WO2012032519A2 - Méthodes de diagnostic de la maladie de parkinson - Google Patents

Méthodes de diagnostic de la maladie de parkinson Download PDF

Info

Publication number
WO2012032519A2
WO2012032519A2 PCT/IL2011/000720 IL2011000720W WO2012032519A2 WO 2012032519 A2 WO2012032519 A2 WO 2012032519A2 IL 2011000720 W IL2011000720 W IL 2011000720W WO 2012032519 A2 WO2012032519 A2 WO 2012032519A2
Authority
WO
WIPO (PCT)
Prior art keywords
gene
subject
disease
parkinson
dbs
Prior art date
Application number
PCT/IL2011/000720
Other languages
English (en)
Other versions
WO2012032519A3 (fr
Inventor
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 (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd., Hadasit Medical Research Services And Development Ltd. filed Critical Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
Publication of WO2012032519A2 publication Critical patent/WO2012032519A2/fr
Publication of WO2012032519A3 publication Critical patent/WO2012032519A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/40—Detecting, measuring or recording for evaluating the nervous system
    • A61B5/4076—Diagnosing or monitoring particular conditions of the nervous system
    • A61B5/4082—Diagnosing or monitoring movement diseases, e.g. Parkinson, Huntington or Tourette
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the present invention in some embodiments thereof, relates to methods of diagnosing Parkinson's disease.
  • Parkinson's Disease 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).
  • DA dopamine
  • SNpc substantia nigra pars compacta
  • DA dopamine
  • SNpc substantia nigra pars compacta
  • 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.
  • a method of diagnosing Parkinson's disease in a subject 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.
  • PJAl praja ring finger 1
  • TAMl translocation associated membrane protein 1
  • PTPN1 protein tyrosine phosphatase 1
  • PCBP2 poly(rc)-binding protein 2
  • NRF1 nuclear receptor subfamily 2, group F, member 1
  • a method of predicting an efficacy of a medicament for treating Parkinson's disease (PD) in a subject 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.
  • PD Parkinson's disease
  • a method of diagnosing Parkinson's disease in a subject 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.
  • a method of diagnosing Parkinson's disease in a subject 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.
  • 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.
  • a method of predicting an efficacy of deep brain stimulation (DBS) for treating Parkinson's disease (PD) in a subject 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.
  • DBS deep brain stimulation
  • 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 polynucle
  • 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.
  • the difference is an increase and the control sample is derived from a non-diseased subject.
  • the difference is a decrease and the control sample is derived from a non-diseased subject.
  • 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.
  • 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
  • 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.
  • the gene is LRRC8C
  • a decrease in an expression level of a variant which encodes exons 2-3 and 4-5 is indicative of Parkinson's disease.
  • an increase in an expression level of a variant which encodes exons 4-5 and 6- 7 is indicative of Parkinson's disease.
  • an increase in an expression level of a variant which encodes a 3' untranslated region (UTR) is indicative of Parkinson's disease.
  • 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.
  • the method further comprises informing the subject of an outcome of the diagnosis.
  • the sample obtained from the subject is a white blood cell sample.
  • control sample is age and sex-matched.
  • control sample is obtained from a non-diseased subject.
  • the method further comprises corroborating the diagnosis by neurologically examining the subject or imaging a brain of the subject.
  • the analyzing an expression level is effected at the protein level.
  • the analyzing en expression level is effected at the polynucleotide level.
  • 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.
  • 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.
  • 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.
  • 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).
  • FIG. 2 is an experimental and analysis flow.
  • 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).
  • FIGs. 4A-E illustrates that DBS neurosurgery and OFF-stimulus states both reverse the PD leukocyte transcript profiles.
  • HCL Hierarchical clustering
  • 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).
  • RT reaction included nuclease-free water (4 ⁇ ), ImProm-IITM 5X reaction buffer (4 ⁇ ), MgCL2 (Promega, 4 ⁇ ), dNTP ( ⁇ ⁇ , 10 ⁇ each), RNase inhibitor (Rnasin, Promega) ( ⁇ ) and RT enzyme (1 ⁇ ). Random primers added (0.5 ⁇ g), 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 ⁇ ) cDNA (8 ⁇ , 1 : 10), SYBR green (sigma, 10 ⁇ ), the appropriate primers (10 ⁇ , 1 ⁇ 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.
  • 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.
  • 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).
  • 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).
  • NFkB p50/p65 proteins NFkB structures
  • 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.
  • UPD Unified Parkinson's Disease Rating Scale
  • LDE Levodopa Equivalent Dose
  • 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).
  • HCL Hierarchical classification
  • 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.
  • FIG. 13 is a scatter plot illustrating that pre-DBS alternative splicing patterns correlates with DBS efficacy.
  • the Normalizes Root Mean Square (NRMS) correlated with UPDRS-III score (R square:, p 0.046,).
  • 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.
  • 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.
  • the present invention in some embodiments thereof, relates to a method of diagnosing and treating Parkinson's disease.
  • 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).
  • a method of diagnosing Parkinson's disease 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.
  • diagnosis 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.
  • 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.
  • PD Parkinson's disease
  • 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.
  • the control samples are taken from age and se -matched healthy subjects.
  • 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.
  • 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.
  • a preferred detection methodology is one in which the resulting detection values are above the minimum detection limit of the methodology utilized.
  • the genes listed in Tables 1-6 were identified in white blood cells.
  • the sample obtained from the individual is preferably a white blood sample or any sample which includes blood cells such as T-cells.
  • the sample is blood, thymus, spleen, lymph, pus, or bone marrow.
  • 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.
  • 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.
  • 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.
  • genes presented herein are referred to by a gene symbol number.
  • probe sequence data may be obtained from sources such as Affymetrix. Since the lists of genes were obtained using GeneChipTM 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.
  • 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.
  • 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.
  • ATP11B ATP11B
  • LRRC8C LING04
  • DDI2 FAM46C
  • CCDC5 ARNT2
  • ARNT2 ARNT2
  • AMIG03 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.
  • 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.
  • the diagnostic method of the present invention preferably utilizes a marker set that can range anywhere from 1 gene to 200 genes.
  • 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.
  • 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.
  • 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.
  • each of PJA1, TRAM1, PTPN1, PCBP2, NR2F1 or HNRPDL are analyzed for the diagnosis of Parkinson's disease.
  • 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.
  • the oligonucleotide is a probe.
  • probe refers to an oligonucleotide which hybridizes to one of the gene sequences listed herein to provide a detectable signal under experimental conditions
  • 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).
  • the array comprises probes for detection of at least 6 genes - (e.g. PJA1, TRAM1, PTPN1, PCBP2, NR2F1 and HNRPDL).
  • genes - e.g. PJA1, TRAM1, PTPN1, PCBP2, NR2F1 and HNRPDL.
  • the array comprises probes for detection of the genes listed in Table 4.
  • 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.
  • the oligonucleotide is a primer of a primer pair.
  • 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)).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Such a template directed synthesis is also called "primer extension”.
  • 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
  • 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.
  • 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.
  • hybridization of short nucleic acids 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 ⁇ g/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 m
  • 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.
  • 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.
  • oligonucleotides may be prepared using an automated, solid-phase procedure based on the phosphoramidite approach.
  • 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.
  • DMT dimethoxytriyl
  • 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.).
  • 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 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).
  • 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.
  • chemical degradation A. M. Maxam and W. Gilbert, Methods of Enzymology, 1980, 65: 499-560
  • MALDI-TOF matrix-assisted laser desorption ionization time-of-flight
  • mass spectrometry U. Pieles et al., Nucleic Acid
  • 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.
  • the oligonucleotide sequences of the present invention may also be modified with a label.
  • 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.
  • the detection probes are labeled with a detectable agent.
  • 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.
  • 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).
  • 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.
  • 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.
  • ULS Universal Linkage System
  • 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
  • the inventive detection probes are fluorescently labeled.
  • 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,
  • fluorescein and fluorescein dyes e.g., fluorescein isothiocyanine or FITC, nap
  • 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 FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BOD
  • 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.).
