WO2018045162A1 - Biomarqueurs prédictifs de la sclérose en plaques progressive primaire et leurs utilisations - Google Patents

Biomarqueurs prédictifs de la sclérose en plaques progressive primaire et leurs utilisations Download PDF

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WO2018045162A1
WO2018045162A1 PCT/US2017/049599 US2017049599W WO2018045162A1 WO 2018045162 A1 WO2018045162 A1 WO 2018045162A1 US 2017049599 W US2017049599 W US 2017049599W WO 2018045162 A1 WO2018045162 A1 WO 2018045162A1
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subject
nucleic acid
gene
genetic polymorphism
sample
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PCT/US2017/049599
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WO2018045162A8 (fr
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Paola BRONSON
Kd NGUYEN
Aaron DAY-WILLIAMS
Karl GUTWIN
Karol ESTRADA
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Biogen Ma Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

  • MS Multiple sclerosis
  • RRMS Relapsing-Remitting MS
  • SPMS Secondary-Progressive MS
  • SPMS Secondary-Progressive MS
  • PPMS Primary- Progressive MS
  • PRMS Progressive-Relapsing MS
  • MS research has primarily focused on RRMS and SPMS.
  • genetic markers associated with RRMS have been identified, including the HLA Class II allele HLA-DRBl *15:01. Accordingly, there is a need for the identification of genetic markers identifying susceptibility to other forms of MS, e.g., PPMS, to allow for early diagnosis and treatment.
  • the present invention provides, at least in part, methods, assays and kits for the identification, assessment and/or treatment of a subject having multiple sclerosis (MS) (e.g. , a subject with Primary Progressive MS (PPMS)).
  • MS multiple sclerosis
  • PPMS Primary Progressive MS
  • the genetic biomarker is a LIX1L gene variant, e.g., one or more genetic polymorphisms in the LIX1L gene.
  • the genetic biomarker is the HLA allele HLA- A*02:01.
  • the gene signature can be acquired from a whole blood sample, e.g., peripheral blood, from the subject.
  • the invention can have prognostic, diagnostic and therapeutic applications, including, for example, (i) to diagnose a subject with MS or at risk of MS; (ii) to stratify a subject, e.g., as having or not having MS (e.g., PPMS), or as being likely or unlikely to develop MS; and (iii) to treat a subject having MS or at risk of having MS.
  • the invention features a method, or assay, for evaluating, monitoring, stratifying, or treating, a subject.
  • the method includes:
  • PPMS Primary Progressive Multiple Sclerosis
  • MS multiple sclerosis
  • the invention features a method of treating Primary Progressive Multiple Sclerosis (PPMS).
  • PPMS Primary Progressive Multiple Sclerosis
  • PPMS Primary Progressive Multiple Sclerosis
  • the invention features a method, or assay, for evaluating a subject's likelihood of developing multiple sclerosis (MS).
  • MS multiple sclerosis
  • identifying a subject as being a blood relative of an individual with MS e.g., a sibling, or a descendant
  • the invention features a method, or assay, for evaluating, monitoring, stratifying, or treating, a subject, comprising:
  • PPMS Primary Progressive Multiple Sclerosis
  • MS multiple sclerosis
  • the invention features a kit for evaluating a subject's likelihood of developing multiple sclerosis (MS).
  • MS multiple sclerosis
  • nucleic acid reagents for detecting a Primary Progressive Multiple Sclerosis (PPMS)-associated genetic polymorphism in an LIXIL gene
  • the nucleic acid reagents comprise: a) a probe complimentary to one or more genetic polymorphisms in the LIXIL gene; or b) allele-specific primers or primer pairs that provides for detection of the one or more genetic polymorphisms in the LIXIL gene.
  • the invention features a nucleic acid encoding an LIXIL gene variant, e.g., a genetic polymorphism in the LIXIL gene chosen from one or more of R114H, V173L, S286C, and L322F, e.g., relative to SEQ ID NO: l, or a combination of two, three, four or all of Rl 14H, V173L, S286C, and L322F.
  • an LIXIL gene variant e.g., a genetic polymorphism in the LIXIL gene chosen from one or more of R114H, V173L, S286C, and L322F, e.g., relative to SEQ ID NO: l, or a combination of two, three, four or all of Rl 14H, V173L, S286C, and L322F.
  • the invention features an LIXIL polypeptide variant, e.g., a polypeptide encoded by a genetic polymorphism in the LIXIL gene.
  • the LIXIL polypeptide variant comprises an amino acid substitution chosen from one or more of R114H, V173L, S286C, and L322F, e.g., relative to SEQ ID NO: l, or a
  • vectors and host cells comprising the aforesaid nucleic acid encoding the LIXIL gene variants, as well as detection reagents (e.g., probes, primers and antibodies) that specifically bind to the LIXIL gene and polypeptide
  • nucleic acids or polypeptides include one or more of the following.
  • any of the aforesaid method, assays, or kits detect a genetic biomarker, e.g., one or more LIXIL variants, or an HLA-A*02:01 allele.
  • the LIXIL variant comprises a genetic polymorphism in the LIXIL gene.
  • the genetic polymorphism in the LIXIL gene comprises single nucleotide polymorphisms.
  • the genetic polymorphism in the LIXIL gene comprises one or more of: a stop gain, a splice donor, a splice acceptor, and/or a missense variants in the LIXIL sequence, or a combination thereof.
  • the genetic polymorphism in the LIXIL gene comprises one or more of R114H, V173L, S286C, and L322F, e.g., relative to SEQ ID NO: l, or a combination of two, three, four or all of Rl 14H, V173L, S286C, and L322F.
  • the genetic polymorphism comprises Rl 14H.
  • the genetic polymorphism comprises V173L.
  • the genetic polymorphism comprises
  • polymorphism comprises S286C. In further embodiments, the genetic polymorphism comprises L322F. In some embodiments, the genetic polymorphism comprises one or both of Rl 14H and V173L. In certain embodiments, the genetic polymorphism
  • the genetic polymorphism comprises one or both of Rl 14H and S286C. In further embodiments, the genetic polymorphism comprises one or both of Rl 14H and L322F. In some embodiments, the genetic polymorphism comprises one or both of V173L and S286C. In certain embodiments,
  • the genetic polymorphism comprises one or both of V173L and L322F. In further embodiments, the genetic polymorphism comprises one or both of S286C and L322F. In some embodiments, the genetic polymorphism comprises one or more of Rl 14H, V173L, and S286C, or a combination of at least two or three thereof. In certain embodiments, the genetic polymorphism comprises one or more of Rl 14H, V173L, and L322F, or a combination of at least two or three thereof. In further embodiments, the genetic polymorphism comprises one or more of Rl 14H, S286C, and L322F, or a combination of at least two or three thereof. In some embodiments, the genetic
  • polymorphism comprises one or more of V173L, S286C, and L322F, or a combination of at least two or three thereof.
  • the genetic polymorphism comprises one or more of Rl 14H, V173L, S286C, and L322F, or a combination of at least two, three or all thereof.
  • the invention features a method of determining the presence of a polymorphism in the LIXIL gene.
  • the method includes acquiring a genotype, or acquiring knowledge, e.g., directly acquiring knowledge, that a nucleic acid molecule or polypeptide having the genetic polymorphism in the LIX1L gene (e.g., one or more of the
  • polymorphisms described herein is present in a sample from a subject.
  • acquiring a genotype comprises acquiring a sequence (e.g., by sequencing) for a position in the LIX1L gene, thereby determining if the polymorphism in the LIX1L gene is present.
  • the sequence acquired is compared to a reference sequence, or a wild type reference sequence.
  • acquiring a genotype comprises hybridizing one or more probes to the nucleic acid sequence in the biological sample, wherein the probes are complimentary to the genetic polymorphism in the LIX1L gene, and detecting the polymorphism, e.g., in the biological sample.
  • the genetic polymorphism comprises one or more single nucleotide polymorphisms.
  • the genetic polymorphism in the LIX1L gene is detected in a nucleic acid molecule.
  • the method comprises one or more of: sequencing (e.g., next generation sequencing), nucleic acid hybridization assay, amplification-based assays, PCR-RFLP assay, real-time PCR, screening analysis, FISH, spectral karyotyping or MFISH, comparative genomic hybridization, in situ
  • a polypeptide having the genetic polymorphism in the LIX1L gene is detected.
  • the method comprises contacting a protein sample with a reagent which specifically binds to a polypeptide having the genetic polymorphism in the LIX1L gene; and detecting the formation of a complex of the polypeptide and the reagent.
  • the reagent is labeled with a detectable group to facilitate detection of the bound and unbound reagent.
  • the reagent is an antibody molecule.
  • the method, or assay further includes the step of acquiring, e.g., obtaining, a sample, e.g., a biological sample, from the subject.
  • the method, or assay includes the step of acquiring, e.g., obtaining, whole blood, e.g., peripheral blood, from the subject.
  • the sample comprises whole blood.
  • the sample comprises a predominantly non-cellular fraction of a body fluid from the subject.
  • the non-cellular fraction can be plasma, serum, or other non-cellular body fluid.
  • the sample is a serum sample.
  • the blood can be further processed to obtain plasma or serum.
  • the sample contains a tissue, cells (e.g., peripheral blood mononuclear cells (PBMC)).
  • the sample can be a fine needle biopsy sample, an archival sample (e.g., an archived sample with a known diagnosis and/or treatment history), a histological section (e.g., a frozen or formalin-fixed section, e.g., after long term storage), among others.
  • a sample can include any material obtained and/or derived from a biological sample, including a nucleic acid (e.g., genomic DNA, cDNA, RNA) purified or processed from the sample. Purification and/or processing of the sample can include one or more of extraction, concentration, sorting, concentration, fixation, addition of reagents and the like.
  • the sample contains the biomarkers described herein.
  • the acquiring step of the methods described herein include acquiring a genetic biomarker, e.g., genotype, e.g., of a LIX1L gene, from a sample, e.g., a sample of whole or peripheral blood, plasma, serum, or other non-cellular body fluid; or a cellular sample (e.g., a PBMC sample).
  • a genetic biomarker e.g., genotype, e.g., of a LIX1L gene
  • a sample e.g., a sample of whole or peripheral blood, plasma, serum, or other non-cellular body fluid
  • a cellular sample e.g., a PBMC sample.
  • the acquired genotype is compared to a specified parameter (e.g., a reference sequence; a control sample; or a sample obtained from a healthy subject), to thereby diagnose, evaluate, or identify a patient.
  • methods of the invention relate to acquiring a genetic biomarker e.g., genotype, e.g., of a LIXIL gene, from whole blood or peripheral blood, or plasma or serum of the subject, wherein the plasma or serum is obtained from the blood of the subject, for example.
  • a genetic biomarker e.g., genotype, e.g., of a LIXIL gene
  • a change in the genotype (e.g., sequence) of the genetic biomarker relative to a reference value is indicative of the presence of MS, e.g., Primary Progressive MS.
