WO1993005783A1

WO1993005783A1 - Diagnosis and therapy for parkinson's disease

Info

Publication number: WO1993005783A1
Application number: PCT/US1992/006746
Authority: WO
Inventors: Xandra O. Breakefield; Gokhan S. Hotamisligil
Original assignee: The General Hospital Corporation
Priority date: 1991-09-20
Filing date: 1992-08-17
Publication date: 1993-04-01
Also published as: EP0666745A1; EP0666745A4; CA2116130A1; JPH07501518A; AU2492192A

Abstract

Compositions and methods for the diagnosis and treatment of Parkinson's disease are provided. Particularly, monoamineoxidase A haplotypes are correlated with Parkinson's disease. Compositions and methods for treatment of the disease are also provided.

Description

DIAGNOSIS AND THERAPY FOR PARKINSON'S DISEASE

Field of the Invention

The invention relates to the detection and treatment of Parkinson's Disease. Background of the Invention

Parkinson's disease (PD) is a debilitating movement disorder affecting 1% of the population over 50 yrs of age (Adams and Victor, 1985). The disease is a neurological syndrome manifested by any combination of tremor at rest, rigidity, bradykinesia, and loss of postural reflexes. Onset of Parkinsonian symptoms, including stiffness, slowness of voluntary movements, tremor at rest, stooped posture and fixed facial expression, occurs after about 80% of dopaminergic neurons in the substantia nigra have degenerated (Fahn, S. NY Acad. Sci.570:186 (1989)). Current assessment of numbers of dopaminergic neurons (Flood el al., Neurobiol. Aging 9:453 (1988)) and dopa uptake (Sawle el al., Annals.

Neurol 28:799 (1990)) into the substantia nigra suggest no dramatic decrease during normal human aging. Thus death of these neurons in Parkinson's Disease appears to be an exceptional phenomena in some individuals.

PD is a progressive degenerative disorder of the central nervous system in which there is loss of monoamine neurons in the brain stem nuclei associated with eosinophilic cytoplasmic inclusion bodies in these neurons. Accompanying this loss of cells is a marked reduction of monoamines in the brain (S. Fahn supra (1989)).

Evidence implicating monoamine oxidase (MAO), a degradative enzyme for biogenic amines, in Parkinson's Disease stems from its ability to convert 1-methyl-4-phenyl-1,2,3,6-tetrahydroρyridine (MPTP) to a toxin that can kill nigral neurons, whose loss in turn can cause Parkinsonian symptoms in animals and humans largely through activity of the B isozyme, MAO-B (Langston el al., Science 219:919 (1983); Singer el al, J. Neurochem. 49:1 (1987); Burns el al., Proc. Nail. Acad. Sci. USA 80:4546 (1983); Langston et al., Brain Res. 292:390 (1984); Heikkila et al., Science 224:1451 (1984)). Activity of the A isozyme, MAO-A, is high in these same nigral neurons and has been thought to generate toxic free radicals and hydrogen peroxide during normal oxidative functions (Fahn, Ann. NY Acad. Sci 570:186 (1989); Riederer el al., Ada Neurol Scad. Suppl 126:41 (1989); Cohen, Adv. Neurol 45:119 (1987)). MAO oxidizes primary aromatic amines, and at lower rates, primary aliphatic, secondary, and tertiary amines. For a review, see, Beckmann and Riederer, eds. (1983) Modem Problems of Pharmacopsychiatry, Vol. 19: Monoamine Oxidase and Its Selective Inhibitors; Mondovi, B. (1985) Structure and Function of Amine Qxidases. CRG Press, Boca Raton, Florida; Glover and Sandier, Cell Biochem. Fund. 4:89-97 (1986); and Weyler el al. J. Pharmacol Ther.

(1989).