  • identification of target sequences may be carried out using an amplification reaction.
  • 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 include, but are not limited to, polymerase chain reaction (PCR) and ligase chain reaction (LCR).
  • PCR polymerase chain reaction
  • LCR ligase chain 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.
  • 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.
  • 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
  • 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.
  • forward primer and “forward amplification primer” are used herein interchangeably, and refer to a primer that hybridizes (or anneals) to the target (template strand).
  • 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.
  • amplification conditions 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.
  • 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.
  • 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 aquerative PCR (ISSR),
  • PCR polymerase chain reaction
  • 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).
  • 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 results in the exponential accumulation of a specific DNA fragment
  • PCR Protocols A Guide to Methods and Applications
  • PCR Strategies M. A. Innis (Ed.), 1995, Academic Press: New York
  • Polymerase chain reaction basic principles and automation in PCR: A Practical Approach
  • the termini of the amplified fragments are defined as the 5' ends of the primers.
  • 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.
  • RT-PCR PCR
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • conditions sufficient for enzymatic production of primer extension product i.e., conditions sufficient for enzymatic production of primer extension product.
  • 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.
  • 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.
  • UV ultraviolet
  • the amplification and quantification of the amplification product may be effected in real-time (qRT-PCR).
  • QRT-PCR methods use double stranded DNA detecting molecules to measure the amount of amplified product in real time.
  • double stranded DNA detecting molecule refers to a double stranded DNA interacting molecule that produces a quantifiable signal (e.g., fluorescent signal).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a reverse transcriptase enzyme such as an MMLV-RT
  • primers such as, oligo dT, random hexamers or gene specific primers.
  • 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 a
  • RNA in situ hybridization stain DNA or RNA probes are attached to the RNA molecules present in the cells.
  • 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.
  • formamide and salts e.g., sodium chloride and sodium citrate
  • 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.
  • 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.
  • such arrays are about 2 x 2 cm and contain about individual nucleic acids 6000 spots.
  • 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.
  • 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).
  • labeled oligonucleotide probes e.g., 5'-biotinylated probes
  • cDNA complementary DNA
  • cRNA RNA
  • 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).
  • RT reverse transcriptase
  • DNA ligase DNA polymerase I
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention contemplates use of probes that span the bridging regions of these exons, in order to confirm the disease.
  • primers that may be used to analyze SNCA are set forth in SEQ ID NOs: 45 and 46; and 47 and 48.
  • primers that may be used to analyze PARK7 are set forth in SEQ ID NOs: 49 and 50.
  • the present invention further contemplates additional sequences which are common to these variants, but absent in other SNCA variants.
  • the present invention further contemplates detection of additional sequences which are common to these variants, but absent in other PARK7 variants.
  • an increase in an expression level of a variant which encodes a non-truncated 3' untranslated region (UTR) of ASF 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.
  • 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 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 In one version, this method involves precipitation of the desired protein (i.e., the substrate) with a specific antibody and radiolabeled antibody binding protein (e.g., protein A labeled with I 125 ) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of substrate.
  • a specific antibody and radiolabeled antibody binding protein e.g., protein A labeled with I 125
  • 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 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.
  • genes listed herein may be used for predicting an efficacy of a medicament for treating Parkinson's disease (PD).
  • PD Parkinson's disease
  • 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).
  • DBS deep brain stimulation
  • a method of predicting an efficacy of deep brain stimulation (DBS) for treating Parkinson's disease (PD) in a subject 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.
  • DBS deep brain stimulation
  • 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.
  • AChE acetylcholinesterase
  • 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.
  • 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.
  • STN subthalamic nucleus
  • GPi globus pallidus interna
  • the method may be effected ex vivo (following removal of a sample from the subject) or in vivo.
  • 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.
  • PET Positron emission tomography
  • a subject may be treated with a particular drug or treatment such as those described herein above.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • LTRs long terminal repeats
  • such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention.
  • the construct may also include a signal that directs polyadenylation., as well as one or more restriction sites and a translation termination sequence.
  • a signal that directs polyadenylation 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.
  • 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.
  • the agents may be the polypeptides themselves.
  • the polypeptides may be recombinant polypeptides.
  • 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.
  • 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.
  • active ingredient refers to the polypeptide or antibody preparation, which is accountable for the biological effect.
  • pharmaceutically acceptable carrier refers 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).
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients examples include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • 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.
  • 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.
  • 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.
  • 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.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • 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).
  • 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.
  • 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.
  • filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • 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.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • 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.
  • compositions 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.
  • 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.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • 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.
  • 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.
  • the therapeutically effective amount or dose can be estimated initially from in vitro assays.
  • 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].
  • 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.
  • compositions, 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.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • 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.
  • 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.
  • 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.
  • 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 kitTM (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-ReagentTM, Ambion) into l-bromo-3-chloropropane-containing 15 ml tubes and centrifuged.
  • TRI-ReagentTM Tri-ReagentTM, Ambion
  • 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 GeneChipTM Exon_1.0_ST_Array (Affymetrix, Santa Clara, CA, USA) microarrays, and results were scanned (GeneChip scanner 30007G, 27 CEL files).
  • 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].
  • exon arrays 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.
  • 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)).
  • qRT-PCR quantitative real-time Polymerase Chain Reaction
  • STN-DBS affects a wide range of transcripts including disease-modified ones
  • 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).
  • 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:
  • the Post-DBS stimulation state differs from healthy controls
  • the present inventors proceeded to test if the leukocyte post-DBS transcript profiles regained similarity to those of healthy controls.
  • 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.
  • 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.
  • 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.
  • 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).
  • RNA samples extracted one hour after the electrical stimulation was turned off 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.
  • the major disease symptoms rapidly re-occur.
  • a PD patient pre-operation and in OFF-state both lacking the DBS stimulation, but with different medication doses
  • the OFF-state was accompanied by differential expression of 351 transcripts (Table 3).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Neurology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Neurosurgery (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Physiology (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Surgery (AREA)
  • Medical Informatics (AREA)
  • Biotechnology (AREA)
  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • Developmental Disabilities (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une méthode visant à diagnostiquer la maladie de Parkinson chez un sujet. La méthode consiste à déterminer un taux d'expression d'une pluralité de gènes dans un échantillon prélevé chez le sujet, la pluralité de gènes comprenant PJA1, TRAM1, PTPN1, PCBP2, NR2F1 et HNRPDL. Une différence importante d'un point de vue statistique entre les taux d'expression de la pluralité de gènes dans l'échantillon prélevé chez le sujet et les taux d'expression de la pluralité de gènes dans un échantillon témoin indique que le sujet est atteint de la maladie de Parkinson.
PCT/IL2011/000720 2010-09-07 2011-09-07 Méthodes de diagnostic de la maladie de parkinson WO2012032519A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US38036010P 2010-09-07 2010-09-07
US61/380,360 2010-09-07