  • the method of treatment includes an MS therapy, e.g. , an MS therapy that comprises one or more of an IFN- ⁇ 1 molecule; a polymer of glutamic acid, lysine, alanine and tyrosine, e.g., glatiramer (e.g., Copaxone®); an antibody or fragment thereof against alpha-4 integrin, e.g., natalizumab (e.g., Tysabri®); an anthracenedione molecule, e.g., mitoxantrone (e.g., Novantrone®); a fingolimod, e.g., FTY720 (e.g., Gilenya®); a dimethyl fumarate, e.g., an oral dimethyl fumarate (e.g., Tecfidera®); an antibody to the alpha subunit of the IL-2 receptor of T cells (CD25), e.g., daclizumab;
  • the method further includes the use of a symptom management therapies, including one or more of an antidepressant, analgesic, anti-tremor agent, among others.
  • a symptom management therapies including one or more of an antidepressant, analgesic, anti-tremor agent, among others.
  • the subject has been administered a MS therapy, e.g. , prior to, or at the time of, acquiring the value.
  • the subject treated, or the subject from which the sample is obtained is a subject having, or at risk of having MS, e.g. PPMS, at any stage of treatment.
  • the subject is an MS patient (e.g. , a patient with PPMS) prior to administration of an MS therapy described herein.
  • the subject is a newly diagnosed PPMS patient.
  • the subject is an MS patient (e.g., an PPMS patient) after administration of an MS therapy described herein.
  • the subject is an MS patient after administration of the MS therapy for one, two weeks, one month, two months, three months, four months, six months, one year or more.
  • the methods described herein further comprises treating, or preventing in, the subject having MS one or more symptoms associated with MS by administering to a subject an MS therapy, in an amount sufficient to reduce one or more symptoms associated with MS.
  • said treating or preventing comprises reducing, retarding or preventing, a relapse, or the worsening of the disease, in the MS subject.
  • the methods further comprise one or more of: performing a neurological examination, evaluating the subject' s status on the Expanded Disability Status Scale (EDSS), or detecting the subject's lesion status as assessed using an MRI.
  • EDSS Expanded Disability Status Scale
  • FIG. 1 is a Manhattan plot of p-values from PPMS cases vs. controls.
  • FIG. 2 is a Manhattan plot of p-values from RRMS cases vs. controls.
  • FIG. 3 is a graphical representation of a comparison of the HLA signal in PPMS cases and RRMS cases.
  • FIG. 4 is a diagram showing the location of rare, probably damaging variants in LIX1L that were over-represented in PPMS.
  • the present invention provides, at least in part, methods, assays, and kits for the identification, assessment and/or treatment of a subject having MS ⁇ e.g. , a subject with PPMS).
  • a subject having MS e.g., a subject with PPMS.
  • the genetic biomarker evaluated comprises a LIX1 variant, e.g., genetic polymorphisms in the LIX1L gene.
  • the genetic biomarker evaluated comprises a LIX1 variant, e.g., genetic polymorphisms in the LIX1L gene.
  • the biomarker evaluated comprises an HLA allele, e.g., HLA-A*02:01. Said biomarkers can discriminate a subject with PPMS from a healthy subject.
  • the genetic biomarkers can be acquired from a whole blood sample, e.g., peripheral blood, from the subject.
  • the invention can have prognostic, diagnostic and therapeutic applications. Definitions
  • the articles “a” and “an” refer to one or to more than one (e.g., to at least one) of the grammatical object of the article, unless indicated to the contrary or otherwise evident from the context.
  • Acquire or “acquiring” as the terms are used herein, refer to obtaining possession of a physical entity, or a value, e.g., a numerical value, by “directly acquiring” or “indirectly acquiring” the physical entity or value.
  • Directly acquiring means performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value.
  • “Indirectly acquiring” refers to receiving the physical entity or value from another party or source (e.g., a third party laboratory that directly acquired the physical entity or value).
  • Directly acquiring a physical entity includes performing a process that includes a physical change in a physical substance, e.g., a starting material. Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond.
  • Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which
  • an analytical method includes a physical change in a substance, e.g., a sample, analyte, or reagent (sometimes referred to herein as "physical analysis"), performing an analytical method, e.g., a method which includes one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or fragment or other derivative thereof, with another substance, e.g., a buffer, solvent, or reactant; or changing the structure of an analyte, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the analyte; or by changing the structure of a reagent, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom
  • Acquiring a sequence refers to obtaining possession of a nucleotide sequence or amino acid sequence, by “directly acquiring” or “indirectly acquiring” the sequence.
  • Directly acquiring a sequence means performing a process (e.g., performing a synthetic or analytical method) to obtain the sequence, such as performing a sequencing method (e.g., a Next Generation Sequencing (NGS) method).
  • NGS Next Generation Sequencing
  • Indirectly acquiring a sequence refers to receiving information or knowledge of, or receiving, the sequence from another party or source (e.g., a third party laboratory that directly acquired the sequence).
  • the sequence acquired need not be a full sequence, e.g., sequencing of at least one nucleotide, or obtaining information or knowledge, that identifies a LIX1L
  • polymorphism or HLA-A*02:01 allele disclosed herein as being present in a subject constitutes acquiring a sequence.
  • Directly acquiring a sequence includes performing a process that includes a physical change in a physical substance, e.g., a starting material, such as a tissue sample, e.g., a biopsy, or an isolated nucleic acid (e.g., DNA or RNA) sample.
  • a starting material such as a tissue sample, e.g., a biopsy, or an isolated nucleic acid (e.g., DNA or RNA) sample.
  • Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, such as a genomic DNA fragment; separating or purifying a substance (e.g., isolating a nucleic acid sample from a tissue); combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non- covalent bond.
  • Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance as described above.
  • “Complementary” refers to sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
  • a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. Each divergent sequence is termed an allele, and can be part of a gene or located within an intergenic or non-genic sequence.
  • a diallelic polymorphism has two alleles, and a triallelic polymorphism has three alleles. Diploid organisms can contain two alleles and may be homozygous or heterozygous for allelic forms.
  • SNP single polynucleotide polymorphism
  • genotype refers to a description of the alleles of a gene or genes contained in an individual or a sample. Diploid individuals have a genotype that
  • genotype of an individual comprises two different sequences (heterozygous) or one sequence (homozygous) at a polymorphic site.
  • genotype of an individual comprises two different sequences (heterozygous) or one sequence (homozygous) at a polymorphic site.
  • no distinction is made between the genotype of an individual and the genotype of a sample originating from the individual.
  • a genotype is determined from samples of diploid cells
  • a genotype can be determined from a sample of haploid cells, such as a sperm cell.
  • genes include coding sequences and/or the regulatory sequences required for their expression.
  • gene refers to a nucleic acid fragment that expresses mRNA, functional RNA, or specific protein, including regulatory sequences.
  • Genes also include non- expressed DNA segments that, for example, form recognition sequences for other proteins.
  • Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
  • nucleotide sequence in the context of nucleotide sequence, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity.
  • the term "functional variant” refers to polypeptides that have a substantially identical amino acid sequence to the naturally-occurring sequence, or are encoded by a substantially identical nucleotide sequence, and are capable of having one or more activities of the naturally-occurring sequence.
  • the sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the respective programs ⁇ e.g.,
  • XBLAST and NBLAST can be used. See http://www.ncbi.nlm.nih.gov.
  • molecules of the present invention may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions.
  • “Sequencing" a nucleic acid molecule requires determining the identity of at least 1 nucleotide in the molecule. In embodiments, the identity of less than all of the nucleotides in a molecule are determined. In other embodiments, the identity of a majority or all of the nucleotides in the molecule is determined.
  • next-generation sequencing or NGS or NG sequencing refers to any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules ⁇ e.g., in single molecule sequencing) or clonally expanded proxies for individual nucleic acid molecules in a highly parallel fashion ⁇ e.g., greater than 10 5 molecules are sequenced simultaneously).
  • the relative abundance of the nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences in the data generated by the sequencing experiment.
  • Next generation sequencing methods are known in the art, and are described, e.g., in Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46, incorporated herein by reference. Next generation sequencing can detect a variant present in less than 5% of the nucleic acids in a sample.
  • LIX1L gene refers to a gene encoding the LIXl-like protein.
  • the human amino acid and nucleic acid sequences can be found in a public database, such as GenBank and UniProt.
  • the mRNA sequence can be found at Accession No. NM_153713.2 (provided herein as SEQ ID NO:2) and the protein sequence can be found at Accession No. NP_714924.1 (provided herein as SEQ ID
  • LIX1L variant refers to any alteration in the nucleic acid sequence of LIX1L, including in the promoter sequence and termination sequence.
  • the LIX1L variant comprises one or more polymorphisms, e.g., one, two, three, four, or more, polymorphisms, in the LIX1 gene.
  • the one or more polymorphisms comprise single nucleotide polymorphisms.
  • the polymorphisms result in missense mutations, nonsense mutations, read-through mutations, splice donor and acceptor site mutations, which alter the protein sequence.
  • a LIX1 variant comprises one or more polymorphisms that encodes one or more amino acid substitutions relative to a wild type sequence selected from R114H, V173L, S286C, and L322F.
  • a “biomarker,” “marker” or “genetic biomarker signature” is a genomic DNA sequence (e.g., an LIX1L genetic polymorphism described herein or the HLA-A*02:01 allele) that are positively or negatively associated with MS, e.g., PPMS.
  • the genomic DNA sequence can be in a biological sample (e.g., a whole blood or peripheral sample) obtained from a subject having MS.
  • a healthy control or a healthy subject population (e.g., a control)
  • such genomic DNA sequences are positively or negatively associated with presence of a disease state, such as MS.
  • a marker of the invention which is associated with having PPMS can be present in the genomic DNA in a biological sample obtained from a subject having, or suspected of having, PPMS as compared to a biological sample obtained from a healthy subject.
  • MS Multiple Sclerosis
  • MS is “treated,” “inhibited” or “reduced,” if at least one symptom of the disease is reduced, alleviated, terminated, slowed, or prevented.
  • MS is also “treated,” “inhibited,” or “reduced,” if recurrence or relapse of the disease is reduced, slowed, delayed, or prevented.
  • Exemplary clinical symptoms of MS that can be used to aid in determining the disease status in a subject can include optic neuritis, diplopia, nystagmus, ocular dysmetria, internuclear opthalmoplegia, movement and sound phosphenes, afferent pupillary defect, paresis, monoparesis, paraparesis, hemiparesis, quadraparesis, plegia, paraplegia, hemiplegia, tetraplegia, quadraplegia, spasticity, dysarthria, muscle atrophy, spasms, cramps, hypotonia, clonus, myoclonus, myokymia, restless leg syndrome, footdrop, dysfunctional reflexes, paraesthesia,
  • neuralgia neuropathic and neurogenic pain
  • l'hermitte's proprioceptive dysfunction
  • trigeminal neuralgia ataxia
  • intention tremor dysmetria, vestibular ataxia, vertigo
  • speech ataxia dystonia
  • dysdiadochokinesia frequent micturation
  • bladder spasticity flaccid bladder
  • detrusor-sphincter dyssynergia erectile dysfunction
  • cognitive dysfunction dementia, mood swings, emotional lability, euphoria, bipolar syndrome, anxiety, aphasia, dysphasia, fatigue, Uhthoff s symptom, gastroesophageal reflux, and sleeping disorders.
  • therapy e.g., MS therapy or MS treatment
  • treatment e.g., MS therapy or MS treatment
  • PPMS Primary Progressive Multiple Sclerosis
  • EDSS Extended Disability Status Scale
  • EDSS is a rating system that is frequently used for classifying and standardizing MS.
