The invention provides methods/kits for assessing levels of trait or state anxiety in a subject by comparing genotypes and/or expression patterns at the ACHE, PON1 and/or BChE genes to the genotype and/or expression pattern of the genes in a reference population whose genotype and/or expression pattern of the genes is known or by correlating AChE levels activity to those of PON.

Description of the Invention

SUMMARY OF THE INVENTION

The invention is based in part on the discovery that genomic polymorphisms in the acetylcholinesterase (ACHE) and the paraoxonase (PON1) genes (also known as the ACHE-PON1 locus) and corresponding changes in serum AChE and PON activities can serve as objective predictors of anxiety in human subjects.

Accordingly, in one aspect, the invention features a method of assessing trait anxiety in a subject. The method includes providing from the subject a test sample, which can be any biological fluid, cell sample, or tissue, as long as it contains genomic DNA from the subject, and/or RNA transcribed from the ACHE, PON1, or BCHE genes. The test sample is assayed to determine whether it contains a polymorphism in an acetylcholinesterase (ACHE) gene, a paraoxonase (PON1) gene, and/or a butyrylcholinesterase (BCHE), which is associated with a known level of trait anxiety, e.g., whether it is associated with a specific trait anxiety score. The presence of the polymorphism in the sample indicates the subject has the corresponding level of anxiety.

In some embodiments, the subject is a human. In other embodiments, the subject is a non-human primate, dog, cat, cow, horse, pig, goat, sheep, or rodent (including a rat or mouse), or other mammal.

In some embodiments, the polymorphism is compared to the polymorphism in a reference sample derived from one or more individuals whose trait anxiety level is known, to assess trait anxiety in the subject.

In some embodiments, the subject is a human (including a healthy human or a diseased human). In some embodiments, polymorphisms are identified in humans that are members of certain racial or ethnic groups, e.g., Caucasians, Blacks, Asians, or Hispanics.

Suitable ACHE gene polymorphisms include, e.g., the 1682 C/T (P446) ACHE polymorphism in a human ACHE gene.

Suitable PON1 gene polymorphisms include, e.g., a polymorphism in a PON1 gene promoter polymorphism. PON1 promoter polymorphisms can include, e.g., -162A/G PON1, -126 G/C PON1, or -108 C/T PON1 substitutions in a human PON1 gene.

Alternatively, the PON1 polymorphism can be in a PON1 coding sequence. PON1 coding sequence polymorphisms can include, e.g., a polymorphism that results in a L55M or Q192R substitution in a human PON1 coding sequence.

In some embodiments, the BCHE polymorphism results in a D70G substitution in a human BCHE coding sequence.

In some embodiments, the method includes identifying and comparing at least two polymorphic pairs in the ACHE, PON1, and/or BCHE genes. Suitable polymorphic pairs include, e.g., ACHE P446 and PON1108; ACHE P446 and PON1192; PON1108 and PON1126; or PON1126 and PON1162.

In another aspect, the invention provides a method of assessing trait anxiety in a subject. The method includes providing a test sample from the subject and identifying acetylcholinesterase (AChE) activity in the test sample. AChE activity in the test sample is compared to AChE activity in a reference sample derived from one or more individuals whose trait anxiety level is known. The individual or individuals in the reference sample are similar to the subject in at least one or more of the traits gender, age, race, ethnic group, and body mass index. In some embodiments, the subject and individual or individuals are similar in two, three, four or five of these traits. In some embodiments, AChE activity in the test sample is compared to an average AChE activity in the individual or individuals in the reference sample. A higher level of AChE activity in the test sample compared to AChE activity in the reference sample indicates the subject has less trait anxiety than the trait anxiety level of the one or more individuals from which the reference sample was derived.

Any biological fluid, cell sample, or tissue can be used in the test sample, as long as it contains, or is suspected of containing active AChE protein. A preferred test sample includes serum.

In some embodiments, the subject is a human (including a healthy human or a diseased human). In some embodiments, polymorphisms are identified in humans that are members of certain racial or ethnic groups, e.g., Caucasians, Blacks, Asians or Hispanics.

Also within the invention is a method of assessing trait anxiety in a subject. The method includes providing a test sample from the subject and identifying acetylcholinesterase (AChE) monomeric forms in the test sample. The amount of AChE monomeric forms in the test sample is compared to the amount of AChE monomeric forms in a reference sample derived from one or more individuals whose trait anxiety level is known in order to assess trait anxiety in the subject. A higher amount of AChE monomeric forms in the test sample compared to AChE monomeric forms in the reference sample indicates the subject has less trait anxiety than the trait anxiety level of the one or more individuals from which the reference sample was derived.

In some embodiments, the AChE monomeric forms are detected using non-denaturing gel electrophoresis.

Any biological fluid, cell sample, or tissue can be used in the test sample, as long as it contains, or is suspected of containing AChE monomeric forms. A preferred test sample is serum.

In some embodiments, the subject is a human (including a healthy human or a diseased human). In some embodiments, polymorphisms are identified in humans that are- members-of-certain racial or ethnic groups, e.g., Caucasians, Blacks, Asians, or Hispanics.

The individual or individuals in the reference sample are similar to the subject in at least one or more of the traits gender, age, race, ethnic group, and body mass index. In some embodiments, the subject and individual or individuals are similar in two, three, four or five of these traits.

Also featured by the invention is a method of determining susceptibility to state anxiety in a subject. The method includes providing a test sample from the subject and identifying PON activity in the subject. PON activity in the test sample is compared to the amount of PON activity in a reference sample derived from one or more individuals whose state anxiety level is known.

In some embodiments, lower PON activity in the test sample relative to the reference sample indicates the subject is at increased susceptibility for developing state anxiety than the one or more individuals in the reference sample.

The individual or individuals in the reference sample are similar to the subject in at least one trait selected from the traits gender, age, race, ethnic group, and body mass index. In some embodiments, the subject and individual or individuals are similar in two, three, four or five of these traits.

In some embodiments, the method includes identifying AChE activity in the subject and comparing the AChE activity to AChE activity in the one or more individuals.

In some embodiments, the subject is a human (including a healthy human or a diseased human). In some embodiments, polymorphisms are identified in humans that are members of certain racial or ethnic groups, e.g., Caucasians, Blacks, or Hispanics. Any biological fluid, cell sample, or tissue can be used in the test sample, as long as it contains, or is suspected of containing AChE monomeric forms. A preferred test sample is serum.

In a further aspect, the invention provides a method of determining susceptibility to state anxiety in a subject by providing a test sample from the subject and identifying AChE activity in the test sample. AChE activity in the test sample is compared to the amount of AChE activity in a reference sample derived from one or more individuals whose state anxiety level is known. The individual or individuals in the reference sample are similar to the subject in one or more of the traits gender, age, race, ethnic group, or body mass index. In some embodiments, the subject and individual or individuals are similar in two, three, four or five of these traits.

In some embodiments, a higher level of AChE in the test sample compared to the reference sample indicates the subject has increased susceptibility to state anxiety compared to the one or more individuals in the reference sample.

