WO2012032519A2

WO2012032519A2 - Methods of diagnosing parkinson's disease

Info

Publication number: WO2012032519A2
Application number: PCT/IL2011/000720
Authority: WO
Inventors: Hermona Soreq; Hagai Bergman; David S. Greenberg; Zvi Israel; Lilach Soreq
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.; Hadasit Medical Research Services And Development Ltd.
Priority date: 2010-09-07
Filing date: 2011-09-07
Publication date: 2012-03-15
Also published as: WO2012032519A3

Abstract

A method of diagnosing Parkinson's disease in a subject is disclosed. The method comprises determining an expression level of a plurality of genes in a sample obtained from the subject, the plurality of genes comprising PJA1, TRAM1, PTPN1, PCBP2, NR2F1 and HNRPDL, wherein a statistically significant difference between expression levels of the plurality of genes in the sample obtained from the subject and expression levels of the plurality of genes in a control sample is indicative of Parkinson's disease.

Description

METHODS OF DIAGNOSING PARKINSON'S DISEASE

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to methods of diagnosing Parkinson's disease.

Parkinson's Disease (PD) is a progressive and incurable neurological disease most often beginning in the sixth decade of life. PD afflicts an estimated 4 million people worldwide is the most common neurodegenerative movement disorder and the second most common neurodegenerative disorder affecting more than 0.1% of the population over 40 years of age. Annual health care costs in the United States associated with PD have been estimated to be in excess of $6B. The core motor features of PD include bradykinesia (slowness of movement), akinesia (difficulty initiating movement), rigidity, tremor, and loss of postural reflexes. The progressive neurodegeneration is the result of a steep decline in the number of neurons in the substantia nigra pars compacta (SNpc); this brain structure is responsible for generating dopamine (DA). When the amount of DA produced falls below 80% of normal, disruption occurs in various DA mediated brain circuits that involve the basal ganglia and medial premotor cortex and the first observed features of PD appear. These brain regions are critically involved in higher order aspects of movement control, cognitive functioning (e.g., memory, attention), and emotions. PD patients indicate a deficit in generating complex sequences of movements in the absence of an environmental cue. This deficit is present at the level of organizing sequential finger movements of the same effector and at the level of coordinating multiple effectors or body segments. Patients show particular deficits in performing sequential and simultaneous movements that require added planning, execution time or timing processes.

There is a great need for the development of a reliable diagnostic tool to improve promptness of diagnosis, definition of disease subtypes, and to monitor disease progression and demonstrate treatment efficacy in the case of disease modifying therapies and enable earlier treatment which may affect disease progression. Current biomarkers range from objective clinical tools, to neuroimaging, to 'wet' markers involving blood and cerebrospinal fluid. To date, all candidate biomarkers for PD have failed to be developed into a clinically useful tool. Ideally, a combination of sensitive markers will be needed, not only to predict the onset of PD, but also to help in subtype classification and to follow progression and to enable earlier treatment.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of diagnosing Parkinson's disease in a subject, the method comprising determining an expression level of a plurality of genes in a sample obtained from the subject, the plurality of genes comprising praja ring finger 1 (PJAl), translocation associated membrane protein 1 (TRAMl), protein tyrosine phosphatase 1 (PTPN1), poly(rc)-binding protein 2 (PCBP2), nuclear receptor subfamily 2, group F, member 1 (NR2F1) and heterogeneous nuclear ribonucleoprotein D-like (HNRPDL), wherein a statistically significant difference between expression levels of the plurality of genes in the sample obtained from the subject and expression levels of the plurality of genes in a control sample is indicative of Parkinson's disease.

According to an aspect of some embodiments of the present invention there is provided a method of predicting an efficacy of a medicament for treating Parkinson's disease (PD) in a subject, the method comprising comparing an expression level of a plurality of genes in a sample obtained from the subject prior to and following administration of the medicament, the plurality of genes comprising PJAl, TRAMl, PTPN1, PCBP2, NR2F1 and HNRPDL, wherein a statistically significant difference between expression levels of the plurality of genes in the sample obtained from the subject prior to administration of the medicament and expression levels of the plurality of genes in the sample obtained from the subject following administration of the medicament is indicative of an efficacious medicament.

According to an aspect of some embodiments of the present invention there is provided a method of diagnosing Parkinson's disease in a subject, the method comprising determining an expression of at least one gene in a sample'obtained from the subject being selected from the group consisting of SNCA, PARK7 and ASF (SFRS1), wherein a statistically significant difference between expression of a variant of the at least one gene in the sample obtained from the subject and expression of the variant of the at least one gene in a control sample is indicative of Parkinson's disease. According to an aspect of some embodiments of the present invention there is provided a method of diagnosing Parkinson's disease in a subject, the method comprising determining an expression of at least one gene in a sample obtained from the subject as set forth in Table 1, wherein a statistically significant difference between expression levels of the at least one gene in the sample obtained from the subject and an expression level of the identical gene in a control sample is indicative of Parkinson's disease.

According to an aspect of some embodiments of the present invention there is provided a method of treating Parkinson's in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent which increases an amount or an activity of at least one polypeptide encoded by a gene listed in Table 2 or 6.

According to an aspect of some embodiments of the present invention there is provided a method of predicting an efficacy of deep brain stimulation (DBS) for treating Parkinson's disease (PD) in a subject, the method comprising analyzing an expression level of at least one gene listed in Table 3 and/or acetylcholineasterase, wherein a statistically significant upregulation between an expression level of the at least one gene in a sample obtained from the subject and an expression level of the at least one gene in a control sample obtained from a non-diseased subject is indicative that DBS is efficacious for the treatment of Parkinson's disease in the subject.

According to an aspect of some embodiments of the present invention there is provided a polynucleotide array comprising at least 6 and no more than 100 polynucleotide sequences for determining a gene expression profile of a biological sample, wherein at least one of the sequences is selected capable of hybridizing with a transcription product of a polynucleotide sequence of PJA1, at least one of the sequences is selected capable of hybridizing with a transcription product of a polynucleotide sequence of TRAMl, at least one of the sequences is selected capable of hybridizing with a transcription product of a polynucleotide sequence of PTPN1, at least one of the sequences is selected capable of hybridizing with a transcription product of a polynucleotide sequence of PCBP2, at least one of the sequences is selected capable of hybridizing with a transcription product of a polynucleotide sequence of NR2F1 and at least one of the sequences is selected capable of hybridizing with a transcription product of a polynucleotide sequence of HNRPDL.

According to an aspect of some embodiments of the present invention there is provided a array comprising at least 6 and no more than 100 antibodies or antibody fragments for determining a gene expression profile of a biological sample, wherein at least one of the antibodies or antibody fragments is selected capable of binding with a protein product of PJA1, at least one of the antibodies or antibody fragments is selected capable of binding with a protein product of TRAM1, at least one of the antibodies or antibody fragments is selected capable of binding with a protein product of PTPN1, at least one of the antibodies or antibody fragments is selected capable of binding with a protein product of PCPB2, at least one of the antibodies or antibody fragments is selected capable of binding with a protein product of NR2F1 and at least one of the antibodies or antibody fragments is selected capable of binding with a protein product of HNRPDL.

According to some embodiments of the invention, when the gene is TRAM1,

PTPN1 or PCBP2 the difference is an increase and the control sample is derived from a non-diseased subject.

According to some embodiments of the invention, when the gene is PJA1, NR2F1 or HNRPDL, the difference is a decrease and the control sample is derived from a non-diseased subject.

According to some embodiments of the invention, the method further comprises analyzing an expression level of at least one additional gene set forth in Table 1, wherein a statistically significant difference between an expression level of the at least one additional gene in the sample obtained from the subject and an expression level of the additional gene in the control sample is further indicative of Parkinson's disease or an efficacious treatment.

According to some embodiments of the invention, the method further comprises analyzing an expression level of at least one additional gene selected from the group consisting of ATPase, class VI, type 11B (ATP11B), leucine rich repeat containing 8 family, member C (LRRC8C), Leucine rich repeat and Ig domain containing 4 (LING04), DNA-damage inducible 1, homolog 2 (DDK), family with sequence similarity 46, member C (FAM46C), coiled-coil domain containing 5 (CCDC5), aryl- hydrocarbon receptor nuclear translocator 2 (ARNT2), olfactory receptor, family 52, subfamily N, member 5 (OR52N5), adhesion molecule with Ig-like domain 3 (AMIG03), calmodulin binding transcription activator 1 (CAMTA1), oculomedin (OCLM), solute carrier family 26, member 8 (SLC26A8), chorionic somatomammotropin hormone-like 1 (CSHL1), leucine-rich repeat, immunoglobulin- like and transmembrane domains 1 (LRIT1), catenin beta-like 1 (CTNNBL1), nerve growth factor (NGF), G protein-coupled receptor 61 (GPR61), cyclin dependent kinase 10 (CDK10), zinc finger CCCH-type containing 7A (ZC3H7A), fumarylacetoacetate hydrolase (FAH), N-terminal EF-hand calcium binding protein 1 (NECAB1), carbonic anhydrase VII (CA7), SEC13 homolog (SEC13), LY6/PLAUR domain containing 6B (LYPD6B), EP400 N-terminal like (EP400NL), ATP-binding cassette, sub-family C (CFTR/MRP), member 13, pseudogene (ABCC13), transcript (AF130358.5), KIAA2026, zinc finger protein 257 (ZNF257), cyclin D binding myb-like transcription factor 1 (DMTF1), adaptor-related protein complex 1, sigma 2 subunit (AP1S2), vacuolar protein sorting 37 homolog A (VPS37A), MYB binding protein (P160) la (MYBBP1A), LA16c-60G3.8, discs, large (Drosophila) homolog-associated protein 5 (DLGAP5), cytoplasmic linker associated protein 1 (CLASP 1), phosphodiesterase 3 A, cGMP-inhibited (PDE3A), transketolase line 1 (TKTL1), MYCBP associated protein (MYCBPAP), USOl vesicle docking protein homolog (USOl) and capicua homolog (Drosophila) pseudogene 13 (CICP13), wherein a statistically significant difference between an expression level of the at least one additional gene in the sample obtained from the subject and an expression level of the additional gene in the control sample is further indicative of Parkinson's disease or an efficacious treatment.

According to some embodiments of the invention, the gene is ATP 1 IB, LING04, DD12, FAM46C, CCDC5, ARNT2, OR52N5, AMIG03, CAMTA1, OCLM, CTNNBL1, NGF, GPR61, CDK10, NECAB1, CA7, SEC13, LYPD6B, EP400NL, AF130358.5, KIAA2026, ZNF257, DMTF1, AP1S2, VPS37A, LRRC8C, MYBBP1A, LA16c-60G3.8, DLGAP5, the difference is a decrease and the control sample is derived from a non-diseased subject.

According to some embodiments of the invention, the gene is LRRC8C,

SLC26A8, CSHL1, LRIT1, ZC3H7A, FAH, CLASP1, PDE3A, TKTL1, MYCBPAP, USOl and CICP13 the difference is an increase and the control sample is derived from a non-diseased subject.

According to some embodiments of the invention, when the at least one gene is SNCA, a decrease in an expression level of a variant which encodes exons 2-3 and 4-5 is indicative of Parkinson's disease.

According to some embodiments of the invention, when the at least one gene is PARK7, an increase in an expression level of a variant which encodes exons 4-5 and 6- 7 is indicative of Parkinson's disease.

According to some embodiments of the invention, when the at least one gene is ASF, an increase in an expression level of a variant which encodes a 3' untranslated region (UTR) is indicative of Parkinson's disease.

According to some embodiments of the invention, the method further comprises analyzing an expression level of at least one additional gene set forth in Table 1, wherein a statistically significant difference between an expression level of the at least one additional gene in the sample obtained from the subject and an expression level of the additional gene in the control sample is further indicative of Parkinson's disease.

According to some embodiments of the invention, the method further comprises informing the subject of an outcome of the diagnosis.

According to some embodiments of the invention, the sample obtained from the subject is a white blood cell sample.

According to some embodiments of the invention, the control sample is age and sex-matched.

According to some embodiments of the invention, the control sample is obtained from a non-diseased subject.

According to some embodiments of the invention, the method further comprises corroborating the diagnosis by neurologically examining the subject or imaging a brain of the subject.

According to some embodiments of the invention, the analyzing an expression level is effected at the protein level.

According to some embodiments of the invention, the analyzing en expression level is effected at the polynucleotide level. According to some embodiments of the invention, at least one of the sequences is selected capable of hybridizing with a transcription product of a polynucleotide sequence with a gene selected from the group consisting of ATP11B, LR C8C, LING04, DDI2, FAM46C, CCDC5, ARNT2, OR52N5, AMIG03,CAMTA1, OCLM, SLC26A8, CSHL1, LRIT1, CTNNBL1, NGF, GPR61, CDK10, ZC3H7A, FAH, NECAB1, CA7 and SEC13.

According to some embodiments of the invention, at least one of the antibodies is selected capable of hybridizing with a protein product of a gene selected from the group consisting of ATP11B, LRRC8C, LING04, DDI2, FAM46C, CCDC5, ARNT2, OR52N5, AMIG03 ,C AMTA1 , OCLM, SLC26A8, CSHL1, LRIT1, CTNNBL1, NGF, GPR61, CDK10, ZC3H7A, FAH, NECABl, CA7 and SEC13.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testin of embodiments of the invention, exemplary methods and/or 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 are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGs. 1A-C illustrates the experimental design and workflow (A) Study participant's clinical parameters. Clinical parameters of age, white and red blood cells count and BMI were measured. (B) For patients, the average UPDRS-III (motor) score and levodopa equivalent dose (LDE) are given prior to, and following, DBS and following 1 hour stimulation cessation (note that LDE is identical both on and off stimulation). (C) RNA was hybridized to human exon_l_0_ST arrays. Leukocytes were fractionated from blood samples of HC (1) (n=6) and PD patients (n=7): (2) one day before undergoing DBS neurosurgery; (3) upon symptoms stabilization 6-18 weeks post-DBS while being on electrical stimulation; (4) following 1 hour discontinued stimulation.

FIG. 2 is an experimental and analysis flow. Statistical analyses included permutation (n=1000) t-test and was followed by functional post-hoc enrichment analysis. Bioinformatic validation included comparison of the detected genes to the results of identical analysis that was performed on the published 3' array data set GSE6613 whole blood transcripts in an early PD cohort, which included both healthy and neurological control samples. Functional ad-hoc gene-list independent GO analysis included Kolmogorov-Smirnov and discrete hypergeometric Fisher exact tests for detection of changed GO terms. Quantitative real time PCR served as validation for selected genes.

FIGs. 3A-B are graphs illustrating the proportion of increased and decreased genes and pathways in patients, post stimulation and upon off stimulation. The ratio of genes up- or down-regulated in patients compared to controls (left column) is inversed in stim-ON to more decreases than increases (middle column) and shows similar increases to decreases in stim-OFF samples (right column). (B) Larger numbers of BP and MF terms were enriched between stim-ON and -OFF than in patients compared to controls.

FIGs. 4A-E illustrates that DBS neurosurgery and OFF-stimulus states both reverse the PD leukocyte transcript profiles. (A) Hierarchical clustering (HCL) analysis on the genes detected as modified between PD patients pre-DBS to HC subjects (n=173, Table 1) classified correctly all samples according to sample type. Post-hoc functional analysis of the six top gene clusters revealed enriched GO BP terms. The majority of genes increased in PD compared to HC. Scales: green-to-purple, down- and up- regulation. Color scale denotes normalized signal expression intensity. Orange-yellow gradient: severity of UPDRS-III scale. Blue-red gradient: levels of levodopa equivalent dose medications (color is standardized to maximal value of 900). Rows and columns clustering achieved with Spearman's rank distance and average linkage method. (B) 22 (13%) of the genes detected as changed in PD patients pre-DBS compared to controls were also detected as changed following DBS during stimulation as compared with pre- DBS state (presented in A).(C) HCL analysis of the genes detected as differentially expressed following DBS (in stim-ON state) compared to pre-DBS state (n=465 genes). All the samples were clustered accurately by clinical state. The top level clusters were found as enriched in functional terms. The majority of genes decreased following STN- DBS surgery. Left bars: both the medication dosage (Levodopa Equivalent Dosage - LDE) and UPDRS motor scale decreased following DBS (t-test p<0.05). (D) HCL separated patients upon 1 hour stim-OFF from 1 hour earlier stim-ON state. Color scale denotes normalized signal expression intensity. The top dendrogram sub-clusters reflect enriched GO BP terms. See bottom marks for major modified terms. (E) Reversibility of transcript changes upon 1 hour stimulation discontinuation is reflected in 30% overlap between the genes detected as changed upon stim-ON and -OFF. Of the 351 OFF stimulation detected genes, 105 (30%) were genes identified as changed following STN- DBS stim-ON as compared with pre-DBS state.

FIGs. 5A-C are results of Quantitative real time PCR validation. (A) Schematic structure of the SNCA gene on chromosome 4. Strands are indicated by arrows and (+) and (-) signs; constitutive and alternative exons are noted by open and closed top cases. Regions amplified by qRT-PCR are marked above in red. Fold change and standard error is given for qRT-PCR of the changed gene area for exons 2-3 and 4-5 junctions. Human beta actin served as normalization control. Relative fold change is given for pre- STN-DBS patients (left column, gray), patients post-DBS Stim-ON (middle column, black) and post-DBS Stim-off (right column, white). (B) Scheme drawn to scale and qRT-PCR data for Exon 4-5 and Exon 6-7 regions of the PARK7 gene on chr. 1. Note Exon 4-5 increases from pre-DBS to stim-ON and more to stim-OFF (*: t-test p < 0.05), and Exon 6-7 decrease upon stim-OFF. (C) The SFRS1/ASF chr.17 gene and 3'-UTR qRT-PCR validation. Note increases from pre-DBS to stim-ON which persist under stim-OFF conditions. 0.5-1 μg (in Ι μΐ) from each RNA sample was converted to cDNA. 15 -μΐ amplification reactions conducted using Reverse transcriptase (ImProm-IITM, Promega, Madison, WI, USA). Each RT reaction included nuclease-free water (4 μΐ), ImProm-IITM 5X reaction buffer (4 μΐ), MgCL2 (Promega, 4 μΐ), dNTP (Ι μΐ, 10 μηι each), RNase inhibitor (Rnasin, Promega) (Ιμΐ) and RT enzyme (1 μΐ). Random primers added (0.5 μg), sample incubated in 70 °C (5 minutes), chilled and reaction mix added. Cycle program: 25 °C (5 minutes), 42 °C (1 hour) and 70 °C (15 minutes). Real time PCR contained (final volume 20 μΐ) cDNA (8 μΐ, 1 : 10), SYBR green (sigma, 10 μΐ), the appropriate primers (10 μηι, 1 μΐ each). qRT-PCR performed with ABi 7300 cycler and SDS software (Applied Biosystems, Inc.) on 4 biological and 3-6 technical repeats. Human beta-actin served as internal control.

FIGs. 6A-B illustrate that Ad-hoc GO analysis detects disease-associated and stimulus-reversible pathways. BP and MF GO terms were detected as significantly changed by gene-list independent functional analysis of exon arrays in either cumulative KS or discrete hypergeometric Fisher exact test (2-fold change threshold) (p<0.05). Three comparisons were conducted: PD pre-DBS compared to HC, PD stim-ON compared to pre-DBS state, and stim-OFF compared to stim-ON states. (A) (1) Ratio of up- to down-regulated detected terms in patients compared to controls (left column), patients pre-DBS compared to stim-ON state (middle column) and stim-OFF compared to stim-ON state (right column). (2) The percent of BP and (3) MF terms which changed in more than one state comparison. Categories uniquely changed: dark blue. Categories shared between each of the 3 comparisons or all of them together: pink, green, blue or lilac. (B) MF and BP categories that changed in all three comparisons: PD/HC, PD/stim-ON and stim-OFF/stim-ON. Note double inverse pattern changes (increases versus decreases). Purple: up-regulation; green: down-regulation. Gray: both up- and down-regulation (threshold test).

FIGs. 7A-C: 29 Transcripts signature based on stim-ON and stim-OFF effects (A) 29 transcripts changed between stim-ON and healthy control (HC), PD patients pre- DBS to stim-ON and stim-OFF to stim-ON (p<0.01). Those served for hierarchical classification (rows distance: Spearman's rank, column distance: Manhattan) which classified PD pre-STN-DBS treatment together with stim-OFF and stim-ON state with HC samples. Two controls and one stim-OFF patient were misclassified. (B) PCA discriminated stim-ON and HC samples together (green) as distinct from the pre-DBS and stim-OFF samples (pink) samples which were separately grouped together. (C) Average personal transcript fold change (of the 29 genes) correlated with the relative motor improvement as measured by the UPDRS-III scale (linear regression R = 0.902, ANOVA P = 0.005). Validation in whole blood 3' arrays of an early PD cohort (GSE6613) identified 6 of the genes (bold letters) as changed in the independent data set. Stars denote genes which were also modified in early PD patients (2) compared to HC (2 stars) or to patients with other neurological diseases that served as neurological controls (NC) (3 stars).

FIGs. 8A-B illustrate that a six-transcript signature classifies early PD patients from healthy controls and other neurological diseases. Shown is HCL based on the 6 signature genes detected as differentially expressed between all of the currently tested clinical states and which were also identified as changed in a larger cohort of early PD patients, at first diagnosis (starred in Figures 7A-C). (A) HCL discriminated advanced PD (n=7) from healthy control (n=6) subjects of the current study. (B) A less rigorous discrimination of advanced PD from stim-ON state (n=7) of the current study observed.

FIG. 9 illustrates the implicated mechanisms of action based on the modified transcript categories using a model based on the gene-list independent Kolmogorov- Smirnov and Fisher exact test functional analysis results. Shown are the four tested groups. The arrows below reflect increased intensity of cholinergic activities in PD, suppression of these activities following DBS ON-stimulus and their re-enhancement under OFF-stimulus. Consequent changes in the levels of acetylcholine (chemical structure) modulate its capacity to block transcriptional activation by the NFkB p50/p65 proteins (PDB structures) of interferons and pro-inflammatory cytokines (e.g. IL1).

FIGs. 10A-B illustrate that alternatively spliced exons discriminate PD patients from controls. Splicing index values normalized to the constitutive gene level expression served to classify the samples using Hierarchical classification. (A) PD patients were classified apart from control subjects correctly based on the normalized SI values of the 163 alternatively spliced probe-sets which interrogate 150 distinct genes (Splicing-Index t-test Benjamini and Hochberg FDR p<0.05). (B) Further restriction on the detected events to the highly significant ones (FDR adjusted p<0.005 and a 2-fold change) yielded 18 evens in 18 different genes the majority of which decreased in PD patients as compared with control samples.

FIG. 11 illustrates experimental design and patient parameters for Example 10. Seven male PD patients' blood leukocyte mRNA expression was measured using exon microarrays. Patients were samples pre- and post- DBS neurosurgery, and following one hour stimulation cessation. The motor Unified Parkinson's Disease Rating Scale (UPDRS) improved in all patients post-DBS (t-test p<0.05), and Levodopa Equivalent Dose (LDE) decreased (t-test p<0.05) post-DBS. Total white and red blood cell count did not differ pre- from post-DBS.

FIGs. 12A-B are pictorial splicing-index based classifications of post- from pre- DBS patients and OFF- from ON-Stim states. The splicing index values normalized to the constitute gene level expression of the detected exons (SI FDR p<0.05 and/or MiDAS p<0.05) served to classify the samples using Hierarchical classification (HCL) (with Euclidean distance metric and average linkage). (A) PD patients pre-DBS state was classified from post-DBS on stimulation state (Stim-ON) based on the SI signals of the 102 alternatively spliced probe-sets. In all the patients, the UPDRS-III score was improved, and medication dosage reduced post-DBS (left bars) (B) PD patients post- DBS on stimulation were fully classified apart from 1 hour off stimulation state (Stim- OFF). The medication dosage remained low, and UPDRS-III scale worsened following OFF stimulation (left bar).