  • the accepted scores range from 0 (normal) to 10 (death due to MS).
  • patients having an EDSS score of about 4-6 will have moderate disability (e.g., limited ability to walk), whereas patients having an EDSS score of about 7 or 8 will have severe disability (e.g., will require a wheelchair).
  • EDSS scores in the range of 1-3 refer to an MS patient who is fully ambulatory, but has some signs in one or more functional systems; EDSS scores in the range higher than 3 to 4.5 show moderate to relatively severe disability; an EDSS score of 5 to 5.5 refers to a disability impairing or precluding full daily activities; EDSS scores of 6 to 6.5 refer to an MS patient requiring intermittent to constant, or unilateral to bilateral constant assistance (cane, crutch or brace) to walk; EDSS scores of 7 to 7.5 means that the MS patient is unable to walk beyond five meters even with aid, and is essentially restricted to a wheelchair; EDSS scores of 8 to 8.5 refer to patients that are restricted to bed; and EDSS scores of 9 to 10 mean that the MS patient is confined to bed, and progressively is unable to communicate effectively or eat and swallow, until death due to MS.
  • Responsiveness to “respond” to treatment, and other forms of this verb, as used herein, refer to the reaction of a subject to treatment with an MS therapy.
  • a subject responds to treatment with an MS therapy if at least one symptom of multiple sclerosis in the subject is reduced or retarded by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.
  • a subject responds to treatment with an MS therapy, if at least one symptom of multiple sclerosis in the subject is reduced by about 5%, 10%, 20%, 30%, 40%, 50% or more as determined by any appropriate
  • a subject responds to treatment with an MS therapy, if the subject experiences a life expectancy extended by about 5%, 10%, 20%, 30%, 40%, 50% or more beyond the life expectancy predicted if no treatment is administered.
  • a subject responds to treatment with an MS therapy, if the subject has an increased disease-free survival, overall survival or increased time to progression.
  • probe or “detection reagent” refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example a biomarker as described herein. Probes or detection reagents can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes can be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes or detection reagents include, but are not limited to, RNA, DNA, proteins, antibodies, and organic monomers.
  • tissue sample each refers to a biological sample obtained from a tissue or bodily fluid of a subject or patient.
  • the source of the tissue sample can be solid tissue as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate; blood (e.g., whole blood or peripheral blood) or any blood constituents (e.g., serum, plasma); bodily fluids such as urine, cerebral spinal fluid, whole blood, plasma and serum.
  • the sample can include a non-cellular fraction (e.g., urine, plasma, serum, or other non-cellular body fluid).
  • the body fluid from which the sample is obtained from an individual comprises blood (e.g., whole blood).
  • the blood can be further processed to obtain plasma or serum.
  • the sample contains a tissue, cells (e.g., peripheral blood mononuclear cells (PBMC)).
  • PBMC peripheral blood mononuclear cells
  • the sample can be a fine needle biopsy sample, an archival sample (e.g., an archived sample with a known diagnosis and/or treatment history), a histological section (e.g., a frozen or formalin-fixed section, e.g., after long term storage), among others.
  • PBMC peripheral blood mononuclear cells
  • the term sample includes any material obtained and/or derived from a biological sample, including a polypeptide, and nucleic acid (e.g., genomic DNA, cDNA, RNA) purified or processed from the sample.
  • Purification and/or processing of the sample can involve one or more of extraction, concentration, antibody isolation, sorting, concentration, fixation, addition of reagents and the like.
  • the sample can contain compounds that are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like.
  • LIX1L Limb expression 1-like (LIX1L) encodes a 36.5 KDa protein and is located on chromosome lq21.1.
  • LIXl-like protein (the protein encoded by LIX1L) is a putative RNA binding protein with an RNA binding motif. It is expressed on cancer cells and is thought to be involved with apoptosis inhibition. (Nakamura et al., 2015 Scientific Reports 5: 13474) LIX1L expression increases cell proliferation (which may reflect a decrease in apoptosis). (Nakamura et al. supra)
  • the LIX1 variants include LIXIL gene polymorphisms, e.g., single nucleotide polymorphisms. Approximately 90% of all polymorphisms in the human genome are SNPs. SNPs are single base positions in DNA at which different alleles, or alternative nucleotides, exist in a population. The SNP position
  • SNP SNP site
  • SNP locus SNP marker
  • SNP marker SNP marker
  • SNPs can be in the gene coding region (i.e., exons) or in introns, in upstream and downstream sequence. Missense mutations, nonsense mutations, and read-through mutations are types of SNPs in the exons.
  • the LIXIL variant comprises a missense mutation. A missense mutation changes a codon coding for one amino acid to a codon coding for a different amino acid.
  • the LIXIL variant comprises a nonsense mutation. A nonsense mutation results in a type of codon change in which a stop codon is formed, thereby leading to premature termination of a polypeptide chain and a truncated protein.
  • the LIXIL variant comprises a read-through mutation.
  • a read-through mutation is another type of codon change that causes the destruction of a stop codon, thereby resulting in an extended polypeptide product.
  • Other types of SNPs can include splice donor and acceptor site mutations, mutations in promoter sequences, mutations in termination signals and mutations in ribosome binding sites.
  • the LIXIL variant comprises a mutation in a splice donor or acceptor site. Splice donor or acceptor site mutations alter splicing resulting in a mis-spliced protein.
  • the LIXIL variant comprises a SNP in a promoter sequence.
  • the LIXIL variant comprises a SNP in a termination signal.
  • the LIXIL variant comprises a SNP in a ribosome binding site.
  • the LIXIL variant comprises a single polymorphism. In another embodiment, the LIXIL variant comprises two polymorphisms. In another embodiment, the LIXIL variant comprises three or more polymorphisms. In another embodiment, the LIXIL variant comprises four or more polymorphisms.
  • the LIXIL variant results in an altered level of LIXIL.
  • LIXIL transcription levels are downregulated 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100% relative to a wild-type sequence.
  • LIXIL transcription levels are upregulated 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100% relative to a wild-type sequence.
  • LIXIL translation levels are downregulated 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100% relative to a wild-type sequence.
  • LIXIL translation levels are upregulated 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100% relative to a wild-type sequence.
  • the human leukocyte antigen (HLA) system is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans. It encodes cell surface molecules specialized to present antigenic peptides to the T-cell receptor (TCR) on T cells.
  • MHC major histocompatibility complex
  • TCR T-cell receptor
  • the HLA gene complex is located within a 3 Mbp region within chromosome 6p21.
  • the HLA genes are highly polymorphic.
  • the HLA- A gene encodes class I MHC molecules.
  • Class I MHC molecules are transmembrane glycoproteins on the surface of all nucleated cells that present peptides from inside the cell.
  • the HLA-A*02:01 allele has been shown to be associated with a decreased risk of multiple sclerosis in cohorts of patients having multiple sclerosis compared to healthy controls (Brynedal et al., 2007 PLOS One 2(7): e664). These studies do not evaluate the role of different forms of MS.
  • the HLA-A*02:01 allele is more highly associated with RRMS than PPMS.
  • the odds ratio for HLA-A*02:01 in RRMS is 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, or 0.69.
  • the odds ratio for HLA-A*02:01 in PPMS is 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, or 0.85.
  • the LIX1L variant or HLA-A*02:01 allele is detected in a nucleic acid molecule by a method chosen from one or more of: nucleic acid hybridization assay, amplification-based assays (e.g., polymerase chain reaction (PCR)),
  • PCR polymerase chain reaction
  • PCR-RFLP assay real-time PCR, sequencing, screening analysis (including metaphase cytogenetic analysis by standard karyotype methods, FISH (e.g., break away FISH), spectral karyotyping or MFISH, comparative genomic hybridization), in situ
  • Additional exemplary methods include, traditional "direct probe” methods such as Southern blots or in situ hybridization (e.g., fluorescence in situ hybridization (FISH) and FISH plus SKY), and “comparative probe” methods such as comparative genomic hybridization (CGH), e.g., cDNA-based or oligonucleotide-based CGH, can be used.
  • FISH fluorescence in situ hybridization
  • CGH comparative genomic hybridization
  • the methods can be used in a wide variety of formats including, but not limited to, substrate (e.g., membrane or glass) bound methods or array-based approaches.
  • the evaluation methods include the probes/primers described herein.
  • nucleic acid molecules of the invention include those nucleic acid molecules which reside in genomic regions identified herein. Isolated nucleic acid molecules of the invention also include nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules that correspond to a marker of the invention and fragments of such nucleic acid molecules, e.g., those suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single- stranded or double- stranded; in certain embodiments the nucleic acid molecule is double-stranded DNA.
  • an “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an “isolated” nucleic acid molecule is free of sequences (such as protein-encoding sequences) which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kB, less than about 4 kB, less than about 3 kB, less than about 2 kB, less than about 1 kB, less than about 0.5 kB or less than about 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the nucleic acids are isolated from a e.g., blood sample or peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • nucleic acid molecule that is substantially free of cellular material includes preparations of nucleic acid molecule having less than about 30%, less than about 20%, less than about 10%, or less than about 5% (by dry weight) of other cellular material or culture medium.
  • nucleic acid molecule of the present invention can be isolated using standard molecular biology techniques and the sequence information in the database records described herein. Using all or a portion of such nucleic acid sequences, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • a nucleic acid molecule of the invention can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid molecules so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which has a nucleotide sequence complementary to the nucleotide sequence of a nucleic acid corresponding to a marker of the invention.
  • a nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex.
  • nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence, wherein the full length nucleic acid sequence comprises a marker of the invention.
  • nucleic acid molecules can be used, for example, as a probe or primer.
  • the probe/primer typically is used as one or more substantially purified oligonucleotides.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 7, at least about 15, at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1 kb, at least about 2 kb, at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 15 kb, at least about 20 kb, at least about 25 kb, at least about 30 kb, at least about 35 kb, at least about 40 kb, at least
  • Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts (e.g., mRNA) or genomic sequences corresponding to one or more markers of the invention.
  • the probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as part of a diagnostic test kit for identifying cells or tissues have the nucleic acid sequence of the markers described herein, such as by measuring presence or levels of a nucleic acid molecule in a sample of cells from a subject, e.g., detecting genomic DNA or mRNA levels.
  • labels, dyes, or labeled probes and/or primers are used to detect genomic DNA, amplified or unamplified mRNAs.
  • detection methods are appropriate based on the sensitivity of the detection method and the abundance of the target.
  • amplification may or may not be required prior to detection.
  • detection methods where mRNA amplification is preferred.
  • oligonucleotide probes or primers present in an amplification reaction are suitable for monitoring the amount of amplification product produced as a function of time.
  • probes having different single stranded versus double stranded character are used to detect the nucleic acid.
  • oligonucleotide probes or primers present in an amplification reaction are suitable for detecting the presence or absence of a particular polymorphism, with polymorphism specific probes only allowing for amplification when the polymorphism is present.
  • Probes include, but are not limited to, the 5'-exonuclease assay (e.g., TaqManTM) probes (see U.S. Pat. No. 5,538,848), stem-loop molecular beacons (see, e.g., U.S. Pat. Nos. 6,103,476 and
  • stemless or linear beacons see, e.g., WO 9921881, U.S. Pat. Nos. 6,485,901 and 6,649,349), peptide nucleic acid (PNA) Molecular Beacons (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, e.g. U.S. Pat. No. 6,329,144), non- FRET probes (see, e.g., U.S. Pat. No. 6,150,097), SunriseTM/AmplifluorBTM probes (see, e.g., U.S. Pat. No.
