# Patent

## Title
Methods of predicting clinical course and treating multiple sclerosis

## Abstract

Provided are methods and kits for classifying a subject as being more likely to have benign multiple sclerosis (BMS) or as being more likely to have typical relapsing remitting multiple sclerosis (RRMS). Classification of multiple sclerosis disease course is performed by comparing a level of expression of at least one gene involved in the RNA polymerase I pathway in a cell of the subject to a reference expression data of said at least one gene obtained from a cell of at least one subject pre-diagnosed as having BMS and/or from a cell of at least one subject pre-diagnosed as having typical RRMS, thereby classifying the subject as being more likely to have BMS or as being more likely to have typical RRMS. Also provided are methods of diagnosing and treating multiple sclerosis and methods of monitoring treatment efficiency.

## Background
RELATED APPLICATIONS
This application is a division of U.S. patent application Ser. No. 13/260,573 filed on Sep. 27, 2011, which is a National Phase of PCT Patent Application No. PCT/IB2010/051344 having International filing date of Mar. 28, 2010, which claims the benefit of priority of U.S. Provisional Patent Application No. 61/202,703 filed on Mar. 30, 2009. The contents of the above applications are all incorporated herein by reference.


SEQUENCE LISTING STATEMENT
The ASCII file, entitled 62191SequenceListing.txt, created on Sep. 27, 2011, comprising 7,168,844 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to methods of classifying a subject as having benign multiple sclerosis or typical relapsing remitting multiple sclerosis and, more particularly, but not exclusively, to methods of treating multiple sclerosis based on same.
Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system (CNS) affecting young adults (disease onset between 20 to 40 years of age) and is the third leading cause for disability after trauma and rheumatic diseases, with an estimated annual cost 34,000 USD per patient (total life time cost of 2.2 million USD per patient).
The disease is characterized by destruction of myelin, associated with death of oligodendrocytes and axonal loss. The main pathologic finding in MS is the presence of infiltrating mononuclear cells, predominantly T lymphocytes and macrophages, which surpass the blood brain barrier and induce an active inflammation within the brain and spinal cord. The neurological symptoms that characterize MS include complete or partial vision loss, diplopia, sensory symptoms, motor weakness that can worsen to complete paralysis, bladder dysfunction and cognitive deficits, which eventually may lead to a significant disability. The associated multiple inflammatory foci lead to myelin destruction, plaques of demyelination, gliosis and axonal loss within the brain and spinal cord and are the reasons contribute to the clinical manifestations of neurological disability.
The etiology of MS is not fully understood. The disease develops in genetically predisposed subjects exposed to yet undefined environmental factors and the pathogenesis involves autoimmune mechanisms associated with autoreactive T cells against myelin antigens. It is well established that not one dominant gene determines genetic susceptibility to develop MS, but rather many genes, each with different influence, are involved.
Clinically, in 85% of MS patients the illness is initiated with a relapsing-remitting course (RRMS), and in about 10-15% of MS patients have an a-priori primary progressive course (PPMS) without relapses. RRMS is characterized by inflammatory attacks associated with neurological deficits with periods of remissions between the relapses that vary in time. After a period of 10 years, about 50% of RRMS patients will progress to a secondary progressive MS (SPMS) course, characterized by permanent neurological dysfunction, with or without relapses and progressive disability.
Benign M S (BMS) is a clinical variant of RRMS in which the patients develop low neurological disability if at all after a disease duration of at least 10 years. Accordingly, this group of MS patients do not experience devastating accumulating disability over-time and when these patients are examined neurologically and scored by the Expanded Disability Status Scale (EDSS) they receive a score that is equal to or lower than 3.0. This low EDSS score signifies mild disability and when this low disability occurs more than 10 years after disease onset, the course of MS is defined as benign (Pittock S J and Rodriguez M, 2008; Costelloe, L., et al., 2008). Prediction of patients that will have BMS is currently impossible and the definition of these patients is retrospective. The molecular events accountable for the BMS variant of disease are not understood.
Diterpenoid triepoxide Triptolide (TPT), isolated from the Chinese herb Tripterygium wilfordii (Leuenroth S J and Crews C M. Triptolide-induced transcriptional arrest is associated with changes in nuclear substructure. Cancer Res. 2008; 68:5257-5266) has various anti-inflammatory effects (Liu Y, et al. Triptolide, a component of Chinese herbal medicine, modulates the functional phenotype of dendritic cells. Transplantation. 2007; 84:1517-1526), it modulates T-cell inflammatory responses and ameliorates Experimental Autoimmune Encephalomyelitis (Wang Y, et al. Triptolide modulates T-cell inflammatory responses and ameliorates experimental autoimmune encephalomyelitis. J Neurosci Res. 2008; 86:2441-2449). Derivatives of TPT were suggested for treating autoimmune diseases (EP 0983256, PCT/US1998/008562; WO9852933A1).
Cycloheximide, inhibits the phosphorylation of RRN3 and causes its dissociation from RNA polymerase I. RRN3 interacts with the rpa43 subunit of RNA polymerase I, and treatment with cycloheximide inhibits the formation of a RRN3/rpa43 complex in vivo (Alice H. Cavanaugh, et al., 2002. Rrn3 Phosphorylation is a regulatory checkpoint for ribosome biogenesis J. Biol. Chem., 2002; 277: 27423-27432).
PCT Application No. PCT/IL2007/32856 discloses methods and kits for predicting prognosis of multiple sclerosis.
PCT Application No. PCT/IL2007/001617 discloses methods and kits for predicting the prognosis of a subject diagnosed with multiple sclerosis and methods of selecting a treatment regimen of a subject diagnosed with multiple sclerosis.
Achiron A, et al., 2007 (Clinical and Experimental Immunology, 149: 235-242) describe genes of the zinc-ion binding and cytokine activity regulation pathways which predict outcome in relapsing—remitting multiple sclerosis.
Additional background art includes PCT Pub. No. WO03081201A2.
SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a method of classifying a subject as being more likely to have benign multiple sclerosis (BMS) or as being more likely to have typical relapsing remitting multiple sclerosis (RRMS), the method comprising comparing a level of expression of at least one gene involved in the RNA polymerase I pathway in a cell of the subject to a reference expression data of the at least one gene obtained from a cell of at least one subject pre-diagnosed as having BMS and/or from a cell of at least one subject pre-diagnosed as having typical RRMS, thereby classifying the subject as being more likely to have BMS or as being more likely to have typical RRMS.
According to an aspect of some embodiments of the present invention there is provided a method of diagnosing a subject pre-diagnosed with multiple sclerosis (MS) as having benign multiple sclerosis (BMS) or typical relapsing remitting multiple sclerosis (RRMS), the method comprising:
(a) classifying the subject as being more likely to have BMS or as being more likely to have typical RRMS according to the method of the present invention,
(i) wherein when the subject is classified as being more likely to have the BMS then the subject is diagnosed as having BMS;
(ii) wherein when the subject is classified as being more likely to have the typical RRMS, then the subject is diagnosed as having typical RRMS; and
(c) informing the subject of the diagnosis,
thereby diagnosing the subject pre-diagnosed with the MS as having the BMS or the typical RRMS.
According to an aspect of some embodiments of the present invention there is provided a method of monitoring an efficiency of an anti multiple sclerosis (MS) drug in treating a subject diagnosed with a typical relapsing remitting multiple sclerosis (RRMS) course, the method comprising:
(a) treating the subject with the anti MS drug; and
(b) comparing a level of expression of least one gene involved in the RNA polymerase I pathway in a cell of the subject following the treating with the anti MS drug to a level of expression of the at least one gene in a cell of the subject prior to the treating the subject with the anti MS drug,
(i) wherein a decrease above a predetermined threshold in the level of expression of the at least one gene following the treating with the anti MS drug relative to the level of expression of the at least one gene prior to the treating with the anti MS drug indicates that the anti MS drug is efficient for treating the subject;
(ii) wherein an increase above a predetermined threshold in the level of expression of the at least one gene following the treating with the anti MS drug relative to the level of expression of the at least one gene prior to the treating with the anti MS drug indicates that the anti MS drug is not efficient for treating the subject; or
(iii) wherein when a level of expression of the at least one gene following the treating with the anti MS drug is identical or changed below a predetermined threshold as compared to prior to the treating with the anti MS drug then the treatment is not efficient for treating the subject.
thereby monitoring the efficiency of the anti multiple sclerosis (MS) drug in treating the subject diagnosed with the typical RRMS course.
According to an aspect of some embodiments of the present invention there is provided an in vitro method of predicting an efficiency of an anti multiple sclerosis (MS) drug for treatment of a subject diagnosed with a typical relapsing remitting multiple sclerosis (RRMS), the method comprising:
(a) contacting cells of the subject with a therapeutically effective amount of the anti MS drug; and
(b) comparing a level of expression in the cells of at least one gene involved in the RNA polymerase I pathway following the contacting with the anti MS drug to a level of expression of the at least one gene in the cells prior to the contacting with the anti MS drug,
(i) wherein a decrease above a predetermined threshold in the level of expression of the at least one gene following the contacting with the anti MS drug relative to the level of expression of the at least one gene prior to the contacting with the anti MS drug indicates that the treatment is efficient for treating the subject;
(ii) wherein an increase above a predetermined threshold in the level of expression of the at least one gene following the contacting with the anti MS drug relative to the level of expression of the at least one gene prior to the contacting with the anti MS drug indicates that the treatment is not efficient for treating the subject; or
(iii) wherein when a level of expression of the at least one gene following the contacting with the anti MS drug is identical or changed below a predetermined threshold as compared to prior to the contacting with the anti MS drug then the treatment is not efficient for treating the subject.
thereby predicting the efficiency of the anti MS drug for treatment of the subject diagnosed with the typical RRMS.
According to an aspect of some embodiments of the present invention there is provided a method of treating a subject diagnosed with multiple sclerosis, the method comprising
(a) classifying the subject as being more likely to have BMS or typical RRMS according to the method of the present invention,
(b) selecting a treatment regimen based on classification results of step (a); thereby treating the subject diagnosed with multiple sclerosis.
According to an aspect of some embodiments of the present invention there is provided a method of treating a subject diagnosed with multiple sclerosis, the method comprising:
(a) diagnosing a typical relapsing remitting multiple sclerosis (RRMS) according to the method of the present invention,
(b) administering to the subject a therapeutically effective amount of diterpenoid triepoxide Triptolide (TPT) or a derivative thereof, thereby treating the subject.
According to an aspect of some embodiments of the present invention there is provided a probeset comprising a plurality of oligonucleotides and no more than 50 oligonucleotides, wherein an oligonucleotide of the plurality of oligonucleotides specifically recognizes a polynucleotide of at least one gene involved in the RNA polymerase pathway.
According to an aspect of some embodiments of the present invention there is provided a kit for classifying a disease course in a subject diagnosed with multiple sclerosis (MS), comprising the probeset of the present invention.
According to an aspect of some embodiments of the present invention there is provided a method of selecting a drug for treating a typical relapsing remitting multiple sclerosis (RRMS) in a subject, the method comprising:
contacting cells of a subject classified as having a typical RRMS with a plurality of drug molecules,
identifying at least one drug molecule which downregulates a level of expression of at least one gene involved in the RNA polymerase I pathway, the at least one drug molecule is suitable for treating the typical RRMS in the subject,
thereby selecting the drug for treating the typical RRMS in the subject.
According to some embodiments of the invention, a decrease above a predetermined threshold in the level of expression of the at least one gene in the cell of the subject relative to the reference expression data of the at least one gene obtained from the at least one subject having the typical RRMS classifies the subject as being more likely to have the BMS.
According to some embodiments of the invention, an increase above a predetermined threshold in the level of expression of the at least one gene in the cell of the subject relative to the reference expression data of the at least one gene obtained from the at least one subject having the BMS classifies the subject as being more likely to have the typical RRMS.
According to some embodiments of the invention, a level of expression of the at least one gene in the cell of the subject is identical or changed below a predetermined threshold as compared to the reference expression data of the at least one gene obtained from the at least one subject having the BMS, then the subject is classified as being more likely to have the BMS.
According to some embodiments of the invention, a level of expression of the at least one gene in the cell of the subject is identical or changed below a predetermined threshold as compared to the reference expression data of the at least one gene obtained from the at least one subject having the typical RRMS, then the subject is classified as being more likely to have the typical RRMS.
According to some embodiments of the invention, when the subject being more likely to have typical RRMS then the treatment regimen comprises administering to the subject an agent which downregulates the level of expression of the at least one gene involved in the RNA polymerase I pathway.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway is selected from the group consisting of POLR1D, LRPPRC, RRN3 and NCL.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway comprises the POLR1D, LRPPRC, RRN3 and NCL genes.
According to some embodiments of the invention, the agent is selected from the group consisting of an siRNA, an antisense, an antibody and a small molecule.
According to some embodiments of the invention, the small molecule is Cycloheximide
According to some embodiments of the invention, the at least one gene is RRN3, and whereas the downregulating is effected using diterpenoid triepoxide Triptolide (TPT) or a derivative thereof.
According to some embodiments of the invention, the at least one gene is RRN3, and whereas the downregulating is effected using Cycloheximide
According to some embodiments of the invention, the kit further comprising a positive control for an expression level of the at least one gene involved in the RNA polymerase pathway.
According to some embodiments of the invention, each of the plurality of oligonucleotides is bound to a solid support.
According to some embodiments of the invention, the plurality of oligonucleotides are bound to the solid support in an addressable location.
According to some embodiments of the invention, the level of expression is determined using an RNA detection method.
According to some embodiments of the invention, the level of expression is determined using a protein detection method.
According to some embodiments of the invention, the cell is a blood cell.
According to some embodiments of the invention, the method further comprising administering to the subject a therapeutically effective amount of an anti MS agent.
According to some embodiments of the invention, the anti-MS agent is selected from the group consisting of Diterpenoid triepoxide Triptolide (TPT), Adderall, Ambien, Avonex, Baclofen, Beta interferon, Betaseron, Celexa, Clonazepam, Copaxone, Corticosteroids, Cymbalta, Cytoxan, Dexamethasone, Effexor, Elavil, Gabapentin, Hydrocodone, Lexapro, Lyrica, Mitoxantrone, Naltrexone, Neurontin, Novantrone, Prednisone, Provigil, Rebif, Solumedrol, Symmetrel, Topamax, Tysabri, Wellbutrin, Xanax, Zanaflex, Zoloft, fingolimod, laquinimod, Mylinax (cladribine), teriflunomide, BG-12 (Biogen Idec's), firategrast (GSK/Mitsubishi Tanabe Pharma), ibudilast (MediciNova's), and CDP323 (Biogen/UCB).
According to an aspect of some embodiments of the present invention there is provided a method of predicting a benign multiple sclerosis course in a subject diagnosed with multiple sclerosis, the method comprising: (a) determining in a biological sample of the subject a level of expression of at least one polynucleotide selected from the group consisting of C22orf8, TLK1, HNRPH1, PLXDC1, TLK1, PKN2, ALS2CR8, FLJ12547, ZNF238, PDPR, NT5E, PASK, HPGD, IL6ST, JARID1A, PASK, LEF1, FLJ10246, MTUS1, FLJ14011, VSIG4, MARCH-VI, FLJ10613, EWSR1, ATP8A1, SLC4A7, FLJ21127, HNRPH1, ABLIM1, ITGA6, ADCY9, CROCC, SH3YL1, SMA4, SPTBN1, DPEP3, PDE3B, AF5Q31, NRCAM, DOCK9, IPW, FLJ20152, SIRPB2, GALNT4, CD28, TXK, ETS1, DGCR5, ZNF192, TCF7, CAMK4, SIM2, MGEA5, TGFBR2, RET, MAPK8IP3, RRN3, DKFZp547H025, FBXW11, ZNF423, DLG1, MGC17330, CD164L1, REPS1, ACHE, ITGB1BP2, LOC94431, LTK, RUNX1, EVER1, KIAA2010, CEACAM7, STX16, SLC4A5, CRTAP, RECQL5, MAGEF1, VIPR1, FLJ10979, TTC3, CRSP2, BAZ2A, GTF2I, MGC50853, KIAA0508, BPHL, LTBP4, FN3KRP, SCARB1, MGC17330, HYAL4, DGKA, FLJ11196, DHRS6, EPHB4, IDI2/GTPBP4, SNTG2, SLC7A6, PMS2L2, KIAA0436, TOSO, THRAP3, T3JAM, LOC283232, LOC92482, PTER, ATM, NUCB2, PIK3R2, MGC1136, CD59, JARID1A, FLJ39616, ABLIM1, PBP, MAPK8IP3, FTS, LHX5, TNFRSF7, MYC, PBXIP1, DATF1, HTF9C, PUS1, KIAA0924, C6orf4, KIAA0372, WDR42A, CRYZL1, TERE1, LTBP4, TTC3, NFATC1, POM121/LOC340318, TOSO, LOC348926/MGC16279/SB153/FLJ10661, SPOCK2, KIAA0515, SLC37A4, CD44, SMARCA2, SPTBN1, C6orf130, TTC3, DLG1, SLC35E2, MCCC1, PMS2L11, RCN3, STX16, FLJ20618, STAT5B, SMARCA2, SATB1, POLR1D, ASXL1, REV1L, PMS2L2/PMS2L5, FLJ12355, CCNB1IP1, FLJ12270, KIAA0692, MCM7, GPSN2, STX16, MMS19L, GTF2I/GTF2IP1, AKAP7, ZNF444, SLC35A3, MGEA5, RUTBC3, C20orf36, RAD17, ALG12, LOC112869, C6orf48, CUTC, LGTN, DEF6, WAC, HNRPH3, NS, KIAA0892, LRPPRC, HMG20A, DDX42, TINP1, ZDHHC17, C19orf2, EIF4B, LOC376745, DKFZP434C171, TH1L, C19orf13, RPL22, PHF15, EWSR1, EIF4B, FAM48A, YT521, NEK9, EIF3S7, RPS6, RPL35A, EEF2, RPL3, RPS6, UBA52, RPL6, RPS6, RPL13, AL353949, AL580863, AF052160, AW128846, AW974481, N92920, BG178274, AW303460, BF057458, AL050035, M59917, AK025422, AI693985, AU158442, AK021460, AL023773, NM_003790, AC005011, M90355, AL353580, U38964, D50683 and BE967207, wherein downregulation below a predetermined threshold in the level of expression relative to a level of expression of the at least one polynucleotide in a biological sample of a subject diagnosed with typical relapsing-remitting multiple sclerosis (RRMS) is indicative of the prediction of the benign multiple sclerosis course of the subject; (b) informing the subject of the prediction of the benign multiple sclerosis course; thereby predicting the benign multiple sclerosis course in the subject diagnosed with the multiple sclerosis.
According to an aspect of some embodiments of the present invention there is provided a method of predicting a benign multiple sclerosis course in a subject diagnosed with multiple sclerosis, the method comprising: (a) determining in a biological sample of the subject a level of expression of at least one polynucleotide selected from the group consisting of YWHAB, ATP6V1E1, UBB, MRLC2/MRCL3, UQCR, MRLC2, RTN4, UBE2A, RTN4, WDR1, PSMA6, C14orf123, PP1201, TBK1, CAST, CAST, RSN, PSME1, SDF2, GSTO1, CAST, DNCL1, SQRDL, ADIPOR2, ICMT, NDUFA6, NDUFA6, COX17, HIF1AN, FLJ20257, TBPL1, RAPGEF2, CRSP8, APOL1, PAOX, CNDP2, ETFA, DPP3, KPNA1, MGC3036, TUBB2, PDCL, CCL5, CDS2, RAP1GDS1, ATP6V1D, OBRGRP/LEPR, SF4, GCLC, MGST3, BICD2, BRF1, CHST12, EXOSC7, TOR1B, ZFP95, ILK, UNC13A, MTHFD2, CASP10, FLJ45850, CMRF-35H, ARF3, NDOR1, DUSP10, AP1M2, VRK2, GSN, PTRF, RBM19, RABGAP1L, ATP5S, STOM, TFPI2, SLCO3A1, PTPN12, CSF1, SIGLEC6, KIRREL, OBRGRP, TP53AP1, SUHW1, NUP98, IL15RA, MICB, CMRF-35H, SPHK1, TNFRSF6, FLJ1301, LRP5, STOM, EPHA2, SRC, FLJ11301, PSTPIP2, EBP, MCPH1, PTPRF, LIMK2, FSTL4, CBR1, MGC2654, MYCT1, NOL3, MITF, ATP10B, FBXO31, TBX21, LSS, SLC17A3, MNAB, CHPPR, GIF, VAMPS, ABCG2, KIF1B, LOH11CR2A, NID2, RBBP8, ETV7, CTSL, RUFY1, RSU1, PARD3, APOB, ACOX3, DAB2, LDLR, TJP2, GNAS, PARD3, NCKAP1, TAP2, HDGFRP3, LDLR, PIK3R3, HTR2B, GAS2L1, FER1L3, C3orf14, TP53TG3, LEPR, CLIC5, PDE4DIP, ATP9A, ITGB1BP1, INDO, SELP, FHL2, FER1L3, EGF, SIAT8A, HDGFRP3, LRAP, VWF, FLJ10134, IMP-3, DMN, MCTP1, FSTL1, CTNNAL1, RAB27B, THBS1, PROS1, MMRN1, CTTN, AL078596, AI148659, U00956 and M29383, wherein upregulation above a predetermined threshold in the level of expression relative to a level of expression of the at least one polynucleotide in a biological sample of a subject diagnosed with typical relapsing-remitting multiple sclerosis (RRMS) is indicative of the prediction of the benign multiple sclerosis course of the subject; (b) informing the subject of the prediction of the benign multiple sclerosis course; thereby predicting the benign multiple sclerosis course in the subject diagnosed with the multiple sclerosis.
According to an aspect of some embodiments of the present invention there is provided a method of monitoring treatment with an anti MS drug in a subject in need thereof, the method comprising: (a) treating the subject with the anti MS drug; and (b) determining in a biological sample of the subject a level of expression of at least one polynucleotide selected from the group consisting of YWHAB, ATP6V1E1, UBB, MRLC2/MRCL3, UQCR, MRLC2, RTN4, UBE2A, RTN4, WDR1, PSMA6, C14orf123, PP1201, TBK1, CAST, CAST, RSN, PSME1, SDF2, GSTO1, CAST, DNCL1, SQRDL, ADIPOR2, ICMT, NDUFA6, NDUFA6, COX17, HIF1AN, FLJ20257, TBPL1, RAPGEF2, CRSP8, APOL1, PAOX, CNDP2, ETFA, DPP3, KPNA1, MGC3036, TUBB2, PDCL, CCL5, CDS2, RAP1GDS1, ATP6V1D, OBRGRP/LEPR, SF4, GCLC, MGST3, BICD2, BRF1, CHST12, EXOSC7, TOR1B, ZFP95, ILK, UNC13A, MTHFD2, CASP10, FLJ45850, CMRF-35H, ARF3, NDOR1, DUSP10, AP1M2, VRK2, GSN, PTRF, RBM19, RABGAP1L, ATP5S, STOM, TFPI2, SLCO3A1, PTPN12, CSF1, SIGLEC6, KIRREL, OBRGRP, TP53AP1, SUHW1, NUP98, IL15RA, MICB, CMRF-35H, SPHK1, TNFRSF6, FLJ11301, LRP5, STOM, EPHA2, SRC, FLJ11301, PSTPIP2, EBP, MCPH1, PTPRF, LIMK2, FSTL4, CBR1, MGC2654, MYCT1, NOL3, MITF, ATP10B, FBXO31, TBX21, LSS, SLC17A3, MNAB, CHPPR, GIF, VAMPS, ABCG2, KIF1B, LOH11CR2A, NID2, RBBP8, ETV7, CTSL, RUFY1, RSU1, PARD3, APOB, ACOX3, DAB2, LDLR, TJP2, GNAS, PARD3, NCKAP1, TAP2, HDGFRP3, LDLR, PIK3R3, HTR2B, GAS2L1, FER1L3, C3orf14, TP53TG3, LEPR, CLIC5, PDE4DIP, ATP9A, ITGB1BP1, INDO, SELP, FHL2, FER1L3, EGF, SIAT8A, HDGFRP3, LRAP, VWF, FLJ10134, IMP-3, DMN, MCTP1, FSTL1, CTNNAL1, RAB27B, THBS1, PROS1, MMRN1, CTTN, AL078596, AI148659, U00956 and M29383, wherein an upregulation above a predetermined threshold in the level of expression relative to a level of expression of the at least one polynucleotide in a biological sample of a subject diagnosed with typical relapsing-remitting multiple sclerosis (RRMS) is indicative of treatment efficacy.
According to some embodiments of the invention, step (b) is effected also prior to step (a) and wherein the upregulation is with respect to a level of the at least one polynucleotide prior to the treating.
According to an aspect of some embodiments of the present invention there is provided a method of monitoring treatment with an anti MS drug in a subject in need thereof, the method comprising: (a) treating the subject with the anti MS drug; and (b) determining in a biological sample of the subject a level of expression of at least one polynucleotide selected from the group consisting of C22orf8, TLK1, HNRPH1, PLXDC1, TLK1, PKN2, ALS2CR8, FLJ12547, ZNF238, PDPR, NT5E, PASK, HPGD, IL6ST, JARID1A, PASK, LEF1, FLJ10246, MTUS1, FLJ14011, VSIG4, MARCH-VI, FLJ10613, EWSR1, ATP8A1, SLC4A7, FLJ21127, HNRPH1, ABLIM1, ITGA6, ADCY9, CROCC, SH3YL1, SMA4, SPTBN1, DPEP3, PDE3B, AF5Q31, NRCAM, DOCK9, IPW, FLJ20152, SIRPB2, GALNT4, CD28, TXK, ETS1, DGCR5, ZNF192, TCF7, CAMK4, SIM2, MGEA5, TGFBR2, RET, MAPK8IP3, RRN3, DKFZp547H025, FBXW11, ZNF423, DLG1, MGC17330, CD164L1, REPS1, ACHE, ITGB1BP2, LOC94431, LTK, RUNX1, EVER1, KIAA2010, CEACAM7, STX16, SLC4A5, CRTAP, RECQL5, MAGEF1, VIPR1, FLJ10979, TTC3, CRSP2, BAZ2A, GTF2I, MGC50853, KIAA0508, BPHL, LTBP4, FN3KRP, SCARB1, MGC17330, HYAL4, DGKA, FLJ11196, DHRS6, EPHB4, IDI2/GTPBP4, SNTG2, SLC7A6, PMS2L2, KIAA0436, TOSO, THRAP3, T3JAM, LOC283232, LOC92482, PTER, ATM, NUCB2, PIK3R2, MGC1136, CD59, JARID1A, FLJ39616, ABLIM1, PBP, MAPK8IP3, FTS, LHX5, TNFRSF7, MYC, PBXIP1, DATF1, HTF9C, PUS1, KIAA0924, C6orf4, KIAA0372, WDR42A, CRYZL1, TERE1, LTBP4, TTC3, NFATC1, POM121/LOC340318, TOSO, LOC348926/MGC16279/SB153/FLJ10661, SPOCK2, KIAA0515, SLC37A4, CD44, SMARCA2, SPTBN1, C6orf130, TTC3, DLG1, SLC35E2, MCCC1, PMS2L11, RCN3, STX16, FLJ20618, STAT5B, SMARCA2, SATB1, POLR1D, ASXL1, REV1L, PMS2L2/PMS2L5, FLJ12355, CCNB1IP1, FLJ12270, KIAA0692, MCM7, GPSN2, STX16, MMS19L, GTF2I/GTF2IP1, AKAP7, ZNF444, SLC35A3, MGEA5, RUTBC3, C20orf36, RAD17, ALG12, LOC112869, C6orf48, CUTC, LGTN, DEF6, WAC, HNRPH3, NS, KIAA0892, LRPPRC, HMG20A, DDX42, TINP1, ZDHHC17, C19orf2, EIF4B, LOC376745, DKFZP434C171, TH1L, C19orf13, RPL22, PHF15, EWSR1, EIF4B, FAM48A, YT521, NEK9, EIF3S7, RPS6, RPL35A, EEF2, RPL3, RPS6, UBA52, RPL6, RPS6, RPL13, AL353949, AL580863, AF052160, AW128846, AW974481, N92920, BG178274, AW303460, BF057458, AL050035, M59917, AK025422, AI693985, AU158442, AK021460, AL023773, NM_003790, AC005011, M90355, AL353580, U38964, D50683 and BE967207, wherein downregulation below a predetermined threshold in the level of expression relative to a level of expression of the at least one polynucleotide in a biological sample of a subject diagnosed with typical relapsing-remitting multiple sclerosis (RRMS) is indicative of treatment efficacy.
According to some embodiments of the invention, step (b) is effected also prior to step (a) and wherein the downregulation is with respect to a level of the at least one polynucleotide prior to the treating.
According to an aspect of some embodiments of the present invention there is provided a method of predicting a typical relapsing-remitting multiple sclerosis (RRMS) course in a subject diagnosed with multiple sclerosis, the method comprising: (a) determining in a biological sample of the subject a level of expression of at least one polynucleotide selected from the group consisting of YWHAB, ATP6V1E1, UBB, MRLC2/MRCL3, UQCR, MRLC2, RTN4, UBE2A, RTN4, WDR1, PSMA6, C14orf123, PP1201, TBK1, CAST, CAST, RSN, PSME1, SDF2, GSTO1, CAST, DNCL1, SQRDL, ADIPOR2, ICMT, NDUFA6, NDUFA6, COX17, HIF1AN, FLJ20257, TBPL1, RAPGEF2, CRSP8, APOL1, PAOX, CNDP2, ETFA, DPP3, KPNA1, MGC3036, TUBB2, PDCL, CCL5, CDS2, RAP1GDS1, ATP6V1D, OBRGRP/LEPR, SF4, GCLC, MGST3, BICD2, BRF1, CHST12, EXOSC7, TOR1B, ZFP95, ILK, UNC13A, MTHFD2, CASP10, FLJ45850, CMRF-35H, ARF3, NDOR1, DUSP10, AP1M2, VRK2, GSN, PTRF, RBM19, RABGAP1L, ATP5S, STOM, TFPI2, SLCO3A1, PTPN12, CSF1, SIGLEC6, KIRREL, OBRGRP, TP53AP1, SUHW1, NUP98, IL15RA, MICB, CMRF-35H, SPHK1, TNFRSF6, FLJ11301, LRP5, STOM, EPHA2, SRC, FLJ11301, PSTPIP2, EBP, MCPH1, PTPRF, LIMK2, FSTL4, CBR1, MGC2654, MYCT1, NOL3, MITF, ATP10B, FBXO31, TBX21, LSS, SLC17A3, MNAB, CHPPR, GIF, VAMPS, ABCG2, KIF1B, LOH11CR2A, NID2, RBBP8, ETV7, CTSL, RUFY1, RSU1, PARD3, APOB, ACOX3, DAB2, LDLR, TJP2, GNAS, PARD3, NCKAP1, TAP2, HDGFRP3, LDLR, PIK3R3, HTR2B, GAS2L1, FER1L3, C3orf14, TP53TG3, LEPR, CLIC5, PDE4DIP, ATP9A, ITGB1BP1, INDO, SELP, FHL2, FER1L3, EGF, SIAT8A, HDGFRP3, LRAP, VWF, FLJ10134, IMP-3, DMN, MCTP1, FSTL1, CTNNAL1, RAB27B, THBS1, PROS1, MMRN1, CTTN, AL078596, AI148659, U00956 and M29383, wherein downregulation below a predetermined threshold in the level of expression relative to a level of expression of the at least one polynucleotide in a biological sample of a subject diagnosed with benign multiple sclerosis (BMS) is indicative of the prediction of the relapsing-remitting multiple sclerosis course of the subject; (b) informing the subject of the prediction of the relapsing-remitting multiple sclerosis course; thereby predicting the relapsing-remitting multiple sclerosis course in the subject diagnosed with the multiple sclerosis.
According to an aspect of some embodiments of the present invention there is provided a method of predicting a typical relapsing-remitting multiple sclerosis (RRMS) course in a subject diagnosed with multiple sclerosis, the method comprising: (a) determining in a biological sample of the subject a level of expression of at least one polynucleotide selected from the group consisting of C22orf8, TLK1, HNRPH1, PLXDC1, TLK1, PKN2, ALS2CR8, FLJ12547, ZNF238, PDPR, NT5E, PASK, HPGD, IL6ST, JARID1A, PASK, LEF1, FLJ10246, MTUS1, FLJ14011, VSIG4, MARCH-VI, FLJ10613, EWSR1, ATP8A1, SLC4A7, FLJ21127, HNRPH1, ABLIM1, ITGA6, ADCY9, CROCC, SH3YL1, SMA4, SPTBN1, DPEP3, PDE3B, AF5Q31, NRCAM, DOCK9, IPW, FLJ20152, SIRPB2, GALNT4, CD28, TXK, ETS1, DGCR5, ZNF192, TCF7, CAMK4, SIM2, MGEA5, TGFBR2, RET, MAPK8IP3, RRN3, DKFZp547H025, FBXW11, ZNF423, DLG1, MGC17330, CD164L1, REPS1, ACHE, ITGB1BP2, LOC94431, LTK, RUNX1, EVER1, KIAA2010, CEACAM7, STX16, SLC4A5, CRTAP, RECQL5, MAGEF1, VIPR1, FLJ10979, TTC3, CRSP2, BAZ2A, GTF2I, MGC50853, KIAA0508, BPHL, LTBP4, FN3KRP, SCARB1, MGC17330, HYAL4, DGKA, FLJ11196, DHRS6, EPHB4, IDI2/GTPBP4, SNTG2, SLC7A6, PMS2L2, KIAA0436, TOSO, THRAP3, T3JAM, LOC283232, LOC92482, PTER, ATM, NUCB2, PIK3R2, MGC1136, CD59, JARID1A, FLJ39616, ABLIM1, PBP, MAPK8IP3, FTS, LHX5, TNFRSF7, MYC, PBXIP1, DATF1, HTF9C, PUS1, KIAA0924, C6orf4, KIAA0372, WDR42A, CRYZL1, TERE1, LTBP4, TTC3, NFATC1, POM121/LOC340318, TOSO, LOC348926/MGC16279/SB153/FLJ10661, SPOCK2, KIAA0515, SLC37A4, CD44, SMARCA2, SPTBN1, C6orf130, TTC3, DLG1, SLC35E2, MCCC1, PMS2L11, RCN3, STX16, FLJ20618, STAT5B, SMARCA2, SATB1, POLR1D, ASXL1, REV1L, PMS2L2/PMS2L5, FLJ12355, CCNB1IP1, FLJ12270, KIAA0692, MCM7, GPSN2, STX16, MMS19L, GTF2I/GTF2IP1, AKAP7, ZNF444, SLC35A3, MGEA5, RUTBC3, C20orf36, RAD17, ALG12, LOC112869, C6orf48, CUTC, LGTN, DEF6, WAC, HNRPH3, NS, KIAA0892, LRPPRC, HMG20A, DDX42, TINP1, ZDHHC17, C19orf2, EIF4B, LOC376745, DKFZP434C171, TH1L, C19orf13, RPL22, PHF15, EWSR1, EIF4B, FAM48A, YT521, NEK9, EIF3S7, RPS6, RPL35A, EEF2, RPL3, RPS6, UBA52, RPL6, RPS6, RPL13, AL353949, AL580863, AF052160, AW128846, AW974481, N92920, BG178274, AW303460, BF057458, AL050035, M59917, AK025422, AI693985, AU158442, AK021460, AL023773, NM_003790, AC005011, M90355, AL353580, U38964, D50683 and BE967207, wherein upregulation above a predetermined threshold in the level of expression relative to a level of expression of the at least one polynucleotide in a biological sample of a subject diagnosed with benign multiple sclerosis (BMS) is indicative of the prediction of the relapsing-remitting multiple sclerosis course of the subject; (b) informing the subject of the prediction of the relapsing-remitting multiple sclerosis course; thereby predicting the relapsing-remitting multiple sclerosis course in the subject diagnosed with the multiple sclerosis.
According to an aspect of some embodiments of the present invention there is provided a method of treating a subject diagnosed with multiple sclerosis, the method comprising: (a) determining if the subject is predicted to have a relapsing-remitting multiple sclerosis course according to the method of the invention, (b) selecting a treatment regimen based on the prediction of the relapsing-remitting multiple sclerosis course; thereby treating the subject diagnosed with multiple sclerosis.
According to an aspect of some embodiments of the present invention there is provided a method of treating a subject diagnosed with multiple sclerosis, the method comprising: (a) determining in a biological sample of the subject a level of expression of RRN3, wherein upregulation above a predetermined threshold in the level of expression relative to a level of expression of the RRN3 in a biological sample of a subject diagnosed with benign multiple sclerosis (BMS) is indicative of the prediction of the relapsing-remitting multiple sclerosis course of the subject; (b) administering to the subject a therapeutically effective amount of diterpenoid triepoxide Triptolide (TPT) or a derivative thereof, thereby treating the subject.
According to an aspect of some embodiments of the present invention there is provided a method of treating a subject diagnosed with multiple sclerosis, the method comprising: administering to the subject an agent which downregulates the expression level and/or activity of at least one polynucleotide or polypeptide of the RNA polymerase 1 pathway, with the proviso that the agent is not diterpenoid triepoxide Triptolide (TPT), thereby treating the subject.
According to an aspect of some embodiments of the present invention there is provided a probeset comprising a plurality of oligonucleotides and no more than 500 oligonucleotides, the plurality of oligonucleotides specifically recognizing the polynucleotides of C22orf8, TLK1, HNRPH1, PLXDC1, TLK1, PKN2, ALS2CR8, FLJ12547, ZNF238, PDPR, NT5E, PASK, HPGD, IL6ST, JARID1A, PASK, LEF1, FLJ10246, MTUS1, FLJ14011, VSIG4, MARCH-VI, FLJ10613, EWSR1, ATP8A1, SLC4A7, FLJ21127, HNRPH1, ABLIM1, ITGA6, ADCY9, CROCC, SH3YL1, SMA4, SPTBN1, DPEP3, PDE3B, AF5Q31, NRCAM, DOCK9, IPW, FLJ20152, SIRPB2, GALNT4, CD28, TXK, ETS1, DGCR5, ZNF192, TCF7, CAMK4, SIM2, MGEA5, TGFBR2, RET, MAPK8IP3, RRN3, DKFZp547H025, FBXW11, ZNF423, DLG1, MGC17330, CD164L1, REPS1, ACHE, ITGB1BP2, LOC94431, LTK, RUNX1, EVER1, KIAA2010, CEACAM7, STX16, SLC4A5, CRTAP, RECQL5, MAGEF1, VIPR1, FLJ10979, TTC3, CRSP2, BAZ2A, GTF2I, MGC50853, KIAA0508, BPHL, LTBP4, FN3KRP, SCARB1, MGC17330, HYAL4, DGKA, FLJ11196, DHRS6, EPHB4, IDI2/GTPBP4, SNTG2, SLC7A6, PMS2L2, KIAA0436, TOSO, THRAP3, T3JAM, LOC283232, LOC92482, PTER, ATM, NUCB2, PIK3R2, MGC1136, CD59, JARID1A, FLJ39616, ABLIM1, PBP, MAPK8IP3, FTS, LHX5, TNFRSF7, MYC, PBXIP1, DATF1, HTF9C, PUS1, KIAA0924, C6orf4, KIAA0372, WDR42A, CRYZL1, TERE1, LTBP4, TTC3, NFATC1, POM121/LOC340318, TOSO, LOC348926/MGC16279/SB153/FLJ10661, SPOCK2, KIAA0515, SLC37A4, CD44, SMARCA2, SPTBN1, C6orf130, TTC3, DLG1, SLC35E2, MCCC1, PMS2L11, RCN3, STX16, FLJ20618, STAT5B, SMARCA2, SATB1, POLR1D, ASXL1, REV1L, PMS2L2/PMS2L5, FLJ12355, CCNB1IP1, FLJ12270, KIAA0692, MCM7, GPSN2, STX16, MMS19L, GTF2I/GTF2IP1, AKAP7, ZNF444, SLC35A3, MGEA5, RUTBC3, C20orf36, RAD17, ALG12, LOC112869, C6orf48, CUTC, LGTN, DEF6, WAC, HNRPH3, NS, KIAA0892, LRPPRC, HMG20A, DDX42, TINP1, ZDHHC17, C19orf2, EIF4B, LOC376745, DKFZP434C171, TH1L, C19orf13, RPL22, PHF15, EWSR1, EIF4B, FAM48A, YT521, NEK9, EIF3S7, RPS6, RPL35A, EEF2, RPL3, RPS6, UBA52, RPL6, RPS6, RPL13, AL353949, AL580863, AF052160, AW128846, AW974481, N92920, BG178274, AW303460, BF057458, AL050035, M59917, AK025422, AI693985, AU158442, AK021460, AL023773, NM_003790, AC005011, M90355, AL353580, U38964, D50683, BE967207, YWHAB, ATP6V1E1, UBB, MRLC2/MRCL3, UQCR, MRLC2, RTN4, UBE2A, RTN4, WDR1, PSMA6, C14orf123, PP1201, TBK1, CAST, CAST, RSN, PSME1, SDF2, GSTO1, CAST, DNCL1, SQRDL, ADIPOR2, ICMT, NDUFA6, NDUFA6, COX17, HIF1AN, FLJ20257, TBPL1, RAPGEF2, CRSP8, APOL1, PAOX, CNDP2, ETFA, DPP3, KPNA1, MGC3036, TUBB2, PDCL, CCL5, CDS2, RAP1GDS1, ATP6V1D, OBRGRP/LEPR, SF4, GCLC, MGST3, BICD2, BRF1, CHST12, EXOSC7, TOR1B, ZFP95, ILK, UNC13A, MTHFD2, CASP10, FLJ45850, CMRF-35H, ARF3, NDOR1, DUSP10, AP1M2, VRK2, GSN, PTRF, RBM19, RABGAP1L, ATP5S, STOM, TFPI2, SLCO3A1, PTPN12, CSF1, SIGLEC6, KIRREL, OBRGRP, TP53AP1, SUHW1, NUP98, IL15RA, MICB, CMRF-35H, SPHK1, TNFRSF6, FLJ11301, LRP5, STOM, EPHA2, SRC, FLJ11301, PSTPIP2, EBP, MCPH1, PTPRF, LIMK2, FSTL4, CBR1, MGC2654, MYCT1, NOL3, MITF, ATP10B, FBXO31, TBX21, LSS, SLC17A3, MNAB, CHPPR, GIF, VAMPS, ABCG2, KIF1B, LOH11CR2A, NID2, RBBP8, ETV7, CTSL, RUFY1, RSU1, PARD3, APOB, ACOX3, DAB2, LDLR, TJP2, GNAS, PARD3, NCKAP1, TAP2, HDGFRP3, LDLR, PIK3R3, HTR2B, GAS2L1, FER1L3, C3orf14, TP53TG3, LEPR, CLIC5, PDE4DIP, ATP9A, ITGB1BP1, INDO, SELP, FHL2, FER1L3, EGF, SIAT8A, HDGFRP3, LRAP, VWF, FLJ10134, IMP-3, DMN, MCTP1, FSTL1, CTNNAL1, RAB27B, THBS1, PROS1, MMRN1, CTTN, AL078596, AI148659, U00956 and M29383.
According to an aspect of some embodiments of the present invention there is provided a kit for predicting a benign or relapsing-remitting course in a subject diagnosed with multiple sclerosis, comprising the probeset of the invention.
According to some embodiments of the invention, the kit further comprising a positive control for an expression level of at least one of the polynucleotides.
According to some embodiments of the invention, the at least one polynucleotide comprises the polynucleotides of RRN3, POLR1D and LRPPRC.
According to some embodiments of the invention, the treatment regimen comprises administering to the subject an agent which downregulates the expression level and/or activity of at least one polynucleotide or polypeptide of the RNA polymerase 1 pathway, thereby treating the subject.
According to some embodiments of the invention, the at least one polynucleotide or polypeptide of the RNA polymerase 1 pathway comprises RRN3.
According to some embodiments of the invention, the at least one polynucleotide or polypeptide of the RNA polymerase 1 pathway further comprises POLR1D and LRPPRC.
According to some embodiments of the invention, the method further comprising determining in the biological sample of the subject the level of expression of POLR1D and/or LRPPRC, wherein upregulation above a predetermined threshold in the level of expression relative to a level of expression of the POLR1D and/or LRPPRC in a biological sample of a subject diagnosed with benign multiple sclerosis (BMS) is indicative of the prediction of the relapsing-remitting multiple sclerosis course of the subject.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
Implementation of the method and/or system of embodiments of the invention can involve performing or completing selected tasks manually, automatically, or a combination thereof. Moreover, according to actual instrumentation and equipment of embodiments of the method and/or system of the invention, several selected tasks could be implemented by hardware, by software or by firmware or by a combination thereof using an operating system.
For example, hardware for performing selected tasks according to embodiments of the invention could be implemented as a chip or a circuit. As software, selected tasks according to embodiments of the invention could be implemented as a plurality of software instructions being executed by a computer using any suitable operating system. In an exemplary embodiment of the invention, one or more tasks according to exemplary embodiments of method and/or system as described herein are performed by a data processor, such as a computing platform for executing a plurality of instructions. Optionally, the data processor includes a volatile memory for storing instructions and/or data and/or a non-volatile storage, for example, a magnetic hard-disk and/or removable media, for storing instructions and/or data. Optionally, a network connection is provided as well. A display and/or a user input device such as a keyboard or mouse are optionally provided as well.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
FIG. 1 is a schematic illustration depicting the design of the study for identification of genes which predict benign multiple sclerosis;
FIG. 2 depicts Principal Component Analysis (PCA) based on 406 most informative genes (MIGs);



DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to methods of classifying a subject as being more likely to have BMS or to be more likely to have typical RRMS and, more particularly, but not exclusively, to methods of diagnosing typical RRMS or BMS and treating a subject based on the diagnosis.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
The present inventors have applied a high throughput gene expression technology to identify biomarkers for the diagnosis of benign multiple sclerosis (BMS) and for potential targets for therapeutic interventions in order switch RRMS course of disease to a BMS course of disease.
Thus, as shown in Example 1 of the Examples section which follows, the present inventors have identified 406 genes which are differentially expressed between multiple sclerosis subjects having a benign MS course or an RRMS course (typical RRMS) (Tables 1 and 2). These genes can be used for classification of MS disease course, diagnosis of a typical RRMS course and selecting a suitable treatment regimen for a subject diagnosed with MS which will prevent deterioration in the subject's state while avoiding unnecessary side effects. In addition, as shown in Table 3 (Example 1) and Tables 4A-C (Example 2) the present inventors have uncovered that the expression level of genes which are involved in the RNA polymerase I pathway such as POLR1D, LRPPRC, RRN3 and NCL is downregulated in subjects having a BMS course of MS as compared to the expression level of these genes in subjects having an RRMS course of MS. Moreover, as shown in Tables 5 and 6 (Example 2) the present inventors identified MIGs (most informative genes) discriminating between BMS and typical RRMS and genes which can classify subjects as having a BMS or a typical RRMS. These results suggest the use of genes involved in the RNA polymerase I pathway as diagnostic markers and drug targets for treating and preventing a typical RRMS course in a subject diagnosed with MS.
Thus, according to an aspect of some embodiments of the invention there is provided a method of classifying a subject as being more likely to have benign multiple sclerosis (BMS) or as being more likely to have typical relapsing remitting multiple sclerosis (RRMS), the method comprising comparing a level of expression of at least one gene involved in the RNA polymerase I pathway in a cell of the subject to a reference expression data of the at least one gene obtained from a cell of at least one subject pre-diagnosed as having BMS and/or from a cell of at least one subject pre-diagnosed as having typical RRMS, thereby classifying the subject as being more likely to have BMS or as being more likely to have typical RRMS.
The term “subject” refers to mammal, preferably a human being.
According to some embodiments of the invention, the subject is diagnosed with multiple sclerosis.
As used herein, the phrase “diagnosed with multiple sclerosis” refers to a subject who experienced at least one neurological attack affecting the central nervous system (CNS) accompanied by demyelinating lesions within the brain or spinal cord, which may have, but not necessarily confirmed by magnetic resonance imaging (MRI). The neurological attack can involve acute or sub-acute neurological symptomatology (attack) manifested by various clinical presentations like unilateral loss of vision, vertigo, ataxia, incoordination, gait difficulties, sensory impairment characterized by paresthesia, dysesthesia, sensory loss, urinary disturbances until incontinence, diplopia, dysarthria, various degrees of motor weakness until paralysis, cognitive decline either as a monosymptomatic or in combination. The symptoms usually remain for several days to few weeks, and then partially or completely resolve.
The accepted diagnostic criteria of multiple sclerosis are presented in Hypertext Transfer Protocol://World Wide Web (dot) mult-sclerosis (dot) org/DiagnosticCriteria (dot) html.
According to some embodiments of the invention, the subject is suspected of having multiple sclerosis.
According to some embodiments of the invention, the subject has probable multiple sclerosis.
According to some embodiments of the invention, the subject does not have a primary progressive course (PPMS).
According to some embodiments of the invention, the subject does not have a secondary progressive MS course (SPMS).
As used herein the term “classifying” refers to determining if the subject is more likely to have benign multiple sclerosis (BMS) or typical relapsing remitting multiple sclerosis (RRMS).
As used herein the phrase “being more likely to have” refers to having increased probability to have a certain disease course (classification of disease) than another disease course.
According to some embodiments of the invention, the phrase “being more likely to have” refers to a probability of at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., about 100% that the subject has a certain disease course and not the other disease course, i.e., a BMS or typical RRMS.
As used herein, the phrase “benign multiple sclerosis” refers to a subject having MS and exhibiting an Expanded Disability Status Scale (EDSS) of less than 3.0 following at least 10 years from onset and/or diagnosis of MS.
As used herein, the phrase “typical RRMS” or a “relapsing-remitting multiple sclerosis course”, which is interchangeably used herein, refers to a subject having MS and exhibiting an Expanded Disability Status Scale (EDSS) higher than 3.0 within less than 10 years of disease onset and/or diagnosis.
The phrase “onset of multiple sclerosis (MS)” as used herein refers to the time of occurrence of the first clinical neurological symptomatology suggestive of MS.
The Kurtzke EDSS is a method scale of quantifying disability in MS by scoring eight Functional Systems (FS) (pyramidal, cerebellar, brainstem, sensory, bowel and bladder, visual, cerebral, and other) and allows neurologists to assign a Functional System Score (FSS) in each and to combine the FSS scores into the EDSS score as follows:
EDSS 0.0—Normal neurological examination;
EDSS 1.0—No disability, minimal signs in one FS;
EDSS 1.5—No disability, minimal signs in more than one FS;
EDSS 2.0—Minimal disability in one FS;
EDSS 2.5—Mild disability in one FS or minimal disability in two FS;
EDSS 3.0—Moderate disability in one FS, or mild disability in three or four FS. Fully ambulatory;
EDSS 3.5—Fully ambulatory but with moderate disability in one FS and more than minimal disability in several others;
EDSS 4.0—Fully ambulatory without aid, self-sufficient, up and about some 12 hours a day despite relatively severe disability; able to walk without aid or rest some 500 meters;
EDSS 4.5—Fully ambulatory without aid, up and about much of the day, able to work a full day, may otherwise have some limitation of full activity or require minimal assistance; characterized by relatively severe disability; able to walk without aid or rest some 300 meters;
EDSS 5.0—Ambulatory without aid or rest for about 200 meters; disability severe enough to impair full daily activities (work a full day without special provisions);
EDSS 5.5—Ambulatory without aid or rest for about 100 meters; disability severe enough to preclude full daily activities;
EDSS 6.0—Intermittent or unilateral constant assistance (cane, crutch, brace) required to walk about 100 meters with or without resting;
EDSS 6.5—Constant bilateral assistance (canes, crutches, braces) required to walk about 20 meters without resting;
EDSS 7.0—Unable to walk beyond approximately five meters even with aid, essentially restricted to wheelchair; wheels self in standard wheelchair and transfers alone; up and about in wheelchair some 12 hours a day;
EDSS 7.5—Unable to take more than a few steps; restricted to wheelchair; may need aid in transfer; wheels self but cannot carry on in standard wheelchair a full day, May require motorized wheelchair;
EDSS 8.0—Essentially restricted to bed or chair or perambulated in wheelchair, but may be out of bed itself much of the day; retains many self-care functions; generally has effective use of arms;
EDSS 8.5—Essentially restricted to bed much of day, has some effective use of arms retains some self care functions;
EDSS 9.0—Confined to bed; can still communicate and eat;
EDSS 9.5—Totally helpless bed patient; unable to communicate effectively or eat/swallow;
EDSS 10.0—Death due to MS;
As mentioned, the diagnosis of MS is performed by clinical neurological symptoms and/or findings such as laboratory tests involving evaluation of IgG synthesis and oligoclonal bands (immunoglobulins) in the cerebrospinal fluid (CSF) which provide evidence of chronic inflammation of the central nervous system, and brain or spinal cord MRI according to the McDonald criteria [McDonald W I, Compston A, Edan G, et al., 2001, “Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis”. Ann Neurol. 50 (1): 121-7; Polman C H, Reingold S C, Edan G, et al., 2005, “Diagnostic criteria for multiple sclerosis: 2005 revisions to the “McDonald Criteria””. Ann Neurol. 58 (6): 840-6].
It should be noted that onset of multiple sclerosis and the diagnosis of multiple sclerosis could occur on the same time.
As used herein, the phrase “level of expression” refers to the degree of gene expression and/or gene product activity in a specific cell. For example, up-regulation or down-regulation of various genes can affect the level of the gene product (i.e., RNA and/or protein) in a specific cell.
Sequence information regarding gene products (i.e., RNA transcripts and polypeptide sequences) of the genes of the polynucleotides of the invention such as the genes of RNA polymerase I pathway and of probes which can be used to detect thereof can be found in Tables 1, 2, 3, 5 and 6 of the Examples section which follows.
It should be noted that the level of expression can be determined in arbitrary absolute units, or in normalized units (relative to known expression levels of a control reference). For example, when using DNA chips, the expression levels are normalized according to the chips' internal controls or by using quantile normalization such as RMA.
As used herein the phrase “a cell of the subject” refers to at least one cell (e.g., an isolated cell), cell culture, cell content and/or cell secreted content which contains RNA and/or proteins of the subject. Examples include a blood cell, a cell obtained from any tissue biopsy [e.g., cerebrospinal fluid, (CSF), brain biopsy], a bone marrow cell, body fluids such as plasma, serum, saliva, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, sputum and milk. According to an embodiment of the invention, the cell is a blood cell (e.g., white blood cells, macrophages, B- and T-lymphocytes, monocytes, neutrophiles, eosinophiles, and basophiles) which can be obtained using a syringe needle from a vein of the subject. It should be noted that the cell may be isolated from the subject (e.g., for in vitro detection) or may optionally comprise a cell that has not been physically removed from the subject (e.g., in vivo detection).
According to some embodiments of the invention, the white blood cell comprises peripheral blood mononuclear cells (PBMC). The phrase, “peripheral blood mononuclear cells (PBMCs)” as used herein, refers to a mixture of monocytes and lymphocytes. Several methods for isolating white blood cells are known in the art. For example, PBMCs can be isolated from whole blood samples using density gradient centrifugation procedures. Typically, anticoagulated whole blood is layered over the separating medium. At the end of the centrifugation step, the following layers are visually observed from top to bottom: plasma/platelets, PBMCs, separating medium and erythrocytes/granulocytes. The PBMC layer is then removed and washed to remove contaminants (e.g., red blood cells) prior to determining the expression level of the polynucleotide(s) therein.
The cell or the biological sample comprising same can be obtained from the subject at any time, e.g., immediately after an attack or at any time during remission.
According to some embodiments of the invention, the level of expression of the gene(s) of the invention is determined using an RNA and/or a protein detection method.
According to some embodiments of the invention, the RNA or protein molecules are extracted from the cell of the subject.
Methods of extracting RNA or protein molecules from cells of a subject are well known in the art. Once obtained, the RNA or protein molecules can be characterized for the expression and/or activity level of various RNA and/or protein molecules using methods known in the arts.
Non-limiting examples of methods of detecting RNA molecules in a cell sample include Northern blot analysis, RT-PCR, RNA in situ hybridization (using e.g., DNA or RNA probes to hybridize RNA molecules present in the cells or tissue sections), in situ RT-PCR (e.g., as described in Nuovo G J, et al. Am J Surg Pathol. 1993, 17: 683-90; Komminoth P, et al. Pathol Res Pract. 1994, 190: 1017-25), and oligonucleotide microarray (e.g., by hybridization of polynucleotide sequences derived from a sample to oligonucleotides attached to a solid surface [e.g., a glass wafer) with addressable location, such as Affymetrix microarray (Affymetrix®, Santa Clara, Calif.)].
For example, the level of RRN3 in a sample can be determined by RT-PCR using primers available from Santa Cruz Biotechnology Inc. (sc-106866-PR), or Taqman Gene Expression Assay HS00607907_ml (Applied Biosystems, Foster City, Calif., USA), according to manufacturer's recommendation.
Non-limiting examples of methods of detecting the level and/or activity of specific protein molecules in a cell sample include Enzyme linked immunosorbent assay (ELISA), Western blot analysis, radio-immunoassay (RIA), Fluorescence activated cell sorting (FACS), immunohistochemical analysis, in situ activity assay (using e.g., a chromogenic substrate applied on the cells containing an active enzyme), in vitro activity assays (in which the activity of a particular enzyme is measured in a protein mixture extracted from the cells). For example, in case the detection of the expression level of a secreted protein is desired, ELISA assay may be performed on a sample of fluid obtained from the subject (e.g., serum), which contains cell-secreted content.
As used herein the phrase “reference expression data” refers to the expression level of the gene in a cell of at least one subject who is pre-diagnosed as having BMS or typical RRMS. Such as an expression level can be known from the literature, from the database, or from biological samples comprising RNA or protein molecules obtained from a reference subject who is already diagnosed as having BMS or typical RRMS.
As used herein the phrase “pre-diagnosed” refers to being diagnosed based on the acceptable clinical tools/markers as described above (e.g., by evaluating the EDSS score after 10 years from onset or diagnosis of MS).
According to some embodiments of the invention, the reference expression data is obtained from at least subject who is pre-diagnosed as having BMS, e.g., from at least 2, from at least 3, from at least 4, from at least 5, from at least 6, from at least 7, from at least 8, from at least 9, from at least 10, from at least 20, from at least 30, from at least 40, from at least 50, from at least 100 or more subjects who are pre-diagnosed as having BMS.
According to some embodiments of the invention, the reference expression data is obtained from at least one subject who is pre-diagnosed as having typical RRMS, e.g., from at least 2, from at least 3, from at least 4, from at least 5, from at least 6, from at least 7, from at least 8, from at least 9, from at least 10, from at least 20, from at least 30, from at least 40, from at least 50, from at least 100 or more subjects who are pre-diagnosed as having typical RRMS.
It should be noted that when more than one reference subjects (i.e., a subject who is pre-diagnosed as having BMS or typical RRMS) is used, the reference expression data may comprise an average of the expression level of several or all subjects, and those of skills in the art are capable of averaging expression levels from 2 or more subject, using e.g., normalized expression values.
According to some embodiments of the invention, a decrease above a predetermined threshold in the level of expression of the at least one gene in the cell of the subject relative to the reference expression data of the at least one gene obtained from a cell of the at least one subject having the typical RRMS classifies the subject as being more likely to have the BMS.
As used herein the phrase “a decrease above a predetermined threshold” refers to a decrease in the level of expression in the cell of the subject relative to the reference expression data obtained from a cell of the at least one subject having the typical RRMS which is higher than a predetermined threshold such as a about 10%, e.g., higher than about 20%, e.g., higher than about 30%, e.g., higher than about 40%, e.g., higher than about 50%, e.g., higher than about 60%, higher than about 70%, higher than about 80%, higher than about 90%, higher than about 2 times, higher than about three times, higher than about four time, higher than about five times, higher than about six times, higher than about seven times, higher than about eight times, higher than about nine times, higher than about 20 times, higher than about 50 times, higher than about 100 times, higher than about 200 times, higher than about 350, higher than about 500 times, higher than about 1000 times, or more relative to the reference expression data obtained from a cell of the at least subject having the typical RRMS.
According to some embodiments of the invention, an increase above a predetermined threshold in the level of expression of the at least one gene in the cell of the subject relative to the reference expression data of the at least one gene obtained from a cell of the at least one subject having the BMS classifies the subject as being more likely to have the typical RRMS.
As used herein the phrase “an increase above a predetermined threshold” refers to an increase in the level of expression in the cell of the subject relative to the reference expression data obtained from a cell of the at least one subject having the BMS which is higher than a predetermined threshold such as a about 10%, e.g., higher than about 20%, e.g., higher than about 30%, e.g., higher than about 40%, e.g., higher than about 50%, e.g., higher than about 60%, higher than about 70%, higher than about 80%, higher than about 90%, higher than about 2 times, higher than about three times, higher than about four time, higher than about five times, higher than about six times, higher than about seven times, higher than about eight times, higher than about nine times, higher than about 20 times, higher than about 50 times, higher than about 100 times, higher than about 200 times, higher than about 350, higher than about 500 times, higher than about 1000 times, or more relative to the reference expression data obtained from a cell of the at least one subject having the BMS.
According to some embodiments of the invention, when a level of expression of the at least one gene in the cell of the subject is identical or changed below a predetermined threshold as compared to the reference expression data of the at least one gene obtained from a cell of the at least one subject having the BMS, then the subject is classified as being more likely to have the BMS.
As used herein the phrase “changed below a predetermined threshold as compared to the reference expression data . . . subject having the BMS” refers to an increase or a decrease in the level of expression in the cell of the subject relative to the reference expression data obtained from a cell of the at least one subject having the BMS which is lower than a predetermined threshold, such as lower than about 10 times, e.g., lower than about 9 times, e.g., lower than about 8 times, e.g., lower than about 7 times, e.g., lower than about 6 times, e.g., lower than about 5 times, e.g., lower than about 4 times, e.g., lower than about 3 times, e.g., lower than about 2 times, e.g., lower than about 90%, e.g., lower than about 80%, e.g., lower than about 70%, e.g., lower than about 60%, e.g., lower than about 50%, e.g., lower than about 40%, e.g., lower than about 30%, e.g., lower than about 20%, e.g., lower than about 10%, e.g., lower than about 9%, e.g., lower than about 8%, e.g., lower than about 7%, e.g., lower than about 6%, e.g., lower than about 5%, e.g., lower than about 4%, e.g., lower than about 3%, e.g., lower than about 2%, e.g., lower than about 1% relative to the reference expression data obtained from a cell of the at least one subject having the BMS.
According to some embodiments of the invention, when a level of expression of the at least one gene in the cell of the subject is identical or changed below a predetermined threshold as compared to the reference expression data of the at least one gene obtained from a cell of the at least one subject having the typical RRMS, then the subject is classified as being more likely to have the typical RRMS.
As used herein the phrase “changed below a predetermined threshold as compared to the reference expression data . . . subject having the typical RRMS” refers to an increase or a decrease in the level of expression in the cell of the subject relative to the reference expression data obtained from a cell of the one subject having the typical RRMS which is lower than a predetermined threshold, such as lower than about 10 times, e.g., lower than about 9 times, e.g., lower than about 8 times, e.g., lower than about 7 times, e.g., lower than about 6 times, e.g., lower than about 5 times, e.g., lower than about 4 times, e.g., lower than about 3 times, e.g., lower than about 2 times, e.g., lower than about 90%, e.g., lower than about 80%, e.g., lower than about 70%, e.g., lower than about 60%, e.g., lower than about 50%, e.g., lower than about 40%, e.g., lower than about 30%, e.g., lower than about 20%, e.g., lower than about 10%, e.g., lower than about 9%, e.g., lower than about 8%, e.g., lower than about 7%, e.g., lower than about 6%, e.g., lower than about 5%, e.g., lower than about 4%, e.g., lower than about 3%, e.g., lower than about 2%, e.g., lower than about 1% relative to the reference expression data obtained from the at least one subject having the typical RRMS.
Non-limiting examples of genes involved in the RNA polymerase I pathway which can be used according to the method of the invention are provided in Table 3 along with representative polynucleotides thereof and probes which can be used to detect thereof (Example 1 of the Examples section which follows; e.g., RRN3, LRPPRC, POLR1B, POLR1C, POLR1D, POLR2A, POLR2B, POLR2C, POLR2D, POLR2E, POLR2E, POLR2F, POLR2G, POLR2H, POLR2I, POLR2J, POLR2J2, MGC13098, POLR2K, POLR2L, POLR3B, POLR3C, POLR3D, POLR3E, POLR3F, POLR3G, POLR3K, POLRMT, POLRMT and POLS).
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway is selected from the group consisting of POLR1D, LRPPRC, RRN3 and NCL.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway is RRN3.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway is LRPPRC.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway is POLR1D.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway comprises RRN3 and POLR1D.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway comprises RRN3 and LRPPRC.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway comprises POLR1D and LRPPRC.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway comprises RRN3, LRPPRC and POLR1D.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway is RRN3 and NCL.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway is LRPPRC and NCL.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway is POLR1D and NCL.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway comprises RRN3, POLR1D and NCL.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway comprises RRN3, LRPPRC and NCL.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway comprises POLR1D, LRPPRC and NCL.
According to some embodiments of the invention, the at least one gene involved in the RNA polymerase 1 pathway comprises RRN3, LRPPRC, POLR1D and NCL.
Tables 4A-C in the Examples section which follows demonstrate exemplary combinations of genes of the RNA polymerase I pathway along with their classification rates for BMS and typical RRMS.
The prediction of the MS course is important in terms of monitoring the clinical state of the subject (e.g., how often does the patient need to be evaluated for the disease progression in terms of neurological evaluation and EDSS), planning of subject's future life (e.g., making decisions regarding marriage, having children, being involved in high risk activities, getting a life-insurance, etc.) and planning the treatment regimen of the subject.
For example, a subject who is more likely to have BMS may be advised to reduce the frequency of neurological clinical evaluations to no more than once per year; to avoid frequent MRI examinations; to not be included in treatment schedule of MS; and/or to avoid receiving immunomodulatory drugs which have side effects or adverse events that can be even life-threatening [e.g., progressive multifocal leukoencephalopathy (PML) in MS patients treated with natalizumab (Tysabri®, Biogen-Idec); Hypertext Transfer Protocol://World Wide Web (dot) va (dot) gov/MS/pressreleases/Treating_Natalizumab_and_Risk_of_PML (dot) asp].
On the other hand, a subject who is more likely to have typical RRMS may be advised to have neurological clinical evaluations at a higher frequency, e.g., about 3-4 times per year; to have frequent MRI examinations; to be included in treatment schedule of MS; and/or to receive immunomodulatory drugs.
It should be noted that the classification of the subject as being more likely to have BMS or typical RRMS can be used to diagnose the subject as having BMS or typical RRMS.
As used herein the term “diagnosing” refers to determining presence or absence of a pathology (e.g., a disease, disorder, condition or syndrome) and/or likelihood of same, classifying a pathology or a symptom, determining a severity of the pathology, monitoring pathology progression, forecasting an outcome of a pathology and/or prospects of recovery and screening of a subject for a specific disease.
According to some embodiments of the invention the method of diagnosing is effected by (a) classifying the subject as being more likely to have BMS or as being more likely to have typical RRMS according to the method of the invention,
(i) wherein when the subject is classified as being more likely to have the BMS then the subject is diagnosed as having BMS;
(ii) wherein when the subject is classified as being more likely to have the typical RRMS, then the subject is diagnosed as having typical RRMS; and
(c) informing the subject of the diagnosis,
thereby diagnosing the subject pre-diagnosed with the MS as having the BMS or the typical RRMS.
According to some embodiments of the invention, the subject is pre-diagnosed with multiple sclerosis (MS), i.e., has a confirmed diagnosis of MS without knowing the disease course, e.g., typical RRMS, BMS.
As used herein the term “informing” refers to providing to the subject the results of the diagnosis of the disease sub-class (i.e., BMS or typical RRMS). The results may be provided as a computer output and/or oral conversation with the subject.
The teachings of the invention can be also used to determine efficiency anti multiple sclerosis drugs by determining the effect of the drug(s) on the expression level of the at least one gene of the RNA polymerase I pathway.
Thus, according to an aspect of some embodiments of the invention, there is provided a method of monitoring an efficiency of an anti multiple sclerosis (MS) drug in treating a subject diagnosed with a typical relapsing remitting multiple sclerosis (RRMS) course, the method is effected by:
(a) treating the subject with the anti MS drug; and
(b) comparing a level of expression of least one gene involved in the RNA polymerase I pathway in a cell of the subject following treating with the anti MS drug to a level of expression of the at least one gene in a cell of the subject prior to the treating the subject with the anti MS drug,
(i) wherein a decrease above a predetermined threshold in the level of expression of the at least one gene following the treating with the anti MS drug relative to the level of expression of the at least one gene prior to the treating with the anti MS drug indicates that the anti MS drug is efficient for treating the subject;
(ii) wherein an increase above a predetermined threshold in the level of expression of the at least one gene following the treating with the anti MS drug relative to the level of expression of the at least one gene prior to the treating with the anti MS drug indicates that the anti MS drug is not efficient for treating the subject; or
(iii) wherein when a level of expression of the at least one gene following the treating with the anti MS drug is identical or changed below a predetermined threshold as compared to prior to the treating with the anti MS drug then the treatment is not efficient for treating the subject.
thereby monitoring the efficiency of the anti multiple sclerosis (MS) drug in treating the subject diagnosed with the typical RRMS course.
As used herein the phrase “treating” refers to inhibiting or arresting the development of pathology [multiple sclerosis, e.g., typical RRMS] and/or causing the reduction, remission, or regression of a pathology and/or optimally curing the pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of the pathology.
According to some embodiments of the invention, treating the subject refers to changing the disease course of the subject from a typical RRMS course to a BMS course.
According to some embodiments of the invention, treating the subject refers to preventing a typical RRMS course.
As used herein the phrase “following treating with the anti MS drug” refers to any time period after administering the anti MS drug to the subject, e.g., from a few minutes to hours, or from a few days to weeks or months after drug administration.
According to some embodiments of the invention the level of expression is determined following the first dose of the anti MS drug.
According to some embodiments of the invention the level of expression is determined following any dose of the anti MS drug.
As used herein the phrase “prior to treating with the anti MS drug” refers to any time period prior administering the anti MS drug to the subject, e.g., from a few minutes to hours, or from a few days to weeks or months prior to drug administration.
According to some embodiments of the invention the level of expression is determined prior any dose of the anti MS drug (e.g., when the subject is naïve to treatment).
According to some embodiments of the invention prior to treating refers to when the subject is first diagnosed with multiple sclerosis.
According to some embodiments of the invention prior to treating refers to when the subject is suspected of having multiple sclerosis, or diagnosed with probable multiple sclerosis.
According to some embodiments of the invention prior to treating refers to upon MS disease onset.
According to some embodiments of the invention the effect of the treatment on the subject can be evaluated by monitoring the level of expression of at least one of the polynucleotides described hereinabove. For example, downregulation in the level of RRN3 in the subject following treatment can be indicative of the positive effect of the treatment on the subject, i.e., switching from a typical RRMS to a BMS course of disease.
The teachings of the invention can be also used to predict efficiency of a drug in vitro.
Thus, according to an aspect of some embodiments of the invention there is provided an in vitro method of predicting an efficiency of an anti multiple sclerosis (MS) drug for treatment of a subject diagnosed with a typical relapsing remitting multiple sclerosis (RRMS), the method is effected by:
(a) contacting cells of the subject with a therapeutically effective amount of the anti MS drug; and
(b) comparing a level of expression in the cells of at least one gene involved in the RNA polymerase I pathway following the contacting with the anti MS drug to a level of expression of the at least one gene in the cells prior to the contacting with the anti MS drug,
(i) wherein a decrease above a predetermined threshold in the level of expression of the at least one gene following the contacting with the anti MS drug relative to the level of expression of the at least one gene prior to the contacting with the anti MS drug indicates that the treatment is efficient for treating the subject;
(ii) wherein an increase above a predetermined threshold in the level of expression of the at least one gene following the contacting with the anti MS drug relative to the level of expression of the at least one gene prior to the contacting with the anti MS drug indicates that the treatment is not efficient for treating the subject; or
(iii) wherein when a level of expression of the at least one gene following the contacting with the anti MS drug is identical or changed below a predetermined threshold as compared to prior to the contacting with the anti MS drug then the treatment is not efficient for treating the subject.
thereby predicting the efficiency of the anti MS drug for treatment of the subject diagnosed with the typical RRMS.
Contacting cells with the anti MS drug can be performed by any in vitro conditions including for example, adding the anti MS drug to cells derived from a subject (e.g., a primary cell culture, a cell line) or to a biological sample comprising same (e.g., a fluid, liquid which comprises the cells) such that the drug is in direct contact with the cells. According to some embodiments of the invention, the cells of the subject are incubated with the anti MS drug. The conditions used for incubating the cells are selected for a time period/concentration of cells/concentration of drug/ratio between cells and drug and the like which enable the drug to induce cellular changes, such as changes in transcription and/or translation rate of specific genes, proliferation rate, differentiation, cell death, necrosis, apoptosis and the like.
Methods of monitoring cellular changes induced by the drugs are known in the art and include for example, the MTT test which is based on the selective ability of living cells to reduce the yellow salt MTT (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (Sigma, Aldrich St Louis, Mo., USA) to a purple-blue insoluble formazan precipitate; the BrDu assay [Cell Proliferation ELISA BrdU colorimetric kit (Roche, Mannheim, Germany]; the TUNEL assay [Roche, Mannheim, Germany]; the Annexin V assay [ApoAlert® Annexin V Apoptosis Kit (Clontech Laboratories, Inc., CA, USA)]; the Senescence associated-β-galactosidase assay (Dimri G P, Lee X, et al. 1995. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 92:9363-9367); as well as various RNA and protein detection methods (which detect level of expression and/or activity) which are further described hereinabove.
According to some embodiments of the invention, the cells are incubated under conditions which enable the effect of the drug on cellular processes such as downregulation of the at least one gene of the RNA polymerase I pathway.
According to an aspect of some embodiments of the invention there is provided a method of treating a subject diagnosed with multiple sclerosis, the method is effected by: (a) classifying the subject as being more likely to have BMS or typical RRMS according to the method of the invention, (b) selecting a treatment regimen based on classification results of step (a); thereby treating the subject diagnosed with multiple sclerosis.
As used herein the phrase “treatment regimen” refers to a treatment plan that specifies the type of treatment, dosage, schedule and/or duration of a treatment provided to a subject in need thereof (e.g., a subject diagnosed with MS). The selected treatment regimen can be an aggressive one which is expected to result in the best clinical outcome (e.g., complete cure of the pathology) or a more moderate one which may relief symptoms of the pathology yet results in incomplete cure of the pathology. It will be appreciated that in certain cases the more aggressive treatment regimen may be associated with some discomfort to the subject or adverse side effects (e.g., a damage to healthy cells or tissue). The dosage, schedule and duration of treatment can vary, depending on the severity of pathology and the selected type of treatment, and those of skills in the art are capable of adjusting the type of treatment with the dosage, schedule and duration of treatment.
For example, when the subject is classified as being more likely to have typical RRMS (or being diagnosed with typical RRMS) then the treatment regimen is an aggressive therapy such as an immunomodulation therapy, e.g., a high dosage of interferon beta 1a [Rebif, which can be administered subcutaneously, at a dosage of e.g., 44 μg, three times a week].
MS drugs which can be administered to a subject predicted to have an RRMS course of disease according to the present teachings include, but are not limited to Diterpenoid triepoxide Triptolide (TPT), Adderall; Ambien; Avonex; Baclofen; Beta interferon; Betaseron; Celexa; Clonazepam; Copaxone; Corticosteroids; Cymbalta; Cytoxan; Dexamethasone; Effexor; Elavil; Gabapentin; Hydrocodone; Lexapro; Lyrica; Mitoxantrone; Naltrexone; Neurontin; Novantrone; Prednisone; Provigil; Rebif; Solumedrol; Symmetrel; Topamax; Tysabri; Wellbutrin; Xanax; Zanaflex; Zoloft; Novartis' fingolimod [sphingosine 1-phosphate receptor (S1P-R) modulator]; Teva's laquinimod; Merck KGaA's Mylinax (cladribine); Sanofi-aventis' teriflunomide; Biogen Idec's BG-12 (Phase III); GSK/Mitsubishi Tanabe Pharma's firategrast; MediciNova's ibudilast; Biogen/UCB's CDP323 (Phase II).
One the other hand, when the subject is classified as being more likely to have BMS (or is diagnosed with BMS) then the aggressive treatment is not recommended, and these patients would not be treated or treatment can be delayed.
In addition, knowing the prediction or classification of MS disease course (BMS or typical RRMS) is highly beneficial in terms of saving un-necessary costs to the health system.
According to an aspect of some embodiments of the invention there is provided a method of treating a subject diagnosed with multiple sclerosis, the method is effected by (a) diagnosing a typical relapsing remitting multiple sclerosis (RRMS) according to the method of the invention, and (b) administering to the subject a therapeutically effective amount of diterpenoid triepoxide Triptolide (TPT) or a derivative thereof, thereby treating the subject
TPT derivatives and preparation thereof are described in WO9852933A1, which is fully incorporated herein by reference. Non-limiting examples of TPT derivatives include, compounds of the general formulas.