The two isozymes of MAO, MAO-A and MAO-B, are encoded in homologous genes near each other in the pll.3 region of the human X chromosome (Pintar el al., J. NeuroscL 1:166 (1981); Levy el al., Genomics 5368 (1989); Sims el al., Neuron 2:1069 (1989); Lan el al., Genomics 4:552

(1989)). These isozymes show different kinetics for inhibitors and substrates and are differentially distributed throughout the body (For a review see Weyler el al., J. Pharmacol Therapeut.47:391 (1990). MAO-A is highest in liver and placenta; and it is the predominant form in catecholaminergic neurons throughout the nervous system (Thorpe el al.,

Histochem. Cytochem. 35:23 (1987); Fowler el al., Science 255:481 (1987)). MAO-B is also highest in the liver and predominates in platelets and lymphocytes, as well as in glial cells and serotonergic neurons in the central nervous system (ibid.; Levitt el al., Proc Natl Acad. Sci. USA 79:6385 (1982)). These isozymes are differentially regulated during the lifetime of the individual with MAO-A appearing first in development (Tsang el al., Dev. Neurosci. 8:243 (1986); Lewinsohn el al., Biochem. Pharmacol 29:1221 (1980); Diez el al., Brain Res. 128:187 (1977)) and increasing with age in some studies (Edelstein el al., Cell Mol. Neurobiol 6:121 (1986);

Shih, in Monoamine Oxidase: Structure, Function and Altered Function, T.P.Singer, R.W. Von Korff, D.L. Murphy, Eds. (Academic Press, New York, (1979) pp. 413-422. The only known function of these enzymes is the oxidative deamination of biogenic amine transmitters. MAO inhibitors have been used in humans to treat hypertension and depression, but many of these drugs, especially those that selectively inhibit MAO-A, have toxic side effects due to failure to degrade amines that can act as false transmitters and thereby disrupt cardiovascular function (Murphy, Adv. Pharmacol and Chemother. 14:72 (1977)).

MAO-A and MAO-B activities can be measured in cultured skin jgbroblasts and platelets, respectively (Breakefield el al., Science 192:1018 (1976); Donnelly el al., Pharmacol. 26:853 (1977)). Under carefully controlled growth and assay conditions these activities are stable for an individual and are largely genetically determined; levels among normal individuals vary over 50-fold (Murphy el al., Biochem. Med. 16:254 (1976);

Gershon el al., in Enzymes and Neurolransmiiters in Mental Disease, E. Usdin, P. Sourkes, M.B.H. Youdin Eds. (London) 281-299 (1980); Breakefield el al., Psychiatry Res. 2:307 (1980)); for review see Hsu el al., J. Neurochem. 53:12 (1989)). Low normal levels of MAO-B activity have been reported in individuals with a number of neuropsychiatric diseases, including alcoholism (Tabakoff el al., N. Engl. J. Med. 318:134 (1988); Sullivan etal, Biol Psych. 27:911 (1990)) and schizophrenia (Murphy Adv. Pharmacol and Chemother. 14:12 (1977)). High normal levels of MAO-B activity have been reported in Parkinson's Disease patients (Danielczyk el al., Acta Psychiatr. Scand. 75:730 (1988); Steventon et al., 1989). Current therapy for the symptoms of Parkinson's Disease focuses on increasing dopaminergic transmission by the surviving neurons through administration of the precursor, L-dopa, and by giving drugs that act as dopamine agonists at postsynaptic sites (Jankovic and Marsden, in Parkinson's Disease and Movement Disorders, J. Jankovic, E. Tolosa Eds.

(Urban and Schwarzenberg, Baltimore-Munich, 95-119 (1988); Youdim et al., Adv. Neurol 45:121 (1986); Knoll, el al., Adv. Neurol 45:107 (1986)). To tiy to protect nigral neurons from further degeneration in Parkinson's Disease, a selective inhibitor of MAO-B, deprenyl, has been given to patients which appears to slow the progression of the disease (Birkmayer el al., Mod. Probl. Pharmacopsychiatr. 19:170 (1983); Golbe el al., Clin. Neuropharmacol 11:45 (1988); Tetrad el al., Science 245:519 (1989); Parkinson Study Group, New Engl J. Med. 321:1364 (1989)). In order to maximize therapy it will be necessary to identify individuals early in the course of the disease and try to prevent neuronal death prior to onset of symptoms. This strategy will require identifying those individuals at risk and the factors that lead to neuronal death.