Publications (2)

Publication Number Publication Date
WO2012032519A2 true WO2012032519A2 (fr) 2012-03-15
WO2012032519A3 WO2012032519A3 (fr) 2012-07-05

Family

ID=44898119

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2011/000720 WO2012032519A2 (fr) 2010-09-07 2011-09-07 Méthodes de diagnostic de la maladie de parkinson

Country Status (1)

Country Link
WO (1) WO2012032519A2 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2773779A4 (fr) * 2011-11-04 2015-09-30 Population Diagnotics Inc Méthodes et compositions pour le diagnostic, le pronostic et le traitement d'états neurologiques
WO2016078546A1 (fr) * 2014-11-20 2016-05-26 华中科技大学 Médicament prévenant la maladie d'alzheimer
CN106021915A (zh) * 2016-05-17 2016-10-12 华南师范大学 基于大数据面向自动诊疗的医疗数据分析系统和装置
EP3091087A1 (fr) * 2015-05-08 2016-11-09 Fundació Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol Procédé de diagnostic in vitro de synucléinopathies utilisant des transcriptions du gène de l'alpha-synucléine
CN109545370A (zh) * 2018-10-27 2019-03-29 平安医疗健康管理股份有限公司 基于样本处理的病种确定方法、装置及终端
US11142570B2 (en) 2017-02-17 2021-10-12 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof
CN115957349A (zh) * 2022-10-08 2023-04-14 华中农业大学 激活猪的pja1基因表达的制剂在制备猪抗猪流行性腹泻病毒感染药物中的应用

