WO2015172008A1

WO2015172008A1 - Transglutaminadase 6 as a biomarker for multiple sclerosis

Info

Publication number: WO2015172008A1
Application number: PCT/US2015/029865
Authority: WO
Inventors: Saud A. Sadiq; Massimiliano CRISTOFANILLI
Original assignee: Multiple Sclerosis Research Center Of New York
Priority date: 2014-05-09
Filing date: 2015-05-08
Publication date: 2015-11-12

Abstract

Disclosed are methods to diagnose multiple sclerosis (MS) in a subject by measuring the level of transglutaminadase 6 (TGM6) in a biological sample from the subject. Further disclosed are methods to monitor the activity and/or progression of MS in a subject with MS, by measuring the level of TGM6 over time in the subject. Also disclosed are methods to determine the MS subtype of a subject with MS, by comparing the level of TGM6 in a sample from the subject to control levels correlating TGM6 levels with identified MS subtypes, and determining the MS subtype of the subject by determining the range within which the TGM6 level of the sample falls.

Description

TRANSGLUTAMINADASE 6 AS A BIOMARKER FOR MULTIPLE SCLEROSIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application 61/991,120, filed May 9, 2014, which is incorporated herein in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The Sequence Listing in the ASCII text file, named as 31072_sequence.txt of 1800 KB, created on May 6, 2015, and submitted to the United States Patent and Trademark Office via EFS-Web, is incorporated herein by reference.

BACKGROUND OF THE DISCLOSURE

[0003] Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) with an unpredictable stepwise or chronic progressive evolution of inflammation, demyelination, axonal injury and oligodendrocyte death intertwined with the nervous system's attempts to repair damage and regain homeostasis (Lassmann H., Brain: a journal of neurology 135:2904-2905 (2012)). Worldwide, MS affects about 2.1 million people

(Compston A. et al., Lancet 372: 1502-1517 (2008)). In the majority of patients the disease is characterized clinically by relapses and remissions when the disease is pathologically inflammatory and treatment responsive, but over time the disease course becomes progressive in nature. Approximately 65% of relapsing remitting (RRMS) patients develop secondary progressive MS (SPMS), characterized by treatment-resistant functional deterioration. In a minority of patients (15%), the disease course is progressive from onset without recognizable remissions and is referred to as primary progressive MS (PPMS) (Compston A. et al., Lancet 372: 1502-1517 (2008)).

[0004] MS is associated with the development of discrete lesions in the CNS characterized by demyelination of neurons and infiltration of leucocytes. Ferguson et al., Brain 120:393- 399 (1997), categorizes human MS lesions as acute, active chronic, or chronic. Acute MS lesions are typified by areas of local demyelination, with CD3⁺ T-cells and CD68⁺ macrophages present throughout the lesion. In some cases inclusions containing myelin debris are seen in the interior of macrophages. Acute lesions are further characterized by positive staining for amyloid precursor protein (APP), a marker for axonal damage, throughout the lesion. Active chronic lesions are characterized by T-cells, macrophages, and APP staining present more around the border of the lesion than within. Chronic lesions are characterized by few or no T-cells or macrophages in the lesion, no or low APP staining within or around the lesion, and reduced axon density. In addition, MS and MS lesions are characterized by the presence of B cells, including CD 19+ B cells and CD 138+ plasma cells (Gilden et al., Mult. Scler. 2: 179-183 (1996); Ritchie et al., J. Immunol. 173:649-656 (2004)); and astrogliosis (activation of astrocytes; Gudi et al., Front. Cell. Neurosci. 8(73): 1-24 (2014)).

[0005] Diagnosing MS is challenging because no single test with a high diagnostic accuracy is available (Rudick RA. et al., Neurology 78: 1904-1906 (2012)). Rather, diagnosis is made on the basis of patients' histories, physical exam findings, supporting evidence from a battery of tests and the exclusion of other neuroinflammatory disorders. The two major diagnostic techniques used for MS diagnosis are magnetic resonance imaging (MRI) of the brain and spinal cord, and cerebrospinal fluid (CSF) analysis. MRI uses gadolinium-based contrast to detect areas of demyelination and to distinguish old lesions from new, active lesions. It is currently routinely used for MS diagnosis. However, the diagnosis of MS cannot be made solely on the basis of MRI because other pathologies can present with similar CNS lesions. Lumbar puncture for CSF analysis is routinely performed to aid in MS diagnosis. Intrathecal synthesis of immunoglobulin G (IgG) is associated with oligoclonal bands (OCB) which indicate an immune response within the CNS. They are found in the spinal fluid of about 90- 95% of people with MS. While the presence of OCBs in the CSF can be supportive of a diagnosis of MS, OCBs are seen in other diseases as well. Hence, they cannot be relied on as positive proof of MS (Link H. et al., J. Neuroimmunology 180: 17-28 (2006)).

