US20120020954A1

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Publication number: US20120020954A1
Application number: US13/260,573
Authority: US
Inventors: Anat Achiron; Michael Gurevich
Original assignee: Individual
Current assignee: Tel HaShomer Medical Research Infrastructure and Services Ltd
Priority date: 2009-03-30
Filing date: 2010-03-28
Publication date: 2012-01-26
Also published as: EP2414542A1; WO2010113096A1; EP2414542B1; IL215392A; US20150315646A1; US9758831B2; IL215392A0; DK2414542T3; US20170362657A1; US10738361B2

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.

Description

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 MS (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 herbTripterygium 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 ofclaim1,

(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 ofclaim1,

(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 ofclaim2,

(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 claim7.

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 theRNA 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 theRNA 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, FLJ11301, LRP5, STOM, EPHA2, SRC, FLJ11301, PSTPIP2, EBP, MCPH1, PTPRF, LIMK2, FSTL4, CBR1, MGC2654, MYCT1, NOL3, MITF, ATP10B, FBXO31, TBX21, LSS, SLC17A3, MNAB, CHPPR, G1F, 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, G1F, 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, G1F, 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 theRNA 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, G1F, 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 theRNA polymerase 1 pathway, thereby treating the subject.

According to some embodiments of the invention, the at least one polynucleotide or polypeptide of theRNA polymerase 1 pathway comprises RRN3.

According to some embodiments of the invention, the at least one polynucleotide or polypeptide of theRNA 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 DRAWINGS

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

In the drawings:

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) multi-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.

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.

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.

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.

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 theRNA polymerase 1 pathway is RRN3.

According to some embodiments of the invention, the at least one gene involved in theRNA polymerase 1 pathway is LRPPRC.

According to some embodiments of the invention, the at least one gene involved in theRNA polymerase 1 pathway is POLR1D.

According to some embodiments of the invention, the at least one gene involved in theRNA polymerase 1 pathway comprises RRN3 and POLR1D.

According to some embodiments of the invention, the at least one gene involved in theRNA polymerase 1 pathway comprises RRN3 and LRPPRC.

According to some embodiments of the invention, the at least one gene involved in theRNA polymerase 1 pathway comprises POLR1D and LRPPRC.

According to some embodiments of the invention, the at least one gene involved in theRNA polymerase 1 pathway comprises RRN3, LRPPRC and POLR1D.

According to some embodiments of the invention, the at least one gene involved in theRNA polymerase 1 pathway is RRN3 and NCL.

According to some embodiments of the invention, the at least one gene involved in theRNA polymerase 1 pathway is LRPPRC and NCL.

According to some embodiments of the invention, the at least one gene involved in theRNA polymerase 1 pathway is POLR1D and NCL.

According to some embodiments of the invention, the at least one gene involved in theRNA polymerase 1 pathway comprises RRN3, POLR1D and NCL.

According to some embodiments of the invention, the at least one gene involved in theRNA polymerase 1 pathway comprises RRN3, LRPPRC and NCL.

According to some embodiments of the invention, the at least one gene involved in theRNA polymerase 1 pathway comprises POLR1D, LRPPRC and NCL.

According to some embodiments of the invention, the at least one gene involved in theRNA 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,

(c) informing the subject of the diagnosis,

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,

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:

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;
R₁and R₂each 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 theinteger 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 inE. colior 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 asE. 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)₃and 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 down-regulate 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 dsRNA specifically designed not to induce the interferon and PKR pathways for down-regulating 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 21mers with a central 19 by 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 21mers 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 (27mer) instead of a product (21mer) 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 [Tuschl 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-abl 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 inPhase 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 oligonucleotides (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 oligonucleotides, 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, inrtaperitoneal, 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, G1F, 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™ 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 inFIG. 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

				p-value	Log Fold
				BMS	Change
Affymetrix	SEQ	Representative	SEQ	vs.	(BMS vs.
Probeset	ID	Public	ID	typical	typical	Gene
ID	NO:	ID	NO:	RRMS	RRMS)	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