In a further aspect, the invention provides a method of assessing state anxiety in a subject. The method includes providing a plurality of test samples, with the test samples taken from the subject at different times. PON activity is identified in the plurality of test samples and the PON activity of two or more test samples from the plurality of test samples is compared.

In some embodiments, comparing PON activity includes comparing PON activity of one or more test samples taken at one or more timepoints before administering an anxiety treatment and one or more test samples taken at one or more timepoints after administering an anxiety treatment. In other embodiments, comparing PON activity includes comparing PON activity of one or more test samples taken at one or more timepoints during an anxiety attack and one or more test samples taken at one or more timepoints after an anxiety attack.

In another aspect, the invention provides a method of assessing state anxiety in a subject including providing a plurality of test samples from the subject taken at different times from the subject. AChE activity in the plurality of test samples is identified and AChE activity of two or more test samples from the plurality of test samples is compared.

In some embodiments, comparing AChE activity includes comparing AChE activity of one or more test samples taken at one or more timepoints before administering an anxiety treatment and one or more test samples taken at one or more timepoints after administering anxiety treatment. In other embodiments, comparing AChE activity includes comparing AChE activity of one or more test samples taken at one or more timepoints during an anxiety attack and one or more test samples taken at one or more timepoints after an anxiety attack.

The anxiety can be either a primary, non-situational anxiety, or a secondary, situational anxiety. In some embodiments, the situational anxiety can be depression-related anxiety or hypertension related anxiety.

In another aspect, the invention provides a method for monitoring treatment outcome of an anti-anxiety therapy in a subject, comprising comparing cholinergic enzyme activity of one or more test samples taken at one or more timepoints before administering an anxiety treatment and one or more test samples taken at one or more time points after administering anxiety treatment.

In another embodiment, monitoring changes in cholinergic enzymes in depression-related anxiety is used for monitoring treatment outcome of an anti-depression therapy in a subject, comprising comparing cholinergic enzyme activity of one or more test samples taken at one or more timepoints before administering an anti-depression treatment and one or more test samples taken at one or more timepoints after administering anti-depression treatment.

Also provided are kits for monitoring treatment outcome of an anti-anxiety therapy in a subject, comprising reagents for determining in a biological sample of the subject activity levels of at least one of cholinesterase (ChE) and/or paraoxonase (PON1). In some embodiments, the kits further comprise means for comparing between the activity levels at different time points.

Also provided are kits for monitoring treatment outcome of an anti-depression therapy in a subject, comprising reagents for determining in a biological sample of the subject activity levels of at least one of cholinesterase (ChE) and/or paraoxonase (PON1). In some embodiments, the kits further comprise means for comparing between the activity levels at different time points.

Further, the invention provides for kits for assessing trait or state anxiety in a subject, the kits comprising reagents for determining in a biological sample of the subject activity levels of at least one of cholinesterase (ChE) and/or paraoxonase (PON1). In some embodiments, the kits further comprise means for comparing between the activity levels of the at least one of cholinesterase and/or said paraoxonase and a reference standard of known levels of at least one of cholinesterase (ChE) and/or paraoxonase (PON1) activity.

The present invention successfully addresses the shortcomings of the presently known configurations by providing a biomolecular or biochemical diagnostic tool which enables accurate and rapid diagnosis of subjects afflicted with anxiety or being predisposed thereto.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a method which can be utilized to quickly and accurately asses an anxiety state or predisposition in a subject. Specifically, the present invention can be used to diagnose anxiety in a subject using highly accurate commonly used biochemical and/or molecular techniques.

The invention provides methods for assessing levels of trait or state anxiety in a subject by comparing genotypes and/or expression patterns at the ACHE, PON1 and/or BChE genes to the genotype and/or expression pattern of the genes in a reference population whose genotype and/or expression pattern of the genes is known.

The term "state anxiety" refers to anxiety that is experienced by an individual at a certain time. The term "trait anxiety" refers to a general susceptibility to anxiety in an individual. Anxiety disorders include, but are not limited to, panic attack, agoraphobia, panic disorder without agoraphobia, panic disorder with agoraphobia, agoraphobia without history of panic disorder, specific phobia, social phobia, obsessive-compulsive disorder, posttraumatic stress disorder, acute stress disorder, generalized anxiety disorder, anxiety disorder due to a general medical condition, substance induced anxiety disorder, separation anxiety disorder, sexual aversion disorder and anxiety disorder not otherwise specified. See Diagnostic and Statistical Manual for Mental Disorders, 4.sup.th Ed (1994) pp. 393-444.

The physiological manifestations that accompany anxiety may include intense fear, racing heart, turning red or blushing, excessive sweating, dry throat and mouth, trembling, swallowing with difficulty, and muscle twitches. The psychological manifestations may include feelings of impending danger, powerlessness, apprehension and tension. Commonly used indices of anxiety include the STAI, ASQ, Cornell, and compound indices (for assessment of anxiety and related conditions) such as the DASS (Depression, Anxiety Stress Scale).

It will be appreciated that anxiety is an important component of many normal behaviors, such as fright, physical pain or trauma, etc. As an adaptive behavior, an appropriate level of anxiety in such instances can serve a constructive purpose. However, abnormal or misdirected anxiety, inappropriate relative to the severity of the circumstance, is counterproductive, and may become a disorder in and of itself. A chronically recurring case of anxiety that has a serious affect on your life may be clinically diagnosed as an anxiety disorder. The most common anxiety disorders are Generalized anxiety disorder, Panic disorder, Social anxiety disorder, phobias, Obsessive-compulsive disorder, and posttraumatic stress disorder (PTSD). Thus, one may distinguish between situational anxiety, secondary to pain, another underlying medical condition, a hormone-secreting tumor, or a side effect of medication or other primary factors, and the pathological, primary non-situational anxiety unrelated to disease, disorder, etc.

The term "non-situational anxiety" refers to anxiety that does not stem from, or does not occur simultaneously with (associated) another physiological or psychological disorder or disease.

The term "situational anxiety" refers to anxiety that does stem from, or does not occur simultaneously with (associated) another physiological or psychological disorder or disease.

A non-limiting list of conditions and factors which can provoke such secondary anxiety includes poorly controlled pain; abnormal metabolic states such as hypoxia, pulmonary embolus, sepsis, delirium, hypoglycemia, bleeding coronary occlusion, heart failure and electrolyte imbalance; endocrine disorders such as hyperthyroidism and pheochromocytoma; and use of anxiety producing drugs, such as corticosteroids, thyroxine, bronchodilators, beta-adrenergic stimulants and antihistamines.

While reducing the present invention to practice, it was uncovered that genotypes and/or expression patterns of genes encoding cholinergic enzymes, such as AChE, BChE, arylesterase and PON1, are correlated with levels of anxiety in both primary, non-disease-related and secondary, disease-related anxiety.

The ACHE, PON1 and/or BCHE genes encode polypeptides involved in neurotransmission mediated by the neurotransmitter acetylcholine (ACh). ACh contributes to numerous physiologic functions, including motor activity and secretion processes as well as cognition and behavioral states, including memory, learning and panic responses. Anxiety is known to provoke cholinergic hyper-arousal (e.g. sweating, intestinal or gastric constrictions etc.). In addition, AChE is a target of pesticides and human exposure to them, or to the closely related chemical warfare agents, depletes the catalytic activity of both AChE and the homologous enzyme butyrylcholinesterase (BChE).