[0006] Due to the lack of accurate diagnostic markers, the diagnosis of MS is based on subjective assessments and frequently takes months or years after the onset of disease symptoms (Rudick RA. et al., Neurology 78: 1904-1906 (2012)). False positive diagnoses expose patients unnecessarily to chronic and expensive therapy that might actually enhance their underlying condition. As the early phase of multiple sclerosis is considered a "window of therapeutic opportunity", a delayed diagnosis diminishes the efficacy of first-line therapeutics in MS (Jones JL. et al., Experimental neurology 225:34-39 (2010)). [0007] Transglutaminase 6 (TGM6) is a member of the transglutaminase enzyme family found predominantly in the central nervous system (CNS), mainly expressed by neuronal cells under physiological conditions. The main function of transglutaminase enzymes is to covalently cross-link or modify proteins by formation of an isopeptide bond between a peptide-bound glutamine residue and a primary amine. Transglutaminase 2 (TGM2) also known as tissue transglutaminase, a relative of TGM6 found distributed in the endomysium of the human gut, is the primary auto-antigen in the T-cell mediated autoimmune mediated disorder celiac disease (CD). Antibodies against TGM2 are considered a very reliable diagnostic indicator of the disease. In addition, TGM2 has been studied and proposed as a cerebrospinal fluid (CSF) biomarker in a few different neurodegenerative disorders, but not multiple sclerosis (MS).

[0008] Transglutaminase activity as measured by the levels of free gamma-glutamylamines has been studied in CSF of Huntington's disease (HD) patients (Jeitner TM et al., J

Neurochem. 106(l):37-44 (2008)) and Parkinson's disease (PD) patients (Vermes I et al., Mov Disord. 19(10): 1252-4 (2004)). Furthermore, one group has recently studied TGM2 in the context of MOG-induced EAE (the classical animal model of MS) in mice, and found that it exacerbates the disease through positive regulation of T cell differentiation and

inflammation (Oh K et al., Clin Immunol. 145(2): 122-32 (2012)).

[0009] TGM6 has been proposed as the autoimmune target in gluten-sensitive patients with neurological symptoms such as cerebellar ataxia, independent of gastrointestinal involvement (Hadjivassiliou M et al., Ann Neurol. 64(3):332-43 (2008)). Since then, a few groups have investigated the levels of IgG and IgA antibodies against TGM6 in the sera of gluten-caused ataxic patients versus genetically ataxic patients, as well as in the sera of schizophrenic patients. However, TGM6 has not been studied in the context of MS.

BRIEF SUMMARY OF THE DISCLOSURE

[0010] Contemplated herein are methods to diagnose multiple sclerosis in a subject with at least one neurological symptom. The methods include measuring the level of

transglutaminadase 6 (TGM6) in a biological sample from the subject, comparing the level of TGM6 in the biological sample with a control representing the levels of TGM6 in a subject without MS, and diagnosing MS if an elevated level of TGM6 is present in the sample, relative to control levels. The biological sample can be a sample of cerebrospinal fluid, brain, or spinal cord. The level of TGM6 can be measured by measuring the levels of TGM6 nucleic acid or TGM6 protein, such as by PCR, real-time PCR, RNA sequencing, microarray hybridization, Southern-blot, Northern-blot, Western-blot; immunoassay, ELISA,

immunocytochemistry, or immunohistochemistry. The methods can further involve correlating TGM6 measurements with one or more additional methods for diagnosis or detection of MS selected from magnetic resonance imaging, neurological testing, and measurement of at least one additional biomarker for MS. Additional biomarkers include from fetuin-A, chemokine (C-X-C motif) ligand 13, neurofilament, hepatocyte growth factor, osteopontin, isoprostane 8-iso-prostaglandin F_2a, Chitinase 3-like 1, and miR-29 miRNA.

[0011] The at least one neurological symptom in the subject is indicative of MS, and/or is consistent with a diagnosis of MS. The at least one neurological symptom can be any one or more symptoms selected from: numbness or weakness in one or more limbs; partial or complete loss of central vision in one eye; partial or complete loss of central vision in both eyes; optic neuritis; double vision or blurring of vision; tingling or pain in parts of the body; electric -shock sensations occurring with certain head movements; tremors; walking, balance, or coordination problems; slurred speech; fatigue; dizziness; bladder or bowel dysfunction; cognitive dysfunction; emotional changes; sexual dysfunction; depression; spasticity; speech or swallowing problems; hearing loss; seizures; respiration or breathing problems; and itching. The at least one neurological symptom can be one, two, three, four, or five or more neurological symptoms.