To date there is no diagnostic available to recognize a propensity for PD until after the onset of symptoms. Furthermore, currently there is no effective therapy to slow or prevent the progressive worsening of the disease. Thus, methods to diagnose and treat symptoms of PD are needed.

Related Literature

Research on the monoamine oxidase genes has been reported in Ozehus et al. Genomics Vol. 3:53-58 (1988); HSU et al. Journal of

Neurochemistry Vol. 53:12-18 (1989); and, PCT Publication No.

WO90/00195. Msp I RFLP for human MAO-A gene was reported by

OzeHus et al. Nucleic Acids Research 17:10516 (1989). Black et al. Nucleic Acids Research Vol. 19:689 (1991) report on a dinucleotide repeat polymorphism at the MAO-A locus.

SUMMARY OF THE INVENTION

Methods and compositions for the diagnosis and treatment of Parkinson's Disease are disclosed. Utilizing methods to determine MAO allele status of an individual, alleles associated with activity states of MAO and with Parkinson's Disease (PD) is provided. Therefore, individuals at risk for developing Parkinson's Disease can be determined. This is the first assessment of MAO-A in Parkinson's Disease and predicts that high activity or some other genetically determined property of this isozyme is protective against Parkinson's disease, possibly by degrading a neurotoxic compound taken in from the environment or generated internally.

Therapies are also provided for the prevention and/or treatment of Parkinson's Disease. DETAILED DESCRIPTION OF THE INVENTION

The present invention is drawn to methods to determine the propensity of an individual for developing Parkinson's Disease. By determining the inherited levels of MAO activity in an individual, the propensity to developing Parkinson's Disease can be assessed. The levels of MAO-A activity are largely determined by ailelic variations in the structural gene on the X chromosome. Results indicate that MAO-A activity levels are correlated with PD. Thus, methods and haplotypes for correlating MAO-A allele status with PD are provided. The methods are useful for determining the role of MAO-A and MAO-B in the onset and development of PD.

Levels of MAO-A activity can be determined, for example, from cultured human skin fibroblasts and placenta. See Breakefield el al., Science 192:1018-1020 (1976); Weyler and Salach, J. Biol. Chem. 260:13199-13207 (1985); and Edelstein el al., J. Neurochem. 31:1247-1254 (1978).

MAO-A alleles that are predictive of high activity are more frequent in normal individuals as compared to Parkinson's patients.

Accordingly, by determining the allelic status associated with high activity of MAO-A, markers can be determined which will be useful to predict the propensity for Parkinson's Disease.

Genetic probes or markers for the MAO-A allele can be determined by a variety of methods known in the art. Generally, any method capable of detecting polymorphisms in the nucleic acid molecule can be used. Techniques such as amplification of a desired region of the chromosome coupled with direct sequencing, or detection of base pair alterations by a variety of techniques, e.g., denaturing gradient gel electrophoresis, chemical mismatch cleavage, single strand conformation polymorphisms or differential hybridization to oligonucleotides or cleavage by restriction enzymes, or locations of polymorphisms on the chromosome by radiolabelling, fluorescent labelling, or enzyme labelling can also be utilized. As described below, (GT)n polymorphisms are highly informative in identifying markers. Also, restriction fragment length polymorphisms (RFLP's) can be used. RFLP's can be detected which correlate with high or low levels of MAO-A activity.

Once a genetic marker has been determined which is predictive for

MAO-A activity and/or Parkinson's Disease, one can screen an individual to determine the MAO-A allele status and susceptibility to PD. The presence of a particular allele status will be correlated with susceptibility or development of Parkinson's Disease.

The gene for MAO-A has been mapped to human chromosome X.

