Methods for diagnosing or predicting neurological disorders

Abstract

To provide a method of diagnosing or predicting a neurological disorder in a subject.SOLUTION: A method comprises: determining a quantitative or qualitative level of one or more biomarkers in a biological sample from a subject; and diagnosing or predicting a neurological disorder in the subject based on the quantitative or qualitative level of a specific biomarker or each biomarker in the biological sample.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method of diagnosing or predicting a neurological disorder in a subject, the method comprising determining the quantitative or qualitative level of one or more biomarkers in a biological sample of the subject, and diagnosing or predicting the neurological disorder in the subject based on the quantitative or qualitative level of the or each biomarker in the biological sample. [Background technology]

[0002] Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is a fatal motor neuron disorder that causes progressive degeneration and death of upper and lower motor neurons, protein aggregation, severe muscle atrophy, and respiratory failure. Median survival is 2-5 years from onset of symptoms. ALS manifests as either familial ALS (FALS; -10% of cases) or sporadic ALS (SALS; -90% of cases). The incidence of ALS is estimated to be 0.3-2.6 cases per 100,000 (2.16 / 100,000 / year in Europe and 2.0 / 100,000 / year in Ireland).

[0003] Studies in animal models and ALS patients indicate that motor neuron degeneration begins at the neuromuscular junction and that postsynaptic muscle changes may play an active role. Studies have shown that skeletal muscle has functional secretory activity. The muscle secretome contains exosomes, which are vesicles that carry out intercellular transport of functional proteins, mRNA, and miRNA. Exosomes may act in an autocrine / paracrine manner on muscle cells or other types of cells, contributing to muscle growth and regeneration, metabolism throughout the body, and other functions.

[0004] Pathological aggregation of misfolded proteins such as SOD1, TDP-43, or FUS proteins in affected neurons and adjacent glia can be considered a hallmark of ALS. Several recent studies have shown that these intracellular proteins are released via vesicle-mediated exocytosis and then phagocytosed by neighboring cells, which allows for self-perpetuating transmission to neighboring motor neurons. Furthermore, mutations in genes involved in the autophagy pathway and multivesicular biogenesis (such as ALSIN, VAPB, CHMP2B, and VCP) have been identified in familial ALS. Taken together, these data strongly suggest a disruption of the endosomal and lysosomal pathways in sporadic and familial ALS patients - both of which are involved in the generation of exosomes.

[0005] Diagnosis takes time, often requiring patients to see multiple specialists over a period of several months, and the average time from onset of symptoms to confirmation of diagnosis is 13-18 months. Thus, the problem / need is to diagnose ALS more quickly. Summary of the Invention [Problem to be solved by the invention]

[0006] According to a first aspect of the present invention, there is provided a method of diagnosing or predicting a neurological disorder in a subject, the method comprising the steps of: (a) determining a quantitative or qualitative level of one or more biomarkers in a biological sample from a subject; (b) diagnosing or predicting a neurological disorder in a subject based on a quantitative or qualitative level of the or each biomarker in a biological sample, wherein the or each biomarker is PDIA3;CCDC80;HSPA8;SERPINE2;PLEC;HSPA5;NCL;TPM1;TNC;IGFBP3;COL5A1;CLEC11A;MAP1B;NACA;PSMA2;RPL5;MYL6;CTSB;HSPD1;PTRF;NUCB1;PDIA4;P4HB;ACTN4;EEF1D;TPI1;SSBP1;PSMB8;PSAP;PSMB7;CAPZA1;PRDX1;PODN;MMP1;NUCB2;HMGB1;AHNAK;FNDC1;DENR;PENK;S100A11;RPL29;RPL27;RPL8;SRSF2;SF3B4;S BSN;LYAR;NUDC;CRLF1;TGFB1;CAT;CTSD;DLD;EPB41L2;PSMB9;RPS14;RPL7A;COLEC12;SMARCC2;TAGLN2;CCT4 ;ASPH;GPS1;CDK13;SH3GL2;HBA1;APEX1;PSMB5;TXN;AXL;RPS8;SET;RPS18;CFL1;DMKN;RPL22;RPL6;FKBP10; Selected from PAFAH1B1; S100A13; PSMA6; SERPINE1; CAPRIN1; SEPT7; VTN; ENPP2; NEO1; DKC1; CHI3L1; SKOR1; SSRP1; COL4A2; NME1; CFB; EIF2S3; DPYSL2; NUMA1; KTN1; CHID1; EFEMP1; YWHAZ; LDHB; SDCBP; TLN1; DES; APP; and DAG1.

[0007] Preferably, the or each biomarker is selected from PDIA3; CCDC80; HSPA8; SERPINE2; PLEC; HSPA5; NCL; TPM1; TNC; IGFBP3; COL5A1; CLEC11A; MAP1B; NACA; PSMA2; RPL5; MYL6; CTSB; HSPD1; PTRF; NUCB1; PDIA4; P4HB; ACTN4; and CAPRIN1.

[0008] More preferably, the or each biomarker is selected from PDIA3; CCDC80; HSPA8; SERPINE2; PLEC; HSPA5; NCL; TPM1; TNC; IGFBP3; COL5A1; CLEC11A; MAP1B; NACA; PSMA2; RPL5; MYL6; CTSB; HSPD1; PTRF; NUCB1; PDIA4; P4HB; ACTN4; CAPRIN1; DES; APP; and DAG1.

[0009] More preferably, the or each biomarker is selected from: PDIA3; CCDC80; HSPA8; SERPINE2; PLEC; DES; APP; and DAG1.