FIG. 13 is a scatter plot illustrating that pre-DBS alternative splicing patterns correlates with DBS efficacy. Positive correlation between microelectrode recording NRMS during the neurosurgery and the alternative splicing changes pre-DBS as compared with HC detected (R square = 0.645, p=0.03). The Normalizes Root Mean Square (NRMS) correlated with UPDRS-III score (R square:, p=0.046,). NRMS were averages for the left and right trajectories. Values were calculated for each patient (n=7). The correlation was computed using linear regression.

FIGs. 14A-B are graphs illustrating that motor improvement post-DBS and DBS efficacy correlates with post-DBS alternative splicing changes. Correlation between the UPDRS-III relative improvement post- compared to pre-DBS (R Square: 0.503, A) and the microelectrode recording NRMS (R square: 0.58, p=0.046, B) to the relative alternative splicing change magnitude in the detections post- compared to pre-DBS. NRMS were averages for the left and right trajectories. Values were calculated for each patient (n=7). The correlation was computed using linear regression.

FIG. 15 is a graph illustrating the activity of acetylcholinesterase prior to and following DBS treatment in 7 samples from Parkinson's patients and 6 control samples. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to a method of diagnosing and treating Parkinson's disease.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

The present inventors analyzed in vivo changes in mRNA leukocyte samples from human control samples and paired sample of Parkinson's patients pre- and post- surgery using high resolution exon arrays. The results allowed the present inventors to compose a list of genes that could be used as a molecular diagnostic signature and also as a screen for monitoring disease progression and efficacy of therapy. Specifically, the present inventors have found that analysis of a minimum set of six genes may be carried out so to accurately predict if a patient has Parkinson's disease (Figures 8A-B).

In addition, changes in paired mRNA blood samples from patients undergoing a neurological intervention procedure (prior to, and following deep brain stimulation (DBS) treatment, ON- and OFF-electrical stimulation) were investigated. Multiple transcripts were changed by the disease, upon stimulation and upon stimulation cessation. The present inventors deduced that knowledge of such transcripts allow for the prediction of which genes may be advantageously regulated for the treatment of Parkinsons' disease.

Thus, according to one aspect of the present invention, there is provided a method of diagnosing Parkinson's disease. The method comprises determining an expression level of one or more genes in a sample obtained from the subject, wherein a statistically significant difference (upregulation or downregulation) between expression levels of the plurality of genes in the sample obtained from the subject and expression levels of the plurality of genes in a control sample is indicative of Parkinson's disease.

As used herein, the term "diagnosing" refers to classifying Parkinson's disease (PD, determining a severity of PD (stage), monitoring PD progression, forecasting an outcome of the PD and/or prospects of recovery.

According to a particular embodiment, the genes listed herein may be used for predicting an efficacy of a medicament for treating Parkinson's disease (PD) in a subject, the method comprising comparing an expression level of a plurality of genes in a sample obtained from the subject prior to and following administration of the medicament, wherein a statistically significant difference between expression levels of the plurality of genes in the sample obtained from the subject prior to administration of the medicament and expression levels of the plurality of genes in the sample obtained from the subject following administration of the medicament is indicative of an efficacious medicament.

Control sample may be taken from isolated or cultured white blood cells (fresh or de-frosted, after having being frozen (-80°C) for about up to a year), or samples obtained from individuals not affected with Parkinson's. According to one embodiment, the control samples are taken from age and se -matched healthy subjects. According to another embodiment, the samples comprise white blood cells. Methods of isolating white blood cells are known in the art (see for example, the Examples section below). A substantial difference is preferably of a magnitude that is statistically significant. In particularly preferred embodiments, the marker gene is increased or decreased relative to control samples by at least 1.5-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold or more. Similarly, one skilled in the art will be well aware of the fact that a preferred detection methodology is one in which the resulting detection values are above the minimum detection limit of the methodology utilized.

As is further described in the Examples section which follows, the genes listed in Tables 1-6 were identified in white blood cells. As such, the sample obtained from the individual is preferably a white blood sample or any sample which includes blood cells such as T-cells. In a preferred embodiment, the sample is blood, thymus, spleen, lymph, pus, or bone marrow. However, it will be apparent to one skilled in the art that white blood cells may be present as an infiltrate in many other tissues, and that such tissues may also serve as samples in which the presence, activity, and/or quantity of the markers of the invention may be assessed. The tissue samples containing one or more of the markers themselves may be useful in the methods of the invention, and one skilled in the art will be well aware of methods by which such samples may be conveniently obtained, stored, preserved and processed. For further description relating to collection and processing of blood samples please see the Examples section which follows. As is detailed in the Examples section below, analysis of white blood cell genes differentially expressed in Parkinson's disease_^ or following deep brain stimulation, according to the methods described herein, revealed groups of genes of specific interest to Parkinson's disease.

The genes presented herein are referred to by a gene symbol number.

Knowledge of the gene sequence may be gleaned from various sources including online sources such as Ensembl Genome Browser, Genbank, Genecards etc.

Additional sequence data may be obtained from sources such as Affymetrix. Since the lists of genes were obtained using GeneChip™ Exon_1.0_ST_Array (Catalogue No. 900649-51), probe sequence data may be obtained from Affymetrix. Typically, the genes referred to herein are capable of hybridizing to at least one of these probes. Probe sequences are available online in the HuEx-l_0-st-v2.r2.pgf file.

According to one embodiment, when PJAl, TRAM1, PTPN1, PCBP2, NR2F1 or HNRPDL is differentially expressed, this is indicative of Parkinson's disease.

More specifically, Parkinson's disease may be diagnosed when the expression of any of the genes TRAM1, PTPN1 or PCBP2 is increased (e.g. by at least 1.5 fold, 2 fold, 5 fold or more) compared to a control sample derived from a non-diseased subject and the expression of any of the PJAl, NR2F1 and HNRPDL (e.g. by at least 1.5 fold, 2 fold, 5 fold or more) is decreased compared to a control sample derived from a non- diseased subject.

Other contemplated genes that may be analyzed include ATP11B, LRRC8C, LING04, DDI2, FAM46C, CCDC5, ARNT2, OR52N5, AMIG03, CAMTA1, OCLM, SLC26A8, CSHL1, LRIT1, CTNNBL1, NGF, GPR61, CDK10, ZC3H7A, FAH, NECABl, CA7, SEC13, LYPD6B, EP400NL, AF130358.5, KIAA2026, ZNF257, DMTF1, AP1S2, VPS37A, MYBBP1A, LA16c-60G3.8, DLGAP5, CLASP1, PDE3A, TKTL1, MYCBPAP, USOl or CICP13.

More specifically, when the gene is ATP11B, LING04, DD12, FAM46C, CCDC5, ARNT2, OR52N5, AMIG03, CAMTA1, OCLM, CTNNBL1, NGF, GPR61, CDK10, NECABl, CA7, SEC13, LYPD6B, EP400NL, AF130358.5, KIAA2026, ZNF257, DMTF1, AP1S2, VPS37A, LRRC8C, MYBBP1A, LA16c-60G3.8 or DLGAP5, a decrease in expression compared to a sample from a non-diseased subject is indicative of Parkinson's; and when the gene is LRRC8C, SLC26A8, CSHL1, LRIT1, ZC3H7A, FAH, CLASP1, PDE3A, TKTL1, MYCBPAP, USOl or CICP13 an increase in expression compared to a sample from a non-diseased subject is indicative of Parkinson's disease.

Other contemplated genes that may be analyzed according to the present invention for the diagnosis of Parkinson's disease are listed in Table 1 of the Examples section herein below.

It will be appreciated that although a single marker can be used for diagnosis, diagnostic accuracy typically increases with an increase in the number of markers utilized.

As such, the diagnostic method of the present invention preferably utilizes a marker set that can range anywhere from 1 gene to 200 genes. For example, the present method can utilize at least 2, 3, 4, 5, 6, 10, at least 50, at least 100, at least 200 genes each independently selected from the group consisting of the genes listed in the Examples section herein below.

The markers sets utilized can be selected according to a statistical significance or fold change thereof, a higher significance and higher fold change indicating higher probability of marker accuracy. Alternatively, markers can be selected according to shared features of the marker gene. For example, gene markers of similar cellular function (e.g., genes of a signaling pathway such as apoptosis) or markers displaying similar activity (e.g., enzymes of the same enzyme family) can be grouped into specific marker sets. See for example Figure 6A which illustrates functions that changed inversely in pre-deep brain stimulation DBS patients, following DBS and upon stimulation cessation.

Each marker set may be considered individually, although it is within the scope of the invention to provide combinations of two or more marker sets for use in the methods of the invention to increase the confidence of the analysis.

Thus, according to one embodiment, each of PJA1, TRAM1, PTPN1, PCBP2, NR2F1 or HNRPDL (as a single marker set) are analyzed for the diagnosis of Parkinson's disease.

According to another embodiment, each of the genes listed in Table 4 are analyzed for the diagnosis of Parkinson's disease. It will be appreciated that the methods described in the present application can be effected on the RNA or protein level.

Oligonucleotides based on the nucleotide sequence of a marker gene or of a nucleic acid molecule encoding a marker polypeptide of the invention can be used to detect transcripts or genomic sequences corresponding to the marker gene(s) and/or marker polypeptide(s) of the invention or to particular target sequences on the marker gene as further detailed herein below.

According to one embodiment, the oligonucleotide is a probe. As used herein, the term "probe" refers to an oligonucleotide which hybridizes to one of the gene sequences listed herein to provide a detectable signal under experimental conditions Preferably, the probe does not hybridize to non relevant sequences to provide a detectable signal under identical experimental conditions.

The probes of this embodiment of this aspect of the present invention may be, for example, affixed to a solid support (e.g., arrays or beads).

According to a particular embodiment, the array comprises probes for detection of at least 6 genes - (e.g. PJA1, TRAM1, PTPN1, PCBP2, NR2F1 and HNRPDL).

According to still another embodiment, the array comprises probes for detection of the genes listed in Table 4.

Preferably, the arrays of the present invention comprise probes for detecting no more than 40 genes, 50 genes, 60 genes, 70 genes, 80 genes, 90 genes or even no more than 100 genes.

According to another embodiment, the oligonucleotide is a primer of a primer pair. As used herein, the term "primer" refers to an oligonucleotide which acts as a point of initiation of a template-directed synthesis using methods such as PCR (polymerase chain reaction) or LCR (ligase chain reaction) under appropriate conditions (e.g., in the presence of four different nucleotide triphosphates and a polymerization agent, such as DNA polymerase, RNA polymerase or reverse-transcriptase, DNA ligase, etc, in an appropriate buffer solution containing any necessary co-factors and at suitable temperature(s)). Such a template directed synthesis is also called "primer extension". For example, a primer pair may be designed to amplify a region of DNA using PCR. Such a pair will include a "forward primer" and a "reverse primer" that hybridize to complementary strands of a DNA molecule and that delimit a region to be 000720

18

synthesized/amplified. A primer of this aspect of the present invention is capable of amplifying, together with its pair (e.g. by PCR) one of the gene sequences listed herein to provide a detectable signal under experimental conditions and which does not amplify non relevant sequences to provide a detectable signal under identical experimental conditions.

According to additional embodiments, the oligonucleotide is about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. While the maximal length of a probe can be as long as the target sequence to be detected, depending on the type of assay in which it is employed, it is typically less than about 50, 60, 65, or 70 nucleotides in length. In the case of a primer, it is typically less than about 30 nucleotides in length. In a specific preferred embodiment of the invention, a primer or a probe is within the length of about 18 and about 28 nucleotides. It will be appreciated that when attached to a solid support, the probe may be of about 25-70, 75, 80, 90, 100, or more nucleotides in length.

The oligonucleotide of this aspect of the present invention need not reflect the exact sequence of the gene sequence (i.e. need not be fully complementary), but must be sufficiently complementary to hybridize with the gene specific nucleic acid sequence under the particular experimental conditions. Accordingly, the sequence of the oligonucleotide typically has at least 70 % homology, preferably at least 80 %, 90 %, 95 %, 97 , 99 % or 100 % homology, for example over a region of at least 13 or more contiguous nucleotides with the target nucleic acid sequence. The conditions are selected such that hybridization of the oligonucleotide to the target nucleic acid sequence is favored and hybridization to other non-target nucleic acid sequences is minimized.

By way of example, hybridization of short nucleic acids (below 200 bp in length, e.g. 13-50 bp in length) can be effected by the following hybridization protocols depending on the desired stringency; (i) hybridization solution of 6 x SSC and 1 % SDS or 3 M TMAC1, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 μg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 1 - 1.5 °C below the Tm, final wash solution of 3 M TMAC1, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 °C below the Tm (stringent hybridization conditions) (ii) hybridization solution of 6 x SSC and 0.1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 μ^πιΐ denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 2 - 2.5 °C below the Tm, final wash solution of 3 M TMAC1, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 °C below the Tm, final wash solution of 6 x SSC, and final wash at 22 °C (stringent to moderate hybridization conditions); and (iii) hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 g/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature at 2.5-3 °C below the Tm and final wash solution of 6 x SSC at 22 °C (moderate hybridization solution).

Various considerations must be taken into account when selecting the stringency of the hybridization conditions. For example, the more closely the oligonucleotide reflects a sequence that is present in the non-target nucleic acid, the higher the stringency of the assay conditions should be, although the stringency must not be too high so as to prevent hybridization of the oligonucleotides to the target specific nucleic acid sequence. Further, the lower the homology of the oligonucleotide to the target specific nucleic acid sequence, the lower the stringency of the assay conditions should be, although the stringency must not be too low to allow hybridization to non target specific nucleic acid sequences.

Oligonucleotides of the invention may be prepared by any of a variety of methods (see, for example, J. Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989, 2.sup.nd Ed., Cold Spring Harbour Laboratory Press: New York, N.Y.; "PCR Protocols: A Guide to Methods and Applications", 1990, M. A. Innis (Ed.), Academic Press: New York, N.Y.; P. Tijssen "Hybridization with Nucleic Acid Probes- -Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II)", 1993, Elsevier Science; "PCR Strategies", 1995, M. A. Innis (Ed.), Academic Press: New York, N.Y.; and "Short Protocols in Molecular Biology", 2002, F. M. Ausubel (Ed.), 5.sup.th Ed., John Wiley & Sons: Secaucus, N.J.). For example, oligonucleotides may be prepared using any of a variety of chemical techniques well-known in the art, including, for example, chemical synthesis and polymerization based on a template as described, for example, in S. A. Narang et al., Meth. Enzymol. 1979, 68: 90-98; E. L. Brown et al., Meth. Enzymol. 1979, 68: 109-151; E. S. Belousov et al., Nucleic Acids Res. 1997, 25: 3440-3444; D. Guschin et al., Anal. Biochem. 1997, 250: 203-211; M. J. Blommers et al., Biochemistry, 1994, 33: 7886-7896; and K. Frenkel et al., Free Radic. Biol. Med. 1995, 19: 373-380; and U.S. Pat. No. 4,458,066.

For example, oligonucleotides may be prepared using an automated, solid-phase procedure based on the phosphoramidite approach. In such a method, each nucleotide is individually added to the 5'-end of the growing oligonucleotide chain, which is attached at the 3'-end to a solid support. The added nucleotides are in the form of trivalent 3'- phosphoramidites that are protected from polymerization by a dimethoxytriyl (or DMT) group at the 5 '-position. After base-induced phosphoramidite coupling, mild oxidation to give a pentavalent phosphotriester intermediate and DMT removal provides a new site for oligonucleotide elongation. The oligonucleotides are then cleaved off the solid support, and the phosphodiester and exocyclic amino groups are deprotected with ammonium hydroxide. These syntheses may be performed on oligo synthesizers such as those commercially available from Perkin Elmer/ Applied Biosystems, Inc. (Foster City, Calif.), DuPont (Wilmington, Del.) or Milligen (Bedford, Mass.). Alternatively, oligonucleotides can be custom made and ordered from a variety of commercial sources well-known in the art, including, for example, the Midland Certified Reagent Company (Midland, Tex.), ExpressGen, Inc. (Chicago, 111.), Operon Technologies, Inc. (Huntsville, Ala.), and many others.

Purification of the oligonucleotides of the invention, where necessary or desirable, may be carried out by any of a variety of methods well-known in the art. Purification of oligonucleotides is typically performed either by native acrylamide gel electrophoresis, by anion-exchange HPLC as described, for example, by J. D. Pearson and F. E. Regnier (J. Chrom., 1983, 255: 137-149) or by reverse phase HPLC (G. D. McFarland and P. N. Borer, Nucleic Acids Res., 1979, 7: 1067-1080).

The sequence of oligonucleotides can be verified using any suitable sequencing method including, but not limited to, chemical degradation (A. M. Maxam and W. Gilbert, Methods of Enzymology, 1980, 65: 499-560), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (U. Pieles et al., Nucleic Acids Res., 1993, 21: 3191-3196), mass spectrometry following a combination of alkaline phosphatase and exonuclease digestions (H. Wu and H. Aboleneen, Anal. Biochem., 2001, 290: 347-352), and the like. As already mentioned above, modified oligonucleotides may be prepared using any of several means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc), or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc). Oligonucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc), intercalators (e.g., acridine, psoralen, etc), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc), and alkylators. The oligonucleotide may also be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the oligonucleotide sequences of the present invention may also be modified with a label.

In certain embodiments, the detection probes or amplification primers or both probes and primers are labeled with a detectable agent or moiety before being used in amplification/detection assays. In certain embodiments, the detection probes are labeled with a detectable agent. Preferably, a detectable agent is selected such that it generates a signal which can be measured and whose intensity is related (e.g., proportional) to the amount of amplification products in the sample being analyzed.

The association between the oligonucleotide and detectable agent can be covalent or non-covalent. Labeled detection probes can be prepared by incorporation of or conjugation to a detectable moiety. Labels can be attached directly to the nucleic acid sequence or indirectly (e.g., through a linker). Linkers or spacer arms of various lengths are known in the art and are commercially available, and can be selected to reduce steric hindrance, or to confer other useful or desired properties to the resulting labeled molecules (see, for example, E. S. Mansfield et al., Mol. Cell. Probes, 1995, 9: 145- 156).

Methods for labeling nucleic acid molecules are well-known in the art. For a review of labeling protocols, label detection techniques, and recent developments in the field, see, for example, L. J. Kricka, Ann. Clin. Biochem. 2002, 39: 114-129; R. P. van Gijlswijk et al., Expert Rev. Mol. Diagn. 2001, 1: 81-91; and S. Joos et al., J. Biotechnol. 1994, 35: 135-153. Standard nucleic acid labeling methods include: incorporation of radioactive agents, direct attachments of fluorescent dyes (L. M. Smith et al., Nucl. Acids Res., 1985, 13: 2399-2412) or of enzymes (B. A. Connoly and O. Rider, Nucl. Acids. Res., 1985, 13: 4485-4502); chemical modifications of nucleic acid molecules making them detectable immunochemically or by other affinity reactions (T. R. Broker et al., Nucl. Acids Res. 1978, 5: 363-384; E. A. Bayer et al., Methods of Biochem. Analysis, 1980, 26: 1-45; R. Langer et al., Proc. Natl. Acad. Sci. USA, 1981, 78: 6633-6637; R. W. Richardson et al., Nucl. Acids Res. 1983, 11: 6167-6184; D. J. Brigati et al., Virol. 1983, 126: 32-50; P. Tchen et al., Proc. Natl. Acad. Sci. USA, 1984, 81: 3466-3470; J. E. Landegent et al., Exp. Cell Res. 1984, 15: 61-72; and A. H. Hopman et al., Exp. Cell Res. 1987, 169: 357-368); and enzyme-mediated labeling methods, such as random priming, nick translation, PCR and tailing with terminal transferase (for a review on enzymatic labeling, see, for example, J. Temsamani and S. Agrawal, Mol. Biotechnol. 1996, 5: 223-232). More recently developed nucleic acid labeling systems include, but are not limited to: ULS (Universal Linkage System), which is based on the reaction of mono-reactive cisplatin derivatives with the N7 position of guanine moieties in DNA (R. J. Heetebrij et al., Cytogenet. Cell. Genet. 1999, 87: 47-52), psoralen-biotin, which intercalates into nucleic acids and upon UV irradiation becomes covalently bonded to the nucleotide bases (C. Levenson et al., Methods Enzymol. 1990, 184: 577-583; and C. Pfannschmidt et al., Nucleic Acids Res. 1996, 24: 1702-1709), photoreactive azido derivatives (C. Neves et al., Bioconjugate Chem. 2000, 11: 51-55), and DNA alkylating agents (M. G. Sebestyen et al., Nat. Biotechnol. 1998, 16: 568-576).

Any of a wide variety of detectable agents can be used in the practice of the present invention. Suitable detectable agents include, but are not limited to, various ligands, radionuclides (such as, for example, 32P, 35S, 3H, 14C, .sup.1251, 1311, and the like); fluorescent dyes (for specific exemplary fluorescent dyes, see below); chemiluminescent agents (such as, for example, acridinium esters, stabilized dioxetanes, and the like); spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper and platinum) or nanoclusters; enzymes (such as, for example, those used in an ELISA, i.e., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase); colorimetric labels (such as, for example, dyes, colloidal gold, and the like); magnetic labels (such as, for example, Dynabeads.TM.); and biotin, dioxigenin or other haptens and proteins for which antisera or monoclonal antibodies are available.

In certain embodiments, the inventive detection probes are fluorescently labeled. Numerous known fluorescent labeling moieties of a wide variety of chemical structures and physical characteristics are suitable for use in the practice of this invention. Suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxy- fluorescein, 6 carboxyfluorescein or FAM), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X- rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine or TMR), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin. hydroxycoumarin and aminomethylcoumarin or AMCA), Oregon Green Dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514), Texas Red, Texas Red-X, Spectrum Red.TM., Spectrum Green.TM., cyanine dyes (e.g., Cy-3.TM., Cy-5.TM., Cy-3.5.TM., Cy- 5.5.TM.), Alexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), IRDyes (e.g., IRD40, IRD 700, IRD 800), and the like. For more examples of suitable fluorescent dyes and methods for linking or incorporating fluorescent dyes to nucleic acid molecules see, for example, "The Handbook of Fluorescent Probes and Research Products", 9th Ed., Molecular Probes, Inc., Eugene, Oreg. Fluorescent dyes as well as labeling kits are commercially available from, for example, Amersham Biosciences, Inc. (Piscataway, N.J.), Molecular Probes Inc. (Eugene, Oreg.), and New England Biolabs Inc. (Berverly, Mass.).

According to one embodiment, identification of target sequences may be carried out using an amplification reaction.

As used herein, the term "amplification" refers to a process that increases the representation of a population of specific nucleic acid sequences in a sample by producing multiple (i.e., at least 2) copies of the desired sequences. Methods for nucleic acid amplification are known in the art and include, but are not limited to, polymerase chain reaction (PCR) and ligase chain reaction (LCR). In a typical PCR amplification reaction, a nucleic acid sequence of interest is often amplified at least fifty thousand fold in amount over its amount in the starting sample. A "copy" or "amplicon" does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable but not complementary to the template), and/or sequence errors that occur during amplification.

A typical amplification reaction is carried out by contacting a forward and reverse primer (a primer pair) to the sample DNA together with any additional amplification reaction reagents under conditions which allow amplification of the target sequence.

The terms "forward primer" and "forward amplification primer" are used herein interchangeably, and refer to a primer that hybridizes (or anneals) to the target (template strand). The terms "reverse primer" and "reverse amplification primer" are used herein interchangeably, and refer to a primer that hybridizes (or anneals) to the complementary target strand. The forward primer hybridizes with the target sequence 5' with respect to the reverse primer.