Table 1: 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
	ID
PROBESET	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 herbTripterygium 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 herbTripterygium wilfordiiHook 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 herbTripterygium wilfordiiHook 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™ 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 Discovery 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
	POLRID	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		Represen-		vs	(BMS vs	Gene
ProbSet	SEQ	tative	SEQ ID	typical	typical	Sym-
ID	NO ID:	Public ID	NO:	RRMS	RRMS)	bol	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	CD 164 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
							proteinkinase 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/reduc-
							tase (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
						TRD @	diversity 3 /// T cell
							receptor delta locus
217143_s_at	1468	X06557	1511	0.00187286	−1.83	TRDD3 ///	T cell receptor delta
						TRD @	diversity 3 /// T cell
							receptor delta locus

Table 5. 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

	SEQ	Gene	P	BMS	Sequence	SEQ		Full Length	SEQ
Probeset	ID	Sym-	value	vs	Derived	ID		Ref.	ID
ID	NO:	bol	(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					histocompatibili-
							ty complex,
							class II,DQ
							beta
1 precursor
209823_x_at	1400	HLA-			M17955	1518	major	NM_002123	1699
		DQB1					histocompatibili-
							ty complex,
							class II, DQ
							beta 1 precursor
209480_at	1398	HLA-			M16276	1521	major	NM_002123	1699
		DQB1					histocompatibili-
							ty complex,
							class II,DQ
							beta
1 precursor
211656_x_at	1420	HLA-			M32577	1520	major	NM_002123	1699
		DQB1					histocompatibili-
							ty complex,
							class II,DQ
							beta
1 precursor
211654_x_at	1419	HLA-			M17565	1519	major	NM_002123	1699
		DQB1					histocompatibili-
							ty complex,
							class II,DQ
							beta
1 precursor
210747_at	1412	HLA-			M24364	1525	major	NM_002123	1699
		DQB1					histocompatibili-
							ty 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/calmod-	NM_006549	1590
							ulin-dependent
							protein kinase
							kinase
2,beta
							isoform
1
214209_s_at	1452	CAMKK2			BE504895	1683	calcium/calmod-	NM_006549	1590
							ulin-dependent
							protein kinase
							kinase
2,beta
							isoform
1
213812_s_at	1446	CAMKK2			AK024748	1565	calcium/calmod-	NM_006549	1590
							ulin-dependent
							protein kinase
							kinase
2,beta
							isoform
1
210787_s_at	1413	CAMKK2			AF140507	1546	calcium/calmod-	NM_006549	1590
							ulin-dependent
							protein kinase
							kinase
2,beta
							isoform
1
212252_at	1425	CAMKK2			AA181179	1660	calcium/calmod-	NM_006549	1590
							ulin-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_002	1609
							70 kDa protein 6	155
							(HSP70B′)
213418_at	1445	HSPA6			NM_002155	1609	heat shock	NM_002	1609
							70 kDa protein 6	155
							(HSP70B′)
200610_s_at	1326	NCL	8.9*10−5	(−)	NM_005381	1613	nucleolin	NM_005	1613
								381
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-	NM_153186	//1697
							containing
							protein

Table 6. 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 ofMycobacterium 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 CO₂incubator 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 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 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.

2. 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 ofclaim 1,

(i) wherein when the subject is classified as being more likely to have said BMS then the subject is diagnosed as having BMS;

(ii) wherein when the subject is classified as being more likely to have said typical RRMS, then the subject is diagnosed as having typical RRMS; and

(c) informing the subject of the diagnosis,

3. 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 said treating with the anti MS drug to a level of expression of said at least one gene in a cell of the subject prior to said treating the subject with the anti MS drug,

(i) wherein a decrease above a predetermined threshold in said level of expression of said at least one gene following said treating with the anti MS drug relative to said level of expression of said at least one gene prior to said 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 said level of expression of said at least one gene following said treating with the anti MS drug relative to said level of expression of said at least one gene prior to said 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 said at least one gene following said treating with the anti MS drug is identical or changed below a predetermined threshold as compared to prior to said treating with the anti MS drug then the treatment is not efficient for treating the subject

4. 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:

(b) comparing a level of expression in said cells of at least one gene involved in the RNA polymerase I pathway following said contacting with the anti MS drug to a level of expression of said at least one gene in said cells prior to said contacting with the anti MS drug,