Wherein



represents a single or double bond;

R1 and R2 each independently is H or —OR5;

R3 is H, —C(═O)(CH2)nCO2H or a suitable amino acid; R, is H or —OH; R, is H, —C(═O)(CH2)nCO2H or a suitable amino acid;

n is the integer 2, 3, 4, 5 or 6;

and the stereoisomers, enantiomers and pharmaceutically acceptable salts thereof; provided that R, and R2 are H when R, is other than H (for further details see WO9852933A1).

A commercially available preparation of Triptolide which can be used according to the teachings of the invention is Trisoxireno(4b,5:6,7:8a,9)phenanthro(1,2-c)furan-1(3H)-one, 3b,4,4a,6,6a,7a,7b,8b,9,10-decahydro-6-hydroxy-8b-methyl-6a-(1-methylethyl)-, (3bS,4aS,5aS,6R,6aR,7aS,7bS,8aS,8bS)-[CAS No.: 38748-32-2; PG490, Chengdu Biopurify Phytochemicals Ltd. Chengdu, Sichuan, China].
According to some embodiments of the invention, when the subject is more likely to have typical RRMS then the treatment regimen comprises administering to the subject an agent which downregulates the level of expression of the at least one gene involved in the RNA polymerase I pathway.
According to some embodiments of the invention, treating the subject is effected by downregulating the expression level and/or activity (RNA and/or polypeptide encoded thereby) of at least one polynucleotide of the polymerase I pathway (for details see the list of genes/polynucleotide in Table 3 in Example 1 of the Examples section which follows).
Following is a list of downregulating agents which can decrease the expression level of the gene product (RNA or protein molecules) of at least one of the polynucleotides of the polymerase I pathway.
Downregulation can be effected on the genomic and/or the transcript level using a variety of molecules which interfere with transcription and/or translation (e.g., RNA silencing agents, Ribozyme, DNAzyme and antisense), or on the protein level using e.g., an antibody, antagonists, enzymes that cleave the polypeptide and the like.
One example, of an agent capable of downregulating a polypeptide-of-interest is an antibody or antibody fragment capable of specifically binding the polypeptide-of-interest. Preferably, the antibody specifically binds at least one epitope of the polypeptide-of-interest. As used herein, the term “epitope” refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
The term “antibody” as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab′)2, and Fv that are capable of binding to macrophages. These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab′, the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (3) (Fab′)2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (“SCA”), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).
Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab′ fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides (“minimal recognition units”) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).
In case the target antigen (the protein which is detected by the antibody) is presented within the cell, the antibody of the invention can be expressed within the cell intracellular antibodies (also known as “intrabodies”) or a particular compartment thereof. Intrabodies are essentially SCA to which intracellular localization signals have been added (e.g., ER, mitochondrial, nuclear, cytoplasmic). This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors and to inhibit a protein function within a cell (See, for example, Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Deshane et al., 1994, Gene Ther. 1: 332-337; Marasco et al., 1998 Human Gene Ther 9: 1627-42; Shaheen et al., 1996 J. Virol. 70: 3392-400; Werge, T. M. et al., 1990, FEBS Letters 274:193-198; Carlson, J. R. 1993 Proc. Natl. Acad. Sci. USA 90:7427-7428; Biocca, S. et al., 1994, Bio/Technology 12: 396-399; Chen, S-Y. et al., 1994, Human Gene Therapy 5:595-601; Duan, L et al., 1994, Proc. Natl. Acad. Sci. USA 91:5075-5079; Chen, S-Y. et al., 1994, Proc. Natl. Acad. Sci. USA 91:5932-5936; Beerli, R. R. et al., 1994, J. Biol. Chem. 269:23931-23936; Mhashilkar, A. M. et al., 1995, EMBO J. 14:1542-1551; PCT Publication No. WO 94/02610 by Marasco et al.; and PCT Publication No. WO 95/03832 by Duan et al.).
To prepare an intracellular antibody expression vector, the cDNA encoding the antibody light and heavy chains specific for the target protein of interest are isolated, typically from a hybridoma that secretes a monoclonal antibody specific for the marker. Hybridomas secreting anti-marker monoclonal antibodies, or recombinant monoclonal antibodies, can be prepared using methods known in the art. Once a monoclonal antibody specific for the marker protein is identified (e.g., either a hybridoma-derived monoclonal antibody or a recombinant antibody from a combinatorial library), DNAs encoding the light and heavy chains of the monoclonal antibody are isolated by standard molecular biology techniques. For hybridoma derived antibodies, light and heavy chain cDNAs can be obtained, for example, by PCR amplification or cDNA library screening. For recombinant antibodies, such as from a phage display library, cDNA encoding the light and heavy chains can be recovered from the display package (e.g., phage) isolated during the library screening process and the nucleotide sequences of antibody light and heavy chain genes are determined. For example, many such sequences are disclosed in Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 and in the “Vbase” human germline sequence database. Once obtained, the antibody light and heavy chain sequences are cloned into a recombinant expression vector using standard methods.
For cytoplasmic expression of the light and heavy chains, the nucleotide sequences encoding the hydrophobic leaders of the light and heavy chains are removed. An intracellular antibody expression vector can encode an intracellular antibody in one of several different forms. For example, in one embodiment, the vector encodes full-length antibody light and heavy chains such that a full-length antibody is expressed intracellularly. In another embodiment, the vector encodes a full-length light chain but only the VH/CH1 region of the heavy chain such that a Fab fragment is expressed intracellularly. In another embodiment, the vector encodes a single chain antibody (scFv) wherein the variable regions of the light and heavy chains are linked by a flexible peptide linker [e.g., (Gly4Ser)3 and expressed as a single chain molecule. To inhibit marker activity in a cell, the expression vector encoding the intracellular antibody is introduced into the cell by standard transfection methods, as discussed hereinbefore.
Once antibodies are obtained, they may be tested for activity, for example via ELISA.
Downregulation of the polynucleotide-of-interest can be also achieved by RNA silencing. As used herein, the phrase “RNA silencing” refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or “silencing” of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.
As used herein, the term “RNA silencing agent” refers to an RNA which is capable of inhibiting or “silencing” the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs. In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression.
The RNA silencing agent can be directed to a specific compartment within the cells, such as to the nucleus (see e.g., Shim M S and Kwon Y J. 2009, “Controlled cytoplasmic and nuclear localization of plasmid DNA and siRNA by differentially tailored polyethylenimine”; J. Control Release. 133:206-13, Epub 2008 Nov. 1), nucleoli, and the like.
RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla. Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA.
The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.
Accordingly, the present invention contemplates use of dsRNA to downregulate protein expression from mRNA.
According to one embodiment, the dsRNA is greater than 30 bp. The use of long dsRNAs (i.e. dsRNA greater than 30 bp) has been very limited owing to the belief that these longer regions of double stranded RNA will result in the induction of the interferon and PKR response. However, the use of long dsRNAs can provide numerous advantages in that the cell can select the optimal silencing sequence alleviating the need to test numerous siRNAs; long dsRNAs will allow for silencing libraries to have less complexity than would be necessary for siRNAs; and, perhaps most importantly, long dsRNA could prevent viral escape mutations when used as therapeutics.
Various studies demonstrate that long dsRNAs can be used to silence gene expression without inducing the stress response or causing significant off-target effects—see for example [Strat et al., Nucleic Acids Research, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res. Protoc. 2004; 13:115-125; Diallo M., et al., Oligonucleotides. 2003; 13:381-392; Paddison P. J., et al., Proc. Natl Acad. Sci. USA. 2002; 99:1443-1448; Tran N., et al., FEBS Lett. 2004; 573:127-134].
In particular, the present invention also contemplates introduction of long dsRNA (over 30 base transcripts) for gene silencing in cells where the interferon pathway is not activated (e.g. embryonic cells and oocytes) see for example Billy et al., PNAS 2001, Vol 98, pages 14428-14433. and Diallo et al, Oligonucleotides, Oct. 1, 2003, 13(5): 381-392. doi:10.1089/154545703322617069.
The present invention also contemplates introduction of long ds-RNA specifically designed not to induce the interferon and PKR pathways for downregulating gene expression. For example, Shinagwa and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP, to express long double-strand RNA from an RNA polymerase II (Pol II) promoter. Because the transcripts from pDECAP lack both the 5′-cap structure and the 3′-poly(A) tail that facilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP does not induce the interferon response.
Another method of evading the interferon and PKR pathways in mammalian systems is by introduction of small inhibitory RNAs (siRNAs) either via transfection or endogenous expression.
The term “siRNA” refers to small inhibitory RNA duplexes (generally between 18-30 basepairs) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21 mers with a central 19 bp duplex region and symmetric 2-base 3′-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100-fold increase in potency compared with 21 mers at the same location. The observed increased potency obtained using longer RNAs in triggering RNAi is theorized to result from providing Dicer with a substrate (27 mer) instead of a product (21 mer) and that this improves the rate or efficiency of entry of the siRNA duplex into RISC.
It has been found that position of the 3′-overhang influences potency of an siRNA and asymmetric duplexes having a 3′-overhang on the antisense strand are generally more potent than those with the 3′-overhang on the sense strand (Rose et al., 2005). This can be attributed to asymmetrical strand loading into RISC, as the opposite efficacy patterns are observed when targeting the antisense transcript.
The strands of a double-stranded interfering RNA (e.g., an siRNA) may be connected to form a hairpin or stem-loop structure (e.g., an shRNA). Thus, as mentioned the RNA silencing agent of the present invention may also be a short hairpin RNA (shRNA).
A non-limiting example for an siRNA which can be used to down regulate RRN3 (RNA polymerase I transcription factor homolog) expression level in a cell of a subject is Rrn3 siRNA (h): sc-106866 (Santa Cruz Biotechnology, Inc. Santa Cruz, Calif., USA). In addition, downregulation of RRN3 can be achieved by Rrn3 shRNA plasmid (h): sc-106866-SH and Rrn3 shRNA (h) Lentiviral Particles: sc-106866-V ((Santa Cruz Biotechnology, Inc. Santa Cruz, Calif., USA).
The term “shRNA”, as used herein, refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that can be used to form the loop include 5′-UUCAAGAGA-3′ (Brummelkamp, T. R. et al. (2002) Science 296: 550) and 5′-UUUGUGUAG-3′ (Castanotto, D. et al. (2002) RNA 8:1454). It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem-loop or hairpin structure comprising a double-stranded region capable of interacting with the RNAi machinery.
According to another embodiment the RNA silencing agent may be a miRNA. miRNAs are small RNAs made from genes encoding primary transcripts of various sizes. They have been identified in both animals and plants. The primary transcript (termed the “pri-miRNA”) is processed through various nucleolytic steps to a shorter precursor miRNA, or “pre-miRNA.” The pre-miRNA is present in a folded form so that the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA (the strand that will eventually basepair with the target) The pre-miRNA is a substrate for a form of dicer that removes the miRNA duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex. It has been demonstrated that miRNAs can be transgenically expressed and be effective through expression of a precursor form, rather than the entire primary form (Parizotto et al. (2004) Genes & Development 18:2237-2242 and Guo et al. (2005) Plant Cell 17:1376-1386).
Unlike, siRNAs, miRNAs bind to transcript sequences with only partial complementarity (Zeng et al., 2002, Molec. Cell 9:1327-1333) and repress translation without affecting steady-state RNA levels (Lee et al., 1993, Cell 75:843-854; Wightman et al., 1993, Cell 75:855-862). Both miRNAs and siRNAs are processed by Dicer and associate with components of the RNA-induced silencing complex (Hutvagner et al., 2001, Science 293:834-838; Grishok et al., 2001, Cell 106: 23-34; Ketting et al., 2001, Genes Dev. 15:2654-2659; Williams et al., 2002, Proc. Natl. Acad. Sci. USA 99:6889-6894; Hammond et al., 2001, Science 293:1146-1150; Mourlatos et al., 2002, Genes Dev. 16:720-728). A recent report (Hutvagner et al., 2002, Sciencexpress 297:2056-2060) hypothesizes that gene regulation through the miRNA pathway versus the siRNA pathway is determined solely by the degree of complementarity to the target transcript. It is speculated that siRNAs with only partial identity to the mRNA target will function in translational repression, similar to an miRNA, rather than triggering RNA degradation.
Synthesis of RNA silencing agents suitable for use with the present invention can be effected as follows. First, the mRNA sequence of the polynucleotide-of-interest is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3′ adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tusch1 ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5′ UTR mediated about 90% decrease in cellular GAPDH mRNA and completely abolished protein level (www.ambion.com/techlib/tn/91/912.html).
Second, potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www.ncbi.nlm nih gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.
Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55%. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
It will be appreciated that the RNA silencing agent of the present invention need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.
In some embodiments, the RNA silencing agent provided herein can be functionally associated with a cell-penetrating peptide. As used herein, a “cell-penetrating peptide” is a peptide that comprises a short (about 12-30 residues) amino acid sequence or functional motif that confers the energy-independent (i.e., non-endocytotic) translocation properties associated with transport of the membrane-permeable complex across the plasma and/or nuclear membranes of a cell. The cell-penetrating peptide used in the membrane-permeable complex of the present invention preferably comprises at least one non-functional cysteine residue, which is either free or derivatized to form a disulfide link with a double-stranded ribonucleic acid that has been modified for such linkage. Representative amino acid motifs conferring such properties are listed in U.S. Pat. No. 6,348,185, the contents of which are expressly incorporated herein by reference. The cell-penetrating peptides of the present invention preferably include, but are not limited to, penetratin, transportan, pIsl, TAT(48-60), pVEC, MTS, and MAP.
mRNAs to be targeted using RNA silencing agents include, but are not limited to, those whose expression is correlated with an undesired phenotypic trait. Exemplary mRNAs that may be targeted are those that encode truncated proteins i.e. comprise deletions. Accordingly the RNA silencing agent of the present invention may be targeted to a bridging region on either side of the deletion. Introduction of such RNA silencing agents into a cell would cause a down-regulation of the mutated protein while leaving the non-mutated protein unaffected.
Another agent capable of downregulating the polynucleotide-of-interest is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or DNA sequence of the polynucleotide-of-interest. DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R. R. and Joyce, G. Chemistry and Biology 1995; 2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997; 943:4262) A general model (the “10-23” model) for the DNAzyme has been proposed. “10-23” DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions (Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, L M [Curr Opin Mol Ther 4:119-21 (2002)].
Examples of construction and amplification of synthetic, engineered DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al, 20002, Abstract 409, Ann Meeting Am Soc Gen Ther World Wide Web (dot) asgt (dot) org). In another application, DNAzymes complementary to bcr-ab1 oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL.
Downregulation of the polynucleotide-of-interest can also be effected by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the polynucleotide-of-interest.
Design of antisense molecules which can be used to efficiently downregulate the polynucleotide-of-interest must be effected while considering two aspects important to the antisense approach. The first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.
The prior art teaches of a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types [see, for example, Luft J Mol Med 76: 75-6 (1998); Kronenwett et al. Blood 91: 852-62 (1998); Rajur et al. Bioconjug Chem 8: 935-40 (1997); Lavigne et al. Biochem Biophys Res Commun 237: 566-71 (1997) and Aoki et al. (1997) Biochem Biophys Res Commun 231: 540-5 (1997)].
In addition, algorithms for identifying those sequences with the highest predicted binding affinity for their target mRNA based on a thermodynamic cycle that accounts for the energetics of structural alterations in both the target mRNA and the oligonucleotide are also available [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9 (1999)].
Such algorithms have been successfully used to implement an antisense approach in cells. For example, the algorithm developed by Walton et al. enabled scientists to successfully design antisense oligonucleotides for rabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNF alpha) transcripts. The same research group has more recently reported that the antisense activity of rationally selected oligonucleotides against three model target mRNAs (human lactate dehydrogenase A and B and rat gp130) in cell culture as evaluated by a kinetic PCR technique proved effective in almost all cases, including tests against three different targets in two cell types with phosphodiester and phosphorothioate oligonucleotide chemistries.
In addition, several approaches for designing and predicting efficiency of specific oligonucleotides using an in vitro system were also published (Matveeva et al., Nature Biotechnology 16: 1374-1375 (1998)].
Several clinical trials have demonstrated safety, feasibility and activity of antisense oligonucleotides. For example, antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Holmund et al., Curr Opin Mol Ther 1:372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz Curr Opin Mol Ther 1:297-306 (1999)].
More recently, antisense-mediated suppression of human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model [Uno et al., Cancer Res 61:7855-60 (2001)].
Thus, the current consensus is that recent developments in the field of antisense technology which, as described above, have led to the generation of highly accurate antisense design algorithms and a wide variety of oligonucleotide delivery systems, enable an ordinarily skilled artisan to design and implement antisense approaches suitable for downregulating expression of known sequences without having to resort to undue trial and error experimentation.
Another agent capable of downregulating the polynucleotide-of-interest is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding the polynucleotide-of-interest. Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)]. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al., Clin Diagn Virol. 10:163-71 (1998)]. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials. ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models. HEPTAZYME, a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated—WEB home page).
An additional method of regulating the expression of the polynucleotide-of-interest in cells is via triplex forming oligonuclotides (TFOs). Recent studies have shown that TFOs can be designed which can recognize and bind to polypurine/polypirimidine regions in double-stranded helical DNA in a sequence-specific manner. These recognition rules are outlined by Maher III, L. J., et al., Science, 1989; 245:725-730; Moser, H. E., et al., Science, 1987; 238:645-630; Beal, P. A., et al, Science, 1992; 251:1360-1363; Cooney, M., et al., Science, 1988; 241:456-459; and Hogan, M. E., et al., EP Publication 375408. Modification of the oligonuclotides, such as the introduction of intercalators and backbone substitutions, and optimization of binding conditions (pH and cation concentration) have aided in overcoming inherent obstacles to TFO activity such as charge repulsion and instability, and it was recently shown that synthetic oligonucleotides can be targeted to specific sequences (for a recent review see Seidman and Glazer, J Clin Invest 2003; 112:487-94).
Another agent capable of downregulating the expression level of the polynucleotide-of-interest is a small molecule which inhibits the activity, level and/or interactions of the gene product of polynucleotide-of-interest, such as by interfering with the pathway in which the gene product of the polynucleotide-of-interest is involved.
Non-limiting examples of small molecules which can be used along with the method of the invention to treat the subject include Cycloheximide and diterpenoid triepoxide Triptolide (TPT) or a derivative thereof.
For example, when RRN3 is upregulated in the typical RRMS subject then the treatment can be with diterpenoid triepoxide Triptolide (TPT) or a derivative thereof; and/or with Cycloheximide or a derivative thereof.
Any of the downregulating agents described hereinabove (e.g., the agent which downregulates the gene of the RNA polymerase I pathway, e.g., siRNA, antibody) can be provided to the subject in need thereof along with any of the known multiple sclerosis therapies (e.g., the anti MS drugs) described hereinabove (combination therapy) and/or with Triptolide or a derivative thereof.
Any of the downregulating agents described above can be administered to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
Herein the term “active ingredient” refers to the downregulating agent accountable for the biological effect.
Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.
Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
For example, the pharmaceutical composition may be administered using a dermal patch which releases the active ingredient [e.g., Diterpenoid triepoxide Triptolide (TPT) has a molecular weight of 360.40 and it will thus be suitable to be used in a dermal patch].
Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.
Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (the downregulating agent) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., RRMS) or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. 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, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).
Dosage amount and interval may be adjusted individually to provide levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
Compositions 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 comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
According to an aspect of some embodiments of the invention, there is provided a method of selecting a drug for treating a typical relapsing remitting multiple sclerosis (RRMS) in a subject, the method is effected by (a) contacting cells of a subject classified as having a typical RRMS with a plurality of drug molecules, (b) identifying at least one drug molecule which downregulates a level of expression of at least one gene involved in the RNA polymerase I pathway, the at least one drug molecule is suitable for treating the typical RRMS in the subject, thereby selecting the drug for treating the typical RRMS in the subject.
The plurality of drug molecules can be peptides, RNA, DNA, aptamers and small molecules.
According to some embodiments of the invention the polynucleotides described hereinabove (e.g., oligonucleotides) can form a part of a probeset.
According to some embodiments of the invention, the probeset comprises a plurality of oligonucleotides and no more than 50 oligonucleotides (e.g., no more than about 40, no more than about 30, e.g., no more than about 20, e.g., no more than about 15, e.g., no more than about 10 oligonucleotides) wherein an oligonucleotide of the plurality of oligonucleotides specifically recognizes a polynucleotide of at least one gene involved in the RNA polymerase pathway. For example, each of the oligonucleotides can specifically recognize a polynucleotide of the RNA polymerase I pathway.
According to some embodiments of the invention the probeset comprises a plurality of oligonucleotides and no more than 500 oligonucleotides wherein each of the plurality of oligonucleotides is capable of specifically recognizing at least one polynucleotide sequence selected from the group consisting of C22orf8, TLK1, HNRPH1, PLXDC1, TLK1, PKN2, ALS2CR8, FLJ12547, ZNF238, PDPR, NT5E, PASK, HPGD, IL6ST, JARID1A, PASK, LEF1, FLJ10246, MTUS1, FLJ14011, VSIG4, MARCH-VI, FLJ10613, EWSR1, ATP8A1, SLC4A7, FLJ21127, HNRPH1, ABLIM1, ITGA6, ADCY9, CROCC, SH3YL1, SMA4, SPTBN1, DPEP3, PDE3B, AF5Q31, NRCAM, DOCK9, IPW, FLJ20152, SIRPB2, GALNT4, CD28, TXK, ETS1, DGCR5, ZNF192, TCF7, CAMK4, SIM2, MGEA5, TGFBR2, RET, MAPK8IP3, RRN3, DKFZp547H025, FBXW11, ZNF423, DLG1, MGC17330, CD164L1, REPS1, ACHE, ITGB1BP2, LOC94431, LTK, RUNX1, EVER1, KIAA2010, CEACAM7, STX16, SLC4A5, CRTAP, RECQL5, MAGEF1, VIPR1, FLJ10979, TTC3, CRSP2, BAZ2A, GTF2I, MGC50853, KIAA0508, BPHL, LTBP4, FN3KRP, SCARB1, MGC17330, HYAL4, DGKA, FLJ11196, DHRS6, EPHB4, IDI2/GTPBP4, SNTG2, SLC7A6, PMS2L2, KIAA0436, TOSO, THRAP3, T3JAM, LOC283232, LOC92482, PTER, ATM, NUCB2, PIK3R2, MGC1136, CD59, JARID1A, FLJ39616, ABLIM1, PBP, MAPK8IP3, FTS, LHX5, TNFRSF7, MYC, PBXIP1, DATF1, HTF9C, PUS1, KIAA0924, C6orf4, KIAA0372, WDR42A, CRYZL1, TERE1, LTBP4, TTC3, NFATC1, POM121/LOC340318, TOSO, LOC348926/MGC16279/SB153/FLJ10661, SPOCK2, KIAA0515, SLC37A4, CD44, SMARCA2, SPTBN1, C6orf130, TTC3, DLG1, SLC35E2, MCCC1, PMS2L11, RCN3, STX16, FLJ20618, STAT5B, SMARCA2, SATB1, POLR1D, ASXL1, REV1L, PMS2L2/PMS2L5, FLJ12355, CCNB1IP1, FLJ12270, KIAA0692, MCM7, GPSN2, STX16, MMS19L, GTF2I/GTF2IP1, AKAP7, ZNF444, SLC35A3, MGEA5, RUTBC3, C20orf36, RAD17, ALG12, LOC112869, C6orf48, CUTC, LGTN, DEF6, WAC, HNRPH3, NS, KIAA0892, LRPPRC, HMG20A, DDX42, TINP1, ZDHHC17, C19orf2, EIF4B, LOC376745, DKFZP434C171, TH1L, C19orf13, RPL22, PHF15, EWSR1, EIF4B, FAM48A, YT521, NEK9, EIF3S7, RPS6, RPL35A, EEF2, RPL3, RPS6, UBA52, RPL6, RPS6, RPL13, AL353949, AL580863, AF052160, AW128846, AW974481, N92920, BG178274, AW303460, BF057458, AL050035, M59917, AK025422, AI693985, AU158442, AK021460, AL023773, NM_003790, AC005011, M90355, AL353580, U38964, D50683, BE967207, YWHAB, ATP6V1E1, UBB, MRLC2/MRCL3, UQCR, MRLC2, RTN4, UBE2A, RTN4, WDR1, PSMA6, C14orf123, PP1201, TBK1, CAST, CAST, RSN, PSME1, SDF2, GSTO1, CAST, DNCL1, SQRDL, ADIPOR2, ICMT, NDUFA6, NDUFA6, COX17, HIF1AN, FLJ20257, TBPL1, RAPGEF2, CRSP8, APOL1, PAOX, CNDP2, ETFA, DPP3, KPNA1, MGC3036, TUBB2, PDCL, CCL5, CDS2, RAP1GDS1, ATP6V1D, OBRGRP/LEPR, SF4, GCLC, MGST3, BICD2, BRF1, CHST12, EXOSC7, TOR1B, ZFP95, ILK, UNC13A, MTHFD2, CASP10, FLJ45850, CMRF-35H, ARF3, NDOR1, DUSP10, AP1M2, VRK2, GSN, PTRF, RBM19, RABGAP1L, ATP5S, STOM, TFPI2, SLCO3A1, PTPN12, CSF1, SIGLEC6, KIRREL, OBRGRP, TP53AP1, SUHW1, NUP98, IL15RA, MICB, CMRF-35H, SPHK1, TNFRSF6, FLJ11301, LRP5, STOM, EPHA2, SRC, FLJ11301, PSTPIP2, EBP, MCPH1, PTPRF, LIMK2, FSTL4, CBR1, MGC2654, MYCT1, NOL3, MITF, ATP10B, FBXO31, TBX21, LSS, SLC17A3, MNAB, CHPPR, GIF, VAMPS, ABCG2, KIF1B, LOH11CR2A, NID2, RBBP8, ETV7, CTSL, RUFY1, RSU1, PARD3, APOB, ACOX3, DAB2, LDLR, TJP2, GNAS, PARD3, NCKAP1, TAP2, HDGFRP3, LDLR, PIK3R3, HTR2B, GAS2L1, FER1L3, C3orf14, TP53TG3, LEPR, CLIC5, PDE4DIP, ATP9A, ITGB1BP1, INDO, SELP, FHL2, FER1L3, EGF, SIAT8A, HDGFRP3, LRAP, VWF, FLJ10134, IMP-3, DMN, MCTP1, FSTL1, CTNNAL1, RAB27B, THBS1, PROS1, MMRN1, CTTN, AL078596, AI148659, U00956 and M29383.
It will be appreciated that the isolated nucleic acid sequences included in the kit or the probeset of the present invention can be bound to a solid support e.g., a glass wafer in a specific order, i.e., in the form of a microarray. Alternatively, isolated nucleic acid sequences can be synthesized directly on the solid support using well known prior art approaches (Seo T S, et al., 2004, Proc. Natl. Acad. Sci. USA, 101: 5488-93.). In any case, the isolated nucleic acid sequences are attached to the support in a location specific manner such that each specific isolated nucleic acid sequence has a specific address on the support (i.e., an addressable location) which denotes the identity (i.e., the sequence) of that specific isolated nucleic acid sequence.
The kit may further include a positive control for an expression level of at least one of the polynucleotides of the invention (e.g., which involves in the RNA polymerase I pathway). The positive control can be any biological sample derived from a reference subject (i.e., a subject with a known course of MS, i.e., BMS or typical RRMS), a biological sample with known amount/concentration of the gene product (i.e., RNA or protein) of at least one of the polynucleotides of the invention; or a pre-determined level (amount/concentration) of purified, chemically synthesized or recombinantly generated RNA or protein molecules (gene products) of the at least one polynucleotide of the invention. The kit may further comprise instructions for use in classifying a subject as being more like to have BMS or typical RRMS, to diagnose BMS or typical RRMS, to monitor treatment efficiency, to select a treatment regimen, to treat a subject having multiple sclerosis and/or to select for drugs suitable for treating multiple sclerosis.
As used herein the term “about” refers to ±10%.
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
The term “consisting of means “including and limited to”.
The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
General Materials and Experimental Methods
Subjects—
31 patients (age 44.5±1.5; female to male ratio 24:7) with BMS were characterized by mean EDSS 1.95±0.15, disease duration 17.0±1.3 years, annual EDSS rate 0.13±0.01, annual relapse rate 0.23±0.04. 36 patients (age 40.3±1.8; female to male ratio 8:3) with typical RRMS were characterized by mean EDSS 3.54±0.23, disease duration 10.9±1.4 years, annual EDSS rate 0.45±0.06, annual relapse rate 0.64±0.09.
RNA Isolation and Microarray Expression Profiling—
Peripheral blood mononuclear cells (PBMC) were separated on ficoll-hypaque gradient. Total RNA was isolated using the TRIzol Reagent (Invitrogen, Carlsbad, Calif.), and cDNA was synthesized, labeled and hybridized to HG-U133A-2 array (Affymetrix, Inc, Santa Clara, Calif.) containing 22,215 gene-transcripts, washed and scanned (Hewlett Packard, GeneArray-TM scanner G2500A) according to manufacturer's protocol Affymetrix (Inc, Santa Clara, Calif.).
Data Analysis—
Data analysis was performed using the Partek Genomics Solution software [World Wide Web (dot) partek (dot) com]. Expression values were computed from raw CEL (cell) files by applying the Robust Multi-Chip Average (RMA) background correction algorithm. The RMA correction included: 1) values background correction; 2) quantile normalization; 3) log 2 transformation; 4) median polish summarization. In order to avoid the noise caused by variable set effects each set was normalized to pre-saved distribution pattern of a well balanced set used as a reference distribution. To reduce batch effect ANOVA multiple model analysis was applied. Source of variation was analyzed; nuisance batches effects such as working batch, patient age, gender and treatment were eliminated. Most informative genes (MIGs) were defined as genes with p<0.01 by ANOVA linear contrasts model. For samples classification, principal component analyses (PCA) were performed.
Gene functional annotation, enrichment and pathway analysis were performed using functional classification tools, David Bioinformatics Resources [Hypertext Transfer Protocol://david (dot) abcc (dot) ncifcrf (dot) gov/home (dot) jsp], and Ingenuity Pathways Analysis web-software [World Wide Web (dot) ingenuity (dot) com]. Enrichment was defined as significantly (p<0.05) higher proportion of genes in a given gene set than expected by chance analysis. The study design is demonstrated in FIG. 1.
Example 1
Experimental Results
Identification of Differentiating Genes Between Patients with BMS and Patients with Typical RRMS—
BMS patients differentiated from typical RRMS by 406 MIGs (most informative genes), 171 genes were over-expressed (upregulated) and 235 were down-expressed (downregulated), with the log fold change ranged from −3.1 to 3.3 (FIG. 2).
Table 1 hereinbelow provides the differentiating genes between BMS and typical RRMS patients.