Citations (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3791932A (en) 1971-02-10 1974-02-12 Akzona Inc Process for the demonstration and determination of reaction components having specific binding affinity for each other
US3839153A (en) 1970-12-28 1974-10-01 Akzona Inc Process for the detection and determination of specific binding proteins and their corresponding bindable substances
US3850578A (en) 1973-03-12 1974-11-26 H Mcconnell Process for assaying for biologically active molecules
US3850752A (en) 1970-11-10 1974-11-26 Akzona Inc Process for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specifically
US3853987A (en) 1971-09-01 1974-12-10 W Dreyer Immunological reagent and radioimmuno assay
US3867517A (en) 1971-12-21 1975-02-18 Abbott Lab Direct radioimmunoassay for antigens and their antibodies
US3879262A (en) 1972-05-11 1975-04-22 Akzona Inc Detection and determination of haptens
US3901654A (en) 1971-06-21 1975-08-26 Biological Developments Receptor assays of biologically active compounds employing biologically specific receptors
US3935074A (en) 1973-12-17 1976-01-27 Syva Company Antibody steric hindrance immunoassay with two antibodies
US3984533A (en) 1975-11-13 1976-10-05 General Electric Company Electrophoretic method of detecting antigen-antibody reaction
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4034074A (en) 1974-09-19 1977-07-05 The Board Of Trustees Of Leland Stanford Junior University Universal reagent 2-site immunoradiometric assay using labelled anti (IgG)
US4098876A (en) 1976-10-26 1978-07-04 Corning Glass Works Reverse sandwich immunoassay
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4666828A (en) 1984-08-15 1987-05-19 The General Hospital Corporation Test for Huntington's disease
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4800159A (en) 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
US4801531A (en) 1985-04-17 1989-01-31 Biotechnology Research Partners, Ltd. Apo AI/CIII genomic polymorphisms predictive of atherosclerosis
US4879219A (en) 1980-09-19 1989-11-07 General Hospital Corporation Immunoassay utilizing monoclonal high affinity IgM antibodies
US5011771A (en) 1984-04-12 1991-04-30 The General Hospital Corporation Multiepitopic immunometric assay
US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
US5272057A (en) 1988-10-14 1993-12-21 Georgetown University Method of detecting a predisposition to cancer by the use of restriction fragment length polymorphism of the gene for human poly (ADP-ribose) polymerase
US5281521A (en) 1992-07-20 1994-01-25 The Trustees Of The University Of Pennsylvania Modified avidin-biotin technique
US5322770A (en) 1989-12-22 1994-06-21 Hoffman-Laroche Inc. Reverse transcription with thermostable DNA polymerases - high temperature reverse transcription
US5464764A (en) 1989-08-22 1995-11-07 University Of Utah Research Foundation Positive-negative selection methods and vectors
US5475610A (en) 1990-11-29 1995-12-12 The Perkin-Elmer Corporation Thermal cycler for automatic performance of the polymerase chain reaction with close temperature control
US5538871A (en) 1991-07-23 1996-07-23 Hoffmann-La Roche Inc. In situ polymerase chain reaction
US5612473A (en) 1996-01-16 1997-03-18 Gull Laboratories Methods, kits and solutions for preparing sample material for nucleic acid amplification

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7595159B2 (en) * 2004-11-03 2009-09-29 The Brigham And Women's Hospital, Inc. Prediction of Parkinson's disease using gene expression levels of peripheral blood samples

Patent Citations (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3850752A (en) 1970-11-10 1974-11-26 Akzona Inc Process for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specifically
US3839153A (en) 1970-12-28 1974-10-01 Akzona Inc Process for the detection and determination of specific binding proteins and their corresponding bindable substances
US3791932A (en) 1971-02-10 1974-02-12 Akzona Inc Process for the demonstration and determination of reaction components having specific binding affinity for each other
US3901654A (en) 1971-06-21 1975-08-26 Biological Developments Receptor assays of biologically active compounds employing biologically specific receptors
US3853987A (en) 1971-09-01 1974-12-10 W Dreyer Immunological reagent and radioimmuno assay
US3867517A (en) 1971-12-21 1975-02-18 Abbott Lab Direct radioimmunoassay for antigens and their antibodies
US3879262A (en) 1972-05-11 1975-04-22 Akzona Inc Detection and determination of haptens
US3850578A (en) 1973-03-12 1974-11-26 H Mcconnell Process for assaying for biologically active molecules
US3935074A (en) 1973-12-17 1976-01-27 Syva Company Antibody steric hindrance immunoassay with two antibodies
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4034074A (en) 1974-09-19 1977-07-05 The Board Of Trustees Of Leland Stanford Junior University Universal reagent 2-site immunoradiometric assay using labelled anti (IgG)
US3984533A (en) 1975-11-13 1976-10-05 General Electric Company Electrophoretic method of detecting antigen-antibody reaction
US4098876A (en) 1976-10-26 1978-07-04 Corning Glass Works Reverse sandwich immunoassay
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4879219A (en) 1980-09-19 1989-11-07 General Hospital Corporation Immunoassay utilizing monoclonal high affinity IgM antibodies
US5011771A (en) 1984-04-12 1991-04-30 The General Hospital Corporation Multiepitopic immunometric assay
US4666828A (en) 1984-08-15 1987-05-19 The General Hospital Corporation Test for Huntington's disease
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (fr) 1985-03-28 1990-11-27 Cetus Corp
US4801531A (en) 1985-04-17 1989-01-31 Biotechnology Research Partners, Ltd. Apo AI/CIII genomic polymorphisms predictive of atherosclerosis
US4683195B1 (fr) 1986-01-30 1990-11-27 Cetus Corp
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4800159A (en) 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
US5272057A (en) 1988-10-14 1993-12-21 Georgetown University Method of detecting a predisposition to cancer by the use of restriction fragment length polymorphism of the gene for human poly (ADP-ribose) polymerase
US5464764A (en) 1989-08-22 1995-11-07 University Of Utah Research Foundation Positive-negative selection methods and vectors
US5487992A (en) 1989-08-22 1996-01-30 University Of Utah Research Foundation Cells and non-human organisms containing predetermined genomic modifications and positive-negative selection methods and vectors for making same
US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
US5322770A (en) 1989-12-22 1994-06-21 Hoffman-Laroche Inc. Reverse transcription with thermostable DNA polymerases - high temperature reverse transcription
US5475610A (en) 1990-11-29 1995-12-12 The Perkin-Elmer Corporation Thermal cycler for automatic performance of the polymerase chain reaction with close temperature control
US5602756A (en) 1990-11-29 1997-02-11 The Perkin-Elmer Corporation Thermal cycler for automatic performance of the polymerase chain reaction with close temperature control
US5538871A (en) 1991-07-23 1996-07-23 Hoffmann-La Roche Inc. In situ polymerase chain reaction
US5281521A (en) 1992-07-20 1994-01-25 The Trustees Of The University Of Pennsylvania Modified avidin-biotin technique
US5612473A (en) 1996-01-16 1997-03-18 Gull Laboratories Methods, kits and solutions for preparing sample material for nucleic acid amplification