While not wishing to be bound by theory, the invention described herein was developed in part by investigating the hypothesis that anxiety trait scores reflect inherited genotype properties combined with the corresponding enzyme activities, as affected by demographic parameters. It was also postulated that the capacity to respond to changing conditions by increasing serum AChE levels would be more limited in subjects with high basal activity of serum AChE, because there is a maximal expression level for this gene that is likely independent of demographic parameters. PON activity may determine the requirement for AChE overproduction. Therefore, the prediction of state anxiety was tested for association with the difference between the observed and predicted activity values of AChE and PON.

Measurements of serum AChE-PON enzyme activities were performed in samples from 451 healthy subjects participating in the HERITAGE Family Study. The HERITAGE Family Study was originally designed to evaluate the role of genetic and non-genetic factors in cardiovascular, metabolic, and hormonal responses to aerobic exercise training. For a description of the study, see Bouchard et al. Medicine and Science in Sports and Exercise 27:721-29, 1995.

Measurements of serum AChE-PON enzyme activities, when corrected for demographic parameters, revealed interrelated inverse associations with state anxiety scores, supporting the notion of corresponding enzyme relationships. These results indicate that a significant source of anxiety feelings involves inherited and acquired parameters of acetylcholine regulation that can be readily quantified, providing an independent tool for assessing anxiety measures. The findings reveal previously non-perceived interrelationships between anxiety feelings, serum AChE, BChE and PON activities, and their corresponding genotypes.

While not wishing to be bound by theory, it is postulated that polymorphisms in the corresponding ACHE and BCHE genes can affect both the environmental and the experience-related elements of anxiety. Furthermore, the paraoxonase PON1 gene is adjacent to the AChE gene, ACHE on chromosome 7. Also, the PON1 protein product can affect AChE activity by destroying environmental toxins that target AChE. Polymorphisms in the ACHE, BCHE and PON1 genes therefore affect both the environmental and the experience-related elements of anxiety.

Assessing Anxiety by Identifying ACHE-PON1 and/or BCHE Polymorphisms

Trait anxiety is assessed by determining whether a test sample from a subject contains a polymorphic form of a ACHE, PON1 and/or BCHE gene that is associated with a particular level of anxiety (trait or state). For example, the polymorphism can be associated with a particular anxiety score or scoring range in the STAI index. In general, any polymorphism in an ACHE, PON1, and/or BCHE gene that correlates with a particular level of trait or state anxiety can be used.

ACHE polymorphisms include promoter and coding sequence polymorphisms. The extended human ACHE promoter includes a functional glucocorticoid response element (GRE). In one ACHE1 polymorphism, a region of this element is deleted (Shapira et al., Hum. Mol. Genet. 9:1273-81, 2000). A second polymorphism in the AChE gene is the P446-polymorphism (Bartels et al. Am. J. Hu. Genet. 52:928-36, 1993).

Suitable PON1 promoter polymorphisms include (indicated by the distance in nucleotides from the transcription start site at 0): -108 C/T, which contributes to 22.4% of the variation in PON1 expression, possibly by eliminating a potential SP1 transcription factor binding site (Suehiro et al., Atherosclerosis 150:295-98, 2000); -162 G/C, which contributes to 2.4% of this variation (Brophy et al., Am. J. Human Genet. 68:1428-36, 2001) and -126 C/G (Costa et al. Ann. Rev Med 54:371-92, 2003). Suitable polymorphisms in the PON1 coding region include those changing the encoded polypeptide sequence, e.g., the PON1 polymorphisms can include (indicated by amino acid number and symbol) L55M (CTG into ATG) (Garin et al., J. Clin. Invest. 99:62-66, 1997) and Q192R (CAA into CGA) (Davies et al., Nat. Genet. 14:334-36, 1996).

Suitable BCHE polymorphisms include the D70G (Neville et al., J. Biol. Chem. 265:20735-38, 1990).

The test sample can be any biological fluid, cell sample, or tissue, as long as it contains genomic DNA from the subject, and/or RNA transcribed from the ACHE, PON1, or BCHE genes. Thus, a suitable test sample includes one obtained from any nucleated cell of the body, such as those present in peripheral blood, urine, saliva, buccal samples, surgical specimen, and autopsy specimens.

Methods of preparing nucleic acids in a form that is suitable for mutation detection is well known in the art. The DNA may be used directly or may be amplified enzymatically in vitro through use of PCR (Saiki et al., Science 239:487-91, 1988) or other in vitro amplification methods such as the ligase chain reaction (LCR) (Wu et al., Genomics 4:560-69 (1989), strand displacement amplification (SDA) (Walker et al. Proc. Natl. Acad. Sci. U.S.A, 89:392-96, 1992), self-sustained sequence replication (3SR), prior to mutation analysis.

Individuals carrying polymorphic alleles may be detected by using a variety of techniques that are well known in the art. Strategies for identification and detection are described in e.g., EP 730,663, EP 717,113, and PCT US97/02102. The present methods usually employ pre-characterized polymorphisms. That is, the genotyping location and nature of polymorphic forms present at a site have already been determined. The availability of this information allows sets of probes to be designed for specific identification of the known polymorphic forms.

Detection can include amplification of the starting nucleic acid (DNA or RNA) from the target samples. This can be accomplished by e.g., PCR. See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202.

The detection of polymorphisms in specific DNA sequences, can be accomplished by a variety of methods including, but not limited to, restriction-fragment-length-polymorphism detection based on allele-specific restriction-endonuclease cleavage, hybridization with allele-specific oligonucleotide probes (Wallace et al., Nucl. Acids Res. 6:3543-3557, 1978), including immobilized oligonucleotides (Saiki et al., Proc. Natl. Acad. Sci. USA, 86:6230-6234, 1969) or oligonucleotide arrays (Maskos et al., Nucl. Acids Res 21:2269-2270, 1993), allele-specific PCR (Newton et al., Nucl Acids Res. 17:2503-16, 1989), mismatch-repair detection (MRD) (Faham et al., Genome Res. 5:474-482, 1995), binding of MutS protein (Wagner et al., Nucl. Acids Res. 23:3944-48, 1995), denaturing-gradient gel electrophoresis (DGGE) (Fisher et al., Proc. Natl. Acad. Sci. USA 80:1579-83, 1983), single-strand-conformation-polymorphism detection (Orita et al., Genomics 5:874-879, 1983), RNAase cleavage at mismatched base-pairs (Myers et al., Science 230:1242, 1985), chemical (Cotton et al., Proc. Natl. Sci. U.S.A 85:4397-4401, 1988) or enzymatic (Youil et al., Proc. Natl. Acad. Sci. USA 92:87-91, 1995) cleavage of heteroduplex DNA, methods based on allele specific primer extension (Syvanen et al., Genomics 8:684-92, 1990), genetic bit analysis (GBA) (Nikiforov et al., Nucleic Acids Res. 22:4167-4175 (1994), the oligonucleotide-ligation assay (OLA) (Landegren et al., Science 241:1077, 1988), the allele-specific ligation chain reaction (LCR) (Barrany, Proc. Natl. Acad. Sci. USA 88:189-193, 1991), gap-LCR (Abravaya et al., Nucl Acids Res 23:675-682, 1995), radioactive and/or fluorescent DNA sequencing using standard procedures well known in the art, and peptide nucleic acid (PNA) assays (Orum et al., Nucl. Acids Res, 21:5332-5356, 1993; Thiede et al., Nucl. Acids Res. 24:983-984, 1996).