TABLE 1







Genes which are differentially expressed in blood samples of benign multiple sclerosis


(BMS) and typical relapsing-remitting multiple sclerosis (RRMS) subjects



















Log Fold








p-value
Change





SEQ

SEQ
BMS vs.
(BMS vs.




Affymetrix
ID
Representative
ID
typical
typical




Probeset ID
NO:
Public ID
NO:
RRMS
RRMS)
Gene Symbol
Gene Title

















216683_at
1
AL353949
407
6.85E−04
−1.129
—
—


219629_at
2
NM_017911
408
5.51E−03
−1.117
C22orf8
chromosome 22









open reading frame 8


210379_s_at
3
AI469203
409
7.42E−03
−1.083
TLK1
tousled-like kinase 1


213472_at
4
AI022387
410
4.25E−03
−1.081
HNRPH1
heterogeneous









nuclear









ribonucleoprotein









H1 (H)


219700_at
5
NM_020405
411
5.56E−05
−1.081
PLXDC1
plexin domain









containing 1


211077_s_at
6
Z25421
412
9.89E−04
−1.080
TLK1
tousled-like kinase 1









/// tousled-like









kinase 1


210969_at
7
AF118089
413
4.20E−04
−1.078
PKN2
protein kinase N2


216298_at
8
AL580863
414
3.57E−03
−1.075
—
Similar to T-cell









receptor gamma









chain V region PT-









gamma-1/2









precursor /// Simil


219834_at
9
NM_024744
415
1.20E−03
−1.069
ALS2CR8
amyotrophic lateral









sclerosis 2 (juvenile)









chromosome region,









candidate 8


215262_at
10
AF052160
416
2.29E−04
−1.068
—
Clone 24629 mRNA









sequence


220715_at
11
NM_024992
417
1.53E−04
−1.067
FLJ12547
hypothetical protein









FLJ12547


207164_s_at
12
NM_006352
418
4.17E−03
−1.067
ZNF238
zinc finger protein









238


220236_at
13
NM_017990
419
7.14E−04
−1.067
PDPR
pyruvate









dehydrogenase









phosphatase









regulatory subunit


203939_at
14
NM_002526
420
1.84E−03
−1.065
NT5E
5′-nucleotidase, ecto









(CD73)


216945_x_at
15
U79240
421
2.45E−03
−1.063
PASK
PAS domain









containing









serine/threonine









kinase


203913_s_at
16
NM_000860
422
4.09E−03
−1.062
HPGD
hydroxyprostaglandin









dehydrogenase 15-









(NAD)


204864_s_at
17
BE856546
423
9.98E−03
−1.060
IL6ST
interleukin 6 signal









transducer (gp130,









oncostatin M









receptor)


215698_at
18
AF007135
424
3.73E−03
−1.057
JARID1A
Jumonji, AT rich









interactive domain









1A (RBBP2-like)


213534_s_at
19
D50925
425
3.86E−03
−1.056
PASK
PAS domain









containing









serine/threonine









kinase


210948_s_at
20
AF294627
426
1.98E−03
−1.056
LEF1
lymphoid enhancer-









binding factor 1


220458_at
21
NM_018038
427
8.30E−03
−1.055
FLJ10246
hypothetical protein









FLJ10246


212093_s_at
22
AI695017
428
8.37E−03
−1.052
MTUS1
mitochondrial tumor









suppressor 1


207120_at
23
NM_022103
429
1.75E−03
−1.050
FLJ14011
hypothetical zinc









finger protein









FLJ14011


204787_at
24
NM_007268
430
3.61E−03
−1.050
VSIG4
V-set and









immunoglobulin









domain containing 4


215512_at
25
AK000970
431
1.74E−03
−1.049
MARCH-VI
membrane-









associated RING-









CH protein VI


46947_at
26
T87245
432
1.23E−03
−1.049
FLJ10613
Hypothetical protein









FLJ10613


210012_s_at
27
BC000527
433
7.10E−03
−1.048
EWSR1
Ewing sarcoma









breakpoint region 1


210192_at
28
AB013452
434
7.32E−03
−1.048
ATP8A1
ATPase,









aminophospholipid









transporter (APLT),









Class I, type 8A,









member 1


207603_at
29
NM_003615
435
2.30E−03
−1.047
SLC4A7
solute carrier family









4, sodium









bicarbonate









cotransporter,









member 7


218584_at
30
NM_024549
436
7.79E−03
−1.047
FLJ21127
hypothetical protein









FLJ21127


213470_s_at
31
BF983406
437
8.91E−03
−1.047
HNRPH1
heterogeneous









nuclear









ribonucleoprotein









H1 (H)


210461_s_at
32
BC002448
438
1.63E−03
−1.047
ABLIM1
actin binding LIM









protein 1


217656_at
33
AW128846
439
5.27E−04
−1.045
—
—


215177_s_at
34
AV733308
440
4.01E−04
−1.045
ITGA6
integrin, alpha 6


204498_s_at
35
NM_001116
441
3.72E−04
−1.043
ADCY9
adenylate cyclase 9


216419_at
36
AK026910
442
7.82E−03
−1.043
CROCC
ciliary rootlet









coiled-coil, rootletin


204019_s_at
37
NM_015677
443
1.11E−03
−1.043
SH3YL1
SH3 domain









containing, Ysc84-









like 1 (S. cerevisiae)


214850_at
38
X75940
444
4.48E−03
−1.043
SMA4
SMA4


200672_x_at
39
NM_003128
445
1.59E−03
−1.043
SPTBN1
spectrin, beta, non-









erythrocytic 1


220179_at
40
NM_022357
446
1.40E−03
−1.042
DPEP3
dipeptidase 3


208591_s_at
41
NM_000922
447
6.54E−05
−1.042
PDE3B
phosphodiesterase









3B, cGMP-inhibited


219199_at
42
NM_014423
448
5.72E−03
−1.042
AF5Q31
ALL1 fused gene









from 5q31


216959_x_at
43
U55258
449
1.65E−03
−1.041
NRCAM
neuronal cell









adhesion molecule


215041_s_at
44
BE259050
450
6.36E−03
−1.040
DOCK9
dedicator of









cytokinesis 9


213447_at
45
AI672541
451
5.32E−05
−1.040
IPW
imprinted in Prader-









Willi syndrome


218532_s_at
46
NM_019000
452
4.80E−03
−1.040
FLJ20152
hypothetical protein









FLJ20152


220485_s_at
47
NM_018556
453
1.56E−03
−1.039
SIRPB2
signal-regulatory









protein beta 2


220442_at
48
NM_003774
454
8.94E−03
−1.039
GALNT4
UDP-N-acetyl-









alpha-D-









galactosamine:









polypeptide N-









acetylgalactosaminyltransferase


211856_x_at
49
AF222341
455
9.84E−03
−1.039
CD28
CD28 antigen









(Tp44)


206828_at
50
NM_003328
456
1.62E−03
−1.039
TXK
TXK tyrosine kinase


214447_at
51
NM_005238
457
3.28E−03
−1.038
ETS1
v-ets









erythroblastosis









virus E26 oncogene









homolog 1 (avian)


215244_at
52
AI479306
458
6.97E−03
−1.038
DGCR5
DiGeorge syndrome









critical region gene









5 (non-coding)


206579_at
53
NM_006298
459
5.64E−03
−1.038
ZNF192
zinc finger protein









192


205254_x_at
54
AW027359
460
3.05E−03
−1.038
TCF7
transcription factor









7 (T-cell specific,









HMG-box)


210349_at
55
L24959
461
3.07E−03
−1.037
CAMK4
calcium/calmodulin-









dependent protein









kinase IV


208157_at
56
NM_009586
462
9.47E−03
−1.037
SIM2
single-minded









homolog 2









(Drosophila)


214972_at
57
AU144791
463
4.25E−03
−1.037
MGEA5
Meningioma









expressed antigen 5









(hyaluronidase)


207334_s_at
58
NM_003242
464
2.70E−03
−1.037
TGFBR2
transforming growth









factor, beta receptor









II (70/80 kDa)


217666_at
59
AW974481
465
1.06E−03
−1.037
—
—


215771_x_at
60
X15786
466
1.14E−03
−1.036
RET
ret proto-oncogene









(multiple endocrine









neoplasia and









medullary thyroid









carcinoma


216139_s_at
61
AL031718
467
7.40E−03
−1.036
MAPK8IP3
mitogen-activated









protein kinase 8









interacting protein 3


216902_s_at
62
AF001549
468
4.86E−05
−1.036
RRN3
RRN3 RNA









polymerase I









transcription factor









homolog (yeast)


208265_at
63
NM_020161
469
6.06E−04
−1.036
DKFZp547H025
hypothetical protein









DKFZp547H025


209456_s_at
64
AB033281
470
3.49E−03
−1.036
FBXW11
F-box and WD-40









domain protein 11


217237_at
65
Y10615
471
7.72E−03
−1.035
ZNF423
Zinc finger protein









423


217208_s_at
66
AL121981
472
2.91E−03
−1.035
DLG1
discs, large homolog









1 (Drosophila)


221757_at
67
BE042976
473
1.83E−04
−1.035
MGC17330
HGFL gene ///









HGFL gene


213481_at
68
N92920
474
4.47E−03
−1.035
—
—


219025_at
69
NM_020404
475
2.84E−03
−1.035
CD164L1
CD164 sialomucin-









like 1


215201_at
70
AW166925
476
9.15E−03
−1.034
REPS1
RALBP1 associated









Eps domain









containing 1


205378_s_at
71
NM_015831
477
6.79E−03
−1.034
ACHE
acetylcholinesterase









(YT blood group)


219829_at
72
NM_012278
478
6.24E−03
−1.033
ITGB1BP2
integrin beta 1









binding protein









(melusin) 2


216908_x_at
73
AF001549
468
6.22E−04
−1.033
LOC94431
similar to RNA









polymerase I









transcription factor









RRN3


217184_s_at
74
X52213
479
4.40E−03
−1.033
LTK
leukocyte tyrosine









kinase


211181_x_at
75
AF312386
480
4.63E−03
−1.033
RUNX1
runt-related









transcription factor









1 (acute myeloid









leukemia 1; aml1









oncogene)


214958_s_at
76
AK021738
481
1.62E−03
−1.033
EVER1
epidermodysplasia









verruciformis 1


220369_at
77
NM_017936
482
7.90E−03
−1.033
KIAA2010
KIAA2010


211848_s_at
78
AF006623
483
8.77E−04
−1.033
CEACAM7
carcinoembryonic









antigen-related cell









adhesion molecule 7


221638_s_at
79
AF008937
484
8.81E−03
−1.033
STX16
syntaxin 16


221723_s_at
80
AF243499
485
8.08E−03
−1.033
SLC4A5
solute carrier family









4, sodium









bicarbonate









cotransporter,









member 5


201380_at
81
NM_006371
486
8.45E−04
−1.032
CRTAP
cartilage associated









protein


34063_at
82
AB006533
487
1.03E−03
−1.032
RECQL5
RecQ protein-like 5


214757_at
83
BG178274
488
5.20E−03
−1.032
—
Hypothetical gene









supported by









AK024602


218176_at
84
NM_022149
489
4.04E−03
−1.032
MAGEF1
melanoma antigen,









family F, 1


221977_at
85
AW303460
490
7.35E−03
−1.032
—
—


214161_at
86
BF057458
491
9.56E−03
−1.031
—
—


214857_at
87
AL050035
492
6.21E−03
−1.031
—
MRNA; cDNA









DKFZp566H0124









(from clone









DKFZp566H0124)


216230_x_at
88
M59917
493
1.74E−03
−1.031
—
—


205019_s_at
89
NM_004624
494
3.56E−03
−1.031
VIPR1
vasoactive intestinal









peptide receptor 1


221707_s_at
90
BC006116
495
7.60E−03
−1.031
FLJ10979
hypothetical protein









FLJ10979 ///









hypothetical protein









FLJ10979


208664_s_at
91
AU131711
496
1.15E−04
−1.030
TTC3
tetratricopeptide









repeat domain 3


215167_at
92
BE567032
497
8.69E−03
−1.030
CRSP2
cofactor required for









Sp1 transcriptional









activation, subunit









2, 150 kDa


215437_x_at
93
BE513659
498
4.06E−03
−1.030
BAZ2A
bromodomain









adjacent to zinc









finger domain, 2A


210892_s_at
94
BC004472
499
3.77E−03
−1.029
GTF2I
general transcription









factor II, i


212400_at
95
AL043266
500
1.57E−03
−1.029
MGC50853
hypothetical protein









MGC50853


215137_at
96
H92070
501
5.46E−03
−1.029
KIAA0508
KIAA0508 protein


205750_at
97
NM_004332
502
1.95E−03
−1.028
BPHL
biphenyl hydrolase-









like (serine









hydrolase; breast









epithelial mucin-









associated an


210628_x_at
98
AF051344
503
1.93E−03
−1.028
LTBP4
latent transforming









growth factor beta









binding protein 4


218210_at
99
NM_024619
504
1.42E−03
−1.028
FN3KRP
fructosamine-3-









kinase-related









protein


216784_at
100
AK025422
505
2.68E−03
−1.028
—
Transcribed locus,









weakly similar to









XP_375174.1









hypothetical gene









supported by


201819_at
101
NM_005505
506
7.48E−03
−1.028
SCARB1
scavenger receptor









class B, member 1


214312_at
102
AI693985
507
2.93E−03
−1.028
—
—


215556_at
103
AU158442
508
3.90E−03
−1.028
—
—


221756_at
104
BE042976
473
2.52E−03
−1.027
MGC17330
HGFL gene ///









HGFL gene


216909_at
105
AK021460
509
5.34E−03
−1.027
—
—


220249_at
106
NM_012269
510
8.17E−03
−1.027
HYAL4
hyaluronoglucosaminidase 4


211272_s_at
107
AF064771
511
3.46E−03
−1.027
DGKA
diacylglycerol









kinase, alpha 80 kDa


218651_s_at
108
NM_018357
512
7.30E−03
−1.027
FLJ11196
acheron


218285_s_at
109
NM_020139
513
2.93E−03
−1.027
DHRS6
dehydrogenase/reductase









(SDR family)









member 6


217385_at
110
AL023773
514
2.79E−03
−1.027
—
—


202894_at
111
NM_004444
515
1.52E−03
−1.026
EPHB4
EPH receptor B4


217631_at
112
AI081107
516
6.97E−03
−1.026
IDI2 ///
isopentenyl-








GTPBP4
diphosphate delta









isomerase 2 /// GTP









binding protein 4


220487_at
113
NM_018968
517
9.34E−03
−1.026
SNTG2
syntrophin, gamma 2


203579_s_at
114
AI660619
518
1.98E−03
−1.026
SLC7A6
solute carrier family









7 (cationic amino









acid transporter, y+









system), member 6


215412_x_at
115
AB017007
519
7.10E−04
−1.026
PMS2L2
postmeiotic









segregation









increased 2-like 2


212217_at
116
AU154782
520
1.32E−03
−1.026
KIAA0436
putative prolyl









oligopeptidase


221602_s_at
117
AI084226
521
3.58E−04
−1.026
TOSO
regulator of Fas-









induced apoptosis ///









regulator of Fas-









induced apoptosis


217847_s_at
118
NM_005119
522
8.43E−03
−1.025
THRAP3
thyroid hormone









receptor associated









protein 3


215275_at
119
AW963138
523
9.31E−03
−1.025
T3JAM
TRAF3-interacting









Jun N-terminal









kinase (JNK)-









activating modulator


221951_at
120
AI739035
524
2.65E−03
−1.025
LOC283232
hypothetical protein









LOC283232


213224_s_at
121
AK025724
525
7.84E−05
−1.024
LOC92482
Hypothetical protein









LOC92482


218967_s_at
122
BF112019
526
5.08E−03
−1.024
PTER
phosphotriesterase









related


208442_s_at
123
NM_000051
527
9.87E−04
−1.024
ATM
ataxia telangiectasia









mutated (includes









complementation









groups A, C and D)


203675_at
124
NM_005013
528
5.38E−03
−1.024
NUCB2
nucleobindin 2


207105_s_at
125
NM_005027
529
2.15E−03
−1.023
PIK3R2
phosphoinositide-3-









kinase, regulatory









subunit 2 (p85 beta)


219144_at
126
NM_024025
530
3.23E−03
−1.023
MGC1136
hypothetical protein









MGC1136


212463_at
127
BE379006
531
9.58E−03
−1.023
CD59
CD59 antigen p18-20









(antigen









identified by









monoclonal









antibodies 16.3A5,









EJ16, E


202040_s_at
128
NM_005056
532
8.73E−03
−1.023
JARID1A
Jumonji, AT rich









interactive domain









1A (RBBP2-like)


64432_at
129
W05463
533
4.53E−03
−1.023
FLJ39616
apoptosis related









protein PNAS-1


200965_s_at
130
NM_006720
534
2.98E−03
−1.022
ABLIM1
actin binding LIM









protein 1


205353_s_at
131
NM_002567
535
9.67E−03
−1.022
PBP
prostatic binding









protein


213177_at
132
AB028989
536
9.13E−03
−1.022
MAPK8IP3
mitogen-activated









protein kinase 8









interacting protein 3


218373_at
133
NM_022476
537
6.35E−03
−1.022
FTS
fused toes homolog









(mouse)


208333_at
134
NM_022363
538
4.11E−03
−1.022
LHX5
LIM homeobox 5


206150_at
135
NM_001242
539
8.17E−03
−1.022
TNFRSF7
tumor necrosis









factor receptor









superfamily,









member 7 /// tumor









necrosis factor r


202431_s_at
136
NM_002467
540
7.39E−03
−1.022
MYC
v-myc









myelocytomatosis









viral oncogene









homolog (avian)