The term "amplification conditions", as used herein, refers to conditions that promote annealing and/or extension of primer sequences. Such conditions are well- known in the art and depend on the amplification method selected. Thus, for example, in a PCR reaction, amplification conditions generally comprise thermal cycling, i.e., cycling of the reaction mixture between two or more temperatures. In isothermal amplification reactions, amplification occurs without thermal cycling although an initial temperature increase may be required to initiate the reaction. Amplification conditions encompass all reaction conditions including, but not limited to, temperature and temperature cycling, buffer, salt, ionic strength, and pH, and the like.

As used herein, the term "amplification reaction reagents", refers to reagents used in nucleic acid amplification reactions and may include, but are not limited to, buffers, reagents, enzymes having reverse transcriptase and/or polymerase activity or exonuclease activity, enzyme cofactors such as magnesium or manganese, salts, nicotinamide adenine dinuclease (NAD) and deoxynucleoside triphosphates (dNTPs), such as deoxyadenosine triphospate, deoxyguanosine triphosphate, deoxycytidine triphosphate and thymidine triphosphate. Amplification reaction reagents may readily be selected by one skilled in the art depending on the amplification method used.

According to this aspect of the present invention, the amplifying may be effected using techniques such as polymerase chain reaction (PCR), which includes, but is not limited to Allele-specific PCR, Assembly PCR or Polymerase Cycling Assembly (PCA), Asymmetric PCR, Helicase-dependent amplification, Hot-start PCR, Intersequence- specific PCR (ISSR), Inverse PCR, Ligation-mediated PCR, Methylation-specific PCR (MSP), Miniprimer PCR, Multiplex Ligation-dependent Probe Amplification, Multiplex-PCR,Nested PCR, Overlap-extension PCR, Quantitative PCR (Q-PCR), Reverse Transcription PCR (RT-PCR), Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge PCR (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thennal 'step' to favour solid support priming), Thermal asymmetric interlaced PCR (TAIL-PCR), Touchdown PCR (Step-down PCR), PAN-AC and Universal Fast Walking.

The PCR (or polymerase chain reaction) technique is well-known in the art and has been disclosed, for example, in K. B. Mullis and F. A. Faloona, Methods Enzymol., 1987, 155: 350-355 and U.S. Pat. Nos. 4,683,202; 4,683,195; and 4,800,159 (each of which is incorporated herein by reference in its entirety). In its simplest form, PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences, using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA. A plurality of reaction cycles, each cycle comprising: a denaturation step, an annealing step, and a polymerization step, results in the exponential accumulation of a specific DNA fragment ("PCR Protocols: A Guide to Methods and Applications", M. A. Innis (Ed.), 1990, Academic Press: New York; "PCR Strategies", M. A. Innis (Ed.), 1995, Academic Press: New York; "Polymerase chain reaction: basic principles and automation in PCR: A Practical Approach", McPherson et al. (Eds.), 1991, IRL Press: Oxford; R. K. Saiki et al., Nature, 1986, 324: 163-166). The termini of the amplified fragments are defined as the 5' ends of the primers. Examples of DNA polymerases capable of producing amplification products in PCR reactions include, but are not limited to: E. coli DNA polymerase I, Klenow fragment of DNA polymerase I, T4 DNA polymerase, thermostable DNA polymerases isolated from Thermus aquaticus (Taq), available from a variety of sources (for example, Perkin Elmer), Thermus thermophilus (United States Biochemicals), Bacillus stereothermophilus (Bio-Rad), or Thermococcus litoralis ("Vent" polymerase, New England Biolabs). RNA target sequences may be amplified by reverse transcribing the mRNA into cDNA, and then performing PCR (RT-PCR), as described above. Alternatively, a single enzyme may be used for both steps as described in U.S. Pat. No. 5,322,770.

The duration and temperature of each step of a PCR cycle, as well as the number of cycles, are generally adjusted according to the stringency requirements in effect. Annealing temperature and timing are determined both by the efficiency with which a primer is expected to anneal to a template and the degree of mismatch that is to be tolerated. The ability to optimize the reaction cycle conditions is well within the knowledge of one of ordinary skill in the art. Although the number of reaction cycles may vary depending on the detection analysis being performed, it usually is at least 15, more usually at least 20, and may be as high as 60 or higher. However, in many situations, the number of reaction cycles typically ranges from about 20 to about 40.

The denaturation step of a PCR cycle generally comprises heating the reaction mixture to an elevated temperature and maintaining the mixture at the elevated temperature for a period of time sufficient for any double-stranded or hybridized nucleic acid present in the reaction mixture to dissociate. For denaturation, the temperature of the reaction mixture is usually raised to, and maintained at, a temperature ranging from about 85 °C. to about 100 °C, usually from about 90 °C to about 98 °C, and more usually from about 93 °C to about 96 °C for a period of time ranging from about 3 to about 120 seconds, usually from about 5 to about 30 seconds.

Following denaturation, the reaction mixture is subjected to conditions sufficient for primer annealing to template DNA present in the mixture. The temperature to which the reaction mixture is lowered to achieve these conditions is usually chosen to provide optimal efficiency and specificity, and generally ranges from about 50 °C to about °C, usually from about 55 °C. to about 70 °C, and more usually from about 60 °C to about 68 °C. Annealing conditions are generally maintained for a period of time ranging from about 15 seconds to about 30 minutes, usually from about 30 seconds to about 5 minutes.

Following annealing of primer to template DNA or during annealing of primer to template DNA, the reaction mixture is subjected to conditions sufficient to provide for polymerization of nucleotides to the primer's end in a such manner that the primer is extended in a 5' to 3' direction using the DNA to which it is hybridized as a template, (i.e., conditions sufficient for enzymatic production of primer extension product). To achieve primer extension conditions, the temperature of the reaction mixture is typically raised to a temperature ranging from about 65°C to about 75 °C, usually from about 67 °C. to about 73 °C, and maintained at that temperature for a period of time ranging from about 15 seconds to about 20 minutes, usually from about 30 seconds to about 5 minutes.

The above cycles of denaturation, annealing, and polymerization may be performed using an automated device typically known as a thermal cycler or thermocycler. Thermal cyclers that may be employed are described in U.S. Pat. Nos. 5,612,473; 5,602,756; 5,538,871; and 5,475,610 (each of which is incorporated herein by reference in its entirety). Thermal cyclers are commercially available, for example, from Perkin Elmer-Applied Biosystems (Norwalk, Conn.), BioRad (Hercules, Calif.), Roche Applied Science (Indianapolis, Ind.), and Stratagene (La Jolla, Calif.).

Amplification products obtained using primers of the present invention may be detected using agarose gel electrophoresis and visualization by ethidium bromide staining and exposure to ultraviolet (UV) light or by sequence analysis of the amplification product.

According to one embodiment, the amplification and quantification of the amplification product may be effected in real-time (qRT-PCR). Typically, QRT-PCR methods use double stranded DNA detecting molecules to measure the amount of amplified product in real time. As used herein the phrase "double stranded DNA detecting molecule" refers to a double stranded DNA interacting molecule that produces a quantifiable signal (e.g., fluorescent signal). For example such a double stranded DNA detecting molecule can be a fluorescent dye that (1) interacts with a fragment of DNA or an amplicon and (2) emits at a different wavelength in the presence of an amplicon in duplex formation than in the presence of the amplicon in separation. A double stranded DNA detecting molecule can be a double stranded DNA intercalating detecting molecule or a primer- based double stranded DNA detecting molecule.

A double stranded DNA intercalating detecting molecule is not covalently linked to a primer, an amplicon or a nucleic acid template. The detecting molecule increases its emission in the presence of double stranded DNA and decreases its emission when duplex DNA unwinds. Examples include, but are not limited to, ethidium bromide, YO- PRO-1, Hoechst 33258, SYBR Gold, and SYBR Green I. Ethidium bromide is a fluorescent chemical that intercalates between base pairs in a double stranded DNA fragment and is commonly used to detect DNA following gel electrophoresis. When excited by ultraviolet light between 254 nm and 366 nm, it emits fluorescent light at 590 nm. The DNA-ethidium bromide complex produces about 50 times more fluorescence than ethidium bromide in the presence of single stranded DNA. SYBR Green I is excited at 497 nm and emits at 520 nm. The fluorescence intensity of SYBR Green I increases over 100 fold upon binding to double stranded DNA against single stranded DNA. An alternative to SYBR Green I is SYBR Gold introduced by Molecular Probes Inc. Similar to SYBR Green I, the fluorescence emission of SYBR Gold enhances in the presence of DNA in duplex and decreases when double stranded DNA unwinds. However, SYBR Gold's excitation peak is at 495 nm and the emission peak is at 537 nm. SYBR Gold reportedly appears more stable than SYBR Green I. Hoechst 33258 is a known bisbenzimide double stranded DNA detecting molecule that binds to the AT rich regions of DNA in duplex. Hoechst 33258 excites at 350 nm and emits at 450 nm. YO-PRO-1, exciting at 450 nm and emitting at 550 nm, has been reported to be a double stranded DNA specific detecting molecule. In a particular embodiment of the present invention, the double stranded DNA detecting molecule is SYBR Green I.

A primer-based double stranded DNA detecting molecule is covalently linked to a primer and either increases or decreases fluorescence emission when amplicons form a duplex structure. Increased fluorescence emission is observed when a primer-based double stranded DNA detecting molecule is attached close to the 3' end of a primer and the primer terminal base is either dG or dC. The detecting molecule is quenched in the proximity of terminal dC-dG and dG-dC base pairs and dequenched as a result of duplex formation of the amplicon when the detecting molecule is located internally at least 6 nucleotides away from the ends of the primer. The dequenching results in a substantial increase in fluorescence emission. Examples of these type of detecting molecules include but are not limited to fluorescein (exciting at 488 nm and emitting at 530 nm), FAM (exciting at 494 nm and emitting at 518 nm), JOE (exciting at 527 and emitting at 548), HEX (exciting at 535 nm and emitting at 556 nm), TET (exciting at 521 nm and emitting at 536 nm), Alexa Fluor 594 (exciting at 590 nm and emitting at 615 nm), ROX (exciting at 575 nm and emitting at 602 nm), and TAMRA (exciting at 555 nm and emitting at 580 nm). In contrast, some primer-based double stranded DNA detecting molecules decrease their emission in the presence of double stranded DNA against single stranded DNA. Examples include, but are not limited to, rhodamine, and BODIPY-FI (exciting at 504 nm and emitting at 513 nm). These detecting molecules are usually covalently conjugated to a primer at the 5' terminal dC or dG and emit less fluorescence when amplicons are in duplex. It is believed that the decrease of fluorescence upon the formation of duplex is due to the quenching of guanosine in the complementary strand in close proximity to the detecting molecule or the quenching of the terminal dC-dG base pairs.

According to one embodiment, the primer-based double stranded DNA detecting molecule is a 5' nuclease probe. Such probes incorporate a fluorescent reporter molecule at either the 5' or 3' end of an oligonucleotide and a quencher at the opposite end. The first step of the amplification process involves heating to denature the double stranded DNA target molecule into a single stranded DNA. During the second step, a forward primer anneals to the target strand of the DNA and is extended by Taq polymerase. A reverse primer and a 5' nuclease probe then anneal to this newly replicated strand.

In this embodiment, at least one of the primer pairs or 5' nuclease probe should hybridize with the target sequence. The polymerase extends and cleaves the probe from the target strand. Upon cleavage, the reporter is no longer quenched by its proximity to the quencher and fluorescence is released. Each replication will result in the cleavage of a probe. As a result, the fluorescent signal will increase proportionally to the amount of amplification product.

Other exemplary methods for detecting the genes of the present invention are listed below.

Methods of detecting the expression level of RNA

The expression level of an RNA of the present invention can be determined using methods known in the arts. Isolation, extraction or derivation of RNA may be carried out by any suitable method. Isolating RNA from a biological sample generally includes treating a biological sample in such a manner that the RNA present in the sample is extracted and made available for analysis. Any isolation method that results in extracted RNA may be used in the practice of the present invention. It will be understood that the particular method used to extract RNA will depend on the nature of the source.

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, colorimetric 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 nonspecific 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 colorimetric reaction which reveals signals generated using enzyme-linked labeled probes.

In situ RT-PCR stain: This method is described in Nuovo GJ, ef 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, CA).

DNA microarrayslDNA chips:

The expression of thousands of genes may be analyzed simultaneously using DNA microarrays, allowing analysis of the complete transcriptional program of an organism during specific developmental processes or physiological responses. DNA microarrays consist of thousands of individual gene sequences attached to closely packed areas on the surface of a support such as a glass microscope slide. Various methods have been developed for preparing DNA microarrays. In one method, an approximately 1 kilobase segment of the coding region of each gene for analysis is individually PC amplified. A robotic apparatus is employed to apply each amplified DNA sample to closely spaced zones on the surface of a glass microscope slide, which is subsequently processed by thermal and chemical treatment to bind the DNA sequences to the surface of the support and denature them. Typically, such arrays are about 2 x 2 cm and contain about individual nucleic acids 6000 spots. In a variant of the technique, multiple DNA oligonucleotides, usually 20 nucleotides in length, are synthesized from an initial nucleotide that is covalently bound to the surface of a support, such that tens of thousands of identical oligonucleotides are synthesized in a small square zone on the surface of the support. Multiple oligonucleotide sequences from a single gene are synthesized in neighboring regions of the slide for analysis of expression of that gene. Hence, thousands of genes can be represented on one glass slide. Such arrays of synthetic oligonucleotides may be referred to in the art as "DNA chips", as opposed to "DNA microarrays", as described above [Lodish et al. (eds.). Chapter 7.8: DNA Microarrays: Analyzing Genome-Wide Expression. In: Molecular Cell Biology, 4th ed., W. H. Freeman, New York. (2000)] .

Oligonucleotide microarray - In this method oligonucleotide probes capable of specifically hybridizing with the polynucleotides of the present invention are attached to a solid surface (e.g., a glass wafer). Each oligonucleotide probe is of approximately 20- 25 nucleic acids in length. To detect the expression pattern of the polynucleotides of the present invention in a specific cell sample (e.g., blood cells), RNA is extracted from the cell sample using methods known in the art (using e.g., a TRIZOL solution, Gibco BRL, USA). Hybridization can take place using either labeled oligonucleotide probes (e.g., 5'-biotinylated probes) or labeled fragments of complementary DNA (cDNA) or RNA (cRNA). Briefly, double stranded cDNA is prepared from the RNA using reverse transcriptase (RT) (e.g., Superscript II RT), DNA ligase and DNA polymerase I, all according to manufacturer's instructions (Invitrogen Life Technologies, Frederick, MD, USA). To prepare labeled cRNA, the double stranded cDNA is subjected to an in vitro transcription reaction in the presence of biotinylated nucleotides using e.g., the BioArray High Yield RNA Transcript Labeling Kit (Enzo, Diagnostics, Affymetix Santa Clara CA). For efficient hybridization the labeled cRNA can be fragmented by incubating the RNA in 40 mM Tris Acetate (pH 8.1), 100 mM potassium acetate and 30 mM magnesium acetate for 35 minutes at 94 °C. Following hybridization, the microarray is washed and the hybridization signal is scanned using a confocal laser fluorescence scanner which measures fluorescence intensity emitted by the labeled cRNA bound to the probe arrays.

For example, in the Affymetrix microarray (Affymetrix®, Santa Clara, CA) each gene on the array is represented by a series of different oligonucleotide probes, of which, each probe pair consists of a perfect match oligonucleotide and a mismatch oligonucleotide. While the perfect match probe has a sequence exactly complimentary to the particular gene, thus enabling the measurement of the level of expression of the particular gene, the mismatch probe differs from the perfect match probe by a single base substitution at the center base position. The hybridization signal is scanned using the Agilent scanner, and the Microarray Suite software subtracts the non-specific signal resulting from the mismatch probe from the signal resulting from the perfect match probe.

The present inventors utilized exon arrays for studying genes involved in Parkinson's disease, and showed that changes in expression of particular genes are exon specific.

Table 4 (SEQ ID NOs: 1-29) provides exemplary target sequences of the genes against which probes may be prepared in order to determine gene expression of those genes. It will be appreciated that these target sequences may be present in one particular variant of the gene or be present in a number of variants of the same gene.

The present inventors have further shown that expression of a particular variant of a gene is indicative of Parkinson's.

Thus, for example a decrease in an expression level of a variant which encodes exons 2-3 and 4-5 of the SNCA (ENSG00000145335) gene is indicative of Parkinson's disease (i.e. the variant is such that exon 3 is immediately placed after exon 2 in the transcript and exon 5 is placed immediately after exon 4 in the transcript); an increase in an expression level of a variant which encodes exons 4-5 and 6-7 of the PARK7 (ENS G00000116288) gene is indicative of Parkinson's disease. Thus, the present invention contemplates use of probes that span the bridging regions of these exons, in order to confirm the disease. Examples of primers that may be used to analyze SNCA are set forth in SEQ ID NOs: 45 and 46; and 47 and 48. Examples of primers that may be used to analyze PARK7 are set forth in SEQ ID NOs: 49 and 50.

Furthermore, since the bridging regions of exons 2-3 and 4-5 of SNCA are present in variants 1, 2, 3, 6, 7, 8, 10 and 11 (SEQ ID NOs: 34-41), the present invention further contemplates additional sequences which are common to these variants, but absent in other SNCA variants.

Furthermore, since the bridging regions of exons 4-5 and 6-7 of PARK7 are present in variants 1-4 (SEQ ID NOs: 42-45), the present invention further contemplates detection of additional sequences which are common to these variants, but absent in other PARK7 variants.

As a further example, an increase in an expression level of a variant which encodes a non-truncated 3' untranslated region (UTR) of ASF (ENSG00000136450) is indicative of Parkinson's disease. The polynucleotide sequence of such a variant is presented in SEQ ID NO: 33. An example of a primer set that may be used to analyze ASF is presented in SEQ ID NOs: 51 and 52. It will be appreciated that the present invention contemplates analyzing any ASF sequence that is specific to the variant having the non-truncated 3 'UTR.

It will be appreciated that detecting changes in expression of particular genes may also be effected on the protein level (see for example Figure 15). Methods for detecting expression and/or activity of proteins are further described herein 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 colorimetric 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 I¹²⁵) 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.

Jmmunohistochemical 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 colorimetric 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.

As mentioned, herein above, the genes listed herein may be used for predicting an efficacy of a medicament for treating Parkinson's disease (PD).

Exemplary medicaments that may be tested include but are not limited to levodopa, carbidopa, a Catechol-O-methyl Transferase Inhibitor, a dopamine agonist, a monoamine oxidase inhibitor, an anticholinergic agent and Amantadine. The present invention also contemplates predicting efficacy of other types of treatments, including but not limited to deep brain stimulation (DBS).

Thus, according to another aspect of the present invention, there is provided a method of predicting an efficacy of deep brain stimulation (DBS) for treating Parkinson's disease (PD) in a subject, the method comprising analyzing an expression level of at least one gene listed in Table 3, wherein a statistically significant upregulation between an expression level of the at least one gene in a sample obtained from the subject and an expression level of the at least one gene in a control sample obtained from a non-diseased subject is indicative that DBS is efficacious for the treatment of Parkinson's disease in the subject.

According to another embodiment, predicting efficacy ,of DBS is effected by determining an amount of acetylcholinesterase (AChE) in the blood, wherein a statistically significant upregulation of expression of AChE compared to its expression in a control sample obtained from a non-diseased subject is indicative that DBS is efficacious for the treatment of Parkinson's disease in the subject.

DBS works by sending high frequency electrical impulses into specific areas of the brain wherein it can mitigate symptoms and/or directly diminish the side effects induced by Parkinsonian medications, allowing a decrease in medications, and/or making a medication regimen more tolerable. Typically DBS is performed by inserting electrodes into the brain with the aid of a stereotactic frame. The implantation may be unilateral (having only one side symptoms) or bilateral.

There are a few sites in the brain that can be targeted to achieve differing results, so each patient is typically assessed individually, and a site will be chosen based on their needs. Traditionally, the two most common sites are the subthalamic nucleus (STN) and the globus pallidus interna (GPi), but other sites, such as the caudal zona incerta and the pallidofugal fibers medial to the STN, may be selected.

It will be appreciated that when the medicament refers to an agent and not a treatment such as DBS, the method may be effected ex vivo (following removal of a sample from the subject) or in vivo.

Following diagnosis or predicting efficiency of a treatment, typically the subjects are informed (either verbally or via a written document) of the results of the test.

Additional tests may be carried out to corroborate the findings of the tests described herein. According to a particular embodiment, additional tests may be carried out to rule out conditions with similar symptoms. For instance, blood tests may be performed to check for abnormal thyroid hormone levels or liver damage. An imaging test (such as a CT scan or an MRI) may be used to check for signs of a stroke or brain tumor.

A Positron emission tomography (PET) may be performed to corroborate the findings of the diagnostic tests described herein.

According to the results of the diagnostic tests of the present invention, a subject may be treated with a particular drug or treatment such as those described herein above.

As mentioned above, using the chips described in the Examples section below, the present inventors have predicted which genes may be advantageously regulated for the treatment of Parkinson's disease. Thus, according to still another aspect of the present invention, there is provided a method of treating Parkinson's in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent which increases an amount or an activity of at least one polypeptide encoded by a gene listed in Table 2 or 6 in the brain.

As demonstrated in Example 12, the agent may be one which increases the amount or activity or Acetylcholinesterase (AChE) in the brain. The AChE is typically the AChE-R form the enzyme.

Agents capable of upregulating the polypeptides of the present invention may comprise the isolated polynucleotides and/or the polypeptides themselves.

Such polynucleotide sequences are typically inserted into expression vectors to enable expression of the recombinant polypeptide. The expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). Typical cloning vectors contain transcription and translation initiation sequences (e.g., promoters, enhances) and transcription and translation terminators (e.g., polyadenylation signals).

In addition to the elements already described, the expression vector of the present invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA. For example, a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid. Examples of mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205. Other exemplary vectors include pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

Recombinant viral vectors may also be used to synthesize the polynucleotides of the present invention. Viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. Bone marrow cells can be targeted using the human T cell leukemia virus type I (HTLV-I).

Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. Useful lipids for lipid- mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)]. The most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, Ientiviruses, or retroviruses. A viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct. In addition, such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention. Optionally, the construct may also include a signal that directs polyadenylation., as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof. Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.

Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

Introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.

As mentioned, the agents may be the polypeptides themselves. The polypeptides may be recombinant polypeptides.

The above described agents of the present invention can be provided to the individual per se, or as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier. It will be appreciated that the agents of the invention may be administered directly to the subject and/or via ex vivo administration. As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the polypeptide or antibody preparation, which is accountable for the biological effect.

Hereinafter, the phrases "physiologically acceptable carrier" and

"pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979).

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.

Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in

"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the preparation in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p.l].

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.

As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et ah, "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference. MATERIALS AND METHODS FOR EXAMPLES 1-8

Patient recruitment, DBS neurosurgery and clinical evaluation: 7 PD male patients nominated for bilateral STN-DBS neurosurgery and 6 healthy age-matched male controls (HC) (age, disease duration and BMI) were recruited. Levodopa equivalent dosage prior to, and following, STN-DBS was recorded. Subjects were assessed for their clinical background and state and fulfilled detailed medical history questionnaires. Patients with other medical conditions were excluded, including depression and past and current DSM Axis I and II psychological disorders (SM), chronic inflammatory disease, coagulation irregularities, previous malignancies or cardiac events, or any surgical procedure up to one year pre -DBS. Exceptions were hyperlipidemia (2 patients and 3 controls), hypertension and diabetes (2 patients each). All patients went through bilateral STN-DBS electrode implantation (Medtronics, USA) and were under dopamine replacement therapy (DRT) both pre- and post-DBS (on significantly reduced dosage post-DBS with t-test p<0.01, the last medication administered at least 5 hours pre-sampling. Patients exhibited similar total white and red blood cell counts pre- and post-DBS. Two patients received anti-hypertension medication and one-hyperlypidemia treatment. The clinical severity of disease symptoms was assessed by a neurologist for Unified PD Rating Scale (UPDRS-III) [57] section 3 (motor section) at 3 states: (1) pre STN-DBS, (2) post-DBS stim-ON and (3) post-DBS stim-OFF. Blood samples were collected from each patient at three time points: (1) one day pre -DBS upon hospitalization, with medication (2) post-DBS (range 6 - 18 weeks), when reaching optimal clinical state as evaluated by a neurologist and on a lower DRT dose, stim-ON and (3) Stim-OFF, following 60 minutes OFF electrical stimulation (counted from stage 2). Controls were recruited among volunteers. Exclusion criteria included smoking, chronic inflammatory diseases, drug or alcohol use, major depression, previous cardiac events and past year hospitalizations. One control subject received anti-hypertension and one anti-hyperlypidemia medication.