"Specific hybridization" or "selective hybridization" refers to the binding, or duplexing, of a nucleic acid molecule only to a second particular nucleotide sequence to which the nucleic acid is complementary, under suitably stringent conditions when that sequence is present in a complex mixture (e.g., total cellular DNA or RNA). "Stringent conditions" are conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and are different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter ones. Generally, stringent conditions are selected such that the temperature is about 5.degree. C. lower than the thermal melting point (Tm) for the specific sequence to which hybridization is intended to occur at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the target sequence hybridizes to the complementary probe at equilibrium. Typically, stringent conditions include a salt concentration of at least about 0.01 to about 1.0 M Na ion concentration (or other salts), at pH 7.0 to 8.3. The temperature is at least about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For example, conditions of 5.times.SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30.degree. C. are suitable for allele-specific probe hybridizations.

The invention also provides methods for identifying polymorphisms in ACHE, PON1, and BCHE genes that are associated with anxiety levels. The association of a polymorphic form and anxiety levels is compared. A statistically significant association between a polymorphic form of the gene and anxiety levels indicates the polymorphism is associated with a particular anxiety level. The presence of this polymorphism in a subject therefore, indicates the subject has a particular anxiety level. In some embodiments, the anxiety level is determined with reference to a mean STAI score.

Assessing Anxiety by Measuring AChE Activity or Expression

The invention is based in part on the discovery that AChE activity, when corrected for demographic parameters, is inversely associated with trait anxiety. Thus, state anxiety in a subject can be assessed by comparing the amount of AChE activity in a test sample from a subject with the level of activity in a reference sample whose amount of trait anxiety is known. A lower level of AChE activity in the subject as compared to the amount of AChE activity in the reference sample indicates the subject has more trait anxiety than the individual or individuals that constitute the reference sample. Conversely, a higher level of AChE activity in the subject as compared to the amount of AChE activity in the reference sample indicates the subject has less trait anxiety than the level of trait anxiety in the individual or individuals that constitute the reference sample.

Methods of assessing AChE activity are well known (see, e.g., Ellman et al., Biochem. Pharmacol. 7:88-95, 1961). In addition to serum, the test sample can alternatively be any biological fluid, cell sample, or tissue, as long as it includes active AChE protein. Activity is measured in the presence of a BChE inhibitor, such as iso-OMPA at 5.10.sup.-5M.

Anxiety levels can alternatively, or in addition, be assessed by examining relative levels of AChE monomeric forms relative to other AChE forms. As is discussed in detail below, AChE monomeric forms are over represented in subjects with low trait anxiety scores. Alternative splicing of AChE gene products yields at least three distinct proteins with acetylcholine hydrolytic activity. Of these, the primary AChE-S variant forms tetramers, the erythrocytic AChE-E protein appears as glycophosphoinositide-bound dimers and the stress-induced AChE-R variant remains monomeric (Soreq et al., Nat. Rev. Neurosci. 2:294-302, 2001). These forms of AChE can be resolved using methods known in the art, such as non-denaturing polyacrylamide gel electrophoresis. The resolved AChE can be visualized using various method, such as staining for activity or by immunoblot analysis.

A higher level of monomeric forms of AChE relative to the other AChE forms in the test sample indicates the subject has less trait anxiety than the individual or individuals that constitute the reference sample. A lower level of monomeric forms reveals greater trait anxiety in the subject than in the individual or individuals that constitute the reference sample.

Susceptibility to state anxiety is determined by identifying PON activity and/or AChE activity in a subject and comparing the activity to a reference sample derived from one or more individuals whose state anxiety level is known. Assays for PON activity are known in the art and are described in, e.g., Furlong et al., Anal Biochem 180:242-7, 1989. The individual or individuals in the reference sample are similar to the subject in at least one trait selected from gender, age, race, ethnic group, and body mass index. A higher level of PON in said test sample compared to said reference sample indicates the subject has increased susceptibility to state anxiety compared to said one or more individuals in said reference sample.

Further, assessing the activity of more than one enzyme may provide a more accurate measurement of susceptibility to trait anxiety relative to assessing the activity of one enzyme. As is describe in the Examples section and hereinbelow it is postulated that the functional relationship between AChE, PON and BChE activity results in the activity of all three enzymes correlating with susceptibility to trait anxiety.

Referring to FIG. 12 (see Original Patent), there are several modes of AChE and PON1 interaction at the protein level. As shown in the upper portion of FIG. 12, the first mode of AChE-PON1 interaction(s) relates to the broad antioxidative properties of PON1. PON1 protects LDL from oxidation and is an established protection factor in atherosclerosis (Mackness et al., Atheroscler Suppl, 3(4):49-55, 2002). This ability may reflect the capacity to hydrolyze lipid peroxides and hydroperoxides and to hydrolyze hydrogen peroxide (Aviram et al., J Clin Invest, 101(8):1581-90, 1998), as well as its ability to reduce oxidative stress in macrophages, including decrease in superoxide anion release (Rozenberg et al., Free Radic Biol Med, 34(6):774-84, 2003). Reciprocally, AChE is known to be particularly sensitive to oxidative stress and is inactivated under oxidative conditions (Weiner et al., Biochem Biophys Res Commun, 198(3):915-22, 1994). By reducing oxidative stress, PON1 can therefore protect plasma AChE and AChE inactivation under low PON1 levels can elevate acetylcholine levels, initiating AChE overproduction and increasing the levels of plasma AChE-R monomers.

Another mode of interaction relates to the age-dependent changes in arylesterase activity, likely mediated through the L55M position yet involving the H1, H2 helices (FIG. 12). Increased arylesterase activity may reflect changes in HDL composition, which in turn involves oxidative stress damages to AChE. This alleviates the need for AChE overproduction, suggesting that PON1 L55 carriers are better protected from cholinergic insults. That AChE-R accumulates under LPS exposure (Cohen et al., J Mol Neurosci, 21(3):199-212, 2003) may reflect the reciprocal decrease in PON1 under such exposure (Feingold et al., Atherosclerosis, 139(2):307-15, 1998.) supporting this notion. That PON1 and AChE polymorphisms were shown to predict anxiety state and trait, and that traumatic experiences can increase the anxiety level, makes carriers of debilitating PON1/ACHE polymorphisms yet more prone to adverse reactions.

Consequently, even though PON activity is inversely associated with susceptibility to state anxiety, PON activity also has an indirect inverse association with susceptibility to trait anxiety. This indirect association results from the paraoxonase, peroxidase and arylesterase activities of PON protecting AChE from oxidative stress. As a result, assessing the activity of both AChE and PON may provide a more accurate measurement of susceptibility to trait anxiety than only assessing the activity of AChE.

