P12 N₂2 P22 Nj Pi N₂ P

OPRDl 01

AN_1 98 32 .33 43 .44 23 .24 107 .55 89 .45

AN_2 83 29 .35 39 .47 15 .18 97 .58 69 .42

All AN 181 61 .34 82 .45 38 .21 204 .56 158 .44

COR 98 26 .27 48 .49 24 .24 100 .51 96 .49

EAF 80 18 .23 39 .49 23 .29 75 .47 85 .53

OPRDl 03

AN_1 98 11 .11 42 .43 45 .46 64 .33 132 .67

AN_2 83 10 .12 29 .35 44 .53 49 .30 117 .71

A11 AN 181 21 .12 71 .39 89 .49 113 .31 249 .69

COR 98 9 .09 42 .43 47 .48 60 .31 136 .69

EAF 89 15 .17 41 .46 33 .37 71 .40 107 .60

OPRDl 05

AN_1 97 32 .33 48 .50 17 .18 112 .58 82 .42

AN_2 84 30 .36 41 .49 13 .16 101 .60 67 .40 AN 181 62 .34 89 .49 30 .17 213 .59 149 .41

COR 95 39 .41 43 .45 13 .14 121 .64 69 .36

EAF 87 41 .47 35 .40 11 .13 117 .67 57 .33

OPRDl 07

AN_1 95 30 .32 47 .50 18 .19 107 .56 83 .44

AN_2 81 28 .35 41 .51 12 .15 97 .60 65 .40

All AN 176 58 .33 88 .50 30 .17 204 .58 148 .42

COR 94 38 .40 43 .46 13 .14 119 .63 69 .37

EAF 82 41 .50 32 .39 9 .11 114 .70 50 .31

A _1 restricting anorectics; AN_2 purging anorectics; All AN all aaorectic subjects combined. COR-- Coriell Cell Resposhorics Human Diversity Panel (Caucasian) samples of~N=100 (49 females/ 51 males). EAF - European-American female sample ofN-125 (subset of samples that genotyped). N total sample size; _{1 1} number of observations for genotype 11; _j ι percentage of observations for genotype 11; idem for genotypes 12 and 22. N_t number of alleles 1; ^pl percentage of alleles 1; idem for allele 2. 3. Association analysis of OPRDl genotypes and alleles to DSM-IV AN phenotype

There is evidence for statistically significant association of OPRDl alleles to DSM- IV anorexia nervosa phenotype at three (OPRDl-01, OPRDl-03, OPRDl-07) of the five OPRDl polymoφhisms genotyped in this study. Specifically, statistically significant association was observed between allele frequencies of these polymoφhisms and DSM-IV phenotype in the AN_2 subgroup and the All AN group when compared to the EAF sample. A significant association of OPRDl-07 allele frequency in the AN_1 sample compared to the EAF sample was observed, while a trend towards significant association of OPRD 1-05 allele frequency in the AN_1 sample compared to the EAF sample was observed.

Significant rank correlations were observed between AN_2 and All AN phenotype and genotype and allele frequency at OPRDl-01 and OPRDl-03 and between AN_1 phenotype and genotype and allele frequency at OPRD-07, while a trend for significant rank correlation was observed between AN_1 phenotype and genotype and allele frequencies at OPRD 1-05 and between AN 2 phenotype and genotype and allele frequencies at OPRDl- 07. There were no trends or significant associations or correlations between DSM-IV anorexia nervosa samples and the COR sample. No corrections for multiple comparisons were performed at this level of analysis.

Table 4. Results of association analyses of genotypes and alleles¹. Note: includes the AN_3 cases incoφorated in the AN_2 subgroup.