[0012] Further contemplated are methods to monitor the activity and/or progression of MS in a subject with MS, comprising measuring the level of TGM6 in a biological sample from the subject and comparing the measured level of TGM6 in the sample with the level of TGM6 in a biological sample taken from the subject at an earlier time point, wherein an increase in the level of TGM6 relative to the level of TGM6 at an earlier time point indicates that MS in the subject is increasing in severity. The biological sample can be a sample of cerebrospinal fluid, brain, or spinal cord. The level of TGM6 can be measured by measuring the levels of TGM6 nucleic acid or TGM6 protein, such as by PCR, real-time PCR, RNA sequencing, microarray hybridization, Southern-blot, Northern-blot, Western-blot; immunoassay, ELISA, immunocytochemistry, or immunohistochemistry. The methods can further involve correlating TGM6 measurements with one or more additional methods for diagnosis or detection of MS selected from magnetic resonance imaging, neurological testing, and measurement of at least one additional biomarker for MS. Additional biomarkers include from fetuin-A, chemokine (C-X-C motif) ligand 13, neurofilament, hepatocyte growth factor, osteopontin, isoprostane 8-iso-prostaglandin F_2a, Chitinase 3-like 1, and miR-29 miRNA.

[0013] Also contemplated are methods to determine the MS subtype of a subject with MS, comprising measuring the level of TGM6 in a biological sample from the subject, comparing the level of TGM6 in the sample with a range representing the levels of TGM6 in subjects with identified MS subtypes, and determining the MS subtype of the subject by determining the range within which the TGM6 level of the sample falls. The MS subtype can be primary progressive MS, secondary progressive MS, or relapsing remitting MS. The biological sample can be a sample of cerebrospinal fluid, brain, or spinal cord. The level of TGM6 can be measured by measuring the levels of TGM6 nucleic acid or TGM6 protein, such as by PCR, real-time PCR, RNA sequencing, microarray hybridization, Southern-blot, Northern- blot, Western-blot; immunoassay, ELISA, immunocytochemistry, or immunohistochemistry. The methods can further involve correlating TGM6 measurements with one or more additional methods for diagnosis or detection of MS selected from magnetic resonance imaging, neurological testing, and measurement of at least one additional biomarker for MS. Additional biomarkers include from fetuin-A, chemokine (C-X-C motif) ligand 13, neurofilament, hepatocyte growth factor, osteopontin, isoprostane 8-iso-prostaglandin F_2a, Chitinase 3-like 1, and miR-29 miRNA.

BRIEF DESCRIPTION OF THE FIGURES

[0014] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0015] FIGS. 1A-1E. ELISA for TGM6-IgG on CSF samples. (A), significant increase (P = 0.0055) of TGM6-IgG in the CSF of MS patients compared to controls. (B), PP group had the highest TGM6-IgG level, followed by the SP and the RR groups. (C), MS patient with an active disease had on average significant higher level TGM6-IgG (P = 0.0015) compared to patient with stable disease course. (D), no significant differences (P = 0.3753) between treated and untreated patient CSF. (E), western blot using CSF as source of primary antibody to bind a recombinant humanTGM6 protein shows that CSF samples with high levels of TGM6-IgG ("high") showed a stronger binding to recombinant TGM6 protein relative to CSF samples with low levels of TGM6-IgG ("low") at equal dilutions.

[0016] FIGS. 2A-2X. Immunohistochemical staining (IHC) for TGM6 on a series of adjacent paraffin embedded brain sections containing areas of MS plaques (lesion) as well as normal appearing white matter (NAWM) identified by luxol fast blue (LFB) and PLP staining. (A-D), lesioned areas lacking LFB. (E-H), lesioned areas lacking PLP. (I-L), lesioned areas lacking LFB and PLP signal contain TGM6 expressing cells which are particularly abundant around blood vessels (marked by red or blue arrows). (M-P), TGM6 expressing cells have astrocyte morphology and express GFAP. (Q-R and U-V), GFAP⁺ cells. (T, X), GFAP⁺ cells are present around perivascular areas of the NAWM. (S, W), GFAP⁺ cells do not express TGM6.