(i) wherein a decrease above a predetermined threshold in said level of expression of said at least one gene following said contacting with the anti MS drug relative to said level of expression of said at least one gene prior to said 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 said level of expression of said at least one gene following said contacting with the anti MS drug relative to said level of expression of said at least one gene prior to said 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 said at least one gene following said contacting with the anti MS drug is identical or changed below a predetermined threshold as compared to prior to said contacting with the anti MS drug then the treatment is not efficient for treating the subject

5. 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 ofclaim 1,

6. 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 ofclaim 2,

7. A probeset comprising a plurality of oligonucleotides and no more than 50 oligonucleotides, wherein an oligonucleotide of said plurality of oligonucleotides specifically recognizes a polynucleotide of at least one gene involved in the RNA polymerase pathway.

8. A kit for classifying a disease course in a subject diagnosed with multiple sclerosis (MS), comprising the probeset ofclaim 7.

9. A method of selecting a drug for treating a typical relapsing remitting multiple sclerosis (RRMS) in a subject, the method comprising:

identifying at least one drug molecule which downregulates a level of expression of at least one gene involved in the RNA polymerase I pathway, said 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.

10. The method ofclaim 1, wherein a decrease above a predetermined threshold in said level of expression of said at least one gene in said cell of the subject relative to said reference expression data of said at least one gene obtained from said at least one subject having said typical RRMS classifies the subject as being more likely to have the BMS.

11. The method ofclaim 1, wherein an increase above a predetermined threshold in said level of expression of said at least one gene in said cell of the subject relative to said reference expression data of said at least one gene obtained from said at least one subject having said BMS classifies the subject as being more likely to have the typical RRMS.

12. The method ofclaim 1, wherein when a level of expression of said at least one gene in said cell of the subject is identical or changed below a predetermined threshold as compared to said reference expression data of said at least one gene obtained from said at least one subject having said BMS, then the subject is classified as being more likely to have the BMS.

13. The method ofclaim 1, wherein when a level of expression of said at least one gene in said cell of the subject is identical or changed below a predetermined threshold as compared to said reference expression data of said at least one gene obtained from said at least one subject having said typical RRMS, then the subject is classified as being more likely to have the typical RRMS.

14. The method ofclaim 5, wherein when the subject being more likely to have typical RRMS then said treatment regimen comprises administering to the subject an agent which downregulates the level of expression of said at least one gene involved in said RNA polymerase I pathway.

15. The method ofclaim 1, wherein said at least one gene involved in said RNA polymerase 1 pathway is selected from the group consisting of POLR1D, LRPPRC, RRN3 and NCL.

16. The method ofclaim 1, wherein said at least one gene involved in said RNA polymerase 1 pathway comprises the POLR1D, LRPPRC, RRN3 and NCL genes.

17. The method ofclaim 6, wherein said agent is selected from the group consisting of an siRNA, an antisense, an antibody and a small molecule.

18. The method ofclaim 17, wherein said small molecule is Cycloheximide.

19. The method ofclaim 6, wherein said at least one gene is RRN3, and whereas said downregulating is effected using diterpenoid triepoxide Triptolide (TPT) or a derivative thereof.

20. The method ofclaim 6, wherein said at least one gene is RRN3, and whereas said downregulating is effected using Cycloheximide.

21. The kit ofclaim 8, further comprising a positive control for an expression level of said at least one gene involved in the RNA polymerase pathway.

22. The probeset ofclaim 7, wherein each of said plurality of oligonucleotides is bound to a solid support.

23. The probeset wherein said plurality of oligonucleotides are bound to said solid support in an addressable location.

24. The method ofclaim 1, wherein said level of expression is determined using an RNA detection method.

25. The method ofclaim 1, wherein said level of expression is determined using a protein detection method.

26. The method ofclaim 1, wherein said cell is a blood cell.

27. The method ofclaim 5, further comprising administering to the subject a therapeutically effective amount of an anti MS agent.

28. The method ofclaim 3, wherein said 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).

29. The probeset ofclaim 7, wherein said at least one gene involved in said RNA polymerase 1 pathway comprises the POLR1D, LRPPRC, RRN3 and NCL genes.