[0010] Optionally, the determining step (a) comprises determining the quantitative or qualitative levels of two or more biomarkers in a biological sample from the subject.

[0011] Further optionally, the determining step (a) comprises determining the quantitative or qualitative levels of 3, 4, 5, 8, 10, 20, 25, 28, 30, 40, 50, 60, 70, 80, 90, 100 or more biomarkers in a biological sample from the subject.

[0012] Optionally, the determining step (a) comprises determining the quantitative or qualitative levels of all of the biomarkers in a biological sample from the subject.

[0013] Optionally, or additionally, the determining step (a) comprises determining the quantitative or qualitative level of each of the biomarkers in a biological sample from the subject.

[0014] Optionally, the or each biomarker is a gene. Further optionally, the or each biomarker is a nucleic acid. Further optionally, the or each biomarker is a deoxyribonucleic acid.

[0015] Optionally, the or each biomarker is a translation product of a gene.

[0016] Optionally, the or each biomarker is selected from the group consisting of PDIA3;CCDC80;HSPA8;SERPINE2;PLEC;HSPA5;NCL;TPM1;TNC;IGFBP3;COL5A1;CLEC11A;MAP1B;NACA;PSMA2;RPL5;MYL6;CTSB;HSPD1;PTRF;NUCB1;PDIA4;P4HB;ACTN4;EEF1D;TPI1;SSBP1;PSMB8;PSAP;PSMB7;CAPZA1;PRDX1;PODN;MMP1;NUCB2;HMGB1;AHNAK;FNDC1;DENR;PENK;S100A11;RPL29;RPL27;RPL8;SRSF2;SF3B4;SBSN;LYAR;NUDC;CRLF1;TGFB1;CAT;CTSD;DLD;EPB 41L2;PSMB9;RPS14;RPL7A;COLEC12;SMARCC2;TAGLN2;CCT4;ASPH;GPS1;CDK13;SH3GL2;HBA1;APEX1;PSMB5;TXN;AXL;RPS8;SET;RPS18;CFL1;DMKN;RPL22;RPL6;FKBP10;PAFAH1B1;S100A13;PSMA6;SERPINE1;CAPRIN1;SEPT7;VTN;ENPP2;NEO1;DKC1;CHI3L1;SKOR1;SSRP1;COL4A2;NME1;CFB;EIF2S3;DPYSL2;NUMA1;KTN1;CHID1;EFEMP1;YWHAZ;LDHB;SDCBP;TLN1;DES;APP; and DAG1.

[0017] Preferably, the or each biomarker is a translation product of a gene selected from PDIA3; CCDC80; HSPA8; SERPINE2; PLEC; HSPA5; NCL; TPM1; TNC; IGFBP3; COL5A1; CLEC11A; MAP1B; NACA; PSMA2; RPL5; MYL6; CTSB; HSPD1; PTRF; NUCB1; PDIA4; P4HB; ACTN4; and CAPRIN1.

[0018] More preferably, the or each biomarker is a translation product of a gene selected from PDIA3; CCDC80; HSPA8; SERPINE2; PLEC; HSPA5; NCL; TPM1; TNC; IGFBP3; COL5A1; CLEC11A; MAP1B; NACA; PSMA2; RPL5; MYL6; CTSB; HSPD1; PTRF; NUCB1; PDIA4; P4HB; ACTN4; CAPRIN1; DES; APP; and DAG1.

[0019] More preferably, the or each biomarker is a translation product of a gene selected from: PDIA3; CCDC80; HSPA8; SERPINE2; PLEC; DES; APP; and DAG1.

[0020] Optionally, the or each biomarker is a ribonucleic acid.

[0021] Optionally, the or each biomarker is a ribonucleic acid having a miRBase accession number selected from MIMAT0000244; ENSG00000239126; MIMAT0016865; MIMAT0002813; MIMAT0004697; MIMAT0000245; MIMAT0000737; MIMAT0000088; MIMAT0000414; MIMAT0004955; MIMAT0000076; MIMAT0000067; MIMAT0018084; and MIMAT0004813.

[0022] Optionally, the or each biomarker is a ribonucleic acid having a nucleic acid sequence selected from any one or more of SEQ ID NOs: 109-122.

[0023] Optionally, the or each biomarker is a protein. Further optionally, the or each biomarker is a peptide. Further optionally, the or each biomarker is a polypeptide.

[0024] Optionally, the or each biomarker is selected from the group consisting of PDIA3;CCDC80;HSPA8;SERPINE2;PLEC;HSPA5;NCL;TPM1;TNC;IGFBP3;COL5A1;CLEC11A;MAP1B;NACA;PSMA2;RPL5;MYL6;CTSB;HSPD1;PTRF;NUCB1;PDIA4;P4HB;ACTN4;EEF1D;TPI1;SSBP1;PSMB8;PSAP;PSMB7;CAPZA1;PRDX1;PODN;MMP1;NUCB2;HMGB1;AHNAK;FNDC1;DENR;PENK;S100A11;RPL29;RPL27;RPL8;SRSF2;SF3B4;SBSN;LYAR;NUDC;CRLF1;TGFB1;CAT;CTSD;DLD;EPB41L2; Proteins encoded by genes selected from PSMB9;RPS14;RPL7A;COLEC12;SMARCC2;TAGLN2;CCT4;ASPH;GPS1;CDK13;SH3GL2;HBA1;APEX1;PSMB5;TXN;AXL;RPS8;SET;RPS18;CFL1;DMKN;RPL22;RPL6;FKBP10;PAFAH1B1;S100A13;PSMA6;SERPINE1;CAPRIN1;SEPT7;VTN;ENPP2;NEO1;DKC1;CHI3L1;SKOR1;SSRP1;COL4A2;NME1;CFB;EIF2S3;DPYSL2;NUMA1;KTN1;CHID1;EFEMP1;YWHAZ;LDHB;SDCBP;TLN1;DES;APP; and DAG1.