  • one or more of the primers in an amplification reaction can include a label.
  • different probes or primers comprise detectable labels that are distinguishable from one another.
  • a nucleic acid, such as the probe or primer may be labeled with two or more
  • a label is attached to one or more probes and has one or more of the following properties: (i) provides a detectable signal; (ii) interacts with a second label to modify the detectable signal provided by the second label, e.g., FRET (Fluorescent Resonance Energy Transfer); (iii) stabilizes hybridization, e.g., duplex formation; and (iv) provides a member of a binding complex or affinity set, e.g., affinity, antibody- antigen, ionic complexes, hapten-ligand (e.g., biotin-avidin).
  • use of labels can be accomplished using any one of a large number of known techniques employing known labels, linkages, linking groups, reagents, reaction conditions, and analysis and purification methods.
  • Labels include, but are not limited to: light-emitting, light-scattering, and light- absorbing compounds which generate or quench a detectable fluorescent,
  • Fluorescent reporter dyes useful as labels include, but are not limited to, fluoresceins (see, e.g. U.S. Pat. Nos. 5,188,934, 6,008,379, and
  • the fluorescent label is selected from SYBR-Green, 6-carboxyfluorescein ("FAM”), TET, ROX, VICTM, and JOE.
  • FAM 6-carboxyfluorescein
  • TET TET
  • ROX ROX
  • VICTM VICTM
  • JOE JOE
  • labels are different fluorophores capable of emitting light at different, spectrally-resolvable wavelengths (e.g., 4-differently colored
  • fluorophores certain such labeled probes are known in the art and described above, and in U.S. Pat. No. 6,140,054.
  • a dual labeled fluorescent probe that includes a reporter fluorophore and a quencher fluorophore is used in some embodiments. It will be appreciated that pairs of fluorophores are chosen that have distinct emission spectra so that they can be easily distinguished.
  • labels are hybridization-stabilizing moieties which serve to enhance, stabilize, or influence hybridization of duplexes, e.g., intercalators and intercalating dyes (including, but not limited to, ethidium bromide and SYBR-Green), minor-groove binders, and cross-linking functional groups (see, e.g., Blackburn et al., eds. "DNA and RNA Structure” in Nucleic Acids in Chemistry and Biology (1996)).
  • intercalators and intercalating dyes including, but not limited to, ethidium bromide and SYBR-Green
  • minor-groove binders include, but not limited to, ethidium bromide and SYBR-Green
  • cross-linking functional groups see, e.g., Blackburn et al., eds. "DNA and RNA Structure” in Nucleic Acids in Chemistry and Biology (1996)).
  • the invention further encompasses nucleic acid molecules that are substantially homologous to the genes or gene products described herein such that they are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or greater.
  • the invention further encompasses nucleic acid molecules that are substantially homologous to the genes or gene products described herein such that they differ by only or at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, at least
  • an isolated nucleic acid molecule of the invention is at least 7, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 550, at least 650, at least 700, at least 800, at least 900, at least 1000, at least 1200, at least 1400, at least 1600, at least 1800, at least 2000, at least 2200, at least 2400, at least 2600, at least 2800, at least 3000, at least 3500, at least 4000, at least 4500, or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule corresponding to a marker of the invention or to a nucle
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% identical to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in e.g., sections 6.3.1-6.3.6 of Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).
  • Another, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50-65°C.
  • the methods described herein can also include molecular beacon nucleic acid molecules having at least one region which is complementary to a nucleic acid molecule of the invention, such that the molecular beacon is useful for quantitating the presence of the nucleic acid molecule of the invention in a sample.
  • a "molecular beacon" nucleic acid is a nucleic acid molecule comprising a pair of complementary regions and having a fluorophore and a fluorescent quencher associated therewith. The fluorophore and quencher are associated with different portions of the nucleic acid in such an orientation that when the complementary regions are annealed with one another, fluorescence of the fluorophore is quenched by the quencher.
  • the invention also includes methods of sequencing nucleic acids.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence at least a portion of a LIX1L variant or HLA-A*02:01 allele.
  • the LIX1L variant or HLA-A*02:01 allele sequence is compared to a corresponding reference (control) sequence.
  • the sequence of the LIX1L variant or HLA-A*02:01 allele nucleic acid molecule is determined by a method that includes one or more of: hybridizing an oligonucleotide, e.g., an allele specific oligonucleotide for one alteration described herein to said nucleic acid; hybridizing a primer, or a primer set (e.g., a primer pair), that amplifies a region comprising the LIX1L variant polymorphism(s) or HLA-A*02:01 allele;
  • amplifying e.g., specifically amplifying, a region comprising the LIX1L variant
  • the sequence is determined by a method that comprises one or more of: determining the nucleotide sequence from an individual nucleic acid molecule, e.g., where a signal corresponding to the sequence is derived from a single molecule as opposed, e.g., from a sum of signals from a plurality of clonally expanded molecules;
  • pyrosequencing real-time sequencing comprising imaging the continuous incorporation of dye-labeling nucleotides during DNA synthesis; nanopore sequencing; sequencing by hybridization; nano-transistor array based sequencing; polony sequencing; scanning tunneling microscopy (STM) based sequencing; or nanowire-molecule sensor based sequencing.
  • STM scanning tunneling microscopy
  • sequencing reactions include those based on techniques developed by Maxam and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al. (1977) Proc. Nat. Acad. Sci 74:5463). Any of a variety of automated sequencing procedures can be utilized when performing the assays (Biotechniques (1995) 19:448), including sequencing by mass spectrometry (see, for example, U.S. Patent Number 5,547,835 and international patent application Publication Number WO 94/16101, entitled DNA Sequencing by Mass
  • Sequencing of nucleic acid molecules can also be carried out using next-generation sequencing (NGS).
  • Next-generation sequencing includes any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules or clonally expanded proxies for individual nucleic acid molecules in a highly parallel fashion (e.g., greater than 10 5 molecules are sequenced simultaneously).
  • the relative abundance of the nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences in the data generated by the sequencing experiment.
  • Next generation sequencing methods are known in the art, and are described, e.g., in Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46, incorporated herein by reference.
  • next-generation sequencing allows for the determination of the nucleotide sequence of an individual nucleic acid molecule (e.g., Helicos Biosciences' HeliScope Gene Sequencing system, and Pacific Biosciences' PacBio RS system).
  • an individual nucleic acid molecule e.g., Helicos Biosciences' HeliScope Gene Sequencing system, and Pacific Biosciences' PacBio RS system.
  • the sequencing method determines the nucleotide sequence of clonally expanded proxies for individual nucleic acid molecules (e.g., the Solexa sequencer, Illumina Inc., San Diego, Calif; 454 Life Sciences (Branford, Conn.), and Ion Torrent), e.g., massively parallel short-read sequencing (e.g., the Solexa sequencer, Illumina Inc., San Diego, Calif.), which generates more bases of sequence per sequencing unit than other sequencing methods that generate fewer but longer reads.
  • Other methods or machines for next-generation sequencing include, but are not limited to, the sequencers provided by 454 Life Sciences (Branford, Conn.), Applied Biosystems (Foster City, Calif.; SOLiD
  • Platforms for next-generation sequencing include, but are not limited to, Roche/454' s
  • GS Genome Sequencer
  • GA Genome Analyzer
  • NGS technologies can include one or more of steps, e.g., template preparation, sequencing and imaging, and data analysis.
  • Methods for template preparation can include steps such as randomly breaking nucleic acids ⁇ e.g., genomic DNA or cDNA) into smaller sizes and generating sequencing templates ⁇ e.g., fragment templates or mate-pair templates).
  • the spatially separated templates can be attached or immobilized to a solid surface or support, allowing massive amounts of sequencing reactions to be performed simultaneously.
  • Types of templates that can be used for NGS reactions include, e.g., clonally amplified templates originating from single DNA molecules, and single DNA molecule templates.
  • Methods for preparing clonally amplified templates include, e.g., emulsion PCR (emPCR) and solid-phase amplification.
  • emPCR emulsion PCR
  • solid-phase amplification emulsion PCR
  • EmPCR can be used to prepare templates for NGS.
  • a library of nucleic acid fragments is generated, and adapters containing universal priming sites are ligated to the ends of the fragment.
  • the fragments are then denatured into single strands and captured by beads. Each bead captures a single nucleic acid molecule.
  • a large amount of templates can be attached or immobilized in a polyacrylamide gel on a standard microscope slide (e.g., Polonator), chemically
  • NGS reaction can be performed.
  • an amino-coated glass surface e.g., Life/APG; Polonator
  • PTP PicoTiterPlate
  • Solid-phase amplification can also be used to produce templates for NGS.
  • forward and reverse primers are covalently attached to a solid support.
  • the surface density of the amplified fragments is defined by the ratio of the primers to the templates on the support.
  • Solid-phase amplification can produce hundreds of millions spatially separated template clusters (e.g., Illumina/Solexa). The ends of the template clusters can be hybridized to universal sequencing primers for NGS reactions.
  • MDA Multiple Displacement Amplification
  • Template amplification methods such as PCR can be coupled with NGS platforms to target or enrich specific regions of the genome (e.g., exons).
  • exemplary template enrichment methods include, e.g., microdroplet PCR technology (Tewhey R. et al., Nature Biotech. 2009, 27: 1025-1031), custom-designed oligonucleotide microarrays (e.g.,
  • MIPs molecular inversion probes
  • Single-molecule templates are another type of templates that can be used for NGS reaction.
  • Spatially separated single molecule templates can be immobilized on solid supports by various methods.
  • individual primer molecules are covalently attached to the solid support.
  • Adapters are added to the templates and templates are then hybridized to the immobilized primers.
  • single-molecule templates are covalently attached to the solid support by priming and extending single-stranded, single- molecule templates from immobilized primers. Universal primers are then hybridized to the templates.
  • single polymerase molecules are attached to the solid support, to which primed templates are bound.
  • Isolated nucleic acid samples can be fragmented or sheared by practicing routine techniques.
  • genomic DNA can be fragmented by physical shearing methods, enzymatic cleavage methods, chemical cleavage methods, and other methods well known to those skilled in the art.
  • a nucleic acid library is prepared for nucleic acid sequencing.
  • the nucleic acid library can contain all or substantially all of the complexity of the genome. The term "substantially all" in this context refers to the possibility that there can in practice be some unwanted loss of genome complexity during the initial steps of the procedure.
  • nucleic acid library is a portion of the genome, i.e., where the complexity of the genome is reduced by design.
  • any selected portion of the genome can be used with the methods described herein.
  • the entire exome or a subset thereof is isolated.
  • Methods can further include isolating a nucleic acid sample to provide a library ⁇ e.g., a nucleic acid library).
  • the nucleic acid sample includes whole genomic, subgenomic fragments, or both.
  • the isolated nucleic acid samples can be used to prepare nucleic acid libraries.
