US 20160244833A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0244833 A1 POTASHKN et al. (43) Pub. Date: Aug. 25, 2016

(54) METHODS AND KITS FOR DIAGNOSING, Publication Classification PROGNOSING AND MONITORING PARKINSONS DISEASE (51) Int. Cl. CI2O I/68 (2006.01) (71) Applicant: Rosalind Franklin University of (52) U.S. Cl. Medicine and Science, North Chicago, CPC ...... CI2O I/6883 (2013.01); C12O 2600/158 IL (US) (2013.01); C12O 2600/1 18 (2013.01); C12O 2600/1 12 (2013.01) (72) Inventors: Judith Ann POTASHKIN, North (57) ABSTRACT Chicago, IL (US); Jose Alfredo Network-based meta-analysis of four independent microar SANTIAGO, North Chicago, IL (US) ray studies identified the hepatocyte nuclear factor (HNF4A), a transcription factor associated with gluconeogenesis and (73) Assignee: Rosalind Franklin University of diabetes, as a central regulatory hub gene upregulated in Medicine and Science, North Chicago, blood of PD patients. In parallel, the polypyrimidine tract binding protein 1 (PTBP1), involved in the stabilization and IL (US) mRNA translation of insulin, was identified as the most downregulated gene. Using both markers, PD patients were (21) Appl. No.: 14/870,960 classified with 90% sensitivity and 80% specificity. Longitu dinal performance analysis demonstrated that relative abun dance of HNF4A and PTBP1 mRNAssignificantly decreased (22) Filed: Sep. 30, 2015 and increased, respectively, in PD patients during 3 years follow up period. The inverse regulation of HNF4A and PTBP1 provides a molecular rationale for the altered insulin Related U.S. Application Data signaling observed in PD patients. The longitudinally (60) Provisional application No. 62/120,848, filed on Feb. dynamic biomarkers identified in this study may be useful for 25, 2015. monitoring disease-modifying therapies for PD. Patent Application Publication Aug. 25, 2016 Sheet 1 of 8 US 2016/0244833 A1

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METHODS AND KITS FOR DIAGNOSING, would to dopamine. An antiviral drug, amantadine, also PROGNOSING AND MONITORING appears to reduce symptoms. In May 2006, the FDA approved PARKINSONS DISEASE rasagiline to be used along with levodopa for patients with advanced PD or as a single-drug treatment for early PD. CROSS REFERENCE 0005. In some cases, surgery may be appropriate if the disease does not respond to drugs. Atherapy called deep brain 0001. This application is related to U.S. provisional patent stimulation (DBS) has now been approved by the U.S. Food application No. 62/120,848, filed Feb. 25, 2015, the disclo and Drug Administration. In DBS, electrodes are implanted sure of which is incorporated by reference herein in its into the brain and connected to a small electrical device called entirety. The sequence listing Submitted herewith is incorpo a pulse generator that can be externally programmed. DBS rated by reference in its entirety. can reduce the need for levodopa and related drugs, which in turn decreases the involuntary movements called dyskinesias STATEMENT REGARDING FEDERALLY that are a common side effect of levodopa. It also helps to SPONSORED RESEARCH alleviate fluctuations of symptoms and to reduce tremors, 0002 This invention was made with U.S. government Sup slowness of movements, and gait problems. DBS requires port by the US Army Medical Research and Materiel Com careful programming of the stimulator device in order to work mand under awards number W81XWH-09-0708 and correctly. W81XWH13-1-0O25. 0006. There is a need in the art for a better understanding of the underlying disease mechanism and methods to facili BACKGROUND OF THE DISCLOSURE tate the discovery of accurate biomarkers and therapeutic 0003 Parkinson's disease (PD; also known as idiopathic targets for Parkinson's disease. or primary parkinsonism, hypokinetic rigid syndrome (HRS), or paralysis agitans) belongs to a group of conditions called SUMMARY OF THE DISCLOSURE motor system disorders, which are the result of the loss of 0007. This disclosure demonstrates that network-based dopamine-producing brain cells. The four primary symptoms meta-analysis of four independent microarray studies pro of PD are tremor, or trembling in hands, arms, legs, jaw, and vides a useful framework to identify candidate biomarkers, face; rigidity, or stiffness of the limbs and trunk; bradykine and that expression of highly ranked genes identified can be sia, or slowness of movement; and postural instability, or used as diagnostic and prognostic biomarkers for PD. impaired balance and coordination. As these symptoms 0008. In one aspect, the disclosure provides a method for become more pronounced, patients may have difficulty walk diagnosing, prognosing or monitoring Parkinson's Disease ing, talking, or completing other simple tasks. PD usually (PD) in a human Subject, comprising: (a) obtaining a blood affects people over the age of 60. Early symptoms of PD are sample from a human Subject Suspected of having PD; (b) Subtle and occur gradually. In some people the disease determining the expression level of at least one gene in the progresses more quickly than in others. As the disease blood sample from the human Subject Suspected of having progresses, the shaking, or tremor, which affects the majority PD, wherein the at least one gene is selected from: HNF4A, of people with PD may begin to interfere with daily activities. THY1, SPEF1, SF3A2, SEMA6B, EN2, RTN3, BCAM, Other symptoms may include depression and other emotional SPATA2L and TPSG1; and (c) comparing the expression changes; difficulty in Swallowing, chewing, and speaking; level of the at least one gene expressed in the blood sample to urinary problems or constipation: skin problems; and sleep the expression level of the at least one gene expressed in a disruptions. There are currently no blood or laboratory tests non-PD, healthy control sample, whereby the increased that have been proven to help in diagnosing sporadic PD. expression level of the at least one gene expressed in the blood Therefore the diagnosis is based on medical history and a sample from the human Subject Suspected of having PD as neurological examination, but the disease can be difficult to compared to the non-PD sample is indicative of PD, thereby diagnose accurately. Doctors may sometimes request brain diagnosing the human Subject as having PD. scans or laboratory tests in order to rule out other diseases. 0009. In another aspect, the disclosure provides a method 0004 At present, there is no cure for PD, but a variety of of treating a human subject for Parkinson's Disease (PD), the medications provide dramatic relief from the symptoms. Usu method comprising: (a) obtaining a diagnosis identifying a ally, affected individuals are given levodopa (L-DOPA: human Subject as having PD, wherein the diagnosis was SINEMETTM, PARCOPATM, ATAMETTM, STALEVOTM, obtained by: (i) obtaining a blood sample from a human MADOPARTM, and PROLOPATM) combined with carbidopa Subject Suspected of having PD; (ii) determining the expres (LODOSYNTM) (products containing a combination of sion level of at least one gene selected from: HNF4A,THY1. levodopa and carbidopa include DUOPAR) and RYTARYR). SPEF1, SF3A2, SEMA6B, EN2, RTN3, BCAM, SPATA2L Carbidopa delays the conversion of levodopa into dopamine and TPSG1; and (iii) comparing the expression level of the at until it reaches the brain. Nerve cells can use levodopa to least one gene expressed in the blood sample to the expression make dopamine and replenish the brain's dwindling Supply. level of the at least one gene expressed in a non-PD, healthy Although levodopa helps at least three-quarters of PD cases, control sample, whereby the increased expression level of the not all symptoms respond equally to the drug. Bradykinesia at least one gene expressed in the blood sample from the and rigidity typically respond best, while tremor may be only human Subject Suspected of having PD as compared to the marginally reduced. Problems with balance and other symp non-PD sample is indicative of PD, thereby diagnosing the toms may not be alleviated at all. Anticholinergics may help human Subject as having PD; and (b) administering to the control tremor and rigidity. Dopamine agonists, such as bro subject a PD treatment regimen. mocriptine, pergolide, pramipexole, ropinirole, piribedil, 0010. In yet another aspect, the disclosure provides a Par cabergoline, apomorphine and lisuride, mimic the role of kinson's Disease (PD) diagnosis, prognosis or monitoring kit, dopamine in the brain, causing the neurons to react as they consisting of a set of probes suitable for the detection and US 2016/0244833 A1 Aug. 25, 2016