See, Ozelius et al. Genomics 3:53-58 (1988), herein incorporated by reference. Particularly, the gene has been mapped to chromosome position Xp p11.3. The complete nucleotide sequence and corresponding amino acid sequence of the human MAO-A cDNA is disclosed in PCT US89/02901, herein incorporated by reference. Accordingly, utilizing the nucleic acid sequence probes can be made to identify RFLP's after restriction endonuclease digestion. The use of RFLP's for genetic testing is well known in the art. For example, see Gusella, US 4,666,828, and

Donis-Keller el al., Cell. Vol. 51:319-337 (1987).

Generally, the method for analysis using RFLP's is as follows. A sample of the genetic material from individuals being studied is obtained. Preferably, a blood sample is used as a source of genetic material and analyzed. For detection of RFLP's, the DNA in the sample is digested with a given restriction endonuclease. Alternatively, the DNA sample may be digested by more than one enzyme. If necessary, the DNA may be amplified prior to RFLP analysis. After the digest is obtained, and the sample is separated by a standard technique, such as, for example, agarose gel electrophoresis, the separated bands are probed with the DNA fragment coding for the RFLP sequence.

For convenience, DNA probes may be synthesized utilizing the sequence of the MAO-A gene. It is noted that DNA or RNA probes can be utilized. Generally, a nucleotide probe will comprise about 5 to about 50 nucleotides, more generally about 10 to 30 nucleotides, preferably 10-

18 nucleotides.

(GT)n polymorphisms are discussed in Webber, J.L. Genomics 7:524-530 (1990). To determine (GT)n polymorphisms, the MAO-A gene or gene region is digested with a restriction endonuclease, subjected to gel electrophoresis and hybridized to a GT oligonucleotide. Those bands which hybridize with the GT oligonucleotide are subcloned and sequenced. Primers are made which correspond to the unique sequences flanking GT repeat regions. These primers are utilized in a potymerase chain reaction with DNA from an individual to determine the presence or absence of a particular polymorphism/marker. The polymerase chain reaction is well known in the art. (See, Mullis et al. Coldspring Symp. Quant. Biol 51:263-273 (1987), Earhlich et al. EP 50,424; EP 258017; EP 237, 362; Mullis, K. EP 201,184; Mullis el al. US 4,683,202; and, Loh et al, Science 243:211-222).

If the genetic sample to be analyzed is present in limited amounts, the nucleic acid within such sample may be amplified so as to increase the signal for subsequent analysis. Li el al., Nature 335:414-417 (1988), has reported the use of the polym erase chain reaction to co-amplify two genetic loci from a single sperm to levels capable of genetic analysis.

DNA and/or RNA may also be amplified using an amplifiable RNA sequence as a probe and Qβ -replicase (Chu el al., Nucl Acids Res.

14:5591-5603 (1986)).

Probes can be labeled by standard labeling techniques which are generally well known in the art. Labels such as radiolabel, enzyme label, fluorescent label, bϊotϊn-avidin label, and the like, can be utilized which allow for detection after hybridization. For example, see Leary el al.,

Proc Natl. Acad. Sci, USA 80:4045 (1983).

Allelic markers are then correlated with activity levels of MAO-A. In this manner, it is possible to identify MAO alleles which correspond to different activity levels in the normal population and MAO alleles which correspond with the development of Parkinson's disease. Furthermore, knowledge of the gene structure will allow elucidation of differences in sequence which determine levels of activity directly.

Once haplotypes are identified which correlate with the onset or development of Parkinson's disease, individuals in the population can be screened for the particular haplotype or marker associated with the disease. Thus, the MAO allele status of an individual can be determined by a blood test, which indicates whether the individual may be at risk for developing Parkinson's disease.

Generally, to determine the allele status, blood will be collected from the individual, and the nucleic acid analyzed for the presence or absence of a particular marker associated with Parkinson's disease. The present work has indicated a role for MAO-A in Parkinson's disease. This is the first indication that activity levels of the MAO-A enzyme may be correlated with the onset or development of Parkinson's disease. However, now that the correlation has been discovered, therapies can be developed to prevent the progression of symptoms of the disease.