Non-Patent Citations (136)

* Cited by examiner, † Cited by third party
Title
"Animal Cell Culture", 1986
"Basic and Clinical Immunology", 1994, APPLETON & LANGE
"Cell Biology: A Laboratory Handbook", vol. I-III, 1994
"Current Protocols in Immunology", vol. I-III, 1994
"Current Protocols in Molecular Biology", vol. I-III, 1994
"Genome Analysis: A Laboratory Manual Series", vol. 1-4, 1998, COLD SPRING HARBOR LABORATORY PRESS
"Immobilized Cells and Enzymes", 1986, IRL PRESS
"Methods in Enzymology", vol. 1-317, ACADEMIC PRESS
"Molecular Cell Biology", 2000, W. H. FREEMAN, article "DNA Microarrays: Analyzing Genome-Wide Expression"
"Nucleic Acid Hybridization", 1985
"Oligonucleotide Synthesis", 1984
"Partek@. Genomics Suite Software", 1993, PARTEK INCORPORATED
"PCR Protocols: A Guide To Methods And Applications", 1990, ACADEMIC PRESS
"PCR Strategies", 1995, ACADEMIC PRESS
"Polymerase chain reaction: basic principles and automation in PCR: A Practical Approach", 1991, IRL PRESS
"Remington's Pharmaceutical Sciences", MACK PUBLISHING CO.
"Selected Methods in Cellular Immunology", 1980, W. H. FREEMAN AND CO.
"Short Protocols in Molecular Biology", 2002, JOHN WILEY & SONS
"Transcription and Translation", 1984
"Vectors: A Survey of Molecular Cloning Vectors and Their Uses", 1988, BUTTERWORTHS
A. H. HOPMAN ET AL., EXP. CELL RES., vol. 169, 1987, pages 357 - 368
A. M. MAXAM, W. GILBERT, METHODS OF ENZYMOLOGY, vol. 65, 1980, pages 499 - 560
ALEXA A, RAHNENFUHRER J, LENGAUER T.: "Improved scoring of functional groups from gene expression data by decorrelating GO graph structure", BIOINFORMATICS, vol. 22, 2006, pages 1600 - 7
ASHBURNER M, BALL CA, BLAKE JA ET AL.: "Gene ontology: tool for the unification of biology. The Gene Ontology Consortium", NAT GENET., vol. 25, 2000, pages 25 - 9
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1989, JOHN WILEY AND SONS
B. A. CONNOLY, O. RIDER, NUCL. ACIDS. RES., vol. 13, 1985, pages 4485 - 4502
BAIROCH A, APWEILER R, WU CH ET AL.: "The Universal Protein Resource (UniProt", NUCLEIC ACIDS RES., vol. 33, 2005, pages D154 - 9
BENABID AL, CHABARDES S, MITROFANIS J ET AL.: "Deep brain stimulation of the subthalamic nucleus for the treatment of Parkinson's disease", LANCET NEUROL., vol. 8, 2009, pages 67 - 81, XP025838468, DOI: doi:10.1016/S1474-4422(08)70291-6
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)., vol. 58, 1993, pages 39 - 44
BENJAMINI Y, HOCHBERG Y, JOURNAL OF THE ROYAL STATISTICAL SOCIETY, vol. 57, 1995, pages 289 - 300
BEN-SHAUL Y, BENMOYAL-SEGAL L, BEN-ARI S ET AL.: "Adaptive acetylcholinesterase splicing patterns attenuate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice", EUR J NEUROSCI., vol. 23, 2006, pages 2915 - 22, XP002667115, DOI: doi:10.1111/J.1460-9568.2006.04812.X
BEN-SHAUL Y, BERGMAN H, SOREQ H.: "Identifying subtle interrelated changes in functional gene categories using continuous measures of gene expression", BIOINFORMATICS, vol. 21, 2005, pages 1129 - 37
BERGMAN H, WICHMANN T, DELONG MR.: "Reversal of experimental parkinsonism by lesions of the subthalamic nucleus", SCIENCE, vol. 249, 1990, pages 1436 - 8
BONIFATI V, RIZZU P, VAN BAREN MJ ET AL.: "Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism", SCIENCE, vol. 299, 2003, pages 256 - 9, XP002989501, DOI: doi:10.1126/science.1077209
C. LEVENSON ET AL., METHODS ENZYMOL., vol. 184, 1990, pages 577 - 583
C. NEVES ET AL., BIOCONJUGATE CHEM., vol. 11, 2000, pages 51 - 55
C. PFANNSCHMIDT ET AL., NUCLEIC ACIDS RES., vol. 24, 1996, pages 1702 - 1709
CHANG ET AL.: "Somatic Gene Therapy", 1995, CRC PRESS
CLARK TA, SUGNET CW, ARES M, SCIENCE, vol. 296, 2002, pages 907
D. GUSCHIN ET AL., ANAL. BIOCHEM., vol. 250, 1997, pages 203 - 211
D. J. BRIGATI ET AL., VIROL., vol. 126, 1983, pages 32 - 50
DENNIS G, JR., SHERMAN BT, HOSACK DA ET AL.: "DAVID: Database for Annotation, Visualization, and Integrated Discovery", GENOME BIOL., vol. 4, 2003, pages 3
DEUSCHL G, SCHADE-BRITTINGER C, KRACK P ET AL.: "A randomized trial of deep- brain stimulation for Parkinson's disease", N ENGL J MED., vol. 355, 2006, pages 896 - 908
DUDOIT S, GENTLEMAN RC, QUACKENBUSH J.: "Open source software for the analysis of microarray data", BIOTECHNIQUES, 2003, pages 45 - 51
E. A. BAYER ET AL., METHODS OF BIOCHEM. ANALYSIS, vol. 26, 1980, pages 1 - 45
E. L. BROWN ET AL., METH. ENZYMOL., vol. 68, 1979, pages 109 - 151
E. S. BELOUSOV ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3440 - 3444
E. S. MANSFIELD ET AL., MOL. CELL. PROBES, vol. 9, 1995, pages 145 - 156
EDGAR R, DOMRACHEV M, LASH AE.: "Gene Expression Omnibus: NCBI gene expression and hybridization array data repository", NUCLEIC ACIDS RES., vol. 30, 2002, pages 207 - 10
FAHN S ER: "Members of the UPDRS Development Committee", UNIFIED PARKINSON'S DISEASE RATING SCALE, 1987
FAHN S.: "Description of Parkinson's disease as a clinical syndrome", ANN N YACAD SCI., vol. 991, 2003, pages 1 - 14
FINGL ET AL.: "The Pharmacological Basis of Therapeutics", 1975, pages: 1
FRESHNEY: "Culture of Animal Cells - A Manual of Basic Technique", 1994, WILEY-LISS
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., vol. 22, 2008, pages 1327 - 34
FUJIMURA K, KATAHIRA J, KANO F ET AL.: "Selective localization of PCBP2 to cytoplasmic processing bodies", BIOCHIM BIOPHYS ACTA, vol. 1793, 2009, pages 878 - 87, XP026106500, DOI: doi:10.1016/j.bbamcr.2009.02.002
G. D. MCFARLAND, P. N. BORER, NUCLEIC ACIDS RES., vol. 7, 1979, pages 1067 - 1080
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, vol. 7, 2006, pages 325, XP021022290, DOI: doi:10.1186/1471-2164-7-325
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, vol. 39, 1982, pages 1157 - 67
GILBOA, BIOTECHNIQUES, vol. 4, no. 6, 1986, pages 504 - 512
GORLICH D, HARTMANN E, PREHN S ET AL.: "A protein of the endoplasmic reticulum involved early in polypeptide translocation", NATURE, vol. 357, 1992, pages 47 - 52
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, vol. 13, 2008, pages 717 - 28
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.
GRISARU D, PICK M, PERRY C ET AL.: "Hydrolytic and nonenzymatic functions of acetylcholinesterase comodulate hemopoietic stress responses", J IMMUNOL., vol. 176, 2006, pages 27 - 35
GUREVICH M, TULLER T, RUBINSTEIN U ET AL.: "Prediction of acute multiple sclerosis relapses by transcription levels of peripheral blood cells", BMC MED GENOMICS, vol. 2, 2009, pages 46, XP021060673, DOI: doi:10.1186/1755-8794-2-46
H. WU, H. ABOLENEEN, ANAL. BIOCHEM., vol. 290, 2001, pages 347 - 352
HENCHCLIFFE C, BEAL MF.: "Mitochondrial biology and oxidative stress in Parkinson disease pathogenesis", NAT CLIN PRACT NEUROL., vol. 4, 2008, pages 600 - 9
HOSACK DA, DENNIS G, JR., SHERMAN BT ET AL.: "Identifying biological themes within lists of genes with EASE", GENOME BIOL., vol. 4, 2003, pages 70
J. D. PEARSON, F. E. REGNIER, J. CHROM., vol. 255, 1983, pages 137 - 149
J. E. LANDEGENT ET AL., EXP. CELL RES., vol. 15, 1984, pages 61 - 72
J. SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOUR LABORATORY PRESS
J. TEMSAMANI, S. AGRAWAL, MOL. BIOTECHNOL., vol. 5, 1996, pages 223 - 232