quantification of the nucleic acid expression of at least one (Meta-DE) and those from each individual microarray analy gene selected from: HNF4A, THY1, SPEF1, SF3A2, sis (Individual-DE). FIG. 1C.. Heat map representation of the SEMA6B, EN2, RTN3, BCAM, SPATA2L and TPSG1. top 50 differentially expressed genes across different 0011. In a second aspect, the disclosure provides a method microarrays identified from the meta-analysis (row-wise for diagnosing, prognosing or monitoring Parkinson's Dis comparison). The heat map was re-scaled to prevent domina ease (PD) in a human Subject, comprising: (a) obtaining a tion by study-specific effects. PD denotes Parkinson's disease blood sample from a human subject suspected of having PD; and HC denotes healthy controls. (b) determining the expression level of at least one gene in the 0016 FIGS. 2A and 2B show the network analysis of blood sample from the human Subject Suspected of having differentially expressed genes in blood of PD. FIG. 2A.Zero PD, wherein the at least one gene is selected from: PTBP1, order interaction network of genes upregulated in blood of PD SLC4A1, DAZAP2, EPB42, HELZ, SELENBP1, NUDT4, (Red). FIG. 2B. Zero-order interaction network of genes CA1, AHSP and ALAS2; and (c) comparing the expression downregulated in blood of PD (Green). level of the at least one gene expressed in the blood sample to the expression level of the at least one gene expressed in a 0017 FIGS. 3A, 3B, 3C and 3D show the evaluation of non-PD, healthy control sample, whereby the decreased HNF4A and PTBP1 mRNAS as biomarkers for PD at base expression level of the at least one gene expressed in the blood line. FIG.3A. Relative abundance of HNF4A mRNA in blood sample from the human Subject Suspected of having PD as of PD patients (black circles) compared to HC (white circles) compared to the non-PD sample is indicative of PD, thereby in samples from the PROBE cohort. FIG. 3B. Replication of diagnosing the human Subject as having PD. biomarker expression in an independent set of Samples from 0012. In another aspect, the disclosure provides a method patients enrolled in the Harvard Biomarker Study (HBS) of treating a human subject for Parkinson's Disease (PD), the study. FIG. 3C. Relative abundance of PTBP1 mRNA in method comprising: (a) obtaining a diagnosis identifying a blood of PD patients compared to HC in samples from the human Subject diagnosed as having PD, wherein the diagno PROBE cohort. FIG. 3D. Replication of PTBP1 mRNA sis was obtained by: (i) obtaining a blood sample from a expression in an independent set of samples from patients human Subject Suspected of having PD; (ii) determining the enrolled in the HBS study. Relative abundance of each biom expression level of at least one gene in the blood sample from arker was calculated using GAPDH as a reference gene and the human subject suspected of having PD selected from: HC as calibrator. A student t-test (two-tailed) was used to PTBP1, SLC4A1, DAZAP2, EPB42, HELZ, SELENBP1, estimate the significance between PD cases and controls. NUDT4, CA1, AHSP and ALAS2; and (iii) comparing the Post-hoc pair-wise comparisons were performed using a expression level of the at least one gene expressed in the blood Tukey test of significance. Error bars represent 95% confi sample to the expression level of the at least one gene dence intervals. expressed in a non-PD, healthy control sample, whereby the 0018 FIGS. 4A and 4B show the biomarker correlation decreased expression level of the at least one gene expressed analysis. FIG. 4A. Pearson correlation analysis between in the blood sample from the human subject suspected of HNF4A mRNA and PTBP1 mRNA in blood of PD patients having PD as compared to the non-PD sample is indicative of (black circles) and HC (white circles) in samples from PD, thereby diagnosing the human Subject as having PD; and PROBE and HBS. FIG. 4B. Correlation analysis between (b) administering to the Subject a PD treatment regimen. HNF4A mRNA and Hoehn and Yahr scale in PD patients 0013. In yet another aspect, the disclosure provides a Par from both cohorts. Error bars represent 95% confidence inter kinson's Disease (PD) diagnosis, prognosis or monitoring kit, vals. consisting of a set of probes suitable for the detection and (0019 FIGS. 5A, 5B, 5C and 5D show the longitudinal quantification of the nucleic acid expression of at least one performance of HNF4A and PTBP1 mRNAs in the HBS gene: PTBP1, SLC4A1, DAZAP2, EPB42, HELZ, study. FIG. 5A. Individual trajectories for the relative abun SELENEP1, NUDT4, CA1, AHSP and ALAS2. dance of HNF4A mRNA over time for HC (blue) and PD 0014. These and other features and advantages of the patients (red) in the HBS study. FIG. 5B. Average rate of present disclosure will be more fully understood from the HNF4A mRNA in PD patients compared to HC calculated via following detailed description of the invention taken together linear mixed effects regression analysis adjusting for sex, age with the accompanying claims. It is noted that the scope of the and BMI. FIG. 5C. Individual trajectories for the relative claims is defined by the recitations therein and not by the abundance of PTBP1 mRNA over time for HC and PD specific discussion of features and advantages set forth in the patients. FIG.5D. Average rate of PTBP1 mRNA expression present description. in PD patients compared to HC calculated via linear mixed effects regression analysis. Red and blue lines denote PD and BRIEF DESCRIPTION OF THE DRAWINGS HC, respectively. T0 and T2 indicate baseline and 3 years follow up period, respectively. Post-hoc pair-wise compari 0015 FIGS. 1A, 1B and 1C show the network-based and Sons were performed using a Tukey test of significance transcriptomic meta-analysis. FIG. 1A. Four independent (**p=0.0001, p=0.001). Error bars represent standard error. microarray datasets were downloaded from the Gene Expres sion Ommibus (GEO) and preprocessed in INMEX where 0020 FIG. 6 shows the network-based meta-analysis of meta-analysis was undertaken using the Fisher's method. microarray studies in blood of PD. Network-based meta Datasets were subsequently uploaded into NetworkAnalyst analysis identified HNF4A and GATA1 as the most promi to perform network and functional analysis and to identify nent hub genes across the four microarrays datasets. key regulatory hub genes across the multiple microarray stud 0021 FIGS. 7A and 7B show the dysregulated splicing ies. Finally, the most significant genes were evaluated as factors in blood of PD. FIG. 7A. Upregulated splicing factors biomakers for PD in blood samples obtained from two inde in blood of PD identified across the four microarray datasets. pendent cohorts of patients. FIG. 1B. Venn diagram of differ FIG. 7B. Downregulated splicing factors in blood of PD entially expressed genes identified from the meta-analysis identified across the four microarray datasets. US 2016/0244833 A1 Aug. 25, 2016

0022 FIGS. 8A, 8B and 8C show the ROC analysis of plex interaction between environmental and genetic factors in HNF4A and PTBP1 mRNAs. FIG. 8A. ROC analysis to sporadic PD, four independent microarray studies were inte evaluate the performance of HNF4A mRNA as a diagnostic grated from patients harboring a mutation in the LRRK2 gene biomarker in samples from PROBE and HBS studies. FIG. (G2019S; glycine to serine substitution at amino acid 2019), 8B. ROC analysis to evaluate the performance of PTBP1 sporadic, and untreated PD patients in order to identify a mRNA as a diagnostic biomarker in PROBE and HBS. FIG. universal signature in blood associated with PD. A transcrip 8C. ROC analysis combining HNF4A and PTBP1 mRNAs as tomic and network-based meta-analysis was performed to diagnostic biomarkers in PROBE and HBS. AUC denotes identify key regulators and potential diagnostic biomarkers. area under the curve. This is a powerful approach to integrate gene expression data and to gain insight into complex diseases. The utility of net DETAILED DESCRIPTION OF THE work biology to identify biologically relevant biomarkers for DISCLOSURE neurodegenerative diseases has been demonstrated recently 0023 The present disclosure demonstrates that network (Santiago & Potashkin (2014) A network approach to clinical based meta-analysis of four independent microarray studies intervention in neurodegenerative diseases. Trends Mol Med provides a useful framework to identify candidate biomark 20(12):694-703; Santiago & Potashkin (2013) Integrative ers, and that expression of highly ranked genes identified can network analysis unveils convergent molecular pathways in be used as diagnostic and prognostic biomarkers for PD. Parkinson's disease and diabetes. PLoS One 8(12):e83940; 0024. The development of therapeutic strategies for Par Santiago & Potashkin (2014) A network approach to diag kinson's disease (PD) is hampered by the lack of reliable nostic biomarkers in progressive Supranuclear palsy. Mov biomarkers to identify patients at early stages of the disease Disord 29(4):550-555). and track the therapeutic effect of potential drugs and neuro (0027 Network-based meta-analysis identified HNF4A protective agents. Readily accessible biomarkers capable of and PTBP1, previously implicated in diabetes, as the most providing information about disease status are expected to significant up and downregulated genes in blood of PD accelerate this progress. Network-based meta-analysis iden patients. These results were confirmed in blood samples from tified promising blood biomarkers for recognizing early stage two independent clinical trials. Relative abundance of PD patients with high diagnostic accuracy. Longitudinal HNF4A mRNA correlated with disease severity in PD analysis demonstrated that HNF4A and PTBP1 are longitu patients. Moreover, longitudinal analysis of HNF4A and dinally dynamic biomarkers that provide insights into the PTBP1 revealed that their relative abundance changed over molecular mechanisms underlying the altered insulin signal time indicating their potential use to track the clinical course ing in PD patients and may enable novel therapeutic strate of PD patients. gies. Further, HNF4A was identified as a potential biomarker 0028. It should be understood that the embodiments to monitor disease severity. described herein, are provided for explanatory purposes, and 0025. Substantial efforts have been devoted to the devel are not intended to be limiting. opment of diagnostic strategies for PD. In particular, changes 0029 All publications, patents and patent applications in mRNA from cellular whole blood can facilitate the identi cited herein are hereby expressly incorporated by reference fication of dysregulated processes and diagnostic biomarkers for all purposes to the extent they are consistent with this for PD. Several molecular signatures in blood have been disclosure. identified. For example, 22 unique genes were found differ entially expressed in blood of PD patients compared to 0030 Methods well known to those skilled in the art can healthy controls (Scherzer et al., (2007) Molecular markers of be used to construct expression vectors and recombinant bac early Parkinson's disease based on gene expression in blood. terial cells according to this disclosure. These methods Proc Natl AcadSci U.S.A. 104(3):955-960). Likewise, spe include invitro recombinant DNA techniques, synthetic tech cific splice variants in blood were associated with PD in niques, in vivo recombination techniques, and PCR tech samples obtained from two independent clinical trials (Pot niques. See, for example, techniques as described in Maniatis ashkin et al., (2012) Biosignatures for Parkinson's disease et al., 1989, MOLECULAR CLONING: A LABORATORY and atypical parkinsonian disorders patients. PLoS One 7(8): MANUAL, Cold Spring Harbor Laboratory, New York: e43595, and Santiago et al., (2013) Specific splice variants Ausubel et al., 1989, CURRENT PROTOCOLS IN are associated with Parkinson's disease. Mov Disord 28(12): MOLECULAR BIOLOGY, Greene Publishing Associates 1724-1727). In addition, altered expression of the vitamin D and Wiley Interscience, New York, and PCR Protocols: A receptor (VDR) in blood and reduced plasma levels of 25-hy Guide to Methods and Applications (Innis et al., 1990, Aca droxy vitamin D, have been associated with PD (Ding et al. demic Press, San Diego, Calif.). (2013) Unrecognized vitamin D3 deficiency is common in 0031. Before describing the present invention in detail, a Parkinson disease: Harvard Biomarker Study. Neurology number of terms will be defined. As used herein, the singular 81 (17): 1531-1537). Furthermore, plasma levels of the epider forms “a”, “an’, and “the include plural referents unless the mal growth factor (EGF) have been associated with cognitive context clearly dictates otherwise. For example, reference to decline in PD (Chen-Plotkin et al. (2011) Plasma epidermal a “nucleic acid means one or more nucleic acids. growth factor levels predict cognitive decline in Parkinson 0032. It is noted that terms like “preferably”, “com disease. Ann Neurol 69(4):655-663). monly', and “typically are not utilized herein to limit the 0026 Environmental stressors and genetic factors are Scope of the claimed invention or to imply that certain fea most likely involved in the pathogenesis of PD. Among the tures are critical, essential, or even important to the structure genetic factors associated with PD, mutations in the gene or function of the claimed invention. Rather, these terms are encoding leucine-rich repeat kinase 2 (LRRK2) are the most merely intended to highlight alternative or additional features common cause of autosomal dominant PD and a considerable that can or cannot be utilized in a particular embodiment of risk factor in idiopathic forms of the disease. Given the com the present invention. US 2016/0244833 A1 Aug. 25, 2016