[0025] Preferably, the or each biomarker is a protein encoded by a gene selected from PDIA3; CCDC80; HSPA8; SERPINE2; PLEC; HSPA5; NCL; TPM1; TNC; IGFBP3; COL5A1; CLEC11A; MAP1B; NACA; PSMA2; RPL5; MYL6; CTSB; HSPD1; PTRF; NUCB1; PDIA4; P4HB; ACTN4; and CAPRIN1.

[0026] More preferably, the or each biomarker is a protein encoded by a gene selected from PDIA3; CCDC80; HSPA8; SERPINE2; PLEC; HSPA5; NCL; TPM1; TNC; IGFBP3; COL5A1; CLEC11A; MAP1B; NACA; PSMA2; RPL5; MYL6; CTSB; HSPD1; PTRF; NUCB1; PDIA4; P4HB; ACTN4; CAPRIN1; DES; APP; and DAG1.

[0027] More preferably, the or each biomarker is a protein encoded by a gene selected from: PDIA3; CCDC80; HSPA8; SERPINE2; PLEC; DES; APP; and DAG1.

[0028] Optionally, the or each biomarker is selected from the group consisting of P30101;Q76M96;P11142;P07093;Q15149;P11021;P19338;P09493;P24821;P17936;P20908;Q9Y240;P46821;Q13765;P25787;P46777;P60660;P07858;P10809;Q6NZI2;Q02818;P13667;P07237;O43707;P29692;P60174; Q04837;P28062;P07602;Q99436;P52907;Q06830;Q7Z5L7;P03956;P80303;P09429;Q09666;Q4ZHG4;O43583;P01210;P31949 ;P47914;P61353;P62917;Q01130;Q15427;Q6UWP8;Q9NX58;Q9Y266;O75462;P01137;P04040;P07339;P09622;O43491;P28065 ;P62263;P62424;Q5KU26;Q8TAQ2;P37802;P50991;Q12797;Q13098;Q14004;Q99962;P69905;P27695;P28074;P10599;P3053 0;P62241;Q01105;P62269;P23528;Q6E0U4;P35268;Q02878;Q96AY3;P43034;Q99584;P60900;P05121;Q14444;Q16181;P040 and Q14118.

[0029] Preferably, the or each biomarker is a protein having UniProtKB / Swiss-Prot accession numbers selected from P30101; Q76M96; P11142; P07093; Q15149; P11021; P19338; P09493; P24821; P17936; P20908; Q9Y240; P46821; Q13765; P25787; P46777; P60660; P07858; P10809; Q6NZI2; Q02818; P13667; P07237; O43707; and Q14444.

[0030] More preferably, the or each biomarker is a protein having UniProtKB / Swiss-Prot accession numbers selected from: P30101; Q76M96; P11142; P07093; Q15149; P11021; P19338; P09493; P24821; P17936; P20908; Q9Y240; P46821; Q13765; P25787; P46777; P60660; P07858; P10809; Q6NZI2; Q02818; P13667; P07237; O43707; Q14444; P17661; P05067; and Q14118.

[0031] More preferably, the or each biomarker is a protein having a UniProtKB / Swiss-Prot accession number selected from: P30101; Q76M96; P11142; P07093; Q15149; P17661; P05067; and Q14118.

[0032] Optionally, the or each biomarker is a protein having an amino acid sequence selected from any one or more of SEQ ID NOs: 1-108.

[0033] Optionally, the neurological disorder is a neurodegenerative disorder.

[0034] Optionally, the neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease; motor neuron disease; MND), hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), pseudobulbar palsy, spinal and bulbar muscular atrophy (SBMA), and spinal muscular atrophy.

[0035] Optionally, the diagnosing or predicting step (b) comprises comparing the quantitative or qualitative level of the, or each, biomarker in a biological sample from the subject with the quantitative or qualitative level of the biomarker, or each individual biomarker, in a normal sample.

[0036] Optionally, the normal sample is a biological sample from a subject not suffering from a neurological disorder, optionally a neurodegenerative disorder, further optionally a neurodegenerative disorder selected from amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease; motor neuron disease; MND), hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), pseudobulbar palsy, spinal and bulbar muscular atrophy (SBMA), and spinal muscular atrophy.

[0037] Optionally, a quantitative or qualitative level of the or each individual biomarker in the biological sample from the subject that is greater than the quantitative or qualitative level of the biomarker or each individual biomarker in a normal sample indicates a quantitative or qualitative level of a neurological disorder, optionally a neurodegenerative disorder, further optionally a neurodegenerative disorder selected from amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease; motor neuron disease; MND), hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), pseudobulbar palsy, spinal and bulbar muscular atrophy (SBMA), and spinal muscular atrophy.

[0038] Optionally, a quantitative or qualitative level of the biomarker or each individual biomarker in a biological sample from the subject that is greater than the quantitative or qualitative level of the biomarker or each individual biomarker in a normal sample indicates the quantitative or qualitative presence of a neurological disorder, optionally a neurodegenerative disorder, further optionally a neurodegenerative disorder selected from amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease; motor neuron disease; MND), hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), pseudobulbar palsy, spinal and bulbar muscular atrophy (SBMA), and spinal muscular atrophy.