Sample 1 Sample 2 N X P OR (95% CI) P P

OPRDl-01

Genotypic analyses

AN_1 EAF 178 2.32 .31 .11 .16

AN_2 EAF 163 4.21 .12 .16 .04

All AN EAF 261 3.87 .14 .12 .048

AN_1 COR 196 .92 .63 .05 .48

AN_2 COR 181 1.94 .38 .10 .16

All AN COR 279 1.58 .45 .07 .23

Allelic ; analyses

AN_1 EAF 356 2.10 .15 1.36 .90-2.07 .08 .15

AN_2 EAF 326 4.37 .04 1.59 1.03-2.47 .12 .04

All AN EAF 522 4.01 .0453 1.46 1.01-2.13 .09 .046

AN_1 COR 392 .50 .48 1.15 .78-1.72 .04 .48

AN_2 COR 362 1.99 .16 1.35 .89-2.05 .07 .16

All AN COR 558 1.46 .23 1.24 .88-1.76 .05 .23

OPRDl-03

Genotypic analyses

AN_1 EAF 187 2.05 .36 -.10 .16

AN_2 EAF 172 4.43 .11 -.15 .04

All AN EAF 270 3.84 .15 -.12 .0496

AN_1 COR 196 .24 .89 .03 .70

AN_2 COR 181 1.30 .52 -.03 .69

All AN COR 279 .57 .75 .00 .98

Allelic analyses

AN_1 EAF 374 2.12 .15 .73 .48-1.12 -.08 .15

AN_2 EAF 344 4.07 .04 .63 .40- -.99 -.11 .04

All AN EAF 540 4.00 .046 .68 .47- -.99 -.09 .049

AN_1 COR 392 .19 .66 1.10 .72-1.68 .02 .66

AN_2 COR 362 .05 .82 .95 .61-1.49 -.01 .82

All AN COR 558 .02 .88 1.03 .71-1.50 .01 .88

OPRD 1-05

Genotypic analyses

AN_1 EAF 184 3.90 .14 -.14 .054

AN_2 EAF 171 2.29 .32 -.11 .15

All AN EAF 268 4.14 .13 -.12 .054

AN_1 COR 192 1.48 .48 -.09 .23

AN_2 COR 179 .55 .76 -.05 .47

All AN COR 276 1.32 .52 -.07 .26

Allelic analyses

AN_1 EAF 368 3.53 .06 .67 .44-1.02 -.10 .058

AN_2 EAF 342 1.88 .17 .73 .47-1.14 -.07 .17

All AN EAF 536 3.51 .06 .70 .48-1.02 -.08 .06

AN_1 COR 384 1.43 .23 .78 .52-1.17 -.06 .23

AN_2 COR 358 .48 .49 .86 .56-1.32 -.04 .49

All AN COR 552 1.22 .27 .82 .57-1.17 -.05 .27

OPRDl-07

Genotypi ic analyses

AN_1 EAF 177 6.63 .07 -.19 .01

AN_2 EAF 163 3.98 .14 -.15 .055

All AN EAF 258 7.05 .03 -.16 .01

AN_1 COR 189 1.92 .38 -.10 .16

AN_2 COR 175 .64 .73 -.05 .48

All AN COR 270 1.59 .45 -.08 .22

Allelic analyses

AN_1 EAF 354 6.54 .01 .57 .37- ^■.88 -.14 .01

AN_2 EAF 326 3.31 .07 .66 .41-1.03 -.10 .07

All AN EAF 516 6.32 .01 .61 .41- ^■.90 -.11 .01

AN_1 COR 378 1.92 .17 .75 .50-1.13 -.07 .17

AN_2 COR 350 .43 .51 .87 .56-1.33 -.04 .51

All AN COR 540 1.46 .23 .80 .56-1.15 -.05 .22

¹ AN_1 restricting anorectics, AN_2 purging anorectics, All AN all anorectic subjects combined

COR - Coriell Cell Respositoπes Human Diversity Panel (Caucasian) samples of N=100 ( 9 females/ ₅1 males)

EAF - European-American female sample of N=12₅ (subset of samples that genotyped)