Blood sample collection and RNA extraction: Blood collection was conducted between 10AM-14PM. Collected venous blood (9 ml blood using 4.5 ml EDTA (anticoagulant) tubes) was immediately filtered using the LeukoLock fractionation and stabilization kit™ (Ambion, Applied Biosystems, Inc., Foster City, CA) and incubated in RNALater (Ambion) [58]. Stabilized filters and serum samples were stored at -80 °C. RNA extraction followed the manufacturers' alternative protocol instructions. Briefly, cells were flushed (TRI-Reagent™, Ambion) into l-bromo-3-chloropropane-containing 15 ml tubes and centrifuged. 0.5 and 1.25 volume water and ethanol were added to the aqueous phase. Samples were filtered through spin cartridges stored in pre-heated 150 μΐ EDTA, RNA was quantified in Bioanalyzer 2100 (Agilent) and frozen at -80 °C

Microarray sample preparation, hybridization and scanning: of total RNA was labeled using the Affymetrix exon array using whole transcripts sense targeting labeling assay according to the manufacturers' instructions; cDNA samples were hybridized to GeneChip™ Exon_1.0_ST_Array (Affymetrix, Santa Clara, CA, USA) microarrays, and results were scanned (GeneChip scanner 30007G, 27 CEL files).

Microarray analysis: Quality assurance, normalization (quantile) and probe set summation (DABG iter-PLIER) were conducted using expression console (EC) 1.0 (Affymetrix, Santa Clara, CA). Only core level probe sets (n=284,241) were included to summarize probe set level expression for 22,011 exon array transcript clusters (iter- PLIER, EC). Filtering out transcript clusters with no annotated gene symbols maintained 17,658 of 22,011 transcripts. Matlab R2008B (7.7.0.471) Bioinformatics toolbox served for differential expression analyses. 15^th percentile variance-based filtering was conducted maintaining 15,892 transcript clusters for analysis. Iterative permutation t-test conducted on median gene level on the four examined contrasts. This was followed by minimal list selection (among 1,000 repeated iterations) to reduce both false positive and false non detection outcomes. Three major contrasts were defined for detection of both differentially expressed genes and changed Gene Ontology (GO) terms: (1) patients prior to surgery compared to healthy controls (HC) (2) patients prior to treatment compared to post-DBS on stimulation and (3) patients post-DBS during stimulation compared to one hour after the electrical signal has been switched off (stim- OFF state). An additional contrast was applied to compare differential gene expression also between PD patients. An earlier PD cohort [7,8] of 3' whole blood transcript signals served for comparison. T-test detected genes were subjected to blind hierarchical clustering (HCL) analysis using Spearmen's rank distance metric for rows and columns. PCA was conducted using Partek Genomics Suite [21] Post-hoc functional analysis (Expression Analysis Systematic Explorer, EASE [23]) covered GO [59], KEGG pathways [60], the NIH Clusters of Orthologous Groups of proteins (COG) database [61] and the UniProt databases [62].

Statistical analysis: Ad-hoc GO analysis involved S and Fisher exact test (one tailed) using custom Matlab software on median gene level, core exon array signals. Linear regression p-value was assessed by Student's t-test (two-tailed) (Statistical Package for the Social Sciences, SPSS sotware). Statistical significance was P<0.05. Significance of microarray pair-wise comparisons was derived by iterative permutation (n=1000 each) 2-tailed t-test (Matlab) for selection of a minimal list of transcripts with P<0.05 detected among all the permutation tests iterations.

EXAMPLE 1

Gene expression patterns distinguish Parkinson 's status

To detect gene expression patterns associated with Parkinson's disease, leukocyte mRNA of seven male PD patients and of six age- matching male healthy controls (HC) was examined using exon arrays [16]. Patients' blood samples were taken in three states: prior to DBS neurosurgery ("pre-DBS"), following DBS ON electrical stimulation ("post-DBS", 2.2 +/- 0.9 months after DBS neurosurgery, upon disease motor symptoms stabilization) and following one hour of OFF stimulation (See Figures 1A-C for experimental design and study workflow). Exon arrays contain three main annotation levels for each probe set: core, extended and full. The core probe sets correspond to well-annotated exons and only those were analyzed; in the present study, the specific genes represented by these core exons are referred to as unities.

Prior to the statistical analysis, two filtering steps were conducted on all samples: first, removal of all un-annotated transcripts, and then variance-based filtration (var) to remove those transcripts with variance less than the 15th percentile that were of no interest. Calculating the median expression signal from all the core probe sets that interrogate each gene yielding gene level signals, and differentially expressed genes were identified by t-tests. To select those changes which would not be found under random shuffling of the treatment and the control samples, the t-test was repeated 1,000 times by permuting the columns of the gene expression data matrix [17] (Figure 2). Of the resultant gene lists, the shortest one was selected; this identified 173 transcripts as non-randomly differentially expressed between PD patients to controls (Table 1, herein below).

Table 1

The 173 genes differentially expressed between

PD patients pre-DBS and HC

t score DFs p val gene symbol

2.743546 9,579115 0.019566 CAMTA1

-2.60419 8.345678 0.025237 PARK7

2.438541 8.045268 0.034061 CDC20

-2.23832 10.95097 0.048846 CDKN2C

4.893988 8.634325 0.000452 TMEM61

-3.21482 10.43125 0.008206 MGC34796

-2.50905 11 0.029993 LRRC8C

2.504138 10.91195 0.030257 FAM46C

2.644876 7.166442 0.023445 DTL

-2.48323 7.283139 0.031415 CNIH4

-2.47678 9.143062 0.031782 SSU72

-2.44551 6.991461 0.033633 PLA2G2E

-3.12202 10.13741 0.009723 CAMK2N1

-2.3495 8.366262 0.040014 SYF2

-2.60863 10.24556 0.025033 TMSL1

-2.41146 10.53469 0.03577 SLC16A4

-2.23444 10.60355 0.049191 SIPA1L2

-2.3913 10.3125 0.037083 SLC3A1

-2.4545 10.05882 0.033086 C2orf34

-2.66106 8.121868 0.022758 SPTBN1

-2.38075 7.943393 0.037815 SMYD5 .

2.37455 9.682711 0.038242 IL1F10

-2.28854 10.99802 0.044641 FAM130A2

2.232068 9.937724 0.0494 SLC4A3

2.265008 5.320302 0.04655 COPS7B

2.602398 10.48216 0.025316 FAM150B

2.478556 10.72639 0.031679 VPS54 -2.29989 10.99972 0.043738 IRS1

2.38447 10.7092 0.037549 XYLB

2.334022 10.99122 0.041132 ENDOGL1

-2.80456 10.56481 0.017482 ATP11B

2.267594 10.99496 0.046345 OSTalpha

-2.47688 9.586889 0.031777 LRCH3

-2.24511 9.063052 0.048242 SEC13

2.397723 10.26538 0.036663 ANKRD28

2.388469 10.65098 0.037279 AMIG03

2.349254 10.46618 0.040032 SFMBT1

2.430709 10.43808 0.034549 LRIG1

-2.39213 10.31614 0.037024 RYBP

-2.28218 10.99732 0.045144 IQCB1

-2.44512 10.06291 0.033657 H1FX

3.025675 6.546909 0.011602 SLC25A31

2.568662 10.40881 0.026925 RNF212

-2.28053 8.247411 0.045281 CSN2

2.292324 5.830433 0.044339 HNRPDL

2.579055 10.98404 0.026413 SNCA

-2.82985 10.20781 0.016698 MANBA

-2.28671 10.44329 0.044786 NDUFC1

-2.5293 9.900632 0.028914 ACSL1

-2.27305 10.2655 0.045896 NR2F1

-2.37492 7.201893 0.038216 PRRC1

-2.57847 10.53918 0.026443 PCDHB1

2.291279 10.58255 0.04442 KCNIPl

-2.55355 10.79815 0.027682 ADAMTS6

2.450232 8.335389 0.033349 ETF1

-3.1511 9.950203 0.009221 BPHL

2.842961 5.062939 0.016298 UNQ9364

-2.489 7.484235 0.031085 PRSS16

2.251041 10.30233 0.047725 ZNF165

-2.40504 6.466259 0.036184 LST1

-2.60046 10.59067 0.025408 C6orf48

-2.62515 8.253356 0.024308 SLC26A8

-2.35592 10.98259 0.039557 B3GAT2

-2.27485 9.36988 0.045748 RNGTT

-2.503 10.76006 0.030321 TAAR1

2.613583 9.666661 0.024809 TXLNB

2.226539 10.36712 0.049897 C6orfll8

-2.32908 10.99552 0.041498 AKAP9

2.23588 8.928468 0.049064 GATAD1

-2.4557 9.616352 0.033018 CPSF4 2.571098 8.80462 0.026807 GSTK1

-2.44049 7.475201 0.033943 ZNF680

-2.44471 9.347598 0.033683 LOC285908

2.565723 5.24549 0.027078 NSUN5

3.508626 10.73382 0.004812 SLC26A5

-2.44929 10.99847 0.033404 SLC25A37

3.0875 10.11343 0.01036 FAM110B

-2.38349 10.8267 0.037617 NKAIN3

-2.33049 10.82909 0.041394 NECAB1

2.305243 9.408945 0.043322 ODF1

-2.32062 6.567718 0.042146 CYP7B1

-2.61275 10.54893 0.024847 TRAM1

-2.68926 10.36549 0.021614 TPD52

2.565305 8.122242 0.027099 PSKH2

-3.04594 10.91955 0.011193 DMRT3

-2.78302 10.99 0.018179 C9orf6

-2.23362 10.32383 0.049261 GLDC

-5.77213 8.859068 0.000115 APTX

2.706019 10.97391 0.020948 DOLK

-3.26568 10.90046 0.007476 NOTCH1

-2.7965 10.32066 0.017738 MASTL

-3.25239 10.10784 0.007664 TFAM

-2.24758 9.644969 0.048028 HHEX

-2.38662 10.57059 0.037398 LRIT1

-3.11621 9.876399 0.009828 LIPA

-3.38307 8.302871 0.006049 OR51L1

2.434078 10.17397 0.034335 OR52E5

2.647367 10.8861 0.023342 OR2AG1

-2.43917 9.165202 0.034023 FU46266

-2.83083 10.69219 0.016669 OR51G1

2.31541 6.505434 0.042532 OR52N5

2.440375 6.441969 0.03395 F 33790

-2.74744 10.90844- 0.019428 MAP6

-2.49132 6.84467 0.030954 FZD4

-2.81962 10.48807 0.017007 DDX47

-2.66376 7.799728 0.022647 PCBP2

2.273798 8.378747 0.045835 COPZ1

2.608058 5.728955 0.025059 BIN2

-2.26905 9.827759 0.046224 PPTC7

2.802429 10.40339 0.017547 DIABLO

-2.25288 10.77452 0.04757 FUT8

2.377058 10.45897 0.038069 SFRS5

2.600573 6.469937 0.025402 NRL 3.302437 7.738804 0.006997 C14orf39

-2.39073 10.79785 0.037123 C15orf55

2.683612 9.647965 0.02184 SNAP23

-3.91855 10.39782 0.002304 SNX33

-2.4716 10.61652 0.032075 FAH

2.313202 9.469705 0.0427 ARNT2

2.352072 10.96694 0.03983 PLCB2

-2.33627 8.167982 0.040971 HERO

-2.727 10.92076 0.02017 STRA6

-2.33756 9.836493 0.040879 MESDC2

-2.2614 10.58813 0.046857 FLJ14154

2.786895 6.095615 0.018049 NUPR1

-2.41888 10.22096 0.035298 CMTM2

-2.68699 10.7287 0.021701 CA7

-2.31043 10.52823 0.042921 PSMD7

-2.7413 10.78474 0.019652 CDK10

-2.46932 7.200872 0.032207 C16orf72

-2.50179 8.387502 0.030384 ZC3H7A

2.613642 10.74238 0.024806 SLC6A8

-2.39581 9.997625 0.036786 CHST6

-2.3107 10.86801 0.0429 RNMTL1

-2.65528 10.52327 0.023002 HIGD1B

-2.51987 8.649957 0.029412 ACBD4

-2.47418 10.82804 0.031929 FMNL1

2.341133 8.541062 0.040625 KRBA2

-2.709 10.26831 0.020837 SEZ6

2.785593 10.54238 0.018092 NR1D1

-2.31793 10.22679 0.042343 KRT37

-2.33727 10.22126 0.0409 CSHL1

-2.70362 10.62887 0.021043 GGA3

2.49938 8.427701 0.030511 CCDC5

2.230873 10.89044 0.049508 C18orfl2

-3.65043 10.54171 0.003732 HMHA1

-3.34301 8.083239 0.006496 C19orf60

-2.2691 10.21674 0.04622 CCNE1

-3.0908 10.38042 0.010298 ZNF575

-2.54563 10.98687 0.028086 C20orf72

-2.47581 10.62584 0.031837 ASIP

-2.65754 8.292665 0.022908 CTNNBLl

-2.27396 6.75176 0.045821 SEMG2

-2.68449 10.9987 0.021803 PTPN1

-2.35762 9.117041 0.039437 GGTLC1

-2.36378 7.544394 0.039006 C21orf82 2.370218 8.840093 0.038549 ADAMTS5

2.067336 5.715143 0.022507 N6AMT1

2.249265 6.730664 0.047882 MRPL40

-2.33682 9.947499 0.040932 CCT8L2

2.852912 7.047584 0.016 C22orf39

3.419168 9.970944 0.005663 SSTR3

2.408856 10.91974 0.035933 GYG2

2.642365 8.218962 0.023553 ARMCX3

-2.23925 10.99846 0.048763 RHOXF2

-2.86394 10.86345 0.015688 TKTL1

2.312229 5.259147 0.042776 RPL10

2.286718 5.68402 0.044786 BRCC3

-3.17202 10.94995 0.008878 FAM47A

2.43173 10.90817 0.034484 USP51

-2.86869 10.45358 0.015557 PJA1

2.378881 10.32843 0.037945 TEX11

-2.42048 9.30935 0.035194 SETD8

The differentially expressed transcripts included the PD-associated genes SNCA (also designated PARK1) [18,19] and PARK7 [13,20] in which mutations are linked to early appearance of PD. Also, the current results were compared with identical analysis flow results of a previously published independent cohort of 98 samples out of 105 early PD patients, neurological and healthy controls [7,8] (of which microarray data sets passed quality control assessment). The full lists of differentially expressed transcripts were then subjected to Post-hoc and Ad-hoc Gene Ontology Classifications and to biological validation by quantitative real-time Polymerase Chain Reaction (qRT-PCR) (Figures 3A-B)).

EXAMPLE 2

Transcript profiles of PD patients differ from controls

To test if the identified transcript modifications would distinguish PD patients from controls, two clustering approaches were applied on the expression signals of the detected genes in a manner blind to clinical diagnosis. Unsupervised classification by hierarchical classifier (HCL) segregated correctly all of the samples by the modified expression signals (Figure 4A) using the expression signals of the 173 detected transcripts. Principal component analysis (PCA) [21], classified as well all the samples correctly by type (patients/controls). Additionally, the HCL clustering segregated all the detected transcripts by their expression pattern (Figure 4A, right side dendrogram). To examine functional relevance of the gene-level classification post-hoc functional analysis [22] was applied on the 6 top level gene clusters created by the classifier. This identified enriched neuron development function [22] (Figure 4A), suggesting detection of functional changes in the parkinsonian brain as reflected in patient leukocytes. Of note, the modified PD leukocyte transcripts (as compared with matched controls) did not predict the clinical outcome of the DBS surgery or the subsequent DBS-induced expression changes).

EXAMPLE 3

STN-DBS affects a wide range of transcripts including disease-modified ones Next, the present inventors identified PD patient genes that are differentially expressed between post- to pre-DBS states (while being ON electrical stimulation post- DBS). Following exhaustive permutation tests, 465 genes were found to be differentially expressed after DBS surgery (Table 2).

Table 2

The 465 genes differentic illy expressed between PD patients post- DBS (ON Stim) as coma pred with pre-DBS