[0039] Optionally, the determining step (a) comprises determining a quantitative or qualitative level of one or more subsets of one or more biomarkers in a biological sample from the subject.

[0040] Optionally, the determining step (a) comprises determining the quantitative or qualitative levels of two or more subsets of the one or more biomarkers in a biological sample from the subject.

[0041] Optionally, the determining step (a) comprises determining the quantitative or qualitative levels of a subset of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the one or more biomarkers in the biological sample.

[0042] Optionally, the determining step (a) comprises determining a quantitative or qualitative level of one or more of a first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, or fourteenth subset of one or more biomarkers in a biological sample from the subject.

[0043] Optionally, the first subset comprises one or more biomarkers selected from: PDIA3; and CCDC80.

[0044] Optionally, the second subset includes HSPA8.

[0045] Optionally, the third subset comprises one or more biomarkers selected from: SERPINE2; and PLEC.

[0046] Optionally, the fourth subset comprises one or more biomarkers selected from: HSPA5; and NCL.

[0047] Optionally, the fifth subset comprises one or more biomarkers selected from: TPM1; and TNC.

[0048] Optionally, the sixth subset includes IGFBP3.

[0049] Optionally, the seventh subset comprises one or more biomarkers selected from COL5A1; CLEC11A; MAP1B; NACA; and PSMA2.

[0050] Optionally, the eighth subset comprises one or more biomarkers selected from RPL5; MYL6; CTSB; HSPD1; PTRF; NUCB1; PDIA4; P4HB; and ACTN4.

[0051] Optionally, the ninth subset includes one or more biomarkers selected from EEF1D; TP11; SSBP1; PSMB8; PSAP; PSMB7; CAPZA1; PRDX1; PODN; MMP1; NUCB2; HMGB1; AHNAK; and FNDC1.

[0052] Optionally, the tenth subset is DENR;PENK;S100A11;RPL29;RPL27;RPL8;SRSF2;SF3B4;SBSN;LYAR;NUDC;CRLF1;TGFB1;CAT;CTSD;DLD;EPB41L2;PSMB9;RPS14;RPL7A;COLEC12;SMARCC2;TAGLN2;CCT4;ASPH;GPS1;CDK13;SH3GL2;HBA1;APEX1;PSMB5;TXN;AXL;RPS8;SET;RP The present invention includes one or more biomarkers selected from S18; CFL1; DMKN; RPL22; RPL6; FKBP10; PAFAH1B1; S100A13; PSMA6; SERPINE1; CAPRIN1; SEPT7; VTN; ENPP2; NEO1; DKC1; CHI3L1; SKOR1; SSRP1; COL4A2; NME1; CFB; EIF2S3; DPYSL2; NUMA1; KTN1; CHID1; EFEMP1; YWHAZ; LDHB; SDCBP; and TLN1.

[0053] Optionally, the eleventh subset comprises one or more biomarkers selected from: DES; APP; and DAG1.

[0054] Optionally, the twelfth subset includes one or more biomarkers selected from PDIA3; CCDC80; HSPA8; SERPINE2; PLEC; HSPA5; NCL; TPM1; TNC; IGFBP3; COL5A1; CLEC11A; MAP1B; NACA; PSMA2; RPL5; MYL6; CTSB; HSPD1; PTRF; NUCB1; PDIA4; P4HB; ACTN4; and CAPRIN1.

[0055] Optionally, the thirteenth subset includes one or more biomarkers selected from PDIA3; CCDC80; HSPA8; SERPINE2; PLEC; HSPA5; NCL; TPM1; TNC; IGFBP3; COL5A1; CLEC11A; MAP1B; NACA; PSMA2; RPL5; MYL6; CTSB; HSPD1; PTRF; NUCB1; PDIA4; P4HB; ACTN4; CAPRIN1; DES; APP; and DAG1.

[0056] Optionally, the fourteenth subset comprises one or more biomarkers selected from PDIA3; CCDC80; HSPA8; SERPINE2; PLEC; DES; APP; and DAG1.

[0057] Optionally, the determining step (a) comprises determining the quantitative or qualitative levels of all of the biomarkers in one or more of the one or more of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, or fourteenth subsets.

[0058] Optionally, the determining step (a) comprises determining a quantitative or qualitative level of each of the biomarkers in one or more of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, or fourteenth subsets.

[0059] Optionally, the diagnosing or predicting step (b) comprises comparing the quantitative or qualitative level of the or each biomarker in the subset or each subset in a biological sample from the subject with the quantitative or qualitative level of the biomarker or each individual biomarker in a normal sample.

[0060] Optionally, the normal sample is a biological sample from a subject not suffering from a neurological disorder, optionally a neurodegenerative disorder, further optionally a neurodegenerative disorder selected from amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease; motor neuron disease; MND), hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), pseudobulbar palsy, spinal and bulbar muscular atrophy (SBMA), and spinal muscular atrophy.

[0061] Optionally, a quantitative or qualitative level of the or each biomarker in the subset or each subset in the biological sample from the subject that is greater than the quantitative or qualitative level of the or each individual biomarker in a normal sample indicates a quantitative or qualitative level of a neurological disorder, optionally a neurodegenerative disorder, further optionally a neurodegenerative disorder selected from amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease; motor neuron disease; MND), hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), pseudobulbar palsy, spinal and bulbar muscular atrophy (SBMA), and spinal muscular atrophy.