[0017] FIGS. 3A-3T. EAE induction in C57/BL6 mice and TGM6 expression. (A), EAE induced in C57/BL6 mice using the MOG-35-55 peptide. The black line indicates disability score of 1-5 using Stromnes-Goverman scale for al mice in the study; the red line indicates disability score for animals sacrificed to measure TGM6 protein levels in the brain and spinal cord by ELISA and western blot. The blue line represents mice sacrificed to analyze the distribution of TGM6 and GFAP protein in the spinal cord by immunohistochemistry and by immunofluorescence. (B), ELISA analysis for spinal cord proteins extracted at different time points during EAE revealed a statistically significant correlation between TGM6 expression and disease score of sacrificed animals (red line in (3A)). (C), correlation between TGM6 expression and disease score of sacrificed animals confirmed by Western blot. (D), correlation between the GFAP and TGM6 signals during EAE. (E-H),

Immunohistochemistry (IHC) signal for TGM6 in the mouse spinal cord during EAE. (I-L), TGM6 IHC in naive mice. (M-P), Immunofluorescent (IF) signal for TGM6 (green) in the mouse spinal cord during EAE. Myelin is stained by MBP in red. (Q-T), IF colocalization (yellow) of TGM6 (green) and GFAP (red) signals.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0018] The inventors have identified for the first time the involvement of transglutaminase 6 (TGM6) in the development of multiple sclerosis (MS). [0019] Contemplated herein are methods to diagnose multiple sclerosis in a subject with a neurological condition by measuring the level of transglutaminadase 6 (TGM6) in a biological sample from the subject. The level of TGM6 in the sample are compared to the level of TGM6 in a control representing the levels of TGM6 in a subject without MS, and MS is diagnosed if an elevated level of TGM6 is present in the sample, relative to control levels.

[0020] TGM6 has two identified transcript variants, generated by alternative splicing of the TGM6 gene. Transcript variant 1 has the National Center for Biotechnology Information (NCBI) Accession No. NM_198994. The DNA and amino acid sequences of transcript variant 1 of TGM6 are presented as SEQ ID NO: 1 (DNA) and SEQ ID NO: 2 (amino acid). Transcript variant 2 has the NCBI Accession No. NM_198994. The DNA and amino acid sequences of transcript variant 2 of TGM6 are presented as SEQ ID NO: 3 (DNA) and SEQ ID NO: 4 (amino acid). As used herein, reference to "TGM6" encompasses either or both transcript variants, and encompasses any of SEQ ID NOS: 1-4. Additional transcript variants of TGM6, and TGM6 homologs having 85%, 90%, 93%, or 95% or greater identity to any of SEQ ID NOS: 1-4, as can be determined, for example, using a sequence comparison program such as BLAST, are also encompassed by this disclosure.

[0021] Multiple sclerosis (MS) is an inflammatory disease in which the myelin sheathes surrounding neuronal projections in the CNS (central nervous system) are damaged. Within the CNS, the immune system attacks myelin (the fatty substance that surrounds and insulates axons), as well as the axons themselves. The damaged myelin forms lesions which result in scar tissue (sclerosis) formation, which distorts or interrupts neuronal signaling, leading to a variety of neurological symptoms.

[0022] MS can be categorized as progressive MS (PMS, in which symptoms and disease course steadily worsen over time with little or no remission) or relapsing-remitting MS (RRMS). RRMS is characterized by defined attacks of worsening neurologic function, also called relapses, flare-ups or exacerbations, followed by partial or complete recovery periods (remissions), during which symptoms improve partially or completely and there is no apparent progression of disease. The majority of people with MS are initially diagnosed with RRMS. Progressive MS (PMS) encompasses secondary progressive MS (SPMS) and primary progressive MS (PPMS). Most people who are initially diagnosed with RRMS will eventually transition to SPMS, where the disease progresses more steadily (although not necessarily more quickly), with or without relapses. In contrast to SPMS, primary

progressive MS (PPMS) is characterized by steadily worsening neurologic function from the first appearance of symptoms. Although the rate of progression may vary over time with occasional plateaus and temporary, minor improvements, there are no distinct relapses or remissions. About 10 percent of people with MS are diagnosed with PPMS.

[0023] A subject is defined as a person diagnosed with MS, a person suspected of having MS, or a person having at least one neurological symptom. In one embodiment, the subject is a person with "active" MS disease as defined by the presence of at least one of the following criteria in the 6 months preceding sample collection: (1) one or more relapses documented by a neurologist's examination; (2) a change in 0.5 points or greater in the EDSS score; and (3) change in MRI, such as a change in the number or size of lesions or the presence of gadolinium-enhancing lesions. In some embodiments, the subject has not been treated for MS; in other embodiments, the subject has been treated for MS.