Similarly, through its capacity as a general scavenger of anti-AChEs, BChE has an inverse association with trait anxiety. Increasing BChE activity can protect AChE by scavenging anti-AChEs resulting in a decrease in susceptibility to trait anxiety. Consequently, assessing the activity of AChE and BChE may provide a more accurate measurement of susceptibility to trait anxiety than only assessing the activity of AChE. Further, as there is a functional relationship between all three enzymes, assessing the activity of AChE, BChE and PON may provide a more accurate measurement of susceptibility to trait anxiety than only assessing the activity of two of the enzymes.

State anxiety and/or trait anxiety can be assessed at multiple time points by measuring PON and/or AChE activity in two or more samples taken at two or more time points. Relevant time points for comparing anxiety levels include, e.g., before and after administering an anxiety treatment, as well as during and after an anxiety attack.

Treatment outcome for anxiety therapies can be assessed and predicted by measuring cholinergic enzyme activities; FIGS. 13A-13D and 14A-14D (see Original Patent) show that serum cholinergic enzyme activities correlate with anxiety scores in subjects suffering from situational (such as disease-related) anxiety, as well as primary, non-situational anxiety. Thus, it will be appreciated, that measurement of cholinergic enzyme activities can be used to determine the response of a patient to anti-anxiety treatment. Presently, such assessment of anti-anxiety treatment is typically dependent on observation of improvement in clinical anxiety symptoms and/or scores a considerable time after initiation of treatment. Thus, monitoring and adjusting the anxiety therapy to the individual needs of the patient often becomes an unduly protracted and complex process. However, in view of the correlation between cholinergic enzyme levels and anxiety, as described herein, response to anxiety treatment can be assessed biochemically, with superior accuracy and far in advance of observable clinical changes, by objective measuring cholinergic enzyme activity. Such biochemical assessment of response to treatment can allow rapid adjustment of treatment parameters such as choice of drug, dosage, regimen, etc. Assessing or monitoring response to anti-anxiety treatment, or predicting outcome of anti-anxiety treatment can be performed by measuring, in the patient, cholinergic enzyme activity, such as PON1, AChE, or monomeric AChE levels, as described herein. Measurement can be performed at single or multiple time points prior to initiation of treatment, and comparatively continued at single or multiple time points after initiation of treatment. Changes in cholinergic enzyme levels, compared to levels prior to initiation of therapy, can indicate an effect, or degree of effect of the treatment. Such monitoring can be used to adjust drug dosage, type of anti-anxiety therapy, etc. Such assessment can be performed for short- medium- or long-term monitoring of anxiety in the patient.

Such assessment of treatment outcome and efficacy by measurement of cholinergic enzymes in the patient is suitable for use with all anti-anxiety treatments. In one preferred embodiment, the anti anxiety therapy is for primary, non-situational anxiety. In another embodiment, the anti-anxiety therapy is for secondary, situational anxiety. Following is a non-limiting list of suitable therapies for anxiety: benzodiazepines such as alprazolam, clonazepam, diazepam, lorazepam, halazepam, oxazepam, and chlordiazepam; beta blockers such as propranolol, nadolol, pindolol and atenolol; tricyclic antidepressants such as imipramine, desipramine, nortriptyline, amitriptyline, doxepin, clomipramine, trazodone, and venlafaxine; monoamine oxidase inhibitors such as phenelzine, tranylcypromine, fluoxetine, fluvoxamine, sertraline, paroxetine, escitalopram oxalate and citalopram; mild tranquilizers such as buspirone; and anticonvulsants such as valproate. It will be appreciated that the progress and efficacy of non-pharmaceutical anti-anxiety therapies, such as bio-feedback, psychotherapy, etc, can be assessed and monitored using measurement of cholinergic enzymes in the patient.

While reducing the present invention to practice, it was shown that depressed patients receiving anti-depression therapy showed consistent and sigificant changes in cholinergic enzyme activity (see FIGS. 14 and 15 (see Original Patent)). Thus, measurement of changes in cholinergic enzymes, as described herein, can be used to monitor, assess and/or predict treatment outcomes of anti-depressant therapy. Following is a non-limiting list of anti-depression therapies suitable for use with the methods of the present invention: SSRIs such as citalopram, escitalopram oxalate, fluvoxamine, paroxetine, fluoxetine and sertraline; MAOIs such as phenelzine and tranylcypromine; Tricyclics such as doxepin, clomipramine, amitriptyline, maprotiline, desipramine, nortryptyline, trimipramine, imipramine and protriptyline; buspirone, duloxetine, trazodone, venlafaxine, reboxetine, mirtazapine, nefazodone and bupropion.

AChE/BChE and PON polymorphism can be identified using a variety of methods. One option is to determine the entire gene sequence of a PCR reaction product. Alternatively, a given segment of nucleic acid may be characterized on several other levels. At the lowest resolution, the size of the molecule can be determined by electrophoresis by comparison to a known standard run on the same gel. A more detailed picture of the molecule may be achieved by cleavage with combinations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map. The presence of specific sequences within the fragment can be detected by hybridization of a labeled probe, or the precise nucleotide sequence can be determined by partial chemical degradation or by primer extension in the presence of chain-terminating nucleotide analogs.

Following is a non-limiting list of detection methods which can be used along with the present invention.

Restriction Fragment Length Polymorphism (RFLP): This method uses a change in a single nucleotide (the SNP nucleotide) which modifies a recognition site for a restriction enzyme resulting in the creation or destruction of an RFLP. Single nucleotide mismatches in DNA heteroduplexes are also recognized and cleaved by some chemicals, providing an alternative strategy to detect single base substitutions, generically named the "Mismatch Chemical Cleavage" (MCC) (Gogos et al., Nucl. Acids Res., 18:6807-6817, 1990). However, this method requires the use of osmium tetroxide and piperidine, two highly noxious chemicals which are not suited for use in a clinical laboratory.

Allele specific oligonucleotide (ASO): In this method an allele-specific oligonucleotides (ASOs) is designed to hybridize in proximity to the polymorphic nucleotide, such that a primer extension or ligation event can be used as the indicator of a match or a mis-match. Hybridization with radioactively labeled allelic specific oligonucleotides (ASO) also has been applied to the detection of specific SNPs (Conner et al., Proc. Natl. Acad. Sci., 80:278-282, 1983). The method is based on the differences in the melting temperature of short DNA fragments differing by a single nucleotide. Stringent hybridization and washing conditions can differentiate between mutant and wild-type alleles.