  • the methods featured in the invention further include isolating a nucleic acid sample to provide a library ⁇ e.g., a nucleic acid library as described herein). Protocols for isolating and preparing libraries from whole genomic or subgenomic fragments are known in the art ⁇ e.g., Illumina's genomic DNA sample preparation kit).
  • the genomic or subgenomic DNA fragment is isolated from a subject's sample.
  • the nucleic acid sample used to generate the nucleic acid library is less than 5, less than 1 microgram, less than 500 ng, less than 200 ng, less than 100 ng, less than 50 ng or less than 20 ng (e.g., 10 ng or less).
  • the nucleic acid sample used to generate the library includes RNA or cDNA derived from RNA.
  • the RNA includes total cellular RNA.
  • certain abundant RNA sequences e.g., ribosomal RNAs
  • the poly(A)-tailed mRNA fraction in the total RNA preparation has been enriched.
  • the cDNA is produced by random-primed cDNA synthesis methods.
  • the cDNA synthesis is initiated at the poly(A) tail of mature mRNAs by priming by oligo(dT)-containing oligonucleotides. Methods for depletion, poly(A) enrichment, and cDNA synthesis are well known to those skilled in the art.
  • the method can further include amplifying the nucleic acid sample (e.g., DNA or RNA sample) by specific or non-specific nucleic acid amplification methods that are well known to those skilled in the art.
  • the nucleic acid sample is amplified, e.g., by whole-genome amplification methods such as random-primed strand- displacement amplification.
  • the nucleic acid sample is fragmented or sheared by physical or enzymatic methods and ligated to synthetic adapters, size-selected (e.g., by preparative gel electrophoresis) and amplified (e.g., by PCR).
  • the fragmented and adapter-ligated group of nucleic acids is used without explicit size selection or amplification prior to hybrid selection.
  • the isolated DNA (e.g., the genomic DNA) is fragmented or sheared.
  • the library includes less than 50% of genomic DNA, such as a subtraction of genomic DNA that is a reduced representation or a defined portion of a genome, e.g., that has been subfractionated by other means.
  • the library includes all or substantially all genomic DNA.
  • the library includes less than 50% of genomic DNA, such as a subfraction of genomic DNA that is a reduced representation or a defined portion of a genome, e.g., that has been subfractionated by other means.
  • the library includes all or substantially all genomic DNA.
  • Protocols for isolating and preparing libraries from whole genomic or subgenomic fragments are known in the art (e.g., Illumina's genomic DNA sample preparation kit).
  • Alternative DNA shearing methods can be more automatable and/or more efficient (e.g. , with degraded FFPE samples).
  • Alternatives to DNA shearing methods can also be used to avoid a ligation step during library preparation.
  • the methods described herein can be performed using a small amount of nucleic acids, e.g. , when the amount of source DNA is limiting (e.g. , even after whole-genome amplification).
  • the nucleic acid comprises less than about 5 ⁇ g, 4 ⁇ g, 3 ⁇ g, 2 ⁇ g, 1 ⁇ g, 0.8 ⁇ g, 0.7 ⁇ g, 0.6 ⁇ g, 0.5 ⁇ g, or 400 ng, 300 ng, 200 ng, 100 ng, 50 ng, or 20 ng or less of nucleic acid sample.
  • 500 ng of hybridization-ready nucleic acids one typically begins with 3 ⁇ g of genomic DNA.
  • a library is generated using DNA (e.g., genomic DNA) from a sample tissue, and a corresponding library is generated with RNA (or cDNA) isolated from the same sample tissue.
  • DNA e.g., genomic DNA
  • RNA or cDNA
  • Exemplary sequencing and imaging methods for NGS include, but are not limited to, cyclic reversible termination (CRT), sequencing by ligation (SBL), single-molecule addition (pyrosequencing), and real-time sequencing.
  • CRT cyclic reversible termination
  • SBL sequencing by ligation
  • pyrosequencing single-molecule addition
  • CRT uses reversible terminators in a cyclic method that minimally includes the steps of nucleotide incorporation, fluorescence imaging, and cleavage.
  • a DNA polymerase incorporates a single fluorescently modified nucleotide corresponding to the complementary nucleotide of the template base to the primer.
  • DNA synthesis is terminated after the addition of a single nucleotide and the unincorporated nucleotides are washed away. Imaging is performed to determine the identity of the incorporated labeled nucleotide. Then in the cleavage step, the terminating/inhibiting group and the fluorescent dye are removed.
  • Exemplary NGS platforms using the CRT method include, but are not limited to,
  • G Illumina/Solexa Genome Analyzer
  • TIRF total internal reflection fluorescence
  • Hcos BioSciences/HeliScope which uses the single-molecule template method coupled with the one-color CRT method detected by TIRF.
  • SBL uses DNA ligase and either one-base-encoded probes or two-base-encoded probes for sequencing.
  • a fluorescently labeled probe is hybridized to its complementary sequence adjacent to the primed template.
  • DNA ligase is used to ligate the dye-labeled probe to the primer. Fluorescence imaging is performed to determine the identity of the ligated probe after non-ligated probes are washed away.
  • the fluorescent dye can be removed by using cleavable probes to regenerate a 5'-P0 4 group for subsequent ligation cycles.
  • a new primer can be hybridized to the template after the old primer is removed.
  • Exemplary SBL platforms include, but are not limited to,
  • Life/APG/SOLiD support oligonucleotide ligation detection, which uses two-base-encoded probes.
  • Pyrosequencing method is based on detecting the activity of DNA polymerase with another chemiluminescent enzyme.
  • the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step.
  • the template DNA is immobile, and solutions of A, C, G, and T nucleotides are sequentially added and removed from the reaction. Light is produced only when the nucleotide solution complements the first unpaired base of the template.
  • the sequence of solutions which produce chemiluminescent signals allows the determination of the sequence of the template.
  • Exemplary pyrosequencing platforms include, but are not limited to, Roche/454, which uses DNA templates prepared by emPCR with 1-2 million beads deposited into PTP wells.
  • Real-time sequencing involves imaging the continuous incorporation of dye-labeled nucleotides during DNA synthesis.
  • exemplary real-time sequencing platforms include, but are not limited to, Pacific Biosciences platform, which uses DNA polymerase molecules attached to the surface of individual zero-mode waveguide (ZMW) detectors to obtain sequence information when phospholinked nucleotides are being incorporated into the growing primer strand; Life/VisiGen platform, which uses an engineered DNA polymerase with an attached fluorescent dye to generate an enhanced signal after nucleotide
  • FRET fluorescence resonance energy transfer
  • NGS nanopore sequencing
  • sequencing by hybridization nano-transistor array based sequencing
  • polony sequencing polony sequencing
  • STM scanning tunneling microscopy
  • Nanopore sequencing involves electrophoresis of nucleic acid molecules in solution through a nano- scale pore which provides a highly confined space within which single- nucleic acid polymers can be analyzed. Exemplary methods of nanopore sequencing are described, e.g., in Branton D. et al, Nat Biotechnol. 2008; 26(10): 1146-53.
  • Sequencing by hybridization is a non-enzymatic method that uses a DNA microarray.
  • a single pool of DNA is fluorescently labeled and hybridized to an array containing known sequences.
  • Hybridization signals from a given spot on the array can identify the DNA sequence.
  • the binding of one strand of DNA to its complementary strand in the DNA double-helix is sensitive to even single -base mismatches when the hybrid region is short or is specialized mismatch detection proteins are present.
  • Exemplary methods of sequencing by hybridization are described, e.g., in Hanna G.J. et ah, J. Clin. Microbiol. 2000; 38 (7): 2715-21; and Edwards J.R. et al, Mut. Res. 2005; 573 (1-2): 3-12.
  • Polony sequencing is based on polony amplification and sequencing -by- synthesis via multiple single-base-extensions (FISSEQ).
  • Polony amplification is a method to amplify DNA in situ on a polyacrylamide film. Exemplary polony sequencing methods are described, e.g. , in US Patent Application Publication No. 2007/0087362.
  • Nano-transistor array based devices such as Carbon NanoTube Field Effect
  • CNTFET CNTFET Transistor
  • DNA molecules are stretched and driven over nanotubes by micro-fabricated electrodes. DNA molecules sequentially come into contact with the carbon nanotube surface, and the difference in current flow from each base is produced due to charge transfer between the DNA molecule and the nanotubes. DNA is sequenced by recording these differences.
  • Exemplary Nano- transistor array based sequencing methods are described, e.g. , in U.S. Patent Application Publication No. 2006/0246497.
  • Scanning tunneling microscopy can also be used for NGS.
  • STM uses a piezoelectric-controlled probe that performs a raster scan of a specimen to form images of its surface.
  • STM can be used to image the physical properties of single DNA molecules, e.g. , generating coherent electron tunneling imaging and spectroscopy by integrating scanning tunneling microscope with an actuator-driven flexible gap.
  • Exemplary sequencing methods using STM are described, e.g. , in U.S. Patent Application Publication No. 2007/0194225.
  • a molecular-analysis device which is comprised of a nanowire-molecule sensor can also be used for NGS. Such device can detect the interactions of the nitrogenous material disposed on the nanowires and nucleic acid molecules such as DNA.
  • a molecule guide is configured for guiding a molecule near the molecule sensor, allowing an interaction and subsequent detection. Exemplary sequencing methods using nanowire-molecule sensor are described, e.g. , in U.S. Patent Application Publication No. 2006/0275779.
  • Double ended sequencing methods can be used for NGS.
  • Double ended sequencing uses blocked and unblocked primers to sequence both the sense and antisense strands of DNA. Typically, these methods include the steps of annealing an unblocked primer to a first strand of nucleic acid; annealing a second blocked primer to a second strand of nucleic acid; elongating the nucleic acid along the first strand with a polymerase; terminating the first sequencing primer; deblocking the second primer; and elongating the nucleic acid along the second strand.
  • Exemplary double ended sequencing methods are described, e.g., in U.S. Patent Serial No. 7,244,567.
  • NGS reads After NGS reads have been generated, they can be aligned to a known reference sequence or assembled de novo.
  • identifying genetic variations such as single-nucleotide polymorphism and structural variants in a sample can be accomplished by aligning NGS reads to a reference sequence (e.g., a wild-type sequence).
  • a reference sequence e.g., a wild-type sequence.
  • de novo assemblies are described, e.g., in Warren R. et ah,
  • Sequence alignment or assembly can be performed using read data from one or more NGS platforms, e.g., mixing Roche/454 and Illumina/Solexa read data.
  • Amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • nucleic acid sequence based amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification
  • NASBA RNA amplification technique
  • Nucleic acid primers useful for amplifying sequences from a gene locus are able to specifically hybridize with a portion of the gene locus that flank a target region of said locus, said target region being altered in subjects having PPMS. Examples of such target regions are polymorphisms in the LIXIL gene, e.g., nucleic acids encoding the R114H, V173L, S286C, and L322F mutations in the LIXIL protein sequence.
  • nucleic acid primer useful for amplifying sequences from the gene or locus including surrounding regions.
  • Such primers are preferably complementary to, and hybridize specifically to nucleic acid sequences in the gene locus.
  • Particular primers are able to specifically hybridize with a portion of the gene locus that flank a target region of said locus, said target region being altered in in subjects having PPMS.
  • the invention also relates to a nucleic acid primer, said primer being
  • primers of this invention are specific for altered sequences in the gene.