[0024] The disclosed methods can be used to diagnose multiple sclerosis in a subject with at least one neurological symptom. A "neurological symptom" as encompassed herein includes: numbness or weakness in one or more limbs; partial or complete loss of central vision in one eye; partial or complete loss of central vision in both eyes; pain during eye movement (optic neuritis); double vision or blurring of vision; tingling or pain in parts of the body; electric- shock sensations occurring with certain head movements; tremors; walking (gait), balance, or coordination problems; slurred speech; fatigue; dizziness; bladder or bowel dysfunction; cognitive dysfunction; emotional changes; sexual dysfunction; depression; spasticity; speech or swallowing problems; hearing loss; seizures; respiration or breathing problems; and itching. In some embodiments, the subject has at least two, at least three, at least four, or at least five neurological symptoms.

[0025] The biological sample can be a sample of cerebrospinal fluid (CSF), or a sample of brain or spinal cord of the subject, such as a tissue biopsy. CSF can be collected by standard methods, such as by lumbar puncture or by access port aspiration of implanted pumps.

Methods to extract brain or spinal cord samples by tissue biopsy are known in the art.

[0026] The levels of TGM6 can be measured by measuring the level or levels of TGM6 nucleic acid or the level or levels of TGM6 protein product. The level of expression of the TGM6 gene or TGM6 RNA expression can be measured by any technique known in the art, including but not limited to PCR, real-time PCR, RNA sequencing, microarray hybridization, Southern-blot, and Northern-blot.

[0027] The level of TGM6 protein can be measured by any technique known in the art, including but not limited to Western-Blot; immunoassays, such as ELISA;

immunocytochemistry; and immunohistochemistry. Each of these formats utilizes a binding agent, such as an antibody, that recognizes TGM6. Blotting techniques are known to those of ordinary skill in the art, and may be performed as, for example electro -blots, semidry-blots, vacuum-blots or dot-blots. Immunocyto/histochemical staining procedures are known to those of skill in the art, and may comprise binding agent-mediated detection of polypeptides as well as in situ hybridisation techniques.

[0028] In one embodiment, the methods disclosed herein are combined with other methods for diagnosis of MS, such as magnetic resonance imaging (MRI), evoked potential (EP), visual evoked potential (VEP), oligoclonal band analysis, or blood tests. Such additional methods can be performed prior to, subsequent to, or concurrently with TGM6 testing.

[0029] The level of TGM6 in a sample is compared with a control representing the level of TGM6 in a subject without MS. The control level represents the average expression of TGM6 in a healthy person without MS. "Elevated" or "increased" levels of TGM6, compared to control levels, can be an increase of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% or greater relative to control levels.

[0030] In one embodiment, MS is diagnosed if elevated levels of TGM6 are present in the sample relative to control levels. In another embodiment, MS is diagnosed based on increased levels of TGM6, and a result positive for MS in another diagnostic test selected from neurological testing, magnetic resonance imaging (MRI), evoked potential (EP), visual evoked potential (VEP), oligoclonal band analysis, or blood/CSF test for one or more additional biomarkers utilized in the diagnosis of MS.

[0031] Biomarkers that can be used to diagnose MS, or to determine if MS is progressing in a subject, or to subtype MS in a subject, include fetuin-A (alpha-2-HS-glycoprotein);

chemokine (C-X-C motif) ligand 13 (CXCL13); neurofilament; hepatocyte growth factor (HGF); osteopontin; isoprostane 8-iso-prostaglandin F_2a (8-iso-PGF_2a); Chitinase 3-like 1 (CHI3L1); and miRNAs, such as miR-29 miRNA (for review, see Harris, VH, and Sadiq, SA, Mol Diagn Ther. 18(6): 605-617 (2014), the contents of which are incorporated by reference). In general, levels of these additional biomarkers are elevated in MS, and become increasingly elevated as MS progresses. Levels of these additional biomarkers also increase between primary progressive MS, secondary progressive MS, and relapsing remitting MS. Thus, correlation of elevated levels of MS with elevated levels of one or more additional biomarkers can confirm a diagnosis of MS, confirm that MS is progressing, and/or confirm that the subject has primary progressive MS, secondary progressive MS, or relapsing remitting MS subtype.

[0032] Further contemplated are methods to monitor the activity and/or progression of MS in a subject with MS. In these methods, CSF from a subject diagnosed with MS is repeatedly obtained, such as every two, four, six, or eight months; once a year; once every two, three, or four years; or any combination of these times. The subject can be undergoing treatment for MS, or untreated.