0033. In one aspect, the disclosure provides a method for fluorescence by a sequence detection system following a diagnosing, prognosing or monitoring Parkinson's Disease quantitative, real-time polymerase chain reaction (PCR) (PD) in a human Subject, comprising: (a) obtaining a blood assay. sample from a human Subject Suspected of having PD; (b) 0038. In yet another embodiment, an increased HNF4A determining the expression level of at least one gene in the expression level of the human Subject at a later time point blood sample from the human Subject Suspected of having compared to the HNF4A expression level at an initial time PD, wherein the at least one gene is selected from: HNF4A, point or expression levels the same as a healthy control indi THY1, SPEF1, SF3A2, SEMA6B, EN2, RTN3, BCAM, cates an improved or steady prognosis of PD in the Subject. SPATA2L and TPSG1; and (c) comparing the expression level of the at least one gene expressed in the blood sample to 0039. In a second aspect, the disclosure provides a method the expression level of the at least one gene expressed in a for diagnosing, prognosing or monitoring Parkinson's Dis non-PD, healthy control sample, whereby the increased ease (PD) in a human Subject, comprising: (a) obtaining a expression level of the at least one gene expressed in the blood blood sample from a human subject suspected of having PD; sample from the human Subject Suspected of having PD as (b) determining the expression level of at least one gene in the compared to the non-PD sample is indicative of PD, thereby blood sample from the human Subject Suspected of having diagnosing the human Subject as having PD. PD, wherein the at least one gene is selected from: PTBP1, SLC4A1, DAZAP2, EPB42, HELZ, SELENBP1, NUDT4, 0034. In another aspect, the disclosure provides a method CA1, AHSP and ALAS2; and (c) comparing the expression of treating a human subject for Parkinson's Disease (PD), the level of the at least one gene expressed in the blood sample to method comprising: (a) obtaining a diagnosis identifying a the expression level of the at least one gene expressed in a human Subject as having PD, wherein the diagnosis was non-PD, healthy control sample, whereby the decreased obtained by: (i) obtaining a blood sample from a human expression level of the at least one gene expressed in the blood Subject Suspected of having PD; (ii) determining the expres sample from the human Subject Suspected of having PD as sion level of at least one gene selected from: HNF4A,THY1. compared to the non-PD sample is indicative of PD, thereby SPEF1, SF3A2, SEMA6B, EN2, RTN3, BCAM, SPATA2L diagnosing the human Subject as having PD. and TPSG1; and (iii) comparing the expression level of the at 0040. In another aspect, the disclosure provides a method least one gene expressed in the blood sample to the expression of treating a human subject for Parkinson's Disease (PD), the level of the at least one gene expressed in a non-PD, healthy method comprising: (a) obtaining a diagnosis identifying a control sample, whereby the increased expression level of the human Subject diagnosed as having PD, wherein the diagno at least one gene expressed in the blood sample from the sis was obtained by: (i) obtaining a blood sample from a human Subject Suspected of having PD as compared to the human Subject Suspected of having PD; (ii) determining the non-PD sample is indicative of PD, thereby diagnosing the expression level of at least one gene in the blood sample from human Subject as having PD; and (b) administering to the the human subject suspected of having PD selected from: subject a PD treatment regimen. PTBP1, SLC4A1, DAZAP2, EPB42, HELZ, SELENBP1, 0035. In an embodiment, the methods disclosed herein NUDT4, CA1, AHSP and ALAS2; and (iii) comparing the typically involve determining expression levels of at least one expression level of the at least one gene expressed in the blood gene in a biological sample obtained from a human Subject sample to the expression level of the at least one gene suspected of having PD. The methods may involve determin expressed in a non-PD, healthy control sample, whereby the ing expression levels of at least 2, at least 3, at least 4, at least decreased expression level of the at least one gene expressed 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least in the blood sample from the human subject suspected of 20, at least 30, at least 40, at least 50, at least 60, at least 70, having PD as compared to the non-PD sample is indicative of at least 80, at least 100, at least 200 or more genes in a PD, thereby diagnosing the human Subject as having PD; and biological sample obtained from an individual, wherein the (b) administering to the Subject a PD treatment regimen. genes are selected from: HNF4A, THY1, SPEF1, SF3A2, 0041. In an embodiment, the methods disclosed herein SEMA6B, EN2, RTN3, BCAM, SPATA2L and TPSG1; or typically involve determining expression levels of at least one the genes listed in Table 2 or Table 3. gene in a biological sample obtained from a human Subject 0036. In one embodiment, the at least one gene is HNF4A suspected of having PD. The methods may involve determin hepatocyte nuclear factor 4, alpha (official symbol: HNF4A: ing expression levels of at least 2, at least 3, at least 4, at least and official full name: hepatocyte nuclear factor 4, alpha; 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least provided by HGNC). HNF4A is also known as: TCF; HNF4; 20, at least 30, at least 40, at least 50, at least 60, at least 70, MODY FRTS4; MODY1; NR2A1; TCF 14; HNF4a7; at least 80, at least 100, at least 200 or more genes in a HNF4a8; HNF4a9; NR2A21; HNF4alpha. Nucleic acid biological sample obtained from an individual, wherein the accession number:Dgi385298689|refNM 000457.4| Homo genes are selected from: PTBP1, SLC4A1, DAZAP2, sapiens hepatocyte nuclear factor 4, alpha (HNF4A), tran EPB42, HELZ, SELENBP1, NUDT4, CA1, AHSP and script variant 2, mRNA (SEQID NO: 07). Protein accession ALAS2; or the genes listed in Table 2 or Table 3. number: >gi31077205 reflNP 000448.3| Homo sapiens 0042. In one embodiment, the at least one gene is PTBP1 hepatocyte nuclear factor 4-alpha isoform HNF4alpha2 (SEQ polypyrimidine tract binding protein 1 (official symbol: ID NO: 08). PTBP1; official full name: polypyrimidine tract binding pro 0037. In another embodiment, the expression level is tein 1; provided by HGNC). PTBP1 is also known as: PTB; determined by detecting messenger RNA of the at least one PTB2; PTB3; PTB4; pPTB; HNRPI; PTB-1; PTB-T: gene. If mRNA is determined, then the method may further HNRNPI; HNRNP-I). Nucleic acid accession number: comprise reverse transcription of the messenger RNA prior to >gi209870087|refNM 002819.4| Homo sapiens polypyri detecting. In an embodiment, determining the expression midine tract binding protein 1 (PTBP1), transcript variant 1, level of the at least one gene is by measuring a level of mRNA (SEQ ID NO: 09). Protein accession number: US 2016/0244833 A1 Aug. 25, 2016

>gi4506243 refNP 0028.10.1| Homo sapiens polypyrimi 0048. As used herein, the terms “nucleic acid”, “poly dine tract-binding protein 1 isoform a (SEQID NO: 10). nucleotide”, “nucleotide', and "oligonucleotide' can be used 0043. In another embodiment, the expression level is interchangeably to refer to single stranded or double Stranded, determined by detecting messenger RNA of the at least one nucleic acid comprising DNA, RNA, derivatives thereof, or gene. If mRNA is determined, then the method may further combinations thereof. comprise reverse transcription of the messenger RNA prior to 0049 Expression of the genes disclosed herein can be detecting. In an embodiment, determining the expression measured at the RNA level using any method known in the art. level of the at least one gene is by measuring a level of For example, expression can be measured using Real-Time fluorescence by a sequence detection system following a PCR assays (RT-PCR), e.g., using primers specific for the quantitative, real-time polymerase chain reaction (PCR) differentially expressed sequences. As used herein, the term assay. “real-time PCR (also called quantitative real-time poly 0044. In yet another embodiment, an increased PTBP1 merase chain reaction) refers to a method for the detection expression level of the human Subject at a later time point and quantitation of an amplified PCR product based on incor compared to the PTBP1 expression level at an initial time poration of a fluorescent reporter dye; the fluorescent signal point indicates a worse prognosis of PD in the subject. Fur increases in direct proportion to the amount of PCR product thermore, a decreased PTBP1 expression level of the human produced and is monitored at each cycle, in real-time. Such subject after treatment compared to the PTBP1 expression that the time point at which the first significant increase in the amount of PCR product correlates with the initial amount of level prior to treatment or PTB1 expression levels similar to target template. In one embodiment, real-time PCR can be the healthy control sample indicates a better prognosis of PD preceded by reverse-transcription of the messenger RNA. in the subject. RNA can also be quantified using, for example, other target 0045. As used herein, a “sample' is a biological sample amplification methods (e.g., TMA, SDA, NASBA), or signal isolated from a subject and can include, but is not limited to, amplification methods (e.g., b)NA). Alternatively, northern whole blood, serum, plasma, blood cells, endothelial cells, hybridization analysis using probes which specifically recog tissue biopsies, lymphatic fluid, ascites fluid, interstitial fluid, nize one or more of the sequences or each gene can be used to bone marrow, cerebrospinal fluid (CSF), saliva, mucous, spu determine gene expression. tum, Sweat, urine, or any other secretion, excretion, or other 0050. The difference in the level of biomarker between bodily fluids. A “blood sample” refers to whole blood or any normal and abnormal is preferably statistically significant fraction thereof, including blood cells, serum and plasma. and may be an increase in biomarker expression level or a 0046. As used herein, the terms “diagnosis”, “diagnostic', decrease in biomarker expression level, and without any limi "diagnosing, refer to an identification of PD or to a predis tation of the method, achieving statistical significance, and position of developing PD, based on a detection of at least one thus the preferred analytical and clinical accuracy, generally gene. The terms “prognosis”, “prognostic”, “prognosing. but not always requires that combinations of several biomar refer to the ability of predicting, forecasting or correlating a kers be used together in panels and combined with math given detection or measurement with a future outcome of PD ematical algorithms in order to achieve a statistically signifi in the patient (e.g., severity, likelihood of Successfully treat cant biomarker index. ing, or Survival). The disclosure also relates to monitoring the 0051. As used herein, the term “primer set' or “primers' influence of agents, treatments or therapies for PD (e.g., refers to a pair of PCR primers that include a forward primer drugs, compounds). As used herein, "monitoring refers to and reverse primer used in a PCR reaction and allows the determining the regression, progression, course and/or onset generation of an amplicon. Numerous primers used in the of and/or prognoses of PD before any treatment or during context of the present disclosure can be readily determined by treatment in order to assess the PD patient’s improvement or a person of ordinary skill in the art to which the present lack thereof over time. invention pertains. Non-limiting examples of primers are 0047. As used herein, the term “control sample' or shown in SEQ ID NO: 3-6. A person skilled in the art can “healthy control” refers to a sample from a subject that does design numerous other primers based on the teachings herein not have PD or subject that does not have PD. In a particular and the common general knowledge. As used herein, the term embodiment, the control sample or healthy control does not “probes’ refers to a nucleic acid molecule which typically have PD or is indicative of the absence of PD. Control ranges in size from about 8 nucleotides to several hundred samples can be obtained from patients/individuals not nucleotides in length. Such a molecule is typically used to afflicted with PD. Other types of control samples may also be identify a target nucleic acid sequence in a sample by hybrid used. In a related facet, a control reaction may be designed to izing to such target nucleic acid sequence under stringent control the method itself (e.g., cell extraction, the capture, the hybridization conditions. Generally, an oligonucleotide use amplification reaction or detection method, number of cells ful as a probe or primer that selectively hybridizes to a present in the sample, a combination thereof or any step selected nucleotide sequence is at least about 15 nucleotides which could be monitored to positively validate that the in length, usually at least about 18 nucleotides, and particu absence of a signal (e.g., the expression level of a gene) is not larly about 21 nucleotides in length or more in length. Primer the result of a defect in one or more of the steps). Once a and probe design software programs are also commercially cut-off value is determined, a control sample giving a signal available, including without limitation, Primer Detective characteristic of the predetermined cut-off value can also be (ClonTech, Palo Alto, Calif.), Lasergene, (DNASTAR, Inc., designed and used in the methods of the present invention. Madison, Wis.); and Oligo software (National Biosciences, Diagnosis/prognosis tests are commonly characterized by the Inc., Plymouth, Minn.) and iOligo (Caesar Software, Ports following 4 performance indicators: sensitivity (Se), speci mouth, N.H.). ficity (Sp), positive predictive value (PPV), and negative pre 0052. As used herein, a “sequence detection system” is dictive value (NPV). any computational method in the art that can be used to US 2016/0244833 A1 Aug. 25, 2016