TC id exon t-scores DFs p value gene symbol

2318455 2.687381 8.842979 0.019378 ATAD3C

2320472 -2.96707 8.676032 0.011304 CAMTA1

2321911 -2.95147 9.193813 0.01165 CLCN6

2330723 -3.7333 11.96494 0.00259 DDI2

2350818 2.832477 10.83191 0.014645 DNALI1

2355591 2.481336 9.199611 0.0288 GPR61

2356300 -2.21868 10.45249 0.04725 FLJ21272

2363992 -2.23342 10.21127 0.045973 PIAS3

2365933 -2.37096 11.08206 0.035478 LOC646470

2372169 -2.51807 11.2439 0.026849 LOC100128751

2372858 -3.56661 11.57228 0.003557 OCLM

2378068 3.405939 11.95993 0.004825 RGS2

2382781 2.294956 6.467827 0.040959 G0S2

2394608 -2.32921 7.946522 0.038404 SRP9

2401753 2.215389 11.96348 0.047524 GPR153

2408041 2.196212 9.837044 0.049223 IL22RA1

2420642 -2.22333 10.51838 0.046839 HPCAL4

2429466 -2.61631 9.875002 0.022213 MCOLN2

2435251 2.498135 11.97705 0.027902 NGF

2439373 -2.53979 9.34142 0.025754 LING04 2448971 -2.4525 8.834709 0.03Ό41 SPTA1

2459352 2.2608 9.137021 0.043682 UCHL5

2460325 -2.22874 8.590058 0.046376 WNT9A

2461654 2.471455 9.248674 0.029346 Clorfl98

2461786 -2.30693 11.74625 0.040058 PP2672

2462589 3.123276 11.36156 0.008369 ARID4B

2484841 -2.32857 10.75209 0.03845 MT1H

2492783 -2.22775 9.630455 0.046463 B3GNT2

2497082 -3.13489 8.710757 0.008186 THNSL2

2516780 -2.2465 8.003239 0.044876 IL1RL1

2525272 2.46527 10.09194 0.02969 HOXD13

2537109 2.303124 11.87091 0.040337 PIP5K3

2544484 -2.89713 11.21767 0.012933 SH3YL1

2544781 -3.07153 7.89964 0.009248 ADCY3

2558483 2.261997 9.286057 0.043581 DTNB

2562729 -2.44618 8.881176 0.030782 C2orf42

2565082 -2.58688 7.700785 0.023533 REEP1

2565262 -2.2959 8.247092 0.040886 ADRA2B

2565592 -2.4468 8.166216 0.030749 ASCC3L1

2576988 -2.98493 10.29296 0.010926 SEMA4C

2582701 2.488293 10.15484 0.028426 LYPD1

2602110 2.854399 8.251484 0.014039 CCDC148

2603544 -2.98504 6.622419 0.010924 COL4A4

2610631 -2.89426 11.48252 0.013004 NMUR1

2620114 -2.28935 9.476566 0.041382 SLC6A1

2635895 2.831809 10.33978 0.014663 ZNF167

2635906 -3.41423 11.99214 0.00475 PHLDB2

2644418 -2.27637 7.831552 0.042423 CLDN18

2654815 2.229889 9.577747 0.046274 ATP11B

2654855 -2.23797 11.1816 0.045589 TPRG1

2657546 2.415868 8.875653 0.032597 SEC13

2662657 -2.62991 10.83795 0.021635 WNT7A

2663714 -2.40501 9.85833 0.033269 SH3BP5

2664209 -2.33639 10.78067 0.03788 EPM2AIP1

2669157 2.277139 11.67053 0.04236 GPX1

2674229 -3.08543 11.80228 0.00901 AMIG03

2674646 -2.30358 10.02934 0.040303 IFT57

2687840 3.313543 9.300882 0.005781 GP5

2711627 -2.21448 11.41839 0.047602 MAEA

2714672 2.230587 9.788342 0.046213 EPGN

2731496 -2.71374 7.395278 0.018404 CXCL13

2732508 -3.766 9.106679 0.002446 Fll

2755154 -2.56737 11.99759 0.024433 GNRHR 2771812 -2.40032 7.935008 0.033568 HNRPDL

2775562 -2.53596 10.64293 0.025956 SNCA

2777714 -2.96694 11.99932 0.011307 GC42105

2808308 -2.53264 7.632173 0.026119 SNX18

2809628 -2.55657 11.85718 0.024952 PDE8B

2816681 -2.65908 10.61843 0.020459 ZCCHC9

2818079 2.433977 8.868621 0.031492 POU5F2

2867284 2.949563 11.92914 0.011692 HNRNPAO

2877141 2.6799 11.97238 0.01966 MGAT1

2890859 2.34899 7.124718 0.036971 BAT2

2902463 -2.87689 11.1191 0.013447 HSPA1B

2902725 -2.75023 10.26467 0.017161 ABCC10

2907943 -2.30946 11.40351 0.03987 C6orfl95

2938895 3.020021 9.328615 0.010217 HIST1H2BJ

2946681 -2.28115 9.280149 0.042039 RP3-377H14.5

2947975 -2.50917 9.777723 0.027308 RGL2

2950590 2.609483 8.64752 0.022516 SLC26A8

2951730 2.538488 8.147827 0.025823 PGC

2953751 -2.21202 9.97713 0.047825 PPIL4

2978876 -2.77542 8.167633 0.016345 TCTE3

2986127 -2.25338 9.824955 0.044309 POR

3009229 -2.2895 11.45268 0.041371 TRIM56

3016098 2.324141 9.674432 0.038774 DUS4L

3018509 -2.38726 10.19856 0.034418 ATP6V1F

3023211 -2.42581 11.9938 0.031986 CSGlcA-T

3031967 -2.37255 8.34374 0.035369 C7orf31

3042012 -2.47846 8.927896 0.028962 FIGNL1

3050367 -2.1983 7.334776 0.049035 SAMD9

3061438 -2.62064 10.67282 0.022019 TRPV6

3077072 -2.32432 9.273682 0.03876 ARP11

3078948 -2.36473 10.7506 0.035903 C8orf45

3101851 2.401914 9.118172 0.033467 NECAB1

3106479 2.997819 11.9946 0.01066 DEFA6

3122703 2.85971 8.663198 0.013895 PPP1R3B

3123675 2.957181 11.88511 0.01152 STC1

3128046 3.400467 10.03738 0.004876 ANK1

3132940 2.27225 11.42088 0.042754 MYST3

3133135 2.781884 10.02949 0.01615 TRAM1

3139882 2.351025 11.99978 0.036837 PXMP3

3141346 2.501086 11.00821 0.027747 BOP1

3158190 2.303367 11.99901 0.040319 C9orf93

3163200 -2.97806 7.632811 0.011072 DNAJB5

3167660 2.557713 7.800924 0.024897 MELK 3168508 2.431624 10.45027 0.031635 SMC5

3174224 2.216403 7.817183 0.047442 PHF2

3179975 2.375517 9.464142 0.035174 PRPF4

3185558 2.302014 7.383442 0.040426 ORM1

3186123 -2.34812 11.98591 0.037029 CEP110

3187577 2.356344 9.621975 0.036472 GSN

3187686 2.224179 11.98236 0.046769 NEK6

3188697 2.505804 11.85689 0.027492 NUP214

3191900 3.037128 11.78333 0.009896 KIAA0515

3192062 2.215298 11.68391 0.047532 KIAA0020

3192117 2.63585 10.4075 0.021397 RFX3

3196691 -2.5232 10.50979 0.026586 KLHL9

3196842 2.573099 11.13542 0.02417 LOC554202

3201277 2.27239 11.84827 0.042743 TAF1

3201345 2.32719 11.25672 0.038549 C9orf23

3203199 -2.28094 8.070694 0.042056 ANKRD18A

3204174 2.953907 10.24583 0.011597 KLF9

3205834 2.244312 6.283292 0.045057 SPTLC1

3208995 2.271314 11.48333 0.04283 C9orfl30

3214582 2.752414 11.8433 0.017087 CTNNAL1

3216023 2.317442 8.937237 0.039266 PTRH1

3219621 -3.4553 11.99991 0.004393 NAIF1

3226005 -2.33095 10.86394 0.038277 MY03A

3226303 2.970263 10.00036 0.011235 CDH23

3226311 2.58237 11.51296 0.023739 KIAA1975

3239584 2.714873 10.18519 0.018366 LZTS2

3251068 -2.25899 8.717396 0.043837 ELOVL3

3256221 3.137218 8.447748 0.008148 NFKB2

3260957 2.805368 10.27597 0.015437 TRUB1

3261532 2.219801 11.95702 0.047152 C10orf96

3261643 2.713085 8.821257 0.018427 PLEKHA1

3265494 2.421772 10.85775 0.03223 LHPP

3265809 2.428059 8.251339 0.031852 INPP5A

3268274 2.713106 9.429853 0.018427 UTF1

3269065 2.374804 11.46751 0.03522 VENTX

3272205 -2.24929 11.07907 0.044637 ANUBL1

3272686 3.738427 11.10821 0.002565 CTGLF3

3272706 4.702964 9.514346 0.000459 TYSND1

3286975 2.727921 11.80154 0.017896 CAMK2G

3288013 2.920814 11.99218 0.012357 LRIT1

3293215 2.348043 8.203857 0.037034 CRTAC1

3294854 2.380531 9.229239 0.034852 C10orf33

3298337 3.573985 11.89034 0.003501 CHUK 3302572 2.229721 9.567079 0.046289 POLL

3302740 3.020729 10.5343 0.010203 PCGF6

3303300 2.333578 11.53731 0.038089 SH3PXD2A

3303870 2.239561 7.153766 0.045457 ODF3

3304718 2.794576 10.23277 0.015763 PSMD13

3304853 2.309149 9.697195 0.039894 DRD4

3304970 2.759213 11.83575 0.016865 LSPl

3315487 -2.42155 9.08212 0.032243 DNHD1

3315549 3.185865 11.91034 0.007414 SLC35C1

3316057 2.812798 8.958326 0.015215 PRDX5

3317071 2.341585 11.50081 0.037498 FADD

3318844 2.239391 11.5351 0.045472 INPPL1

3329018 2.879204 7.684994 0.013387 Cllorf67

3334501 3.427778 11.34313 0.004631 ARHGEF12

3338424 2.873324 10.35801 0.013542 EI24

3339423 2.382808 11.13659 0.034705 APLP2

3341440 -2.49599 10.56834 0.028017 RNH1

3352503 4.791083 9.560219 0.000394 IGF2

3354731 -2.49815 10.98482 0.027902 HBE1

3356115 -2.2518 8.444593 0.044434 OR52N5

3358049 2.321629 8.360396 0.03896 Cllorf56

3359121 2.264898 10.00623 0.043347 HPX

3359134 2.517763 10.40704 0.026865 TMEM9B

3360456 2.312065 7.412314 0.03967 GALNTL4

3360672 2.360941 11.74648 0.036158 E2F8

3360772 3.24951 11.16916 0.006544 OR4C11

3360874 -2.25122 8.312551 0.044478 VPS37C

3362124 2.225271 7.551884 0.046678 PYGM

3363091 2.479748 9.721127 0.028888 PRO1880

3365776 2.881206 10.87378 0.013338 Cllorf59

3373212 -3.1061 11.99585 0.008649 P4HA3

3375147 2.47774 7.566389 0.029003 PAK1

3377016 2.966958 11.77375 0.011307 RAB30

3378671 2.199512 11.34643 0.048929 BTG4

3380980 2.430872 11.25612 0.03168 BCL9L

3381879 4.478718 9.04168 0.00067 THY1

3382861 3.101313 9.786745 0.008734 BICDl

3384321 2.561464 10.79774 0.024717 C12orf54

3390949 2.41236 11.83875 0.032813 ACCN2

3393993 2.759444 10.57226 0.016857 KRT7

3394412 3.362458 11.15111 0.005253 MFSD5

3410445 2.198301 8.594566 0.049035 C12orfl0

3413495 2.807715 9.742399 0.015365 PCBP2 3414351 2.274627 9.943133 0.042565 HOXC11

3415320 2.710783 11.68787 0.018513 GDF11

3415849 -2.41587 11.0047 0.032596 RAB5B

3415937 2.35894 11.68462 0.036294 IL23A

3416036 2.858836 9.230759 0.013918 MBD6

3416278 -3.12855 11.84271 0.008284 SOCS2

3416943 2.421517 9.559918 0.032245 PGAM5

3417161 3.007426 7.659683 0.010466 NRIP2

3417557 2.456544 10.08562 0.030188 RBP5

3418214 -2.25782 9.131393 0.043934 TAS2R49

3426257 2.482353 11.95165 0.028747 VDR

3439195 2.548847 10.91639 0.025322 KRT82

3440568 5.378192 11.72522 0.000143 KRT72

3442579 2.493788 9.918741 0.028132 CCDC59

3444476 2.214504 11.72085 0.0476 GALNT4

3452818 2.188521 11.63386 0.049938 UHRFIBPIL

3455344 -2.58938 9.15942 0.023416 SELPLG

3455651 3.373595 10.84053 0.005138 RBM19

3464000 2.653603 11.99023 0.020675 POP5

3464967 2.51476 11.5347 0.027014 C13orf26

3467637 3.001889 9.309363 0.010577 EXOSC8

3470523 2.438983 11.94014 0.031204 CKAP2

3472468 -2.21847 11.99464 0.047268 CLYBL

3474619 -3.19218 10.2925 0.007319 PCCA

3484165 2.404201 10.73209 0.033319 CUL4A

3485863 -2.31494 10.70808 0.039457 UPF3A

3490655 2.323085 7.842784 0.038856 PSPC1

3498589 2.39041 9.990132 0.034206 ZDHHC20

3498837 2.890072 9.005344 0.013108 MTIF3

3502497 3.739926 11.93842 0.002558 NUFIP1

3503224 3.630341 11.82595 0.003151 EFNB2

3503976 2.874158 11.96109 0.013522 FAM12B

3504691 -2.20837 9.554747 0.048139 DHRS4

3504760 3.13719 11.99322 0.008149 KTN1

3506936 2.304304 11.96452 0.040251 DACT1

3512449 -2.60007 9.259948 0.022938 C14orfl00

3524570 2.551092 11.5029 0.025215 EXDL2

3527655 2.237355 11.36887 0.04564 BATF

3529309 2.876554 11.63641 0.013457 SERPINA4

3536905 2.680109 9.087922 0.019652 SERPINA13

3537030 2.646155 11.97902 0.020974 AK7

3538087 3.238377 7.761395 0.006681 ZNF409

3538324 2.373162 11.55618 0.035328 WDR22 3541937 2.448345 11.53119 0.030658 ACYP1

3544605 2.281326 10.45182 0.042024 ALKBH1

3549708 -2.62361 11.74316 0.021897 GPR68

3549790 2.74086 11.41941 0.017461 SNRPN

3550343 2.335787 11.90394 0.037925 APBA2

3557593 2.702537 11.99772 0.018811 NUSAP1

3569926 2.512758 7.269273 0.027112 FAM148A

3572263 2.53331 11.4916 0.026086 C15orf37

3573229 2.291878 11.99994 0.041187 FAH

3576411 -2.31136 9.338923 0.039723 ARNT2

3584443 2.416228 10.12839 0.032574 DN

3585905 2.191614 9.397078 0.049653 SLC30A4

3590388 2.200797 10.41577 0.048814 C15orfl5

3596894 2.43487 8.42156 0.031439 CLPX

3603840 2.739843 8.721147 0.017496 DPP8

3603932 2.390999 11.87631 0.034168 SNAPC5

3604006 2.498523 8.596158 0.027881 C15orf28

3613725 2.29906 11.08769 0.040643 SEMA7A

3622436 3.040813 10.04257 0.009822 ETFA

3625234 4.32597 8.804398 0.000879 BCL2A1

3629494 2.477015 7.300697 0.029045 KLHL25

3629698 2.370956 11.87253 0.035479 NME4

3630156 2.835991 8.957142 0.014543 OR1F1

3630957 2.205214 7.959234 0.048419 DNAJA3

3632907 3.228865 9.572533 0.006802 SEC14L5

3633794 3.073233 6.698744 0.009221 NDE1

3635198 2.589464 11.92482 0.023412 C16orf52

3637367 2.234507 11.65784 0.045881 LCMT1

3642815 2.328104 10.82692 0.038484 ZNF771

3645764 -2.21052 11.72728 0.047958 RSPRY1

3646164 2.373186 11.91311 0.035327 CA7

3646495 2.46352 8.087777 0.029789 UNQ6484

3649811 2.424427 10.97771 0.03207 C16orf44

3652271 3.321763 11.82658 0.005694 CDK10

3653619 -2.34442 9.941042 0.037295 DEF8

3656191 -2.78322 11.63368 0.016111 AXINl

3662201 2.613284 10.36532 0.022346 RPUSD1

3662612 4.195715 11.55095 0.001109 C16orf42

3664924 2.846602 9.336519 0.014253 MGC45438

3667702 2.371287 10.18534 0.035456 DEXI

3671850 3.040594 11.06921 0.009827 ZC3H7A

3674303 2.688532 11.76923 0.019333 PLA2G10

3674559 2.299141 11.76307 0.040638 KIAA0430 3675047 3.982572 11.82904 0.00163 DNAH3

3675430 2.749264 10.78314 0.017195 C16orf65

3675815 3.776276 11.81752 0.0024 ARHGAP17

3678542 2.306562 10.21363 0.040085 PYCARD

3680130 2.329863 11.96904 0.038357 DNAJA2

3680524 2.269102 11.04105 0.043012 EXOC3L

3681488 3.019408 7.911839 0.010229 ATP6V0D1

3681956 2.479723 11.61172 0.028889 TMEM170

3683879 2.268249 10.28568 0.043082 C16orf74

3684703 2.5263 8.074975 0.026435 PRDM7

3685610 3.672799 10.35606 0.002904 LOC400566

3688311 -2.85127 7.859832 0.014123 OR3A2

3690084 2.339765 9.966986 0.037629 TX DC17

3695450 2.223222 10.452 0.046849 NLGN2

3695699 2.318648 10.91553 0.039176 SPEM1

3699581 2.571049 9.079822 0.024263 CYB5D1

3703129 2.431808 8.703999 0.031623 RANGRF

3705195 2.478607 9.793125 0.028953 FAM18B

3705412 2.193963 10.86307 0.049435 KC J12

3706651 3.082817 8.050919 0.009056 TAOK1

3707990 2.367714 11.41567 0.035699 TAF15

3708553 2.250179 10.10241 0.044563 FKBP10

3708582 2.249305 10.86936 0.044636 TTC25

3708597 2.532233 11.85978 0.026138 ATP6V0A1

3709213 2.909015 11.82322 0.012637 PSME3

3709590 2.591162 10.99755 0.023333 NSF

3713539 -2.87294 7.82034 0.013552 SNX11

3714779 2.528108 11.17015 0.02634 CROP

3716048 2.209606 11.69791 0.048032 NOG

3718791 2.308072 7.316213 0.039974 BCAS3

3721452 2.241769 11.93202 0.04527 METTL2A

3721516 2.834177 10.14054 0.014596 PITPNC1

3721718 2.286582 11.85324 0.0416 KCNJ16

3722152 3.332692 7.06292 0.005572 GPR142

3724197 3.16775 10.82935 0.00768 FAM100B

3725083 2.31169 11.42784 0.039698 FN3KRP

3726772 2.27179 9.357808 0.042791 PFN1

3727928 2.790083 9.658365 0.015901 PIK3R5

3729569 -2.72671 10.07471 0.017936 CDRT1

3730211 -2.21341 11.04728 0.047699 SDF2

3732230 2.226587 8.550482 0.046565 SLC6A4

3733238 2.392737 10.77518 0.034054 C17orf66

3734342 2.402147 11.9816 0.033452 LOC100129395 3735447 2.673258 8.091076 0.019907 MED1

3739108 2.570148 11.70048 0.024306 KRTAP2-4

3741352 2.269108 8.455423 0.043012 GHDC

3742400 2.867261 11.71884 0.013695 DCAKD

3744680 3.182124 11.66266 0.007471 HOXB3

3746809 -2.416 8.488768 0.032588 TOB1

3750939 3.034388 9.206988 0.00995 CSHL1

3751794 5.102821 11.26497 0.000228 SCN4A

3753833 2.209161 11.88722 0.04807 AXIN2

3755316 2.430532 9.383956 0.031701 WIPI1

3755714 3.003104 11.13937 0.010553 C17orf54

3756709 2.416218 9.661541 0.032575 EXOC7

3756723 2.217617 10.54529 0.047341 TK1

3757745 2.40549 11.98173 0.03324 THOC4

3759540 2.835635 9.868233 0.014553 VAPA

3761313 -2.38698 8.507137 0.034437 CCDC5

3762473 2.536543 10.8957 0.025928 MAPK4

3766499 2.859635 11.99137 0.013897 ZFP161

3766549 2.479317 7.325206 0.028913 FBX015

3767465 2.192864 9.971486 0.049536 GNA11

3767480 2.244416 7.808388 0.045048 FZR1

3768474 2.617272 11.39108 0.022172 CCDC94

3770172 2.266723 8.781762 0.043204 MPND

3771336 2.736648 9.094057 0.017599 HSDIIBIL

3772158 2.628462 9.438194 0.021694 EVI5L

3774331 2.485099 10.29148 0.028602 ANGPTL4

3778601 2.283909 10.40858 0.041817 C19orf39

3787005 2.272309 10.97821 0.042749 MAST1

3788097 2.246732 8.856679 0.044857 BTBD14B

3797015 2.759703 7.326015 0.016849 MGC3207

3813198 2.564808 11.98542 0.024554 FLJ25328

3816778 2.192229 8.807381 0.049595 HSH2D

3816803 2.656325 11.42186 0.020566 LSR

3816988 2.431129 10.71693 0.031664 CD22

3817400 4.493603 11.77674 0.000653 MED29

3817464 2.378092 11.68948 0.035007 AXL

3818047 2.691371 8.76654 0.019223 MEGF8

3819200 2.764137 9.242559 0.016699 CD177

3819474 2.25858 9.197938 0.043871 CBLC

3821183 2.22459 10.84829 0.046736 LOC374920

3821937 2.684697 11.68431 0.019477 BAX

3822195 2.548416 9.958701 0.025343 MED25

3822322 3.740018 11.98237 0.002558 KLK2 3823488 2.398332 9.229871 0.033691 CACNG6

3823583 2.33992 8.993619 0.037618 TTYH1

3830246 2.436414 10.3063 0.031353 KIR3DL2

3830353 2.254007 11.93377 0.044253 ZNF581

3830359 2.690148 11.5879 0.01927 FUT5

3832964 2.231066 8.63573 0.046171 CTXNl

3834046 6.200246 10.78376 3.98E-05 ZNF414

3834778 -2.59802 7.631566 0.023027 AP1M2

3834837 2.762335 11.53324 0.016758 SPC24

3835035 2.391721 9.845721 0.034121 ECSIT

3835751 2.551724 10.43987 0.025185 RTBDN

3837664 2.61498 8.870895 0.02227 SAMD1

3838067 2.594687 7.367259 0.023178 ABHD8

3838947 -2.20484 8.582648 0.048452 ZNF100

3839563 -2.90478 8.307597 0.012742 ANKRD27

3841184 2.226478 7.762287 0.046574 ALKBH6

3841439 3.536543 11.94412 0.003756 ECH1

3841777 2.266931 11.99798 0.043187 RPS16

3842301 2.313615 8.769931 0.039555 CNTD2

3847503 2.384094 8.683128 0.034622 SNRPD2

3848644 2.248962 8.387796 0.044665 IRF2BP1

3849022 3.983125 11.90878 0.001629 CABP5

3850457 2.626894 11.71026 0.021757 HSD17B14

3850660 2.388855 7.125221 0.034305 GYS1

3851020 -2.28319 11.94428 0.041873 PTH2

3851801 2.244708 11.93749 0.045023 SHANK1

3852507 2.834727 11.85505 0.01458 KLK12

3854349 2.23577 8.095916 0.045773 ATPBD3

3856554 2.550014 11.83797 0.025266 SIGLEC8

3858659 2.283803 7.419996 0.041825 ZNF614

3860208 2.373034 10.19438 0.035337 ZNF702

3861581 2.738244 9.592086 0.017549 LILRA5

3862018 3.32135 10.57447 0.005699 SAPS1

3862452 2.88468 8.961543 0.013245 IL11

3865568 2.256388 9.17157 0.044051 FU39005

3865776 2.478386 11.97943 0.028966 DEFB127

3866831 2.391091 8.19225 0.034163 C20orfl41

3867443 2.799511 11.436 0.01561 SNAP25

3867538 2.558661 7.604476 0.024851 DEFB123

3867835 2.572419 9.975416 0.024201 ID1

3868587 -2.31894 11.30654 0.039154 MAPRE1

3868857 2.394216 11.24159 0.033962 C20orfl44

3868905 2.513183 7.262181 0.027091 RALY 3869062 2.85274 7.787175 0.014084 PHF20

3869379 2.809961 11.99777 0.0153 CTNNBL1

3869880 2.773945 11.12615 0.01639 C20orfl21

3870758 3.811346 9.771923 0.00225 PKIG

3871256 2.715196 11.99847 0.018354 SNX21

3871406 2.506562 9.389879 0.027451 ZSWIM1

3872905 2.211815 9.550935 0.047844 PCIF1

3873078 2.942769 9.980027 0.011845 PTPN1

3873997 3.378535 8.188196 0.005085 RAB22A

3874008 2.576568 10.29249 0.024005 OGFR

3876245 2.727689 8.380202 0.017903 SDCBP2

3881251 2.209597 9.195056 0.048033 ADAM33

3881391 2.207461 11.24267 0.048221 SLC23A2

3882069 2.539787 8.722476 0.025754 TRMT6

3882614 2.304565 8.078716 0.040233 LOC200261

3882720 -2.19177 8.145759 0.049638 CHD6

3883547 2.306793 7.228467 0.040068 PTPRT

3884324 2.34679 _j 11.56444 0.037121 ZMYND8

3886512 2.538647 10.33045 0.025814 ZFP64

3886532 2.66526 11.538 0.020219 TCFL5

3887069 2.889307 10.08474 0.013126 PRIC285

3887107 2.557915 7.101583 0.024887 TTC3

3887165 2.542234 11.96411 0.025633 ZNF294

3888721 2.216273 10.25243 0.047453 KRTAP21-1

3890870 3.731598 11.31461 0.002598 TFF1

3892941 -2.2124 10.52219 0.04779 S100B

3894545 2.190772 10.15634 0.049731 FLJ38343

3895552 3.134569 10.25221 0.008191 LIMK2

3896078 2.651361 8.351816 0.020763 APOL5

3896524 3.39023 8.893787 0.004972 NCF4

3901085 2.223976 7.08839 0.046786 PSCD4

LL22NC03-

3906160 -3.35595 9.98267 0.00532 5H6.5

3906390 2.211977 8.739955 0.047829 ACR

3908149 3.578257 10.17169 0.003471 DGCR2

3909843 3.502278 9.297152 0.004011 ZDHHC8P

3913483 3.400186 8.181102 0.004879 PITPNB

3913960 2.19785 9.623255 0.049074 MB^;

3920385 2.931729 9.012241 0.0121 SLC16A8

3927949 2.212652 10.86262 0.047767 S100G

3928590 2.236673 11.1693 0.045697 SCML1

3933559 -2.27913 10.2006 0.042204 USP11

3935486 2.270057 11.98287 0.042933 PLP2 3940985 -2.34502 10.34061 0.037252 GSPT2

3942838 3.606806 11.41565 0.003287 GNL3L

3944273 2.490189 10.73106 0.028324 MAGEA4

3944543 2.390398 11.99987 0.034207 SLC9A7

3944690 2.330123 11.64513 0.038338 PFKFB1

3949017 4.130206 11.64458 0.001243 ALAS2

3951117 3.20183 11.99517 0.007178 PJA1

3952453 -3.4747 11.95608 0.004234 INGX

3954691 2.50943 11.98749 0.027294 ESX1

3956290 3.015026 9.685378 0.010313 MORC4

3959166 2.925065 10.16597 0.012253 FGF13

3960337 2.482238 9.834044 0.028753 RPS4Y1

3970111 -3.67067 11.13417 0.002915 DMBT1

3970476 2.745227 11.88237 0.017318 FAM72A

3976163 2.478266 7.052487 0.028973 Clorf222

3977067 2.605492 8.151331 0.022694 APOD

Thus, the DBS stimulus induced almost three times more leukocyte transcriptional changes than the disease itself. HCL classification analysis distinguished all of the pre- DBS samples correctly from the post-DBS ones based on the expression signals of these 465 detected genes (Figure 4C, right side dendrogram). PCA classification as well correctly segregated all of the samples by state. The present inventors then compared the transcripts differentially expressed in PD to control to those modified in the post- DBS compared to pre-DBS samples. 22 (13 %) of the 173 PD modified transcripts, including SNCA were among the 465 DBS-modified transcripts (Figure 4B). The probability that 22 of the 173 transcripts that were detected as changed in PD compared to controls will also change post-DBS was calculated using the binomial coefficient \k with the binomial probability equation:

p(X = k) = Qp^kqⁿ-^k

465 where q=(l-p) and^the P^robabi]ity ^{for a} &^{ene t0} change post-DBS is 15,895 = 0.0293 .

The outcome of the test is p = 6.661 xlO^"9,

demonstrating that it is highly unlikely to observe a random overlap between 13 % of the transcripts modified in PD and those changed following DBS. In all patients, the Unified PD motor Rating Scale (UPDRS-III) improved (i.e decreased) following STN-DBS (t-test p=0.0008, Figure 4C). To better characterize the change, post-hoc GO functional analysis [23] was conducted on the detected genes. Genes that exhibit similar patterns of expression were clustered together by the classifier. Analyzing each of the 10 top level clusters revealed enriched decreases in DNA replication transcripts, T-helper immune response, response to metal ions, mitochondrial transport, interferon-gamma biosynthesis, protein kinase cascade and cation homeostasis (Figure 4C). There was no correlation between pre-operative levodopa responsiveness and STN-DBS efficacy, similar to findings in a larger group of DBS treated PD patients [24].

EXAMPLE 4

The Post-DBS stimulation state differs from healthy controls

Given the improved motor symptoms following DBS, the present inventors proceeded to test if the leukocyte post-DBS transcript profiles regained similarity to those of healthy controls. Surprisingly, PD patients post-DBS on stimulation exhibited distinct expression as compared with healthy controls. Permutation t-tests identified 321 transcripts as changed between PD patients post-DBS to controls, including PARK7 and SFRS7 which maintained their PD-characteristic changes. Nevertheless, all post-DBS samples were correctly classified from controls by both HCL and PCA classifiers. Post- hoc functional analysis revealed enrichment of dopaminergic synaptic transmission in the list of detected transcripts.