* P-values based on Fisher's exact test

4. Pairwise linkage disequilibrium

Pairwise linkage disequilibrium was observed in the AN_1, AN_2, All AN and EAF samples but the pattern of linkage disequilibrium varied between the samples. Because six pairwise estimates of linkage disequilibrium for each sample was performed, significant linkage equilibrium is assumed to be present with a permutation P value of 0.00833. Specifically, the EAF sample exhibited significant or nominally significant linkage disequilibrium between all pairs of OPRDl loci but highly significant LD between the OPRDl-01 and OPRDl-03 and the OPRD 1-05 and OPRDl-07 loci. The AN_1 sample exhibited significant linkage disequilibrium between the OPRDl-01 and OPRDl-03 and the OPRD 1-05 and OPRDl-07 loci and nominally significant linkage disequilibrium between the OPRDl-03 and both the OPRD 1-05 and OPRDl-07 loci. The AN_2 sample exhibited significant linkage disequilibrium between the OPRDl-01 and OPRDl-03 and the OPRDl- 05 and OPRDl-07 loci only. The All AN sample exhibited significant linkage disequilibrium between the OPRDl-01 and OPRDl-03 and the OPRD 1-05 and OPRDl-07 loci and nominally significant linkage disequilibrium between the OPRDl-03 and OPRDl- 05 loci.

Table 5

Table of P values for likelihood ratio test of two locus linkage disequilibrium

OPRDIJ OPRDl_3 OPRDl_5 OPRDl_7 A11AN

OPRDl-01 * O.00000 0.43206 0.55034

OPRDl-03 <0.00000 * 0.00218 0.01857

OPRD 1-05 0.0088 0.00293 * O.00000

OPRDl-07 0.00978 0.00293 O.00000 *

EAF

OPRDIJ OPRDl_3 OPRDl_5 OPRDl_7 AN1

OPRDl-01 * O.00000 0.01955 0.01466

OPRDl-03 O.00000 * 0.00397 0.00684

OPRD 1-05 0.21212 0.17817 * O.00000

OPRDl-07 0.13685 0.58065 O.00000 *

AN2

5. Transmission Disequilibrium Test (TDT) Analyses TDT analysis was performed on the AN family sample consisting of X probands, Y affected relatives and Z parents where X individuals fulfill criteria for Anorexia Nervosa (AN) and Y relatives fulfill criteria for AN, Bulimia Nervosa (BN) or Eating Disorders Not Otherwise Specified (ED-NOS). There are ABC trios in this sample, i.e., A trios where the affected child fulfills criteria for AN, B trios where the affected child fulfills criteria for BN and C trios where the affected child fulfills criteria for ED-NOS. TDT analysis of the four OPRDl genotyped loci obtained evidence for a trend of transmission disequilibrium at the OPRD 1-05 and OPRD-07 loci only. There was no evidence for transmission distortion at OPRDl-01 or OPRDl-03.

Table 6

Table of TDT Results for OPRDl polymoφhisms

Allele b C Chi-Sq P Value W Mean(A) Var(V) z'

OPRDl-01

1 55 54 0.009 0.924 55 54.5 27.25 0

2 54 55 0.009 0.924 54 54.5 27.25 0

OPRDl-03

2 54 45 0.818 0.366 54 49.5 24.75 0.804

1 45 54 0.818 0.366 45 49.5 24.75 0.804

OPRD 1-05

1 59 81 3.457 0.063 59 70 35 1.775

2 81 59 3.457 0.063 81 70 35 1.775

OPRDl-07

1 61 82 3.084 0.079 61 71.5 35.75 1.672

2 82 61 3.084 0.079 82 71.5 35.75 1.672

E. DISCUSSION

The delta opioid receptor is a candidate gene for eating disorders dues to its role in the regulation of feeding in humans and animal models and its regulation of reward circuits in the human brain. Several SNPs have been identified at the locus of the gene on chromosome lp36.1-34.3 in this and other sequencing or scanning studies (www.cshl.org; Gelernter and Kranzler, 2000; Mayer et al, 1997). Genotyping of five of these SNPs in individuals fulfilling criteria for DSM-IV anorexia nervosa and in individuals without apparent mental disorder has identified statistically significant associations between frequencies of alleles and haplotypes and DSM phenotype. Specifically, statistically significant associations between allele frequency and