  • the detection of an amplification product indicates the presence of an alteration in the gene locus.
  • the absence of amplification product indicates that the specific alteration is not present in the sample.
  • the invention also concerns the use of a nucleic acid primer or a pair of nucleic acid primers as described above in a method of detecting the presence of or predisposition to Primary Progressive MS in a subject.
  • Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid sequences that serve to detect nucleic acid sequence alteration(s).
  • a particular detection technique involves the use of a nucleic acid probe specific for wild-type or altered gene or RNA, followed by the detection of the presence of a hybrid.
  • the probe may be in suspension or immobilized on a substrate or support (as in nucleic acid array or chips technologies).
  • the probe is typically labeled to facilitate detection of hybrids.
  • a particular embodiment of this invention comprises contacting the sample from the subject with a nucleic acid probe specific for an altered gene locus, and assessing the formation of a hybrid.
  • the method comprises contacting simultaneously the sample with a set of probes that are specific, respectively, for the wild type gene locus and for various altered forms thereof.
  • a probe refers to a polynucleotide sequence which is complementary to and capable of specific hybridization with a (target portion of a) gene or RNA, and which is suitable for detecting
  • polynucleotide polymorphisms associated with alleles which predispose to or are associated with PPMS are associated with PPMS.
  • the method of the invention employs a nucleic acid probe specific for an altered (e.g., a mutated) gene or RNA, i.e., a nucleic acid probe that specifically hybridizes to said altered gene or RNA and essentially does not hybridize to a gene or RNA lacking said alteration.
  • a nucleic acid probe specific for an altered (e.g., a mutated) gene or RNA i.e., a nucleic acid probe that specifically hybridizes to said altered gene or RNA and essentially does not hybridize to a gene or RNA lacking said alteration.
  • Specificity indicates that hybridization to the target sequence generates a specific signal which can be distinguished from the signal generated through non-specific hybridization. Perfectly complementary sequences are preferred to design probes according to this invention. It should be understood, however, that certain mismatch may be tolerated, as long as the specific signal may be distinguished from non-specific hybridization.
  • probes are nucleic acid sequences complementary to a target portion of the genomic region including the LIX1L gene locus carrying one or more of polymorphisms, e.g., nucleic acids encoding the R114H, V173L, S286C, and L322F mutations in the LIX1L protein sequence.
  • the sequence of the probes can be derived from the sequences of the gene as provided in the present application. Nucleotide substitutions may be performed, as well as chemical modifications of the probe, as described herein.
  • the invention also concerns the use of a nucleic acid probe as described above in a method of detecting the presence of or predisposition to PPMS or an associated disorder in a subject.
  • the LIX1 variant or HLA-A*02:01expression level can also be assayed.
  • LIX1 variant or HLA-A*02:01expression can be assessed by any of a wide variety of methods for detecting expression of a transcribed molecule or protein. Non-limiting examples of such methods include immunological methods for detection of secreted, cell-surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.
  • activity of a particular gene is characterized by a measure of gene transcript (e.g., mRNA), by a measure of the quantity of translated protein, or by a measure of gene product activity.
  • LIX1 variant or HLA-A*02:01 expression can be monitored in a variety of ways, including by detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can involve quantification of the level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can be a qualitative assessment of the level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from the context.
  • mRNA or cDNA made therefrom Methods of detecting and/or quantifying the LIX1 variant or HLA-A*02:01 gene transcript (mRNA or cDNA made therefrom) using nucleic acid hybridization techniques are known to those of skill in the art (see Sambrook et al. supra).
  • one method for evaluating the presence, absence, or quantity of cDNA involves a Southern transfer as described above. Briefly, the mRNA is isolated (e.g., using an acid guanidinium-phenol-chloroform extraction method, Sambrook et al. supra.) and reverse transcribed to produce cDNA. The cDNA is then optionally digested and run on a gel in buffer and transferred to membranes. Hybridization is then carried out using the nucleic acid probes specific for the LIX1 variant or HLA-A*02:01cDNA, e.g., using the probes and primers described herein.
  • LIX1 variant or HLA-A*02:01expression is assessed by preparing genomic DNA or mRNA/cDNA (i.e., a transcribed polynucleotide) from cells in a subject sample, and by hybridizing the genomic DNA or mRNA/cDNA with a reference polynucleotide which is a complement of a polynucleotide comprising the LIX1 variant or HLA-A*02:01, and fragments thereof.
  • cDNA can, optionally, be amplified using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide.
  • Expression of LIX1 variant or HLA- A*02:01 can likewise be detected using quantitative PCR (QPCR) to assess the level of LIX1 variant or HLA-A*02:01expression.
  • the activity or level of a LIX1 variant or HLA-A*02:01polypeptide can also be detected and/or quantified by detecting or quantifying the expressed polypeptide.
  • the LIX1 variant or HLA-A*02:01polypeptide can be detected and quantified by any of a number of means known to those of skill in the art.
  • analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), Immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • ELISAs enzyme-linked immunosorbent assays
  • immunofluorescent assays Western blotting, immunohistochemistry (IHC) and the like.
  • IHC immunohistochemistry
  • Another agent for detecting a LIX1 variant or HLA-A*02:01polypeptide is an antibody molecule capable of binding to a polypeptide corresponding to a marker of the invention, e.g., an antibody with a detectable label.
  • a marker of the invention e.g., an antibody with a detectable label.
  • Techniques for generating antibodies are described herein.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • the antibody is labeled, e.g., a radio-labeled
  • an antibody derivative e.g., an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair ⁇ e.g., biotin-streptavidin ⁇
  • an antibody fragment e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc. which binds specifically with a LIX1 variant or HLA-A*02:01protein, is used.
  • LIX1 variant or HLA-A*02:01polypeptides from cells can be isolated using techniques that are known to those of skill in the art.
  • the protein isolation methods employed can, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
  • Western blot (immunoblot) analysis is used to detect and quantify the presence of a polypeptide in the sample.
  • the polypeptide is detected using an immunoassay.
  • an immunoassay is an assay that utilizes an antibody to specifically bind to the analyte.
  • the immunoassay is thus characterized by detection of specific binding of a polypeptide to an anti-antibody as opposed to the use of other physical or chemical properties to isolate, target, and quantify the analyte.
  • the LIX1 variant or HLA-A*02:01polypeptide is detected and/or quantified using any of a number of immunological binding assays (see, e.g., U.S. Patent Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • kits are any manufacture (e.g., a package or container) comprising at least one reagent, e.g., a probe, primers, or an antibody, for specifically detecting a marker of the invention, the manufacture being promoted, distributed, or sold as a unit for performing the methods of the present invention.
  • reagent e.g., a probe, primers, or an antibody
  • a biomarker comprising one or more of the LIX1L polymorphisms described herein, e.g., Rl 14H, V173L, S286C, and L322F, can be selected such that a positive result is obtained in at least about 20%, at least about 40%, at least about 60%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or in 100% of subjects afflicted with multiple sclerosis, of the corresponding sub-type, of primary progressive nature.
  • kits and methods for assaying serum in a sample e.g., a sample obtained from a subject.
  • a sample e.g., a sample obtained from a subject.
  • kits and methods are substantially the same as those described above, except that, where necessary, the kits and methods are adapted for use with certain types of samples.
  • the sample is a serum sample, it can be necessary to adjust the ratio of compounds in the kits of the invention, or the methods used.
  • Such methods are well known in the art and within the skill of the ordinary artisan.
  • the invention thus includes a kit for evaluating MS, e.g., primary progressive MS in a subject (e.g., in a sample such as a serum sample).
  • the kit can comprise one or more reagents capable of identifying one or more of the genetic polymorphisms in the LIX1L gene described herein, e.g., binding specifically with a nucleic acid corresponding one or more of the genetic polymorphisms in the LIX1L gene described herein.
  • Suitable reagents for binding with a nucleic acid include complementary nucleic acids.
  • the nucleic acid reagents can include oligonucleotides (labeled or non-labeled) fixed to a substrate, labeled oligonucleotides not bound with a substrate, pairs of PCR primers, molecular beacon probes, and the like.
  • the kit of the invention can optionally comprise additional components useful for performing the methods of the invention.
  • the kit can comprise fluids (e.g., SSC buffer) suitable for annealing complementary nucleic acids or for binding an antibody with a protein with which it specifically binds, one or more sample
  • an instructional material which describes performance of a method of the invention, a reference sample for comparison of amplification levels of the biomarkers described herein, and the like.
  • kits of the invention can comprise a reagent useful for determining protein level or protein activity of a marker.
  • MS Multiple sclerosis
  • Patients having MS can be identified by clinical criteria establishing a diagnosis of clinically definite MS as defined by Poser et al., Ann. Neurol. 13:22 ', 1983. Briefly, an individual with clinically definite MS has had two attacks and clinical evidence of either two lesions or clinical evidence of one lesion and paraclinical evidence of another, separate lesion. Definite MS may also be diagnosed by evidence of two attacks and oligoclonal bands of IgG in cerebrospinal fluid or by combination of an attack, clinical evidence of two lesions and oligoclonal band of IgG in cerebrospinal fluid.
  • McDonald criteria can also be used to diagnose MS. (McDonald et al., 2001, Recommended diagnostic criteria for Multiple sclerosis: guidelines from the International Panel on the Diagnosis of Multiple Sclerosis, Ann Neurol 50: 121-127).
  • the McDonald criteria include the use of MRI evidence of CNS impairment over time to be used in diagnosis of MS, in the absence of multiple clinical attacks.
  • Effective treatment of multiple sclerosis may be evaluated in several different ways. The following parameters can be used to gauge effectiveness of treatment. Two exemplary criteria include: EDSS (extended disability status scale), and appearance of exacerbations on MRI (magnetic resonance imaging).
  • the EDSS is a means to grade clinical impairment due to MS (Kurtzke,
  • Exacerbations are defined as the appearance of a new symptom that is attributable to MS and accompanied by an appropriate new neurologic abnormality (IFNB MS Study Group, supra). In addition, the exacerbation must last at least 24 hours and be preceded by stability or improvement for at least 30 days. Briefly, patients are given a standard neurological examination by clinicians. Exacerbations are mild, moderate, or severe according to changes in a Neurological Rating Scale (Sipe et al., Neurology 34: 1368, 1984). An annual exacerbation rate and proportion of exacerbation-free patients are determined.
  • Treatment can be deemed to be effective using a clinical measure if there is a statistically significant difference in the rate or proportion of exacerbation-free or relapse- free patients between the treated group and the placebo group for either of these measurements.
  • time to first exacerbation and exacerbation duration and severity may also be measured.
  • a measure of effectiveness as therapy in this regard is a statistically significant difference in the time to first exacerbation or duration and severity in the treated group compared to control group.
  • An exacerbation-free or relapse-free period of greater than one year, 18 months, or 20 months is particularly noteworthy.
  • Clinical measurements include the relapse rate in one and two-year intervals, and a change in EDSS, including time to progression from baseline of 1.0 unit on the EDSS that persists for six months. On a Kaplan-Meier curve, a delay in sustained progression of disability shows efficacy. Other criteria include a change in area and volume of T2 images on MRI, and the number and volume of lesions determined by gadolinium enhanced images.