JOHNSON JM, CASTLE J, GARRETT-ENGELE P ET AL.: "Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays", SCIENCE, vol. 302, 2003, pages 2141 - 4, XP002458857, DOI: doi:10.1126/science.1090100
K. B. MULLIS, F. A. FALOONA, METHODS ENZYMOL., vol. 155, 1987, pages 350 - 355
K. FRENKEL ET AL., FREE RADIC. BIOL. MED., vol. 19, 1995, pages 373 - 380
KAGAN JC, SU T, HORNG T ET AL.: "TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-beta", NAT IMMUNOL., vol. 9, 2008, pages 361 - 8
KAHANE P, DEPAULIS A.: "Deep brain stimulation in epilepsy: what is next?", CURR OPIN NEUROL.
KANEHISA M, GOTO S, FURUMICHI M ET AL.: "KEGG for representation and analysis of molecular networks involving diseases and drugs", NUCLEIC ACIDS RES., vol. 38, 2010, pages D355 - 60
KAPRANOV P, WILLINGHAM AT, GINGERAS TR.: "Genome-wide transcription and the implications for genomic organization", NAT REV GENET., vol. 8, 2007, pages 413 - 23
KARNOVSKY, ROOTS, J HISTOCHEM CYTOCHEM, vol. 12, 1964, pages 2 19 - 22 1
KAUFER, D., NATURE, vol. 393, 1998, pages 373 - 377
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., vol. 277, 2002, pages 2732 - 9
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., vol. 10, 2009, pages 105, XP021062607, DOI: doi:10.1186/1471-2350-10-105
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., vol. 190, 1994, pages 1017 - 25
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", NEUROBIOL DIS., vol. 28, 2007, pages 122 - 32, XP022263492, DOI: doi:10.1016/j.nbd.2007.07.012
KURIAN SM, LE-NICULESCU H, PATEL SD ET AL.: "Identification of blood biomarkers for psychosis using convergent functional genomics", MOL PSYCHIATRY, 2009
L. J. KRICKA, ANN. CLIN. BIOCHEM., vol. 39, 2002, pages 114 - 129
L. M. SMITH ET AL., NUCL. ACIDS RES., vol. 13, 1985, pages 2399 - 2412
M. G. SEBESTYEN ET AL., NAT. BIOTECHNOL., vol. 16, 1998, pages 568 - 576
M. J. BLOMMERS ET AL., BIOCHEMISTRY, vol. 33, 1994, pages 7886 - 7896
MARSHAK ET AL.: "Strategies for Protein Purification and Characterization - A Laboratory Course Manual", 1996, CSHL PRESS
MILLER G.: "Neuropsychiatry. Rewiring faulty circuits in the brain", SCIENCE, vol. 323, 2009, pages 1554 - 6
NUOVO GJ ET AL.: "Intracellular localization of polymerase chain reaction (PCR)-amplified hepatitis C cDNA", AM J SURG PATHOL., vol. 17, 1993, pages 683 - 90
OLANOW CW, RASCOL O, HAUSER R ET AL.: "A double-blind, delayed-start trial of rasagiline in Parkinson's disease", N ENGL J MED., vol. 361, 2009, pages 1268 - 78
P. TCHEN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 3466 - 3470
P. TIJSSEN: "Hybridization with Nucleic Acid Probes--Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II", 1993, ELSEVIER SCIENCE
PANKRATZ N, WILK JB, LATOURELLE JC ET AL.: "Genomewide association study for susceptibility genes contributing to familial Parkinson disease", HUM GENET., vol. 124, 2009, pages 593 - 605, XP002638618, DOI: doi:10.1007/S00439-008-0582-9
PERBAL, B., A PRACTICAL GUIDE TO MOLECULAR CLONING, 1984
PERBAL: "A Practical Guide to Molecular Cloning", 1988, JOHN WILEY & SONS
R. J. HEETEBRIJ ET AL., CYTOGENET. CELL. GENET., vol. 87, 1999, pages 47 - 52
R. K. SAIKI ET AL., NATURE, vol. 324, 1986, pages 163 - 166
R. LANGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 78, 1981, pages 6633 - 6637
R. P. VAN GIJLSWIJK ET AL., EXPERT REV. MOL. DIAGN., vol. 1, 2001, pages 81 - 91
R. W. RICHARDSON ET AL., NUCL. ACIDS RES., vol. 11, 1983, pages 6167 - 6184
REIZENSTEIN P.: "The haematological stress syndrome", BR J HAEMATOL., vol. 43, 1979, pages 329 - 34
RILEY D, LOZANO A.: "The fourth dimension of stereotaxis: timing of neurosurgery for Parkinson disease", NEUROLOGY, vol. 68, 2007, pages 252 - 3
S. A. NARANG ET AL., METH. ENZYMOL., vol. 68, 1979, pages 90 - 98
S. JOOS ET AL., J. BIOTECHNOL., vol. 35, 1994, pages 135 - 153
SAMBROOK ET AL.: "Molecular Cloning: A laboratory Manual", 1989
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRINGS HARBOR LABORATORY
SAVICA R, ROCCA WA, AHLSKOG JE.: "When does Parkinson disease start?", ARCH NEUROL., vol. 67, 2010, pages 798 - 801
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 U S A., vol. 104, 2007, pages 955 - 60, XP055019562, DOI: doi:10.1073/pnas.0610204104
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., vol. 105, 2008, pages 10907 - 12
SEGMAN RH, GOLTSER-DUBNER T, WEINER I ET AL.: "Blood mononuclear cell gene expression signature of postpartum depression", MOL PSYCHIATRY, vol. 15, 2010, pages 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, vol. 10, 2005, pages 500 - 13,425
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)., vol. 5, 2008, pages 24 - 33
SHAKED I, MEERSON A, WOLF Y ET AL.: "MicroRNA-132 potentiates cholinergic anti-inflammatory signaling by targeting acetylcholinesterase", IMMUNITY, vol. 31, 2009, pages 965 - 73
SOREQ L, GILBOA-GEFFEN A, BERRIH-AKNIN S ET AL.: "Identifying alternative hyper- splicing signatures in MG-thymoma by exon arrays", PLOS ONE, vol. 3, 2008, pages E2392
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., vol. 201-202, 2008, pages 227 - 36, XP025495599, DOI: doi:10.1016/j.jneuroim.2008.06.019
SPILLANTINI MG, SCHMIDT ML, LEE VM ET AL.: "Alpha-synuclein in Lewy bodies", NATURE, vol. 388, 1997, pages 839 - 40, XP002090996, DOI: doi:10.1038/42166
SRINIVASAN K ET AL., METHODS, vol. 37, 2005, pages 345 - 359
SUN S, ZHANG Z, SINHA R ET AL.: "SF2/ASF autoregulation involves multiple layers of post-transcriptional and translational control", NAT STRUCT MOL BIOL., 2010
T. R. BROKER ET AL., NUCL. ACIDS RES., vol. 5, 1978, pages 363 - 384
TATUSOV RL, KOONIN EV, LIPMAN DJ.: "A genomic perspective on protein families", SCIENCE, vol. 278, 1997, pages 631 - 7, XP002939284, DOI: doi:10.1126/science.278.5338.631
TEMPERLI P, GHIKA J, VILLEMURE JG ET AL.: "How do parkinsonian signs return after discontinuation of subthalamic DBS?", NEUROLOGY, vol. 60, 2003, pages 78 - 81, XP009187954
TOFT P, TONNESEN E, HELBO-HANSEN HS ET AL.: "Redistribution of granulocytes in patients after major surgical stress", APMIS., vol. 102, 1994, pages 43 - 8
TONKINSON ET AL., CANCER INVESTIGATION, vol. 14, no. 1, 1996, pages 54 - 65
TRACEY KJ.: "The inflammatory reflex", NATURE, vol. 420, 2002, pages 853 - 9
U. PIELES ET AL., NUCLEIC ACIDS RES., vol. 21, 1993, pages 3191 - 3196
VAN DUIJN CM, DEKKER MC, BONIFATI V ET AL.: "Park7, a novel locus for autosomal recessive early-onset parkinsonism, on chromosome 1p36", AM J HUM GENET., vol. 69, 2001, pages 629 - 34
VEGA ET AL.: "Gene Targeting", 1995, CRC PRESS
WATSON ET AL.: "Recombinant DNA", SCIENTIFIC AMERICAN BOOKS
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, vol. 301, 2009, pages 63 - 73
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, vol. 79, 2002, pages 869 - 74
ZAIDEL A, BERGMAN H, RITOV Y ET AL.: "Levodopa and subthalamic deep brain stimulation responses are not congruent.", MOV DISORD., vol. 25, 2010, pages 2379 - 86
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., vol. 281, 2006, pages 20940 - 8
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 USA., vol. 92, 1995, pages 8586 - 90