Denaturing/Temperature Gradient Gel Electrophoresis (DGGE/TGGE): Two other methods rely on detecting changes in electrophoretic mobility in response to minor sequence changes. One of these methods, termed "Denaturing Gradient Gel Electrophoresis" (DGGE) is based on the observation that slightly different sequences will display different patterns of local melting when electrophoretically resolved on a gradient gel. In this manner, variants can be distinguished, as differences in melting properties of homoduplexes versus heteroduplexes differing in a single nucleotide can detect the presence of SNPs in the target sequences because of the corresponding changes in their electrophoretic mobilities. The fragments to be analyzed, usually PCR products, are "clamped" at one end by a long stretch of G-C base pairs (30-80) to allow complete denaturation of the sequence of interest without complete dissociation of the strands. The attachment of a GC "clamp" to the DNA fragments increases the fraction of mutations that can be recognized by DGGE (Abrams et al., Genomics 7:463-475,1990). Attaching a GC clamp to one primer is critical to ensure that the amplified sequence has a low dissociation temperature (Sheffield et al., Proc. Natl. Acad. Sci., 86:232-236, 1989; and Lerman and Silverstein, Meth. Enzymol., 155:482-501, 1987). Modifications of the technique have been developed, using temperature gradients (Wartell et al., Nucl. Acids Res., 18:2699-2701, 1990), and the method can be also applied to RNA:RNA duplexes (Smith et al., Genomics 3:217-223, 1988).

Limitations on the utility of DGGE include the requirement that the denaturing conditions must be optimized for each type of DNA to be tested. Furthermore, the method requires specialized equipment to prepare the gels and maintain the needed high temperatures during electrophoresis. The expense associated with the synthesis of the clamping tail on one oligonucleotide for each sequence to be tested is also a major consideration. In addition, long running times are required for DGGE. The long running time of DGGE was shortened in a modification of DGGE called constant denaturant gel electrophoresis (CDGE) (Borrensen et al., Proc. Natl. Acad. Sci. USA 88:8405, 1991). CDGE requires that gels be performed under different denaturant conditions in order to reach high efficiency for the detection of SNPs.

A technique analogous to DGGE, termed temperature gradient gel electrophoresis (TGGE), uses a thermal gradient rather than a chemical denaturant gradient (Scholz, et al., Hum. Mol. Genet. 2:2155, 1993). TGGE requires the use of specialized equipment which can generate a temperature gradient perpendicularly oriented relative to the electrical field. TGGE can detect mutations in relatively small fragments of DNA therefore scanning of large gene segments requires the use of multiple PCR products prior to running the gel.

Single-Strand Conformation Polymorphism (SSCP): Another common method, called "Single-Strand Conformation Polymorphism" (SSCP) was developed by Hayashi, Sekya and colleagues (reviewed by Hayashi, PCR Meth. Appl., 1:34-38, 1991) and is based on the observation that single strands of nucleic acid can take on characteristic conformations in non-denaturing conditions, and these conformations influence electrophoretic mobility. The complementary strands assume sufficiently different structures that one strand may be resolved from the other. Changes in sequences within the fragment will also change the conformation, consequently altering the mobility and allowing this to be used as an assay for sequence variations (Orita, et al., Genomics 5:874-879, 1989).

The SSCP process involves denaturing a DNA segment (e.g., a PCR product) that is labeled on both strands, followed by slow electrophoretic separation on a non-denaturing polyacrylamide gel, so that intra-molecular interactions can form and not be disturbed during the run. This technique is extremely sensitive to variations in gel composition and temperature. A serious limitation of this method is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions.

Dideoxy fingerprinting (ddF): The dideoxy fingerprinting (ddF) is another technique developed to scan genes for the presence of mutations (Liu and Sommer, PCR Methods Appli., 4:97, 1994). The ddF technique combines components of Sanger dideoxy sequencing with SSCP. A dideoxy sequencing reaction is performed using one dideoxy terminator and then the reaction products are electrophoresed on nondenaturing polyacrylamide gels to detect alterations in mobility of the termination segments as in SSCP analysis. While ddF is an improvement over SSCP in terms of increased sensitivity, ddF requires the use of expensive dideoxynucleotides and this technique is still limited to the analysis of fragments of the size suitable for SSCP (i.e., fragments of 200-300 bases for optimal detection of mutations).

In addition to the above limitations, all of these methods are limited as to the size of the nucleic acid fragment that can be analyzed. For the direct sequencing approach, sequences of greater than 600 base pairs require cloning, with the consequent delays and expense of either deletion sub-cloning or primer walking, in order to cover the entire fragment. SSCP and DGGE have even more severe size limitations. Because of reduced sensitivity to sequence changes, these methods are not considered suitable for larger fragments. Although SSCP is reportedly able to detect 90% of single-base substitutions within a 200 base-pair fragment, the detection drops to less than 50% for 400 base pair fragments. Similarly, the sensitivity of DGGE decreases as the length of the fragment reaches 500 base-pairs. The ddF technique, as a combination of direct sequencing and SSCP, is also limited by the relatively small size of the DNA that can be screened.

Pyrosequencing.TM. analysis (Pyrosequencing, Inc. Westborough, Mass., USA): This technique is based on the hybridization of a sequencing primer to a single stranded, PCR-amplified, DNA template in the presence of DNA polymerase, ATP sulfurylase, luciferase and apyrase enzymes and the adenosine 5' phosphosulfate (APS) and luciferin substrates. In the second step the first of four deoxynucleotide triphosphates (dNTP) is added to the reaction and the DNA polymerase catalyzes the incorporation of the deoxynucleotide triphosphate into the DNA strand, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide. In the last step the ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5' phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and seen as a peak in a pyrogram.TM.. Each light signal is proportional to the number of nucleotides incorporated.

Acycloprime.TM. analysis (Perkin Elmer, Boston, Mass., USA): This technique is based on fluorescent polarization (FP) detection. Following PCR amplification of the sequence containing the SNP of interest, excess primer and dNTPs are removed through incubation with shrimp alkaline phosphatase (SAP) and exonuclease I. Once the enzymes are heat inactivated, the Acycloprime-FP process uses a thermostable polymerase to add one of two fluorescent terminators to a primer that ends immediately upstream of the SNP site. The terminator(s) added are identified by their increased FP and represent the allele(s) present in the original DNA sample. The Acycloprime process uses AcycloPol.TM., a novel mutant thermostable polymerase from the Archeon family, and a pair of AcycloTerminators.TM. labeled with R110 and TAMRA, representing the possible alleles for the SNP of interest. AcycloTerminator.TM. non-nucleotide analogs are biologically active with a variety of DNA polymerases. Similarly to 2',3'-dideoxynucleotide-5'-triphosphates, the acyclic analogs function as chain terminators. The analog is incorporated by the DNA polymerase in a base-specific manner onto the 3'-end of the DNA chain, and since there is no 3'-hydroxyl, is unable to function in further chain elongation. It has been found that AcycloPol has a higher affinity and specificity for derivatized AcycloTerminators than various Taq mutant have for derivatized 2',3'-dideoxynucleotide terminators.

Reverse dot blot: This technique uses labeled sequence specific oligonucleotide probes and unlabeled nucleic acid samples. Activated primary amine-conjugated oligonucleotides are covalently attached to carboxylated nylon membranes. After hybridization and washing, the labeled probe, or a labeled fragment of the probe, can be released using oligomer restriction, i.e., the digestion of the duplex hybrid with a restriction enzyme. Circular spots or lines are visualized colorimetrically after hybridization through the use of streptavidin horseradish peroxidase incubation followed by development using tetramethylbenzidine and hydrogen peroxide, or via chemiluminescence after incubation with avidin alkaline phosphatase conjugate and a luminous substrate susceptible to enzyme activation, such as CSPD, followed by exposure to x-ray film.