[0033] The activity and/or progression of MS is monitored by measuring the level of TGM6 in a biological sample from the subject and comparing the measured level of TGM6 in the sample with the level of TGM6 in a biological sample taken from the subject at an earlier time point. An increase in the level of TGM6 at a later time point, relative to the level of TGM6 at an earlier time point, indicates that MS in the subject is increasing in severity. Such increase can also indicate the need for the start of therapy, if the subject was not previously undergoing therapy; or can indicate the need for additional, or different, therapeutic methods, if the subject was already undergoing therapeutic treatment.

[0034] Also contemplated are methods to determine the MS subtype of a subject with MS, comprising measuring the level of TGM6 in a biological sample from the subject, comparing the level of TGM6 in the sample with a range representing the levels of TGM6 in subjects with identified MS subtypes, and determining the MS subtype of the subject by determining the range within which the TGM6 level of the sample falls. The MS subtype can be primary progressive MS, secondary progressive MS, or relapsing remitting MS. Relapsing remitting MS would be indicated by increased levels of TGM6 relative to normal controls; secondary progressive MS would be indicated by increased levels of TGM6 relative to relapsing remitting MS, and primary progressive MS would be indicated by increased levels of TGM6 relative to secondary progressive MS.

EXAMPLES

Example 1. Materials and Methods.

[0035] Patient Selection and CSF Collection. CSF was collected with IRB approval and informed consent from 181 patients (138 with clinically definite MS (McDonald WI et al., Ann Neurol. 50(1): 121-7 (2001)) and 43 non-MS controls) seen at the International Multiple Sclerosis Management Practice, the clinical affiliate of the Tisch MS Research Center of New York. Of the 138 MS patients, 41 were primary progressive, 51 secondary progressive, and 46 relapsing remitting. Of the 43 controls 22 samples were obtained for diagnostic purpose from untreated patients with other neurological diseases and 21 from healthy donor. In the MS group 86 patients had active disease, and 52 had inactive disease.

[0036] CSF samples were processed immediately and kept on ice. Samples were centrifuged at 200xg for 15 minutes to remove cells. All samples were confirmed to be free of red blood cell contamination. Aliquots of CSF were stored at -80°C until use.

[0037] Clinical Assessment of MS Patients. All patients in the study had a complete neurological examination at the onset of the study. In addition, routine brain MRI scans were performed on all study subjects 1-2 weeks before CSF collection. Active disease was defined by the presence of any one of the following criteria in the 6 months preceding CSF sample collection: (1) one or more relapses documented by a neurologist's examination; (2) change in 0.5 point or greater in the EDSS score; and (3) change in MRI, specifically a change in the number or size of lesions or the presence of gadolinium-enhancing lesions.

[0038] Human autopsy tissue and immunohistochemistry. Brain samples were provided by the Human Brain and Spinal Fluid Resource Center, Department of Veterans Affairs (Los Angeles, CA). TGM6 protein expression was examined in chronic or chronic/active plaques and in adjacent areas of normal-appearing white and gray matter from 4 brain samples from donors with MS. The average age of donors was 60 years old (range 50 to 79). [0039] EAE induction. All animal experiments were approved by the St. Luke's Roosevelt Hospital Center IACUC. 8 week old female C57BL/6 mice purchased from Jackson

Laboratory (Bar Harbor, ME) were immunized subcutaneously with 200 μg of the myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55; AnaSpec, Fremont, CA) diluted in 100 μΐ PBS and prepared in an equal volume of complete Freund's adjuvant containing 5 mg/ml Mycobacterium tuberculosis (H37Ra) (Difco, Detroit, MI). Mice received i.v.

injection of 200 ng pertussis toxin (List Biological Laboratories, Campbell, CA) on days 0 and 2 post immunization. Mice were weighed and evaluated for neurological disability daily. Disability was scored using the standard 0-5 scale (Stromnes JJVl, Goverman JM. Nat Protoc. 1(4): 1810-9 (2006)).

[0040] Quantitative Analysis of Immunostained Images. Spinal cord blocks were cut into 200 sections at 5μΓη/8εΰίίοη, and 13 sections (1 out of every 25) were stained per mouse. Excluding 3 sections spanning the injection site, a total of ten images per animal spanning 3mm of the corpus callosum were analyzed. Image J software (Image Processing and Analysis in Java) was used to calculate total cell count for BrdU+ and DAPI+ cells and to measure mean intensity for GFAP, Ibal, MBP, and NF stains.

[0041] ELISA and Western blot. Fresh mouse brain and spinal cord were placed in RIPA buffer (Cell Signaling Technologies, Danvers, MA) with Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific, Rockford, IL) and then sonicated for protein extraction. The other half was processed for RNA extraction using QIAzol Lysis Reagent and RNeasy Lipid Tissue Midi Kit (Qiagen, Venlo, Netherlands).