207838_x_at
137
NM_020524
541
3.63E−03
−1.021
PBXIP1
pre-B-cell leukemia









transcription factor









interacting protein 1


218325_s_at
138
NM_022105
542
1.46E−03
−1.021
DATF1
death associated









transcription factor 1


218475_at
139
NM_022727
543
1.53E−03
−1.021
HTF9C
HpaII tiny









fragments locus 9C


218670_at
140
NM_025215
544
1.05E−03
−1.021
PUS1
pseudouridylate









synthase 1


205594_at
141
NM_014897
545
8.79E−03
−1.021
KIAA0924
KIAA0924 protein


215411_s_at
142
AL008730
546
7.21E−03
−1.021
C6orf4
chromosome 6 open









reading frame 4


203048_s_at
143
NM_014639
547
2.14E−03
−1.020
KIAA0372
KIAA0372


216885_s_at
144
AK026481
548
4.07E−04
−1.020
WDR42A
WD repeat domain









42A


219767_s_at
145
NM_005111
549
3.79E−03
−1.020
CRYZL1
crystallin, zeta









(quinone reductase)-









like 1


219131_at
146
NM_013319
550
3.21E−03
−1.020
TERE1
transitional epithelia









response protein


213176_s_at
147
AI910869
551
6.21E−03
−1.020
LTBP4
latent transforming









growth factor beta









binding protein 4


208661_s_at
148
D84294
552
4.85E−03
−1.020
TTC3
tetratricopeptide









repeat domain 3


208196_x_at
149
NM_006162
553
9.44E−03
−1.020
NFATC1
nuclear factor of









activated T-cells,









cytoplasmic,









calcineurin-









dependent 1


212178_s_at
150
AK022555
554
2.95E−03
−1.020
POM121 ///
POM121 membrane








LOC340318
glycoprotein (rat) ///









hypothetical protein









LOC340318


221601_s_at
151
AI084226
521
2.54E−03
−1.020
TOSO
regulator of Fas-









induced apoptosis ///









regulator of Fas-









induced apoptosis


222013_x_at
152
BE348837
555
5.16E−03
−1.019
LOC348926
hypothetical protein








///
LOC348926 ///








MGC16279
hypothetical protein








/// SB153 ///
MGC16279 ///








FLJ10661
hypothetica


202524_s_at
153
AI952009
556
5.43E−03
−1.019
SPOCK2
sparc/osteonectin,









cwcv and kazal-like









domains









proteoglycan









(testican) 2


212068_s_at
154
AB011087
557
9.23E−03
−1.019
KIAA0515
KIAA0515


202830_s_at
155
NM_001467
558
5.82E−03
−1.019
SLC37A4
solute carrier family









37 (glycerol-6-









phosphate









transporter),









member 4


209835_x_at
156
BC004372
559
5.79E−03
−1.019
CD44
CD44 antigen









(homing function









and Indian blood









group system)


212257_s_at
157
AW131754
560
1.51E−03
−1.019
SMARCA2
SWI/SNF related,









matrix associated,









actin dependent









regulator of









chromatin, subf


212071_s_at
158
BE968833
561
2.68E−03
−1.019
SPTBN1
spectrin, beta, non-









erythrocytic 1


213322_at
159
AL031778
562
8.31E−04
−1.019
C6orf130
chromosome 6 open









reading frame 130


210645_s_at
160
D83077
563
2.20E−03
−1.019
TTC3
tetratricopeptide









repeat domain 3


202514_at
161
AW139131
564
4.70E−03
−1.018
DLG1
DKFZP586B0319









protein


217122_s_at
162
AL031282
565
4.05E−03
−1.018
SLC35E2
solute carrier family









35, member E2


218440_at
163
NM_020166
566
9.21E−05
−1.018
MCCC1
methylcrotonoyl-









Coenzyme A









carboxylase 1









(alpha)


210707_x_at
164
U38980
567
3.76E−04
−1.018
PMS2L11
postmeiotic









segregation









increased 2-like 11


61734_at
165
AI797684
568
3.19E−03
−1.018
RCN3
reticulocalbin 3, EF-









hand calcium









binding domain


221500_s_at
166
AK026970
569
1.50E−04
−1.018
STX16
syntaxin 16


219422_at
167
NM_0003790
570
6.24E−03
−1.018
—
—


222244_s_at
168
AK000749
571
5.14E−03
−1.018
FLJ20618
hypothetical protein









FLJ20618


212550_at
169
AI149535
572
1.46E−03
−1.018
STAT5B
signal transducer









and activator of









transcription 5B


206544_x_at
170
NM_003070
573
5.64E−03
−1.018
SMARCA2
SWI/SNF related,









matrix associated,









actin dependent









regulator of









chromatin, subf


216380_x_at
171
AC005011
574
3.97E−03
−1.018
—
—


203408_s_at
172
NM_002971
575
6.31E−03
−1.018
SATB1
special AT-rich









sequence binding









protein 1 (binds to









nuclear









matrix/scaffold-ass


218258_at
173
NM_015972
576
2.71E−03
−1.017
POLR1D
polymerase (RNA) I









polypeptide D,









16 kDa


212234_at
174
AL034550
577
5.94E−03
−1.017
ASXL1
additional sex









combs like 1









(Drosophila)


217461_x_at
175
M90355
578
7.56E−03
−1.017
—
—


218428_s_at
176
NM_016316
579
8.87E−03
−1.017
REV1L
REV1-like (yeast)


215667_x_at
177
AI375694
580
1.00E−03
−1.017
PMS2L2 ///
postmeiotic








PMS2L5
segregation









increased 2-like 2 ///









postmeiotic









segregation









increased


220465_at
178
NM_024988
581
2.08E−03
−1.017
FLJ12355
hypothetical protein









FLJ12355


217988_at
179
NM_021178
582
9.05E−03
−1.017
CCNB1IP1
cyclin B1









interacting protein 1


221981_s_at
180
AA702154
583
3.45E−03
−1.017
FLJ12270
hypothetical protein









FLJ12270


212201_at
181
AW274877
584
5.96E−03
−1.017
KIAA0692
KIAA0692 protein


208795_s_at
182
D55716
585
1.81E−03
−1.017
MCM7
MCM7









minichromosome









maintenance









deficient 7 (S. cerevisiae)


208336_s_at
183
NM_004868
586
5.33E−03
−1.017
GPSN2
glycoprotein,









synaptic 2


221499_s_at
184
AK026970
569
8.49E−03
−1.017
STX16
syntaxin 16


202167_s_at
185
NM_022362
587
8.82E−03
−1.017
MMS19L
MMS19-like









(MET18 homolog,









S. cerevisiae)


201065_s_at
186
NM_001518
588
4.02E−03
−1.017
GTF2I ///
general transcription








GTF2IP1
factor II, i /// general









transcription factor









II, i, pseud


211172_x_at
187
AF161075
589
2.00E−03
−1.017
AKAP7
A kinase (PRKA)









anchor protein 7


218707_at
188
NM_018337
590
7.16E−03
−1.016
ZNF444
zinc finger protein









444


206770_s_at
189
NM_012243
591
9.43E−03
−1.016
SLC35A3
solute carrier family









35 (UDP-N-









acetylglucosamine









(UDP-GlcNAc)









transporter), mem


200898_s_at
190
AK002091
592
2.97E−03
−1.016
MGEA5
meningioma









expressed antigen 5









(hyaluronidase)


215519_x_at
191
AI081779
593
3.72E−03
−1.016
RUTBC3
RUN and TBC1









domain containing 3


212406_s_at
192
AB028973
594
4.48E−03
−1.016
C20orf36
chromosome 20









open reading frame









36


210826_x_at
193
AF098533
595
4.28E−03
−1.015
RAD17
RAD17 homolog (S. pombe)


218444_at
194
NM_024105
596
6.99E−03
−1.015
ALG12
asparagine-linked









glycosylation 12









homolog (yeast,









alpha-1,6-









mannosyltransferase


221822_at
195
BE544663
597
4.91E−03
−1.015
LOC112869
hypothetical protein









BC011981


220755_s_at
196
NM_016947
598
7.95E−04
−1.015
C6orf48
chromosome 6 open









reading frame 48


218970_s_at
197
NM_015960
599
9.75E−03
−1.015
CUTC
cutC copper









transporter homolog









(E. coli)


218253_s_at
198
NM_006893
600
5.75E−03
−1.015
LGTN
ligatin


221293_s_at
199
NM_022047
601
2.42E−03
−1.015
DEF6
differentially









expressed in FDCP









6 homolog (mouse)


217742_s_at
200
NM_016628
602
5.14E−03
−1.015
WAC
WW domain









containing adaptor









with coiled-coil


207127_s_at
201
NM_021644
603
4.06E−03
−1.014
HNRPH3
heterogeneous









nuclear









ribonucleoprotein









H3 (2H9)


217850_at
202
NM_014366
604
5.81E−03
−1.014
NS
nucleostemin


212505_s_at
203
AL110250
605
8.73E−03
−1.014
KIAA0892
KIAA0892


211971_s_at
204
AI653608
606
2.30E−05
−1.014
LRPPRC
leucine-rich PPR-









motif containing


216387_x_at
205
AL353580
607
5.15E−03
−1.014
—
—


218152_at
206
NM_018200
608
8.22E−03
−1.014
HMG20A
high-mobility group









20A


201788_at
207
NM_007372
609
4.77E−03
−1.014
DDX42
DEAD (Asp-Glu-









Ala-Asp) box









polypeptide 42


201922_at
208
NM_014886
610
6.30E−03
−1.014
TINP1
TGF beta-inducible









nuclear protein 1


212982_at
209
AI621223
611
4.57E−03
−1.014
ZDHHC17
zinc finger, DHHC









domain containing









17


214173_x_at
210
AW514900
612
2.20E−04
−1.014
C19orf2
chromosome 19









open reading frame 2


211937_at
211
NM_001417
613
3.24E−03
−1.014
EIF4B
eukaryotic









translation initiation









factor 4B


216843_x_at
212
U38964
614
6.22E−03
−1.014
—
—


212854_x_at
213
AB051480
615
1.60E−03
−1.013
LOC376745
AG1


212886_at
214
AL080169
616
4.76E−03
−1.013
DKFZP434C171
DKFZP434C171









protein


220607_x_at
215
NM_016397
617
3.15E−03
−1.012
TH1L
TH1-like









(Drosophila)


212132_at
216
AL117499
618
5.52E−04
−1.011
C19orf13
chromosome 19









open reading frame









13


214042_s_at
217
AW071997
619
3.11E−03
−1.011
RPL22
ribosomal protein









L22


212660_at
218
AI735639
620
5.22E−03
−1.011
PHF15
PHD finger protein









15


208944_at
219
D50683
621
5.54E−03
−1.011
—
—


210011_s_at
220
BC000527
433
9.77E−03
−1.010
EWSR1
Ewing sarcoma









breakpoint region 1


211938_at
221
BF247371
622
8.55E−03
−1.010
EIF4B
eukaryotic









translation initiation









factor 4B


220408_x_at
222
NM_017569
623
6.06E−03
−1.010
FAM48A
family with









sequence similarity









48, member A


212114_at
223
BE967207
624
9.73E−03
−1.010
—
Similar to









microtubule-









associated proteins









1A/1B light chain 3


212455_at
224
N36997
625
2.91E−03
−1.009
YT521
splicing factor









YT521-B


212299_at
225
AL117502
626
6.14E−03
−1.009
NEK9
NIMA (never in









mitosis gene a)-









related kinase 9


200005_at
226
NM_003753
627
4.23E−03
−1.008
EIF3S7
eukaryotic









translation initiation









factor 3, subunit 7









zeta, 66/67 kDa ///









eukaryo


200081_s_at
227
BE741754
628
1.02E−03
−1.007
RPS6
ribosomal protein









S6 /// ribosomal









protein S6


213687_s_at
228
BE968801
629
8.26E−03
−1.006
RPL35A
ribosomal protein









L35a


204102_s_at
229
NM_001961
630
5.59E−03
−1.006
EEF2
eukaryotic









translation









elongation factor 2


211666_x_at
230
L22453
631
6.17E−03
−1.006
RPL3
ribosomal protein









L3 /// ribosomal









protein L3


209134_s_at
231
BC000524
632
1.77E−03
−1.005
RPS6
ribosomal protein









S6


221700_s_at
232
AF348700
633
2.98E−04
−1.005
UBA52
ubiquitin A-52









residue ribosomal









protein fusion









product 1 ///









ubiquitin A-52 res


200034_s_at
233
NM_000970
634
9.34E−03
−1.005
RPL6
ribosomal protein









L6 /// ribosomal









protein L6


201254_x_at
234
NM_001010
635
7.37E−03
−1.004
RPS6
ribosomal protein









S6


212734_x_at
235
AI186735
636
8.06E−03
−1.003
RPL13
ribosomal protein









L13


217718_s_at
236
NM_014052
637
6.36E−03
1.005
YWHAB
tyrosine 3-









monooxygenase/tryptophan









5-









monooxygenase









activation protein,









beta pol


208678_at
237
BC004443
638
8.57E−03
1.006
ATP6V1E1
ATPase, H+









transporting,









lysosomal 31 kDa,









V1 subunit E









isoform 1


200633_at
238
NM_018955
639
2.94E−03
1.006
UBB
ubiquitin B ///









ubiquitin B


201318_s_at
239
NM_006471
640
1.52E−03
1.007
MRLC2 ///
myosin regulatory








MRCL3
light chain MRLC2









/// myosin









regulatory light









chain MRCL3


202090_s_at
240
NM_006830
641
3.41E−03
1.008
UQCR
ubiquinol-









cytochrome c









reductase (6.4 kD)









subunit


221474_at
241
U26162
642
7.75E−03
1.008
MRLC2
myosin regulatory









light chain MRLC2


214629_x_at
242
AF320999
643
3.29E−03
1.009
RTN4
reticulon 4


200067_x_at
243
AL078596
644
9.11E−03
1.010
—
—


201899_s_at
244
NM_003336
645
8.17E−03
1.010
UBE2A
ubiquitin-









conjugating enzyme









E2A (RAD6









homolog)


210968_s_at
245
AF333336
646
3.86E−03
1.010
RTN4
reticulon 4


200609_s_at
246
NM_017491
647
5.41E−04
1.010
WDR1
WD repeat domain 1


208805_at
247
BC002979
648
3.97E−03
1.011
PSMA6
proteasome









(prosome,









macropain) subunit,









alpha type, 6


218571_s_at
248
NM_014169
649
5.41E−03
1.011
C14orf123
chromosome 14









open reading frame









123


217730_at
249
NM_022152
650
5.26E−03
1.013
PP1201
PP1201 protein


218520_at
250
NM_013254
651
9.23E−03
1.013
TBK1
TANK-binding









kinase 1


208908_s_at
251
AF327443
652
7.99E−03
1.014
CAST
calpastatin


207467_x_at
252
NM_001750
653
8.54E−03
1.014
CAST
calpastatin


201975_at
253
NM_002956
654
5.39E−03
1.015
RSN
restin (Reed-









Steinberg cell-









expressed









intermediate









filament-associated









protein)


200814_at
254
NM_006263
655
2.49E−04
1.015
PSME1
proteasome









(prosome,









macropain) activator









subunit 1 (PA28









alpha)


203090_at
255
NM_006923
656
4.45E−03
1.015
SDF2
stromal cell-derived









factor 2


201470_at
256
NM_004832
657
2.35E−03
1.015
GSTO1
glutathione S-









transferase omega 1


212586_at
257
AA195244
658
6.42E−03
1.016
CAST
calpastatin


200703_at
258
NM_003746
659
5.88E−03
1.016
DNCL1
dynein, cytoplasmic,









light polypeptide 1


217995_at
259
NM_021199
660
9.02E−03
1.016
SQRDL
sulfide quinone









reductase-like









(yeast)


201346_at
260
NM_024551
661
3.81E−03
1.016
ADIPOR2
adiponectin receptor 2


201609_x_at
261
AL578502
662
1.36E−03
1.016
ICMT
isoprenylcysteine









carboxyl









methyltransferase


202000_at
262
BC002772
663
6.15E−03
1.016
NDUFA6
NADH









dehydrogenase









(ubiquinone) 1 alpha









subcomplex, 6,









14 kDa


202001_s_at
263
BC002772
663
7.78E−03
1.017
NDUFA6
NADH









dehydrogenase









(ubiquinone) 1 alpha









subcomplex, 6,









14 kDa


203880_at
264
NM_005694
664
6.24E−03
1.017
COX17
COX17 homolog,









cytochrome c









oxidase assembly









protein (yeast)


218525_s_at
265
NM_017902
665
9.42E−03
1.017
HIF1AN
hypoxia-inducible









factor 1, alpha









subunit inhibitor


219798_s_at
266
NM_019606
666
3.38E−03
1.017
FLJ20257
hypothetical protein









FLJ20257


208398_s_at
267
NM_004865
667
7.76E−03
1.017
TBPL1
TBP-like 1


218358_at
268
NM_024324
668
6.57E−03
1.017
—
—


203097_s_at
269
NM_014247
669
4.66E−03
1.017
RAPGEF2
Rap guanine









nucleotide exchange









factor (GEF) 2


221598_s_at
270
BC002878
670
7.63E−03
1.017
CRSP8
cofactor required for









Sp1 transcriptional









activation, subunit









8, 34 kDa


209546_s_at
271
AF323540
671
6.71E−03
1.018
APOL1
apolipoprotein L, 1


50400_at
272
AI743990
672
7.35E−03
1.018
PAOX
polyamine oxidase









(exo-N4-amino)


217752_s_at
273
NM_018235
673
4.11E−03
1.018
CNDP2
CNDP dipeptidase 2









(metallopeptidase









M20 family)


201931_at
274
NM_000126
674
6.90E−03
1.018
ETFA
electron-transfer-









flavoprotein, alpha









polypeptide









(glutaric aciduria II)


218567_x_at
275
NM_005700
675
1.00E−02
1.019
DPP3
dipeptidylpeptidase 3


202056_at
276
AW051311
676
8.93E−03
1.019
KPNA1
Karyopherin alpha 1









(importin alpha 5)


218907_s_at
277
NM_023942
677
2.62E−03
1.019
MGC3036
hypothetical protein









MGC3036


213726_x_at
278
AA515698
678
2.22E−03
1.019
TUBB2
tubulin, beta, 2


204448_s_at
279
AF031463
679
7.00E−03
1.019
PDCL
phosducin-like


1405_i_at
280
M21121
680
9.54E−03
1.019
CCL5
chemokine (C-C









motif) ligand 5


212864_at
281
Y16521
681
9.17E−03
1.019
CDS2
CDP-diacylglycerol









synthase









(phosphatidate









cytidylyltransferase) 2


209444_at
282
BC001851
682
1.27E−04
1.019
RAP1GDS1
RAP1, GTP-GDP









dissociation









stimulator 1


208898_at
283
AF077614
683
2.37E−03
1.019
ATP6V1D
ATPase, H+









transporting,









lysosomal 34 kDa,









V1 subunit D


202377_at
284
AW026535
684
5.30E−03
1.019
OBRGRP ///
leptin receptor gene-








LEPR
related protein ///









leptin receptor


209547_s_at
285
BC001043
685
4.14E−03
1.020
SF4
splicing factor 4


202922_at
286
BF676980
686
4.19E−03
1.020
GCLC
glutamate-cysteine









ligase, catalytic









subunit


201403_s_at
287
NM_004528
687
3.64E−03
1.020
MGST3
microsomal









glutathione S-









transferase 3


213154_s_at
288
AI934125
688
2.75E−03
1.020
BICD2
bicaudal D homolog









2 (Drosophila)


215676_at
289
N91109
689
8.42E−03
1.021
BRF1
BRF1 homolog,









subunit of RNA









polymerase III









transcription









initiation factor IIIB


218927_s_at
290
BC002918
690
8.09E−03
1.021
CHST12
carbohydrate









(chondroitin 4)









sulfotransferase 12


213648_at
291
AW614427
691
6.11E−03
1.021
EXOSC7
Exosome









component 7


209593_s_at
292
AF317129
692
8.82E−03
1.021
TOR1B
torsin family 1,









member B (torsin B)


203731_s_at
293
NM_014569
693
1.78E−03
1.022
ZFP95
zinc finger protein









95 homolog (mouse)


201234_at
294
NM_004517
694
2.90E−03
1.022
ILK
integrin-linked









kinase


214817_at
295
BE783668
695
8.61E−03
1.022
UNC13A
unc-13 homolog A









(C. elegans)


201761_at
296
NM_006636
696
3.83E−03
1.022
MTHFD2
methylene









tetrahydrofolate









dehydrogenase









(NAD+ dependent),









methenyltetrahydrofol


205467_at
297
NM_001230
697
6.50E−03
1.022
CASP10
caspase 10,









apoptosis-related









cysteine protease


222318_at
298
AI744673
698
9.20E−03
1.022
FLJ45850
FLJ45850 protein


209933_s_at
299
AF020314
699
6.24E−03
1.022
CMRF-35H
leukocyte









membrane antigen


200734_s_at
300
BG341906
700
4.33E−03
1.023
ARF3
ADP-ribosylation









factor 3


219899_x_at
301
NM_014434
701
7.97E−03
1.023
NDOR1
NADPH dependent









diflavin









oxidoreductase 1


221563_at
302
N36770
702
3.41E−03
1.024
DUSP10
dual specificity









phosphatase 10


65517_at
303
AA910946
703
4.00E−03
1.024
AP1M2
adaptor-related









protein complex 1,









mu 2 subunit


205126_at
304
NM_006296
704
8.89E−03
1.024
VRK2
vaccinia related









kinase 2


200696_s_at
305
NM_000177
705
8.30E−03
1.024
GSN
gelsolin









(amyloidosis,









Finnish type)


208790_s_at
306
AF312393
706
4.43E−03
1.025
PTRF
polymerase I and









transcript release









factor


205115_s_at
307
NM_016196
707
8.06E−03
1.025
RBM19
RNA binding motif









protein 19


213982_s_at
308
BG107203
708
8.15E−03
1.025
RABGAP1L
RAB GTPase









activating protein 1-









like


206992_s_at
309
NM_015684
709
2.03E−03
1.025
ATP5S
ATP synthase, H+









transporting,









mitochondrial F0









complex, subunit s









(factor B)


201060_x_at
310
AI537887
710
9.12E−04
1.025
STOM
stomatin


209278_s_at
311
L27624
711
7.18E−03
1.026
TFPI2
tissue factor









pathway inhibitor 2


210542_s_at
312
BC000585
712
4.38E−03
1.026
SLCO3A1
solute carrier









organic anion









transporter family,









member 3A1


202006_at
313
NM_002835
713
3.01E−03
1.026
PTPN12
protein tyrosine









phosphatase, non-









receptor type 12


210557_x_at
314
M76453
714
2.04E−03
1.026
CSF1
colony stimulating









factor 1









(macrophage)


210796_x_at
315
D86359
715
2.95E−03
1.027
SIGLEC6
sialic acid binding









Ig-like lectin 6


220825_s_at
316
NM_018240
716
9.74E−03
1.027
KIRREL
kin of IRRE like









(Drosophila)


202378_s_at
317
NM_017526
717
2.72E−03
1.027
OBRGRP
leptin receptor gene-









related protein


210241_s_at
318
AB007458
718
3.02E−03
1.027
TP53AP1
TP53 activated









protein 1


213069_at
319
AI148659
719
2.90E−03
1.027
—
—


216034_at
320
AA558468
720
9.90E−03
1.027
SUHW1
suppressor of hairy









wing homolog 1









(Drosophila)


210793_s_at
321
U41815
721
2.64E−03
1.029
NUP98
nucleoporin 98 kDa


207375_s_at
322
NM_002189
722
3.71E−03
1.029
IL15RA
interleukin 15









receptor, alpha


206247_at
323
NM_005931
723
2.70E−03
1.029
MICB
MHC class I









polypeptide-related









sequence B


217078_s_at
324
AJ010102
724
9.65E−03
1.029
CMRF-35H
leukocyte









membrane antigen


219257_s_at
325
NM_021972
725
1.62E−03
1.030
SPHK1
sphingosine kinase 1


204781_s_at
326
NM_000043
726
8.44E−03
1.030
TNFRSF6
tumor necrosis









factor receptor









superfamily,









member 6


221536_s_at
327
AL136897
727
2.28E−03
1.030
FLJ11301
hypothetical protein









FLJ11301


209468_at
328
AB017498
728
8.91E−03
1.030
LRP5
low density









lipoprotein receptor-









related protein 5


201061_s_at
329
M81635
729
1.46E−04
1.030
STOM
stomatin


203499_at
330
NM_004431
730
6.49E−03
1.031
EPHA2
EPH receptor A2


213324_at
331
AK024281
731
7.63E−03
1.031
SRC
v-src sarcoma









(Schmidt-Ruppin A-









2) viral oncogene









homolog (avian)


221535_at
332
AL136897
727
3.81E−03
1.031
FLJ11301
hypothetical protein









FLJ11301


219938_s_at
333
NM_024430
732
3.19E−03
1.032
PSTPIP2
proline-serine-









threonine









phosphatase









interacting protein 2


213787_s_at
334
AV702405
733
1.78E−03
1.032
EBP
emopamil binding









protein (sterol









isomerase)


219592_at
335
NM_024596
734
8.43E−03
1.032
MCPH1
microcephaly,









primary autosomal









recessive 1


200637_s_at
336
AI762627
735
5.52E−03
1.033
PTPRF
protein tyrosine









phosphatase,









receptor type, F


202193_at
337
NM_005569
736
4.88E−03
1.033
LIMK2
LIM domain kinase 2


214859_at
338
AI635302
737
8.68E−03
1.034
FSTL4
follistatin-like 4


209213_at
339
BC002511
738
5.69E−03
1.034
CBR1
carbonyl reductase 1


218945_at
340
NM_024109
739
3.75E−03
1.034
MGC2654
hypothetical protein









MGC2654


220471_s_at
341
NM_025107
740
8.29E−03
1.035
MYCT1
myc target 1


59625_at
342
AI912351
741
5.19E−03
1.035
NOL3
nucleolar protein 3









(apoptosis repressor









with CARD









domain)


207233_s_at
343
NM_000248
742
2.93E−03
1.035
MITF
microphthalmia-









associated









transcription factor


214070_s_at
344
AW006935
743
5.14E−03
1.036
ATP10B
ATPase, Class V,









type 10B


219785_s_at
345
NM_024735
744
5.84E−03
1.036
FBXO31
F-box protein 31


220684_at
346
NM_013351
745
3.34E−03
1.037
TBX21
T-box 21


202245_at
347
AW084510
746
7.02E−03
1.037
LSS
lanosterol synthase









(2,3-oxidosqualene-









lanosterol cyclase)


207298_at
348
NM_006632
747
6.68E−03
1.037
SLC17A3
solute carrier family









17 (sodium









phosphate), member 3


220201_at
349
NM_018835
748
3.85E−03
1.038
MNAB
membrane









associated DNA









binding protein


203208_s_at
350
BF214329
749
6.89E−04
1.038
CHPPR
likely ortholog of









chicken chondrocyte









protein with a poly-









proline region


207033_at
351
NM_005142
750
3.42E−04
1.039
GIF
gastric intrinsic









factor (vitamin B









synthesis)


204929_s_at
352
NM_006634
751
6.38E−03
1.039
VAMP5
vesicle-associated









membrane protein 5









(myobrevin)


209735_at
353
AF098951
752
7.05E−03
1.039
ABCG2
ATP-binding









cassette, sub-family









G (WHITE),









member 2


209234_at
354
BF939474
753
3.31E−04
1.039
KIF1B
kinesin family









member 1B


210102_at
355
BC001234
754
5.97E−03
1.040
LOH11CR2A
loss of









heterozygosity, 11,









chromosomal region









2, gene A


204114_at
356
NM_007361
755
4.45E−03
1.041
NID2
nidogen 2









(osteonidogen)


203344_s_at
357
NM_002894
756
1.33E−03
1.041
RBBP8
retinoblastoma









binding protein 8


216891_at
358
U00956
757
5.03E−03
1.042
—
—


221680_s_at
359
AF147782
758
9.08E−03
1.042
ETV7
ets variant gene 7









(TEL2 oncogene)


202087_s_at
360
NM_001912
759
3.63E−03
1.042
CTSL
cathepsin L


218243_at
361
NM_025158
760
6.62E−03
1.043
RUFY1
RUN and FYVE









domain containing 1


201980_s_at
362
NM_012425
761
5.59E−03
1.044
RSU1
Ras suppressor









protein 1


221280_s_at
363
NM_019619
762
4.65E−04
1.044
PARD3
par-3 partitioning









defective 3 homolog









(C. elegans)


205108_s_at
364
NM_000384
763
2.36E−04
1.045
APOB
apolipoprotein B









(including Ag(x)









antigen)


204241_at
365
BF055171
764
6.95E−04
1.045
ACOX3
acyl-Coenzyme A









oxidase 3, pristanoyl


201278_at
366
N21202
765
2.71E−03
1.049
DAB2
Disabled homolog









2, mitogen-









responsive









phosphoprotein









(Drosophila)


202067_s_at
367
AI861942
766
1.95E−03
1.050
LDLR
low density









lipoprotein receptor









(familial









hypercholesterolemia)


202085_at
368
NM_004817
767
3.10E−03
1.051
TJP2
tight junction









protein 2 (zona









occludens 2)


214157_at
369
AA401492
768
4.90E−03
1.053
GNAS
GNAS complex









locus


221526_x_at
370
AF196185
769
2.72E−03
1.053
PARD3
par-3 partitioning









defective 3 homolog









(C. elegans)


207738_s_at
371
NM_013436
770
2.00E−03
1.054
NCKAP1
NCK-associated









protein 1


204769_s_at
372
NM_000544
771
3.15E−03
1.054
TAP2
transporter 2, ATP-









binding cassette,









sub-family B









(MDR/TAP)


216693_x_at
373
AL133102
772
6.34E−03
1.055
HDGFRP3
hepatoma-derived









growth factor,









related protein 3


202068_s_at
374
NM_000527
773
5.72E−03
1.057
LDLR
low density









lipoprotein receptor









(familial









hypercholesterolemia)


202743_at
375
BE622627
774
7.96E−04
1.061
PIK3R3
phosphoinositide-3-









kinase, regulatory









subunit 3 (p55,









gamma)


206638_at
376
NM_000867
775
7.47E−03
1.061
HTR2B
5-









hydroxytryptamine









(serotonin) receptor









2B


31874_at
377
Y07846
776
2.32E−03
1.061
GAS2L1
growth arrest-









specific 2 like 1


201798_s_at
378
NM_013451
777
5.01E−03
1.062
FER1L3
fer-1-like 3,









myoferlin (C. elegans)


219288_at
379
NM_020685
778
6.67E−04
1.063
C3orf14
chromosome 3 open









reading frame 14


220167_s_at
380
NM_015369
779
5.44E−04
1.064
TP53TG3
TP53TG3 protein


211354_s_at
381
U52913
780
1.67E−03
1.066
LEPR
leptin receptor


213317_at
382
AL049313
781
7.15E−03
1.067
CLIC5
Chloride









intracellular channel 5


212390_at
383
AB007923
782
8.15E−03
1.070
PDE4DIP
phosphodiesterase









4D interacting









protein









(myomegalin)


212062_at
384
AB014511
783
8.65E−03
1.070
ATP9A
ATPase, Class II,









type 9A


203337_x_at
385
NM_004763
784
6.03E−03
1.071
ITGB1BP1
integrin beta 1









binding protein 1


210029_at
386
M34455
785
5.97E−03
1.073
INDO
indoleamine-pyrrole









2,3 dioxygenase


206049_at
387
NM_003005
786
8.60E−03
1.074
SELP
selectin P (granule









membrane protein









140 kDa, antigen









CD62)


202949_s_at
388
NM_001450
787
6.99E−03
1.076
FHL2
four and a half LIM









domains 2


211864_s_at
389
AF207990
788
7.27E−03
1.076
FER1L3
fer-1-like 3,









myoferlin (C. elegans)


206254_at
390
NM_001963
789
5.83E−03
1.077
EGF
epidermal growth









factor (beta-









urogastrone)


210073_at
391
L32867
790
5.66E−04
1.077
SIAT8A
sialyltransferase 8A









(alpha-N-









acetylneuraminate:









alpha-2,8-









sialyltransferase,









GD


209524_at
392
AK001280
791
1.03E−03
1.080
HDGFRP3
hepatoma-derived









growth factor,









related protein 3


219759_at
393
NM_022350
792
8.03E−04
1.081
LRAP
leukocyte-derived









arginine









aminopeptidase


202112_at
394
NM_000552
793
1.22E−03
1.082
VWF
von Willebrand









factor


219410_at
395
NM_018004
794
4.81E−03
1.083
FLJ10134
hypothetical protein









FLJ10134


203819_s_at
396
AU160004
795
3.58E−03
1.088
IMP-3
IGF-II mRNA-









binding protein 3


212730_at
397
AK026420
796
3.19E−03
1.090
DMN
desmuslin


220122_at
398
NM_024717
797
5.82E−03
1.090
MCTP1
multiple C2-









domains with two









transmembrane









regions 1


208782_at
399
BC000055
798
2.14E−03
1.095
FSTL1
follistatin-like 1


202468_s_at
400
NM_003798
799
1.36E−03
1.096
CTNNAL1
catenin (cadherin-









associated protein),









alpha-like 1


207018_s_at
401
NM_004163
800
4.02E−03
1.101
RAB27B
RAB27B, member









RAS oncogene









family


210354_at
402
M29383
801
4.60E−03
1.102
—
—


201110_s_at
403
NM_003246
802
9.38E−03
1.111
THBS1
thrombospondin 1


207808_s_at
404
NM_000313
803
2.92E−03
1.114
PROS1
protein S (alpha)


205612_at
405
NM_007351
804
7.97E−03
1.118
MMRN1
multimerin 1


214073_at
406
BG475299
805
2.37E−03
1.121
CTTN
cortactin





Presented are the 406 differentiating genes given by the gene name and description, the Affymetrix probeset identification number, and a representative GenBank Accession number between BMS and typical RRMS patients. p-value - statistical significance by ANOVA; Log Fold Change = refers to the logarithms fold change between the expression level of a polynucleotide in a blood sample of a BMS subject as compared to the expression level in a blood sample of a typical RRMS subject: The (−) sign means that the polynucleotide is downregulated (decreased in level) in BMS as compared to typical RRMS subjects; and the (+) sign means that the polynucleotide is upregulated (increased in level) in BMS as compared to typical RRMS subjects.