[0062] Optionally, a quantitative or qualitative level of the or each biomarker in the subset or each subset in the biological sample from the subject that is greater than the quantitative or qualitative level of the biomarker or each individual biomarker in a normal sample indicates the quantitative or qualitative presence of a neurological disorder, optionally a neurodegenerative disorder, further optionally a neurodegenerative disorder selected from amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease; motor neuron disease; MND), hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), pseudobulbar palsy, spinal and bulbar muscular atrophy (SBMA), and spinal muscular atrophy.

[0063] Optionally or additionally, the determining step (a) further comprises the additional step of determining a quantitative or qualitative level of one or more biomarkers in a biological sample from the subject, wherein the or each biomarker is a ribonucleic acid having a miRBase accession number selected from MIMAT0000244; ENSG00000239126; MIMAT0016865; MIMAT0002813; MIMAT0004697; MIMAT0000245; MIMAT0000737; MIMAT0000088; MIMAT0000414; MIMAT0004955; MIMAT0000076; MIMAT0000067; MIMAT0018084; and MIMAT0004813.

[0064] Optionally, the biological sample is selected from whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid.

[0065] Further optionally, the biological sample is selected from whole blood, serum, and plasma.

[0066] Further optionally, the biological sample is whole blood.

[0067] PDIA3 is a synonym of Erp57. Embodiments of the present invention will now be described with reference to the following non-limiting examples and the accompanying drawings. [Brief description of the drawings]

[0068] [Figure 1] FIG. 1 is a schematic diagram of the ALS signature in muscle stem cells. [Diagram 2] (A) Immunostaining of exosome markers in differentiated muscle stem cells (B) Secretion of exosomes by muscle cells (C) Multivesicular bodies in ALS muscle cells contain more exosomes than controls. [Diagram 3]Representative images of healthy myotubes treated with ALS or control exosomes are shown, where ALS exosomes induce cellular stress. [Figure 4] Box plots showing expression values ​​of non-coding RNA microarrays (raw: left panel; background corrected: middle panel; background corrected and normalized: right panel). [Diagram 5] Box plot showing RLE values ​​for non-coding RNA microarrays (mean values ​​close to 0-pass quality control). [Figure 6] Box plot showing NUSE values ​​for non-coding RNA microarrays (mean values ​​for <1.05 pass quality controls). [Figure 7] DAG1 expression levels in circulating serum exosomes using anti-DAG antibody. Values ​​are mean ± SD. Serum from 8 ALS patients (1 month after diagnosis), 11 healthy subjects and 9 Parkinson's patients. ANOVA 1-factor followed by Tukey post-hoc test was performed. **, significantly different from healthy values, P < 0.01; [Figure 8] Figure 1 shows HSPA8 expression levels in circulating serum exosomes. Values ​​are mean ± SD. Serum from 5 ALS patients (1 month after diagnosis), 9 healthy subjects and 7 Parkinson's patients. ANOVA 1-way followed by Tukey post-hoc test was performed. *, **, significantly different from ALS values, P<0.05 and P<0.01, respectively; [Figure 9] The levels of each biomarker in exosomes isolated from serum of ALS patients are shown, normalized to the amount of total serum exosomes (CD63) and plotted against the rate of progression of the pathology (Delta ALSFRS-R).

[0069] Open biopsies were performed on the deltoid muscle of 18 ALS patients attending the Motor Neuron Diseases center (Pitie Salpetriere, Paris). Twenty-one deltoid muscle biopsies from age- and sex-matched healthy subjects were obtained from the BTR (Bank of Tissues for Research, partner of the U network EuroBioBank) in accordance with European recommendations and French legislation. The protocol (NCT01984957) was approved by the local ethical committee and all subjects completed informed consent in accordance with institutional guidelines. Muscle stem cell extraction and culture

[0070] Muscle biopsies were mechanically dissociated and plated in growth medium as previously described (Mamchaoui et al., Skeletal muscle, 2011, PMID: 22040608). Myogenic cell populations were sorted using a CD56 magnetic bead sorting system (MACS, Miltenyi Biotech) according to the manufacturer's instructions. The myogenicity of the cell cultures was confirmed before and after cell sorting by immunolabeling using an anti-desmin antibody, and cells were differentiated in Dulbecco's modified Eagle's medium (DMEM) for 3 days. A minimum of 80% of the cell population was desmin positive. Exosome extraction from culture medium

[0071] Briefly, 10 million muscle stem cells (ALS and healthy, n=3 / group) were differentiated in DMEM for 3 days. After 3 days, conditioned medium was collected and centrifuged at 300xg for 5 min at room temperature (RT) and 4000g for 20 min at 4°C to remove any dead cells and cell debris. The supernatant was filtered through a 0.22 μm filter to remove particulates. Samples were used for protein and miRNA extraction and stored at -80°C. Protein extraction from exosomes

[0072] A volume of cell culture medium was mixed with 0.5 volume of Total Exosome Isolation Reagent from Cell Culture Media (lifeTechnologies®) and incubated overnight at 4°C. Samples were then centrifuged at 10,000xg for 1 hour at 4°C. Exosome pellets were resuspended in 25μl of 8M urea, 50mM ammonium bicarbonate, pH 8.5 and reduced with dithiothreitol (DTT) for 1 hour at 4°C. Protein concentration was then quantified using the Pierce BCA Protein Assay Kit (ThermoFisher®). Exosomal proteins were kept at -80°C. Proteomic profiles determined by mass spectrometry