analyze the results of a PCR reaction. One example is the using the terms “Parkinson's disease' and “blood” or “tran Applied Biosystems HT7900 fast Real-Time PCR system. In scriptional profiling as of May 31, 2014. Microarray studies certain embodiments, gene expression can be analyzed using, using RNA prepared from human blood with 10 samples or e.g., direct DNA expression in microarray, Sanger sequenc more were included in the study. Only samples from PD ing analysis, Northern blot, the NanoString R technology, patients and healthy controls were analyzed. A total of four serial analysis of gene expression (SAGE), RNA-Seq tissue microarray studies met the inclusion criteria and were con microarray, or protein expression with immunohistochemis sidered for Subsequent analysis. The microarray studies ana try or western blot technique. lyzed in this study are listed in Table 1. GSE6613 included 50 0053. In an embodiment, the methods disclosed here fur predominantly early stage sporadic PD patients with a mean ther comprise determining a treatment regimen for the human Subject. The methods could be used to generate a prescription Hoehn and Yahr stage of 2.3 from which nine were untreated treatment to treat, delay development or prevent progression patients and 22 age and sex matched healthy controls. of PD in an individual identified by the methods disclose GSE8838 included 18 sporadic PD patients treated with dif herein as having PD. Furthermore, the method disclosed ferent PD medications and 12 HC. GSE22491 included 10 PD herein to adapt the correct or most appropriate treatment patients carrying a LRRK2 mutation from which one patient regimen and/or monitor the patient response to therapy. was untreated and seven HC. GSE54536 included 5 untreated 0054. In yet another aspect, the disclosure provides a Par PD patients and 5 HC. A microarray meta-analysis was con kinson's Disease (PD) diagnosis, prognosis or monitoring kit, ducted using the Integrative Meta-Analysis of Expression consisting of a set of probes suitable for the detection and Data (INMEX; Xia et al. (2013) INMEX-a web-based tool quantification of the nucleic acid expression of at least one for integrative meta-analysis of expression data. Nucleic gene selected from: HNF4A, THY1, SPEF1, SF3A2, Acids Res 41 (Web Server issue):W63-70) in accordance with SEMA6B, EN2, RTN3, BCAM, SPATA2L and TPSG1. In the PRISMA guidelines for meta-analysis (Moher et al., another aspect, the disclosure provides a Parkinson's Disease (2009) Preferred reporting items for systematic reviews and (PD) diagnosis, prognosis or monitoring kit, consisting of a meta-analyses: the PRISMA statement. PLoS Med 6(7): set of probes suitable for the detection and quantification of e1000097). All gene probes were converted to a common the nucleic acid expression of at least one gene: PTBP1, Entrez, ID using the gene/probe conversion tool in INMEX. SLC4A1, DAZAP2, EPB42, HELZ, SELENBP1, NUDT4, After matching all probes to a common EntreZID, all datasets CA1, AHSP and ALAS2. were preprocessed using the log-transformation and quantile 0055. In an embodiment, the present disclosure also normalization. Each individual dataset was visualized in box relates to kits containing nucleic acid primers and kits con plots to ensure identical distribution among the samples. Dif taining nucleic acid primers and nucleic acid probes to diag ferential expression analysis was performed with INMEX for nose and prognose PD in a sample of human thought to be each dataset independently using a false discovery rate (FDR) afflicted with PD or known to be afflicted with PD. Such kit of 0.05, a significance of p-0.05 and moderated t-test based generally comprises a first container means having at least on the Limma algorithm. In INMEX, the results from indi one oligonucleotide probe and/or primer that hybridizes to a vidual microarray dataset analyses are only for reference target nucleic acid (e.g., HNF4A or PTBP1 RNA) and a comparison and not required for meta-analysis in the Subse second container means containing at least one other oligo quent steps. After microarray preprocessing steps, a data nucleotide primer and/or probe that hybridizes to the above integrity check was performed before proceeding to the meta mentioned nucleic acid specific sequences. The kit may fur analysis. For meta-analysis, the Fisher's method was used ther include other containers comprising additional with a significance level of p-0.05 to combine p-values from components such as an additional oligonucleotide or primer the multiple datasets. Fisher's method is a widely used sta and/or one or more of the following: buffers, reagents to be tistical approach in meta-analysis to combine p-values from used in the assay (e.g., wash reagents, polymerases, internal different studies independently of the sample size (Xia et al. controls or else) and reagents capable of detecting the pres (2013) INMEX—a web-based tool for integrative meta ence of bound nucleic acid probe(s)/primer(s). Numerous analysis of expression data. Nucleic Acids Res 41 (Web Server embodiments of the kits of the present invention are possible. issue):W63-70; Tsenget al., (2012) Comprehensive literature For example, the different container means can be divided in review and statistical considerations for microarray meta amplifying reagents and detection reagents. In addition, the analysis. Nucleic Acids Res 40(9):3785-3799). Gene ontol kits may further include instructions for practicing the diag ogy and functional analysis was performed using Networ nostic and/or prognostic methods of the present disclosure. kAnalyst (Xia et al., (2014) NetworkAnalyst integrative Such instructions can concern details relating to the experi approaches for protein-protein interaction network analysis mental protocol as well as to the cut-off values for the PD and visual exploration. Nucleic Acids Res 42(Web Server specific biomarker ratio that may be used. issue):W167-74. Epub May 26, 2014). EXAMPLES TABLE 1

Methods Microarray studies in blood used for meta-analysis. GEO Microarray Meta-Analysis accession No. of number samples Description Platform Ref. 0056 Gene expression data from microarrays studies was GSE6613 PD = 50: Early stage sporadic Affymetrix 1 downloaded from the Gene Expression Omnibus (GEO) and HC = 22 PD patients (mean Human Genome GEMMA database (Zoubarev et al. (2012) Gemma: a Hoehn and Yahr = U133A resource for the reuse, sharing and meta-analysis of expres 2.3) sion profiling data. Bioinformatics 28(17):2272-2273) by US 2016/0244833 A1 Aug. 25, 2016

TABLE 1-continued healthy age-matched controls (26 men, 20 women; mean age at enrollment 64.28+10.42) enrolled in the Harvard Biomar Microarray studies in blood used for meta-analysis. ker Study (HBS). Three years follow-up samples from cases GEO and controls enrolled in the HBS were collected and analyzed accession No. of in this study. Diagnosis of cases and controls was assessed at number samples Description Platform Ref. each visit to ensure high diagnostic accuracy. Additional GSE18838 PD = 18; Sporadic PD patients Affymetrix Exon 2 information about the participants enrolled in the HBS clini HC = 12 (treated) Arrays cal trial has been published previously Ding et al. (2011) GSE22491 PD = 10: (G2019S) LRRK2 Agilent Whole 3 Association of SNCA with Parkinson: replication in the Har HC = 7 mutation carriers Human Genome vard NeuroDiscovery Center Biomarker Study. Mov Disord with PD GSE54536 PD = 5; Untreated sporadic PD Ilumina HT-12 4 26(12):2283-2286. HC = S Patients (mean Hoehn V4 and Yahir stage = 1) Quantitative Real Time Polymerase Chain Reactions 0059 Blood was collected and prepared as described 1 - Scherzer et al. (2007) Molecular markers of early Parkinson's disease based on gene expression in blood. Proc Nati AcadSci U.S.A. 104(3): 955-960. using the PAXgene Blood RNA system (Qiagen, Valencia, 2 - Shehadeh et al. (2010) SRRM2, a potential blood biomarker revealing high alternative splicing in Parkinson's disease. PLoS One 5(2): e9104. Calif., USA) (Scherzer et al. (2007) Molecular markers of 3 - Mutez et al. (2011) Transcriptional profile of Parkinson blood mononuclear cells with early Parkinson's disease based on gene expression in blood. LRRK2 mutation. Neurobioi Aging 32(10): 1839-1848. 4- Alieva et al. (2014) Involvement of endocytosis and alternative splicing in the formation Proc Natl AcadSci U.S.A. 104(3):955-960; and Potashkinet of the pathological process in the early stages of Parkinson's disease. Biomed Resini 2014: al., (2012) Biosignatures for Parkinson's disease and atypical 718732. parkinsonian disorders patients. PLoS One 7(8):e43595). Samples with RNA integrity values >7.0 and ratio of absor Network-Based Meta-Analysis bances at 260/280 nm between 1.7 and 2.4 were used in the 0057 Network-based meta-analysis was performed using current study. A total of 1 lug of RNA was reversed transcribed NetworkAnalyst (Xia et al., (2014) NetworkAnalyst inte using the High Capacity RNA transcription kit (Life Tech grative approaches for protein-protein interaction network nologies, Carlsbad, Calif., USA). Primers were designed analysis and visual exploration. Nucleic Acids Res 42(Web using Primer Express software and ordered (Life Technolo Server issue):W167-74. Epub May 26, 2014). Microarray gies, Carlsbad, Calif., USA). The primer sequences used in datasets were processed as described above. Briefly, microar this study are as follows: ray datasets were preprocessed by a log transformation fol lowed by quantile normalization. Duplicate genes were GAPDH forward: replaced by their mean value. A significance value of p-0.05 (SEQ ID NO: O1) and a log fold change of 1 were used as a cut-off value. 5'- CAACGGATTTGGTCGTATTGG-3 '; Network construction was restricted to contain only the origi GAPDH reverse: nal seed proteins. (SEQ ID NO: O2) Information about Study Participants 5'-TGATGGCAACAATATCCACTTTACC-3' 0058. The Institutional Review Boards of Rosalind Fran HNF4A forward: klin University of Medicine and Science approved the study (SEQ ID NO: O3) protocol. Written informed consent was received from all 5' - CAGAATGAGCGGGACCGGATC-3"; participants. Clinical characteristics of the participants used HNF4A reverse: in this study have been reported elsewhere in (Potashkin et al., (SEQ ID NO: O4) (2012) Biosignatures for Parkinson's disease and atypical 5'- CAGCAGCTGCTCCTTCATGGAC-3', parkinsonian disorders patients. PLoS One 7(8):e43595; San tiago et al., (2013) Specific splice variants are associated with PTBP1 forward: Parkinson's disease. Mov Disord 28(12): 1724-1727; and (SEO ID NO: O5) Santiago & Potashkin (2013) Integrative network analysis 5'- GCTCAGGATCATCGTGGAGAA-3"; unveils convergent molecular pathways in Parkinson's dis PTBP1 rewerse: ease and diabetes. PLoS One 8(12):e83940. Briefly, 51 PD (SEQ ID NO: O6) patients (29 men, 22 women; mean age at enrollment s' - ATCTTCAACACTGTGCCGAACTT-3 '' . 63.16+6.42; Hoehn and Yahr scale 2+0.28) and 45 healthy Quantitative PCR assays were carried using 25 ul reactions age-matched controls (24 male, 21 women; mean age at containing Power SYBR Green master mix (Life Technolo enrollment 65.12+8.60) enrolled in the Diagnostic and Prog gies, Carlsbad, Calif., USA), primer at a concentration of 5 nostic Biomarkers for Parkinson's Disease (PROBE) uM and nuclease free water. PCR reactions were amplified (#NCT00653783). Clinical diagnosis of PD was based on the using a DNA engine Opticon 2 Analyzer (Bio-Rad Life Sci United Kingdom Parkinson's Disease Society Brain Bank ences, Hercules, Calif., USA). Amplification conditions and criteria. Healthy individuals had no history of neurological detailed description of qPCR experiments are reported else disease and a Mini-Mental State Examination (MMSE) test where in Potashkin et al., (2012) Biosignatures for Parkin score higher than 27. Inclusion and exclusion criteria for son's disease and atypical parkinsonian disorders patients. patients enrolled in the PROBE study have been reported in PLoS One 7(8):e43595; and Santiago et al., (2013) Specific Potashkinet al., (2012) Biosignatures for Parkinson's disease splice variants are associated with Parkinson's disease. Mov and atypical parkinsonian disorders patients. PLoS One 7(8): Disord 28(12): 1724-1727). e43595. As an independent replication cohort of patients 96 individuals were used, including 50 PD patients (31 men, 19 Statistical Analysis women; Hoehn and Yahr scale 1.97+0.62; mean age at enroll 0060 Network-based microarray meta-analysis was per ment 63.12+8.96: mean age at onset 58.75+10.17) and 46 formed using INMEX and NetworkAnalyst. A student t-test US 2016/0244833 A1 Aug. 25, 2016