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2773779A4 (fr) * 2011-11-04 2015-09-30 Population Diagnotics Inc Méthodes et compositions pour le diagnostic, le pronostic et le traitement d'états neurologiques
WO2016078546A1 (fr) * 2014-11-20 2016-05-26 华中科技大学 Médicament prévenant la maladie d'alzheimer
EP3091087A1 (fr) * 2015-05-08 2016-11-09 Fundació Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol Procédé de diagnostic in vitro de synucléinopathies utilisant des transcriptions du gène de l'alpha-synucléine
WO2016180726A1 (fr) * 2015-05-08 2016-11-17 Fundació Institut D'investigació En Ciències De La Salut Germans Trias I Pujol Procédé de diagnostic in vitro de la démence à corps de lewy à l'aide de transcrits du gène de l'alphasynucléine
CN106021915A (zh) * 2016-05-17 2016-10-12 华南师范大学 基于大数据面向自动诊疗的医疗数据分析系统和装置
US11142570B2 (en) 2017-02-17 2021-10-12 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof
US11827695B2 (en) 2017-02-17 2023-11-28 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof
CN109545370A (zh) * 2018-10-27 2019-03-29 平安医疗健康管理股份有限公司 基于样本处理的病种确定方法、装置及终端
CN109545370B (zh) * 2018-10-27 2023-08-22 平安医疗健康管理股份有限公司 基于样本处理的病种确定方法、装置及终端
CN115957349A (zh) * 2022-10-08 2023-04-14 华中农业大学 激活猪的pja1基因表达的制剂在制备猪抗猪流行性腹泻病毒感染药物中的应用
CN115957349B (zh) * 2022-10-08 2024-04-05 华中农业大学 激活猪的pja1基因表达的制剂在制备猪抗猪流行性腹泻病毒感染药物中的应用