Therapies will be aimed at increasing the MAO-A enzyme activity levels in Parkinson's patients. Thus, possible therapies include but are not limited to supplying MAO-A as a pharmaceutical as well as the transfer of the MAO-A gene into individuals for expression.

Pharmaceutical preparations can be employed to administer to individuals suffering from Parkinson's disease. Such pharmaceutical preparations will contain a therapeutically effective amount of MAO-A. By therapeutically effective amount is intended an amount of the enzyme to 7100 pmol/min/mg protein in the critical tissue, e.g., gut or endothelial. cells lining blood vessels.

Methods useful for administering the pharmaceutical composition include oral administration, subcutaneous, intravenous, grafting of cells with high MAO-A activity and the like. In a simple form, an encased form of MAO-A could be taken orally.

While a variety of oral administrations can be envisioned, one method of oral administration is to administer a recombinant yeast preparation wherein the yeast has been genetically modified to overexpress the MAO-A gene. Such a yeast preparation can be prepared since the MAO-A gene has been identified by methods available in the art. See, for example, Sambrook el al., eds., Molecular Cloning: A

Laboratory Manual, 2nd ed., Vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); U.S. Patent No. 4,775,622; and U.S. Patent No. 4,425,437.

A pharmaceutical composition may comprise in addition to the MAO-A, controlled release preparations, pharmaceutical carriers and the like. Controlled release preparations may be achieved through the use of polymers to complex or absorb MAO-A The controlled delivery may be achieved by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl pyrrolidone, etheylvinylacetate, methylcellulose, carboxymethyicellulose, or protamine sulfate, and the appropriate concentration of macromolecules as well as the methods of incorporation, in order to control release).

Another possible method useful in controlling the duration of action by controlled release preparations is incorporation of the enzyme or its functional derivatives into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic), or ethylene vinylacetate copolymers. Carriers and controlled release substances are discussed in Remington's Pharmaceutical Sciences (1980).

Alternatively, vectors can be developed to transfer the MAO-A gene into individuals with Parkinson's disease where the gene is expressed

For expression, the MAO-A gene can be operably linked into an expression vector and introduced into a host cell to enable the expression of MAO-A. The gene with appropriate regulatory regions will be provided in proper orientation and reading frame to allow for expression. Methods for gene and vector construction are known in the art See, generally, Sambrook et al eds, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, 2nd Edition, Cold Spring Harbor, N.Y. (1989) and the references cited therein.

Herpes, simplex virus vectors and retrovirus vectors may be utilized. See, Breakefield el al., Mol Neurobiol.1:3 39 (1988); Dobson el al., J. Virol

63:3844-3851 (1989); Ho el al., Proc. Natl. Acid Sci. USA 86:1596-1600 (1989); Friedman Science 244:1215-1280; and, Paleloa el al., Gene 80:138 (1989). Recently, an amplicon-type plasma-vector system based on Herpes Simplex Virus I has been used to achieve stable expression of the Lac-Z gene in cultured neurons. See, Staete el al., Proc. Natl. Acad. Sci. USA 82:694 (1985); Geiler el al., Science 241:1661 (1988); Geller el al., Proc. Natl. Acad. Sci. USA 87:1148 (1990).

Adenovirus vectors may also be utilized.

Reviews on retrovirus vectors can be found in: Cepko, C, in Neuromethods: Molecular Neurobiological Techniques, Vol. 16, Boulton el al., eds. (Clifton, NJ: Humana), pp. 177-219; and Gilboa el al., Bio Techniques 4:504-512 (1986).

Friedmann, T., Science 244:1215-1281 (1989), and Breakefield and Geller, Mol Neurobiol 1:339-379 (1987), discuss strategies for gene therapy, including retrovirus vectors.

Methods for grafting genetically modified cells (e.g., endothelial cells which have been genetically modified through transfection or infection to have high MAO-A activity) to the brain are discussed by: Gage el al., Neuroscience 23:795-807 (1987); Danos et al., Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988); Rosenberg el al., Science 242:1515-1518

(1988); Wolff et al., Proc. Natl. Acad. Sci. USA 86:9011-9014 (1989); and Shimohama et al., Molecular Brain Res. 5:271-278 (1989).