It will be appreciated that advances in the field of SNP detection have provided additional accurate, easy, and inexpensive large-scale SNP genotyping techniques, such as dynamic allele-specific hybridization (DASH, Howell, W. M. et al., 1999. Dynamic allele-specific hybridization (DASH). Nat. Biotechnol. 17: 87-8), microplate array diagonal gel electrophoresis [MADGE, Day, I. N. et al., 1995. High-throughput genotyping using horizontal polyacrylamide gels with wells arranged for microplate array diagonal gel electrophoresis (MADGE). Biotechniques. 19: 830-5], the TaqMan system (Holland, P. M. et al., 1991. Detection of specific polymerase chain reaction product by utilizing the 5'.fwdarw.3' exonuclease activity, of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA. 88: 7276-80), as well as various DNA "chip" technologies such as the GeneChip microarrays (e.g., Affymetrix SNP chips) which are disclosed in U.S. Pat. No. 6,300,063 to Lipshutz, et al. 2001, which is fully incorporated herein by reference, Genetic Bit Analysis (GBA.TM.) which is described by Goelet, P. et al. (PCT Appl. No. 92/15712), peptide nucleic acid (PNA, Ren B, et al., 2004. Nucleic Acids Res. 32: e42) and locked nucleic acids (LNA, Latorra D, et al., 2003. Hum. Mutat. 22: 79-85) probes, Molecular Beacons (Abravaya K, et al., 2003. Clin Chem Lab Med. 41: 468-74), intercalating dye [Germer, S. and Higuchi, R. Single tube genotyping without oligonucleotide probes. Genome Res. 9:72-78 (1999)], FRET primers (Solinas A et al., 2001. Nucleic Acids Res. 29: E96), AlphaScreen (Beaudet L, et al., Genome Res. 2001, 11(4): 600-8), SNPstream (Bell P A, et al., 2002. Biotechniques. Suppl.: 70-2, 74, 76-7), Multiplex minisequencing (Curcio M, et al., 2002. Electrophoresis. 23: 1467-72), SnaPshot (Turner D, et al., 2002. Hum Immunol. 63: 508-13), MassEXTEND (Cashman J R, et al., 2001. Drug Metab Dispos. 29: 1629-37), GOOD assay (Sauer S, and Gut I G. 2003. Rapid Commun. Mass. Spectrom. 17: 1265-72), Microarray minisequencing (Liljedahl U, et al., 2003. Pharmacogenetics. 13: 7-17), arrayed primer extension (APEX) (Tonisson N, et al., 2000. Clin. Chem. Lab. Med. 38: 165-70), Microarray primer extension (O'Meara D, et al., 2002. Nucleic Acids Res. 30: e75), Tag arrays (Fan J B, et al., 2000. Genome Res. 10: 853-60), Template-directed incorporation (TDI) (Akula N, et al., 2002. Biotechniques. 32: 1072-8), fluorescence polarization (Hsu T M, et al., 2001. Biotechniques. 31: 560, 562, 564-8), Colorimetric oligonucleotide ligation assay (OLA, Nickerson D A, et al., 1990. Proc. Natl. Acad. Sci. USA. 87: 8923-7), Sequence-coded OLA (Gasparini P, et al., 1999. J. Med. Screen. 6: 67-9), Microarray ligation, Ligase chain reaction, Padlock probes, Rolling circle amplification, Invader assay (reviewed in Shi M M. 2001. Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies. Clin Chem. 47: 164-72), coded microspheres (Rao K V et al., 2003. Nucleic Acids Res. 31: e66) and MassArray (Leushner J, Chiu N H, 2000. Mol Diagn. 5: 341-80).

According to preferred embodiments of the present invention the SNPs used by the present invention are selected from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/SNP/).

As is mentioned hereinabove and described in great detail in the Examples section which follows, the present inventors have uncovered several novel traits which can associated with ChE and PON activity:

(i) PON activity which is lower than predicted (likely due to genotype differences) reflects increased risk to develop state anxiety;

(ii) AChE activity which is lower than predicted reflects elevated trait anxiety; and

(iii) PON, in its capacities as a paraoxonase or peroxidase, protects circulation AChE from oxidative stress, therefore, debilitated PON may further contribute to trait anxiety, albeit indirectly.

In addition, BChE serves to protect circulation AChE by acting as a general scavenger of anti AChEs. As such, triple assays (of AChE, BChE and PON) would be yet more reliable.

Based on these observations, the present inventor postulates that diagnosis which is effected by combined testing (of AChE, and PON and optionally BChE) and in particular, diagnosis which correlates AChE and PON activity or expression can yield more reliable predictions of anxiety parameters than single tests of either of these activities.

The usefulness and accuracy of such combined testing is clearly supported by the finding presented herein which illustrate that anxiety is a multifactorial state which depends upon discrete activities of two or more enzymes as well as the interaction therebetween.

Assessing Anxiety by Correlating ChE Activity or Expression with PON Activity or Expression

As is illustrated in the Examples section which follows, the present inventors also correlated ChE/PON activity/expression levels (herein ChE/PON ratio) and uncovered that individuals predisposed to, or having anxiety display ChE/PON ratios which differ from those displayed by healthy individuals.

Thus, according to another aspect of the present invention there is provided yet another method of assessing state or trait anxiety in a subject.

The method is effected by determining in a biological sample of an individual expression and/or activity levels of at least one cholinesterase (ChE), preferably Acetylcholinesterase (AChE) and paraoxonase (PON1); and correlating between expression and/or activity levels of the at least one cholinesterase and the paraoxonase to thereby obtain the ChE/PON ratio described herein. Such a ratio is then compared with a predetermined threshold (a single value or preferably a value range) predefined for healthy or diseased individuals of a specific group (age, gender BMI, ethnicity, race etc.), to thereby diagnose the subject as healthy, having anxiety or being predisposed thereto. Further description of this ratio is provided in the examples section which follows, and illustrated in FIG. 6 (see Original Patent).

It will be appreciated that quantification of ChE (preferably AChE) and PON expression or activity levels can be facilitated using one of several known approaches.

The above described expression and/or activity levels can be determined by allele typing and correlation of a specific allele with expression/activity levels. Since a correlation between allele types and expression levels can be established, mere typing of an allele can be translated into expression or activity levels of ChE or PON.

Biochemical or molecular analysis of test samples can also be used to determine the above described ratio. Numerous approaches for measuring mRNA or protein levels in a biological sample such as blood are known in the art, most of these approaches are readily adaptable for high throughput automatic screening. Examples of suitable approaches are provided below.

Northern Blot analysis: This method involves the detection of a particular RNA in a mixture of RNAs. An RNA sample is denatured by treatment with an agent (e.g., formaldehyde) that prevents hydrogen bonding between base pairs, ensuring that all the RNA molecules have an unfolded, linear conformation. The individual RNA molecules are then separated according to size by gel electrophoresis and transferred to a nitrocellulose or a nylon-based membrane to which the denatured RNAs adhere. The membrane is then exposed to labeled DNA probes. Probes may be labeled using radio-isotopes or enzyme linked nucleotides. Detection may be using autoradiography, calorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of particular RNA molecules and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the gel during electrophoresis.