Also Published As

Publication number Publication date
WO2012032519A3 (fr) 2012-07-05

Similar Documents

Publication Publication Date Title
Himeda et al. The genetics and epigenetics of facioscapulohumeral muscular dystrophy
Spiteri et al. Identification of the transcriptional targets of FOXP2, a gene linked to speech and language, in developing human brain
WO2012032519A2 (fr) Méthodes de diagnostic de la maladie de parkinson
CA2993142A1 (fr) Signature genique pour immunotherapies dans le cancer
EP2683412B1 (fr) Procédés pour évaluer la sensibilité de cellules cancéreuses au lénalidomide, au thalidomide et/ou au pomalidomide
WO2016046640A2 (fr) Procédés de prédiction de la réactivité à un médicament
Babar et al. Genes of the interleukin-18 pathway are associated with susceptibility to Barrett's esophagus and esophageal adenocarcinoma
US20110166028A1 (en) Methods for predicting treatment response based on the expression profiles of biomarker genes in notch mediated cancers
JP2015512630A (ja) 急性骨髄性白血病の診断、予後、及び治療用の方法及び組成物
KR20240095481A (ko) Rnaset2를 통한 염증성 장 질환의 진단 방법
US20120142544A1 (en) Diagnostic transcriptomic biomarkers in inflammatory cardiomyopathies
US10072296B2 (en) Compositions and methods for sjögren's syndrome
EP2841595A2 (fr) Prédicteurs génétiques de réponse à un traitement avec des antagonistes de crhr1
EP3660172A1 (fr) Procédés de diagnostic de lésions cérébrales traumatiques
EP3011048B1 (fr) Procédé de prédiction de la réponse à un traitement par un antagoniste de v1b chez un patient atteint de symptômes de la dépression et/ou de l'anxiété
US20180044733A1 (en) Circulatory MicroRNAs (miRNAs) as Biomarkers for Diabetic Retinopathy (DR) and Age-Related Macular Degeneration
US11376261B2 (en) Methods for diagnosing and managing treatment of atopic dermatitis
CN108715893B (zh) 一组与放疗引起的放射性脑损伤相关的snp标志物及其应用
EP3277836B1 (fr) Méthode de traitement utilisant les prédicteurs génétiques d'une réponse à un traitement avec ssr-125543
US20160244845A1 (en) Method for determining the prognosis of pancreatic cancer
McMahon et al. Detection of microchromosomal aberrations in refractory epilepsy: a pilot study
WO2007137366A1 (fr) Indicateurs de diagnostic et de pronostic du cancer
US20120208718A1 (en) Schizophrenia treatment response biomarkers
EP2716767A1 (fr) Procédé pour déterminer le pronostic du cancer du pancréas
US20100105054A1 (en) Gene expression in duchenne muscular dystrophy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11770875

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11770875

Country of ref document: EP

Kind code of ref document: A2