The method lends itself readily to the formation of kits which can be utilized in diagnosis. Such a kit would comprise a carrier being compartmentalized to receive in close confinement one or more containers wherein a first container may contain nucleic acid containing coding sequences which are capable of recognizing a given polymorphism, e.g., a RFLP. A second container may contain a different set of sequences coding for a second polymorphism. Other containers may also contain reagents useful in the localization of the labeled probes, such as enzyme substrates. Still other containers may contain restriction enzymes, buffers, primers, and the like.

The following experiments are offered by way of illustration and not by way of limitation. EXPERIMENTAL

Variations in levels of MAO-A activity appear to be determined in large part by allelic variations in the structural gene for this enzyme. Using two restriction fragment length polymorphisms (RFLPs) to mark alleles for MAO-A (Ozelius el al., Genomics, 3:53 (1988) and Ozelius et al., Nucleic Acids Res. 17:10516 (1989) a strong association between certain alleles and levels of enzyme activity in 40 control males has been demonstrated. The number of alleles at this locus has been expanded using a variable (GT)n repeat element (Black el al., Nucleic Acids Res. 19:689 (1991)). The "A(EcoRV-, MspI-)/114" haplotype is more frequent in lines with low activity (0.1-10 pmol/min/mg protein) than those with high activity (12-179 pmol/min/mg protein (X²=4.6, p = 0.03), but it also occurs in several lines with high activity (Table 1). Statistical analysis was carried out using StatView TM SE and graphics program [Abacus Concepts, Inc.].

An even stronger association was found between the "B or C(EcoRV ± Mspl + )/122" between the haplotypes and high MAO-A activity. In 33 control male lines evaluated only 1/15 (7%) with low activities had either of these haplotypes, while 8/20 (40%) of those with high activities had one or the other of them (X²:5.0; p=0.02). Of the four alleles for human MAO-A that have been sequenced to date through most or all of their coding sequence, no differences in deduced amino add composition has been revealed. Further studies will be required to establish whether the sequence differences determining activity levels are in coding or non-coding regions of the mRNA and/or in regulatory regions of the gene. Preliminary studies using a (GT)n for the MAOB gene, have not revealed any significant associations of MAOB alleles with levels of MAO-B activity measured in platelets from control males. A difference in the frequency of alleles for MAOA genes in Parkinson's Disease patients and control subjects could serve as a biologic marker for Parkinson's Disease and at the same time incriminate compounds acted upon by these enzymes as causative agents in this disease. Evaluation of the frequency of alleles for MAOA and MAOB in control and Parkinson's Disease populations allows a rapid means to establish whether variations in the structure of these genes have any role in this disease. While it is recognized that the methods of the invention are not dependent on any particular mechanism, variations in MAO activity could affect the levels or cellular distribution of compounds metabolized by this enzyme that can act as neurotoxins in Parkinson's Disease models.

The allelic status for MAOA and MAOB have been determined using DNA extracted from blood samples obtained from 128 male control subjects and 70 male Parkinson patients with onset over 50 years.

Controls consisted of 77 individuals from 1 to 47 yrs of age and 51 from 50 to 87 yrs. No statistically significant difference was found in the frequency of MAOA and MAOB alleles in these two control populations. All controls filled out a questionnaire about neurologic and psychiatric illness in themselves or their blood relatives and were only used if there was no history of a major disorder; many were also examined by a neurologist to determine that they were free of neurologic problems. Almost all controls were of mixed Caucasian background.