[0073] Approximately 20 μg of exosomal protein was trypsin digested using a SmartDigest column (Thermo) for 2 h at 70 °C and 1400 rpm according to the manufacturer's instructions. Peptides were then fractionated into eight fractions using a high pH reversed-phase spin column (Thermo) according to the manufacturer's instructions. Fractionated peptides were vacuum dried, resuspended, and analyzed by data-dependent mass spectrometry on a Q Exactive HF (Thermo) with the following parameters: positive polarity, m / z 400–2000 MS resolution 70,000, AGC 3e6, 100 ms IT, MS / MS resolution 17,500, AGC 5e5, 50 ms IT, isolation width 3 m / z, and NCE 30, cycle number 15. Database searching and quantification

[0074] The resulting mass spectral files were searched for protein identification using ProteomeDiscoverer (Thermo) against the Uniprot human database of semitryptic peptides and filtered based on a false discovery rate of less than 1%. Proteomic profile analysis

[0075] Proteins were selected as candidates if the range of observed abundance in the three ALS replicates did not overlap with the range in the three healthy control replicates, and these candidates were ranked based on the difference between the median abundance in the three ALS replicates and the median abundance in the healthy replicates. miRNA extraction from exosomes

[0076] A volume of cell culture medium was mixed with 0.5 volume of Total Exosome Isolation Reagent from Cell Culture Media (lifeTechnologies®) and incubated overnight at 4°C. Samples were then centrifuged at 10,000xg for 1 hour at 4°C. The exosome pellet was resuspended in 1 ml of Trizol. After adding 0.2 mL of chloroform, samples were incubated at room temperature for 3 minutes and then centrifuged at 12,000xg for 15 minutes at 4°C. The aqueous upper phase was then mixed with 1 / 3 volume of 100% ethanol and mixed thoroughly. Samples were then placed into a filter cartridge into the collection tube of an Invitrogen™ (LifeTechnologies™) Total RNA and Protein Isolation Kit. miRNA extraction was performed according to the manufacturer's instructions. RNA quality was determined using a Eukaryote Total RNA Nano2.6 (Agilent) and a 2100 bioanalyzer. RNA quantity was determined using a nanodrop, according to the manufacturer's instructions, respectively. miRNA profiling and analysis

[0077] Total RNA (150 ng) was poly(A)-tailed and biotinylated using the FlashTag Biotin HSR RNA labeling kit and hybridized to Affymetrix GeneChip miRNA4.0 arrays for 16 h (48 °C) according to the manufacturer's instructions (ThermoFisher Scientific, Waltham). The hybridization cocktail was then removed, and the arrays were washed and stained on a FluidicsStation450 using the fluidics script for miRNA4.0 arrays. Finally, the arrays were scanned on an Affymetrix GCS3000 7G scanner, and initial quality control data were evaluated using the Affymetrix Expression Console software (both from ThermoFisher Scientific). miRNome profile analysis

[0078] Further quality control of the raw data was performed using the R / Bioconductor packages oligo and pd.mirna.4.0. These packages were used to assess the distribution of expression values, relative log expression (RLE) values, and normalized unscaled standard error (NUSE) for each sample, all of which were consistent with high quality. Expression matrices were then normalized by quantile normalization and detection above background using Affymetrix Expression Console v1.4.1.46. Differentially expressed miRNAs (ALS vs. healthy) were then identified as those with absolute fold changes greater than 2 and one-way unpaired ANOVA p-values ​​less than 0.05. Serum samples from patients

[0079] Blood samples were collected using silica vacutainers and gently inverted 8–10 times. After incubation on ice for 2 h to allow clotting, samples were centrifuged at 1,500 g for 15 min. Aliquots of 100 μl of serum were transferred to cryovials and stored at -80°C. Eight age- and sex-matched ALS patients, 11 healthy subjects, and 9 Parkinson's patients were included (NCT01302600 and NCT02305147). Exosome extraction from serum

[0080] Exosomes were precipitated by adding 6 μl Total Exosome Isolation Reagent, vortexing, and incubating on ice for 30 min. Samples were then centrifuged at 10,000g for 10 min at room temperature. The pellet was resuspended in 13.5 μl urea 4M / RIPA and kept on ice. In parallel, 6.3 μl Total Exosome Isolation Reagent was added to the remaining supernatant, vortexed, and incubated on ice for 30 min. These second mixtures were then centrifuged at 10,000g for 10 min at RT. The second exosome pellet was resuspended in 13.5 μl urea 4M / RIPA. The two exosome protein extracts were then mixed together and used for dot blot analysis. Exosome protein extracts were stored at -80°C. Preparation of samples for dot blot

[0081] 5 μl of exosomal protein extract was loaded onto 1xNuPAGE (Invitrogen™). These non-reduced samples were used for CD63 analysis. The remaining 22 μl of exosomal protein extract was loaded onto 1xNuPAGE (Invitrogen™) supplemented with 1x reducing reagent (Invitrogen™). All samples were then heated at 70°C for 10 min. Dot blot analysis

[0082] 1 μl of reduced or non-reduced sample was applied to a 0.2 μm PVDF (Immobilon®, Sigma-Aldrich®) preactivated with methanol. After drying for 5 min at room temperature, the membrane was quickly reactivated with methanol and stained with Ponceau S (Sigma-Aldrich®) to control for loading. The membrane was then blocked overnight at 4° C. in TBS-0.01% tween (TBS-T) supplemented with 5% milk.

[0083] Primary antibodies against CD63 (TS63, Invitrogen™), beta-dystroglycan (MANDAG2, 7D11, DSHB), and HSPA8 (Millipore®) were immediately conjugated with biotin using the Zenon™ BiotinXX Mouse lgG1 Labeling Kit according to the manufacturer's instructions. Briefly, 1 μg of antibody was first added with 5 μl of conjugate, followed by 5 μl of blocking buffer. Anti-CD63, anti-beta-dystroglycan, and anti-HSPA8 were diluted 1 / 1000, 1 / 133, and 1 / 500, respectively, in TBS-T-5% milk.