Two central PD genes and one splicing factor, two of them detected as changed in patients and two following DBS were selected for qRT-PCR validation: SNCA (PARK1) (changed in patients and following DBS), PARK7 (changed in patients) and SFRS1 (changed following DBS). The SNCA gene consists of 6 exons creating 6 different splice variants [25], 3 of which encode protein. qRT-PCR validated 2-fold disease-associated decreases in both exons 2-3 and 4-5, which are included in different SNCA variants. These decreases were fully correctible by STN-DBS, with similar trends detected by qRT-PCR.

The PARK7 gene up-regulates human tyrosine hydroxylase by inhibiting the splicing factor SFPQ [28]. PARK7 covers 4 splice variants [26] and an additional 5' promoter variant [27]. PARK7 exhibited disease-induced increases and qRT-PCR validated those in both the junctions linking exons 4 to 5 and 6 to 7.

Among several splice factors which changed, SFRSl (ASF/SF2) has 2 ultra- conserved splice variants differing in their 3'-UTR. Only SFRSl transcripts including full-length 3'-UTR encode the intact ASF protein and are rescued from mR A degradation [29]. The arrays detected treatment-associated increases in the SFRSl 3'- UTR as compared to controls, which were validated by qRT-PCR (Figures 5A-C).

EXAMPLE 5

Rapid, immediate, reversal of transcript modifications upon stimulation cessation

The changes observed post-DBS could tentatively be due to the electrical stimulation, to the operation itself or to both. To discriminate between these possibilities, RNA samples extracted one hour after the electrical stimulation was turned off (OFF state) were tested. ON- and OFF-sampIe sets were both derived from patients on the same dose of dopamine replacement therapy, which is considerably lower than that administered to pre-surgery patients. Therefore, the OFF state also served to assess the contribution of medication dose to the observed changes. Under OFF state, the major disease symptoms rapidly re-occur. For example, a PD patient pre-operation and in OFF-state (both lacking the DBS stimulation, but with different medication doses) will have limited mobility, as reflected by a worsened UPDRS-III (Figures 4A-D). The OFF-state was accompanied by differential expression of 351 transcripts (Table 3).

Table 3

The 351 g enes differentially expressed between

PD patien ts post-DBS (OFF tim) as compared

with post DBS (ONstim)

t-scores DFs p value gene_symbol

-3.15264 11.97581 0.008006 B3GALT6

-2.82716 10.66046 0.014955 TARDBP

-3.43697 11.94334 0.004619 DDI2

-2.97849 9.077637 0.011168 RSC1A1

-2.80651 11.94859 0.015552 LRRC8C

2.888939 8.922693 0.013277 GPR61

2.337768 11.69085 0.037884 CHIA

-2.30547 10.96816 0.040249 FAM46C

-2.60082 11.7245 0.023035 RBM8A

3.008814 8.68531 0.010538 LCE2B -2.33243 10.94712 0.034835 OCLM

-2.4003 11.9452 0.03369 CAMSAPILI

-2.56374 11.41111 0.024719 MTR

-2.67293 8.523359 0.020074 CLSTN1

-2.20626 11.68348 0.048391 ATP13A2

-2.31412 10.05018 0.039602 UBR4

-2.24998 11.91102 0.044598 SFPQ

-2.6515 10.60279 0.020891 HIVEP3

-2.53563 11.73342 0.026058 KCNA3

2.25086 11.78346 0.044528 NGF

-3.11498 8.939612 0.008593 ANP32E

-3.45922 7.431826 0.004423 LING04

-3.19882 11.32745 _, 0.007316 NUCKS1

-2.90785 10.30073 0.012802 JMJD4

-3.42031 9.01175 0.004766 MT1H

-2.53329 8.51193 0.026175 CCDC128

-2.57326 10.25497 0.024269 KIF5C

-2.36115 11.57674 0.036244 RAPGEF4

-2.48984 9.175446 0.02846 OBFC2A

-2.44473 7.218875 0.03098 ALS2CR8

-2.38599 11.93262 0.034593 ALS2CR16

-2.31945 11.53578 0.039198 RTN4

-2.37076 10.82113 0.035606 REEP1

-4.15998 11.16289 0.001171 ASCC3L1

-2.45711 8.247427 0.030262 ALS2CR11

-3.09398 10.83076 0.008945 COL4A4

-2.30423 11.79667 0.040343 NMUR1

-2.67896 11.95543 0.019854 C2orf54

-2.38718 11.9999 0.034515 APEH

-2.66192 9.34994 0.020487 MITF

-3.14002 9.240756 0.008204 UNQ6125

-2.33314 10.51525 0.038215 POLR2H

-3.18444 8.241225 0.007525 C3orf48

-2.5303 11.9318 0.026328 EPM2AIP1

-2.39414 8.537521 0.034076 LRRFIP2

-2.28599 11.96524 0.041728 CDC25A

-2.18854 8.137342 0.049999 NME6

-2.20819 8.77389 0.048223 CISH

-2.91209 8.321712 0.012698 RRP9

-2.80634 10.63138 0.015556 RFTl

-2.26802 8.429647 0.043127 STX19

-2.34151 10.62939 0.037615 GPR171

-2.21676 10.76291 0.047464 GHSR -2.2524 11.99604 0.044405 HTT

-2.53895 8.225078 0.025897 HGFAC

-4.3772 11.25586 0.000772 MED28

-2.2469 10.35289 0.044864 KLHL5

2.329208 7.703487 0.038502 CXCL1

-2.50327 8.392535 0.027709 LRIT3

-2.24075 10.60551 0.045388 FAT4

-2.4727 11.19409 0.029388 MRFAPILI

-2.26304 10.41489 0.043531 PF4

-2.51189 10.74156 0.027256 MFSD8

-2.2196 11.22454 0.047207 TTC29

-2.87551 10.93051 0.013624 PET112L

-2.65287 10.45946 0.020839 MTRR

-3.53608 11.09388 0.003808 TRIO

2.410119 9.363897 0.033077 CDH6

-2.2792 9.286289 0.042251 SNX18

-2.64428 11.8844 0.021188 PDE8B

-2.28964 11.40346 0.041449 ZCCHC9

-2.48578 11.99997 0.028671 NR2F1

-2.4964 8.147299 0.028088 ISOC1

-2.29791 11.67649 0.040816 MRPL22

-2.37791 9.507743 0.03514 ΡΓΓΧ1

-2.42559 11.83254 0.032118 UNQ9374

-2.19893 11.15978 0.049046 GMNN

-2.29753 10.58071 0.040846 HSPA1B

2.553728 10.02884 0.02518 GUCA1A

-2.31064 11.93021 0.039857 SLC25A27

-2.20882 10.81616 0.048171 MTOl

-2.28537 11.72266 0.041775 MTHFD1L

-2.58443 7.473402 0.023757 ACAT2

-2.49591 8.406261 0.028114 QKI

-2.3973 11.25085 0.03388 C6orfl95

-2.55292 10.57137 0.025216 GJB7

-2.54886 9.231962 0.025409 MAP3K7

-2.5123 8.859715 0.027235 RTN4IP1

-2.23117 12 0.046211 C6orf60

-3.00322 9.885807 0.010651 PPIL4

-2.26703 11.98493 0.043205 C1GALT1

-2.29278 11.2106 0.041216 DKFZp586I1420

2.519372 11.75307 0.026878 NPSR1

2.236898 9.729039 0.045716 LANCL2

2.425457 10.72786 0.032126 ZNF679

-2.69209 9.691518 0.019371 POR -2.94699 7.435167 0.01187 DPY19L2P4

-2.6141 11.5273 0.022464 DNAJB9

2.37374 7.564004 0.035414 C7orf34

-2.34712 11.99964 0.03723 GCK

-2.35582 9.003204 0.036604 FIGNL1

-2.21129 11.01752 0.04796 TBL2

-2.21757 6.259839 0.04739 SAMD9

-2.60614 11.38099 0.022803 SAMD9L

-3.19861 11.61816 0.007319 POT1

-2.2446 9.523797 0.045061 TRPV6

-2.62935 8.245011 0.021808 TACC1

2.428005 10.86226 0.031973 MFHAS1

3.082943 10.35235 0.009142 GTF2E2

2.481519 11.97935 0.0289 C8orf51

3.010945 11.86175 0.010493 LRRC24

2.643644 10.70436 0.021211 C9orf93

-2.21054 10.26271 0.048025 TLE4

-2.4639 11.83605 0.029878 FOXE1

2.80063 10.80236 0.015731 NEK6

2.453225 9.53278 0.03049 C9orfl63

2.703807 11.86825 0.018951 ADFP

2.671816 10.51995 0.020117 LOC554202

2.377867 10.36854 0.035143 DCTN3

2.494546 10.57458 0.028189 KLF9

2.394113 11.49949 0.034077 KIF12

2.253098 8.333875 0.044347 FAM129B

2.774601 9.599447 0.016546 FIBCDl

3.358212 7.358746 0.005367 RALGDS

2.226716 8.984696 0.046599 CAMSAP1

2.431547 10.95398 0.03176 CALML3

2.393772 8.462613 0.034101 SLC18A3

2.227318 11.26272 0.046546 RGR

-2.60613 10.40713 0.022804 ELOVL3

2.8851 8.631771 0.013376 NFKB2

-2.79825 11.99952 0.015807 TDRD1

2.538304 8.182608 0.025927 LRRC27

2.595803 10.8514 0.023259 UTF1

-3.07326 11.66245 0.009305 ANUBL1

2.24416 11.99999 0.045097 CTGLF3

2.606688 11.4742 0.022781 TYSND1

3.393459 9.462582 0.005019 CAMK2G

-2.25069 9.391925 0.044542 EHF

2.702246 11.4531 0.019005 SLC35C1 2.655306 8.997062 0.020746 ZP1

3.020807 9.27042 0.010291 LOC790955

-2.67952 10.8823 0.019833 CABP4

2.221446 10.67332 0.047045 DNAJB13

-2.86397 10.8637 0.013932 UBE4A

-2.34697 10.51661 0.037241 TMEM25

2.478802 11.94756 0.029044 EI24

-2.73285 9.380572 0.017937 KRTAP5-7

-2.20366 10.81476 0.04863 OSBPL5

2.192198 10.70668 0.04966 OR52R1

2.770426 10.05415 0.016683 OR52E2

-2.72141 11.4657 0.018333 HBE1

2.372541 9.044224 0.035491 OR52E8

2.571458 11.98362 0.024352 OR56A4

2.252495 9.35194 0.044397 GALNTL4

2.863595 9.661784 0.013942 OR5M10

2.518066 8.214831 0.026946 SSRP1

-2.42926 11.149 0.031901 VPS37C

-2.67853 6.904577 0.01987 MTA2

-2.21484 8.341076 0.04764 LOC51035

2.878292 10.50288 0.01355 PYGM

-2.36765 11.97504 0.035813 LTBP3

-2.61787 11.6992 0.022298 P4HA3

3.965362 10.08598 0.001693 PAK1

2.286995 11.0535 0.041652 RAB30

-2.41461 9.476763 0.032803 SYTL2

2.732093 10.85556 0.017963 BCL9L

2.443777 7.623646 0.031037 THY1

3.028369 6.816078 0.010143 OR10G7

2.323942 11.83874 0.038872 KCNJ1

-2.57434 8.447874 0.024222 RAD51AP1

2.4929 11.94851 0.028286 ACVR1B

2.207407 11.00658 0.048288 MFSD5

3.292966 11.96866 0.006095 HOXC11

2.437344 10.00042 0.031416 FAM62A

2.236923 11.9345 0.045714 MARS

-2.37989 11.83928 0.035007 SOCS2

-2.27618 9.154142 0.042488 MYBPC1

-3.19397 8.703052 0.007384 TCTN1

-2.20762 11.9928 0.04827 B3GNT4

2.203762 10.87694 0.04862 PGAM5

2.527141 10.53134 0.026485 VDR

3.591578 11.51638 0.003423 KRT72 3.567575 9.951413 0.003586 KRT78

2.302355 9.842166 0.040484 COROIC

2.266683 8.932296 0.043233 BRAP

2.482905 9.01784 0.028829 RBM19

2.299934 9.454395 0.040665 C12orf49

-2.33969 10.07634 0.037747 FAM124A

2.486031 10.49051 0.02866 CKAP2

-2.53983 11.94789 0.025853 PCCA

2.506357 11.7383 0.027547 PROZ

-3.20202 8.233195 0.007272 UPF3A

-2.3348 9.320066 0.038093 FLJ46358

-2.61051 11.01341 0.022619 UBL3

-2.38339 9.305748 0.034769 TNAP

-2.37331 10.6568 0.035442 EBI2

4.07182 11.84389 0.00138 EFNB2

2.684808 11.41317 0.019634 OR4N2

3.623466 8.969078 0.003218 WDR23

2.448931 11.16179 0.030733 KIAA0323

-2.22697 11.27204 0.046576 AKAP6

4.457541 10.28198 0.000669 KTN1

-2.31272 8.014232 0.039704 C14orfl00

2.372956 11.99996 0.035465 EXDL2

-2.63077 8.048628 0.021746 C14orfl31

2.540646 9.497697 0.025813 CMA1

4.283609 8.895796 0.000925 C14orfl05

-2.34481 8.960762 0.037389 LTBP2

2.602989 9.054002 0.022938 CAPN3

3.029494 7.643386 0.010122 C15orf50

2.869821 8.841919 0.013777 UNQ6190

-2.52023 9.751828 0.026836 TJP1

2.779615 8.950651 0.016388 PLA2G4F

2.687943 10.03488 0.019525 SLC30A4

2.583082 11.16116 0.023823 C15orfl5

-3.16766 9.921538 0.007773 ZNF280D

2.777638 11.99771 0.016453 SEMA7A

4.257292 11.43455 0.000974 ETFA

2.198695 11.9284 0.049067 NME4

2.341189 11.76684 0.037638 RAB40C

2.309337 10.29457 0.039954 DNAJA3

-2.38879 10.96699 0.034416 RBL2

2.324089 11.42439 0.038861 LPCAT2

2.66205 9.104882 0.020482 OGFOD1

2.315836 11.54498 0.039474 POLR2C -3.43881 9.018471 0.004603 CES3

2.962281 11.9464 0.01152 NOL3

-2.34855 11.89766 0.037126 NRN1L

-2.87074 10.52748 0.013753 FLJ14327

3.072113 11.50277 0.009323 ADA 2

2.689745 11.99786 0.019458 C16orf44

2.228075 9.596112 0.046478 FOXL1

3.46919 11.95853 0.004342 C16orf42

-2.22561 11.71305 0.046693 FU11151

2.22293 11.9445 0.046923 THUMPDl

4.212401 11.74763 0.001061 ZNF668

-2.65017^ 8.255299 0.020945 MLKL

2.318355 11.33876 0.039281 TMEM170

2.851627 9.566105 0.014275 PRDM7

2.280243 10.66094 0.042174 ASPA

3.560227 11.54476 0.003636 MED 11

2.302181 11.50212 0.040498 TXNDC17

3.064906 11.88309 0.009451 CENTB1

2.306382 10.71212 0.040177 NLGN2

2.595896 9.221629 0.023255 RA GRF

2.642345 9.250193 0.021263 WDR16

2.676783 8.145207 0.019933 ALDH3A2

-2.72708 9.796941 0.018137 PIPOX

2.833851 11.19719 0.01477 TAOK1

2.657401 10.40955 0.020663 TTC25

2.293903 10.20376 0.041131 NSF

2.526945 9.72008 0.026495 MYL4

2.588938 11.9184 0.023556 CROP

-2.79994 11.2501 0.015753 ANKFN1

2.720267 11.02404 0.018373 EFCAB3

2.492296 11.50094 0.028321 METTL2A

-2.99404 11.98167 0.010836 CACNG4

3.35855 8.143938 0.005363 GPR142

2.881191 11.1237 0.013474 C17orf77

2.304993 8.735181 0.040286 FAM100B

-2.59691 11.52773 0.023209 FOXK2

2.440097 11.13482 0.031249 YBX2

2.388434 11.93293 0.034438 PERI

2.933989 11.40153 0.012176 PIK3R5

2.657696 10.88211 0.020652 STX8

4.095076 8.981093 0.001319 TEKT3

-2.55971 10.07108 0.024908 MFAP4

-2.25299 8.30359 0.044357 DDX52 2.700179 10.1284 0.019077 LOC100129395

2.589302 11.88439 0.023539 KRT38

4.00579 11.92317 0.001563 KRT32

2.398057 11.86292 0.033834 SOST

3.425276 7.971585 0.004723 PHOSPHOl

3.435748 11.81368 0.00463 TK1

-2.26777 10.43131 0.043146 LGALS3BP

2.408853 11.6169 0.033156 SGSH

2.859244 10.22803 0.014063 MAPK4

2.340527 11.33798 0.037685 PSTPIP2

2.384309 8.483423 0.034706 HSH2D

2.277905 11.59762 0.042351 FFAR2

2.323758 10.61099 0.038886 KIAA0841

3.80282 10.69259 0.002285 MED29

2.401176 11.88093 0.033633 CD79A

2.669073 10.63911 0.020215 RELB

2.190506 11.66042 0.049819 SEPW1

2.229715 10.60955 0.046334 DHDH

2.475818 11.903 0.029216 CACNG8

2.68485 11.92622 0.019633 MKNK2

2.817454 11.99276 0.01523 ZNF266

2.492716 9.222246 0.028296 SAMD1

2.731894 11.06964 0.01797 PHLDB3

3.164859 10.88221 0.007815 SNRPD2

2.472514 11.69872 0.029399 IRF2BP1

2.460168 8.821278 0.03009 CCDC8

2.292064 10.9735 0.041268 CABP5

2.267598 11.80095 0.04316 RASIP1

3.229799 11.96878 0.00689 KLK5

3.8255 11.52042 0.002191 KLK12

3.456289 10.87453 0.004448 ATPBD3

2.545149 11.86274 0.025593 SIGLEC8

2.466383 7.459965 0.029741 ZNF577

3.336487 11.31706 0.005605 LILRA5

2.280185 11.87551 0.042178 TMEM86B

2.403743 10.31983 0.033474 SAPSl

2.395143 11.39232 0.03401 FLJ39005

-2.72774 11.32006 0.018116 MAPRE1

3.552367 9.264139 0.003688 RALY

2.88742 11.84745 0.013315 ERGIC3

2.253603 9.063204 0.044305 PHF20

2.988949 7.397837 0.010951 DLGAP4

2.197174 11.78507 0.049202 YWHAB 2.655872 11.57073 0.020723 COL20A1

3.165626 9.895526 0.007803 SDCBP2

3.27983 11.04904 0.006249 SLC23A2

2.249698 11.32604 0.044621 THBD

2.37927 9.735371 0.035048 CST11

2.315423 6.965156 0.039506 RP4-691N24.1

-2.37629 11.84997 0.035248 BCL2L1

-2.34947 8.440905 0.037059 CHD6

2.402312 11.96042 0.033561 SLPI

3.442656 11.65767 0.004569 WFDC8

-2.33761 10.84567 0.037895 KCNG1

2.191338 11.99951 0.049741 RP11-93B14.6

2.441594 11.37721 0.031163 KRTAP20-1

-2.61935 8.521915 0.022232 DYRK1A

3.411175 11.9917 0.00485 C21orfl25

3.061405 9.331558 0.009517 KRTAPlO-7

2.198418 11.75004 0.049092 KRTAP21-2

2.634947 7.184714 0.021578 SFRS15

-2.45223 8.237886 0.030546 DSCR3

2.712869 8.90688 0.01863 PLAC4

2.490871 7.053845 0.028403 TFF1

3.261756 10.29183 0.006479 C21orf29

2.243395 10.06294 0.045159 FTCD

2.564514 8.579893 0.024677 C22orB7

2.245036 9.116502 0.045024 H1F0

2.359382 11.1918 0.036364 RBX1

2.966009 9.936604 0.011444 ZDHHC8P

2.320319 11.98003 0.039133 TMEM184B

-2.31003 9.514439 0.039903 PPP1R2P9

-2.68166 9.060788 0.019751 FAM120C

3.29909 11.99363 0.006023 ALAS2

2.406156 11.88534 0.033324 ZMYM3

-2.28869 11.13402 0.041522 INGX

-2.66354 11.95407 0.020423 TMSL8

3.094487 11.59604 0.008936 TEX13A

-2.78905 8.190486 0.016096 UPF3B

-2.22953 7.372463 0.04635 USP26

2.339679 10.76946 0.037747 PNMA5

3.269566 11.14579 0.006377 ND1

2.797162 8.548408 0.015842 Clorf222 Unsupervised HCL fully distinguished PD patients in stim-ON state from stim-OFF state. The classified clusters were enriched in DNA binding and nucleotide binding (Figure 4D). PCA further segregated all samples correctly.

Globally, the post-DBS state exhibited more transcript decreases than increases compared to pre- DBS patients, inverse to the trend for increases between PD patients and healthy controls (Figure 6A). Furthermore, post- to pre-surgery comparison presented more divergent functional categories than those enriched in PD compared to controls or to OFF-state.

Of the stim-OFF changed transcripts, 105 (30 %) (Figure 6B) also changed following STN-DBS compared to the pre-surgery state, mostly in an inverse direction, reflecting a molecular manifestation of the reversibility of motor function observed with DBS and verifying that the effect of the stimulus was larger than this of the medication dosage. The likelihood for an overlap of 105 common transcripts to occur in random is: pCX = 105)= ( ¾0.O221¹⁰⁵O.77-³⁶⁰ = 9.856 _*tlO-⁷²) . „ .

1Q5* , nearly zero. Considering that the patients received the same reduced medication dose under OFF- and ON-state further attributes the observed change to the cessation of the stimulus alone. Also, comparing the pre-DBS to post-DBS OFF stimulation profiles (once again, using permutation t-tests) identified 114 transcripts as differentially expressed, much less than those 465 transcripts modified following DBS stimulation; demonstrating that the pre- DBS and post-DBS -OFF stimulation states were more similar to one another than any other two states among those tested. Of the above 114 differential transcripts, 27 (24 %) were also modified under DBS stimulation and reduced medication.

Next, multi-comparisons were performed. Of the post-DBS transcripts different from controls 114 (35.5 %), also changed in at least one of the three major comparisons (PD compared to controls, post-DBS compared to pre-DBS or post-DBS stim-OFF compared to stim-ON). Of those, 46 (23%) changed both from PD to post-DBS and to HC, although pre- and post-DBS patients are on different medication dosages, again highlighting the reversible mode of the observed changes and their dependence on the stimulus itself. EXAMPLE 6

Rapid functional reversibility of the DBS stimulus

Post-hoc functional analyses detect robust changes in differentially expressed genes, but disregard subtle changes in functionally related transcript groups which are not robustly modified [30]. To detect subtle increases and decreases in all transcript classes, the expression signal of each gene was calculated based on all of its core- annotated probe sets for both Biological Process (BP) and Molecular Function (MF) terms. Then functional ad-hoc GO analysis was applied by both Kolmogorov-Smirnov (KS) statistics and threshold-dependent Fisher exact test [8]. The KS analysis is not based on pre-defined cutoff by fold-change threshold. Therefore, it is completely gene list-independent and is able to detect subtle expression changes in groups of genes that function together [31]. Each GO term was separately tested for increase and decrease by the above tests. Nine of the reversed terms increased stim-ON and decreased stim-OFF, and 16 terms decreased in stim-ON and increased upon stim-OFF, including the Wnt receptor signaling pathway. Of note, five disease-increased BP terms resisted stim- OFF.

Next, the present inventors searched for GO terms that changed between all of the tested stages (controls to PD, pre-DBS to ON- and ON- to OFF-stimulation). These included 16 MF and 4 BP common terms (of those, 12 MF and 3 BP terms changed in the same direction between controls to PD, as between the DBS-ON to the OFF- stimulation state (Figure 6B). Most of the disease-increased BP terms reversed under stim-ON and reversed again under stim-OFF, reinforcing the observation of a reversible clinical effect of stimulus cessation toward a pre-treatment disease state. Comparing each GO term to its direct GO tree "parents" enabled a zoom-in (magnified) focus on specific changes.