Table 2 hereinbelow discloses additional polynucleotides (RNA alternative transcripts) of the above identified 406 genes which expression level is differentiating between typical RRMS and BMS.











TABLE 2






SEQ





PROBESET
ID NO:
Refseq Ids/SEQ ID NO:
REFSEQ_UNIGENE
REFSEQ_BAND


























216683_at
1
—/







—
—


219629_at
2
NM_017911/







C22ORF8
Hs.265018




997











210379_s_at
3
NM_012290/







TLK1
Hs.470586




948











213472_at
4
—/







—
—


219700_at
5
NM_020405/







PLXDC1
Hs.125036




1022











211077_s_at
6
NM_012290/







TLK1
Hs.470586




948











210969_at
7
—/







—
—


216298_at
8
—/







—
—


219834_at
9
NM_024744/







ALS2CR8
Hs.444982




1051











215262_at
10
—/







—
—


220715_at
11
—/







—
—


207164_s_at
12
NM_205768/
NM_006352/






ZNF238,
Hs.69997,




1103
1133






ZNF238,
Hs.69997,


220236_at
13
NM_017990/







PDPR
Hs.461183




999











203939_at
14
NM_002526/







NT5E
Hs.153952




865











216945_x_at
15
NM_015148/







PASK
Hs.397891




970











203913_s_at
16
NM_000860/







HPGD
Hs.77348




834











204864_s_at
17
NM_175767/
NM_002183/






IL6ST,
Hs.532082,




1088
1189






IL6ST,
Hs.532082,


215698_at
18
—/







—
—


213534_s_at
19
NM_015148/







PASK
Hs.397891




970











210948_s_at
20
NM_016269/







LEF1
Hs.125132




988











220458_at
21
NM_018038/







FLJ10246
Hs.274274




1001











212093_s_at
22
NM_020749/
NM_001001931/
NM_001001925/
NM_001001924/
NM_001001927/



MTUS1,
Hs.7946,




1025
1114
1182
1216
1234



MTUS1,
Hs.7946,












MTUS1,
Hs.7946,












MTUS1,
Hs.7946,












MTUS1,
Hs.7946,


207120_at
23
NM_022103/







FLJ14011
Hs.433473




1033











204787_at
24
NM_007268/







VSIG4
Hs.8904




939











215512_at
25
—/







—
—


46947_at
26
—/







—
—


210012_s_at
27
—/







—
—


210192_at
28
NM_006095/







ATP8A1
Hs.435052




918











207603_at
29
—/







—
—


218584_at
30
NM_024549/







FLJ21127
Hs.211511




1047











213470_s_at
31
NM_005520/







HNRPH1
Hs.202166




912











210461_s_at
32
NM_006720/
NM_002313/
NM_001003407/
NM_001003408/




ABLIM1,
Hs.438236,




930
1120
1187
1232




ABLIM1,
Hs.438236,












ABLIM1,
Hs.438236,












ABLIM1,
Hs.438236,


217656_at
33
—/







—
—


215177_s_at
34
NM_000210/







ITGA6
Hs.133397




826











204498_s_at
35
NM_001116/







ADCY9
Hs.391860




848











216419_at
36
—/







—
—


204019_s_at
37
NM_015677/







SH3YL1
Hs.515951




979











214850_at
38
NM_207331/







LOC153561
Hs.545578




1106











200672_x_at
39
NM_003128/







SPTBN1
Hs.503178




872











220179_at
40
NM_022357/







DPEP3
Hs.302028




1037











208591_s_at
41
NM_000922/







PDE3B
Hs.445711




835











219199_at
42
NM_014423/







AF5Q31
Hs.519313




958











216959_x_at
43
NM_005010/







NRCAM
Hs.21422




899











215041_s_at
44
NM_015296/







DOCK9
Hs.314413




973











213447_at
45
—/







—
—


218532_s_at
46
NM_019000/







FLJ20152
Hs.481704




1016











220485_s_at
47
NM_018556/
NM_080816/






SIRPB2,
Hs.50716,




1011
1150






SIRPB2,
Hs.50716,


220442_at
48
NM_003774/







GALNT4
Hs.534374




881











211856_x_at
49
NM_006139/







CD28
Hs.1987




919











206828_at
50
NM_003328/







TXK
Hs.479669




875











214447_at
51
NM_005238/







ETS1
Hs.369438




907











215244_at
52
—/







—
—


206579_at
53
NM_006298/







ZNF192
Hs.57679




922











205254_x_at
54
NM_201632/
NM_201634/
NM_213648/
NM_003202/




TCF7,
Hs.519580,




1099
1173
1215
1219




TCF7,
Hs.519580,












TCF7,
Hs.519580,












TCF7,
Hs.519580,


210349_at
55
NM_001744/







CAMK4
Hs.220629




856











208157_at
56
NM_009586/







SIM2
Hs.146186




942











214972_at
57
—/







—
—


207334_s_at
58
NM_003242/







TGFBR2
Hs.82028




873











217666_at
59
—/







—
—


215771_x_at
60
NM_020975/
NM_020630/






RET,
Hs.350321,




1026
1143






RET,
Hs.350321,


216139_s_at
61
NM_033392/
NM_015133/






MAPK8IP3,
Hs.207763,




1057
1138






MAPK8IP3,
Hs.207763,


216902_s_at
62
NM_018427/







RRN3
Hs.460078




1010











208265_at
63
NM_020161/







DKFZP547H025
Hs.283092




1019











209456_s_at
64
NM_033645/
NM_033644/
NM_012300/





FBXW11,
Hs.484138,




1059
1148
1193





FBXW11,
Hs.484138,












FBXW11,
Hs.484138,


217237_at
65
—/







—
—


217208_s_at
66
NM_004087/







DLG1
Hs.292549




885











221757_at
67
NM_052880/







MGC17330
Hs.26670




1060











213481_at
68
—/







—
—


219025_at
69
NM_020404/







CD164L1
Hs.195727




1021











215201_at
70
—/







—
—


205378_s_at
71
NM_000665/
NM_015831/






ACHE,
Hs.154495,




833
1139






ACHE,
Hs.154495,


219829_at
72
NM_012278/







ITGB1BP2
Hs.109999




947











216908_x_at
73
NM_018427/
NM_145237/






RRN3,
Hs.460078,




1010
1156






LOC94431,
Hs.546468,


217184_s_at
74
NM_206961/
NM_002344/






LTK,
Hs.434481,




1105
1121






LTK,
Hs.434481,


211181_x_at
75
NM_001001890/
NM_001754/






RUNX1,
Hs.149261,




843
1179






RUNX1,
Hs.149261,


214958_s_at
76
NM_007267/







EVER1
Hs.16165




938











220369_at
77
NM_017936/







KIAA2010
Hs.533887




998











211848_s_at
78
NM_006890/







CEACAM7
Hs.74466




932











221638_s_at
79
NM_003763/
NM_001001434/
NM_001001433/





STX16,
Hs.307913,




880
842
1181





STX16,
Hs.307913,












STX16,
Hs.307913,


221723_s_at
80
NM_021196/
NM_133479/
NM_133478/
NM_033323/




SLC4A5,
Hs.469033,




1028
1152
1201
1223




SLC4A5,
Hs.469033,












SLC4A5,
Hs.469033,












SLC4A5,
Hs.469033,


201380_at
81
NM_006371/







CRTAP
Hs.517888




923











34063_at
82
NM_001003715/
NM_001003716/
NM_004259/





RECQL5,
Hs.514480,




845
1178
1191





RECQL5,
Hs.514480,












RECQL5,
Hs.514480,


214757_at
83
—/







—
—


218176_at
84
NM_022149/







MAGEF1
Hs.306123




1034











221977_at
85
—/







—
—


214161_at
86
—/







—
—


214857_at
87
—/







—
—


216230_x_at
88
NM_000543/
NM_001007593/






SMPD1,
Hs.498173,




829
1118






SMPD1,
Hs.498173,


205019_s_at
89
NM_004624/







VIPR1
Hs.348500




893











221707_s_at
90
NM_018289/







FLJ10979
Hs.461819




1006











208664_s_at
91
NM_001001894/
NM_003316/






TTC3,
Hs.368214,




844
1180






TTC3,
Hs.368214,


215167_at
92
—/







—
—


215437_x_at
93
NM_013449/







BAZ2A
Hs.314263




955











210892_s_at
94
NM_001518/
NM_033001/
NM_033000/
NM_032999/




GTF2I,
Hs.520459,




854
1146
1198
1222




GTF2I,
Hs.520459,












GTF2I,
Hs.520459,












GTF2I,
Hs.520459,


212400_at
95
NM_203305/







EEIG1
Hs.460208




1101











215137_at
96
—/







—
—


205750_at
97
NM_004332/







BPHL
Hs.10136




888











210628_x_at
98
NM_003573/







LTBP4
Hs.466766




877











218210_at
99
NM_024619/







FN3KRP
Hs.31431




1050











216784_at
100
—/







—
—


201819_at
101
NM_005505/







SCARB1
Hs.298813




911











214312_at
102
—/







—
—


215556_at
103
—/







—
—


221756_at
104
NM_052880/







MGC17330
Hs.26670




1060











216909_at
105
—/







—
—


220249_at
106
NM_012269/







HYAL4
Hs.28673




946











211272_s_at
107
NM_201554/
NM_001345/
NM_201445/
NM_201444/




DGKA,
Hs.524488,




1098
1119
1211
1229




DGKA,
Hs.524488,












DGKA,
Hs.524488,












DGKA,
Hs.524488,


218651_s_at
108
NM_018357/







FLJ11196
Hs.416755




1008











218285_s_at
109
NM_020139/







DHRS6
Hs.124696




1018











217385_at
110
—/







—
—


202894_at
111
NM_004444/







EPHB4
Hs.437008




890











217631_at
112
—/







—
—


220487_at
113
NM_018968/







SNTG2
Hs.148819




1015











203579_s_at
114
NM_003983/







SLC7A6
Hs.351571




884











215412_x_at
115
NM_005395/
NM_001003686/
NM_001003687/
NM_174930/
NM_002679/
NM_000535/


PMS2L3,
Hs.549057,




908
1115
1183
1086
867
828


PMS2L3,
Hs.549057,












PMS2L3,
Hs.549057,












PMS2L5,
Hs.397073,












POM121,
Hs.488624,












PMS2,
Hs.487470,


212217_at
116
—/







—
—


221602_s_at
117
NM_005449/







TOSO
Hs.58831




909











217847_s_at
118
NM_005119/







THRAP3
Hs.160211




905











215275_at
119
—/







—
—


221951_at
120
NM_174940/







LOC283232
Hs.448664




1087











213224_s_at
121
—/







—
—


218967_s_at
122
NM_030664/
NM_001001484/






PTER,
Hs.444321,




1055
1113






PTER,
Hs.444321,


208442_s_at
123
NM_138292/
NM_000051/






ATM,
Hs.435561,




1069
1109






ATM,
Hs.435561,


203675_at
124
NM_005013/







NUCB2
Hs.128686




900











207105_s_at
125
NM_005027/







PIK3R2
Hs.371344




901











219144_at
126
NM_024025/







MGC1136
Hs.8719




1042











212463_at
127
NM_000611/
NM_203331/
NM_203329/
NM_203330/




CD59,
Hs.278573,




831
1175
1212
1230




CD59,
Hs.278573,












CD59,
Hs.278573,












CD59,
Hs.278573,


202040_s_at
128
NM_005056/







JARID1A
Hs.76272




902











64432_at
129
NM_016534/







FLJ39616
Hs.333120




991











200965_s_at
130
NM_006720/
NM_002313/
NM_001003407/
NM_001003408/




ABLIM1,
Hs.438236,




930
1120
1187
1232




ABLIM1,
Hs.438236,












ABLIM1,
Hs.438236,












ABLIM1,
Hs.438236,


205353_s_at
131
NM_002567/







PBP
Hs.433863




866











213177_at
132
NM_033392/
NM_015133/






MAPK8IP3,
Hs.207763,




1057
1138






MAPK8IP3,
Hs.207763,


218373_at
133
NM_022476/







FTS
Hs.380897




1040











208333_at
134
NM_022363/







LHX5
Hs.302029




1039











206150_at
135
NM_001242/







TNFRSF7
Hs.355307




851











202431_s_at
136
NM_002467/







MYC
Hs.202453




863











207838_x_at
137
NM_020524/







PBXIP1
Hs.505806




1023











218325_s_at
138
NM_080797/
NM_080796/
NM_022105/





DATF1,
Hs.551527,




1062
1149
1197





DATF1,
Hs.551527,












DATF1,
Hs.551527,


218475_at
139
NM_182984/
NM_022727/






HTF9C,
Hs.549133,




1090
1144






HTF9C,
Hs.549133,


218670_at
140
NM_025215/







PUS1
Hs.507295




1053











205594_at
141
NM_014897/







ZNF652
Hs.463375




967











215411_s_at
142
NM_147200/
NM_147686/






C6ORF4,
Hs.486228,




1076
1160






C6ORF4,
Hs.486228,


203048_s_at
143
NM_014639/







KIAA0372
Hs.482868




962











216885_s_at
144
NM_015726/







WDR42A
Hs.492236




982











219767_s_at
145
NM_005111/
NM_145311/
NM_145858/





CRYZL1,
Hs.352671,




903
1157
1202





CRYZL1,
Hs.352671,












CRYZL1,
Hs.352671,


219131_at
146
NM_013319/







TERE1
Hs.522933




953











213176_s_at
147
NM_003573/







LTBP4
Hs.466766




877











208661_s_at
148
NM_001001894/
NM_003316/






TTC3,
Hs.368214,




844
1180






TTC3,
Hs.368214,


208196_x_at
149
NM_172388/
NM_172389/
NM_172387/
NM_006162/




NFATC1,
Hs.534074,




1083
1164
1206
1220




NFATC1,
Hs.534074,












NFATC1,
Hs.534074,












NFATC1,
Hs.534074,


212178_s_at
150
NM_172020/







POM121
Hs.488624




1081











221601_s_at
151
NM_005449/







TOSO
Hs.58831




909











222013_x_at
152
NM_152563/
NM_032916/
NM_018172/





FLJ10661,
Hs.61142,




1077
1145
1196





MGC16279,
Hs.458413,












FLJ10661,
Hs.61142,


202524_s_at
153
NM_014767/







SPOCK2
Hs.523009




963











212068_s_at
154
—/







—
—


202830_s_at
155
NM_001467/







SLC37A4
Hs.132760




853











209835_x_at
156
NM_001001391/
NM_001001390/
NM_001001389/
NM_000610/
NM_001001392/



CD44,
Hs.502328,




841
1177
1186
1231
1233



CD44,
Hs.502328,












CD44,
Hs.502328,












CD44,
Hs.502328,












CD44,
Hs.502328,


212257_s_at
157
NM_139045/
NM_003070/






SMARCA2,
Hs.298990,




1071
1124






SMARCA2,
Hs.298990,


212071_s_at
158
—/







—
—


213322_at
159
NM_145063/







C6ORF130
Hs.549281




1075











210645_s_at
160
NM_001001894/
NM_003316/






TTC3,
Hs.368214,




844
1180






TTC3,
Hs.368214,


202514_at
161
—/







—
—


217122_s_at
162
NM_014854/







SLC35E2
Hs.551612




964











218440_at
163
NM_020166/







MCCC1
Hs.47649




1020











210707_x_at
164
NM_174930/
NM_002679/
NM_005395/
NM_001003686/
NM_001003687/
NM_000535/


PMS2L5,
Hs.397073,




1086
867
908
1115
1183
828


POM121,
Hs.488624,












PMS2L3,
Hs.549057,












PMS2L3,
Hs.549057,












PMS2L3,
Hs.549057,












PMS2,
Hs.487470,


61734_at
165
NM_020650/







RCN3
Hs.439184




1024











221500_s_at
166
NM_001001434/
NM_003763/
NM_001001433/





STX16,
Hs.307913,




842
880
1181





STX16,
Hs.307913,












STX16,
Hs.307913,


219422_at
167
—/







—
—


222244_s_at
168
NM_017903/







FLJ20618
Hs.551545




996











212550_at
169
NM_012448/







STAT5B
Hs.132864




950











206544_x_at
170
NM_139045/
NM_003070/






SMARCA2,
Hs.298990,




1071
1124






SMARCA2,
Hs.298990,


216380_x_at
171
NM_001031/







RPS28
Hs.546293




847











203408_s_at
172
NM_002971/







SATB1
Hs.517717




870











218258_at
173
NM_015972/







MGC9850
Hs.507584




984











212234_at
174
NM_015338/







ASXL1
Hs.374043




976











217461_x_at
175
NM_001207/







BTF3
Hs.529798




849











218428_s_at
176
NM_016316/







REV1L
Hs.443077




989











215667_x_at
177
NM_002679/
NM_174930/
NM_005395/
NM_001003686/
NM_001003687/



POM121,
Hs.488624,




867
1086
908
1115
1183



PMS2L5,
Hs.397073,












PMS2L3,
Hs.549057,












PMS2L3,
Hs.549057,












PMS2L3,
Hs.549057,


220465_at
178
—/







—
—


217988_at
179
NM_021178/
NM_182852/
NM_182851/
NM_182849/




CCNB1IP1,
Hs.107003,




1027
1169
1208
1227




CCNB1IP1,
Hs.107003,












CCNB1IP1,
Hs.107003,












CCNB1IP1,
Hs.107003,


221981_s_at
180
NM_030581/







FLJ12270
Hs.280951




1054











212201_at
181
—/







—
—


208795_s_at
182
NM_005916/
NM_182776/






MCM7,
Hs.438720,




916
1168






MCM7,
Hs.438720,


208336_s_at
183
NM_004868/
NM_138501/






GPSN2,
Hs.515642,




898
1153






GPSN2,
Hs.515642,


221499_s_at
184
NM_001001434/
NM_003763/
NM_001001433/





STX16,
Hs.307913,




842
880
1181





STX16,
Hs.307913,












STX16,
Hs.307913,


202167_s_at
185
NM_022362/







MMS19L
Hs.500721




1038











201065_s_at
186
NR_002206/
NM_001518/
NM_033001/
NM_033000/
NM_032999/



—,
—,




1107
854
1146
1198
1222



GTF2I,
Hs.520459,












GTF2I,
Hs.520459,












GTF2I,
Hs.520459,












GTF2I,
Hs.520459,


211172_x_at
187
NM_004842/
NM_138633/
NM_016377/





AKAP7,
Hs.486483,




896
1154
1194





AKAP7,
Hs.486483,












AKAP7,
Hs.486483,


218707_at
188
NM_018337/







ZNF444
Hs.24545




1007











206770_s_at
189
NM_012243/







SLC35A3
Hs.448979




945











200898_s_at
190
NM_012215/







MGEA5
Hs.500842




943











215519_x_at
191
NM_015705/







RUTBC3
Hs.474914




981











212406_s_at
192
NM_018257/







C20ORF36
Hs.473317




1005











210826_x_at
193
NM_133341/
NM_133339/
NM_133340/
NM_133344/
NM_133342/
NM_133343/
NM_133338/
NM_002873/
RAD17,
Hs.16184,




1066
1151
1200
1224
1237
1239
1243
1246
RAD17,
Hs.16184,












RAD17,
Hs.16184,












RAD17,
Hs.16184,












RAD17,
Hs.16184,












RAD17,
Hs.16184,












RAD17,
Hs.16184,












RAD17,
Hs.16184,


218444_at
194
NM_024105/







ALG12
Hs.526711




1043











221822_at
195
NM_138414/







LOC112869
Hs.460487




1070











220755_s_at
196
NM_016947/







C6ORF48
Hs.109798




992











218970_s_at
197
NM_015960/







CUTC
Hs.16606




983











218253_s_at
198
NM_006893/







LGTN
Hs.497581




933











221293_s_at
199
NM_022047/







DEF6
Hs.15476




1032











217742_s_at
200
NM_100486/
NM_016628/
NM_100264/





WAC,
Hs.435610,




1063
1140
1199





WAC,
Hs.435610,












WAC,
Hs.435610,


207127_s_at
201
NM_021644/
NM_012207/






HNRPH3,
Hs.499891,




1030
1136






HNRPH3,
Hs.499891,


217850_at
202
NM_206826/
NM_014366/
NM_206825/





GNL3,
Hs.313544,




1104
1137
1213





GNL3,
Hs.313544,












GNL3,
Hs.313544,


212505_s_at
203
NM_015329/







KIAA0892
Hs.112751




974











211971_s_at
204
NM_133259/







LRPPRC
Hs.368084




1065











216387_x_at
205
NM_013269/
NM_001004420/
NM_001004419/
NM_002520/




OCIL,
Hs.268326,




952
1117
1185
1218




OCIL,
Hs.268326,












OCIL,
Hs.268326,












NPM1,
Hs.519452,


218152_at
206
NM_018200/







HMG20A
Hs.69594




1002











201788_at
207
NM_203499/
NM_007372/






DDX42,
Hs.8765,




1102
1135






DDX42,
Hs.8765,


201922_at
208
NM_014886/







TINP1
Hs.482526




966











212982_at
209
NM_015336/







ZDHHC17
Hs.4014




975











214173_x_at
210
NM_134447/
NM_003796/






C19ORF2,
Hs.466391,




1068
1128






C19ORF2,
Hs.466391,


211937_at
211
NM_001417/







EIF4B
Hs.512629




852











216843_x_at
212
NM_000535/
NM_174930/
NM_002679/
NM_005395/
NM_001003686/
NM_001003687/
NM_032958/
NM_032959/
PMS2,
Hs.487470,




828
1086
867
908
1115
1183
1242
1247
PMS2,
Hs.397073,












L5,
Hs.488624,












POM121,
Hs.549057,












PMS2L3,
Hs.549057,












PMS2L3,
Hs.549057,












PMS2L3,
Hs.433879,












POLR2J2,
Hs.433879,












POLR2J2,



212854_x_at
213
NM_032264/
NM_173638/
NM_183372/





AE2,
Hs.325422,




1056
1166
1209





MGC8902,
Hs.512037,












LOC200030,
Hs.515837,


212886_at
214
NM_015621/







DKFZP434C171
Hs.132994




978











220607_x_at
215
NM_016397/
NM_198976/






TH1L,
Hs.517148,




990
1172






TH1L,
Hs.517148,


212132_at
216
NM_015578/







C19ORF13
Hs.407368




977











214042_s_at
217
NM_000983/







RPL22
Hs.515329




839











212660_at
218
NM_015288/







PHF15
Hs.483419




972











208944_at
219
—/







—
—


210011_s_at
220
NM_013986/
NM_005243/






EWSR1,
Hs.374477,




956
1130






EWSR1,
Hs.374477,


211938_at
221
NM_001417/







EIF4B
Hs.512629




852











220408_x_at
222
NM_017569/







FAM48A
Hs.435815




994











212114_at
223
—/







—
—


212455_at
224
NM_133370/







YT521
Hs.175955




1067











212299_at
225
—/







—
—


200005_at
226
NM_003753/







EIF3S7
Hs.55682




879











200081_s_at
227
NM_001010/







RPS6
Hs.408073




846











213687_s_at
228
NM_000996/







RPL35A
Hs.529631




840











204102_s_at
229
NM_001961/







EEF2
Hs.515070




859











211666_x_at
230
NM_000967/







RPL3
Hs.119598




836











209134_s_at
231
NM_001010/







RPS6
Hs.408073




846











221700_s_at
232
NM_003333/







UBA52
Hs.5308




876











200034_s_at
233
NM_000970/







RPL6
Hs.528668




837











201254_x_at
234
NM_001010/







RPS6
Hs.408073




846











212734_x_at
235
NM_000977/
NM_033251/






RPL13,
Hs.410817,




838
1147






RPL13,
Hs.410817,


217718_s_at
236
NM_139323/
NM_003404/






YWHAB,
Hs.279920,




1073
1126






YWHAB,
Hs.279920,


208678_at
237
NM_001696/







ATP6V1E1
Hs.517338




855











200633_at
238
NM_018955/







UBB
Hs.356190




1014











201318_s_at
239
NM_033546/
NM_006471/






MRLC2,
Hs.464472,




1058
1134






MRCL3,
Hs.190086,


202090_s_at
240
NM_006830/







UQCR
Hs.8372




931











221474_at
241
NM_033546/







MRLC2
Hs.464472




1058











214629_x_at
242
NM_153828/
NM_207520/
NM_207521/
NM_020532/
NM_007008/



RTN4,
Hs.429581,




1080
1176
1214
1221
1236



RTN4,
Hs.429581,












RTN4,
Hs.429581,












RTN4,
Hs.429581,












RTN4,
Hs.429581,


200067_x_at
243
NM_152827/
NM_003795/
NM_152828/





SNX3,
Hs.12102,




1078
1127
1203





SNX3,
Hs.12102,












SNX3,
Hs.12102,


201899_s_at
244
NM_181762/
NM_003336/
NM_181777/





UBE2A,
Hs.379466,




1089
1125
1207





UBE2A,
Hs.379466,












UBE2A,
Hs.379466,


210968_s_at
245
NM_153828/
NM_207520/
NM_207521/
NM_020532/
NM_007008/



RTN4,
Hs.429581,




1080
1176
1214
1221
1236



RTN4,
Hs.429581,












RTN4,
Hs.429581,












RTN4,
Hs.429581,












RTN4,
Hs.429581,


200609_s_at
246
NM_005112/
NM_017491/






WDR1,
Hs.128548,




904
1142






WDR1,
Hs.128548,


208805_at
247
NM_002791/







PSMA6
Hs.446260




868











218571_s_at
248
NM_014169/







C14ORF123
Hs.279761




957











217730_at
249
NM_022152/







PP1201
Hs.98475




1035











218520_at
250
NM_013254/







TBK1
Hs.505874




951











208908_s_at
251
NM_173061/
NM_173062/
NM_173060/
NM_001750/




CAST,
Hs.440961,




1085
1165
1084
1217




CAST,
Hs.440961,












CAST,
Hs.440961,












CAST,
Hs.440961,


207467_x_at
252
NM_173061/
NM_173062/
NM_173060/
NM_001750/




CAST,
Hs.440961,




1085
1165
1084
1217




CAST,
Hs.440961,












CAST,
Hs.440961,












CAST,
Hs.440961,


201975_at
253
NM_198240/
NM_002956/






RSN,
Hs.524809,




1093
1123






RSN,
Hs.524809,


200814_at
254
NM_006263/
NM_176783/






PSME1,
Hs.75348,




920
1167






PSME1,
Hs.75348,


203090_at
255
NM_006923/







SDF2
Hs.514036




934











201470_at
256
NM_004832/







GSTO1
Hs.190028




895











212586_at
257
NM_173060/







CAST
Hs.440961




1084











200703_at
258
NM_003746/







DNCL1
Hs.5120




878











217995_at
259
NM_021199/







SQRDL
Hs.511251




1029











201346_at
260
NM_024551/







ADIPOR2
Hs.371642




1048











201609_x_at
261
NM_012405/
NM_170705/






ICMT,
Hs.515688,




949
1162






ICMT,
Hs.515688,


202000_at
262
NM_002490/







NDUFA6
Hs.274416




864











202001_s_at
263
NM_002490/







NDUFA6
Hs.274416




864











203880_at
264
NM_005694/







COX17
Hs.534383




914











218525_s_at
265
NM_017902/







HIF1AN
Hs.500788




995











219798_s_at
266
NM_019606/







FLJ20257
Hs.178011




1017











208398_s_at
267
NM_004865/







TBPL1
Hs.486507




897











218358_at
268
NM_024324/







MGC11256
Hs.211282




1045











203097_s_at
269
—/







—
—


221598_s_at
270
NM_004269/







CRS98
Hs.374262




887











209546_s_at
271
AF323540/







GenBank
Hs.114309




811











50400_at
272
AI743990/







GenBank
Hs.24859




812











217752_s_at
273
NM_018235/







GenBank
Hs.273230




1003











201931_at
274
NM_000126/







GenBank
Hs.169919




825











218567_x_at
275
NM_005700/







GenBank
Hs.22880




915











202056_at
276
AW051311/







GenBank
Hs.169149




816











218907_s_at
277
NM_023942/







GenBank
Hs.284135




1041











213726_x_at
278
AA515698/







GenBank
Hs.251653




806











204448_s_at
279
AF031463/







GenBank
Hs.9302




808











1405_i_at
280
M21121/







GenBank
—




823











212864_at
281
Y16521/







GenBank
Hs.24812




1108











209444_at
282
BC001851/







GenBank
Hs.7940




819











208898_at
283
AF077614/







GenBank
Hs.272630




809











202377_at
284
AW026535/







GenBank
Hs.23581




815











209547_s_at
285
BC001043/







GenBank
Hs.15075




818











202922_at
286
BF676980/







GenBank
Hs.151393




821











201403_s_at
287
NM_004528/







GenBank
Hs.111811




892











213154_s_at
288
AI934125/







GenBank
Hs.17411




814











215676_at
289
N91109/







GenBank
Hs.32935




824











218927_s_at
290
NM_018641/







GenBank
Hs.25204




1012











213648_at
291
AW614427/







GenBank
Hs.182877




817











209593_s_at
292
AF317129/







GenBank
Hs.252682




810











203731_s_at
293
NM_014569/







GenBank
Hs.110839




960











201234_at
294
NM_004517/







GenBank
Hs.6196




891











214817_at
295
BE783668/







GenBank
Hs.175780




820











201761_at
296
NM_006636/







GenBank
Hs.154672




929











205467_at
297
NM_001230/







GenBank
Hs.5353




850











222318_at
298
AI744673/







GenBank
Hs.186970




813











209933_s_at
299
AF020314/







GenBank
Hs.9688




807











200734_s_at
300
BG341906/







GenBank
Hs.119177




822











219899_x_at
301
NM_014434/







NDOR1
Hs.512564




959











221563_at
302
NM_144729/
NM_144728/
NM_007207/





DUSP10,
Hs.497822,




1074
1155
1192





DUSP10,
Hs.497822,












DUSP10,
Hs.497822,


65517_at
303
NM_005498/







AP1M2
Hs.18894




910











205126_at
304
NM_006296/







VRK2
Hs.468623




921











200696_s_at
305
NM_198252/
NM_000177/






GSN,
Hs.522373,




1094
1110






GSN,
Hs.522373,


208790_s_at
306
NM_012232/







PTRF
Hs.437191




944











205115_s_at
307
NM_016196/







RBM19
Hs.7482




987











213982_s_at
308
NM_014857/







RABGAP1L
Hs.495391




965











206992_s_at
309
NM_015684/
NM_001003805/
NM_001003803/





ATP5S,
Hs.438489,




980
1116
1184





ATP5S,
Hs.438489,












ATP5S,
Hs.438489,


201060_x_at
310
NM_198194/
NM_004099/
NM_017723/





STOM,
Hs.253903,




1092
1129
1195





STOM,
Hs.253903,












FLJ20245,
Hs.495541,


209278_s_at
311
NM_006528/







TFPI2
Hs.438231




924











210542_s_at
312
—/







—
—


202006_at
313
NM_002835/







PTPN12
Hs.61812




869











210557_x_at
314
NM_172211/
NM_000757/
NM_172210/
NM_172212/




CSF1,
Hs.173894,




1082
1112
1205
1226




CSF1,
Hs.173894,












CSF1,
Hs.173894,












CSF1,
Hs.173894,


210796_x_at
315
NM_198846/
NM_198845/
NM_001245/





SIGLEC6,
Hs.397255,




1097
1171
1188





SIGLEC6,
Hs.397255,












SIGLEC6,
Hs.397255,


220825_s_at
316
NM_018240/







KIRREL
Hs.272234




1004











202378_s_at
317
NM_017526/







LEPR
Hs.23581




993











210241_s_at
318
NM_007233/







TP53AP1
Hs.274329




936











213069_at
319
—/







—
—


216034_at
320
NM_080740/







SUHW1
Hs.178665




1061











210793_s_at
321
NM_139131/
NM_005387/






NUP98,
Hs.524750,




1072
1131






NUP98,
Hs.524750,


207375_s_at
322
NM_002189/
NM_172200/






IL15RA,
Hs.524117,




860
1163






IL15RA,
Hs.524117,


206247_at
323
NM_005931/







MICB
Hs.211580




917











217078_s_at
324
NM_007261/







CMRF-
Hs.9688




937







35H



219257_s_at
325
NM_021972/
NM_182965/






SPHK1,
Hs.68061,




1031
1170






SPHK1,
Hs.68061,


204781_s_at
326
NM_152876/
NM_152877/
NM_152874/
NM_152875/
NM_152873/
NM_152871/
NM_152872/
NM_000043/
FAS,
Hs.244139,




1079
1161
1204
1225
1238
1240
1244
1245
FAS,
Hs.244139,












FAS,
Hs.244139,












FAS,
Hs.244139,












FAS,
Hs.244139,












FAS,
Hs.244139,












FAS,
Hs.244139,












FAS,
Hs.244139,


221536_s_at
327
NM_018385/







FLJ11301
Hs.518505




1009











209468_at
328
NM_002335/







LRP5
Hs.6347




861











201061_s_at
329
NM_198194/
NM_004099






STOM,
Hs.253903,




1092
1129






STOM,
Hs.253903,


203499_at
330
NM_004431/







EPHA2
Hs.171596




889











213324_at
331
NM_198291/
NM_005417/






SRC,
Hs.195659,




1095
1132






SRC,
Hs.195659,


221535_at
332
NM_018385/







FLJ11301
Hs.518505




1009











219938_s_at
333
NM_024430/







PSTPIP2
Hs.368623




1046











213787_s_at
334
NM_006579/







EBP
Hs.522636




926











219592_at
335
NM_024596/







MCPH1
Hs.550532




1049











200637_s_at
336
NM_130440/
NM_002840/






PTPRF,
Hs.272062,




1064
1122






PTPRF,
Hs.272062,


202193_at
337
NM_005569/
NM_016733/






LIMK2,
Hs.474596,




913
1141






LIMK2,
Hs.474596,


214859_at
338
NM_015082/







FSTL4
Hs.483390




969











209213_at
339
NM_001757/







CBR1
Hs.88778




857











218945_at
340
NM_024109/







MGC2654
Hs.306380




1044











220471_s_at
341
NM_025107/







MYCT1
Hs.18160




1052











59625_at
342
NM_003946/







NOL3
Hs.513667




883











207233_s_at
343
NM_198158/
NM_000248/
NM_198178/
NM_198177/
NM_006722/
NM_198159/