[0084] Primary antibodies against Serpin E2 (TS63, Invitrogen™), ERP57 (PDIA3, Merck-Millipore®), desmin (Y66, Invitrogen™), amyloid precursor protein (abeam), CCDC80 (Invitrogen™), and Plectin (invitrogen™) were immediately conjugated with biotin using the Zenon™ BiotinXX Rabbit IgG Labeling Kit according to the manufacturer's instructions. Briefly, 1 μg of antibody was first added with 5 μl of conjugate, followed by 5 μl of blocking buffer. Anti-SerpinE2, anti-ERP57, anti-desmin, anti-amyloid precursor protein, anti-CCDC80, and anti-Plectin were diluted 1 / 1000 in TBS-T-5% milk.

[0085] The membrane was incubated with diluted primary biotin-conjugated antibody for 45 min at RT. The membrane was then rinsed 3 times for 5 min with TBS-T. The membrane was then incubated for 45 min at RT with streptavidin-HRP (Invitrogen™) diluted 1 / 250 in TBS-T-5% milk. The membrane was then rinsed 3 times for 5 min with TBS-T. The membrane was revealed using SuperSignal™ West Pico PLUS chemiluminescent substrate (ThermoScientific™). Images were acquired every 15 seconds for 15 min using a UVP / ChemiDoc-lt® 2Imager. Images were then analyzed using FIJI software. statistical analysis

[0086] Values ​​are means ± SD. One-way ANOVA followed by Tukey post-hoc test was performed to assess differences in biomarker levels between ALS, healthy, and Parkinson subjects. Correlations between biomarker levels and ALSFRS progression were examined using Pearson correlation analysis. The cutoff for significant differences was set at P<0.05. Example 1 - Identification of ALS signatures in muscle stem cells

[0087] Muscle stem cells were extracted from muscle biopsies of patients and healthy controls (all male, age 30-67 years, n=5 / 6 per group), purified (>80% myogenic cells), and differentiated into myotubes for 3 days. Samples were analyzed using GeneChip Human Exon 1.0ST arrays (Affymetrix, Inc., Cleveland, OH); (A) shows a PCA plot showing a clear separation of ALS patients in the second component (y-axis): ALS: blue, SBMA: pink, SMA-III / IV: red, control: green. (B) shows 30 genes with the strongest specific expression signature for ALS, observed in a subcellular localization associated with exosomes, as analyzed by the CellWhere tool. Example 2 - ALS muscle stem cells accumulate and secrete more exosomes than controls

[0088] With reference to FIG. 2(A), immunostaining of exosomal markers of differentiated muscle stem cells is shown. Left panel: Representative images of cultured muscle stem cells from ALS and SMA-III / IV patients, as well as healthy controls or denervated subjects, labeled with exosomal markers (TSG101, CD63). Right panel: Quantification of normalized CD63 fluorescent signal per myonucleus. (n=8, 4, 2, 5 cell cultures from different ALS, SMA-III / IV, polyneuropathy and control individuals). ***, P<0.001.

[0089] Figure 2(B) shows that exosomes are hypersecreted by ALS muscle cells. Left panel: Representative image of exosome pellet from culture medium. Exosomes were extracted from the culture medium of 800,000 cells from ALS and control individuals. The experiment was repeated eight times with different cultures of muscle cells from ALS patients and controls. Right panel: Electron microscopy confirming the presence of exosomes (cup-shaped vesicles) in the pellet. Exosomes were more abundant in the ALS pellet. Of note, immunostaining performed on these vesicles confirmed that they were exosomes, as they were positive for CD63 and CD82.

[0090] Figure 2(C) shows that multivesicular bodies of ALS muscle cells contain more exosomes than controls. Left panel: Representative electron microscopy images showing the accumulation of exosomes in multivesicular bodies (MVBs, highlighted in green and orange) of ALS muscle cells compared to controls. Scale bar = 1 μm. Right panel: Quantification of the number of exosome-like vesicles in MVBs. Two-sample Kolmogorov-Smirnov tests confirmed the visual impression that MVBs of ALS muscle cells contain more exosome-like vesicles compared to controls (P < 0.05). Example 3 - Exosomes from ALS muscle cells are toxic to healthy myotubes and motor neurons

[0091] With reference to FIG. 3(A), it is shown that ALS exosomes induce muscle cell atrophy. Left panel: Representative images of healthy myotubes treated with ALS or control exosomes. Myotubes are stained with myosin heavy chain (green). Right panel: Quantification of myonuclear domain size (area of ​​myosin heavy chain staining divided by number of nuclei). Myotubes treated with ALS-exosomes have smaller myonuclear domains (*P<0.05; n=6 / group).

[0092] Figure 3(B) shows that ALS exosomes induce cellular stress. Quantification of the number of blebs per healthy myotube treated with ALS or control exosomes over time (n=6 / group). ANOVA 2-way interaction, time and treatment P<0.001.