EXAMPLE 7

Transcripts modified in all comparisons reflect the neurological efficacy of DBS To gain translational utility, the present inventors searched for a selected group of transcripts which could constitute a "molecular signature", and would react both to the disease and to the DBS procedure. 29 transcripts changed (p<0.01) both in PD patients compared to controls and between ON and OFF stimulation (see Table 4, herein below).

Table 4

Chromosome 1: 110,082,494- ENSG0000015 SEQ ID

GPR61 83873 3 110,089,882 6097 NO: 23

Chromosome 16: 89,753,078- ENSG0000018 SEQ ID

CDK10 8558 17 89,762,772 5324 NO: 24

ZC3H7 Chromosome 16: 11,844,442- ENSG0000012 SEQ ID A 29066 2 11,891,099 2299 NO: 25

27670 Chromosome 15: 80,445,122- ENSG0000010 SEQ ID

FAH 0 2 80,479,288 3876 NO: 26

NECAB Chromosome 8: 91,803,778- ENSG0000012 SEQ ID 1 69352 3 91,971,630 3119 NO: 27

Chromosome 16: 66,878,280- ENSG0000016 SEQ ID

CA7 766 3 66,888,052 8748 NO: 28

Chromosome 3: 10,334,815- ENSG0000015 SEQ ID

SEC13 44437 22 10,362,862 7020 NO: 29

Clustering the expression values of those 29 genes, classified DBS-treated patients together with controls, and OFF-stimulus ones with pre-operated patients (Figure 7A). PCA analysis classified these two groups similarly, except for two apparent control samples which were classified with the patients group and one stim- OFF patient maintaining a stim-ON pattern (Figure 7B).

Intriguingly, the 29 classifier sequences included 6 "signature" transcripts which were also identified as changed in an identical analysis flow which was conducted on a web database repository deposited 3' microarray dataset of a larger cohort of early PD patients compared to controls [7,8] (Figure 7A, double stars) or to patients with other neurological diseases (neurological controls, NC) (a minimal list of permutation t-test p<0.05) [8] (Figure 7A, triple stars). Clustering analysis by HCL revealed that these 6 transcripts classify in the early PD cohort data set both PD patients from controls and from neurological control (Figures 8A-B).

The 6 "signature" transcripts were all regulatory in nature: the transcription factor NR2F1 [32]; the PCBP2 poly (rC) binding protein that affects RNA turnover [33]; the PD-associated PTPN1 protein tyrosine phosphatase [34]; the HNRPDL member of the heteronu clear proteins which control exon inclusions [35], the protein ubiquitination controller PJA1 [36]; and the endoplasmic reticulum membrane glycoprotein TRAM1 involved in protein translocation [37], which induces interferon- beta [38] through coupling with Toll-like receptor 4 (TLR4) that mediates bacterial infection responses [39].

Post- DBS patients, either ON- or OFF-stimulus, all showed improved motor function as independently evaluated by a neurologist (based on the UPDRS-III scale), albeit to different extents (25-88.9 % improvement). In each patient, the magnitude of change of all of the DBS-modified transcripts was correlated with the individual neurological improvement, but not with the extent of reduction in dopamine replacement therapy or with the extent of pre-DBS change in gene expression compared to controls. Linear regression test validated this correlation (R =0.679). Furthermore, the expression changes observed in the 29 signature transcripts alone showed tight correlation with the motor clinical improvement in each patient as reflected by their UPDRS-III (R²=0.902, ANOVA p=0.005, Figure 7C), identifying these 29 transcripts as an independent "molecular signature" reflecting treatment success.

EXAMPLE 8

Inverse cholinergic and inflammatory changes

In PD, the dopaminergic-cholinergic balance is impaired by dopamine depletion and acetylcholine increase. Correspondingly, anticholinergics were the first drugs available for the symptomatic treatment of Parkinson's disease [40]. In this context, the present inventors were intrigued to detect disease-associated reductions in the KS analysis of transcript categories of cholinergic signaling. The category of muscarinic acetylcholine signaling and activity, for example, showed DBS-inducible enhancement and OFF-stimulus reduction. Compatible with the reported cholinergic blockade of inflammation responses [39,41], the present inventors further observed an inverse trend of PD-associated increases, DBS-repressible inflammation and immune-related categories and re-induction of those by OFF state, suggesting cholinergic-mediated changes in peripheral inflammation as a mechanism underlying at least part of the observed differences in leukocyte transcripts (Figure 9). The DBS-induced decreases in the T-helper (TH)-l immune response, which was not detectable in pre-surgery PD patients may balance part of this effect.

CONCLUSIONS

The reversible, stimulation-dependent, discriminative gene expression profiles described in this work provide initial evidence that deep brain stimulation may affect not only the clinical features of a neurodegenerative disease but also induce electrical stimulus-dependent peripheral changes. Pending validation in additional cohorts and control by examination of other movement disorders, our current approach may lead to substantially more sensitive diagnostics and prognostics, better follow-up of the neurodegenerative process and a novel efficacy evaluation for tested therapeutics. The disease-modified and DBS-inducible transcription signatures may then provide objective tools for the prediction and post-factum assessment of the molecular mechanisms underlying disease severity and therapeutic efficacy, both in PD and in other diseases. Blood leukocytes can hence yield novel perspectives and serve to extract transcript signatures for diagnosis and gene targets for future intervention with PD.

References for Examples 1-8

Fahn S. Description of Parkinson's disease as a clinical syndrome. Ann N YAcad Sci. 2003; 991: 1-14.

Benabid AL, Pollak P, Seigneuret E, et al. Chronic VIM thalamic stimulation in Parkinson's disease, essential tremor and extra-pyramidal dyskinesias. Acta

Neurochir Suppl (Wien). 1993; 58: 39-44.

Bergman H, Wichmann T, DeLong MR. Reversal of experimental parkinsonism by lesions of the subthalamic nucleus. Science. 1990; 249: 1436-8.

Benabid AL, Chabardes S, Mitrofanis J, et al. Deep brain stimulation of the subthalamic nucleus for the treatment of Parkinson's disease. Lancet Neurol.

2009; 8: 67-81.

Deuschl G, Schade-Brittinger C, Krack P, et al. A randomized trial of deep- brain stimulation for Parkinson's disease. N Engl J Med. 2006; 355: 896-908. Weaver FM, Follett K, Stern M, et al. Bilateral deep brain stimulation vs best medical therapy for patients with advanced Parkinson disease: a randomized controlled trial. JAMA. 2009; 301: 63-73.

Scherzer CR, Eklund AC, Morse LJ, et al. Molecular markers of early

Parkinson's disease based on gene expression in blood. Proc Natl Acad Sci US A. 2007; 104: 955-60.

Soreq L, Israel Z, Bergman H, et al. Advanced microarray analysis highlights modified neuro-immune signaling in nucleated blood cells from Parkinson's disease patients. J Neuroimmunol. 2008; 201-202: 227-36.

Segman RH, Goltser-Dubner T, Weiner I, et al. Blood mononuclear cell gene expression signature of postpartum depression. Mol Psychiatry. 2010; 15: 93- 100, 2.

Segman RH, Shefi N, Goltser-Dubner T, et al. Peripheral blood mononuclear cell gene expression profiles identify emergent post-traumatic stress disorder among trauma survivors. Mol Psychiatry. 2005; 10: 500-13, 425.

Temperli P, Ghika J, Villemure JG, et al. How do parkinsonian signs return after discontinuation of subthalamic DBS? Neurology. 2003; 60: 78-81.

Henchcliffe C, Beal MF. Mitochondrial biology and oxidative stress in

Parkinson disease pathogenesis. Nat Clin Pract Neurol. 2008; 4: 600-9. Kumaran R, Kingsbury A, Coulter I, et al. DJ-1 (PARK7) is associated with 3R and 4R tau neuronal and glial inclusions in neurodegenerative disorders.

NeurobiolDis. 2007; 28: 122-32.

Soreq L, Gilboa-Geffen A, Berrih-Aknin S, et al. Identifying alternative hyper- splicing signatures in MG-thymoma by exon arrays. PLoS ONE. 2008; 3: e2392. Gardina PJ, Clark TA, Shimada B, et al. Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array. BMC Genomics. 2006; 7: 325.

Johnson JM, Castle J, Garrett-Engele P, et al. Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science. 2003; 302: 2141-4.

Dudoit S, Gentleman RC, Quackenbush J. Open source software for the analysis of microarray data. Biotechniques. 2003; Suppl: 45-51.

Spillantini MG, Schmidt ML, Lee VM, et al. Alpha-synuclein in Lewy bodies. Nature. 1997; 388: 839-40.

Scherzer CR, Grass JA, Liao Z, et al. GATA transcription factors directly regulate the Parkinson's disease-linked gene alpha-synuclein. Proc Natl Acad Sci USA. 2008; 105: 10907-12.

van Duijn CM, Dekker MC, Bonifati V, et al. Park7, a novel locus for autosomal recessive early-onset parkinsonism, on chromosome lp36. Am J Hum Genet. 2001; 69: 629-34.

Partek®. Genomics Suite Software. 6.5 Beta, 6.09.1110 ed. St. Louis, MO, USA: Partek Incorporated, 1993-2009.

Hosack DA, Dennis G, Jr., Sherman BT, et al. Identifying biological themes within lists of genes with EASE. Genome Biol. 2003; 4: R70.

Dennis G, Jr., Sherman BT, Hosack DA, et al DAVID: Database for

Annotation, Visualization, and Integrated Discovery. Genome Biol. 2003; 4: P3. Zaidel A, Bergman H, Ritov Y, et al. Levodopa and subthalamic deep brain stimulation responses are not congruent. Mov Disord. 2010; 25: 2379-86.

Fuchs J, Tichopad A, Golub Y, et al. Genetic variability in the SNCA gene influences alpha-synuclein levels in the blood and brain. Faseb J. 2008; 22: 1327-34. 26. Bonifati V, Rizzu P, van Baren MJ, et al. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science. 2003; 299: 256-9.

27. Keyser RJ, van der Merwe L, Venter M, et al. Identification of a novel

functional deletion variant in the 5'-UTR of the DJ-1 gene. BMC Med Genet. 2009; 10: 105.

28. Zhong N, Kim CY, Rizzu P, et al. DJ-1 transcriptionally up-regulates the human tyrosine hydroxylase by inhibiting the sumoylation of pyrimidine tract-binding protein-associated splicing factor. J Biol Chem. 2006; 281: 20940-8.

29. Sun S, Zhang Z, Sinha R, et al. SF2/ASF autoregulation involves multiple layers of post-transcriptional and translational control. Nat Struct Mot Biol. 2010;

Online.

30. Ben-Shaul Y, Bergman H, Soreq H. Identifying subtle interrelated changes in functional gene categories using continuous measures of gene expression.

Bioinformatics. 2005; 21: 1129-37.

31. Alexa A, Rahnenfuhrer J, Lengauer T. Improved scoring of functional groups from gene expression data by decorrelating GO graph structure. Bioinformatics. 2006; 22: 1600-7.

32. Zuo F, Mertz JE. Simian virus 40 late gene expression is regulated by members of the steroid/thyroid hormone receptor superfamily. Proc Natl Acad Sci U SA. 1995; 92: 8586-90.

33. Fujimura K, Katahira J, Kano F, et al. Selective localization of PCBP2 to

cytoplasmic processing bodies. Biochim Biophys Acta. 2009; 1793: 878-87.

34. Pankratz N, Wilk JB, Latourelle JQ et al. Genomewide association study for susceptibility genes contributing to familial Parkinson disease. Hum Genet. 2009; 124: 593-605.

35. Kawamura H, Tomozoe Y, Akagi T, et al. Identification of the

nucleocytoplasmic shuttling sequence of heterogeneous nuclear

ribonucleoprotein D-like protein JKTBP and its interaction with mRNA. J Biol Chem. 2002; 277: 2732-9.

36. Yu P, Chen Y, Tagle DA, et al. PJA1, encoding a RING-H2 finger ubiquitin ligase, is a novel human X chromosome gene abundantly expressed in brain. Genomics. 2002; 79: 869-74. 37. Gorlich D, Hartmann E, Prehn S, et al. A protein of the endoplasmic reticulum involved early in polypeptide translocation. Nature. 1992; 357: 47-52.

38. Kagan JC, Su T, Horng T, et al TRAM couples endocytosis of Toll-like

receptor 4 to the induction of interferon-beta. Nat Immunol. 2008; 9: 361-8. 39. Shaked I, Meerson A, Wolf Y, et al. MicroRNA-132 potentiates cholinergic anti-inflammatory signaling by targeting acetylcholinesterase. Immunity. 2009; 31: 965-73.

40. Ben-Shaul Y, Benmoyal-Segal L, Ben-Ari S, et al. Adaptive

acetylcholinesterase splicing patterns attenuate l-methyl-4-phenyl-l,2,3,6- tetrahydropyridine-induced Parkinsonism in mice. Eur J Neurosci. 2006; 23:

2915-22.

41. Tracey KJ. The inflammatory reflex. Nature. 2002; 420: 853-9.

42. Gurevich M, Tuller T, Rubinstein U, et al. Prediction of acute multiple sclerosis relapses by transcription levels of peripheral blood cells. BMC Med Genomics. 2009; 2: 46.

43. Grisaru D, Pick M, Perry C, et al. Hydrolytic and nonenzymatic functions of acetylcholinesterase comodulate hemopoietic stress responses. J Immunol. 2006; 176: 27-35.

44. Reizenstein P. The haematological stress syndrome. Br J Haematol. 1979; 43:

329-34.

45. Toft P, Tonnesen E, Helbo-Hansen HS, et al. Redistribution of granulocytes in patients after major surgical stress. APMIS. 1994; 102: 43-8.

46. Olanow CW, Rascol O, Hauser R, et al. A double-blind, delayed-start trial of rasagiline in Parkinson's disease. N EnglJ Med. 2009; 361: 1268-78.

47. Riley D, Lozano A. The fourth dimension of stereotaxis: timing of neurosurgery for Parkinson disease. Neurology. 2007; 68: 252-3.

48. Savica R, Rocca WA, Ahlskog JE. When does Parkinson disease start? Arch Neurol. 2010; 67: 798-801.

49. Kapranov P, Willingham AT, Gingeras TR. Genome-wide transcription and the implications for genomic organization. Nat Rev Genet. 2007; 8: 413-23.

50. Miller G. Neuropsychiatry. Rewiring faulty circuits in the brain. Science. 2009;

323: 1554-6. Shah DB, Pesiridou A, Baltuch GH, et al. Functional neurosurgery in the treatment of severe obsessive compulsive disorder and major depression:

overview of disease circuits and therapeutic targeting for the clinician.

Psychiatry (Edgmont). 2008; 5: 24-33.

Kahane P, Depaulis A. Deep brain stimulation in epilepsy: what is next? Curr Opin Neurol.

Graff-Radford J, Foote KD, Mikos AE, et al. Mood and motor effects of thalamic deep brain stimulation surgery for essential tremor. Eur J Neurol. Kurian SM, Le-Niculescu H, Patel SD, et al. Identification of blood biomarkers for psychosis using convergent functional genomics. Mol Psychiatry. 2009. Goshen I, Kreisel T, Ben-Menachem-Zidon O, et al. Brain interleukin-1 mediates chronic stress-induced depression in mice via adrenocortical activation and hippocampal neurogenesis suppression. Mol Psychiatry. 2008; 13: 717-28. Gershon ES, Hamovit J, Guroff JJ, et al. A family study of schizoaffective, bipolar I, bipolar II, unipolar, and normal control probands. Arch Gen

Psychiatry. 1982; 39: 1157-67.

Fahn S ER, Members of the UPDRS Development Committee. Unified

Parkinson's Disease Rating Scale; 1987.

www.affymetrix.com/support/technical/technotes/blood_technote.pdf

Ashburner M, Ball CA, Blake JA, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25: 25-9.

Kanehisa M, Goto S, Furumichi M, et al. KEGG for representation and analysis of molecular networks involving diseases and drugs. Nucleic Acids Res. 2010; 38: D355-60.

Tatusov RL, Koonin EV, Lipman DJ. A genomic perspective on prote n families. Science. 1997; 278: 631-7.

Bairoch A, Apweiler R, Wu CH, et al. The Universal Protein Resource

(UniProt). Nucleic Acids Res. 2005; 33: D154-9.

Edgar R, Domrachev M, Lash AE. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 2002; 30: 207-10.

www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE23676 EXAMPLE 9

Exon arrays reveal alternative splicing aberrations in Parkinson 's disease leukocytes pre- and post- deep brain stimulation

MATERIALS AND METHODS

The same blood leukocyte RNA samples that were hybridized in Examples 1-8 on the Affymetrix GeneChip® Exon_1.0_ST_Array (Affymetrix, Santa Clara, CA, USA) were re-analyzed using alternative-splicing detection targeted analysis. The open source AltAnalyze program (version 2.03, using Ensembl version 62) served for exon level analysis and Matlab R20011A(7.12.0) for downstream analyses (False Discovery Rate calculations and clustering analysis).

RESULTS

Hybridization of leukocyte mRNA from 7 carefully matched PD patients and 6 healthy controls (HC) (Figure 1A) on the Affymetrix chip was re-analyzed.

Differential splicing patterns in PD patients' compared to control leukocytes: The expression profiles of PD patients' leukocytes were compared to that of age- and gender-matched HC volunteers. T-test on splicing-index (SI) calculations detected 163 alternative splicing events in 150 distinct genes (SI t-test p-value<0.05 and detection above background (DABG) p-value<0.05). All the detected genes passed Benjamini and Hochberg FDR test [Benjamini Y, Hochberg Y, Journal of the Royal Statistical Society 1995;57:289-300]. Hierarchical classification (HCL) separated patients from controls based on the gene-normalized SI values (Figures 10A-B). The classification was conducted blind to the clinical condition, using Euclidean distance for both rows (exons) and columns (samples).

Highly alternatively spliced exons with a log ratio between tissues (2-fold SI change) having SI p-value<0.005 and DABG p-value <0.05, included 19 events in 18 genes, including the leucine rich repeat containing LRRC8C and the ATP -binding (AF130358.5) gene (Table 5). Table 5

T-test BH- SI direction

FDR adjuected splice- changed of mida pro adjusted p- Ensemb fold- annotatio exon-exon regulatio s p- bes symb value 1 change n score cluster n value et ol

25

ENSGO 10

000015 0.028 02 LYP

0.00089 0556 -1.05 2 El 9-3 down . 777 4 D6B

34

ENSGO 38

000018 0.048 80 EP40

0.00224 5684 -1.25 3 E9-1 down 127 4 ONL

25

ENSGO 73

000007 0.041 73 CLA

0.00111 4054 2.2 2 E16-1 up 294 2 SP1

34

ENSGO 07

000017 0.013 50 PDE3

0.00005 2572 1.02 2 E17-1 up 459 6 A

39

ENSGO 14 AF13

000021 0.048 79 0358.

0.00235 5557 -1.16 3 ElO-1 down 327 9 5

31

ENSGO 97 KIA

000018 0.038 73 A202

0.0025 3354 -1.05 3 E13-1 down 869 2 6

39

ENSGO 96

000000 0.042 25 TKT

0.0013 7350 1.07 2 E14-2 up 905 4 LI

38

ENSGO 26

000019 0.032 83 ZNF2

0.00132 7134 -1.03 2 E5-5 down 421 1 57

30

ENSGO 11

000013 0.037 27 DMT

0.00126 5164 -1.1 2 E9-2 down 227 3 Fl

37

ENSGO 26 MYC

000013 E14- 0.034 51 BPA

0.00132 6449 1.08 4 2,E15-1 up 731 9 P

27

ENSGO 31

000013 0.034 88 USO

0.00126 8768 1.19 2 E26-2 up 506 2 1

ENSGO 0.049 40 APIS

0.00463 000018 -1.17 3 18-3 down 393 00 2 2287 71

1

30

ENSGO 87

000015 0.044 58 VPS3

0.00492 5975 -1.07 2 E13-1 down 573 2 7A

23

ENSGO 81

000023 0.049 77 CICP

0.00161 4419 1.03 2 El-31 up 858 1 13

23

ENSGO 45

000017 0.045 97 LRR

0.00133 1488 -1.04 3 E7-1 down 038 7 C8C

37

ENSGO 42

000013 0.038 17 MYB

0.00126 2382 -1.09 2 E5-1 down 04 2 BP1A

39 LA16

ENSGO 35 c- 000021 0.046 74 60G3

0.00347 5268 -1.5 2 El-7 down 998 1 .8

35

ENSGO 65

000012 0.029 69 DLG

0.00127 6787 -1.01 2 E7-1 down 599 2 AP5

Post-hoc functional enrichment analysis using EASE indicated functional involvement of the detected genes in alternative splicing and protein transport.

EXAMPLE 10

Alternative splicing aberrations following deep brain stimulation Correlated with Parkinson 's disease motor improvement MATERIALS AND METHODS

Patient recruitment, DBS neurosurgery and clinical evaluation: 7 PD male patients nominated for bilateral STN-DBS neurosurgery (age, disease duration and BMI under Figure 11) were recruited. These are the same patients and microarray samples as in Examples 1-9. Figure 11 also details the levodopa equivalent dosage prior to, and following, STN-DBS. Subjects were assessed for their clinical background and state and fulfilled detailed medical history questionnaires. Patients with other medical conditions were excluded, including depression and past and current DSM Axis I and II psychological disorders (SM), chronic inflammatory disease, coagulation irregularities, previous malignancies or cardiac events, or any surgical procedure up to one year pre- DBS. Exceptions were hyperlipidemia (2 patients), hypertension and diabetes (2 patients each). All patients went through bilateral STN-DBS electrode implantation (Medtronics, USA) and were under dopamine replacement therapy (DRT) both pre- and post-DBS (on significantly reduced dosage post-DBS with t-test p<0.01), the last medication administered at least 5 hours pre-sampling. Patients exhibited similar total white and red blood cell counts pre- and post-DBS. Two patients received anti- hypertension medication and one-hyperlypidemia treatment. Blood samples were collected from each patient at three time points: (1) one day pre-DBS upon hospitalization, with medication (2) post-DBS (range 6 - 18 weeks), when reaching optimal clinical state as evaluated by a neurologist and on a lower DRT dose, stim-ON and (3) Stim-OFF, following 60 minutes OFF electrical stimulation (counted from stage 2). The clinical severity of disease symptoms was assessed by a neurologist for Unified PD Rating Scale (UPDRS-III), section 3 (motor section) at 3 states, as the blood sampling: (1) pre STN-DBS, (2) post STN-DBS, stim-ON and (3) post-DBS stim-OFF. Healthy control samples were collected from six age- and gender-matching volunteers

Blood sample collection and RNA extraction: Blood collection was conducted between 10AM-14PM from seven male PD patients 1 day prior to DBS and 6-18 weeks following DBS while being ON and following 1 hour OFF electrical stimulation and from 6 age- and gender-matching healthy control subjects. Collected venous blood (9 ml blood using 4.5 ml EDTA (anti-coagulant) tubes) was immediately filtered using the LeukoLock fractionation and stabilization kitTM (Ambion, Applied Biosystems, Inc., Foster City, CA) and incubated in RNALater (Ambion). Stabilized filters and serum samples were stored at -80 °C. RNA extraction followed the manufacturers' alternative protocol instructions. Briefly, cells were flushed (TRI-ReagentTM, Ambion) into 1- bromo-3-chloropropane-containing 15 ml tubes and centrifuged. 0.5 and 1.25 volume water and ethanol were added to the aqueous phase. Samples were filtered through spin cartridges, stored in pre-heated 150 μΐ EDTA; RNA was quantified in Bioanalyzer 2100 (Agilent) and frozen.