MITF,
Hs.166017,




1091
1111
1210
1228
1235
1241


MITF,
Hs.166017,












MITF,
Hs.166017,












MITF,
Hs.166017,












MITF,
Hs.166017,












MITF,
Hs.166017,


214070_s_at
344
—/







—
—


219785_s_at
345
—/







—
—


220684_at
346
NM_013351/







TBX21
Hs.272409




954











202245_at
347
NM_002340/







LSS
Hs.517366




862











207298_at
348
NM_006632/







SLC17A3
Hs.327179




927











220201_at
349
NM_018835/







MNAB
Hs.533499




1013











203208_s_at
350
NM_014637/







CHPPR
Hs.521608




961











207033_at
351
NM_005142/







GIF
Hs.110014




906











204929_s_at
352
NM_006634/







VAMP5
Hs.172684




928











209735_at
353
NM_004827/







ABCG2
Hs.480218




894











209234_at
354
NM_015074/







KIF1B
Hs.97858




968











210102_at
355
NM_198315/







LOH11CR2A
Hs.152944




1096











204114_at
356
NM_007361/







NID2
Hs.369840




941











203344_s_at
357
NM_203292/
NM_203291/
NM_002894/





RBBP8,
Hs.546282,




1100
1174
1190





RBBP8,
Hs.546282,












RBBP8,
Hs.546282,


216891_at
358
—/







—
—


221680_s_at
359
NM_016135/







ETV7
Hs.272398




986











202087_s_at
360
NM_001912/
NM_145918/






CTSL,
Hs.418123,




858
1159






CTSL,
Hs.418123,


213324_at
331
NM_198291/
NM_005417/






SRC,
Hs.195659,




1095
1132






SRC,
Hs.195659,


221535_at
332
NM_018385/







FLJ11301
Hs.518505




1009











219938_s_at
333
NM_024430/







PSTPIP2
Hs.368623




1046











213787_s_at
334
NM_006579/







EBP
Hs.522636




926











219592_at
335
NM_024596/







MCPH1
Hs.550532




1049











200637_s_at
336
NM_130440/
NM_002840/






PTPRF,
Hs.272062,




1064
1122






PTPRF,
Hs.272062,


202193_at
337
NM_005569/
NM_016733/






LIMK 2,
Hs.474596,




913
1141






LIMK 2,
Hs.474596,


214859_at
338
NM_015082/







FSTL4
Hs.483390




969











209213_at
339
NM_001757/







CBR1
Hs.88778




857











218945_at
340
NM_024109/







MGC2654
Hs.306380




1044











220471_s_at
341
NM_025107/







MYCT1
Hs.18160




1052











59625_at
342
NM_003946/







NOL3
Hs.513667




883











207233_s_at
343
NM_198158/
NM_000248/
NM_198178/
NM_198177/
NM_006722/
NM_198159/


MITF,
Hs.166017,




1091
1111
1210
1228
1235
1241


MITF,
Hs.166017,












MITF,
Hs.166017,












MITF,
Hs.166017,












MITF,
Hs.166017,












MITF,
Hs.166017,


214070_s_at
344
—/







—
—


219785_s_at
345
—/







—
—


220684_at
346
NM_013351/







TBX21
Hs.272409




954











202245_at
347
NM_002340/







LSS
Hs.517366




862











207298_at
348
NM_006632/







SLC17A3
Hs.327179




927











220201_at
349
NM_018835/







MNAB
Hs.533499




1013











203208_s_at
350
NM_014637/







CHPPR
Hs.521608




961











207033_at
351
NM_005142/







GIF
Hs.110014




906











204929_s_at
352
NM_006634/







VAMP5
Hs.172684




928











209735_at
353
NM_004827/







ABCG2
Hs.480218




894











209234_at
354
NM_015074/







KIF1B
Hs.97858




968











210102_at
355
NM_198315/







LOH11CR2A
Hs.152944




1096











204114_at
356
NM_007361/







NID2
Hs.369840




941











203344_s_at
357
NM_203292/
NM_203291/
NM_002894





RBBP8,
Hs.546282,




1100
1174
1190





RBBP8,
Hs.546282,












RBBP8,
Hs.546282,


216891_at
358
—/







—
—


221680_s_at
359
NM_016135/







ETV7
Hs.272398




986











202087_s_at
360
NM_001912/
NM_145918/






CTSL,
Hs.418123,




858
1159






CTSL,
Hs.418123,


210073_at
391
NM_003034/







SIAT8A
Hs.408614




871











209524_at
392
NM_016073/







HDGFRP3
Hs.513954




985











219759_at
393
NM_022350/







LRAP
Hs.482910




1036











202112_at
394
NM_000552/







VWF
Hs.440848




830











219410_at
395
NM_018004/







FLJ10134
Hs.126598




1000











203819_s_at
396
NM_006547/







IMP-3
Hs.432616




925











212730_at
397
NM_015286/
NM_145728/






DMN,
Hs.207106,




971
1158






DMN,
Hs.207106,


220122_at
398
—/







—
—


208782_at
399
NM_007085/







FSTL1
Hs.269512




935











202468_s_at
400
NM_003798/







CTNNAL1
Hs.58488




882











207018_s_at
401
NM_004163/







RAB27B
Hs.25318




886











210354_at
402
NM_000619/







IFNG
Hs.856




832











201110_s_at
403
NM_003246/







THBS1
Hs.164226




874











207808_s_at
404
NM_000313/







PROS1
Hs.64016




827











205612_at
405
NM_007351/







MMRN1
Hs.268107




940











214073_at
406
—/







—
—









The genes encoding RNA polymerase I transcription factor (RRN3) and leucine-rich PPR-motif containing protein (LRPRC) were found as most significantly down-regulated genes in BMS signature (Table 1, hereinabove). RRN33 (transcription initiation factor TIF-IA), a 72-kDa protein, is essential for ribosomal DNA (rDNA) transcription and acts as a bridge between RNA pol I and the committed rDNA promoter (Hirschler-Laszkiewicz I, et al., 2003; Miller G, et al., 2001; 20:1373-1382). The suppression of polymerase I regulation mechanism is confirmed by down-regulation of polymerase (RNA) I polypeptide D (POLR1D).
LRPPRC is a candidate gene for the French-Canadian type of Leigh syndrome, a form of cytochrome c oxidase deficiency, and plays a role in translation or stability of mitochondrially encoded cytochrome c oxidase (COX) subunits (Mootha V K, et al., 2003). The LRPPC together with POLR1D molecules comprise a complex with NFkBIB protein (Bouwmeester T, et al., 2004) that inhibits proinflammatory NFkB pathway.
Without being bound by any theory, the suppression of molecules involved in polymerase I related mechanism, COXI and NFkB regulation could account for the differences between BMS and typical RRMS patients. In addition, the polymerase I related mechanism can be potential drug targets for the treatment of RRMS aimed to switch RRMS to the BMS variant. One of commercially available drug that has proven effects on polymerase I mechanism is a diterpenoid triepoxide Triptolide (TPT), isolated from the Chinese herb Tripterygium wilfordii (Leuenroth S J and Crews C M. Triptolide-induced transcriptional arrest is associated with changes in nuclear substructure. Cancer Res. 2008; 68:5257-5266). Triptolide has various anti-inflammatory effects (Liu Y, et al. Triptolide, a component of Chinese herbal medicine, modulates the functional phenotype of dendritic cells. Transplantation. 2007; 84:1517-1526), it modulates T-cell inflammatory responses and ameliorates Experimental Autoimmune Encephalomyelitis (Wang Y, et al. Triptolide modulates T-cell inflammatory responses and ameliorates experimental autoimmune encephalomyelitis. J Neurosci Res. 2008; 86:2441-2449).
More specifically TPT demonstrated to suppress of T lymphocyte function including T cell apoptosis induction, inhibition of lymphocyte prolipheration and IFNγ production (Chen B J. 2001. Triptolide, a novel immunosuppressive and anti-inflammatory agent purified from a Chinese herb Tripterygium wilfordii Hook f. Leuk Lymphoma 42:253-265; Qiu D, Kao P N. 2003 Immunosuppressive and anti-inflammatory mechanisms of triptolide, the principal active diterpenoid from the Chinese medicinal herb Tripterygium wilfordii Hook f. Drugs R D 4:1-18; Yang Y, Liu Z, Tolosa E, Yang J, Li L. 1998. Triptolide induces apoptotic death of T lymphocyte. Immunopharmacology 40:139-149; Chan M A, Kohlmeier J E, Branden M, Jung M, Benedict S H. 1999. Triptolide is more effective in preventing T cell proliferation and interferon-gamma production than is FK506. Phytother Res 13:464-467). The TPT decreased IL2 and IL2 receptor expression by inhibiting activation of the purine box regulator of the NFkB of activated T cells (Qiu 1999). Additionally, it was demonstrated that TPT can inhibit the maturation, antigen processing, and presentation of dendritic cells and can suppress tumor necrosis factor (TNF)-a and IL-6 production by activated macrophages (Zhu K J, Shen Q Y, Cheng H, Mao X H, Lao L M, Hao G L. 2005. Triptolide affects the differentiation, maturation and function of human dendritic cells. Int Immunopharmacol 5:1415-1426; Wu Y, Cui J, Bao X, Chan S, Young D O, Liu D, Shen P. 2006. Triptolide attenuates oxidative stress, NF-kappaB activation and multiple cytokine gene expression in murine peritoneal macrophage. Int J Mol Med 17:141-150).
Table 3, hereinbelow, discloses the genes involved in the RNA polymerase I pathway, which are likely to be involved in typical RRMS or BMS pathology.







TABLE 3







Genes involved in the RNA polymerase I pathway












SEQ
Representative




Affymetrix
ID
Public ID/SEQ ID




ProbSet
NO:
NO:
Gene Symbol
Gene Title














216902_s_at
62
AF001549/1284;
RRN3
RRN3 RNA polymerase I




NM_018427/1285

transcription factor homolog


211971_s_at
204
AI653608/1286;
LRPPRC
leucine-rich PPR-motif




NM_133259/1287

containing


220113_x_at
1248
NM_019014/1288
POLR1B
polymerase (RNA) I polypeptide






B, 128 kDa


207515_s_at
1249
NM_004875/1289
POLR1C
polymerase (RNA) I polypeptide






C, 30 kDa


209317_at
1250
AF008442/1290
POLR1C
polymerase (RNA) I polypeptide






C, 30 kDa


218258_at
173
NM_015972/1291
POLR1D
polymerase (RNA) I polypeptide






D, 16 kDa


202725_at
1251
NM_000937/1292
POLR2A
polymerase (RNA) II (DNA






directed) polypeptide A, 220 kDa


217420_s_at
1252
M21610/1293
POLR2A
polymerase (RNA) II (DNA






directed) polypeptide A, 220 kDa


201803_at
1253
NM_000938/1294
POLR2B
polymerase (RNA) II (DNA






directed) polypeptide B, 140 kDa


208996_s_at
1254
BC000409/1295
POLR2C
polymerase (RNA) II (DNA






directed) polypeptide C, 33 kDa


214263_x_at
1255
AI192781/1296
POLR2C
polymerase (RNA) II (DNA






directed) polypeptide C, 33 kDa


216282_x_at
1256
AJ224143/1297
POLR2C
polymerase (RNA) II (DNA






directed) polypeptide C, 33 kDa


203664_s_at
1257
NM_004805/1298
POLR2D
polymerase (RNA) II (DNA






directed) polypeptide D


214144_at
1258
BF432147/1299
POLR2D
polymerase (RNA) II (DNA






directed) polypeptide D


213887_s_at
1259
AI554759/1300
POLR2E
polymerase (RNA) II (DNA






directed) polypeptide E, 25 kDa


217854_s_at
1260
BC004441/1301
POLR2E
polymerase (RNA) II (DNA






directed) polypeptide E, 25 kDa


209511_at
1261
BC003582/1302
POLR2F
polymerase (RNA) II (DNA






directed) polypeptide F


202306_at
1262
NM_002696/1303
POLR2G
polymerase (RNA) II (DNA






directed) polypeptide G


209302_at
1263
U37689/1304
POLR2H
polymerase (RNA) II (DNA






directed) polypeptide H


212955_s_at
1264
AL037557/1305
POLR2I
polymerase (RNA) II (DNA






directed) polypeptide I, 14.5 kDa


212782_x_at
1265
BG335629/1306
POLR2J
polymerase (RNA) II (DNA






directed) polypeptide J, 13.3 kDa


216242_x_at
1266
AW402635/1307
POLR2J2
DNA directed RNA polymerase






II polypeptide J-related gene


214740_at
1267
BE676209/1308
POLR2J2 ///
DNA directed RNA polymerase





MGC13098
II polypeptide J-related gene ///






hypothetical prote


202634_at
1268
AL558030/1309
POLR2K
polymerase (RNA) II (DNA






directed) polypeptide K, 7.0 kDa


202635_s_at
1269
NM_005034/1310
POLR2K
polymerase (RNA) II (DNA






directed) polypeptide K, 7.0 kDa


202586_at
1270
AA772747/1311
POLR2L
polymerase (RNA) II (DNA






directed) polypeptide L, 7.6 kDa


211730_s_at
1271
BC005903/1312
POLR2L
polymerase (RNA) II (DNA






directed) polypeptide L, 7.6 kDa






/// polymerase (RNA) II


219459_at
1272
NM_018082/1313
POLR3B
polymerase (RNA) III (DNA






directed) polypeptide B


209382_at
1273
U93867/1314
POLR3C
polymerase (RNA) III (DNA






directed) polypeptide C (62 kD)


210573_s_at
1274
BC004424/1315
POLR3C
polymerase (RNA) III (DNA






directed) polypeptide C (62 kD)


208361_s_at
1275
NM_001722/1316
POLR3D
polymerase (RNA) III (DNA






directed) polypeptide D, 44 kDa


218016_s_at
1276
NM_018119/1317
POLR3E
polymerase (RNA) III (DNA






directed) polypeptide E (80 kD)


205218_at
1277
NM_006466/1318
POLR3F
polymerase (RNA) III (DNA






directed) polypeptide F, 39 kDa


206653_at
1278
BF062139/1319
POLR3G
Polymerase (RNA) III (DNA






directed) polypeptide G (32 kD)


206654_s_at
1279
NM_006467/1320
POLR3G
polymerase (RNA) III (DNA






directed) polypeptide G (32 kD)


218866_s_at
1280
AF060223/1321
POLR3K
polymerase (RNA) III (DNA






directed) polypeptide K, 12.3 kDa


203782_s_at
1281
NM_005035/1322
POLRMT
polymerase (RNA)






mitochondrial (DNA directed)


203783_x_at
1282
BF057617/1323
POLRMT
polymerase (RNA)






mitochondrial (DNA directed)


202466_at
1283
NM_006999/1324
POLS
polymerase (DNA directed)






sigma









The measurement of RRN3, LRPPRC, POLR1D and other polymerase I mechanism related biomarkers could be used for diagnosis and prediction of BMS. Additionally those markers could be useful for typical RRMS patients to monitor the efficacy of various immunomodulatory drugs for assessment of patients with good response to treatment.
Measurement of BMS biomarkers can be performed on the mRNA level by the quantitative reverse-transcriptase polymerase chain reaction (QRT-PCR) method and on protein level by LUMINEX technology. Possible modification of the invention is developing biomarkers on protein level (e.g., using ELISA, Western Blot analysis and the like) in PBMC and serum.
Multiple sclerosis (MS) is a heterogeneous disease. To better diagnose and treat MS patients the various types of disease have to be distinguished. The teachings of the invention enable, for the first time, to distinguish between BMS and typical RRMS patients using molecular tools, which when combined with accurate clinical information enables to dissect the biological complexity of MS.
In the current study the use of gene expression profiling enabled to diagnose benign MS using a phenotypic approach to differentiate subtypes of the disease. The identified gene expression phenotypes also enable to better understand the biology of benign MS and to develop therapeutics strategies to treat MS.
The gene expression signature generated herein of benign MS enables refining MS to diagnose low risk patients versus high risk patients and accordingly suggest appropriate treatment.
The benign MS patients that represent low risk would not be treated while the high risk relapsing-remitting MS patients will be treated. In addition, the teachings of the invention enable to monitor response to treatment and better use of current approved medications.
The teachings of the invention can be used to develop a kit or device for diagnosis and prediction of typical RRMS clinical outcome, improving medical decision support systems and individualizing patient care. In addition, the teachings of the invention can be used to develop new drugs that will imitate BMS gene expression signature and will result in silencing of the active RRMS.
Example 2
Study Subject and Methods
Subjects—
31 patients (age 44.5±1.5; female to male ratio 24:7) with BMS were characterized by mean EDSS 1.95±0.15, disease duration 17.0±1.3 years, annual EDSS rate 0.13±0.01, annual relapse rate 0.23±0.04. 36 patients (age 40.3±1.8; female to male ratio 8:3) with RRMS were characterized by mean EDSS 3.54±0.23, disease duration 10.9±1.4 years, annual EDSS rate 0.45±0.06, annual relapse rate 0.64±0.09.
RNA Isolation and Microarray Expression Profiling—
Peripheral blood mononuclear cells (PBMC) were separated on ficoll-hypaque gradient. Total RNA was isolated using the TRIzol Reagent (Invitrogen, Carlsbad, Calif.), and cDNA was synthesized, labeled and hybridized to HG-U133A-2 array (Affymetrix, Inc, Santa Clara, Calif.) containing 22,215 gene-transcripts, washed and scanned (Hewlett Packard, GeneArray-TM scanner G2500A) according to manufacturer's protocol Affymetrix (Inc, Santa Clara, Calif.).
Data Analysis—
Data analysis was performed using the Partek Genomics Solution software [World Wide Web (dot) partek (dot) com]. Expression values were computed from raw CEL (cells) files by applying the Robust Multi-Chip Average (RMA) background correction algorithm. The RMA correction included: 1) values background correction; 2) quantile normalization; 3) log 2 transformation; 4) median polish summarization. The gene transcripts were filtered using Affymetrix MASS Present/Absent Detection. Thereafter, 9987 transcript that were detected as Present in 100% microarrays were used for analysis. In order to avoid the noise caused by variable set effects each set was normalized to pre-saved distribution pattern of a well balanced set used as a reference distribution. To reduce batch effect ANOVA multiple model analysis was applied. Source of variation was analyzed; nuisance batches effects such as working batch, patient age, gender and treatment were eliminated. Most informative genes (MIGs) were defined as genes that passed Falls Discavery Rate (FDR) correction with p<0.05 by ANOVA linear contrasts model. Thereafter, predictive algorithm based on two level cross validation method, Super Vector Machine (SVM) and K-Nearest Neighbor algorithms were applied to calculate MIGs classification rates. Only genes which were included in classifiers (from 1 to 10 genes) with more than 70% correct classification rates were analyzed.
Experimental Results
Identification of Classification Rates of Genes Involved in the RNA Polymerase I Pathway Between Typical RRMS and BMS Disease Course—
Tables 4A-C presents the corrected classification rates for all combinations of RRN3, LRPPRC and POLR1D genes of the RNA polymerase I pathway for the entire BMS and typical RRMS group (Table 4A), for the BMS group (Table 4B) and for the typical RRMS group (Table 4C). The best predictive performance for each classifier is presented.










TABLE 4A






Classifiers
Aver. % Correct
St. Err (%)



















RRN3
63.6
11.2



LRPPRC
73.3
9.5



POLR1D
72.7
9.5



RRN3, POLR1D
72.7
9.5



RRN3, LRPPRC
63.6
10.2



POLR1D, LRPPRC
81.8
8.2



RRN3, LRPPRC, POLR1D
77.2
8.9





Average % correct = Average percent of correct classification between BMS and typical RRMS patients using specific classifier;


St. Err. = standard error;















TABLE 4B






Classifiers
BMS % corr








RRN3
67



LRPPRC
80



POLR1D
60



RRN3, POLR1D
60



RRN3, LRPPRC
30



POLR1D, LRPPRC
80



RRN3, LRPPRC, POLR1D
70





BMS % Corr. = Average percent of correct classification for BMS patients using specific classifier;















TABLE 4C






Classifiers
Typical RRMS % corr








RRN3
60



LRPPRC
67



POLR1D
80



RRN3, POLR1D
83



RRN3, LRPPRC
90



POLR1D, LRPPRC
83



RRN3, LRPPRC, POLR1D
83





RRMS % Corr. - Average percent of correct classification for typical RRMS patients using specific classifier.






The results presented in Table 4B demonstrate that for classification of BMS, each of the genes of the RNA polymerase I pathway, i.e., RRN3, LRPPRC and POLR1D exhibits a correct classification rate of 67%, 80% and 60%, respectively. The results presented in Table 4C demonstrate that for classification of typical RRMS, each of RRN3, LRPPRC and POLR1D exhibits a correct classification rate of 60%, 67% and 80%, respectively. In addition, an increased rate of correct classification of typical RRMS, which is a more complex and heterogenous condition, can be achieved using a combination of 2 or 3 genes of the RNA polymerase I pathway. For example, a correct classification rate of 83% is obtained using the combination of RRN3 and POLR1D; a correct classification rate of 83% is obtained using the combination of POLR1D and LRPPRC; and a correct classification rate of 90% is obtained using the combination of RRN3 and LRPPRC (Table 4C, above).
The classification for the combination of RRN3 and LRPPRC resulted in outstanding classification rate of 90% of typical RRMS (TMS) (Table 4C), while showing a low 30% classification for these 2 genes for BMS (Table 4B), further supporting the differentiations between the two disease patterns.
To conclude, correct classification for both groups would be achieved using more than one combination of genes of the RNA polymerase I pathway, for example LRPPRC alone or with POLR1D reached 80% classification rate for BMS and RRN3 and LRPPRC correctly classify 90% of TMS patients.
Identification of Biomarkers for Differentiation of Patients with BMS or Typical RRMS Course of Disease—
BMS patients differentiated from typical RRMS by 177 MIGs (Table 5). The 17 genes with higher classification performance (Table 6) were identified from MIGs by applying predictive algorithms.







TABLE 5







MIGs discriminating between BMS and typical RRMS patients


















p-value
Log Fold








BMS
Change




Affymetrix



vs
(BMS vs




ProbSet
SEQ
Representative
SEQ ID
typical
typical
Gene



ID
NO ID:
Public ID
NO:
RRMS
RRMS)
Symbol
Gene description

















216902_s_at
62
AF001549
468
2.12E−08
−2.01
RRN3
RRN3 RNA









polymerase I









transcription factor









homolog (yeast)


210502_s_at
1410
AF042386
1534
2.30E−08
−1.28
PPIE
peptidylprolyl









isomerase E









(cyclophilin E)


211615_s_at
1418
M92439
1512
2.44E−08
−1.20
LRPPRC
leucine-rich PPR-motif









containing /// leucine-









rich PPR-motif









containing


37950_at
1500
X74496
1526
1.05E−06
1.19
PREP
prolyl endopeptidase


218258_at
173
NM_015972
984
1.92E−06
−1.18
POLR1D
polymerase (RNA) I









polypeptide D, 16 kDa


214439_x_at
1453
AF043899
1535
2.24E−06
−1.62
BIN1
bridging integrator 1


214450_at
1454
NM_001335
1629
3.82E−06
−1.45
CTSW
cathepsin W









(lymphopain) ///









cathepsin W









(lymphopain)


214470_at
1455
NM_002258
1615
3.83E−06
−1.77
KLRB1
killer cell lectin-like









receptor subfamily B,









member 1 /// killer cell









lectin-li


205789_at
1373
NM_001766
1627
4.32E−06
1.62
CD1D
CD1D antigen, d









polypeptide /// CD1D









antigen, d polypeptide


206584_at
1378
NM_015364
1650
5.84E−06
1.59
LY96
lymphocyte antigen 96


212252_at
1425
AA181179
1660
6.14E−06
1.62
CAMKK2
calcium/calmodulin-









dependent protein









kinase kinase 2, beta


212748_at
1431
AB037859
1583
7.92E−06
1.18
MKL1
megakaryoblastic









leukemia









(translocation) 1


211654_x_at
1419
M17565
1519
9.99E−06
2.75
HLA-
major








DQB1
histocompatibility









complex, class II, DQ









beta 1 /// major









histocompatibili


204860_s_at
1370
AI817801
1673
1.21E−05
1.62
BIRC1
baculoviral IAP repeat-









containing 1


211971_s_at
204
AI653608
606
1.62E−05
−1.14
LRPPRC
leucine-rich PPR-motif









containing


202832_at
1347
NM_014635
1601
2.21E−05
−1.33
GCC2
GRIP and coiled-coil









domain containing 2


40446_at
1502
AL021366
1537
2.62E−05
−1.39
PHF1
PHD finger protein 1


210136_at
1404
AW070431
1676
2.71E−05
−1.76
MBP
Myelin basic protein


202441_at
1342
AL568449
1691
3.92E−05
1.37
C10orf69
chromosome 10 open









reading frame 69


213241_at
1442
AF035307
1532
4.12E−05
1.55
PLXNC1
Plexin C1


212978_at
1435
AU146004
1686
4.13E−05
1.56
TA-
T-cell activation








LRRP
leucine repeat-rich









protein


220005_at
1490
NM_023914
1591
4.55E−05
2.43
P2RY13
purinergic receptor









P2Y, G-protein









coupled, 13 ///









purinergic receptor









P2Y, G-pr


218304_s_at
1473
NM_022776
1645
4.89E−05
1.58
OSBPL11
oxysterol binding









protein-like 11


218932_at
1480
NM_017953
1652
5.00E−05
−1.27
FLJ20729
hypothetical protein









FLJ20729


213106_at
1440
AI769688
1670
5.57E−05
−1.51
ATP8A1
ATPase,









aminophospholipid









transporter (APLT),









Class I, type 8A,









member 1


219892_at
1489
NM_023003
1648
5.63E−05
1.86
TM6SF1
transmembrane 6









superfamily member 1


219666_at
1486
NM_022349
1585
6.07E−05
1.81
MS4A6A
membrane spanning 4-









domains, subfamily A,









member 6A


206120_at
1376
NM_001772
1631
6.26E−05
1.58
CD33
CD33 antigen (gp67)


209970_x_at
1403
M87507
1523
6.51E−05
1.33
CASP1
caspase 1, apoptosis-









related cysteine









protease (interleukin 1,









beta, convertase)


200980_s_at
1330
NM_000284
1654
8.59E−05
−1.27
PDHA1
pyruvate









dehydrogenase









(lipoamide) alpha 1


200610_s_at
1326
NM_005381
1613
8.97E−05
−1.17
NCL
nucleolin


213418_at
1445
NM_002155
1609
8.99E−05
2.73
HSPA6
heat shock 70 kDa









protein 6 (HSP70B′)


212421_at
1429
AB023147
1545
9.22E−05
1.66
C22orf9
chromosome 22 open









reading frame 9


210201_x_at
1407
AF001383
1531
9.59E−05
−1.59
BIN1
bridging integrator 1


207000_s_at
1381
NM_005605
1600
9.72E−05
−1.32
PPP3CC
protein phosphatase 3









(formerly 2B), catalytic









subunit, gamma









isoform (calcineur


203139_at
1352
NM_004938
1623
0.000102879
1.78
DAPK1
death-associated









protein kinase 1


211368_s_at
1416
U13700
1527
0.000107311
1.36
CASP1
caspase 1, apoptosis-









related cysteine









protease (interleukin 1,









beta, convertase)