(two-tailed) was used to estimate the significance between TABLE 2-continued PD cases and controls for numerical variables. Post-hoc pair wise comparisons were performed using a Tukey test. Pear Top 10 upregulated and downregulated genes son correlation analysis was used to determine statistical in PD identified by meta-analysis. significance for HNF4A and PTBP1 adjusting for sex, age, Combined Combined Hoehn and Yahr scale in both cohorts and BMI in the HBS Entrez ID Gene Symbol Tstat P-value study. A ROC curve analysis was used to evaluate the diag 124044 SPATA2L 59.655 4S1E-07 nostic accuracy. A step-wise linear discriminant analysis was 25823 TPSG1 59.018 S.41E-07 performed to determine the sensitivity and specificity values Downregulated genes for the linear combination of both biomarkers. Power analy ses of completed experiments were performed to demonstrate 5725 PTBP1 -S8.731 S.6OE-07 6521 SLC4A1 -S8.117 6.77E-07 that the sample size used in this study allowed the detection of 98O2 DAZAP2 -56.939 9.2OE-07 a difference of 0.5 in fold change with a power of 99% and a 2O38 EPB42 -55.377 1.39E-06 significance of 0.05. A p-value less than 0.05 was regarded 9931 HELZ. -54.311 183E-O6 statistically significant. For the longitudinal analysis we used 8991 SELENEBP1 -54.268 183E-O6 a linear mixed effects regression model including Subjects as 11163 NUDT4 -53.277 2.54E-O6 random effects and adjusting for sex, age and BMI. Longitu 759 CA1 -52.77 2.97E-06 dinal data was analyzed using SuperMix (Scientific Software 51327 AHSP -52.673 3.OOE-06 International Inc., IL, USA) and Statistica 12 (StatSoft Inc., 212 ALAS2 -52.324 3.4OE-06 OK, USA). All other statistical analyses were undertaken using Prism4.0 (GraphPad, CA, USA). TABLE 3 Example 1 Complete list of differentially expressed genes. Meta-Analysis of Blood Microarrays in PD Entrez Gene symbol Combined Combined ID (Name) Tstat Pval 0061. In order to identify a common transcriptional signa ture in blood of PD patients, four microarray studies (Table 1) Upregulated genes were analyzed using INMEX, a web interface for the integra 7070 THY1 74.145 5.46E-09 tive meta-analysis. The overall meta-analysis workflow used 3172 HNF4A 66.421 8.49E-08 in this study is shown in FIG. 1A. Meta-analysis using a 25876 SPEF1 65.538 8.49E-08 8175 SF3A2 65.127 8.49E-08 Fisher's test identified a total of 2,781 genes differentially 1OSO1 SEMA6B 61.057 4.23E-07 expressed consistently across four microarray studies. 2020 EN2 60.476 4.23E-07 Among this group, 680 genes were upregulated and 2,101 10313 RTN3 60.224 4.23E-07 were downregulated in PD compared to healthy controls. The 4059 BCAM 60.06 4.23E-07 124044 SPATA2L 59.655 4S1E-07 thy-1 cell surface antigen (THY 1) and HNF4A were the most 2S823 TPSG1 59.018 S.41E-07 significant upregulated genes across the four microarray 1584 CYP11B1 57.882 6.95E-07 datasets. The top ten upregulated and downregulated genes 1152 CKB 57.629 7.23E-07 are listed in Table 2. The complete list of differentially 23S46 SYNGR4 56.785 9.24E-O7 8911 CACNA1I 56.141 1.15E-06 expressed genes is provided in Table 3. There were 921 777 CACNA1E 56.031 1.15E-06 gained genes uniquely identified in the meta-analysis that 4209 MEF2D 55.754 1.24E-O6 show relatively weak, but consistent expression across the 66O1 SMARCC2 54,901 1.59E-06 S430 POLR2A 54.861 1.59E-06 four datasets. A total of 491 genes were classified as lost genes 5586 PKN2 S4471 181E-O6 (i.e., genes identified as differentially expressed genes in 2272 FHIT 53.787 2.18E-O6 individual datasets but not in the meta-analysis). Venn dia 23O38 WDTC1 53.556 2.32E-O6 gram of meta-analysis results is shown in FIG. 1B and heat 168544 ZNF467 53.135 2.61E-O6 64O67 NPAS3 52.247 3.41E-O6 map visualization of the top 50 genes across the different 234.92 CBXT S1876 3.63E-O6 studies is displayed in FIG. 1C. 60SO6 NYX 51.846 3.63E-O6 115703 ARHGAP33 SO.931 S.09E-06 TABLE 2 4654 MYOD1 SO.909 S.09E-06 64,405 CDH22 50.434 6.13E-06 Top 10 upregulated and downregulated genes 81693 AMN 49.975 7.16E-O6 in PD identified by meta-analysis. 3S4 KLK3 49.809 7.37E-06 1SOO94 SIK1 49.518 8.2OE-06 Combined Combined 7356 SCGB1A1 49.43 8.34E-06 Entrez ID Gene Symbol Tstat P-value S1286 CEND1 48.614 1.09E-OS 63940 GPSM3 48.45 1.14E-OS Upregulated genes 6369 CCL24 48.292 1.2OE-OS 387509 GPR153 48.183 1.24E-OS 7070 THY1 74.145 5.46E-09 9653 HS2ST1 47.727 1.46E-OS 3172 HNF4A 66.421 8.49E-08 S4345 SOX18 47.623 1.47E-OS 25876 SPEF1 65.538 8.49E-08 SO24 P2RX3 47.149 1.73E-OS 8175 SF3A2 65.127 8.49E-08 4O1 PHOX2A 46.83 1.96E-OS 1OSO1 SEMA6B 61.057 4.23E-07 1628 DBP 46.744 2.OOE-OS 2020 EN2 60.476 4.23E-07 2128 EVX1 46.S6S 2.07E-OS 103.13 RTN3 60.224 4.23E-07 64321 SOX17 46.262 2.23E-OS 4059 BCAM 60.06 4.23E-07 80O23 NRSN2 46.259 2.23E-OS