  • MRI can be used to measure active lesions using gadolinium-DTPA-enhanced imaging (McDonald et al., Ann. Neurol. 36: 14, 1994) or the location and extent of lesions using T2-weighted techniques. Briefly, baseline MRIs are obtained. The same imaging plane and patient position are used for each subsequent study. Positioning and imaging sequences can be chosen to maximize lesion detection and facilitate lesion tracing. The same positioning and imaging sequences can be used on subsequent studies. The presence, location and extent of MS lesions can be determined by radiologists. Areas of lesions can be outlined and summed slice by slice for total lesion area.
  • Exemplary symptoms associated with multiple sclerosis which can be treated with the methods described herein or managed using symptom management therapies, include: optic neuritis, diplopia, nystagmus, ocular dysmetria, internuclear opthalmoplegia, movement and sound phosphenes, afferent pupillary defect, paresis, monoparesis, paraparesis, hemiparesis, quadraparesis, plegia, paraplegia, hemiplegia, tetraplegia, quadraplegia, spasticity, dysarthria, muscle atrophy, spasms, cramps, hypotonia, clonus, myoclonus, myokymia, restless leg syndrome, footdrop, dysfunctional reflexes, paraesthesia, anaesthesia, neuralgia, neuropathic and neurogenic pain, l'hermitte's, proprioceptive dysfunction, trigeminal neuralgia, ataxia, intention tre
  • symptoms of MS comprise optic neuritis, diplopia, nystagmus, ocular dysmetria, internuclear opthalmoplegia, movement and sound phosphenes, afferent pupillary defect, paresis, monoparesis, paraparesis, hemiparesis, quadraparesis, plegia, paraplegia, hemiplegia, tetraplegia, quadraplegia, spasticity, dysarthria, muscle atrophy, spasms, cramps, hypotonia, clonus, myoclonus, myokymia, restless leg syndrome, footdrop, dysfunctional reflexes, paraesthesia, anaesthesia, neuralgia, neuropathic and neurogenic pain, l'hermitte's, proprioceptive dysfunction, trigeminal neuralgia, ataxia, intention tremor, dysmetria, vestibular ataxia, vertigo, speech ataxia
  • MS relapsing-remitting MS
  • PPMS Primary-progressive MS
  • SPMS Secondary- progressive MS
  • PRMS progressive-relapsing
  • malignant MS defined as a swift and relentless decline resulting in significant disability or even death shortly after disease onset. This decline may be arrested or decelerated by determining the likelihood of the patient to respond to a therapy early in the therapeutic regime and switching the patient to an agent that they have the highest likelihood of responding to.
  • Treatment refers to the administration of an MS therapeutic agent, alone or in combination with one or more symptom management agents, to a subject, e.g., an MS patient, to impede progression of multiple sclerosis, to induce remission, to extend the expected survival time of the subject and or reduce the need for medical interventions (e.g., hospitalizations).
  • treatment can include, but is not limited to, inhibiting or reducing one or more symptoms such as numbness, tingling, muscle weakness; reducing relapse rate, reducing size or number of sclerotic lesions; inhibiting or retarding the development of new lesions; prolonging survival, or prolonging progression-free survival, and/or enhanced quality of life.
  • prevention contemplate an action that occurs before a subject begins to suffer from the a multiple sclerosis relapse and/or which inhibits or reduces the severity of the disease.
  • the terms “manage,” “managing” and “management” encompass preventing the progression of MS symptoms in a patient who has already suffered from the disease, and/or lengthening the time that a patient who has suffered from MS remains in remission. The terms encompass modulating the threshold, development and/or duration of MS, or changing the way that a patient responds to the disease.
  • a “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or management of multiple sclerosis, or to delay or minimize one or more symptoms associated with MS.
  • a therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of MS.
  • the term "therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the disease, or enhances the therapeutic efficacy of another therapeutic agent.
  • a prophylactically effective amount of a compound is an amount sufficient to prevent relapse of MS, or one or more symptoms associated with the disease, or prevent its recurrence.
  • a prophylactically effective amount of a compound means an amount of the compound, alone or in combination with other therapeutic agents, which provides a prophylactic benefit in the prevention of MS relapse.
  • the term "prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
  • the term "patient” or “subject” refers to an animal, typically a human (i.e., a male or female of any age group, e.g., a pediatric patient (e.g., infant, child, adolescent) or adult patient (e.g., young adult, middle-aged adult or senior adult) or other mammal, such as a primate (e.g., cynomolgus monkey, rhesus monkey), that will be or has been the object of treatment, observation, and/or experiment.
  • a primate e.g., cynomolgus monkey, rhesus monkey
  • the subject has PPMS. In other embodiments, the MS subject has at least one newly developed lesion. In other embodiment, the MS subject has at least one pre-existing lesion. In other embodiments, the subject has a baseline EDSS score of 1.5 to 7.
  • the methods described herein permit one of skill in the art to identify a monotherapy that an MS patient is most likely to respond to, thus eliminating the need for administration of multiple therapies to the patient to ensure that a therapeutic effect is observed.
  • combination treatment of an individual with MS is contemplated.
  • the MS therapy can be administered in combination with one or more additional therapies to treat and/or reduce the symptoms of MS described herein, particularly to treat patients with moderate to severe disability (e.g., EDSS score of 5.5 or higher).
  • the pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other additional therapies or therapeutic agents.
  • each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • the additional therapeutic agent utilized in this combination can be administered together in a single composition or administered separately in different compositions.
  • the particular combination to employ in a regimen will take into account compatibility of the pharmaceutical composition with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved.
  • it is expected that additional therapeutic agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
  • Such agents include, but are not limited to, dialkyl fumarates (e.g. , DMF or others of Formula A herein), Beta interferons (e.g., Avonex®, Rebif®, Betaseron®, Betaferon®, among others)), glatiramer (Copaxone®), natalizumab (Tysabri®), and mitoxantrone (Novantrone®).
  • dialkyl fumarates e.g. , DMF or others of Formula A herein
  • Beta interferons e.g., Avonex®, Rebif®, Betaseron®, Betaferon®, among others
  • glatiramer Copaxone®
  • natalizumab Tysabri®
  • mitoxantrone Novantrone®
  • the MS therapies can be administered in combination with one or more additional therapies to treat and/or reduce the symptoms of MS described herein, particularly to treat patients with moderate to severe disability (e.g., EDSS score of 5.5 or higher).
  • the pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other additional therapies or therapeutic agents.
  • each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • the additional therapeutic agent utilized in this combination can be administered together in a single composition or administered separately in different compositions.
  • the particular combination to employ in a regimen will take into account compatibility of the pharmaceutical composition with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved.
  • it is expected that additional therapeutic agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
  • immunomodulatory agents are presently used to modify the course of multiple sclerosis in patients.
  • agents include, but are not limited to, an IFN- ⁇ 1 molecule; a polymer of glutamic acid, lysine, alanine and tyrosine, e.g., glatiramer; an antibody or fragment thereof against alpha-4 integrin, e.g., natalizumab; an
  • anthracenedione molecule e.g., mitoxantrone; a fingolimod, e.g., FTY720; a dimethyl fumarate, e.g., an oral dimethyl fumarate; an antibody to the alpha subunit of the IL-2 receptor of T cells (CD25), e.g., daclizumab; an antibody against CD52, e.g., alemtuzumab; an inhibitor of a dihydroorotate dehydrogenase, e.g., teriflunomide; an antibody to CD20, e.g. , ocrelizumab; a corticosteroid; and anti-Lingo- 1 antibodies.
  • the reparative agents disclosed herein can be used in combination with any of these agents.
  • Interferons are natural proteins produced by the cells of the immune systems of most animals in response to challenges by foreign agents such as viruses, bacteria, parasites and tumor cells. Interferons belong to the large class of glycoproteins known as cytokines. Interferon beta has 165 amino acids. Interferons alpha and beta are produced by many cell types, including T- cells and B-cells, macrophages, fibroblasts, endothelial cells, osteoblasts and others, and stimulate both macrophages and NK cells. Interferon gamma is involved in the regulation of immune and inflammatory responses. It is produced by activated T-cells and Thl cells.
  • Interferon alpha (including forms interferon alpha-2a, interferon alpha- 2b, and interferon alfacon-1) was approved by the United States Food and Drug Administration (FDA) as a treatment for Hepatitis C.
  • FDA United States Food and Drug Administration
  • Interferon beta la (Avonex®) is identical to interferon beta found naturally in humans, and interferon beta lb (Betaseron®) differs in certain ways from interferon beta la found naturally in humans, including that it contains a serine residue in place of a cysteine residue at position 17.
  • interferon beta uses of interferon beta have included treatment of AIDS, cutaneous T-cell lymphoma, Acute Hepatitis C (non-A, non-B), Kaposi's sarcoma, malignant melanoma, hairy cell leukemia, and metastatic renal cell carcinoma.
  • IFNP agents can be administered to the subject by any method known in the art, including systemically (e.g., orally, parenterally, subcutaneously, intravenously, rectally, intramuscularly, intravitreally, intraperitoneally, intranasally, transdermally, or by inhalation or intracavitary installation).
  • systemically e.g., orally, parenterally, subcutaneously, intravenously, rectally, intramuscularly, intravitreally, intraperitoneally, intranasally, transdermally, or by inhalation or intracavitary installation.
  • the IFNP agents are administered subcutaneously, or intramuscularly.
  • IFNP agents can be used as a monotherapy (i.e., as a single "disease modifying therapy") although the treatment regimen can further comprise the use of "symptom management therapies" such as antidepressants, analgesics, anti-tremor agents, etc.
  • the IFNP agent is an IFNP-1A agent (e.g., Avonex®, Rebif®).
  • the INFP agent is an INFP-1B agent (e.g., Betaseron®, Betaferon®, Extavia®).
  • Avonex® an Interferon ⁇ -la
  • Avonex® is indicated for the treatment of patients with relapsing forms of MS to slow the accumulation of physical disability and decrease the frequency of clinical exacerbations.
  • Avonex® (Interferon beta- la) is a 166 amino acid glycoprotein with a predicted molecular weight of approximately 22,500 daltons. It is produced by recombinant DNA technology using genetically engineered Chinese Hamster Ovary cells into which the human interferon beta gene has been introduced. The amino acid sequence of Avonex® is identical to that of natural human interferon beta.
  • the recommended dosage of Avonex® (Interferon beta-la) is 30 meg injected intramuscularly once a week.
  • Avonex® is commercially available as a 30 meg lyophilized powder vial or as a 30 meg prefilled syringe.
  • Interferon beta la (Avonex®) is identical to interferon beta found naturally in humans (AVONEX®, i.e., Interferon beta la (SwissProt Accession No. P01574 and gi:50593016).
  • interferon molecules include, e.g., other interferons and fragments, analogues, homologues, derivatives, and natural variants thereof with substantially similar biological activity.
  • the INFP agent can be modified to increase one or more pharmacokinetic properties.
  • the INFP agent can be a modified form of interferon la to include a pegylated moiety.
  • PEGylated forms of interferon beta la are described in, e.g., Baker, D.P. et al. (2006) Bioconjug Chem 17(1): 179-88; Arduini, RM et al. (2004) Protein Expr Purif 34(2):229-42; Pepinsky, RB et al. (2001) J. Pharmacol. Exp.