DSM-IV Anorexia Nervosa phenotype were identified at OPRDl-01, OPRDl-03 and OPRDl-07. A trend (P Value < 0.10) was observed in the transmission disequilibrium test using DSM-IV eating disorder (Anorexia Nervosa, Bulimia Nervosa and Eating Disorders Not Otherwise Specified) phenotype at OPRD 1-05 and OPRDl-07. Significant linkage disequilibrium was identified both in the AN sample and in the EAF sample, however, the pattern of linkage disequilibrium differs significantly between the AN_1 (restricting) and AN_2 (purging) subtype of anorexia nervosa, where the AN_1 subtype exhibits significant linkage disequilibrium only between OPRDl-01 and OPRDl-03 and between OPRD 1-05 and OPRDl-07, while AN 2 exhibits significant linkage disequilibrium between OPRDl- 01 and OPRDl-03, between OPRDl-03 and both OPRD 1-05 and OPRDl-07 and between OPRD1-05 and OPRDl-07.

Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All cited patents, patent applications and publications referred to in this application are herein incoφorated by reference in their entirety.

Claims

WHAT IS CLAIMED:

1. An isolated nucleic acid molecule encoding an delta opioid receptor variant associated with an eating or energy homeostasis disorder.

2. An isolated nucleic acid molecule of claim 1, wherein the eating or energy homeostasis disorder is anorexia nervosa.

3. An isolated nucleic acid molecule of claim 2, wherein the variant is OPRD1- 01, OPRDl-03, OPRDl-07, OPRDl-01-05, OPRDl-01-07, OPRDl-03-07, OPRDl-05-07, or OPRD 1-01-03-05-07.

4. A delta opioid receptor variant encoded by the nucleic acid molecule of any one of claims 1-3.

5. An isolated antibody the specifically recognizes a delta opioid receptor variant of claim 4.

6. A vector comprising an isolated nucleic acid molecule of any one of claims 1-3.

7. A host cell transformed to contain the nucleic acid molecule of any one of claims 1-3.

8. A host cell comprising a vector of claim 6.

9. A host cell of claim 8, wherein said host is selected from the group consisting of prokaryotic hosts and eukaryotic hosts.

10. A method for producing a polypeptide comprising the step of culturing a host cell transformed with the nucleic acid molecule of any one of claims 1-3 under conditions in which the protein encoded by said nucleic acid molecule is expressed.

11. The method of claim 10, wherein said host cell is selected from the group consisting of prokaryotic hosts and eukaryotic hosts.

12. A method ofidentifying an agent which modulates the expression of a nucleic acid encoding a delta opioid receptor of claim 4, comprising the steps of: exposing cells which express the nucleic acid to the agent; and determining whether the agent modulates expression of said nucleic acid, thereby identifying an agent which modulates the expression of a nucleic acid encoding a delta opioid receptor.

13. A method ofidentifying an agent which modulates at least one activity of a delta opioid receptor of claim 4, comprising the steps of: exposing cells which express the protein to the agent; and determining whether the agent modulates at least one activity of said receptor, thereby identifying an agent which modulates at least one activity of a delta opioid receptor.

14. The method of claim 13, wherein the agent modulates one activity of the protein.

15. A method of diagnosing the genetic predisposition to an eating or energy homeostasis disorder, comprising detecting the presence or absence of a nucleic acid molecule of any one of claims 1-3 in a patient sample.

16. A method of claim 15, wherein the presence or absence of a single nucleotide polymoφhism at a nucleotide position corresponding to the ORDl-01, ORDl-03 or ORDl-07 polymoφhisms.

17. An allele specific primer that detects a polymoφhism in the gene encoding a delta opioid receptor associated with an eating or energy homeostasis disorder.

18. An allele specific primer of claim 17, wherein the eating disorder is anorexia nervosa.

19. A kit comprising a primer of any one of claims 17 or 18.

20. A non-human transgenic animal modified to contain a nucleic acid molecule of any of claims 1-3.

21. The transgenic animal of claim 27, wherein the nucleic acid molecule contains a mutation that prevents expression of the encoded protein.