US 2016/0244833 A1 Aug. 25, 2016 28

TABLE 3-continued clinical trials, PROBE and HBS. Quantitative PCR assays revealed that HNF4A and PTBP1 mRNAs were significantly Complete list of differentially expressed genes. up-and downregulated, respectively, in blood of PD patients Entrez Gene symbol Combined Combined compared to HC in both cohorts of study participants at ID (Name) Tstat Pval baseline (FIG. 3A-D). Analysis of receiver operating charac teristic (ROC) was performed to evaluate the diagnostic accu 55862 ECHDC1 -18.376 O.049843 S4665 RSBN1 -18.373 O.O49885 racy of both biomarkers. ROC analysis for HNF4A and 80778 ZNF34 -18.369 O.O4993S PTBP1 resulted in an area under the curve (AUC) of 0.72 and 0.82, respectively (FIG. 8A-B). Combination of both biom arkers resulted in an AUC value of 0.90 (FIG. 8C). A step wise linear discriminant analysis showed that PD patients can Biological and Functional Analysis be classified with 90% sensitivity and 80% specificity accord 0062. In order to identify the overrepresented biological ing to the following canonical function: D-0.10+0. processes dysregulated in blood of PD patients, we performed 56*X-0.20XM, where D, is the discriminant a gene pathway analysis using NetworkAnalyst. Pathway score value and X is the mRNA expression level for each analysis was performed using the set of up and downregulated biomarker multiplied by its respective canonical coefficient. genes separately. Upregulated genes in blood of PD were 0067 Pearson correlation analysis demonstrated that rela associated with the Kyoto Encyclopedia of Genes (KEGG) tive abundance of HNF4A and PTBP1 was independent of pathways (p<0.05) including bacterial invasion of epithelial other covariates including age (HNF4A: r=-0.25, p=0.9; cells, mitogen-activated protein kinase (MAPK) signaling PTBP1: r=0.09, p=0.59) and sex (HNF4A: r=-0.004, p=0.97; pathway, fructose and mannose metabolism, T-cell receptor PTBP1: r=0.05, p=0.76) in both cohorts of patients and body signaling pathway, mammalian target of rapamycin (mTOR) mass index (BMI) (HNF4A: r=-0.005, p=0.96; PTBP1: signaling pathway, type 2 diabetes mellitus and colorectal r–014, p=0.37) in the HBS cohort. Correlation analysis cancer. The most important hub gene in terms of network revealed a significant negative correlation between HNF4A topology measures, betweeness (BC) and degree of centrality mRNA expression and PTBP1 mRNA (r=-0.20, p=0.008, (DC), was HNF4A (BC–2213; DC-84) (FIG. 2A). FIG. 4A) and Hoehn and Yahr stage (HY) at baseline (r--0. 0063. In parallel, downregulated genes in blood of PD 32, p=0.002, FIG. 4B). patients were associated with the KEGG pathways including protein processing in the endoplasmic reticulum (ER). Longitudinal Performance of HNF4A and PTBP1 Epstein-Barr virus infection, and several types of cancer including prostate, endometrial and lung cancer. The most 0068. In order to determine the longitudinal performance prominent hub gene in terms of network topology measures of HNF4A and PTBP1, the relative abundance of each biom was ubiquitin C (UBC) (BC=495; DC=1630) and PTBP1 was arker in HBS samples collected was measured at two time the most downregulated gene across the four microarray points. The estimated rate of change for each biomarker was determined via a linear mixed-effects model using the two datasets (FIG. 2B, Table 2). time points (baseline and 3 years follow-up) collected repeat Network-Based Meta-Analysis edly between the same subjects adjusting for age, sex, and BMI. Relative abundance of HNF4A mRNA significantly 0064. HNF4A was confirmed as potential key hub gene in decreased over time in PD patients compared to HC (B=-0. blood of PD by network-based meta-analysis implemented in 93, p=0.002) whereas PTBP1 mRNA increased in PD NetworkAnalyst. The most highly ranked node across the patients (B=0.33, p=0.004). Relative abundance of HNF4A four datasets based on network topology measures was and PTBP1 mRNAs was significantly upregulated in PD HNF4A (BC-329; DC–35) followed by GATA1 (BC=10.5; patients compared to HC in the follow up period (FIGS. 5B DC-8). The resulting Zero-order interaction network con and D). Correlation between the relative abundance of each tained 76 nodes and 81 edges (FIG. 6). In addition, network biomarker and HY stage did not reach statistical significance based meta-analysis identified the aberrant expression of sev in the longitudinal analysis. eral splicing factors in PD patients (FIGS. 7A and 7B). Among the splicing factors, PTBP1 was the most signifi Discussion cantly downregulated gene in PD patients identified in the meta-analysis (Table 2, FIG. 2B, FIG. 7B). 0069 Biomarker discovery and validation is a crucial step 0065. In order to confirm the dysregulation of HNF4A and towards the improvement of clinical management of PD. PTBP1 at the protein level, a protein microarray study was Specifically, biomarkers that are useful to track the clinical analyzed in human serum samples of PD (GSE29654) using course of PD are essential to the development of effective NetworkAnalyst. PTBP1 was significantly downregulated in therapeutics. Network analysis offers an unbiased approach PD samples compared to healthy controls (p=0.002). Altered to identify and prioritize biologically meaningful biomarkers expression of HNF4A was not confirmed in this protein for several neurodegenerative diseases. Here, a network microarray. based meta-analysis was performed integrating gene expres Evaluation of HNF4A and PTBP1 mRNAs in Blood of PD sion profiles of untreated, sporadic and PD patients harboring 0.066. In order to validate the results obtained from the a LRRK2 (G2019S) mutation in order to identify conver network-based meta-analysis, the most significant hub gene gence among the different studies in blood of PD. Transcrip in the upregulated network, HNF4A, and the most downregu tomic meta-analysis identified 2.781 genes consistently dif lated gene, PTBP1, were analyzed as potential biomarkers for ferentially expressed in blood of PD across four microarray PD. Relative abundance of HNF4A and PTBP1 mRNAS was studies. measured in whole blood of PD patients compared to healthy (0070 Network-based meta-analysis identified HNF4A as controls (HC) from samples obtained from two independent the most significant hub gene across the four microarrray US 2016/0244833 A1 Aug. 25, 2016 29 datasets, and PTBP1 was identified as the most significant of Parkinson's disease based on disease-specific autoanti downregulated gene across the four microarrays datasets. body profiles in human sera. PLoS One 7(2):e32383) revealed HNF4A and PTBP1 mRNAs were further evaluated as blood that PTBP1 was significantly downregulated in PD patients biomarkers for PD. Relative abundance of HNF4A mRNA compared to controls (p=0.02), but expression of HNF4A was was upregulated whereas PTBP1 mRNA was downregulated not identified. Thus, protein levels of PTBP1 may be also a in blood of PD patients compared to healthy individuals in potential diagnostic biomarker for PD. samples obtained from two independent clinical trials (FIG. 3A-D). Evaluation of biomarker performance showed that 0075. The network of downregulated genes was centered HNF4A and PTBP1 can distinguish PD patients from HC in the polyubiquitin precursor UBC and associated with pro with a 90% sensitivity and 80% specificity (FIG. 8C). The tein processing in the ER (FIG. 2B). In this regard, growing diagnostic performance of these two biomarkers is Superior evidence indicates that ER dysfunction is an early event in the than the one afforded by previously identified risk markers in PD pathogenesis and targeting components of the unfolded blood of PD and current clinical assessment (Santiago et al., protein response may be a potential therapeutic strategy. (2013) Specific splice variants are associated with Parkin sons disease. Mov Disord 28(12): 1724-1727; and Moloch 0076. The results from this meta-analysis also highlight nikov et al. (2012) A molecular signature in blood identifies the dysregulation of several splicing factors in blood of PD early Parkinson's disease. Mol Neurodegener 7:26). The sen patients. As the spliceosome assembles, protein-protein inter sitivity of the two markers alone is also greater than the splice actions are highly dynamic. One of the essential steps in the variant-specific RNA blood biosignature that included 13 risk assembly of the spliceosome is the formation of new protein markers (Potashkin et al., (2012) Biosignatures for Parkin interactions that change the inactive B splicing complex to an son's disease and atypical parkinsonian disorders patients. active complex in which SF3B2, SF3B3 and SF3B5 form new PLoS One 7(8):e43595). interactions with proteins of the U5 small nuclear ribonucleic (0071. HNF4A mRNA relative abundance significantly particles (snRNP). In this context, several of the core factors correlated with PTBP1 mRNA. A significant negative corre of the U2 snRNP were upregulated in PD including SF3A1, lation was found between HNF4A mRNA expression and the SF3A2, SF3B1 and SF3B4, whereas SF3B3 was downregu HY staging. Early PD patients with a low HY scale rating lated (FIG. 2 and FIG. 7). These results suggest that assembly (HY=1) showed a significantly higher upregulation of of the U2 snRNP that binds to the 3' splice site may be HNF4A mRNA compared to patients with a higher HY scale facilitated in PD, but the efficient formation of an active (HY-3) (FIG.4B). This finding suggests that HNF4A mRNA splicing complex in PD is highly unlikely. The results from may be useful to identify patients at very early stages of PD the meta-analysis also revealed that many of the regulatory when therapeutic intervention may be most beneficial and to splicing factors, core factors of the U1, U4, U5 and U6 monitor disease severity. snRNPs, and helicases are downregulated in PD further sup 0072 Longitudinal performance analysis showed that porting the idea that splicing may be both inefficient and relative abundance of each biomarker significantly changed dysregulated in PD (FIG. 7B). In this regard, aberrant alter over time in PD patients. For instance. HNF4A mRNA sig native splicing in blood of PD has been highlighted in numer nificantly decreased whereas PTBP1 mRNA increased in PD ous studies (Potashkin et al., (2012) Biosignatures for Parkin patients during 3 years follow up (FIG. 5). The correlation son's disease and atypical parkinsonian disorders patients. between the relative abundance of both biomarkers with HY PLoS One 7(8):e43595; Santiago et al., (2013) Specific splice stage did not reach statistical significance in follow-up variants are associated with Parkinson's disease. Mov Disord samples, however. One possible explanation is that the HY 28(12): 1724-1727; Soreq et al., (2012) Exon arrays reveal stage did not change in most of the PD patients during the 3 alternative splicing aberrations in Parkinson's disease leuko years period, whereas the relative abundance of the genes did cytes. Neurodegener Dis 10(1-4):203-206: Soreqetal. (2013) change during this time period. These results suggest that the Small RNA sequencing-microarray analyses in Parkinson abundance of HNF4A and PTBP1 mRNAs in blood may be leukocytes reveal deep brain stimulation-induced splicing more sensitive than assessment of motor symptoms for moni changes that classify brain region transcriptomes. Front Mol toring disease progression. The dynamic change in expres Neurosci 6:10; and Soreqetal. (2014) Long non-coding RNA sion over time of both biomarkers suggests that they may be and alternative splicing modulations in Parkinson's leuko useful biomarkers to track the clinical course of PD patients. cytes identified by RNA sequencing. PLOS Comput Biol 0073. One potential caveat is that most of the PD patients 10(3):e 1003517). In addition, heterogeneous nuclear ribo were medicated in this study, therefore, a potential confound nucleoproteins (hnRNPs), cap-binding proteins and proteins ing factor introduced by PD medications cannot be ruled out. of the exon junction complex were downregulated in PD Nevertheless, this finding is interesting in light of the evi Suggesting that other post-transcriptional events such as cap dence that indicates that more than 50% of the PD patients are binding protein complex formation, localization, maturation, glucose intolerant and patients with diabetes that develop PD nonsense-mediated mRNA decay and translation may be usually have a higher HY staging. Moreover, impaired glu inefficient or dysregulated in PD (FIG. 2 and FIG. 7). cose metabolism is Suggested to be an early event in sporadic (0077. The vitamin D receptor (VDR) was also present in PD. Given that HNF4A plays a pivotal role in hepatic gluco the network of downregulated genes thus confirming previ neogenesis and PTBP1 regulates and stabilize mRNA trans ous findings reporting lower levels of VDR in blood and lation of insulin in the pancreas, the inverse regulation of both plasma of PD patients. In addition, a subset of highly co genes provide a molecular rationale for the impairment of expressed genes associated with heme metabolism previ insulin signaling in PD patients and thus may be potential ously identified in blood of two independent populations therapeutic targets. ALAS2, FECH, and BLVRB were also found to be down 0074 Analysis of a previous protein microarray study in regulated in the meta-analysis (FIG. 2B). Further, other PD human serum samples with PD (Han et al., (2012) Diagnosis blood biomarkers HSPA8 and SKP1 were identified in the US 2016/0244833 A1 Aug. 25, 2016 30 meta-analysis. Collectively, these results confirm the pres molecular mechanisms underlying the impairment of insulin ence of a common molecular signature in human blood of PD signaling observed in PD patients. Further, this study under patients. scores the potential of network analysis as a powerful frame 0078. In summary, this study highlights the prominent work to gain insight into the mechanisms underlying PD and convergence among blood microarray studies from sporadic, to identify potential therapeutic targets and biomarkers of de novo and the most common hereditary cause of PD and disease severity. Evaluation of HNF4A and PTBP1 mRNAs confirms the utility of blood as a useful source of biomarkers in a larger prospective study including patients at risk will be for PD. In addition, these results strengthen the association important to assess its clinical utility as a diagnostic tool for between PD and diabetes and provide insights into the PD.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 10