[0042] Statistical Analysis. SPSS was used for statistical analysis. Data were presented as mean + standard error of the mean (SEM), and one or two-way ANOVA with post-hoc analysis (Tukey HSD or Bonferroni Test) or student t-test were used to assess the

significance of the data. P-values < 0.05 were considered statistically significant.

Example 2. Results.

[0043] CSF levels of IgG against TGM6 are higher in MS vs. Controls and in Active vs. Inactive MS. CSF samples were collected from 138 MS patients and 43 controls as described in Methods and their cell count, total protein content, and albumin levels were determined. The control group included 21 healthy subjects, 11 with autoimmune diseases other than MS, 8 with non-autoimmune diseases, and 3 with non-specified neurological conditions.

[0044] To measure the level of autoantibodies against TGM6 in our patient's cohort the inventors performed an ELISA for TGM6-IgG on CSF samples. The inventors found a significant increase (P = 0.0055) of TGM6-IgG in the CSF of MS patients compared to controls (Fig. 1A). A stratification of MS patients by disease's subtype revealed that the PP group had the highest TGM6-IgG level, followed by the SP and the RR groups (Fig. IB). There was no significant difference between healthy controls and individual with other (non- MS) diseases. In addition, MS patient with an active disease had on average significant higher level TGM6-IgG (P = 0.0015) compared to patient with stable disease course (Fig. 1C). Finally, there were no significant differences (P = 0.3753) between treated and untreated patient CSF (Fig. ID).

[0045] To confirm the presence of TGM6 IgG in our patient' s CSF cohort the inventors performed a western blot using CSF as source of primary antibody to bind a recombinant humanTGM6 protein (Fig. IE). Compare to CSF samples with low levels of TGM6-IgG (measured by ELISA), CSF samples with high levels of TGM6-IgG showed a stronger binding to recombinant TGM6 protein.

[0046] TGM6 is highly expressed by reactive astrocytes within MS plaques. To explore the distribution of TGM6 protein in the CNS of MS patient the inventors performed immunohistochemical staining (IHC) for TGM6 on a series of adjacent paraffin embedded brain sections containing areas of MS plaques (lesion) as well as normal appearing white matter (NAWM) previously identified by luxol fast blue (LFB) and PLP staining (Fig. 2). Lesioned areas, lacking LFB (Fig. 2A-D) and PLP (Fig. 2E-H) signal, contain TGM6 expressing cells (Fig. 2 I-L) which are particularly abundant around blood vessels (marked by red or blue arrows). These cells have astrocyte morphology and express GFAP (Fig. 2-M-P) a marker for reactive astrocytes. In the NAWM (Fig. 2Q-R and U-V), GFAP⁺ cells were also present mostly around perivascular areas (Fig. 2T and X) but they did not express TGM6 (Fig. 2S and W).

[0047] TGM6 is upregulated in the mouse CNS during EAE and its expression by reactive astrocytes in the spinal cord correlates with disease course. To investigate a possible implication of TGM6 in the MS pathophysiology, the inventors induced EAE in C57/BL6 mice using the MOG-35-55 peptide (black line in Fig. 3A) and analyzed TGM6 expression during disease course. ELISA analysis for spinal cord proteins extracted at different time point during EAE revealed a striking correlation between TGM6 expression (Fig. 3B) and disease score of sacrificed animals (red line in Fig. 3A). Such correlation was further confirmed by Western blot (Fig. 3C). TGM6 expression in the brain was lower than in the spinal cord for each time point examined. It peaked at 14 days post immunization and then plateau to lower level for the remaining of the disease duration (Fig. 3B-C).

[0048] To elucidate the distribution of TGM6 in the spinal cord during EAE a subgroup of animals (blue line in Fig. 3A) were sacrificed at different time points for IHC (Fig. 3-E-L) and immunofluorescent (IF) (Fig. 3M-T) examinations of paraffin embedded spinal cord sections. During EAE, HIC (Fig. 3E-H) and IF (Fig. 3M-T) staining reveal a strong signal for TGM6 in white matter areas such as: lateral (Fig. 3F and N), dorsal (Fig. 5G and O), and ventral funiculus (Fig. 3H and P). In naive mice, the IHC signal for TGM6 signal was very weak and only localized in the gray matter (Fig. 3I-L). Similar to the findings reported above for the MS plaques, TGM6 signal in the spinal cord of mice with EAE was localized primarily in reactive astrocytes as showed by the colocalization with the GFAP IF signal (Fig. 3Q-T) and the correlation between the two signals during EAE (Fig 3D). Similar to the IHC data, TGM6 IF signal in the white matter of naive animal was virtually absent (data not shown).