  • IFN beta la modified at its N-terminal alpha amino acid to include a PEG moiety, e.g., a 20 kDa mPEG-O-2- methylpropionaldehyde moiety.
  • Pegylated forms of IFN beta la can be administered by, e.g., injectable routes of administration (e.g., subcutaneously).
  • Rebif® is also an Interferon ⁇ -la agent, while Betaseron®, Betaferon®, and
  • Extavia® are Interferon ⁇ -lb agents. Both Rebif® and Betaseron® are formulated for administration by subcutaneous injection.
  • Dosages of IFNP agents to administer can be determined by one of skill in the art, and include clinically acceptable amounts to administer based on the specific interferon-beta agent used.
  • AVONEX® is typically administered at 30 microgram once a week via intramuscular injection.
  • Other forms of interferon beta la, specifically REBIF®, is administered, for example, at 22 microgram three times a week or 44 micrograms once a week, via subcutaneous injection.
  • Interferon beta- 1A can be administered, e.g.,
  • AVONEX® can be administered every five to ten days, e.g., once a week, while Rebif® can be administered three times a week.
  • Anti-VLA4 antibody e.g., Natalizumab (Tysabri®)
  • Natalizumab Tysabri®
  • Anti-VLA4 antibodies inhibit the migration of leukocytes from the blood to the central nervous system. These antibodies bind to VLA-4 (also called ⁇ 4 ⁇ 1) on the surface of activated T-cells and other mononuclear leukocytes. They can disrupt adhesion between the T-cell and endothelial cells, and thus prevent migration of VLA-4 (also called ⁇ 4 ⁇ 1)
  • Natalizumab can decrease the number of brain lesions and clinical relapses and accumulation of disability in patients with relapse remitting multiple sclerosis and relapsing secondary-progressive multiple sclerosis.
  • Natalizumab and related VLA-4 binding antibodies are described, e.g. , in U.S. Pat. No. 5,840,299.
  • Monoclonal antibodies 21.6 and HP1/2 are exemplary murine monoclonal antibodies that bind VLA-4.
  • Natalizumab is a humanized version of murine monoclonal antibody 21.6 (see, e.g., U.S. Pat. No. 5,840,299).
  • a humanized version of HP 1/2 has also been described (see, e.g., U.S. Pat. No. 6,602,503).
  • VLA-4 binding monoclonal antibodies such as HP2/1, HP2/4, L25 and P4C2 are described, e.g., in U.S. Pat. No.
  • DMF Dimethyl fumarate
  • Tecfidera® is a fumaric acid ester.
  • DMF is thought to decrease leukocyte passage through the blood brain barrier and exert neuroprotective effects by the activation of antioxidative pathways, specifically through activation of the Nrf-2 pathway (Lee et al. (2008) Int MS Journal 15: 12-18).
  • BG-12® has the potential to reduce the activity and impact of inflammatory cells on the CNS and induce direct cytoprotective responses in CNS cells. These effects may enhance the CNS cells' ability to mitigate the toxic inflammatory and oxidative stress that plays a role in MS pathophysiology.
  • Copaxone® (glatiramer acetate) consists of the acetate salts of synthetic polypeptides, specifically the four naturally occurring amino acids: L-glutamic acid, L- alanine, L-tyrosine, and L-lysine (Bornstein et al. (1987) N Engl J Med. 317: 408-414). Copaxone® exhibits structural similarity to myelin basic protein and is thought to function as an immune modulator by shifting the T helper cell type 1 response towards a T helper cell type 2 response (Duda et al. (2000) Clin Invest 105: 967-976; Nicholas et al. (2011) Drug Design, Development, and Therapy 5: 255-274). Mitoxantrone (Novantrone®, an anthracenedione molecule)
  • Mitoxantrone is an anthracenedione molecule (l,4-dihydroxy-5,8-bis[2-(2- hydroxyethylamino) ethylamino]-anthracene-9,10-dione) and a type II topoisomerase inhibitor that disrupts DNA synthesis and repair of cells. It is used to treat cancers and MS. Mitoxantrone is used to treat several forms of advancing MS, including secondary progressive MS, progressive relapsing MS, and advanced relapsing-remitting MS.
  • mitoxantrone is effective in slowing the progression of secondary progressive MS and extending the time between relapses in relapsing-remitting MS and progressive relapsing MS (Fox E (2006) Clin Ther 28 (4): 461-74).
  • Fingolimod (Gilenya®; sphingosine 1 -phosphate receptor modulator)
  • Fingolimod is an immunomodulating drug, approved for treating MS. It has reduced the rate of relapses in relapsing-remitting multiple sclerosis by over half, but may have serious adverse effects.
  • Fingolimod is a sphingosine 1-phosphate receptor modulator, which sequesters lymphocytes in lymph nodes, preventing them from moving to the central nervous system for autoimmune responses in MS.
  • Antibodies to the alpha subunit of the IL-2 receptor of T cells (e.g., daclizumab HYP; ZINBRYTA®)
  • CD25 e.g., daclizumab HYP; ZINBRYTA®
  • Daclizumab HYP is a therapeutic humanized monoclonal antibody to the alpha subunit of the IL-2 receptor of T cells (CD25). Daclizumab HYP showed efficacy in reducing lesions and annualized relapse rate in patients with relapsing-remitting multiple sclerosis (Kappos et al. (2015). N. Engl. J. Med. 373 (15): 1418-28).
  • Antibody against CD52 e.g., alemtuzumab
  • Antibodies against CD52 e.g., alemtuzumab (currently under further development as Lemtrada®)
  • bind to CD52 which is a protein present on the surface of mature lymphocytes, but not on stem cells.
  • Phase III studies reported positive results comparing alemtuzumab with Rebif® (high-dose subcutaneous interferon beta- la) in the treatment of patients with relapsing-remitting MS (RRMS).
  • RRMS relapsing-remitting MS
  • Antibody to CD20 e.g., ocrelizumab
  • Antibodies against CD20 e.g., ocrelizumab, rituximab, ofatumumab, target mature B lymphocytes.
  • Phase 2 clinical studies of rituximab and ocrelizumab in relapse remitting MS have demonstrated a statistically significant reduction in disease activity measured by brain lesions (e.g., measured by MRI scans) and relapse rate compared to placebo.
  • Phase 3 studies of ocrelizumab showed both reduction in relapse rate and disability compared to interferon beta- la (e.g., Rebif®).
  • Inhibitors of dihydroorotate dehydrogenase e.g., teriflunomide
  • Inhibitors of dihydroorotate dehydrogenase e.g., teriflunomide
  • teriflunomide also known as A77 1726 or
  • Teriflunomide is an active metabolite of leflunomide.
  • Teriflunomide inhibits rapidly dividing cells, including activated T cells, which are thought to drive the disease process in MS.
  • Teriflunomide was investigated in clinical trials as a medication for treating MS. (Vollmer EMS News (May 28, 2009)).
  • Steroids e.g., corticosteroid, and ACTH agents can be used to treat acute relapses in relapsing-remitting MS or secondary progressive MS.
  • Such agents include, but are not limited to, Depo-Medrol®, Solu-Medrol®, Deltasone®, Delta-Cortef®, Medrol®,
  • Anti-Lingo- 1 antibodies function by restoring myelin. Anti-Lingo- 1 antibodies can be used to treat relapsing MS.
  • the method further includes the use of one or more symptom management therapies, such as antidepressants, analgesics, anti-tremor agents, among others.
  • symptom management therapies such as antidepressants, analgesics, anti-tremor agents, among others.
  • Treatment of a subject with a disease modifying MS therapy can be combined with one or more of the following therapies often used in symptom
  • MARINOL® synthetic cannabinoids
  • TRENT AL® pentoxifylline
  • NEUROFEN® ibuprofen
  • aspirin acetaminophen
  • ATARAX® hydroxyzine
  • PROZAC® (fluoxetine), ZOLOFT® (sertraline), LUSTRAL® (sertraline), EFFEXOR XR® (venlafaxine), CELEXA® (citalopram), PAXIL®, SEROXAT®, DESYREL® (trazodone), TRIALODINE® (trazodone), PAMELOR® (nortriptyline), AVENTYL® (imipramine), PROTHIADEN® (dothiepin), GAMANIL® (lofepramine), PARNATE® (tranylcypromine), MANERIX® (moclobemide), AURORIX® (moclobemide),
  • WELLBUTRIN SR® (bupropion), AMFEBUTAMONE® (bupropion), SERZONE® (nefazodone), REMERON® (mirtazapine), AMBIEN® (Zolpidem), XANAX®
  • DULCOLAX® bisacodyl
  • BISACOLAX® bisacodyl
  • SANI-SUPP® glycerin
  • METAMUCIL® psyllium hydrophilic mucilloid
  • FLEET ENEMA® sodium phosphate
  • COLACE® docusate
  • THEREVAC PLUS® KLONOPIN®
  • RIVOTRIL® clonazepam
  • DANTRIUM® diantrolen sodium
  • ZANAFLEX® (tizanidine), SIRDALUD® (tizanidine), MYSOLINE® (primidone), DIAMOX® (acetozolamide), SINEMET® (levodopa, carbidopa), LANIAZID®
  • PANGLOBULIN® IV immunoglobulin
  • SANDOGLOBULIN® IV immunoglobulin
  • VENOBLOGULIN® IV immunoglobulin
  • pregabalin ziconotide
  • ANERGIX- MS® ANERGIX- MS®.
  • the strongest risk factor for MS is thought to be the human leukocyte antigen (HLA) region in the major histocompatibility complex (MHC) on chromosome 6.
  • HLA human leukocyte antigen
  • MHC major histocompatibility complex
  • PPMS Sanna Nat Genet 2 Primary progressive MS
  • VEP Variants were annotated with the Ensembl Variant Effect Predictor
  • the significance threshold for the single variant tests was P ⁇ 5x10 - " 8.
  • Four rare, damaging LIX1L variants were analyzed, and were carried by four different PPMS cases and no RRMS patients or controls.
  • the rare Rl 1H missense mutation (rs 142637090) has been observed in only 5 out of 60,703 exomes in ExAC, and the other three variants have not been observed in the ExAC database.
  • the variants are Rl 14H, V173L, S286C, and L322F. Only one of the four variants (Rl 14H) is located in the LIX1L putative RNA binding domain.
  • the LIXl (Limb & CNS Expressed 1) gene was initially identified in a screen for genes transiently expressed during early chicken limb development (Swindell et al., 2001, Mechanisms of Development).
  • the limb expression 1 Pfam domain is present in only two human proteins: LIXl and LIXIL.
  • LIXl is specific to brain and spinal cord; LIXIL is expressed in brain and spinal cord but is not specific.
  • the BioPlex protein-protein interaction network indicates that LIXIL interacts with the calpain CAPNS2 and the endogenous calpain inhibitor CAST.
  • the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim.
  • elements are presented as lists, e.g. , in Markush group format or as an alternative, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.

Abstract

La présente invention concerne des procédés, des analyses et des trousses pour l'identification, l'évaluation et/ou le traitement d'un sujet présentant une sclérose en plaques (SP) (par exemple, un patient ayant une sclérose en plaques progressive primaire (SPPP)).
PCT/US2017/049599 2016-09-01 2017-08-31 Biomarqueurs prédictifs de la sclérose en plaques progressive primaire et leurs utilisations WO2018045162A1 (fr)

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