<21 Os SEQ ID NO 1 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide

<4 OOs SEQUENCE: 1

caacggattt gg.tc.gtattgg 21

<21 Os SEQ ID NO 2 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide

<4 OOs SEQUENCE: 2

tgatggcaac aatat coact ttacc 25

<21 Os SEQ ID NO 3 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oliognucleotide

<4 OOs SEQUENCE: 3

Cagaatgagc gggaccggat c 21

<21 Os SEQ ID NO 4 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide

<4 OOs SEQUENCE: 4

cagcagotgc ticcitt catgg ac 22

<21 Os SEQ ID NO 5 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide

<4 OOs SEQUENCE: 5

gct caggat C atcgtggaga a 21

<21 Os SEQ ID NO 6

US 2016/0244833 A1 Aug. 25, 2016 33

- Continued c catggagtg ccgtgggcaa cagtggcc ct t catgggaac aatctgttgg agcagggggt 4 O8O cagttct citg ctgggaatct acccotttct ggaggagaaa cc cattccac cittaataact 414 O ttattgtaat gtgagaaaca caaaacaaag titt acttittt togactictaag ctgacatgat 42OO attagaaaat ct citcqct ct ctitttitttitt tttittttittt tttittggcta cittgagttgt 426 O ggtc.ctaaaa cataaaatct gatggacalaa Cagagggttg Ctggggggac aag.cgtgggc 432O acaatttic cc caccaagaca ccctgat citt cagg.cggg to t caggagctt ctaaaaatcc 438 O gcatggct ct cct gagagtg gacagaggag aggagagggit Cagaaatgaa cqct Cttcta 4 44 O tittcttgtca ttaccaagcc aattacttitt gccaaattitt totgttgat cit gcc ct gatta 4500 agatgaattig talaatttac atcaa.gcaat tat caaag.cg ggctgggtcc cat Cagaacg 456 O acccacatct ttctgtgggt gtgaatgtca ttaggtottg cqctgacccc tdagc.ccc.ca 462O t cactg.ccgc ctdatgggg.c aaagaaacaa aaaacatttic titact cittct gtgttittaac 468O aaaagtttat aaaacaaaat aaatgg.cgca tatgttittct aaaaaaaaaa aaaaaaa 4.737

<210s, SEQ ID NO 8 &211s LENGTH: 474 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 8 Met Arg Lieu. Ser Llys Thr Lieu Val Asp Met Asp Met Ala Asp Tyr Ser 1. 5 1O 15 Ala Ala Lieu. Asp Pro Ala Tyr Thir Thr Lieu. Glu Phe Glu Asn Val Glin 2O 25 3O Val Lieu. Thir Met Gly Asn Asp Thr Ser Pro Ser Glu Gly Thr Asn Lieu. 35 4 O 45 Asn Ala Pro Asn. Ser Lieu. Gly Val Ser Ala Lieu. Cys Ala Ile Cys Gly SO 55 6 O Asp Arg Ala Thr Gly Llys His Tyr Gly Ala Ser Ser Cys Asp Gly Cys 65 70 7s 8O Lys Gly Phe Phe Arg Arg Ser Val Arg Lys Asn His Met Tyr Ser Cys 85 90 95 Arg Phe Ser Arg Glin Cys Val Val Asp Lys Asp Lys Arg Asn Glin Cys 1OO 105 11 O Arg Tyr Cys Arg Lieu Lys Lys Cys Phe Arg Ala Gly Met Lys Lys Glu 115 12 O 125 Ala Val Glin Asn. Glu Arg Asp Arg Ile Ser Thr Arg Arg Ser Ser Tyr 13 O 135 14 O Glu Asp Ser Ser Lieu Pro Ser Ile Asn Ala Lieu. Lieu. Glin Ala Glu Val 145 150 155 160

Lieu. Ser Arg Glin Ile Thir Ser Pro Val Ser Gly Ile Asn Gly Asp Ile 1.65 17O 17s

Arg Ala Lys Lys Ile Ala Ser Ile Ala Asp Val Cys Glu Ser Met Lys 18O 185 19 O

Glu Gln Lieu. Lieu Val Lieu Val Glu Trp Ala Lys Tyr Ile Pro Ala Phe 195 2OO 2O5

Cys Glu Lieu Pro Lieu. Asp Asp Glin Val Ala Lieu. Lieu. Arg Ala His Ala 21 O 215 22O Gly Glu. His Lieu Lleu Lieu. Gly Ala Thir Lys Arg Ser Met Val Phe Lys 225 23 O 235 24 O US 2016/0244833 A1 Aug. 25, 2016 34

- Continued

Asp Val Lieu. Lieu. Lieu. Gly Asn Asp Tyr Ile Val Pro Arg His Cys Pro 245 250 255 Glu Lieu Ala Glu Met Ser Arg Val Ser Ile Arg Ile Lieu. Asp Glu Lieu. 26 O 265 27 O Val Lieu Pro Phe Glin Glu Lieu. Glin Ile Asp Asp Asn. Glu Tyr Ala Tyr 27s 28O 285 Lieu Lys Ala Ile Ile Phe Phe Asp Pro Asp Ala Lys Gly Lieu. Ser Asp 29 O 295 3 OO Pro Gly Lys Ile Lys Arg Lieu. Arg Ser Glin Val Glin Val Ser Lieu. Glu 3. OS 310 315 32O Asp Tyr Ile Asn Asp Arg Glin Tyr Asp Ser Arg Gly Arg Phe Gly Glu 3.25 330 335 Lieu. Leu Lleu Lleu Lleu Pro Thr Lieu. Glin Ser Ile Thr Trp Gln Met Ile 34 O 345 35. O Glu Glin Ile Glin Phe Ile Llys Lieu. Phe Gly Met Ala Lys Ile Asp Asn 355 360 365 Lieu. Lieu. Glin Glu Met Lieu. Lieu. Gly Gly Ser Pro Ser Asp Ala Pro His 37 O 375 38O Ala His His Pro Leu. His Pro His Leu Met Glin Glu. His Met Gly Thr 385 390 395 4 OO Asn Val Ile Val Ala Asn Thr Met Pro Thr His Leu Ser Asn Gly Glin 4 OS 41O 415 Met Cys Glu Trp Pro Arg Pro Arg Gly Glin Ala Ala Thr Pro Glu Thr 42O 425 43 O Pro Gln Pro Ser Pro Pro Gly Gly Ser Gly Ser Glu Pro Tyr Lys Lieu. 435 44 O 445 Lieu Pro Gly Ala Val Ala Thir Ile Val Llys Pro Lieu. Ser Ala Ile Pro 450 45.5 460 Gln Pro Thir Ile Thr Lys Glin Glu Val Ile 465 470

<210s, SEQ ID NO 9 &211s LENGTH: 334 O &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 9 tgcgggcgtc. tcc.gc.cattt ttgagticta taact cqgag ccgttgggt C ggttcCtgct 6 O attic.cggcgc ctic cact.ccg tcc.ccc.gcgg gtctgctctg tdtgc catgg acggcattgt 12 O

Cccagatata gcc.gttggta Caaag.cgggg atctgacgag Cttitt ct ct a cittgttgtcac 18O taacggaccg titt at catga gcagdaactic ggcttctgca gcaaacggala atgacagcaa. 24 O gaagttcaaa ggtgacagcc gaagtgcagg C9tcc cct ct agagtgatcc acatc.cggaa 3OO gctic cc catc gacgtcacgg agggggaagt catct Coctg gggctgc cct ttgggaaggit 360

Caccalacctic ctgatgctga aggggaaaaa C caggccttic atcgagatga acacggagga 42O ggctgccaac accatggtga act act acac CtcggtgacC cct gtgctgc gcggc.ca.gc.c 48O catctacatc cagttcticca accacaagga gctgaagacic gacagct ct c ccaac caggc 54 O gcgggcc.cag gcggcc ctgc aggcggtgaa citcggtc.ca.g. tcggggalacc tigcCttggc 6OO tgcCtcgg.cg gC9gcc.gtgg acgcagggat gcgatggcc gggcagagcc cc.gtgct Cag 660

US 2016/0244833 A1 Aug. 25, 2016 36

- Continued ctitt cogtaa aagcgtgtaa caagggtgta aatatttata atttitttata cctdttgttga 3 OOO gaccc.gaggg gcggcggcgc ggtttittt at ggtgacacaa atgtatattt totalacagc 3 O 6 O aatticcaggc ticagtattgt gaccgcggag ccacagggga CCC cacgcac attic.cgttgc 312 O Cttacccgat ggcttgttgac gcggagagaa ccgattaaaa ccgtttgaga aactic ct coc 318O ttgtctagoc Ctgttgttcgc tigtggacgct gtagaggcag gttggcc agt Ctgtacctgg 324 O actitcgaata aatcttctgt atcct cqctic cqttic cqcct taaaaaaaaa aaaaaaaaaa 33 OO aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa. 334 O

<210s, SEQ ID NO 10 211 LENGTH: 557 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 10 Met Asp Gly Ile Val Pro Asp Ile Ala Val Gly. Thir Lys Arg Gly Ser 1. 5 1O 15 Asp Glu Lieu Phe Ser Thr Cys Val Thr Asn Gly Pro Phe Ile Met Ser 2O 25 3O Ser Asn. Ser Ala Ser Ala Ala Asn Gly Asn Asp Ser Llys Llys Phe Lys 35 4 O 45 Gly Asp Ser Arg Ser Ala Gly Val Pro Ser Arg Val Ile His Ile Arg SO 55 6 O Llys Lieu Pro Ile Asp Val Thr Glu Gly Glu Val Ile Ser Lieu. Gly Lieu. 65 70 7s 8O Pro Phe Gly Llys Val Thr Asn Lieu. Lieu Met Lieu Lys Gly Lys Asn Glin 85 90 95