[0049] Using enzyme-linked immunosorbent assays (ELISA) the inventors found that antibodies raised against TGM6 are elevated in MS patient CSF compared to controls. In addition, our preliminary data suggest that TGM6 protein and antibody against it might be elevated in primary progressive MS compared to control individuals as well as relapsing remitting and secondary progressive MS patients. Based on these findings, the inventors hypothesize that CSF levels of TGM6 protein and antibodies against it might be useful biomarkers to: diagnose MS vs. other neurological conditions; differentiate between MS subtypes; and predict diseases activity and progression.

[0050] To further investigate if changes inTGM6 expression might be pathogenic in vivo, the inventors employed the EAE animal model, which primarily affects the spinal cord via demyelination and astrogliosis. The inventors found that mouse TGM6 protein levels are increased at disease onset and correlate with its course. Furthermore, immuno staining of mouse spinal cord at disease peak revealed strong expression of TGM6 in reactive astrocytes, which was not found in control animals. Based on these findings, the inventors hypothesize that the expression of TGM6 by reactive astrocytes might contribute to disease pathogenesis. Interestingly, in primary progressive patients the spinal cord is most severely affected with pronounced astrogliosis. This opens the opportunity to use TGM6 not only as a biomarker for primary progressive MS but also to study the pathological mechanisms leading to this condition.

Claims

WHAT IS CLAIMED IS:

1. A method to diagnose multiple sclerosis (MS) in a subject with at least one neurological symptom, comprising measuring the level of transglutaminadase 6 (TGM6) in a biological sample from said subject, comparing the level of TGM6 in said sample with a control representing the level of TGM6 in a subject without MS, and diagnosing MS if elevated levels of TGM6 are present in said sample relative to control levels.

2. A method to monitor the activity and/or progression of multiple sclerosis (MS) in a subject with MS, comprising measuring the level of transglutaminadase 6 (TGM6) in a biological sample from said subject and comparing the measured level of TGM6 in said sample with the level of TGM6 in a biological sample taken from said subject at an earlier time point, wherein an increase in the level of TGM6 relative to the level of TGM6 at an earlier time point indicates that MS in said subject is increasing in severity.

3. A method to determine the multiple sclerosis (MS) subtype of a subject with MS, comprising measuring the level of transglutaminadase 6 (TGM6) in a biological sample from said subject, comparing the level of TGM6 in said sample to control levels correlating a range of TGM6 levels with identified MS subtypes, and determining the MS subtype of said subject by determining the range within which the TGM6 level of said sample falls.

4. The method of claim 1, wherein the at least one neurological symptom is selected from: numbness or weakness in one or more limbs; partial or complete loss of central vision in one eye; partial or complete loss of central vision in both eyes; pain during eye movement (optic neuritis); double vision or blurring of vision; tingling or pain in parts of the body; electric- shock sensations occurring with certain head movements; tremors; walking (gait), balance, or coordination problems; slurred speech; fatigue; dizziness; bladder or bowel dysfunction; cognitive dysfunction; emotional changes; sexual dysfunction; depression; spasticity; speech or swallowing problems; hearing loss; seizures; respiration or breathing problems; and itching.

5. The method of any of claims 1-3, wherein said biological sample is a sample of cerebrospinal fluid, brain, or spinal cord.

6. The method of any of claims 1-3, wherein the level of TGM6 is measured by measuring the levels of TGM6 nucleic acid or TGM6 protein.

7. The method of any of claims 1-3, further comprising correlating TGM6 measurements with one or more additional methods for diagnosis or detection of MS selected from magnetic resonance imaging, neurological testing, and measurement of at least one additional biomarker for MS.

8. The method of claim 3, wherein said MS subtype is selected from primary progressive MS, secondary progressive MS, and relapsing remitting MS.

9. The method of claim 5, wherein said biological sample is a sample of cerebrospinal fluid.

10. The method of claim 6, wherein the level of TGM6 is measured by PCR, realtime PCR, RNA sequencing, microarray hybridization, Southern-blot, Northern-blot, Western-blot; immunoassay, ELISA, immunocytochemistry, or immunohistochemistry.

11. The method of claim 7, wherein said at least one additional biomarker for MS is selected from fetuin-A, chemokine (C-X-C motif) ligand 13, neurofilament, hepatocyte growth factor, osteopontin, isoprostane 8-iso-prostaglandin F_2a, Chitinase 3-like 1, and miR- 29 miRNA.