Microarray sample preparation, hybridization and scanning: l\,g of total RNA was labeled using the Affymetrix exon array using whole transcripts sense targeting labeling assay according to the manufacturers' instructions; cDNA samples were hybridized to GeneChip® Exon_1.0_ST_Array (Affymetrix, Santa Clara, CA, USA) microarrays, and results were scanned (GeneChip scanner 30007G, 27 CEL files).

Probe-set summarization: Microarray probe-set summarization of the input CEL files conducted using Affymetrix Power Tools (APT) through AltAnalyze.

Microarray alternative splicing analysis: Splicing targeted analysis of the exon array data conducted using the software AltAnalyze (version V04) and the latest version of Enseml human database (worldwidewebdotensembldotorg). Prior to the analysis, probe-sets with detection above background (DABG) p-value were removed from the input data. Expression data formatted to log. Two splicing analyses methods were applied on the expression data: splicing-index (SI) [Clark TA, Sugnet CW, Ares M (2002) Science 296: 907; Srinivasan K et al. (2005) Methods 37: 345-359] and finding isoforais using robust multichip analysis (FIRMA) [Purdom E et al., 2008, Bioinformatics 24: 1707]. Briefly, the SI represents the log ratio of the exon intensities between two conditions following a normalization to the gene intensities in each sample: SIi=log2((eli/glj)/(e2i/g2j)), for the i-th exon of the j-th gene in condition type 1 or 2. The SI values were subjected to normalization to the constitutive gene level expression. Alternative splicing change was detected using one-way ANOVA (equivalent to t-test) in order to probe for differential inclusion of the exon into the gene. Thus, significance of microarray pair-wise comparisons was derived by 2-tailed t- test. To produce the highest proportion of true positives the analysis included only the following probe sets and genes: (1) probe sets with Detection Above Background (DABG) p-value < 0.05 (2) exons with a gene level normalized log ratio between experimental conditions > 2, (3) t-test p-values < 0.05 (4) FDR p-value < 0.05 (5) maximal absolute gene expression change = 3 and (6) Core level exons. Overall, out of 864,741 probe sets, 485,797 remained for analysis following detection p-values filtering. No filtering based on expression data levels conducted. Overall, 42,340 transcripts were analyzed.

Pathway analysis: Pathway analysis conducted through AltAnalyze by GO- Elite. The analysis included both Gene Ontology (GO) database and Wiki-pathways. Filtering conducted based on raw gene expression p-value, and the maximal gene expression t-test p-value was set to 0.05. The minimal number of changed genes was set to 3, and 2000 permutations were conducted in the over representation analysis (ORA). The microelectrode recording and Root mean square (RMS): Data were acquired with the MicroGuide system (AlphaOmega Engineering, Nazareth, Israel). Neurophysiological activity was recorded via polyimide coated tungsten microelectrodes with impedance mean ± standard deviation (SD): 0.64 ± 0.14 ΜΩ (measured at 1 kHz at the beginning of each trajectory). The signal was amplified by 10,000, band-passed from 250 to 6,000 Hz (using a hardware four-pole Butterworth filter) and sampled at 48 kHz by a 12 bit A/D converter (using ±5 V input range, i.e. -0.25 μν amplitude resolution). One trajectory was mistakenly sampled at 12 kHz. For both the left and right hemispheres, a single trajectory using one or two microelectrodes (separated by 2 mm antero-posteriorly in the parasaggital plane) was made starting at 10 mm above the calculated target (center of the lateral STN). The electrodes were advanced in small discrete steps, towards the estimated center of the lateral STN. Step size (ranging 500μπι down to 50 μηι in our recordings) was controlled by the neurophysiologist in order to achieve optimal unit recording and identification of upper and lower borders of the STN. Typically shorter steps (~100 μπι) were used when the electrode was advanced closer to the presumed location of the STN. Following a 2- second signal stabilization period after electrode movement cessation, multi-unit traces were recorded for a minimum of 5 seconds. Conditions in the operating-room often result in non-stable recordings (e.g. due to further movement of brain tissue in relation to the electrode tip or due to neuronal injury). The data traces were therefore analyzed for stability by custom software. This was done by dividing each data trace into consecutive segments of 50 ms and computing the root mean square (RMS) for each segment. A section of the trace was considered stable when all corresponding segments' RMS values lay within three standard deviations of the median RMS. The longest stable section of the data trace was then selected for further analysis, discarding the rest of the trace. All stable sections included in the analysis were longer than 3 seconds (duration mean ± SD: 10.8 ± 3.4s). Only electrodes that passed through the STN were used for this study (56 in total).

The root mean square (RMS) estimate of the raw multi-unit activity recorded by the microele ach electrode depth is defined as follows

is the vector of the sampled analog signal with mean μ, Xi is each sample, and n is the number of samples. RMS values are susceptible to electrode properties and other external factors (e.g. amplifier gain); hence the RMS requires normalization in order to be an absolute measure. The RMS for each trajectory was therefore normalized by the RMS average of the first ten recordings (assumed to represent an unbiased estimate of the pre-STN baseline activity) creating a normalized RMS (NRMS).

RESULTS

Blood leukocyte exon array mRNA expression of seven participant PD patients one day prior to DBS neurosurgery was compared to the expression of the same patients 3-6 months post-DBS, upon symptoms stabilization, while being on electrical stimulation (Figure 11). Alternative splicing analysis conducted using AltAnalyze software against Ensembl human database recent version (GRCh37) by applying Splicing-Index (SI) analysis method. The analysis detected 102 events in 96 distinct genes (t-test and DABG p<0.05). All the detected genes passed p-value false discovering rate (FDR) correction. HCL on the splicing-index gene-level normalized values successfully classified all the samples correctly by experimental state (pre or post-DBS) (Figures 12A-B). Further restriction of the t-test p-value to 0.005 and the fold-change to more than 2 -fold identified 13 significantly changed probe sets that underwent alternative splicing changes, which interrogates 13 genes (Table 6).

Table 6

midas-p

( corresponding

regulation call symbol ) max dl ensembl ID

down ( E14-1) REPS2 0.03 1.63 ENSG00000169891 down ( E6-3) PCBD2 0.05 1.21 ENSG00000132570 down ( E17-1) APPL1 0.03 1.20 ENSG00000157500 up (E16-2 ) EPB41L4A 0.02 1.19 ENSG00000129595 up (E22-3 ) A2M 0.03 1.00 ENSG00000175899

AC002472.1

up (E12-2 ) 3 0.04 1.10 ENSG00000187905 up (E3-2 ) NUDT6 0.05 1.39 ENSG00000170917 down ( El 1-1) RARB 0.04 1.12 ENSG00000077092 down ( E15-1) TMEM67 0.05 1.19 ENSG00000164953 up (E41-1 ) RTTN 0.04 1.07 ENSG00000176225 down ( E5-2) IKBKE 0.05 1.26 ENSG00000143466 down ( E4-2) YIF1A 0.02 1.22 ENSG00000174851 up (E19-1 ) PPP4R4 0.05 1.96 ENSG00000119698 These included a newly identified leucine rich kinase gene (AC002472.13) and A2M which acts in inflammatory response and IKBKE, which participate in positive regulation of I-kappaB kinase cascade.

Intriguingly, an additional analysis comparing PD patients post-DBS on stimulation to healthy controls detected 219 alternative splicing events which occurred in 200 distinct genes (t-test and DABG p<0.05). Post-hoc functional analysis (EASE) revealed that these were enriched in mitochondrial genes (enrichment score: 0.86) and included metal ion binding genes (enrichment score: 0.67). Of these, 14 genes exhibited more than one alternative splicing event. The detected exons probe-sets fully classified post- DBS stimulated patients from controls. These may contain clues regarding some of the unwelcome adverse events which may occur following DBS which include psychiatric disorders and apathy.

Stimulation cessation for one hour induces rapid alternative splicing alternation: To test if the alternative splicing alterations which were observed in PD patients post-DBS were due to the electrical stimulation treatment per-se, blood samples were taken from the patients one hour after temporary electrical stimulation cessation (OFF state) immediate after the examination on stimulation. As the participant patients agreed and signed informed consents to be tested again for blood mRNA expression following one hour of stimulation cessation, RNA of blood leukocytes from these samples was tested using exon arrays. We uncovered, through analysis of their leukocyte mRNA only one hour after the electrical stimulation cessation, 70 stimulus- dependent alternative splicing events using the SI method. Thus, surprisingly, the stimulation cessation induced rapid mRNA alternative splicing alternations in the patients' blood leukocytes. These occurred in 68 distinct genes. The most significant 8 events (significance p<0.0005, DABG p<0.05 and >2-fold change) occurred in 8 distinct genes (Table 7).

Table 7

midas-p

down exons up exons symbol ( corresponding) max dl AffyGene

E15-1 TMEM67 0.04 1.37 ENSG00000164953

E19-1 DNHD1 0.04 1.07 ENSG00000179532

E23-1 MACROD2 0.02 1.20 ENSG00000172264

E7-3 FAM54A 0.03 1.10 ENSG00000146410

El-1 PLEKHA4 0.03 1.25 ENSG00000105559 E4-2 SUV39H2 0.04 1.14 ENSG00000152455

E21-1 USP13 0.05 1.03 ENSG00000058056

E8-1 BAG1 0.05 1.16 ENSG00000107262

One of the detected genes (TMEM67) was also detected as alternatively spliced post- as compared with pre-DBS with inverse direction of change, suggesting stimulation-dependent alleviation for this gene. The detected genes also included a ubiquitin gene: USP13. Post-hoc functional classification enrichment analysis of the 68 detected genes revealed enrichment in metal ion binding pathway.

A non-trivial comparison between the PD leukocyte RNA expression under OFF state to pre-DBS state revealed that while as compared with pre-DBS state, OFF stimulation state induced only 40 alternative splicing events (in 38 distinct genes). An additional comparison of patients OFF stimulation to healthy controls, exhibited 117 alternative splicing events in 110 distinct genes following the one hour stimulation cessation. These classified accurately OFF stimulation patients from controls.

PD-induced splice changes predict DBS efficacy as measured by NRMS physiological recording: The expression profiles of the participant PD patients' leukocytes were compared to that of six age- and gender-matched HC volunteers. T-test on splicing-index (SI) calculations detected 19 events in 18 genes (SI t-test p- value<0.005 and DABG p-value <0.05), including the leucine rich repeat containing LRRC8C and the ATP-binding (AF130358.5) gene. The average splicing-index difference between patients to controls (across all these detected genes) was positively correlated with the NRMS recordings (Figure 13) (R square = 0.645, p=0.03).

Correlation between both NRMS and UPDRS clinical parameters and blood leukocyte mRNA alternative splicing changes post-surgery: Positive therapeutic response without adverse side effects to STN DBS for Parkinson's PD depends to a large extent on electrode location within the STN. Microelectrode recordings conducted for all the study participant patients during the neurosurgery and trajectories included both the left and right sides for all the patients albeit one (for which only one side recording conducted). These can predict the dorsolateral oscillatory region within the STN. Surprisingly, the alternative splicing change magnitude of detected events post- compared to pre-DBS was significantly correlated physiological parameter measured during the neurosurgery, the Normalized Root Mean Square (NRMS). The correlation (R square = 0.583, p=0.046, Figure 14A). Analysis of the RMS together with power spectral density can De used to define the STN borders and demarcate the ventral boundary of the DLOR in real time during neurosurgery and our data supports that conjunction of RMS measurement conjoint with alternative splicing change post-DBS of the detected genes can support the method prediction. Currently, a major selection criteria for DBS is preoperative levodopa responsiveness however it does not predict the outcome of STN DBS. Intriguingly, the dorsolateral oscillatory region (DLOR) length did not correlate with the observed alternative splicing changes. The third subsection (motor score) of the unified PD rating scale (UPDRS-III) was assessed preoperatively. In all the participate patients, the UPDRS-III improved following DBS treatment (Figure 12A, left bar, t-test p<0.05). To surmise whether the observed motor improvement is correlated with the alternative splicing changes post-DBS, we conducted a correlation analysis. For each patient, the tested variables included the UPDRS relative change post- DBS while being on stimulation as compared with pre- DBS, and the average difference of SI gene-normalized values of the highly detected alternatively spliced exons (having SI FDR p<0.005 and at least 2-fold change). Linear regression analysis yielded a positive correlation (R square = 0.503). R square is the proportion of variance in the dependent variable (UPDRS-III relative change post- compared to pre-DBS) which can be predicted from the independent variable (SI change post- compared to pre-DBS). The adjusted R square (calculated as 1 - ((1 - RjsqaareKCN - i)/(N - k - 1» , where N is the number of observations and k- the number of predictors) was 0.04. The results of the regression analysis resulted in the following estimate: UPDRS_relative_change_predicted = 40.32+39.04* SI relative change. A correlation between the UPDRS-III and the NRMS of the tested patients (R square = 0.515, non-significant) was seen (Figure 14B).

EXAMPLE 11

Analysis of Acetylcholinesterase in DBS treated patients MATERIALS AND METHODS

Patient recruitment, DBS neurosurgery and clinical evaluation: as described for Examples 1-8. Serum analysis: Nondenaturing gel and catalytic activity measurements of AChE were as described (Kaufer, D.,et al.,. (1998) Nature 393, 373-377).

Staining of AChE: as described by Karnovsky and Roots (1964) J Histochem Cytochem 12:2 19-22 1.

RESULTS

AChE activity in the serum of 7 patients who were subjected to deep brain stimulation was compared to controls and to patients after discontinuation of the stimulus for one hour. Activity was measured prior to and 3 months following the neurosurgery and 1 hour after discontinuation of the electrical stimulus.

As illustrated in Figure 15, surgery enhanced AChE- as well as improving the tremor considerably.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:

1. A method of diagnosing Parkinson's disease in a subject, the method comprising determining an expression level of a plurality of genes in a sample obtained from the subject, said plurality of genes comprising praja ring finger 1 (PJA1), translocation associated membrane protein 1 (TRAM1), protein tyrosine phosphatase 1 (PTPN1), poly(rc)-binding protein 2 (PCBP2), nuclear receptor subfamily 2, group F, member 1 (NR2F1) and heterogeneous nuclear ribonucleoprotein D-like (HNRPDL), wherein a statistically significant difference between expression levels of said plurality of genes in said sample obtained from said subject and expression levels of said plurality of genes in a control sample is indicative of Parkinson's disease.

2. A method of predicting an efficacy of a medicament for treating Parkinson's disease (PD) in a subject, the method comprising comparing an expression level of a plurality of genes in a sample obtained from the subject prior to and following administration of said medicament, said plurality of genes comprising PJA1, TRAM1, PTPN1, PCBP2, NR2F1 and HNRPDL, wherein a statistically significant difference between expression levels of said plurality of genes in said sample obtained from said subject prior to administration of said medicament and expression levels of said plurality of genes in said sample obtained from said subject following administration of said medicament is indicative of an efficacious medicament.

3. The method of claim 1 or 2, wherein when said gene is TRAM1, PTPN1 or PCBP2 said difference is an increase and said control sample is derived from a non- diseased subject.

4. The method of claim 1 or 2, wherein when said gene is PJA1, NR2F1 or HNRPDL, said difference is a decrease and said control sample is derived from a non- diseased subject.

5. The method of claim 1 or 2, further comprising analyzing an expression level of at least one additional gene set forth in Table 1, wherein a statistically significant difference between an expression level of said at least one additional gene in said sample obtained from said subject and an expression level of said additional gene in said control sample is further indicative of Parkinson's disease or an efficacious treatment.

6. The method of claim 1 or 2, further comprising analyzing an expression level of at least one additional gene selected from the group consisting of ATPase, class VI, type 11B (ATP11B), leucine rich repeat containing 8 family, member C (LRRC8C), Leucine rich repeat and Ig domain containing 4 (LING04), DNA-damage inducible 1, homolog 2 (DDI2), family with sequence similarity 46, member C (FAM46C), coiled- coil domain containing 5 (CCDC5), aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2), olfactory receptor, family 52, subfamily N, member 5 (OR52N5), adhesion molecule with Ig-like domain 3 (AMIG03), calmodulin binding transcription activator 1 (CAMTA1), oculomedin (OCLM), solute carrier family 26, member 8 (SLC26A8), chorionic somatomammotropin hormone-like 1 (CSHL1), leucine-rich repeat, immunoglobulin-like and transmembrane domains 1 (LRIT1), catenin beta-like 1 (CTNNBL1), nerve growth factor (NGF), G protein-coupled receptor 61 (GPR61), cyclin dependent kinase 10 (CDK10), zinc finger CCCH-type containing 7A (ZC3H7A), fumarylacetoacetate hydrolase (FAH), N-terminal EF-hand calcium binding protein 1 (NECAB1), carbonic anhydrase VII (CA7), SEC13 homolog (SEC13), LY6/PLAUR domain containing 6B (LYPD6B), EP400 N-terminal like (EP400NL), ATP-binding cassette, sub-family C (CFTR/MRP), member 13, pseudogene (ABCC13), transcript (AF130358.5), KIAA2026, zinc finger protein 257 (ZNF257), cyclin D binding myb-like transcription factor 1 (DMTF1), adaptor-related protein complex 1, sigma 2 subunit (AP1S2), vacuolar protein sorting 37 homolog A (VPS37A), MYB binding protein (P160) la (MYBBP1A), LA16c-60G3.8, discs, large (Drosophila) homolog-associated protein 5 (DLGAP5), cytoplasmic linker associated protein 1 (CLASP1), phosphodiesterase 3A, cGMP-inhibited (PDE3A), transketolase line 1 (TKTL1), MYCBP associated protein (MYCBPAP), USOl vesicle docking protein homolog (USOl) and capicua homolog (Drosophila) pseudogene 13 (CICP13), wherein a statistically significant difference between an expression level of said at least one additional gene in said sample obtained from said subject and an expression level of said additional gene in said control sample is further indicative of Parkinson's disease or an efficacious treatment.

7. The method of claim 6, wherein when said gene is ATP11B, LING04, DD12, FAM46C, CCDC5, ARNT2, OR52N5, AMIG03, CAMTA1, OCLM, CTNNBL1, NGF, GPR61, CDK10, NECAB1, CA7, SEC13, LYPD6B, EP400NL, AF130358.5, KIAA2026, ZNF257, DMTF1, AP1S2, VPS37A, LRRC8C, MYBBP1A, LA16c- 60G3.8, DLGAP5, said difference is a decrease and said control sample is derived from a non-diseased subject.

8. The method of claim 6, wherein when said gene is LRRC8C, SLC26A8, CSHL1, LRIT1, ZC3H7A, FAH, CLASPl, PDE3A, TKTL1, MYCBPAP, USOl and CICP13 said difference is an increase and said control sample is derived from a non- diseased subject.

9. A method of diagnosing Parkinson's disease in a subject, the method comprising determining an expression of at least one gene in a sample obtained from the subject being selected from the group consisting of SNCA, PARK7 and ASF (SFRSl), wherein a statistically significant difference between expression of a variant of said at least one gene in said sample obtained from said subject and expression of said variant of said at least one gene in a control sample is indicative of Parkinson's disease.

10. The method of claim 9, wherein when said at least one gene is SNCA, a decrease in an expression level of a variant which encodes exons 2-3 and 4-5 is indicative of Parkinson's disease.

11. The method of claim 9, wherein when said at least one gene is PARK7, an increase in an expression level of a variant which encodes exons 4-5 and 6-7 is indicative of Parkinson's disease.

12. The method of claim 9, wherein when said at least one gene is ASF, an increase in an expression level of a variant which encodes a 3' untranslated region (UTR) is indicative of Parkinson's disease.

13. The method of claim 9, further comprising analyzing an expression level of at least one additional gene set forth in Table 1, wherein a statistically significant difference between an expression level of said at least one additional gene in said sample obtained from said subject and an expression level of said additional gene in said control sample is further indicative of Parkinson's disease.

14. A method of diagnosing Parkinson's disease in a subject, the method comprising determining an expression of at least one gene in a sample obtained from the subject as set forth in Table 1, wherein a statistically significant difference between expression levels of said at least one gene in said sample obtained from said subject and an expression level of the identical gene in a control sample is indicative of Parkinson's disease.

15. The method of any of claims 1, 9 and 14, further comprising informing the subject of an outcome of the diagnosis.

16. The method of any of claims 1, 2, 9 and 14, wherein said sample obtained from the subject is a white blood cell sample.

17. The method of any of claims claim 1, 2, 9 and 14 wherein said control sample is age and sex-matched.

18. The method of any of claims 1, 2, 9 and 14, wherein said control sample is obtained from a non-diseased subject.

19. The method of any of claims 1, 9 and 14, further comprising corroborating the diagnosis by neurologically examining the subject or imaging a brain of the subject.

20. The method of any of claims 1, 9 and 14, wherein said analyzing an expression level is effected at the protein level.

21. The method of any of claims 1, 2, 9 and 14, wherein said analyzing en expression level is effected at the polynucleotide level.

22. A method of treating Parkinson's in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent which increases an amount or an activity of at least one polypeptide encoded by a gene listed in Table 2 or 6.

23. A method of predicting an efficacy of deep brain stimulation (DBS) for treating Parkinson's disease (PD) in a subject, the method comprising analyzing an expression level of at least one gene listed in Table 3 and/or acetylcholineasterase, wherein a statistically significant upregulation between an expression level of said at least one gene in a sample obtained from said subject and an expression level of said at least one gene in a control sample obtained from a non-diseased subject is indicative that DBS is efficacious for the treatment of Parkinson's disease in the subject.

24. A polynucleotide array comprising at least 6 and no more than 100 polynucleotide sequences for determining a gene expression profile of a biological sample, wherein at least one of said sequences is selected capable of hybridizing with a transcription product of a polynucleotide sequence of PJA1, at least one of said sequences is selected capable of hybridizing with a transcription product of a polynucleotide sequence of TRAM1, at least one of said sequences is selected capable of hybridizing with a transcription product of a polynucleotide sequence of PTPN1, at least one of said sequences is selected capable of hybridizing with a transcription product of a polynucleotide sequence of PCBP2, at least one of said sequences is selected capable of hybridizing with a transcription product of a polynucleotide sequence of NR2F1 and at least one of said sequences is selected capable of hybridizing with a transcription product of a polynucleotide sequence of HNRPDL.

25. The polynucleotide array of claim 24, wherein at least one of said sequences is selected capable of hybridizing with a transcription product of a polynucleotide sequence with a gene selected from the group consisting of ATP11B, LRRC8C, LING04, DDI2, FAM46C, CCDC5, ARNT2, OR52N5, AMIG03.CAMTA1, OCLM, SLC26A8, CSHLl, LRITl, CTNNBLl, NGF, GPR61, CDK10, ZC3H7A, FAH, NECABl, CA7 and SEC13.

26. An array comprising at least 6 and no more than 100 antibodies or antibody fragments for determining a gene expression profile of a biological sample, wherein at least one of said antibodies or antibody fragments is selected capable of binding with a protein product of PJA1, at least one of said antibodies or antibody fragments is selected capable of binding with a protein product of TRAMl, at least one of said antibodies or antibody fragments is selected capable of binding with a protein product of PTPN1, at least one of said antibodies or antibody fragments is selected capable of binding with a protein product of PCPB2, at least one of said antibodies or antibody fragments is selected capable of binding with a protein product of NR2F1 and at least one of said antibodies or antibody fragments is selected capable of binding with a protein product of HNRPDL.

27. The array of claim 26, wherein at least one of said antibodies is selected capable of hybridizing with a protein product of a gene selected from the group consisting of ATP11B, LRRC8C, LING04, DDI2, FAM46C, CCDC5, ARNT2, OR52N5, AMIG03,CAMTA1, OCLM, SLC26A8, CSHLl, LRITl, CTNNBLl, NGF, GPR61, CDK10, ZC3H7A, FAH, NECABl, CA7 and SEC13.