Ala Phe Ile Glu Met Asn. Thir Glu Glu Ala Ala Asn. Thir Met Wall Asn 1OO 105 11 O Tyr Tyr Thr Ser Val Thr Pro Val Lieu. Arg Gly Glin Pro Ile Tyr Ile 115 12 O 125 Glin Phe Ser Asn His Lys Glu Lieu Lys Thir Asp Ser Ser Pro Asn Glin 13 O 135 14 O Ala Arg Ala Glin Ala Ala Lieu. Glin Ala Val Asn. Ser Val Glin Ser Gly 145 150 155 160 Asn Lieu Ala Lieu Ala Ala Ser Ala Ala Ala Val Asp Ala Gly Met Ala 1.65 17O 17s Met Ala Gly Glin Ser Pro Val Lieu. Arg Ile Ile Val Glu Asn Lieu. Phe 18O 185 19 O Tyr Pro Val Thr Lieu. Asp Val Lieu. His Glin Ile Phe Ser Llys Phe Gly 195 2OO 2O5 Thr Val Lieu Lys Ile Ile Thr Phe Thr Lys Asn Asn Glin Phe Glin Ala 21 O 215 22O Lieu. Lieu. Glin Tyr Ala Asp Pro Val Ser Ala Gln His Ala Lys Lieu. Ser 225 23 O 235 24 O

Lieu. Asp Gly Glin Asn. Ile Tyr Asn Ala Cys Cys Thr Lieu. Arg Ile Asp 245 250 255

Phe Ser Lys Lieu. Thir Ser Lieu. Asn. Wall Lys Tyr Asn. Asn Asp Llys Ser 26 O 265 27 O

Arg Asp Tyr Thr Arg Pro Asp Lieu Pro Ser Gly Asp Ser Glin Pro Ser 27s 28O 285 US 2016/0244833 A1 Aug. 25, 2016 37

- Continued

Lieu. Asp Glin. Thir Met Ala Ala Ala Phe Gly Ala Pro Gly Ile Ile Ser 29 O 295 3 OO

Ala Ser Pro Tyr Ala Gly Ala Gly Phe Pro Pro Thir Phe Ala Ile Pro 3. OS 310 315

Glin Ala Ala Gly Luell Ser Wall Pro Asn Wall His Gly Ala Luell Ala Pro 3.25 330 335

Lell Ala Ile Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg Ile 34 O 345 35. O

Ala Ile Pro Gly Lell Ala Gly Ala Gly Asn. Ser Wall Lieu Luell Wall Ser 355 360 365

Asn Luell Asn. Pro Glu Arg Wall Thir Pro Glin Ser Leul Phe Ile Lieu. Phe 37 O 375

Gly Wall Tyr Gly Asp Wall Glin Arg Wall Ile Lell Phe Asn Lys 385 390 395 4 OO

Glu Asn Ala Luell Wall Glin Met Ala Asp Gly Asn Glin Ala Glin Lieu Ala 4 OS 415

Met Ser His Luell Asn Gly His Lieu. His Gly Pro Ile Arg Ile 425 43 O

Thir Luell Ser Lys His Glin Asn Wall Glin Leul Pro Arg Glu Gly Glin Glu 435 44 O 445

Asp Glin Gly Lieu. Thir Asp Gly Asn. Ser Pro Leu His Arg Phe 450 45.5 460

Lys Pro Gly Ser Lys Asn Phe Glin Asn Ile Phe Pro Pro Ser Ala 465 470 48O

Thir Lieu. His Lieu. Ser Asn Ile Pro Pro Ser Wal Ser Glu Glu Asp Lieu. 485 490 495

Wall Luell Phe Ser Ser Asn Gly Gly Val Val Gly Phe Phe SOO 505

Phe Glin Lys Asp Arg Met Ala Lieu. Ile Glin Met Gly Ser Wall Glu 515 525

Glu Ala Wall Glin Ala Lell Ile Asp Lieu. His Asn His Asp Luell Gly Glu 53 O 535 54 O

Asn His His Lieu. Arg Wall Ser Phe Ser Ser Thir Ile 5.45 550 555

1. A method for diagnosing, prognosing or monitoring 3. The method of claim 1, wherein the expression level is Parkinson's Disease (PD) in a human Subject, comprising: determined by detecting messenger RNA. (a) obtaining a blood sample from a human Subject Sus 4. The method of claim 1, further comprising reverse tran pected of having PD; Scription of the messenger RNA prior to detecting. 5. The method of claim 1, wherein determining the expres (b) determining the expression level of at least one gene in sion level of the at least one gene is by measuring a level of the blood sample from the human subject suspected of fluorescence by a sequence detection system following a having PD, wherein the at least one gene is selected quantitative, real-time polymerase chain reaction (PCR) from: HNF4A,THY1, SPEF1, SF3A2, SEMA6B, EN2, assay. RTN3, BCAM, SPATA2L and TPSG1; and 6. The method of claim 1, further comprising determining (c) comparing the expression level of the at least one gene a treatment regimen for the human Subject. expressed in the blood sample to the expression level of 7. The method of claim 2, wherein an increased HNF4A the at least one gene expressed in a non-PD, healthy expression level of the human Subject at a later time point control sample, whereby the increased expression level compared to the HNF4A expression level at an initial time of the at least one gene expressed in the blood sample point or expression levels the same as a healthy control indi from the human Subject Suspected of having PD as com cates an improved or steady prognosis of PD in the Subject. pared to the non-PD sample is indicative of PD, thereby 8. A method of treating a human subject for Parkinson's diagnosing the human Subject as having PD. Disease (PD), the method comprising: 2. The method of claim 1, wherein the at least one gene is (a) obtaining a diagnosis identifying a human Subject as HNF4A encoded by SEQID NO: 07. having PD, wherein the diagnosis was obtained by: US 2016/0244833 A1 Aug. 25, 2016 38

(i) obtaining a blood sample from a human Subject Sus 18. The method of claim 14, wherein determining the pected of having PD; expression level of the at least one gene is by measuring a (ii) determining the expression level of at least one gene level of fluorescence by a sequence detection system follow selected from: HNF4A, THY1, SPEF1, SF3A2, ing a quantitative, real-time polymerase chain reaction (PCR) SEMA6B, EN2, RTN3, BCAM, SPATA2L and assay. TPSG1; and 19. The method of claim 14, wherein an increased PTBP1 (iii) comparing the expression level of the at least one expression level of the human Subject at a later time point gene expressed in the blood sample to the expression compared to the PTBP1 expression level at an initial time level of the at least one gene expressed in a non-PD, point indicates a worse prognosis of PD in the Subject. healthy control sample, whereby the increased 20. The method of claim 14, wherein a decreased PTBP1 expression level of the at least one gene expressed in expression level of the human Subject after treatment com the blood sample from the human subject suspected of pared to the PTBP1 expression level prior to treatment or having PD as compared to the non-PD sample is PTBP1 expression levels similar to the healthy control indicative of PD, thereby diagnosing the human sub sample indicates a better prognosis of PD in the subject. ject as having PD; and 21. The method of claim 14, further comprising determin (b) administering to the Subject a PD treatment regimen. ing a treatment regimen for the human Subject. 9. The method of claim 8, wherein the at least one gene is 22. A method of treating a human subject for Parkinson's HNF4A encoded by SEQID NO: 07. Disease (PD), the method comprising: 10. The method of claim 8, wherein the expression level is (a) obtaining a diagnosis identifying a human Subject diag determined by detecting messenger RNA of the at least one nosed as having PD, wherein the diagnosis was obtained gene. by: 11. The method of claim 8, further comprising reverse transcription of the messenger RNA prior to detecting. (i) obtaining a blood sample from a human Subject Sus 12. The method of claim 8, wherein determining the pected of having PD; expression level of the at least one gene is by measuring a (ii) determining the expression level of at least one gene level of fluorescence by a sequence detection system follow in the blood sample from the human Subject Suspected ing a quantitative, real-time polymerase chain reaction (PCR) of having PD selected from: PTBP1, SLC4A1, assay. DAZAP2, EPB42, HELZ, SELENBP1, NUDT4, 13. A Parkinson's Disease (PD) diagnosis, prognosis or CA1, AHSP and ALAS2; and monitoring kit, consisting of a set of probes Suitable for the (iii) comparing the expression level of the at least one detection and quantification of the nucleic acid expression of gene expressed in the blood sample to the expression at least one gene selected from: HNF4A, THY1, SPEF1. level of the at least one gene expressed in a non-PD, SF3A2, SEMA6B, EN2, RTN3, BCAM, SPATA2L and healthy control sample, whereby the decreased TPSG1. expression level of the at least one gene expressed in 14. A method for diagnosing, prognosing or monitoring the blood sample from the human subject suspected of Parkinson's Disease (PD) in a human Subject, comprising: having PD as compared to the non-PD sample is (a) obtaining a blood sample from a human Subject Sus indicative of PD, thereby diagnosing the human sub pected of having PD; ject as having PD; and (b) determining the expression level of at least one gene in (b) administering to the Subject a PD treatment regimen. the blood sample from the human subject suspected of 23. The method of claim 22, wherein the at least one gene having PD, wherein the at least one gene is selected PTBP1 encoded by SEQID NO: 09. from: PTBP1, SLC4A1, DAZAP2, EPB42, HELZ, 24. The method of claim 22, wherein the expression level is SELENEP1, NUDT4, CA1, AHSP and ALAS2; and determined by detecting messenger RNA of the at least one (c) comparing the expression level of the at least one gene gene. expressed in the blood sample to the expression level of 25. The method of claim 22, further comprising reverse the at least one gene expressed in a non-PD, healthy transcription of the messenger RNA prior to detecting. control sample, whereby the decreased expression level of the at least one gene expressed in the blood sample 26. The method of claim 22, wherein determining the from the human Subject Suspected of having PD as com expression level of the at least one gene is by measuring a pared to the non-PD sample is indicative of PD, thereby level of fluorescence by a sequence detection system follow diagnosing the human Subject as having PD. ing a quantitative, real-time polymerase chain reaction (PCR) 15. The method of claim 14, wherein the at least one gene assay. is PTBP1 encoded by SEQID NO: 09. 27. A Parkinson's Disease (PD) diagnosis, prognosis or 16. The method of claim 14, wherein the expression level is monitoring kit, consisting of a set of probes Suitable for the determined by detecting messenger RNA of the at least one detection and quantification of the nucleic acid expression of gene. at least one gene: PTBP1, SLC4A1, DAZAP2, EPB42, 17. The method of claim 14, further comprising reverse HELZ, SELENEP1, NUDT4, CA1, AHSP and ALAS2. transcription of the messenger RNA prior to detecting. k k k k k