All Parkinson's Disease subjects met criteria for idiopathic Parkinson's Disease (Ward el al., Adv. Neurol. 55:245 (1990)) including two of the three cardinal signs (tremor at rest, bradykinesia, rigidity), improvement of symptoms when given L-DOPA (in the form of Sinemet), and no evidence of secondary Parkinsonism. Patients were excluded if they had major involvement of the motor system, other than the extr apyramidal system, suggesting atypical Parkinsonism, or if they had dementia. The most significant difference in the distribution of alleles was observed between total controls and Parkinson's Disease patients, with 33/128 controls (25%) bearing the "C/122" haplotype for MAOA predictive for high MAO-A activity, while only 4/70 (6%) of Parkinson patients had this allele (X² = 11.9; p = 0.0005) (Table 2). The most common MAOA allele in both the total control and Parkinson populations was "A/114", with 48/128 (38%) of controls and 24/70 (34%) of Parkinson patients bearing this allele. This allele is usually associated with low activity in controls, but also occurs with high activity. Further work is necessary to determine sequence variations that can distinguish these two activity states and to evaluate whether more of the Parkinson patients have the low activity form of the "A/114" allele, as would be predicted from the finding of the high activity alleles ("B and C/122") being more common in controls. There were no significant differences observed in the frequency of MAOB alleles between Parkinson and control groups (Table 3). As anticipated from their nearness of MAOA and MAOB genes in the genome (within a few hundred kb), there is a significant association between certain alleles for MAOA and MAOB (data not shown). The differential distribution of MAOA alleles, and not MAOB alleles, in Parkinson's Disease and control groups thus appears to be even more striking.

This finding in Parkinson's Disease subjects of low representation of an MAOA allele associated with high activity has dramatic implications for the etiology and treatment of this disease. First, it strongly implicates an MAO-A-metabolized compound in the degenerative process. Under normal conditions, MAOA is high within dopaminergic neurons of the nigra, as well as in liver and placenta, and detoxifies endogenousry generated neurotoxins that are substrates for it. If Parkinson's Disease were caused by exogenous neurotoxic compounds capable of crossing the blood-brain barrier, MAO-A in the periphery may prevent its access to the nervous system. This latter hypothesis would be consistent with the theories that the Parkinson's Disease-producing compound is an environmental toxin (Barbeau el al., Adv. Neurol. 45:299 (1986); Tanner, ΗNS 12:49 (1989)). Second, this finding shows that individuals with increased risk for Parkinson's Disease can be identified prior to onset of symptoms by determining their MAOA allelic status. Thus, it is possible to undertake preventive therapy, by developing drugs, dietary supplements or gene transfer strategies aimed at increasing MAO-A activity. The data also raise the possibility that administration of MAO inhibitors to Parkinson's Disease patients may be detrimental to survival of dopaminergic neurons, to the extent that these inhibitors depress MAO-A activity. Further, to the extent that these toxins are also metabolized by MAO-B, inhibition of this isozyme might also be detrimental. Further studies at the biochemical, neuroanatomical and genetic levels are needed to understand the role of MAO-A in Parkinson's disease, but this study provides the first incriminatory evidence.

It is noted that the present invention is not limited by any particular theory associated with the development of Parkinson's disease.

a Patients were obtained as follows: LN Bur from Dr. Uta Francke, Yale University School of Medicine, New Haven, CT; 115 and 87 from Dr. Roy Bre g ,

Yale University School of Medicine; GM152, CM537, CM1906, GM1662 and CM2227 from Camden Cell Repository, Camden, NJ; On Ser, To Ser an d

Sal Mat from the American Type Culture Collection, Bethesda, MD; and 53 from Dr. J. Edwin Seegmiller, University of California School of Medicine ,

La Jolla, CA. The Lesch-Nyhan syndrome does not affect MAO activity in fibroblasts (Costa et al., 1980). All cell lines were studies in the proliferativ e

stage of growth in DMEM (GIBCO) with 10% FCS (GIBCO) (Edelstein et al., 1978). Lines were harvested 6-7 days after reaching con fluency.

b MAO activity was measured by a modification of the toluene extraction procedure of Wurtman and Axelrod (1963), as reported by Edelstein et al., 197 8 . Thirty micromoles [ethyl- H tryptamine HCl (25.5 Ci/mmol; NEN)] was used as substrate for MAO. Blank values were determined using clorgyline a s

inhibitor at a concentration of 104 M. The listed values refer to pmol/min/mg protein ±. SEM.