RT-PCR analysis: This method uses PCR amplification of relatively rare RNAs molecules. First, RNA molecules are purified from the cells and converted into complementary DNA (cDNA) using a reverse transcriptase enzyme (such as an MMLV-RT) and primers such as, oligo dT, random hexamers or gene specific primers. Then by applying gene specific primers and Taq DNA polymerase, a PCR amplification reaction is carried out in a PCR machine. Those of skills in the art are capable of selecting the length and sequence of the gene specific primers and the PCR conditions (i.e., annealing temperatures, number of cycles and the like) which are suitable for detecting specific RNA molecules. It will be appreciated that a semi-quantitative RT-PCR reaction can be employed by adjusting the number of PCR cycles and comparing the amplification product to known controls.

RNA in situ hybridization stain: In this method DNA or RNA probes are attached to the RNA molecules present in the cells. Generally, the cells are first fixed to microscopic slides to preserve the cellular structure and to prevent the RNA molecules from being degraded and then are subjected to hybridization buffer containing the labeled probe. The hybridization buffer includes reagents such as formamide and salts (e.g., sodium chloride and sodium citrate) which enable specific hybridization of the DNA or RNA probes with their target mRNA molecules in situ while avoiding non-specific binding of probe. Those of skills in the art are capable of adjusting the hybridization conditions (i.e., temperature, concentration of salts and formamide and the like) to specific probes and types of cells. Following hybridization, any unbound probe is washed off and the slide is subjected to either a photographic emulsion which reveals signals generated using radio-labeled probes or to a calorimetric reaction which reveals signals generated using enzyme-linked labeled probes.

In situ RT-PCR stain: This method is described in Nuovo G J, et al. [Intracellular localization of polymerase chain reaction (PCR)-amplified hepatitis C cDNA. Am J Surg Pathol. 1993, 17: 683-90] and Komminoth P, et al. [Evaluation of methods for hepatitis C virus detection in archival liver biopsies. Comparison of histology, immunohistochemistry, in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and in situ RT-PCR. Pathol Res Pract. 1994, 190: 1017-25]. Briefly, the RT-PCR reaction is performed on fixed cells by incorporating labeled nucleotides to the PCR reaction. The reaction is carried on using a specific in situ RT-PCR apparatus such as the laser-capture microdissection PixCell I LCM system available from Arcturus Engineering (Mountainview, Calif.).

Although cell profiling methods which analyze the genome or transcriptome are preferred for their accuracy and high throughput capabilities, it will be appreciated that the present invention can also utilize protein analysis tools for profiling the cells of the cultures.

Expression and/or activity level of proteins can be determined using any of the methods described below.

Enzyme linked immunosorbent assay (ELISA): This method involves fixation of a sample (e.g., fixed cells or a proteinaceous solution) containing a protein substrate to a surface such as a well of a microtiter plate. A substrate specific antibody coupled to an enzyme is applied and allowed to bind to the substrate. Presence of the antibody is then detected and quantitated by a calorimetric reaction employing the enzyme coupled to the antibody. Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced. A substrate standard is generally employed to improve quantitative accuracy.

Western blot: This method involves separation of a substrate from other protein by means of an acrylamide gel followed by transfer of the substrate to a membrane (e.g., nylon or PVDF). Presence of the substrate is then detected by antibodies specific to the substrate, which are in turn detected by antibody binding reagents. Antibody binding reagents may be, for example, protein A, or other antibodies. Antibody binding reagents may be radiolabeled or enzyme linked as described hereinabove. Detection may be by autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of substrate and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.

Radio-immunoassay (RIA): In one version, this method involves precipitation of the desired protein (i.e., the substrate) with a specific antibody and radiolabeled antibody binding protein (e.g., protein A labeled with I.sup.125) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of substrate.

In an alternate version of the RIA, a labeled substrate and an unlabelled antibody binding protein are employed. A sample containing an unknown amount of substrate is added in varying amounts. The decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.

Fluorescence activated cell sorting (FACS): This method involves detection of a substrate in situ in cells by substrate specific antibodies. The substrate specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.

Immunohistochemical analysis: This method involves detection of a substrate in situ in fixed cells by substrate specific antibodies. The substrate specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective or automatic evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required. It will be appreciated that immunohistochemistry is often followed by counterstaining of the cell nuclei using for example Hematoxyline or Giemsa stain.

In situ activity assay: According to this method, a chromogenic substrate is applied on the cells containing an active enzyme and the enzyme catalyzes a reaction in which the substrate is decomposed to produce a chromogenic product visible by a light or a fluorescent microscope.

In vitro activity assays: In these methods the activity of a particular enzyme is measured in a protein mixture extracted from the cells. The activity can be measured in a spectrophotometer well using calorimetric methods or can be measured in a non-denaturing acrylamide gel (i.e., activity gel). Following electrophoresis the gel is soaked in a solution containing a substrate and colorimetric reagents. The resulting stained band corresponds to the enzymatic activity of the protein of interest. If well calibrated and within the linear range of response, the amount of enzyme present in the sample is proportional to the amount of color produced. An enzyme standard is generally employed to improve quantitative accuracy.

It will be appreciated that anxiety diagnosis obtained via the ChE/PON ratio determination described above, offers several advantages over discrete AChE or PON expression/activity. One notable advantage is a lack of need for control samples. Since a ratio does not rely upon absolute numbers but rather on the relationship therebetween, ratio-determined diagnosis does not necessitate comparison with control samples nor does it necessitate standardization of results with respect to age or gender but rather generation of a single threshold for each tested group. For example, groups of similar ethnic background, age, gender or BMI can be used to generate a threshold ratio which can be used to determine diagnosis of individuals belonging to a specific group.

It will be appreciated that any of the reagents described hereinabove (e.g., AChE or PON PCR primers or probes) can be packaged into a kit which can be used for state or trait anxiety diagnosis, or monitoring treatment or therapy outcomes of situational and non-situational anxiety, or monitoring treatment or therapy outcomes of anti-depressants. The kit for use in the method according to the invention preferably contains the various components needed for carrying out the method packaged in separate containers and/or vials and including instructions for carrying out the method. Thus, for example, some or all of the various reagents and other ingredients needed for carrying out the determination, such as buffers, primers, enzymes, control samples or standards etc can be packaged separately but provided for use in the same box. Instructions for carrying out the method can be included inside the box, as a separate insert, or as a label on the box and/or on the separate vials.
 

Claim 1 of 16 Claims

1. A method of assessing trait anxiety in a subject, the method comprising providing a test sample from said subject; determining acetylcholinesterase (AChE) activity in said test sample; and comparing AChE activity in said test sample to AChE activity in a reference sample derived from one or more individuals whose trait anxiety level is known, wherein said one or more individuals in said reference sample are similar to said subject in at least one trait selected from the group consisting of gender, age, race, ethnic group, and body mass index, and wherein a lower level of AChE activity in said test sample compared to AChE activity in said reference sample indicates said subject has greater trait anxiety than the trait anxiety level of said one or more individuals from which the reference sample was derived, thereby assessing trait anxiety in said subject.

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