212820_at
1433
AB020663
1582
0.000108529
1.70
RC3
rabconnectin-3


216945_x_at
15
U79240
421
0.000111457
−1.86
PASK
PAS domain containing









serine/threonine kinase


209337_at
1395
AF063020
1540
0.000112781
−1.35
PSIP1
PC4 and SFRS1









interacting protein 1


201756_at
1337
NM_002946
1599
0.000114388
−1.41
RPA2
replication protein A2,









32 kDa


221565_s_at
1493
BC000039
1602
0.00011473
1.50
FAM26B
family with sequence









similarity 26, member B


117_at
1325
X51757cds
#N/A
0.000116998
2.01
HSPA6
heat shock 70 kDa









protein 6 (HSP70B′)


43544_at
1503
AA314406
1661
0.000118986
−1.65
THRAP5
thyroid hormone









receptor associated









protein 5


219132_at
1483
NM_021255
1621
0.000121966
1.36
PELI2
pellino homolog 2









(Drosophila)


57715_at
1507
W72694
1657
0.000123909
1.35
FAM26B
family with sequence









similarity 26, member B


220066_at
1491
NM_022162
1569
0.000125076
1.53
CARD15
caspase recruitment









domain family,









member 15


212414_s_at
1428
D50918
1528
0.000139145
−1.53
SEPT6
septin 6


213902_at
1448
AI379338
1666
0.000139994
1.32
ASAH1
N-acylsphingosine









amidohydrolase (acid









ceramidase) 1


212998_x_at
1436
AI583173
1667
0.000143667
2.26
HLA-
major








DQB1
histocompatibility









complex, class II, DQ









beta 1 /// major









histocompatibili


202931_x_at
1350
NM_004305
1584
0.000148834
−1.55
BIN1
bridging integrator 1


204112_s_at
1361
NM_006895
1619
0.00014954
1.81
HNMT
histamine N-









methyltransferase


205467_at
297
NM_001230
850
0.000150498
1.44
CASP10
caspase 10, apoptosis









related cysteine









protease


209199_s_at
1394
N22468
1656
0.000157534
1.51
MEF2C
MADS box









transcription enhancer









factor 2, polypeptide C









(myocyte enhancer









factor


211676_s_at
1421
AF056979
1578
0.000157969
1.40
IFNGR1
interferon gamma









receptor 1 /// interferon









gamma receptor 1


203492_x_at
1355
AA918224
1664
0.000163582
−1.22
KIAA0092
translokin


211776_s_at
1423
BC006141
1577
0.000164075
3.18
EPB41L3
erythrocyte membrane









protein band 4.1-like 3









/// erythrocyte









membrane protein ba


56919_at
1505
AI806628
1672
0.000173847
1.32
KIAA1449
WD repeat endosomal









protein


202521_at
1344
NM_006565
1616
0.0001784
−1.14
CTCF
CCCTC-binding factor









(zinc finger protein)


211727_s_at
1422
BC005895
1576
0.000179886
−1.49
COX11
COX11 homolog,









cytochrome c oxidase









assembly protein









(yeast) /// COX11









homolog,


204222_s_at
1363
NM_006851
1628
0.00018487
1.54
GLIPR1
GLI pathogenesis-









related 1 (glioma)


204839_at
1369
NM_015918
1603
0.000185062
−1.21
POP5
processing of precursor









5, ribonuclease P/MRP









subunit (S. cerevisiae)


39729_at
1501
L19185
1524
0.000185886
−1.62
PRDX2
peroxiredoxin 2


221078_s_at
1492
NM_018084
1646
0.000188529
1.49
KIAA1212
KIAA1212


35156_at
1499
AL050297
1548
0.000196739
−1.24
LOC203069
Hypothetical protein









LOC203069


219630_at
1485
NM_005764
1606
0.000198793
1.94
MAP17
membrane-associated









protein 17


202662_s_at
1345
NM_002223
1625
0.00020557
1.34
ITPR2
inositol 1,4,5-









triphosphate receptor,









type 2


210212_x_at
1409
BC002600
1571
0.000219441
−1.27
MTCP1
mature T-cell









proliferation 1


217925_s_at
1471
NM_022758
1610
0.000224904
−1.21
C6orf106
chromosome 6 open









reading frame 106


218739_at
1477
NM_016006
1643
0.000228499
1.75
ABHD5
abhydrolase domain









containing 5


56197_at
1504
AI783924
1671
0.000231008
−1.24
PLSCR3
phospholipid









scramblase 3


208653_s_at
1388
AF263279
1561
0.00023174
1.46
CD164
CD164 antigen,









sialomucin


213292_s_at
1444
AA908770
1663
0.00023191
1.54
SNX13
sorting nexin 13


201194_at
1331
NM_003009
1608
0.000240228
−1.36
SEPW1
selenoprotein W, 1


201619_at
1336
NM_006793
1596
0.000250445
1.27
PRDX3
peroxiredoxin 3


203569_s_at
1356
NM_003611
1640
0.000272476
−1.45
OFD1
oral-facial-digital









syndrome 1


213979_s_at
1450
BF984434
1689
0.00027541
−3.22
—
—


203814_s_at
1359
NM_000904
1635
0.000281273
1.67
NQO2
NAD(P)H









dehydrogenase,









quinone 2


215118_s_at
1462
AW519168
1680
0.000302493
−2.77
MGC27165
Hypothetical protein









MGC27165


203624_at
1357
NM_005088
1607
0.000330513
−1.38
DXYS155E
DNA segment on









chromosome X and Y









(unique) 155 expressed









sequence


206999_at
1380
NM_001559
1588
0.000344019
2.07
IL12RB2
interleukin 12 receptor,









beta 2


205842_s_at
1374
AF001362
1538
0.000351718
1.85
JAK2
Janus kinase 2 (a









protein tyrosine kinase)


210166_at
1405
AF051151
1536
0.000353968
1.63
TLR5
toll-like receptor 5


219714_s_at
1487
NM_018398
1612
0.000364622
2.39
CACNA2D3
calcium channel,









voltage-dependent,









alpha 2/delta 3 subunit


212311_at
1426
AA522514
1662
0.000370292
−1.36
KIAA0746
KIAA0746 protein


213830_at
1447
AW007751
1675
0.000370999
−1.69
TRD @
T-cell receptor delta









chain HE/801 /// T cell









receptor delta locus


213005_s_at
1438
D79994
1614
0.000376513
2.26
ANKRD15
ankyrin repeat domain









15


202944_at
1351
NM_000262
1639
0.000376931
1.68
NAGA
N-









acetylgalactosaminidase,









alpha-


206011_at
1375
AI719655
1668
0.000379898
1.49
CASP1
caspase 1, apoptosis-









related cysteine









protease (interleukin 1,









beta, convertase)


204332_s_at
1365
M64073
1516
0.000382893
−1.22
AGA
aspartylglucosaminidase


215592_at
1463
AU147620
1687
0.000385413
−1.91
—
Transcribed locus,









weakly similar to









XP_375099.1









hypothetical protein









LOC283585


201798_s_at
378
NM_013451
777
0.000387903
2.24
FER1L3
fer-1-like 3, myoferlin









(C. elegans)


212069_s_at
1424
AK026025
1566
0.000388281
−1.17
KIAA0515
KIAA0515


206255_at
1377
NM_001715
1597
0.000395152
−1.72
BLK
B lymphoid tyrosine









kinase


221932_s_at
1497
AA133341
1658
0.000396589
−1.42
C14orf87
chromosome 14 open









reading frame 87


203246_s_at
1353
NM_006545
1611
0.000402598
−1.33
TUSC4
tumor suppressor









candidate 4


213534_s_at
19
D50925
425
0.000408073
−1.73
PASK
PAS domain containing









serine/threonine kinase


219045_at
1481
NM_019034
1604
0.000412198
−1.33
RHOF
ras homolog gene









family, member F (in









filopodia)


202347_s_at
1340
AB022435
1550
0.000414497
−1.16
HIP2
huntingtin interacting









protein 2


212636_at
1430
AL031781
1543
0.000420707
1.61
QKI
quaking homolog, KH









domain RNA binding









(mouse)


202392_s_at
1341
NM_014338
1598
0.000423758
1.26
PISD
phosphatidylserine









decarboxylase


216950_s_at
1467
X14355
1509
0.000437652
2.62
FCGR1A
Fc fragment of IgG,









high affinity Ia,









receptor for (CD64)


214511_x_at
1457
L03419
1515
0.000447516
2.83
LOC440607
Fc-gamma receptor I








///
B2 /// Fc fragment of








FCGR1A
IgG, high affinity Ia,









receptor for (C


219316_s_at
1484
NM_017791
1641
0.000472922
1.68
C14orf58
chromosome 14 open









reading frame 58


220122_at
398
NM_024717
797
0.000473634
3.36
MCTP1
multiple C2-domains









with two









transmembrane regions 1


64883_at
1508
AI744083
1669
0.000478581
1.51
MOSPD2
motile sperm domain









containing 2


203279_at
1354
NM_014674
1644
0.000479669
−1.24
EDEM1
ER degradation









enhancer, mannosidase









alpha-like 1


208891_at
1390
BC003143
1573
0.000483009
1.98
DUSP6
dual specificity









phosphatase 6


205715_at
1372
NM_004334
1636
0.000490107
1.60
BST1
bone marrow stromal









cell antigen 1


214085_x_at
1451
AI912583
1674
0.000510466
1.67
HRB2
HIV-1 rev binding









protein 2


215000_s_at
1461
AL117593
1553
0.000515959
1.24
FEZ2
fasciculation and









elongation protein zeta









2 (zygin II)


202194_at
1339
AL117354
1556
0.000523169
1.36
CGI-
CGI-100 protein








100



210202_s_at
1408
U87558
1530
0.000532952
−1.46
BIN1
bridging integrator 1


218181_s_at
1472
NM_017792
1560
0.000547382
1.13
MAP4K4
mitogen-activated









protein kinase kinase









kinase kinase 4


204023_at
1360
NM_002916
1595
0.000553752
−1.48
RFC4
replication factor C









(activator 1) 4, 37 kDa


207872_s_at
1384
NM_006863
1551
0.000561178
1.58
LILRA1
leukocyte









immunoglobulin-like









receptor, subfamily A









(with TM domain),









member 1


202878_s_at
1349
NM_012072
1622
0.000583921
1.42
C1QR1
complement









component 1, q









subcomponent,









receptor 1


201285_at
1333
NM_013446
1649
0.000593976
−1.21
MKRN1
makorin, ring finger









protein, 1 /// makorin,









ring finger protein, 1


211612_s_at
1417
U62858
1529
0.000613797
1.80
IL13RA1
interleukin 13 receptor,









alpha 1 /// interleukin









13 receptor, alpha 1


219117_s_at
1482
NM_016594
1647
0.000616534
−1.61
FKBP11
FK506 binding protein









11, 19 kDa


215761_at
1464
AK000156
1557
0.000618215
2.26
RC3
rabconnectin-3


200800_s_at
1328
NM_005345
1642
0.000625103
1.65
HSPA1A
heat shock 70 kDa








///
protein 1A /// heat








HSPA1B
shock 70 kDa protein









1B


202816_s_at
1346
AW292882
1679
0.000641212
1.44
SS18
synovial sarcoma









translocation,









chromosome 18


209440_at
1397
BC001605
1572
0.000646328
−1.25
PRPS1
phosphoribosyl









pyrophosphate









synthetase 1


204221_x_at
1362
U16307
1587
0.000654803
1.51
HRB2
HIV-1 rev binding









protein 2


57082_at
1506
AA169780
1659
0.000658787
−1.41
ARH
LDL receptor adaptor









protein


201887_at
1338
NM_001560
1589
0.000679598
1.52
IL13RA1
interleukin 13 receptor,









alpha 1


215933_s_at
1465
Z21533
1510
0.000692626
1.47
HHEX
hematopoietically









expressed homeobox


208774_at
1389
AV700224
1685
0.000699738
1.20
CSNK1D
Casein kinase 1, delta


201478_s_at
1334
U59151
1533
0.000707429
−1.18
DKC1
dyskeratosis congenita









1, dyskerin


208918_s_at
1391
AI334128
1665
0.000708781
1.38
FLJ13052
NAD kinase


208158_s_at
1386
NM_018030
1581
0.000726407
1.37
OSBPL1A
oxysterol binding









protein-like 1A ///









oxysterol binding









protein-like 1A


202838_at
1348
NM_000147
1651
0.00073801
1.43
FUCA1
fucosidase, alpha-L-1,









tissue


205039_s_at
1371
NM_006060
1633
0.000754874
2.05
ZNFN1A1
zinc finger protein,









subfamily 1A, 1









(Ikaros)


204834_at
1368
NM_006682
1626
0.000760238
1.74
FGL2
fibrinogen-like 2


200701_at
1327
NM_006432
1592
0.000763959
1.41
NPC2
Niemann-Pick disease,









type C2


204254_s_at
1364
NM_000376
1617
0.00077458
1.64
VDR
vitamin D (1,25-









dihydroxyvitamin D3)









receptor


217922_at
1470
AL157902
1562
0.000779841
−1.34
MAN1A2
Mannosidase, alpha,









class 1A, member 2


218888_s_at
1479
NM_018092
1586
0.000801694
1.39
NETO2
neuropilin (NRP) and









tolloid (TLL)-like 2


210947_s_at
1414
J04810
1514
0.000810544
1.30
MSH3
mutS homolog 3 (E. coli)


208923_at
1392
BC005097
1575
0.000816109
1.41
CYFIP1
cytoplasmic FMR1









interacting protein 1


209429_x_at
1396
AF112207
1555
0.000818155
−1.22
—
—


204566_at
1366
NM_003620
1593
0.000823091
1.67
PPM1D
protein phosphatase 1D









magnesium-dependent,









delta isoform


218854_at
1478
NM_013352
1630
0.000823847
1.47
SART2
squamous cell









carcinoma antigen









recognized by T cells 2


213198_at
1441
AL117643
1554
0.000836
1.33
ACVR1B
activin A receptor, type









IB


218642_s_at
1476
NM_024300
1618
0.000849263
−1.21
CHCHD7
coiled-coil-helix-









coiled-coil-helix









domain containing 7


203645_s_at
1358
NM_004244
1632
0.000851877
1.60
CD163
CD163 antigen


208117_s_at
1385
NM_031206
1653
0.000852389
−1.32
FLJ12525
hypothetical protein









FLJ12525 ///









hypothetical protein









FLJ12525


218519_at
1474
NM_017945
1594
0.000854014
1.30
SLC35A5
solute carrier family









35, member A5


217764_s_at
1469
AF183421
1563
0.000871159
1.30
RAB31
RAB31, member RAS









oncogene family


214765_s_at
1459
AK024677
1564
0.000919505
1.50
ASAHL
N-acylsphingosine









amidohydrolase (acid









ceramidase)-like


212799_at
1432
BE217875
1682
0.00093697
1.31
—
Clone 23570 mRNA









sequence


204744_s_at
1367
NM_013417
1624
0.000961709
−1.24
IARS
isoleucine-tRNA









synthetase


218526_s_at
1475
NM_014185
1559
0.000964651
−1.22
RANGNRF
RAN guanine









nucleotide release









factor


211139_s_at
1415
AF045452
1539
0.000972219
1.40
NAB1
NGFI-A binding









protein 1 (EGR1









binding protein 1)


213958_at
1449
AW134823
1677
0.000977101
−1.39
CD6
CD6 antigen /// CD6









antigen


221695_s_at
1494
AF239798
1558
0.000979214
1.37
MAP3K2
mitogen-activated









protein kinase kinase









kinase 2 /// mitogen-









activated protein k


210176_at
1406
AL050262
1549
0.000989413
1.62
TLR1
toll-like receptor 1


212314_at
1427
AB018289
1542
0.00099
−1.34
KIAA0746
KIAA0746 protein


209882_at
1402
AF084462
1544
0.000992617
1.42
RIT1
Ras-like without









CAAX 1


214500_at
1456
AF044286
1541
0.00102256
1.51
H2AFY
H2A histone family,









member Y


213088_s_at
1439
BE551340
1684
0.00104481
−1.17
DNAJC9
DnaJ (Hsp40)









homolog, subfamily C,









member 9


200821_at
1329
NM_013995
1638
0.00104551
1.22
LAMP2
lysosomal-associated









membrane protein 2


210732_s_at
1411
AF342816
1574
0.00106818
1.84
LGALS8
lectin, galactoside-









binding, soluble, 8









(galectin 8)


201224_s_at
1332
AU147713
1688
0.00107129
−1.23
SRRM1
serine/arginine









repetitive matrix 1


202444_s_at
1343
NM_006459
1634
0.00107236
1.72
C10orf69
chromosome 10 open









reading frame 69


206707_x_at
1379
NM_015864
1580
0.00107472
−1.24
C6orf32
chromosome 6 open









reading frame 32


221839_s_at
1496
AK026088
1567
0.00107916
−1.34
UBAP2
ubiquitin associated









protein 2


213279_at
1443
AL050217
1547
0.00108194
1.21
DHRS1
dehydrogenase/reductase









(SDR family)









member 1


214974_x_at
1460
AK026546
1568
0.00109223
2.67
CXCL5
chemokine (C—X—C









motif) ligand 5


209583_s_at
1399
AF063591
1570
0.00109644
1.65
CD200
CD200 antigen


209870_s_at
1401
AW571582
1681
0.00112322
−1.39
APBA2
amyloid beta (A4)









precursor protein-









binding, family A,









member 2 (X11-like)


201494_at
1335
NM_005040
1605
0.0011371
1.27
PRCP
prolylcarboxypeptidase









(angiotensinase C)


219806_s_at
1488
NM_020179
1637
0.00113985
1.36
FN5
FN5 protein


208651_x_at
1387
M58664
1513
0.00119019
−1.38
CD24
CD24 antigen (small









cell lung carcinoma









cluster 4 antigen)


216191_s_at
1466
X72501
1522
0.00132593
−1.84
TRDD3
T cell receptor delta








///
diversity 3 /// T cell








TRD @
receptor delta locus


217143_s_at
1468
X06557
1511
0.00187286
−1.83
TRDD3
T cell receptor delta








///
diversity 3 /// T cell








TRD @
receptor delta locus





vs. = versus.













TABLE 6







Genes which when included in classifiers of no more than 10 genes exhibit at least


70% correct classification rates between BMS and typical RRMS patients
























Full






P
BMS
Sequence
SEQ

Length
SEQ


Probeset
SEQ ID
Gene
value
vs
Derived
ID

Ref.
ID


ID
NO:
Symbol
(min)
RRMS
From
NO:
Gene Title
Sequences
NO:



















222204_s_at
1498
RRN3
2.1 * 10−8
(−)
AL110238
1552
RNA
NM_018427
1010









polymerase I











transcription











factor RRN3




221714_s_at
1495
LOC94431


BC006441
1579
similar to RNA
NM_145237
1156









polymerase I











transcription











factor RRN3




211615_s_at
1418
LRPPRC
2.4 * 10−8
(−)
M92439
1512
leucine-rich
NM_133259
1065









PPR-motif











containing











protein




211971_s_at
204
LRPPRC


AI653608
606
leucine-rich
NM_133259
1065









PPR motif











containing











protein




218258_at
173
POLR1D
1.9 * 10−6
(−)
NM_015972
984
hypothetical
NM_152705
1695









protein











MGC9850




205789_at
1373
CD1D
4.3 * 10−6
(+)
NM_001766
1627
CD1D antigen,
NM_001766
1627









d polypeptide




212999_x_at
1437
HLA-
9.9 * 10−6
(+)
AW276186
1678
major
NM_002123
1699




DQB1




histocompatibility











complex,











class II, DQ











beta 1 precursor




209823_x_at
1400
HLA-


M17955
1518
major
NM_002123
1699




DQB1




histocompatibility











complex,











class II, DQ











beta 1 precursor




209480_at
1398
HLA-


M16276
1521
major
NM_002123
1699




DQB1




histocompatibility











complex,











class II, DQ











beta 1 precursor




211656_x_at
1420
HLA-


M32577
1520
major
NM_002123
1699




DQB1




histocompatibility











complex,











class II, DQ











beta 1 precursor




211654_x_at
1419
HLA-


M17565
1519
major
NM_002123
1699




DQB1




histocompatibility











complex,











class II, DQ











beta 1 precursor




210747_at
1412
HLA-


M24364
1525
major
NM_002123
1699




DQB1




histocompatibility











complex,











class II, DQ











beta 1 precursor




206584_at
1378
LY96
5.8 * 10−6
(+)
NM_015364
1650
lymphocyte
NM_015364
1650









antigen 96; //











MD-2 protein




207359_at
1383
CAMKK2
6.1 * 10−6
(+)
NM_006549
1590
calcium/calmodulin-
NM_006549
1590









dependent











protein kinase











kinase 2, beta











isoform 1




214209_s_at
1452
CAMKK2


BE504895
1683
calcium/calmodulin-
NM_006549
1590









dependent











protein kinase











kinase 2, beta











isoform 1




213812_s_at
1446
CAMKK2


AK024748
1565
calcium/calmodulin-
NM_006549
1590









dependent











protein kinase











kinase 2, beta











isoform 1




210787_s_at
1413
CAMKK2


AF140507
1546
calcium/calmodulin-
NM_006549
1590









dependent











protein kinase











kinase 2, beta











isoform 1




212252_at
1425
CAMKK2


AA181179
1660
calcium/calmodulin-
NM_006549
1590









dependent











protein kinase











kinase 2, beta











isoform 1




214643_x_at
1458
BIN1
2.2 * 10−6
(−)
BG034080
1690
bridging
NM_004305
1693









integrator 1
//
//1694









isoform 8
NM_139343



210202_s_at
1408
BIN1


U87558
1530
bridging
NM_004305
1693









integrator 1
//
//1694









isoform 8
NM_139343











//



214439_x_at
1453
BIN1


AF043899
1535
bridging
NM_004305
1693









integrator 1
//
//1694









isoform 8
NM_139343











//



202931_x_at
1350
BIN1


NM_004305
1584
bridging
NM_004305
1693









integrator 1
//
//1694









isoform 8
NM_139343











//



210201_x_at
1407
BIN1


AF001383
1531
bridging
NM_004305
1693









integrator 1
//
//1694









isoform 8
NM_139343











//



214450_at
1454
CTSW
3.8 * 10−6
(−)
NM_001335
1629
cathepsin W
NM_001335
1629









preproprotein




214470_at
1455
KLRB1
3.8 * 10−6
(−)
NM_002258
1615
killer cell lectin-
NM_002258
1615









like receptor











subfamily B,











member 1




212748_at
1431
MKL1
7.9 * 10−9
(+)
AB037859
1583
megakaryoblastic
NM_020831
1696









leukemia











(translocation) 1




209072_at
1393
MBP
2.7 * 10−5
(−)
M13577
1517
myelin basic
NM_002385
1620









protein




210136_at
1404
MBP


AW070431
1676
myelin basic
NM_002385
1620









protein




207323_s_at
1382
MBP


NM_002385
1620
myelin basic
NM_002385
1620









protein




212978_at
1435
TA-
4.1 * 10−5
(+)
AU146004
1686
T-cell activation
NM_015350
1700




LRRP




leucine repeat-











rich protein




212976_at
1434
TA-


R41498
1655
T-cell activation
NM_015350
1700




LRRP




leucine repeat-











rich protein




117_at
1325
HSPA6
8.9 * 10−5
(+)
X51757
1692
heat shock
NM_002155
1609









70 kDa protein 6











(HSP70B′)




213418_at
1445
HSPA6


NM_002155
1609
heat shock
NM_002155
1609









70 kDa protein 6











(HSP70B′)




200610_s_at
1326
NCL
8.9 * 10−5
(−)
NM_005381
1613
nucleolin
NM_005381
1613


216191_s_at
1466
TRD @
3.7 * 10−4
(−)
X72501
1522
T cell receptor
—
#N/A









delta locus




217143_s_at
1468
TRD @


X06557
1511
T cell receptor
—
#N/A









delta locus




213830_at
1447
TRD @


AW007751
1675
T cell receptor
—
#N/A









delta locus




213005_s_at
1438
KANK
3.7 * 10−4
(+)
D79994
1614
kidney ankyrin
NM_015158
1698









repeat-
//
//1697









containing
NM_153186










protein





Presented are 38 gene transcripts which correspond for 17 human genes which classify BMS and typical RRMS patients.


Genes given by the gene name and description, the Affymetrix probeset identification number, and a representative GenBank Accession numbers.


The (−) sign means that the polynucleotide is downregulated (decreased in level) in BMS as compared to typical RRMS subjects;


and the (+) sign means that the polynucleotide is upregulated (increased in level) in BMS as compared to RRMS subjects.






Examples of classifiers are presented as following:
NCL, MKL1, CTSW, KLRB1 and LRPPRC which results in correct classification rate of 79% of BMS and typical RRMS; and NCL, MKL1, CTSW, KLRB1, POLR1D, CD1D, and CAMKK2 which results in correct classification rate of 77% of BMS and typical RRMS.
Example 3
Testing the Effect of an Anti MS Drug in Experimental Allergic Encephalomyelitis (EAE)—Animal MS Model
Animal Model for Multiple Sclerosis—
EAE is induced in female Lewis rats (N=15; 6-8 weeks old, body weight 180-200 g) by hind footpad subcutaneous inoculation with emulsion of 25 mg guinea-pig MBP (myelin basic protein) in CFA containing 40 mg of Mycobacterium tuberculosis (Difco, Detroit, Mich.) in 0.1 ml oil. Control rats (N=15) are injected with the same emulsion where saline solution replaces MBP. The EAE is scored as follows: 0—No obvious changes in motor function on of the rats in comparison to non-immunized rats; 1—Limp tail; 2—Limp tail and weakness of hind legs; 3—Limp tail and complete paralysis of hind legs, or limp tail with paralysis of one front and one hind leg. Or all of: walking only along the edges of the cage, pushing against the cage wall, pushing against the cage wall, spinning when picked up by the tail; 4—Limp tail, complete hind leg and partial front leg paralysis; 5—Complete hind and complete front leg paralysis, no movement around the cage, or mouse is spontaneously rolling in the cage, mouse is found dead due to paralysis.
Treatment with an Anti MS Drug or with an Agent which Downregulates at Least One Gene of the RNA Polymerase Pathway—
A therapeutically effective amount of a drug or an agent which downregulates the expression level of a gene involved in the RNA polymerase I pathway (e.g., TPT) is administered to the animal on the day of EAE induction and blood samples are drawn before and after treatment, at predetermined time points which include baseline=Time 0, Day 12—peak disease, Day 17 and day 21.
Testing the Level of Expression of Genes Involved in the RNA Polymerase I Pathway—
Blood samples, obtained from the control and treated animals, are tested using Q-RT-PCR for bio-markers of benign multiple sclerosis (e.g., RRN3, POLR1D and LRPPRC). Control animals are compared to animals on the peak of EAE disease and to animals treated by the anti MS drug (e.g., TPT).
Example 4
In Vitro Testing Efficacy of a Drug In Vitro Using Cells of a Multiple Sclerosis Subject
Peripheral blood samples are obtained from female subjects with typical RRMS disease course. All patients are free of immunomodulatory or corticosteroid treatments at least 30 days before blood withdrawal. PBMC are extracted from peripheral blood, separated by Ficoll-Hypaque gradient. 15 ml of peripheral blood from patients is diluted 1:1 with Phosphate Buffered Saline (PBS) (without Ca2+/Mg2+). Blood samples are underlied with 10 ml of Ficoll-Lymphoprep (Axis Chield, Norway) and spinned (Eppendorf centrifuge, Germany) at 2300 RPM for 30 minutes. PBMCs are collected, washed with PBS and counted and incubated at 37° C. in a humidified CO2 incubator with or without anti MS drugs. After incubation, total RNA is extracted using both Trizol (Invitrogen, USA) and Phase-Lock-Gel columns (Eppendorf, Germany) including a DNase digestion step. RNA integrity is assessed by RNA Experion automated electrophoresis system.
Treatment with an Anti MS Drug or with an Agent which Downregulates at Least One Gene of the RNA Polymerase Pathway—
A therapeutically effective amount of a drug or an agent which downregulates the expression level of a gene involved in the RNA polymerase I pathway (e.g., TPT) is incubated during 3 hours with the PBMC (of the subject having typical RRMS) and compared with PBMC (of the subject having typical RRMS) incubated during 3 hours without drug or with placebo excluding therapeutic component.
Testing the Level of Expression of Genes Involved in the RNA Polymerase I Pathway—
Total RNA samples, obtained from PBMC of a subject having typical RRMS trayed in-vitro with anti-RNA polymerase I agents (e.g. TPT), are tested using Q-RT-PCR for bio-markers of benign multiple sclerosis (e.g., RRN3, POLR1D and LRPPRC). The results compared with PBMC from same patients incubated without drag or with placebo.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
REFERENCES
Additional References are Cited in Text


    1. Hirschler-Laszkiewicz I, Cavanaugh A H, Mirza A et al. Rrn3 becomes inactivated in the process of ribosomal DNA transcription. J Biol Chem. 2003; 278:18953-18959;
    2. Miller G, Panov K I, Friedrich J K et al. hRRN3 is essential in the SL1-mediated recruitment of RNA Polymerase I to rRNA gene promoters. Embo J. 2001; 20:1373-1382;
    3. Achiron A, Gurevich M, Snir Y et al Zinc-ion binding and cytokine activity regulation pathways predicts outcome in relapsing-remitting multiple sclerosis. Clin Exp Immunol. 2007; 149:235-242;
    4. Mootha V K, Lepage P, Miller K et al. Identification of a gene causing human cytochrome c oxidase deficiency by integrative genomics. Proc Natl Acad Sci USA. 2003; 100:605-610;
    5. Bouwmeester T, Bauch A, Ruffner H et al. A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway. Nat Cell Biol. 2004; 6:97-105;
    6. Leuenroth S J, Crews C M. Triptolide-induced transcriptional arrest is associated with changes in nuclear substructure. Cancer Res. 2008; 68:5257-5266;
    7. Liu Y, Chen Y, Lamb J R, Tam P K. Triptolide, a component of Chinese herbal medicine, modulates the functional phenotype of dendritic cells. Transplantation. 2007; 84:1517-1526;
    8. Wang Y, Mei Y, Feng D, Xu L. Triptolide modulates T-cell inflammatory responses and ameliorates experimental autoimmune encephalomyelitis. J Neurosci Res. 2008; 86:2441-2449;
    9. Pittock S J, Rodriguez M. Benign multiple sclerosis: a distinct clinical entity with therapeutic implications. Curr Top Microbiol Immunol. 2008; 318:1-17;
    10. L Costelloe, A Thompson, C Walsh, N Tubridy, and M Hutchinson Long-term clinical relevance of criteria for designating multiple sclerosis as benign after 10 years of disease J. Neurol. Neurosurg. Psychiatry, November 2008; 79: 1245-1248.

## Claims

1. A composition comprising a probeset for classifying a disease course in a subject diagnosed with multiple sclerosis comprising a plurality of oligonucleotides and no more than 50 oligonucleotides, wherein said plurality of oligonucleotides comprise oligonucleotides that specifically recognize RRN3, POLR1D and LRPPRC; and a biological sample of a subject diagnosed with multiple sclerosis.

2. The composition of claim 1, wherein said biological sample comprises a blood sample.

3. The composition of claim 1, wherein said plurality of oligonucleotides comprises no more than 25 oligonucleotides.

4. The composition of claim 1, wherein said classifying a disease course comprises distinguishing between benign multiple sclerosis (BMS) and relapsing-remitting multiple sclerosis (RRMS).