c Total genomic DNA was extracted from fibroblast homogenates, as described (Maniatis et al., 1989). For the Mspl RFLP 10 ug genomic DNA wa s

digested to completion with 40 U restriction endonuclease Mspl (New England Biolabs). Electrophoresis, blotting and hybridization to the 1,300 bp Xba l - EcoRI fragment of genomic clone A2R/D7 were carried out as described (Ozelius et al., 1988 and 1989). For the EcoRVI RFLP 500 ng genomic D N A

were incorporated in 50 ul of a reaction mix containing 50 mM KCl, 10 mM Tris (pH 8.3), 1.5 mM MgCl₂, 100 ug/gelatinl/ml, I mM of each dNTP, 20 0

nM of each primer, and 2.5 U of Taq DNA polymerase (Perkin-Elmer/Cetus). Thirty cycles of amplification (94°C for 1 min, 57°C for 2 min, 720 fo r

3 min) were carried out with a final extension time of 10 min (Saiki et al., 1988) using primers designated in Hotamisligil and Breakefield (in press). T h e

PCR-amplified DNA was extracted with cholorfbrm, precipitated with EtOH, and resuspended in H₂O for RFLP analysis. Approximately 300 ng DNA w a s

digested with restriction enzymes EcoRV (New England Biolabs, Beverly, MA). DNA fragments were electrophor esed through 1 % standard (EcoRV) o r

3% low-melting-temperature (FMC Bioproducts) plus 1 % standard agarose gels (Fnu 4HI) at 8.5 V/cm for 3 h. The gels were then stained with ethidiu m

bromide and DNA was visualized under [IV light. The (CT)n polymorphism was visualized by PCR amplification in the presence of [32P]?TP and resolutio n

of fragments on sequencing gels as described (Black et al., 1990). Primers were synthesized on a Milligen-BioSearch/Cyclone DNA synthesizer and purifi e d

by PAGE.

The foregoing description of the preferred embodiments of the invention have been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application to thereby enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto.

Claims

WHAT IS CLAIMED IS:

1. A method for determining a propensity for the development of Parkinson's disease in an individual, said method comprising:

analyzing DNA of said individual to determine the haplotype of MAO alleles, and

correlating said haplotype with development of Parkinson's disease.

2. The method according to claim 1, wherein said MAO allele is MAO-A.

3. The method of claim 1, wherein said nucleic acid is DNA.

4. The method of claim 1, wherein said nucleic acid is RNA.

5. A method for treating Parkinson's disease in an individual, said method comprising increasing the levels of MAO-A activity in said individual.

6. The method according to claim 5, wherein said method further comprises administering an effective amount of a pharmaceutical composition wherein said composition comprises MAO-A.

7. The method of claim 5, wherein said method comprises transferring a MAO-A gene into said individual.

8. The method of claim 7, wherein said MAO-A gene is transferred utilizing a retrovirus vector.

9. The method of claim 7, wherein said MAO-A gene is transferred utilizing a herpes simplex virus vector.

10. The method of claim 5, wherein said method comprises grafting of cells with high MAO-A activity.

11. The method of claim 10, wherein said cells are genetically modified.

12. The method of claim 10, wherein said cells are grafted into the brain of an individual diagnosed as having Parkinson's disease.

13. A method for determining the propensity of an individual to develop Parkinson's disease, said method comprising:

analyzing nucleic acid of chromosome X of said individual to determine the haplotype of said individual, and

comparing the haplotype of said individual with a haplotype which predicted for Parkinsons's disease.

14. A kit for the detection of Parkinson's disease in a subject, said kit comprising a carrier being compartmentalized to receive one or more containers in close confinement therein and further comprising:

a first container containing nucleic acid which is capable of identifying markers associated with Parkinson's disease.

15. The kit of claim 8, wherein said nucleic acid is capable of determining the haplotype of MAO alleles.