[0093] Figure 3(C) shows that ALS exosomes induce cell death. Left panel: Representative immunostaining of H2-Ax in healthy myotubes treated with ALS or control exosomes. Middle panel: Quantification of H2-Ax signal per nucleus. Right panel: Quantification of motor neuron nuclei loss in culture. Example 4 - Identification of specific exosomal contents as biomarkers for ALS

[0094] The contents of exosomes contain various types of biomolecules, including proteins and their peptides, as well as non-coding RNAs, which may be involved in signaling roles. Muscle stem cells (primary myoblasts) were obtained from deltoid muscles of ALS patients and healthy controls and cultured to form differentiated myotubes. Exosomes were purified from the culture medium of 10 million muscle cells after 3 days of culture using total exosome isolation reagent (Invitrogen™ Lifetechnologies™). To identify the contents of exosomes, proteomics and non-coding RNA profiling of purified exosomes were performed. Protein Biomarkers

[0095] Proteomic mass spectrometry data identified peptides of 105 proteins (Table 1) with consistently increased levels in ALS muscle exosomes (n = 3) compared to healthy control muscle exosomes (n = 3).

[0096] It was also noted that because proteomic samples were normalized for protein concentration, proteins that were consistently high in both ALS and healthy muscle exosomes may also be good biomarkers, since they may still be elevated in ALS blood per volume due to our observation that muscle exosomes are secreted in greater amounts from ALS myotubes than healthy controls. In this way, we identified three further candidates that were consistently high in both ALS and healthy muscle exosomes and had other mechanistically interesting features. These were DES, APP, and DAG1. APP is a major component of amyloid plaques in Alzheimer's disease. DES was observed at high levels in all muscle tissue types, but was completely absent in other tissues. DAG1 is a major component of the dystroglycan complex and has been implicated in multiple muscular dystrophies. [Table 1] TIFF0007605930000002.tif226162TIFF0007605930000003.tif242162TIFF0007605930000004.tif236162 [Table 2] TIFF0007605930000006.tif242161Non-coding RNA biomarkers

[0097] Profiling of non-coding RNA species was performed by microarray (using Affymetrix GeneChip® miRNA4.0 arrays) on ALS exosomes (n=5) compared to healthy controls (n=5). After quality control and normalization (see Figures 4-6), one-way unpaired ANOVA identified 14 non-coding RNAs significantly dysregulated in ALS vs. healthy controls (Table 3: 12 upregulated in ALS and 2 downregulated in ALS). Of these, 13 are microRNAs (denoted by miRBase ID hsa-*** and accession number MIMAT***) and one is the small nucleolar RNA snoU13 (ENSG00000239126). [Table 3] Example 6 - Comparison of different biomarkers for different disease groups

[0098] Blood samples were collected from 8 ALS patients, 11 healthy controls, and 9 Parkinson's disease patients. Patient characteristics of selected patients are shown in Table 4. Exosomes were extracted from serum and used for dot blot analysis. The levels of DAG1 in exosomes isolated from serum of patients with ALS disease, Parkinson's disease (disease control), or from healthy subjects are shown in Figure 7, and the levels of HSPA8 in exosomes isolated from serum of patients with ALS disease, patients with Parkinson's disease (disease control), or from healthy subjects are shown in Figure 8. The levels of each biomarker in exosomes isolated from serum of ALS patients, normalized to the amount of total serum exosomes (CD63), are plotted against the rate of progression of the disease (delta ALSFRS-R) and are shown in Figure 9. [Table 4] TIFF0007605930000009.tif198162

[0099] The present invention provides a strong ALS gene expression signature and new drivers of toxicity in muscle cells and muscle-motor neuron interactions in ALS patients. By studying differentiated muscle (myotube) cells from muscle biopsies of sporadic ALS patients, the present invention shows that: (1) Gene expression profiles of myotubes isolated from ALS patients cluster completely separately not only from healthy controls but also from SBMA and SMA-III / IV disease controls (see Figure 1A). The 30 genes that contribute most strongly to this ALS-specific signature include genes that code for proteins secreted or localized in exosomes (see Figure 1B). (2) Both intracellular exosome biogenesis and extracellular exosome secretion are dramatically increased in ALS myotubes (see Figures 2A and B), and exosome-rich multivesicular bodies are observed by electron microscopy in muscle biopsies from ALS patients (Figure 2C); (3) Increased exosome biogenesis and secretion are not observed in myotubes from denervated human subjects (neither healthy, SBMA, nor SMA-III / IV) or from denervated healthy mouse muscles (see Figures 1, 2A and other data not shown), indicating that exosome secretion is not a simple downstream consequence of the disease.

Claims

1. 1. An in vitro method for identifying a neurological disorder in a subject, comprising the steps of: (a) determining a quantitative or qualitative level of a biomarker in a biological sample from said subject; (b) comparing the quantitative or qualitative levels of the biomarkers in the biological sample from the subject with the quantitative or qualitative levels of the respective biomarkers in a normal sample; a quantitative or qualitative level of the biomarker in the biological sample from the subject that is greater than the quantitative or qualitative level of the individual biomarker in a normal sample indicates the quantitative or qualitative presence of a neurological disorder; and The method, wherein the biomarker is DAG1.

2. The in vitro method of claim 1 , wherein the biomarker is a gene.

3. The in vitro method of claim 1 , wherein the biomarker is a protein encoded by the DAG1 gene.

4. 2. The in vitro method of claim 1, wherein the biomarker is a protein having UniProtKB / Swiss-Prot accession number Q14118.

5. The in vitro method according to any one of claims 1 to 4, wherein the neurological disorder is a neurodegenerative disorder.

6. 6. The in vitro method of claim 5, wherein the neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease; motor neuron disease; MND), hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), pseudobulbar palsy, spinal and bulbar muscular atrophy (SBMA), and spinal muscular atrophy.

7. 7. The in vitro method according to any one of claims 1 to 6, wherein the biological sample is selected from whole blood, serum, plasma, urine, interstitial fluid, ascites, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid.

8. The in vitro method according to any one of claims 1 to 7, wherein the biological sample is whole blood.

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